var bibbase_data = {"data":"<img src=\"//bibbase.org/img/ajax-loader.gif\" id=\"spinner\"\n  style=\"display: none;\" alt=\"Loading..\" />\n\n<div id=\"bibbase\">\n\n  <script type=\"text/javascript\">\n    var bibbase = {\n      params: {\"bib\":\"https://raw.githubusercontent.com/rosenzweiglab/rosenzweiglab.github.io/master/docs/references.bib\",\"authorfirst\":\"1\",\"theme\":\"simple\",\"jsonp\":\"1\",\"host\":\"bibbase.org\"},\n      data: [{\"bibtype\":\"article\",\"type\":\"article\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Fattah\"],\"firstnames\":[\"Abdel\",\"Rahman\",\"Abdel\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Daza\"],\"firstnames\":[\"Brian\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rustandi\"],\"firstnames\":[\"Gregorius\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Berrocal-Rubio\"],\"firstnames\":[\"Miguel\",\"Angel\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Gorissen\"],\"firstnames\":[\"Benjamin\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Poovathingal\"],\"firstnames\":[\"Suresh\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Davie\"],\"firstnames\":[\"Kristofer\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Cao\"],\"firstnames\":[\"Xuanye\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"Hadar\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Lei\"],\"firstnames\":[\"Yunping\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Finnell\"],\"firstnames\":[\"Richard\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Verfaillie\"],\"firstnames\":[\"Catherine\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Sampaolesi\"],\"firstnames\":[\"Maurilio\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Dedecker\"],\"firstnames\":[\"Peter\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Van\",\"Oosterwyck\"],\"firstnames\":[\"Hans\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Aerts\"],\"firstnames\":[\"Stein\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ranga\"],\"firstnames\":[\"Adrian\"],\"suffixes\":[]}],\"title\":\"Actuation Enhances Patterning in Human Neural Tube Organoids\",\"elocation-id\":\"2020.09.22.308411\",\"year\":\"2020\",\"doi\":\"10.1101/2020.09.22.308411\",\"publisher\":\"Cold Spring Harbor Laboratory\",\"abstract\":\"Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.Competing Interest StatementRF was CEO of Teratomic Consulting, LLC, which has been dissolved. He is also an associate editor of the journal Reproductive and Developmental Medicine, which provides his travel expenses.\",\"url\":\"https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411\",\"eprint\":\"https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411.full.pdf\",\"journal\":\"bioRxiv\",\"bibtex\":\"@article {Fattah2020.09.22.308411,\\n\\tauthor = {Fattah, Abdel Rahman Abdel and Daza, Brian and Rustandi, Gregorius and Berrocal-Rubio, Miguel Angel and Gorissen, Benjamin and Poovathingal, Suresh and Davie, Kristofer and Cao, Xuanye and Rosenzweig, Derek Hadar and Lei, Yunping and Finnell, Richard and Verfaillie, Catherine and Sampaolesi, Maurilio and Dedecker, Peter and Van Oosterwyck, Hans and Aerts, Stein and Ranga, Adrian},\\n\\ttitle = {Actuation Enhances Patterning in Human Neural Tube Organoids},\\n\\telocation-id = {2020.09.22.308411},\\n\\tyear = {2020},\\n\\tdoi = {10.1101/2020.09.22.308411},\\n\\tpublisher = {Cold Spring Harbor Laboratory},\\n\\tabstract = {Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.Competing Interest StatementRF was CEO of Teratomic Consulting, LLC, which has been dissolved. He is also an associate editor of the journal Reproductive and Developmental Medicine, which provides his travel expenses.},\\n\\tURL = {https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411},\\n\\teprint = {https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411.full.pdf},\\n\\tjournal = {bioRxiv}\\n}\\n\\n\",\"author_short\":[\"Fattah, A. R. A.\",\"Daza, B.\",\"Rustandi, G.\",\"Berrocal-Rubio, M. A.\",\"Gorissen, B.\",\"Poovathingal, S.\",\"Davie, K.\",\"Cao, X.\",\"Rosenzweig, D. H.\",\"Lei, Y.\",\"Finnell, R.\",\"Verfaillie, C.\",\"Sampaolesi, M.\",\"Dedecker, P.\",\"Van Oosterwyck, H.\",\"Aerts, S.\",\"Ranga, A.\"],\"key\":\"Fattah2020.09.22.308411\",\"id\":\"Fattah2020.09.22.308411\",\"bibbaseid\":\"fattah-daza-rustandi-berrocalrubio-gorissen-poovathingal-davie-cao-etal-actuationenhancespatterninginhumanneuraltubeorganoids-2020\",\"role\":\"author\",\"urls\":{\"Paper\":\"https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411\"},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":4,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Following the advancements in microfluidics and lab-on-a-chip (LOC) technologies, a novel biomedical application for microfluidic based devices has emerged in recent years and microengineered cell culture platforms have been created. These micro-devices, known as organ-on-a-chip (OOC) platforms mimic the in vivo like microenvironment of living organs and offer more physiologically relevant in vitro models of human organs. Consequently, the concept of OOC has gained great attention from researchers in the field worldwide to offer powerful tools for biomedical researches including disease modeling, drug development, etc. This review highlights the background of biochip development. Herein, we focus on applications of LOC devices as a versatile tool for POC applications. We also review current progress in OOC platforms towards body-on-a-chip, and we provide concluding remarks and future perspectives for OOC platforms for POC applications.\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Azizipour\"],\"firstnames\":[\"Neda\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Avazpour\"],\"firstnames\":[\"Rahi\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Sawan\"],\"firstnames\":[\"Mohamad\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ajji\"],\"firstnames\":[\"Abdellah\"],\"suffixes\":[]}],\"doi\":\"10.3390/mi11060599\",\"issn\":\"2072666X\",\"journal\":\"Micromachines\",\"keywords\":\"BioMEMS,Lab-on-a-chip,Microengineering,Microfluidics,Organ-on-a-chip,Personalized medicine,Point-of-care\",\"number\":\"6\",\"pages\":\"1–15\",\"title\":\"Evolution of biochip technology: A review from lab-on-a-chip to organ-on-a-chip\",\"volume\":\"11\",\"year\":\"2020\",\"bibtex\":\"@article{Azizipour2020,\\nabstract = {Following the advancements in microfluidics and lab-on-a-chip (LOC) technologies, a novel biomedical application for microfluidic based devices has emerged in recent years and microengineered cell culture platforms have been created. These micro-devices, known as organ-on-a-chip (OOC) platforms mimic the in vivo like microenvironment of living organs and offer more physiologically relevant in vitro models of human organs. Consequently, the concept of OOC has gained great attention from researchers in the field worldwide to offer powerful tools for biomedical researches including disease modeling, drug development, etc. This review highlights the background of biochip development. Herein, we focus on applications of LOC devices as a versatile tool for POC applications. We also review current progress in OOC platforms towards body-on-a-chip, and we provide concluding remarks and future perspectives for OOC platforms for POC applications.},\\nauthor = {Azizipour, Neda and Avazpour, Rahi and Rosenzweig, Derek H. and Sawan, Mohamad and Ajji, Abdellah},\\ndoi = {10.3390/mi11060599},\\nissn = {2072666X},\\njournal = {Micromachines},\\nkeywords = {BioMEMS,Lab-on-a-chip,Microengineering,Microfluidics,Organ-on-a-chip,Personalized medicine,Point-of-care},\\nnumber = {6},\\npages = {1--15},\\ntitle = {{Evolution of biochip technology: A review from lab-on-a-chip to organ-on-a-chip}},\\nvolume = {11},\\nyear = {2020}\\n}\\n\",\"author_short\":[\"Azizipour, N.\",\"Avazpour, R.\",\"Rosenzweig, D. H.\",\"Sawan, M.\",\"Ajji, A.\"],\"key\":\"Azizipour2020\",\"id\":\"Azizipour2020\",\"bibbaseid\":\"azizipour-avazpour-rosenzweig-sawan-ajji-evolutionofbiochiptechnologyareviewfromlabonachiptoorganonachip-2020\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"BioMEMS\",\"Lab-on-a-chip\",\"Microengineering\",\"Microfluidics\",\"Organ-on-a-chip\",\"Personalized medicine\",\"Point-of-care\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"The current gold standard technique for treatment of anterior cruciate ligament (ACL) injury is reconstruction with autograft. These treatments have a relatively high failure and re-tear rate. To overcome this, tissue engineering and additive manufacturing are being used to explore the potential of 3D scaffolds as autograft substitutes. However, mechanically optimal polymers for this have yet to be identified. Here, we use 3D printing technology and various materials with the aim of fabricating constructs better matching the mechanical properties of the native ACL. A fused deposition modeling (FDM) 3D printer was used to microfabricate dog bone-shaped specimens from six different polymers-PLA, PETG, Lay FOMM 60, NinjaFlex, NinjaFlex-SemiFlex, and FlexiFil-at three different raster angles. The tensile mechanical properties of these polymers were determined from stress-strain curves. Our results indicate that no single material came close enough to successfully match reported mechanical properties of the native ACL. However, PLA and PETG had similar ultimate tensile strengths. Lay FOMM 60 displayed a percentage strain at failure similar to reported values for native ACL. Furthermore, raster angle had a significant impact on some mechanical properties for all of the materials except for FlexiFil. We therefore conclude that while none of these materials alone is optimal for mimicking ACL mechanical properties, there may be potential for creating a 3D-printed composite constructs to match ACL mechanical properties. Further investigations involving co-printing of stiff and elastomeric materials must be explored.\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Pitaru\"],\"firstnames\":[\"Audrey\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Lacombe\"],\"firstnames\":[\"Jean\",\"Gabriel\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Cooke\"],\"firstnames\":[\"Megan\",\"E.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Beckman\"],\"firstnames\":[\"Lorne\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Steffen\"],\"firstnames\":[\"Thomas\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Martineau\"],\"firstnames\":[\"Paul\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]}],\"doi\":\"10.3390/MI11090846\",\"issn\":\"2072666X\",\"journal\":\"Micromachines\",\"keywords\":\"3D printing,Elastic,Ligament,Mechanical strain,Polymers,Scaffolds,Tissue engineering\",\"number\":\"9\",\"pages\":\"1–15\",\"title\":\"Investigating commercial filaments for 3D printing of stiff and elastic constructs with ligament-like mechanics\",\"volume\":\"11\",\"year\":\"2020\",\"bibtex\":\"@article{Pitaru2020,\\nabstract = {The current gold standard technique for treatment of anterior cruciate ligament (ACL) injury is reconstruction with autograft. These treatments have a relatively high failure and re-tear rate. To overcome this, tissue engineering and additive manufacturing are being used to explore the potential of 3D scaffolds as autograft substitutes. However, mechanically optimal polymers for this have yet to be identified. Here, we use 3D printing technology and various materials with the aim of fabricating constructs better matching the mechanical properties of the native ACL. A fused deposition modeling (FDM) 3D printer was used to microfabricate dog bone-shaped specimens from six different polymers-PLA, PETG, Lay FOMM 60, NinjaFlex, NinjaFlex-SemiFlex, and FlexiFil-at three different raster angles. The tensile mechanical properties of these polymers were determined from stress-strain curves. Our results indicate that no single material came close enough to successfully match reported mechanical properties of the native ACL. However, PLA and PETG had similar ultimate tensile strengths. Lay FOMM 60 displayed a percentage strain at failure similar to reported values for native ACL. Furthermore, raster angle had a significant impact on some mechanical properties for all of the materials except for FlexiFil. We therefore conclude that while none of these materials alone is optimal for mimicking ACL mechanical properties, there may be potential for creating a 3D-printed composite constructs to match ACL mechanical properties. Further investigations involving co-printing of stiff and elastomeric materials must be explored.},\\nauthor = {Pitaru, Audrey A. and Lacombe, Jean Gabriel and Cooke, Megan E. and Beckman, Lorne and Steffen, Thomas and Weber, Michael H. and Martineau, Paul A. and Rosenzweig, Derek H.},\\ndoi = {10.3390/MI11090846},\\nissn = {2072666X},\\njournal = {Micromachines},\\nkeywords = {3D printing,Elastic,Ligament,Mechanical strain,Polymers,Scaffolds,Tissue engineering},\\nnumber = {9},\\npages = {1--15},\\ntitle = {{Investigating commercial filaments for 3D printing of stiff and elastic constructs with ligament-like mechanics}},\\nvolume = {11},\\nyear = {2020}\\n}\\n\\n  \",\"author_short\":[\"Pitaru, A. A.\",\"Lacombe, J. G.\",\"Cooke, M. E.\",\"Beckman, L.\",\"Steffen, T.\",\"Weber, M. H.\",\"Martineau, P. A.\",\"Rosenzweig, D. H.\"],\"key\":\"Pitaru2020\",\"id\":\"Pitaru2020\",\"bibbaseid\":\"pitaru-lacombe-cooke-beckman-steffen-weber-martineau-rosenzweig-investigatingcommercialfilamentsfor3dprintingofstiffandelasticconstructswithligamentlikemechanics-2020\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"3D printing\",\"Elastic\",\"Ligament\",\"Mechanical strain\",\"Polymers\",\"Scaffolds\",\"Tissue engineering\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":2,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"With the expiration of key patents on additive manufacturing, 3D printing devices have become extremely inexpensive. Therefore, the use of 3D printing in biomedical applications has blossomed in the past decade. Low-cost, high-quality instruments have become widely used in both industrial and academic settings. Fused deposition modeling using polymers such as polylactic acid, polycaprolactone, or other composites are now combined with bioprinting to open new avenues to cutting-edge research toward musculoskeletal repair and regeneration such as prevascularized bone or soft-tissue constructs. Most importantly, these tools are being widely used to generate composite scaffolds representing matrices on which to culture cells of various tissue types such as bone, cartilage, cardiac, and nervous tissue. 3D printed composite matrix scaffolds are being tested in sophisticated in vitro and in vivo preclinical models, paving the way for future clinical translation where the ultimate goal is to generate functional replacement tissues.\",\"annote\":\"cited By 0\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ahangar\"],\"firstnames\":[\"Pouyan\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]}],\"doi\":\"10.1016/j.cobme.2019.06.002\",\"issn\":\"24684511\",\"journal\":\"Current Opinion in Biomedical Engineering\",\"keywords\":\"3D printed,Bioprinted,Composite,Hydrogels,Matrix scaffold,Tissue engineering\",\"pages\":\"142–148\",\"title\":\"Advancements in 3D printed scaffolds to mimic matrix complexities for musculoskeletal repair\",\"volume\":\"10\",\"year\":\"2019\",\"bibtex\":\"@article{Haglund2019142,\\n  abstract = {With the expiration of key patents on additive manufacturing, 3D printing devices have become extremely inexpensive. Therefore, the use of 3D printing in biomedical applications has blossomed in the past decade. Low-cost, high-quality instruments have become widely used in both industrial and academic settings. Fused deposition modeling using polymers such as polylactic acid, polycaprolactone, or other composites are now combined with bioprinting to open new avenues to cutting-edge research toward musculoskeletal repair and regeneration such as prevascularized bone or soft-tissue constructs. Most importantly, these tools are being widely used to generate composite scaffolds representing matrices on which to culture cells of various tissue types such as bone, cartilage, cardiac, and nervous tissue. 3D printed composite matrix scaffolds are being tested in sophisticated in vitro and in vivo preclinical models, paving the way for future clinical translation where the ultimate goal is to generate functional replacement tissues.},\\n  annote = {cited By 0},\\n  author = {Haglund, Lisbet and Ahangar, Pouyan and Rosenzweig, Derek H.},\\n  doi = {10.1016/j.cobme.2019.06.002},\\n  issn = {24684511},\\n  journal = {Current Opinion in Biomedical Engineering},\\n  keywords = {3D printed,Bioprinted,Composite,Hydrogels,Matrix scaffold,Tissue engineering},\\n  pages = {142--148},\\n  title = {{Advancements in 3D printed scaffolds to mimic matrix complexities for musculoskeletal repair}},\\n  volume = {10},\\n  year = {2019}\\n  }\\n  \",\"author_short\":[\"Haglund, L.\",\"Ahangar, P.\",\"Rosenzweig, D. H.\"],\"key\":\"Haglund2019142\",\"id\":\"Haglund2019142\",\"bibbaseid\":\"haglund-ahangar-rosenzweig-advancementsin3dprintedscaffoldstomimicmatrixcomplexitiesformusculoskeletalrepair-2019\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"3D printed\",\"Bioprinted\",\"Composite\",\"Hydrogels\",\"Matrix scaffold\",\"Tissue engineering\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 mum), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 mum pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 mum pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 mum pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.\",\"annote\":\"cited By 7\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Fairag\"],\"firstnames\":[\"Rayan\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ramirez-Garcialuna\"],\"firstnames\":[\"Jose\",\"L.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.1021/acsami.9b02502\",\"issn\":\"19448252\",\"journal\":\"ACS Applied Materials and Interfaces\",\"keywords\":\"3D printing,PLA,bone defect,bone repair,human osteoblasts,low-cost,mesenchymal stem cells,tissue engineering\",\"number\":\"17\",\"pages\":\"15306–15315\",\"title\":\"Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bone-like Matrix Deposition in Vitro\",\"volume\":\"11\",\"year\":\"2019\",\"bibtex\":\"@article{Fairag201915306,\\n  abstract = {Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 mum), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 mum pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 mum pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 mum pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.},\\n  annote = {cited By 7},\\n  author = {Fairag, Rayan and Rosenzweig, Derek H. and Ramirez-Garcialuna, Jose L. and Weber, Michael H. and Haglund, Lisbet},\\n  doi = {10.1021/acsami.9b02502},\\n  issn = {19448252},\\n  journal = {ACS Applied Materials and Interfaces},\\n  keywords = {3D printing,PLA,bone defect,bone repair,human osteoblasts,low-cost,mesenchymal stem cells,tissue engineering},\\n  number = {17},\\n  pages = {15306--15315},\\n  title = {{Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bone-like Matrix Deposition in Vitro}},\\n  volume = {11},\\n  year = {2019}\\n  }\\n  \",\"author_short\":[\"Fairag, R.\",\"Rosenzweig, D. H.\",\"Ramirez-Garcialuna, J. L.\",\"Weber, M. H.\",\"Haglund, L.\"],\"key\":\"Fairag201915306\",\"id\":\"Fairag201915306\",\"bibbaseid\":\"fairag-rosenzweig-ramirezgarcialuna-weber-haglund-threedimensionalprintedpolylacticacidscaffoldspromotebonelikematrixdepositioninvitro-2019\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"3D printing\",\"PLA\",\"bone defect\",\"bone repair\",\"human osteoblasts\",\"low-cost\",\"mesenchymal stem cells\",\"tissue engineering\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Additive manufacturing (AM) has emerged over the past four decades as a cost-effective, on-demand modality for fabrication of geometrically complex objects. The ability to design and print virtually any object shape using a diverse array of materials, such as metals, polymers, ceramics and bioinks, has allowed for the adoption of this technology for biomedical applications in both research and clinical settings. Current advancements in tissue engineering and regeneration, therapeutic delivery, medical device fabrication and operative management planning ensure that AM will continue to play an increasingly important role in the future of healthcare. In this review, we outline current biomedical applications of common AM techniques and materials.\",\"annote\":\"cited By 11\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Ahangar\"],\"firstnames\":[\"Pouyan\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Cooke\"],\"firstnames\":[\"Megan\",\"E.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]}],\"doi\":\"10.3390/app9081713\",\"issn\":\"20763417\",\"journal\":\"Applied Sciences (Switzerland)\",\"keywords\":\"3D printing,Additive manufacturing,Biomedical devices,Bioprinting,Rapid prototyping,Regenerative medicine,Tissue engineering\",\"number\":\"8\",\"title\":\"Current biomedical applications of 3D printing and additive manufacturing\",\"volume\":\"9\",\"year\":\"2019\",\"bibtex\":\"@article{Ahangar2019,\\n  abstract = {Additive manufacturing (AM) has emerged over the past four decades as a cost-effective, on-demand modality for fabrication of geometrically complex objects. The ability to design and print virtually any object shape using a diverse array of materials, such as metals, polymers, ceramics and bioinks, has allowed for the adoption of this technology for biomedical applications in both research and clinical settings. Current advancements in tissue engineering and regeneration, therapeutic delivery, medical device fabrication and operative management planning ensure that AM will continue to play an increasingly important role in the future of healthcare. In this review, we outline current biomedical applications of common AM techniques and materials.},\\n  annote = {cited By 11},\\n  author = {Ahangar, Pouyan and Cooke, Megan E. and Weber, Michael H. and Rosenzweig, Derek H.},\\n  doi = {10.3390/app9081713},\\n  issn = {20763417},\\n  journal = {Applied Sciences (Switzerland)},\\n  keywords = {3D printing,Additive manufacturing,Biomedical devices,Bioprinting,Rapid prototyping,Regenerative medicine,Tissue engineering},\\n  number = {8},\\n  title = {{Current biomedical applications of 3D printing and additive manufacturing}},\\n  volume = {9},\\n  year = {2019}\\n  }\\n  \",\"author_short\":[\"Ahangar, P.\",\"Cooke, M. E.\",\"Weber, M. H.\",\"Rosenzweig, D. H.\"],\"key\":\"Ahangar2019\",\"id\":\"Ahangar2019\",\"bibbaseid\":\"ahangar-cooke-weber-rosenzweig-currentbiomedicalapplicationsof3dprintingandadditivemanufacturing-2019\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"3D printing\",\"Additive manufacturing\",\"Biomedical devices\",\"Bioprinting\",\"Rapid prototyping\",\"Regenerative medicine\",\"Tissue engineering\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Background: Bisphosphonates (BPs) including zoledronate (zol) have become standard care for bone metastases as they effectively inhibit tumor-induced osteolysis and associated pain. Several studies have also suggested that zol has direct anti-tumor activity. Systemic administration at high doses is the current approach to deliver zol, yet it has been associated with debilitating side effects. Local therapeutic delivery offers the ability to administer much lower total dosage, while at the same time maintaining sustained high-local drug concentration directly at the target treatment site. Here, we aimed to assess effects of lower doses of zol on bone metastases over a longer time. Methods: Prostate cancer cell line LAPC4 and prostate-induced bone metastasis cells were treated with zol at 1, 3 and 10 muM for 7 days. Following treatment, cell proliferation was assessed using Almarblue(R), Vybrant MTT(R), and Live/Dead(R) viability/cytotoxicity assays. Additionally, cell migration and invasion were carried out using Falcon™ cell culture inserts and Cultrex(R) 3D spheroid cell invasion assays respectively. Results: We show that treatment with 3-10 muM zol over 7-days significantly decreased cell proliferation in both the prostate cancer cell line LAPC4 and cells from spine metastases secondary to prostate cancer. Using the same low-dose and longer time course for treatment, we demonstrate that 10 muM zol also significantly inhibits tumor cell migration and 3D-cell growth/invasion. Conclusions: This project harnesses the potential of using zol at low doses for longer treatment periods, which may be a viable treatment modality when coupled with biomaterials or biodevices for local delivery.\",\"annote\":\"cited By 1\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Akoury\"],\"firstnames\":[\"Elie\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ahangar\"],\"firstnames\":[\"Pouyan\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Nour\"],\"firstnames\":[\"Antone\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Lapointe\"],\"firstnames\":[\"Jacques\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Gu'erard\"],\"firstnames\":[\"Karl\",\"Philippe\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]}],\"doi\":\"10.1186/s12935-019-0745-x\",\"issn\":\"14752867\",\"journal\":\"Cancer Cell International\",\"keywords\":\"Bone metastases secondary to prostate,Cellular assays,Direct in vitro treatment,Low doses,Zoledronate\",\"number\":\"1\",\"title\":\"Low-dose zoledronate for the treatment of bone metastasis secondary to prostate cancer\",\"volume\":\"19\",\"year\":\"2019\",\"bibtex\":\"@article{Akoury2019,\\n  abstract = {Background: Bisphosphonates (BPs) including zoledronate (zol) have become standard care for bone metastases as they effectively inhibit tumor-induced osteolysis and associated pain. Several studies have also suggested that zol has direct anti-tumor activity. Systemic administration at high doses is the current approach to deliver zol, yet it has been associated with debilitating side effects. Local therapeutic delivery offers the ability to administer much lower total dosage, while at the same time maintaining sustained high-local drug concentration directly at the target treatment site. Here, we aimed to assess effects of lower doses of zol on bone metastases over a longer time. Methods: Prostate cancer cell line LAPC4 and prostate-induced bone metastasis cells were treated with zol at 1, 3 and 10 muM for 7 days. Following treatment, cell proliferation was assessed using Almarblue{(R)}, Vybrant MTT{(R)}, and Live/Dead{(R)} viability/cytotoxicity assays. Additionally, cell migration and invasion were carried out using Falcon™ cell culture inserts and Cultrex{(R)} 3D spheroid cell invasion assays respectively. Results: We show that treatment with 3-10 muM zol over 7-days significantly decreased cell proliferation in both the prostate cancer cell line LAPC4 and cells from spine metastases secondary to prostate cancer. Using the same low-dose and longer time course for treatment, we demonstrate that 10 muM zol also significantly inhibits tumor cell migration and 3D-cell growth/invasion. Conclusions: This project harnesses the potential of using zol at low doses for longer treatment periods, which may be a viable treatment modality when coupled with biomaterials or biodevices for local delivery.},\\n  annote = {cited By 1},\\n  author = {Akoury, Elie and Ahangar, Pouyan and Nour, Antone and Lapointe, Jacques and Gu{'{e}}rard, Karl Philippe and Haglund, Lisbet and Rosenzweig, Derek H. and Weber, Michael H.},\\n  doi = {10.1186/s12935-019-0745-x},\\n  issn = {14752867},\\n  journal = {Cancer Cell International},\\n  keywords = {Bone metastases secondary to prostate,Cellular assays,Direct in vitro treatment,Low doses,Zoledronate},\\n  number = {1},\\n  title = {{Low-dose zoledronate for the treatment of bone metastasis secondary to prostate cancer}},\\n  volume = {19},\\n  year = {2019}\\n  }\\n  \",\"author_short\":[\"Akoury, E.\",\"Ahangar, P.\",\"Nour, A.\",\"Lapointe, J.\",\"Gu'erard, K. P.\",\"Haglund, L.\",\"Rosenzweig, D. H.\",\"Weber, M. H.\"],\"key\":\"Akoury2019\",\"id\":\"Akoury2019\",\"bibbaseid\":\"akoury-ahangar-nour-lapointe-guerard-haglund-rosenzweig-weber-lowdosezoledronateforthetreatmentofbonemetastasissecondarytoprostatecancer-2019\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Bone metastases secondary to prostate\",\"Cellular assays\",\"Direct in vitro treatment\",\"Low doses\",\"Zoledronate\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"conference\",\"type\":\"conference\",\"abstract\":\"Zoledronate (Zol) is a bone-preserving/ anti-Tumoral drug that is widely used for the treatment of many cancers including spinal bone metastases. High systemic Zol doses required to elicit an adequate effect in the spine often lead to significant side effects, limiting its prolonged use and effectiveness. Here, we aim to provide an alternative strategy to locally deliver Zol at the tumor site. We show that nanoporous 3D-printed scaffolds can be loaded with Zol and possess the ability to release Zol (10-28%) over a sustained period. Additionally, we demonstrate that Zol-impregnated scaffolds, mostly Gel Lay, impair the proliferation of the prostate cancer cell line LAPC4 and the prostate-induced bone metastasis cells in vitro. 3D-printed nanoporous polymers offer a novel and versatile opportunity for potential local delivery of drugs in future clinical settings. These polymers can decrease systemic exposure and related side effects of Zol while at the same time concentrating the drug effect at the tumor site thereby inhibiting tumor proliferation. Also, these scaffolds could be co-printed or coupled with other materials to produce custom implants that offer better structural support for bone growth at the tumor site following resection.\",\"annote\":\"cited By 1\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Akoury\"],\"firstnames\":[\"Elie\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]}],\"booktitle\":\"MRS Advances\",\"doi\":\"10.1557/adv.2019.156\",\"issn\":\"20598521\",\"keywords\":\"3D printing,nanostructure\",\"number\":\"21\",\"pages\":\"1245–1251\",\"title\":\"3D-Printed Nanoporous Scaffolds Impregnated with Zoledronate for the Treatment of Spinal Bone Metastases\",\"volume\":\"4\",\"year\":\"2019\",\"bibtex\":\"@conference{Akoury20191245,\\n  abstract = {Zoledronate (Zol) is a bone-preserving/ anti-Tumoral drug that is widely used for the treatment of many cancers including spinal bone metastases. High systemic Zol doses required to elicit an adequate effect in the spine often lead to significant side effects, limiting its prolonged use and effectiveness. Here, we aim to provide an alternative strategy to locally deliver Zol at the tumor site. We show that nanoporous 3D-printed scaffolds can be loaded with Zol and possess the ability to release Zol (10-28%) over a sustained period. Additionally, we demonstrate that Zol-impregnated scaffolds, mostly Gel Lay, impair the proliferation of the prostate cancer cell line LAPC4 and the prostate-induced bone metastasis cells in vitro. 3D-printed nanoporous polymers offer a novel and versatile opportunity for potential local delivery of drugs in future clinical settings. These polymers can decrease systemic exposure and related side effects of Zol while at the same time concentrating the drug effect at the tumor site thereby inhibiting tumor proliferation. Also, these scaffolds could be co-printed or coupled with other materials to produce custom implants that offer better structural support for bone growth at the tumor site following resection.},\\n  annote = {cited By 1},\\n  author = {Akoury, Elie and Weber, Michael H. and Rosenzweig, Derek H.},\\n  booktitle = {MRS Advances},\\n  doi = {10.1557/adv.2019.156},\\n  issn = {20598521},\\n  keywords = {3D printing,nanostructure},\\n  number = {21},\\n  pages = {1245--1251},\\n  title = {{3D-Printed Nanoporous Scaffolds Impregnated with Zoledronate for the Treatment of Spinal Bone Metastases}},\\n  volume = {4},\\n  year = {2019}\\n  }\\n  \",\"author_short\":[\"Akoury, E.\",\"Weber, M. H.\",\"Rosenzweig, D. H.\"],\"key\":\"Akoury20191245\",\"id\":\"Akoury20191245\",\"bibbaseid\":\"akoury-weber-rosenzweig-3dprintednanoporousscaffoldsimpregnatedwithzoledronateforthetreatmentofspinalbonemetastases-2019\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"3D printing\",\"nanostructure\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"The spine is the most common site of bone metastasis, often originating from prostate, lung, and breast cancers. High systemic doses of chemotherapeutics such as doxorubicin (DOX), cisplatin, or paclitaxel often have severe side effects. Surgical removal of spine metastases also leaves large defects which cannot spontaneously heal and require bone grafting. To circumvent these issues, we designed an approach for local chemotherapeutic delivery within 3D-printed scaffolds which could also potentially serve as a bone substitute. Direct treatment of prostate cancer cell line LAPC4 and patient derived spine metastases cells with 0.01 muM DOX significantly reduced metabolic activity, proliferation, migration, and spheroid growth. We then assessed uptake and release of DOX in a series of porous 3D-printed scaffolds on LAPC4 cells as well as patient-derived spine metastases cells. Over seven days, 60-75% of DOX loaded onto scaffolds could be released, which significantly reduced metabolic activity and proliferation of both LAPC4 and patient derived cells, while unloaded scaffolds had no effect. Porous 3D-printed scaffolds may provide a novel and inexpensive approach to locally deliver chemotherapeutics in a patient-specific manner at tumor resection sites. With a composite design to enhance strength and promote sustained drug release, the scaffolds could reduce systemic negative effects, enhance bone repair, and improve patient outcomes.\",\"annote\":\"cited By 5\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Ahangar\"],\"firstnames\":[\"Pouyan\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Akoury\"],\"firstnames\":[\"Elie\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Luna\"],\"firstnames\":[\"Ana\",\"Sofia\",\"Ramirez\",\"Garcia\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Nour\"],\"firstnames\":[\"Antone\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]}],\"doi\":\"10.3390/ma11091485\",\"issn\":\"19961944\",\"journal\":\"Materials\",\"keywords\":\"Bone metastases,Bone substitute,Doxorubicin,Local delivery,Low-cost 3D printing,Nanoporous filament,Prostate cancer\",\"number\":\"9\",\"title\":\"Nanoporous 3D-printed scaffolds for local doxorubicin delivery in bone metastases secondary to prostate cancer\",\"volume\":\"11\",\"year\":\"2018\",\"bibtex\":\"@article{Ahangar2018,\\n  abstract = {The spine is the most common site of bone metastasis, often originating from prostate, lung, and breast cancers. High systemic doses of chemotherapeutics such as doxorubicin (DOX), cisplatin, or paclitaxel often have severe side effects. Surgical removal of spine metastases also leaves large defects which cannot spontaneously heal and require bone grafting. To circumvent these issues, we designed an approach for local chemotherapeutic delivery within 3D-printed scaffolds which could also potentially serve as a bone substitute. Direct treatment of prostate cancer cell line LAPC4 and patient derived spine metastases cells with 0.01 muM DOX significantly reduced metabolic activity, proliferation, migration, and spheroid growth. We then assessed uptake and release of DOX in a series of porous 3D-printed scaffolds on LAPC4 cells as well as patient-derived spine metastases cells. Over seven days, 60-75% of DOX loaded onto scaffolds could be released, which significantly reduced metabolic activity and proliferation of both LAPC4 and patient derived cells, while unloaded scaffolds had no effect. Porous 3D-printed scaffolds may provide a novel and inexpensive approach to locally deliver chemotherapeutics in a patient-specific manner at tumor resection sites. With a composite design to enhance strength and promote sustained drug release, the scaffolds could reduce systemic negative effects, enhance bone repair, and improve patient outcomes.},\\n  annote = {cited By 5},\\n  author = {Ahangar, Pouyan and Akoury, Elie and Luna, Ana Sofia Ramirez Garcia and Nour, Antone and Weber, Michael H. and Rosenzweig, Derek H.},\\n  doi = {10.3390/ma11091485},\\n  issn = {19961944},\\n  journal = {Materials},\\n  keywords = {Bone metastases,Bone substitute,Doxorubicin,Local delivery,Low-cost 3D printing,Nanoporous filament,Prostate cancer},\\n  number = {9},\\n  title = {{Nanoporous 3D-printed scaffolds for local doxorubicin delivery in bone metastases secondary to prostate cancer}},\\n  volume = {11},\\n  year = {2018}\\n  }\\n  \",\"author_short\":[\"Ahangar, P.\",\"Akoury, E.\",\"Luna, A. S. R. G.\",\"Nour, A.\",\"Weber, M. H.\",\"Rosenzweig, D. H.\"],\"key\":\"Ahangar2018\",\"id\":\"Ahangar2018\",\"bibbaseid\":\"ahangar-akoury-luna-nour-weber-rosenzweig-nanoporous3dprintedscaffoldsforlocaldoxorubicindeliveryinbonemetastasessecondarytoprostatecancer-2018\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Bone metastases\",\"Bone substitute\",\"Doxorubicin\",\"Local delivery\",\"Low-cost 3D printing\",\"Nanoporous filament\",\"Prostate cancer\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":1,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Study Design.Meta-analysis of published randomized controlled trials (RCTs).Objective.To determine whether anterior cervical discectomy and fusion (ACDF), cervical disc replacement (CDR), or minimally invasive posterior cervical foraminotomy (MI-PCF) provides the best outcomes for patients with symptomatic single-level, single-side, and cervical radiculopathy.Summary of Background Data.The surgical treatment of cervical radiculopathy is still controversial. ACDF has been widely used as a \\\"gold standard.\\\" CDR has evolved and become a motion-preserving alternative with a potentially lower incidence of adjacent segment disease. However, both techniques require anterior neck dissection that carries a potential for serious morbidity. MI-PCF is a motion-preserving technique that can be performed with minimal invasiveness but has not gained universal acceptance.Methods.Electronic database search for RCTs comparing the efficacy and effectiveness of ACDF, CDR, and MI-PCF was performed. Meta-analysis was done for secondary surgical procedures and adverse events.Results.A total of 358 studies were retrieved, of which four RCT reports met the inclusion criteria for this study. Three studies present clinical data comparing ACDF and CDR, and one study presents data comparing ACDF and MI-PCF. Available data from the RCTs analyzed concluded that ACDF, CDR, and MI-PCF result in significant improvements in relevant symptoms, clinical, and functional outcomes in patients with single-level, single side cervical radiculopathy refractory to nonoperative treatment. CDR had the lowest percentage of secondary surgical procedures (P=0.0178) whereas MICPF had the lowest percentage of adverse events (P<0.0001).Conclusion.All three techniques are effective in treating cervical radicular symptoms. MI-PCF has the lowest rate of adverse events whereas CDR has the lowest rate of secondary procedures. There is insufficient evidence to show which technique is the most effective and provides the longest-lasting symptom relief.Level of Evidence.\",\"annote\":\"cited By 14\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Gutman\"],\"firstnames\":[\"Gabriel\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Golan\"],\"firstnames\":[\"Jeff\",\"D.\"],\"suffixes\":[]}],\"doi\":\"10.1097/BRS.0000000000002324\",\"issn\":\"15281159\",\"journal\":\"Spine\",\"keywords\":\"anterior cervical discectomy and fusion,cervical disc arthroplasty,cervical radiculopathy,minimally invasive posterior cervical foraminotomy,spine surgery\",\"number\":\"6\",\"pages\":\"E365–E372\",\"title\":\"Surgical Treatment of Cervical Radiculopathy\",\"volume\":\"43\",\"year\":\"2018\",\"bibtex\":\"@article{Gutman2018E365,\\n  abstract = {Study Design.Meta-analysis of published randomized controlled trials (RCTs).Objective.To determine whether anterior cervical discectomy and fusion (ACDF), cervical disc replacement (CDR), or minimally invasive posterior cervical foraminotomy (MI-PCF) provides the best outcomes for patients with symptomatic single-level, single-side, and cervical radiculopathy.Summary of Background Data.The surgical treatment of cervical radiculopathy is still controversial. ACDF has been widely used as a \\\"gold standard.\\\" CDR has evolved and become a motion-preserving alternative with a potentially lower incidence of adjacent segment disease. However, both techniques require anterior neck dissection that carries a potential for serious morbidity. MI-PCF is a motion-preserving technique that can be performed with minimal invasiveness but has not gained universal acceptance.Methods.Electronic database search for RCTs comparing the efficacy and effectiveness of ACDF, CDR, and MI-PCF was performed. Meta-analysis was done for secondary surgical procedures and adverse events.Results.A total of 358 studies were retrieved, of which four RCT reports met the inclusion criteria for this study. Three studies present clinical data comparing ACDF and CDR, and one study presents data comparing ACDF and MI-PCF. Available data from the RCTs analyzed concluded that ACDF, CDR, and MI-PCF result in significant improvements in relevant symptoms, clinical, and functional outcomes in patients with single-level, single side cervical radiculopathy refractory to nonoperative treatment. CDR had the lowest percentage of secondary surgical procedures (P=0.0178) whereas MICPF had the lowest percentage of adverse events (P<0.0001).Conclusion.All three techniques are effective in treating cervical radicular symptoms. MI-PCF has the lowest rate of adverse events whereas CDR has the lowest rate of secondary procedures. There is insufficient evidence to show which technique is the most effective and provides the longest-lasting symptom relief.Level of Evidence.},\\n  annote = {cited By 14},\\n  author = {Gutman, Gabriel and Rosenzweig, Derek H. and Golan, Jeff D.},\\n  doi = {10.1097/BRS.0000000000002324},\\n  issn = {15281159},\\n  journal = {Spine},\\n  keywords = {anterior cervical discectomy and fusion,cervical disc arthroplasty,cervical radiculopathy,minimally invasive posterior cervical foraminotomy,spine surgery},\\n  number = {6},\\n  pages = {E365--E372},\\n  title = {{Surgical Treatment of Cervical Radiculopathy}},\\n  volume = {43},\\n  year = {2018}\\n  }\\n  \",\"author_short\":[\"Gutman, G.\",\"Rosenzweig, D. H.\",\"Golan, J. D.\"],\"key\":\"Gutman2018E365\",\"id\":\"Gutman2018E365\",\"bibbaseid\":\"gutman-rosenzweig-golan-surgicaltreatmentofcervicalradiculopathy-2018\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"anterior cervical discectomy and fusion\",\"cervical disc arthroplasty\",\"cervical radiculopathy\",\"minimally invasive posterior cervical foraminotomy\",\"spine surgery\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1rho magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1rho values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture setup, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.\",\"annote\":\"cited By 4\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"D.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Fairag\"],\"firstnames\":[\"R.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Mathieu\"],\"firstnames\":[\"A.\",\"P.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Li\"],\"firstnames\":[\"L.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Eglin\"],\"firstnames\":[\"D.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"D'este\"],\"firstnames\":[\"M.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Steffen\"],\"firstnames\":[\"T.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"M.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ouellet\"],\"firstnames\":[\"J.\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.22203/eCM.v036a15\",\"issn\":\"14732262\",\"journal\":\"European Cells and Materials\",\"keywords\":\"Autologous cell implantation,Bioreactors,Human intervertebral disc,Hydrogel,Nucleus pulposus,T1rho magnetic resonance imaging,Tissue engineering\",\"pages\":\"200–217\",\"pmid\":\"30370912\",\"title\":\"Thermoreversi ble hyaluronan-hydrogel and autologous nucleus pulposus cell deli very regenerates human i ntervertebral di scs i n an ex vi vo, physi ologi cal organ culture model\",\"volume\":\"36\",\"year\":\"2018\",\"bibtex\":\"@article{Rosenzweig2018200,\\n  abstract = {Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1rho magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1rho values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture setup, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.},\\n  annote = {cited By 4},\\n  author = {Rosenzweig, D. H. and Fairag, R. and Mathieu, A. P. and Li, L. and Eglin, D. and D'este, M. and Steffen, T. and Weber, M. H. and Ouellet, J. A. and Haglund, Lisbet},\\n  doi = {10.22203/eCM.v036a15},\\n  issn = {14732262},\\n  journal = {European Cells and Materials},\\n  keywords = {Autologous cell implantation,Bioreactors,Human intervertebral disc,Hydrogel,Nucleus pulposus,T1rho magnetic resonance imaging,Tissue engineering},\\n  pages = {200--217},\\n  pmid = {30370912},\\n  title = {{Thermoreversi ble hyaluronan-hydrogel and autologous nucleus pulposus cell deli very regenerates human i ntervertebral di scs i n an ex vi vo, physi ologi cal organ culture model}},\\n  volume = {36},\\n  year = {2018}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Fairag, R.\",\"Mathieu, A. P.\",\"Li, L.\",\"Eglin, D.\",\"D'este, M.\",\"Steffen, T.\",\"Weber, M. H.\",\"Ouellet, J. A.\",\"Haglund, L.\"],\"key\":\"Rosenzweig2018200\",\"id\":\"Rosenzweig2018200\",\"bibbaseid\":\"rosenzweig-fairag-mathieu-li-eglin-deste-steffen-weber-etal-thermoreversiblehyaluronanhydrogelandautologousnucleuspulposuscelldeliveryregenerateshumanintervertebraldiscsinanexvivophysiologicalorganculturemodel-2018\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Autologous cell implantation\",\"Bioreactors\",\"Human intervertebral disc\",\"Hydrogel\",\"Nucleus pulposus\",\"T1rho magnetic resonance imaging\",\"Tissue engineering\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1beta and nerve growth factor gene expression following treatment with Pam2CSK4 (TLR2/6 agonist) but not Pam3CSK4 (TLR1/2 agonist). Challenging NP cells with Pam2CSK4 or 30 kDa fibronectin fragments (FN-f, an endogenous TLR2 and TLR4 alarmin) increased secretion of proinflammatory cytokines. We then investigated the effect of TLR activation in intact, non-degenerate, ex vivo human discs. Discs were injected with PBS, Pam2CSK4 and FN-f, and cultured for 28 days. TLR activation increased proteoglycan and ECM protein release into the culture media and decreased proteoglycan content in the NP. Proteases, including MMP3, 13 and HTRA1, are secreted at higher levels following TLR activation. In addition, proinflammatory cytokine levels, including IL-6, TNFalpha and IFNgamma, increased following TLR activation. These results indicate that TLR activation induces degeneration in human discs. Therefore, TLRs are potential disease-modifying therapeutic targets to slow disc degeneration.\",\"annote\":\"cited By 7\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Krock\"],\"firstnames\":[\"Emerson\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Currie\"],\"firstnames\":[\"J.\",\"Brooke\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Bisson\"],\"firstnames\":[\"Daniel\",\"G.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ouellet\"],\"firstnames\":[\"Jean\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.1038/s41598-017-17472-1\",\"issn\":\"20452322\",\"journal\":\"Scientific Reports\",\"number\":\"1\",\"title\":\"Toll-like receptor activation induces degeneration of human intervertebral discs\",\"volume\":\"7\",\"year\":\"2017\",\"bibtex\":\"@article{Krock2017,\\n  abstract = {Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1beta and nerve growth factor gene expression following treatment with Pam2CSK4 (TLR2/6 agonist) but not Pam3CSK4 (TLR1/2 agonist). Challenging NP cells with Pam2CSK4 or 30 kDa fibronectin fragments (FN-f, an endogenous TLR2 and TLR4 alarmin) increased secretion of proinflammatory cytokines. We then investigated the effect of TLR activation in intact, non-degenerate, ex vivo human discs. Discs were injected with PBS, Pam2CSK4 and FN-f, and cultured for 28 days. TLR activation increased proteoglycan and ECM protein release into the culture media and decreased proteoglycan content in the NP. Proteases, including MMP3, 13 and HTRA1, are secreted at higher levels following TLR activation. In addition, proinflammatory cytokine levels, including IL-6, TNFalpha and IFNgamma, increased following TLR activation. These results indicate that TLR activation induces degeneration in human discs. Therefore, TLRs are potential disease-modifying therapeutic targets to slow disc degeneration.},\\n  annote = {cited By 7},\\n  author = {Krock, Emerson and Rosenzweig, Derek H. and Currie, J. Brooke and Bisson, Daniel G. and Ouellet, Jean A. and Haglund, Lisbet},\\n  doi = {10.1038/s41598-017-17472-1},\\n  issn = {20452322},\\n  journal = {Scientific Reports},\\n  number = {1},\\n  title = {{Toll-like receptor activation induces degeneration of human intervertebral discs}},\\n  volume = {7},\\n  year = {2017}\\n  }\\n  \",\"author_short\":[\"Krock, E.\",\"Rosenzweig, D. H.\",\"Currie, J. B.\",\"Bisson, D. G.\",\"Ouellet, J. A.\",\"Haglund, L.\"],\"key\":\"Krock2017\",\"id\":\"Krock2017\",\"bibbaseid\":\"krock-rosenzweig-currie-bisson-ouellet-haglund-tolllikereceptoractivationinducesdegenerationofhumanintervertebraldiscs-2017\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Bisphosphonates (BPs) have recently been shown to have direct anti-tumor properties. Systemic treatment with BPs can have multiple adverse effects such as osteonecrosis of the jaw and BP induced bone fracturing and spine instability. While benefits of systemic BP treatments may outweigh risks, local treatment with BPs has been explored as an alternate strategy to reduce unwarranted risk. In the present study, we examined whether local delivery of BPs inhibits tumor-induced osteolysis and tumor growth more effectively than systemic treatment in an animal model of tumor-induced bone disease. Following establishment of an intra-tibial model of bone metastases in athymic mice, the experimental group was treated by local administration of zoledronate into the tibial lesion. A comparison of the effect of local versus systemic delivery of zoledronate on the formation of tumor-induced osteolysis was also carried out. A significant increase in mean bone volume/tissue volume % (BV/TV) of the locally treated group (12.30±2.80%) compared to the control group (7.13±1.22%) (P<0.001). Additionally, there was a significant increase in the BV/TV (10.90±1.25%) in the locally treated group compared to the systemically treated group (7.53±0.75%) (P=0.005). These preliminary results suggest that local delivery of BPs outperforms both systemic and control treatments to inhibit tumor-induced osteolysis.\",\"annote\":\"cited By 3\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Nooh\"],\"firstnames\":[\"Anas\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Zhang\"],\"firstnames\":[\"Yu\",\"Ling\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Sato\"],\"firstnames\":[\"Daisuke\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Tabari`es\"],\"firstnames\":[\"S'ebastien\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Siegel\"],\"firstnames\":[\"Peter\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Barralet\"],\"firstnames\":[\"Jake\",\"E.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]}],\"doi\":\"10.1016/j.jbo.2017.01.001\",\"issn\":\"22121374\",\"journal\":\"Journal of Bone Oncology\",\"keywords\":\"Bisphosphonates,Bone metastases,Cancer pain,Local treatment,Osteolysis,Xenograft\",\"pages\":\"8–15\",\"title\":\"Intra-tumor delivery of zoledronate mitigates metastasis-induced osteolysis superior to systemic administration\",\"volume\":\"6\",\"year\":\"2017\",\"bibtex\":\"@article{Nooh20178,\\n  abstract = {Bisphosphonates (BPs) have recently been shown to have direct anti-tumor properties. Systemic treatment with BPs can have multiple adverse effects such as osteonecrosis of the jaw and BP induced bone fracturing and spine instability. While benefits of systemic BP treatments may outweigh risks, local treatment with BPs has been explored as an alternate strategy to reduce unwarranted risk. In the present study, we examined whether local delivery of BPs inhibits tumor-induced osteolysis and tumor growth more effectively than systemic treatment in an animal model of tumor-induced bone disease. Following establishment of an intra-tibial model of bone metastases in athymic mice, the experimental group was treated by local administration of zoledronate into the tibial lesion. A comparison of the effect of local versus systemic delivery of zoledronate on the formation of tumor-induced osteolysis was also carried out. A significant increase in mean bone volume/tissue volume % (BV/TV) of the locally treated group (12.30±2.80%) compared to the control group (7.13±1.22%) (P<0.001). Additionally, there was a significant increase in the BV/TV (10.90±1.25%) in the locally treated group compared to the systemically treated group (7.53±0.75%) (P=0.005). These preliminary results suggest that local delivery of BPs outperforms both systemic and control treatments to inhibit tumor-induced osteolysis.},\\n  annote = {cited By 3},\\n  author = {Nooh, Anas and Zhang, Yu Ling and Sato, Daisuke and Rosenzweig, Derek H. and Tabari{`{e}}s, S{'{e}}bastien and Siegel, Peter and Barralet, Jake E. and Weber, Michael H.},\\n  doi = {10.1016/j.jbo.2017.01.001},\\n  issn = {22121374},\\n  journal = {Journal of Bone Oncology},\\n  keywords = {Bisphosphonates,Bone metastases,Cancer pain,Local treatment,Osteolysis,Xenograft},\\n  pages = {8--15},\\n  title = {{Intra-tumor delivery of zoledronate mitigates metastasis-induced osteolysis superior to systemic administration}},\\n  volume = {6},\\n  year = {2017}\\n  }\\n  \",\"author_short\":[\"Nooh, A.\",\"Zhang, Y. L.\",\"Sato, D.\",\"Rosenzweig, D. H.\",\"Tabari`es, S.\",\"Siegel, P.\",\"Barralet, J. E.\",\"Weber, M. H.\"],\"key\":\"Nooh20178\",\"id\":\"Nooh20178\",\"bibbaseid\":\"nooh-zhang-sato-rosenzweig-tabaries-siegel-barralet-weber-intratumordeliveryofzoledronatemitigatesmetastasisinducedosteolysissuperiortosystemicadministration-2017\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Bisphosphonates\",\"Bone metastases\",\"Cancer pain\",\"Local treatment\",\"Osteolysis\",\"Xenograft\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD) degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP) phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD) gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.\",\"annote\":\"cited By 7\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"D.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Tremblay Gravel\"],\"firstnames\":[\"J.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Bisson\"],\"firstnames\":[\"D.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ouellet\"],\"firstnames\":[\"J.\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"M.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.22203/eCM.v033a18\",\"issn\":\"14732262\",\"journal\":\"European Cells and Materials\",\"keywords\":\"Cell culture,Elastic culture surfaces,Intervertebral disc,Nucleus pulposus cells,Tissue engineering\",\"pages\":\"240–251\",\"title\":\"Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications\",\"volume\":\"33\",\"year\":\"2017\",\"bibtex\":\"@article{Rosenzweig2017240,\\n  abstract = {Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD) degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP) phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD) gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.},\\n  annote = {cited By 7},\\n  author = {Rosenzweig, D. H. and {Tremblay Gravel}, J. and Bisson, D. and Ouellet, J. A. and Weber, M. H. and Haglund, Lisbet},\\n  doi = {10.22203/eCM.v033a18},\\n  issn = {14732262},\\n  journal = {European Cells and Materials},\\n  keywords = {Cell culture,Elastic culture surfaces,Intervertebral disc,Nucleus pulposus cells,Tissue engineering},\\n  pages = {240--251},\\n  title = {{Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications}},\\n  volume = {33},\\n  year = {2017}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Tremblay Gravel, J.\",\"Bisson, D.\",\"Ouellet, J. A.\",\"Weber, M. H.\",\"Haglund, L.\"],\"key\":\"Rosenzweig2017240\",\"id\":\"Rosenzweig2017240\",\"bibbaseid\":\"rosenzweig-tremblaygravel-bisson-ouellet-weber-haglund-comparativeanalysisincontinuousexpansionofbovineandhumanprimarynucleuspulposuscellsfortissuerepairapplications-2017\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Cell culture\",\"Elastic culture surfaces\",\"Intervertebral disc\",\"Nucleus pulposus cells\",\"Tissue engineering\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity but more recently were found to be activated by endogenous alarmins such as fragmented extracellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1beta (control) treatment increased NGF, brain-derived neurotrophic factor (BDNF), and IL-1beta gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1beta treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2, and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-kappaB, and to a lesser extent p38, but not ERK1/2 activity, blocked TLR2-driven NGF up-regulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-kappaB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-kappaB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.\",\"annote\":\"cited By 26\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Krock\"],\"firstnames\":[\"Emerson\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Currie\"],\"firstnames\":[\"J.\",\"Brooke\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ouellet\"],\"firstnames\":[\"Jean\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Stone\"],\"firstnames\":[\"Laura\",\"S.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.1074/jbc.M115.675900\",\"issn\":\"1083351X\",\"journal\":\"Journal of Biological Chemistry\",\"number\":\"7\",\"pages\":\"3541–3551\",\"title\":\"Nerve growth factor is regulated by toll-like receptor 2 in human intervertebral discs\",\"volume\":\"291\",\"year\":\"2016\",\"bibtex\":\"@article{Krock20163541,\\n  abstract = {Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity but more recently were found to be activated by endogenous alarmins such as fragmented extracellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1beta (control) treatment increased NGF, brain-derived neurotrophic factor (BDNF), and IL-1beta gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1beta treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2, and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-kappaB, and to a lesser extent p38, but not ERK1/2 activity, blocked TLR2-driven NGF up-regulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-kappaB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-kappaB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.},\\n  annote = {cited By 26},\\n  author = {Krock, Emerson and Currie, J. Brooke and Weber, Michael H. and Ouellet, Jean A. and Stone, Laura S. and Rosenzweig, Derek H. and Haglund, Lisbet},\\n  doi = {10.1074/jbc.M115.675900},\\n  issn = {1083351X},\\n  journal = {Journal of Biological Chemistry},\\n  number = {7},\\n  pages = {3541--3551},\\n  title = {{Nerve growth factor is regulated by toll-like receptor 2 in human intervertebral discs}},\\n  volume = {291},\\n  year = {2016}\\n  }\\n  \",\"author_short\":[\"Krock, E.\",\"Currie, J. B.\",\"Weber, M. H.\",\"Ouellet, J. A.\",\"Stone, L. S.\",\"Rosenzweig, D. H.\",\"Haglund, L.\"],\"key\":\"Krock20163541\",\"id\":\"Krock20163541\",\"bibbaseid\":\"krock-currie-weber-ouellet-stone-rosenzweig-haglund-nervegrowthfactorisregulatedbytolllikereceptor2inhumanintervertebraldiscs-2016\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent nanotransducers for converting longer wavelength near-infrared (NIR) light to shorter wavelengths spanning the ultraviolet (UV) to the visible (Vis) regions of the spectrum via a multiphoton absorption process, known as upconversion. Here, we report the development of NIR to UV-Vis-NIR UCNPs consisting of LiYF4:Yb3+/Tm3+@SiO2 individually coated with a 10 ± 2 nm layer of chitosan (CH) hydrogel cross-linked with a photocleavable cross-linker (PhL). We encapsulated fluorescent-bovine serum albumin (FITC-BSA) inside the gel. Under 980 nm excitation, the upconverted UV emission cleaves the PhL cross-links and instantaneously liberates the FITC-BSA under 2 cm thick tissue. The release is immediately arrested if the excitation source is switched off. The upconverted NIR light allows for the tracking of particles under the tissue. Nucleus pulposus (NP) cells cultured with UCNPs are viable both in the presence and in the absence of laser irradiation. Controlled drug delivery of large biomolecules and deep tissue imaging make this system an excellent theranostic platform for tissue engineering, biomapping, and cellular imaging applications.\",\"annote\":\"cited By 104\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Jalani\"],\"firstnames\":[\"Ghulam\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Naccache\"],\"firstnames\":[\"Rafik\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Vetrone\"],\"firstnames\":[\"Fiorenzo\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Cerruti\"],\"firstnames\":[\"Marta\"],\"suffixes\":[]}],\"doi\":\"10.1021/jacs.5b12357\",\"issn\":\"15205126\",\"journal\":\"Journal of the American Chemical Society\",\"number\":\"3\",\"pages\":\"1078–1083\",\"title\":\"Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery\",\"volume\":\"138\",\"year\":\"2016\",\"bibtex\":\"@article{Jalani20161078,\\n  abstract = {Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent nanotransducers for converting longer wavelength near-infrared (NIR) light to shorter wavelengths spanning the ultraviolet (UV) to the visible (Vis) regions of the spectrum via a multiphoton absorption process, known as upconversion. Here, we report the development of NIR to UV-Vis-NIR UCNPs consisting of LiYF4:Yb3+/Tm3+@SiO2 individually coated with a 10 ± 2 nm layer of chitosan (CH) hydrogel cross-linked with a photocleavable cross-linker (PhL). We encapsulated fluorescent-bovine serum albumin (FITC-BSA) inside the gel. Under 980 nm excitation, the upconverted UV emission cleaves the PhL cross-links and instantaneously liberates the FITC-BSA under 2 cm thick tissue. The release is immediately arrested if the excitation source is switched off. The upconverted NIR light allows for the tracking of particles under the tissue. Nucleus pulposus (NP) cells cultured with UCNPs are viable both in the presence and in the absence of laser irradiation. Controlled drug delivery of large biomolecules and deep tissue imaging make this system an excellent theranostic platform for tissue engineering, biomapping, and cellular imaging applications.},\\n  annote = {cited By 104},\\n  author = {Jalani, Ghulam and Naccache, Rafik and Rosenzweig, Derek H. and Haglund, Lisbet and Vetrone, Fiorenzo and Cerruti, Marta},\\n  doi = {10.1021/jacs.5b12357},\\n  issn = {15205126},\\n  journal = {Journal of the American Chemical Society},\\n  number = {3},\\n  pages = {1078--1083},\\n  title = {{Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery}},\\n  volume = {138},\\n  year = {2016}\\n  }\\n  \",\"author_short\":[\"Jalani, G.\",\"Naccache, R.\",\"Rosenzweig, D. H.\",\"Haglund, L.\",\"Vetrone, F.\",\"Cerruti, M.\"],\"key\":\"Jalani20161078\",\"id\":\"Jalani20161078\",\"bibbaseid\":\"jalani-naccache-rosenzweig-haglund-vetrone-cerruti-photocleavablehydrogelcoatedupconvertingnanoparticlesamultifunctionaltheranosticplatformfornirimagingandondemandmacromoleculardelivery-2016\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Low back pain originating from intervertebral disc (IVD) degenerationaffectsthequalityoflifeformillionsof people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivomodels of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. Abioreactor wasdevelopedthat facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.6 and high 0.1-1.2 MPa. Cell viability was maintained > 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility oftissuerepairstrategieswithcellsupplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.\",\"annote\":\"cited By 10\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"D.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Gawri\"],\"firstnames\":[\"R.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Moir\"],\"firstnames\":[\"J.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Beckman\"],\"firstnames\":[\"L.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Eglin\"],\"firstnames\":[\"D.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Steffen\"],\"firstnames\":[\"T.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Roughley\"],\"firstnames\":[\"P.\",\"J.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ouellet\"],\"firstnames\":[\"J.\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.22203/eCM.v031a03\",\"issn\":\"14732262\",\"journal\":\"European Cells and Materials\",\"keywords\":\"Bioreactor,Cell therapy,Dynamic loading,Hydrogel,Intervertebral discs,Organ culture,Tissue regeneration\",\"pages\":\"26–39\",\"pmid\":\"26728497\",\"title\":\"Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor\",\"volume\":\"31\",\"year\":\"2016\",\"bibtex\":\"@article{Rosenzweig201626,\\n  abstract = {Low back pain originating from intervertebral disc (IVD) degenerationaffectsthequalityoflifeformillionsof people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivomodels of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. Abioreactor wasdevelopedthat facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.6 and high 0.1-1.2 MPa. Cell viability was maintained > 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility oftissuerepairstrategieswithcellsupplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.},\\n  annote = {cited By 10},\\n  author = {Rosenzweig, D. H. and Gawri, R. and Moir, J. and Beckman, L. and Eglin, D. and Steffen, T. and Roughley, P. J. and Ouellet, J. A. and Haglund, Lisbet},\\n  doi = {10.22203/eCM.v031a03},\\n  issn = {14732262},\\n  journal = {European Cells and Materials},\\n  keywords = {Bioreactor,Cell therapy,Dynamic loading,Hydrogel,Intervertebral discs,Organ culture,Tissue regeneration},\\n  pages = {26--39},\\n  pmid = {26728497},\\n  title = {{Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor}},\\n  volume = {31},\\n  year = {2016}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Gawri, R.\",\"Moir, J.\",\"Beckman, L.\",\"Eglin, D.\",\"Steffen, T.\",\"Roughley, P. J.\",\"Ouellet, J. A.\",\"Haglund, L.\"],\"key\":\"Rosenzweig201626\",\"id\":\"Rosenzweig201626\",\"bibbaseid\":\"rosenzweig-gawri-moir-beckman-eglin-steffen-roughley-ouellet-etal-dynamicloadingmatrixmaintenanceandcellinjectiontherapyofhumanintervertebraldiscsculturedinabioreactor-2016\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Bioreactor\",\"Cell therapy\",\"Dynamic loading\",\"Hydrogel\",\"Intervertebral discs\",\"Organ culture\",\"Tissue regeneration\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"To design a biodegradable hydrogel as cell support, one should know its in vivo degradation rate. A technique commonly used to track gel degradation is fluorescence spectroscopy. However, the fluorescence from conventional fluorophores quickly decays, and the fluorophores are often moderately cytotoxic. Most importantly, they require ultraviolet or visible (UV-Vis) light as the excitation source, which cannot penetrate deeply through biological tissues. Lanthanide-doped upconverting nanoparticles (UCNPs) are exciting alternatives to conventional fluorophores because they can convert near-infrared (NIR) to UV-Vis-NIR light via a sequential multiphoton absorption process referred to as upconversion. NIR light can penetrate up to few cm inside tissues, thus making these UCNPs much better probes than conventional fluorophores for in vivo monitoring. Also, UCNPs have narrow emission bands, high photoluminescence (PL) signal-to-noise ratio, low cytotoxicity and good physical and chemical stability. Here, we show a nanocomposite system consisting of a biodegradable, in situ thermogelling injectable hydrogel made of chitosan and hyaluronic acid encapsulating silica-coated LiYF4:Yb3+/Tm3+ UCNPs. We use these UCNPs as photoluminescent tags to monitor the gel degradation inside live, cultured intervertebral discs (IVDs) over a period of 3 weeks. PL spectroscopy and NIR imaging show that NIR-to-NIR upconversion of LiYF4:Yb3+/Tm3+@SiO2 UCNPs allows for tracking of the gel degradation in living tissues. Both in vitro and ex vivo release of UCNPs follow a similar trend during the first 5 days; after this time, ex vivo release becomes faster than in vitro, indicating a faster gel degradation ex vivo. Also, the amount of released UCNPs in vitro increases continuously up to 3 weeks, while it plateaus after 15 days inside the IVDs showing a homogenous distribution of UCNPs throughout the IVD tissue. This non-invasive optical method for real time, live tissue imaging holds great potential for tissue analysis, biomapping and bioimaging applications.\",\"annote\":\"cited By 33\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Jalani\"],\"firstnames\":[\"Ghulam\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Naccache\"],\"firstnames\":[\"Rafik\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Lerouge\"],\"firstnames\":[\"Sophie\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Vetrone\"],\"firstnames\":[\"Fiorenzo\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Cerruti\"],\"firstnames\":[\"Marta\"],\"suffixes\":[]}],\"doi\":\"10.1039/c5nr02482j\",\"issn\":\"20403372\",\"journal\":\"Nanoscale\",\"number\":\"26\",\"pages\":\"11255–11262\",\"title\":\"Real-time, non-invasive monitoring of hydrogel degradation using LiYF4:Yb3+/Tm3+ NIR-to-NIR upconverting nanoparticles\",\"volume\":\"7\",\"year\":\"2015\",\"bibtex\":\"@article{Jalani201511255,\\n  abstract = {To design a biodegradable hydrogel as cell support, one should know its in vivo degradation rate. A technique commonly used to track gel degradation is fluorescence spectroscopy. However, the fluorescence from conventional fluorophores quickly decays, and the fluorophores are often moderately cytotoxic. Most importantly, they require ultraviolet or visible (UV-Vis) light as the excitation source, which cannot penetrate deeply through biological tissues. Lanthanide-doped upconverting nanoparticles (UCNPs) are exciting alternatives to conventional fluorophores because they can convert near-infrared (NIR) to UV-Vis-NIR light via a sequential multiphoton absorption process referred to as upconversion. NIR light can penetrate up to few cm inside tissues, thus making these UCNPs much better probes than conventional fluorophores for in vivo monitoring. Also, UCNPs have narrow emission bands, high photoluminescence (PL) signal-to-noise ratio, low cytotoxicity and good physical and chemical stability. Here, we show a nanocomposite system consisting of a biodegradable, in situ thermogelling injectable hydrogel made of chitosan and hyaluronic acid encapsulating silica-coated LiYF4:Yb3+/Tm3+ UCNPs. We use these UCNPs as photoluminescent tags to monitor the gel degradation inside live, cultured intervertebral discs (IVDs) over a period of 3 weeks. PL spectroscopy and NIR imaging show that NIR-to-NIR upconversion of LiYF4:Yb3+/Tm3+@SiO2 UCNPs allows for tracking of the gel degradation in living tissues. Both in vitro and ex vivo release of UCNPs follow a similar trend during the first 5 days; after this time, ex vivo release becomes faster than in vitro, indicating a faster gel degradation ex vivo. Also, the amount of released UCNPs in vitro increases continuously up to 3 weeks, while it plateaus after 15 days inside the IVDs showing a homogenous distribution of UCNPs throughout the IVD tissue. This non-invasive optical method for real time, live tissue imaging holds great potential for tissue analysis, biomapping and bioimaging applications.},\\n  annote = {cited By 33},\\n  author = {Jalani, Ghulam and Naccache, Rafik and Rosenzweig, Derek H. and Lerouge, Sophie and Haglund, Lisbet and Vetrone, Fiorenzo and Cerruti, Marta},\\n  doi = {10.1039/c5nr02482j},\\n  issn = {20403372},\\n  journal = {Nanoscale},\\n  number = {26},\\n  pages = {11255--11262},\\n  title = {{Real-time, non-invasive monitoring of hydrogel degradation using LiYF4:Yb3+/Tm3+ NIR-to-NIR upconverting nanoparticles}},\\n  volume = {7},\\n  year = {2015}\\n  }\\n  \",\"author_short\":[\"Jalani, G.\",\"Naccache, R.\",\"Rosenzweig, D. H.\",\"Lerouge, S.\",\"Haglund, L.\",\"Vetrone, F.\",\"Cerruti, M.\"],\"key\":\"Jalani201511255\",\"id\":\"Jalani201511255\",\"bibbaseid\":\"jalani-naccache-rosenzweig-lerouge-haglund-vetrone-cerruti-realtimenoninvasivemonitoringofhydrogeldegradationusingliyf4yb3tm3nirtonirupconvertingnanoparticles-2015\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (NP) cells were cultured on ABS and PLA scaffolds for three weeks. Both cell types proliferated well, showed high viability, and produced ample amounts of proteoglycan and collagen type II on both scaffolds. NP generated more matrix than chondrocytes; however, no difference was observed between scaffold types. Mechanical testing revealed sustained scaffold stability. This study demonstrates that chondrocytes and NP cells can proliferate on both ABS and PLA scaffolds printed with a simplistic, inexpensive desktop 3D printer. Moreover, NP cells produced more proteoglycan than chondrocytes, irrespective of thermoplastic type, indicating that cells maintain individual phenotype over the three-week culture period. Future scaffold designs covering larger pore sizes and better mimicking native tissue structure combined with more flexible or resorbable materials may provide implantable constructs with the proper structure, function, and cellularity necessary for potential cartilage and disc tissue repair in vivo.\",\"annote\":\"cited By 128\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Carelli\"],\"firstnames\":[\"Eric\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Steffen\"],\"firstnames\":[\"Thomas\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Jarzem\"],\"firstnames\":[\"Peter\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.3390/ijms160715118\",\"issn\":\"14220067\",\"journal\":\"International Journal of Molecular Sciences\",\"keywords\":\"3D printing,ABS,Chondrocyte,Intervertebral disc,Nucleus pulposus,PLA,Tissue engineering\",\"number\":\"7\",\"pages\":\"15118–15135\",\"title\":\"3D-printed ABS and PLA scaffolds for cartilage and nucleus pulposustissue regeneration\",\"volume\":\"16\",\"year\":\"2015\",\"bibtex\":\"@article{Rosenzweig201515118,\\n  abstract = {Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (NP) cells were cultured on ABS and PLA scaffolds for three weeks. Both cell types proliferated well, showed high viability, and produced ample amounts of proteoglycan and collagen type II on both scaffolds. NP generated more matrix than chondrocytes; however, no difference was observed between scaffold types. Mechanical testing revealed sustained scaffold stability. This study demonstrates that chondrocytes and NP cells can proliferate on both ABS and PLA scaffolds printed with a simplistic, inexpensive desktop 3D printer. Moreover, NP cells produced more proteoglycan than chondrocytes, irrespective of thermoplastic type, indicating that cells maintain individual phenotype over the three-week culture period. Future scaffold designs covering larger pore sizes and better mimicking native tissue structure combined with more flexible or resorbable materials may provide implantable constructs with the proper structure, function, and cellularity necessary for potential cartilage and disc tissue repair in vivo.},\\n  annote = {cited By 128},\\n  author = {Rosenzweig, Derek H. and Carelli, Eric and Steffen, Thomas and Jarzem, Peter and Haglund, Lisbet},\\n  doi = {10.3390/ijms160715118},\\n  issn = {14220067},\\n  journal = {International Journal of Molecular Sciences},\\n  keywords = {3D printing,ABS,Chondrocyte,Intervertebral disc,Nucleus pulposus,PLA,Tissue engineering},\\n  number = {7},\\n  pages = {15118--15135},\\n  title = {{3D-printed ABS and PLA scaffolds for cartilage and nucleus pulposustissue regeneration}},\\n  volume = {16},\\n  year = {2015}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Carelli, E.\",\"Steffen, T.\",\"Jarzem, P.\",\"Haglund, L.\"],\"key\":\"Rosenzweig201515118\",\"id\":\"Rosenzweig201515118\",\"bibbaseid\":\"rosenzweig-carelli-steffen-jarzem-haglund-3dprintedabsandplascaffoldsforcartilageandnucleuspulposustissueregeneration-2015\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"3D printing\",\"ABS\",\"Chondrocyte\",\"Intervertebral disc\",\"Nucleus pulposus\",\"PLA\",\"Tissue engineering\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"2015 Bentham Science Publishers. Intervertebral disc degeneration is directly linked to chronic low back pain, a condition that affects multitudes of people world-wide and presents tremendous direct and indirect health costs. Water- loss, inflammation and disruption of the extracellular matrix ultimately result in loss of tissue function and associated pain. Cytokines present in degenerate tissue can upregulate protease activity and directly causes pain. Non-invasive therapies provide limited efficacy for pain management, and surgical intervention is therefore often required to treat chronic low back pain. Disc removal can offer immediate pain-relief, however degeneration of adjacent segments can occur and pain can return. To circumvent the caveats of recurring pain and invasive surgeries, stem cell therapy is currently being investigated as a promising means to repair degenerating discs. However, while initial studies have shown promise, few studies have addressed whether stem cell therapies can modulate the inflammatory microenvironment or whether cytokines can affect the ability of the implanted cells to repair damaged tissue. This review focuses briefly on mechanisms of disc degeneration, with more attention given to the role of inflammatory milieu in this process. Cytokine upregulation in disc degeneration, the potential role of tolllike receptor signaling, and effects of these inflammatory factors on stem cells will be discussed. We find that while stem cell differentiation can be negatively influenced by inflammatory cytokines, stem cells can potentially have anti-inflammatory effects. We conclude that further investigation of stem cell interactions with the inflammatory microenvironment is required, and that priming of stem cells under various conditions may be necessary for optimal therapeutic value for intervertebral disc repair and pain reduction.\",\"annote\":\"cited By 20\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Krock\"],\"firstnames\":[\"Emerson\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.2174/1574888x10666150211161956\",\"issn\":\"1574888X\",\"journal\":\"Current Stem Cell Research & Therapy\",\"number\":\"4\",\"pages\":\"317–328\",\"title\":\"The Inflammatory Milieu of the Degenerate Disc: Is Mesenchymal Stem Cell-based Therapy for Intervertebral Disc Repair a Feasible Approach?\",\"volume\":\"10\",\"year\":\"2015\",\"bibtex\":\"@article{Krock2015317,\\n  abstract = { 2015 Bentham Science Publishers. Intervertebral disc degeneration is directly linked to chronic low back pain, a condition that affects multitudes of people world-wide and presents tremendous direct and indirect health costs. Water- loss, inflammation and disruption of the extracellular matrix ultimately result in loss of tissue function and associated pain. Cytokines present in degenerate tissue can upregulate protease activity and directly causes pain. Non-invasive therapies provide limited efficacy for pain management, and surgical intervention is therefore often required to treat chronic low back pain. Disc removal can offer immediate pain-relief, however degeneration of adjacent segments can occur and pain can return. To circumvent the caveats of recurring pain and invasive surgeries, stem cell therapy is currently being investigated as a promising means to repair degenerating discs. However, while initial studies have shown promise, few studies have addressed whether stem cell therapies can modulate the inflammatory microenvironment or whether cytokines can affect the ability of the implanted cells to repair damaged tissue. This review focuses briefly on mechanisms of disc degeneration, with more attention given to the role of inflammatory milieu in this process. Cytokine upregulation in disc degeneration, the potential role of tolllike receptor signaling, and effects of these inflammatory factors on stem cells will be discussed. We find that while stem cell differentiation can be negatively influenced by inflammatory cytokines, stem cells can potentially have anti-inflammatory effects. We conclude that further investigation of stem cell interactions with the inflammatory microenvironment is required, and that priming of stem cells under various conditions may be necessary for optimal therapeutic value for intervertebral disc repair and pain reduction.},\\n  annote = {cited By 20},\\n  author = {Krock, Emerson and Rosenzweig, Derek and Haglund, Lisbet},\\n  doi = {10.2174/1574888x10666150211161956},\\n  issn = {1574888X},\\n  journal = {Current Stem Cell Research & Therapy},\\n  number = {4},\\n  pages = {317--328},\\n  title = {{The Inflammatory Milieu of the Degenerate Disc: Is Mesenchymal Stem Cell-based Therapy for Intervertebral Disc Repair a Feasible Approach?}},\\n  volume = {10},\\n  year = {2015}\\n  }\\n  \",\"author_short\":[\"Krock, E.\",\"Rosenzweig, D.\",\"Haglund, L.\"],\"key\":\"Krock2015317\",\"id\":\"Krock2015317\",\"bibbaseid\":\"krock-rosenzweig-haglund-theinflammatorymilieuofthedegeneratediscismesenchymalstemcellbasedtherapyforintervertebraldiscrepairafeasibleapproach-2015\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Injectable hydrogels are extensively used in drug delivery and tissue engineering to administer drugs, genes, growth factors and live cells. We report a method to produce tough, in-situ thermogelling, non-toxic, injectable hydrogels made of chitosan and hyaluronic acid co-crosslinked with carboxytetramethylrhodamine-glycerophophate and genipin. The gels are highly homogeneous and form within 32 min, i.e., faster than gels crosslinked with either genipin or beta-glycerophophate. The shear strength of co-crosslinked hydrogels is 3.5 kPa, higher than any chitosan-based gel reported. Chondrocytes and nucleus pulposus cells thrive inside the gels and produce large amounts of collagen II. Injection in rats shows that the gels form in-vivo within a short time and remain well localized for more than one week while the rats remain healthy and active. The excellent mechanical properties, fast in-situ gelation, good biocompatibility and the ability to encapsulate live cells at physiological conditions make these hydrogels ideal for tissue engineering, especially cartilage regeneration. Homogenous, insitu thermogelling, highly tough, non-toxic injectable hydrogels from chitosan and hyaluronic acid co-crosslinked with beta-glycerophophate and genipin are produced. The co-crosslinked gels form faster than genipin-only crosslinked gels and are 2.5 times stronger than ionically crosslinked gels. Homogenously dispersed cells express enhanced amount of collagen II, showing the great potential of these gels for cartilage tissue engineering.\",\"annote\":\"cited By 15\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Jalani\"],\"firstnames\":[\"Ghulam\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Makhoul\"],\"firstnames\":[\"Georges\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Abdalla\"],\"firstnames\":[\"Sherif\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Cecere\"],\"firstnames\":[\"Renzo\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Vetrone\"],\"firstnames\":[\"Fiorenzo\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Cerruti\"],\"firstnames\":[\"Marta\"],\"suffixes\":[]}],\"doi\":\"10.1002/mabi.201400406\",\"issn\":\"16165195\",\"journal\":\"Macromolecular Bioscience\",\"keywords\":\"crosslinking,genipin,hydrogels,in-situ thermogelling,injectable\",\"number\":\"4\",\"pages\":\"473–480\",\"title\":\"Tough, in-situ thermogelling, injectable hydrogels for biomedical applications\",\"volume\":\"15\",\"year\":\"2015\",\"bibtex\":\"@article{Jalani2015473,\\n  abstract = {Injectable hydrogels are extensively used in drug delivery and tissue engineering to administer drugs, genes, growth factors and live cells. We report a method to produce tough, in-situ thermogelling, non-toxic, injectable hydrogels made of chitosan and hyaluronic acid co-crosslinked with carboxytetramethylrhodamine-glycerophophate and genipin. The gels are highly homogeneous and form within 32 min, i.e., faster than gels crosslinked with either genipin or beta-glycerophophate. The shear strength of co-crosslinked hydrogels is 3.5 kPa, higher than any chitosan-based gel reported. Chondrocytes and nucleus pulposus cells thrive inside the gels and produce large amounts of collagen II. Injection in rats shows that the gels form in-vivo within a short time and remain well localized for more than one week while the rats remain healthy and active. The excellent mechanical properties, fast in-situ gelation, good biocompatibility and the ability to encapsulate live cells at physiological conditions make these hydrogels ideal for tissue engineering, especially cartilage regeneration. Homogenous, insitu thermogelling, highly tough, non-toxic injectable hydrogels from chitosan and hyaluronic acid co-crosslinked with beta-glycerophophate and genipin are produced. The co-crosslinked gels form faster than genipin-only crosslinked gels and are 2.5 times stronger than ionically crosslinked gels. Homogenously dispersed cells express enhanced amount of collagen II, showing the great potential of these gels for cartilage tissue engineering.},\\n  annote = {cited By 15},\\n  author = {Jalani, Ghulam and Rosenzweig, Derek H. and Makhoul, Georges and Abdalla, Sherif and Cecere, Renzo and Vetrone, Fiorenzo and Haglund, Lisbet and Cerruti, Marta},\\n  doi = {10.1002/mabi.201400406},\\n  issn = {16165195},\\n  journal = {Macromolecular Bioscience},\\n  keywords = {crosslinking,genipin,hydrogels,in-situ thermogelling,injectable},\\n  number = {4},\\n  pages = {473--480},\\n  title = {{Tough, in-situ thermogelling, injectable hydrogels for biomedical applications}},\\n  volume = {15},\\n  year = {2015}\\n  }\\n  \",\"author_short\":[\"Jalani, G.\",\"Rosenzweig, D. H.\",\"Makhoul, G.\",\"Abdalla, S.\",\"Cecere, R.\",\"Vetrone, F.\",\"Haglund, L.\",\"Cerruti, M.\"],\"key\":\"Jalani2015473\",\"id\":\"Jalani2015473\",\"bibbaseid\":\"jalani-rosenzweig-makhoul-abdalla-cecere-vetrone-haglund-cerruti-toughinsituthermogellinginjectablehydrogelsforbiomedicalapplications-2015\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"crosslinking\",\"genipin\",\"hydrogels\",\"in-situ thermogelling\",\"injectable\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Osteoarthritis (OA) is a debilitating joint disorder resulting from an incompletely understood combination of mechanical, biological, and biochemical processes. OA is often accompanied by inflammation and pain, whereby cytokines associated with chronic OA can up-regulate expression of neurotrophic factors such as nerve growth factor (NGF). Several studies suggest a role for cytokines and NGF in OA pain, however the effects of changing mechanical properties in OA tissue on chondrocyte metabolism remain unclear. Here, we used high-extension silicone rubber membranes to examine if high mechanical strain (HMS) of primary articular chondrocytes increases inflammatory gene expression and promotes neurotrophic factor release. HMS cultured chondrocytes displayed up-regulated NGF, TNFalpha and ADAMTS4 gene expression while decreasing TLR2 expression, as compared to static controls. HMS culture increased p38 MAPK activity compared to static controls. Conditioned medium from HMS dynamic cultures, but not static cultures, induced significant neurite sprouting in PC12 cells. The increased neurite sprouting was accompanied by consistent increases in PC12 cell death. Low-frequency high-magnitude mechanical strain of primary articular chondrocytes in vitro drives factor secretion associated with degenerative joint disease and joint pain. This study provides evidence for a direct link between cellular strain, secretory factors, neo-innervation, and pain in OA pathology. (C) 2014 by the authors; licensee MDPI, Basel, Switzerland.\",\"annote\":\"cited By 13\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglun\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.3390/ijms150814427\",\"issn\":\"14220067\",\"journal\":\"International Journal of Molecular Sciences\",\"keywords\":\"Articular cartilage,Inflammation,Mechanical stretch,Nerve growth factor,Osteoarthritis,Pain,p38 MAPK\",\"number\":\"8\",\"pages\":\"14427–14441\",\"title\":\"Low-frequency high-magnitude mechanical strain of articular chondrocytes activates p38 MAPK and induces phenotypic changes associated with osteoarthritis and pain\",\"volume\":\"15\",\"year\":\"2014\",\"bibtex\":\"@article{Rosenzweig201414427,\\n  abstract = {Osteoarthritis (OA) is a debilitating joint disorder resulting from an incompletely understood combination of mechanical, biological, and biochemical processes. OA is often accompanied by inflammation and pain, whereby cytokines associated with chronic OA can up-regulate expression of neurotrophic factors such as nerve growth factor (NGF). Several studies suggest a role for cytokines and NGF in OA pain, however the effects of changing mechanical properties in OA tissue on chondrocyte metabolism remain unclear. Here, we used high-extension silicone rubber membranes to examine if high mechanical strain (HMS) of primary articular chondrocytes increases inflammatory gene expression and promotes neurotrophic factor release. HMS cultured chondrocytes displayed up-regulated NGF, TNFalpha and ADAMTS4 gene expression while decreasing TLR2 expression, as compared to static controls. HMS culture increased p38 MAPK activity compared to static controls. Conditioned medium from HMS dynamic cultures, but not static cultures, induced significant neurite sprouting in PC12 cells. The increased neurite sprouting was accompanied by consistent increases in PC12 cell death. Low-frequency high-magnitude mechanical strain of primary articular chondrocytes in vitro drives factor secretion associated with degenerative joint disease and joint pain. This study provides evidence for a direct link between cellular strain, secretory factors, neo-innervation, and pain in OA pathology. (C) 2014 by the authors; licensee MDPI, Basel, Switzerland.},\\n  annote = {cited By 13},\\n  author = {Rosenzweig, Derek H. and Quinn, Thomas M. and Haglun, Lisbet},\\n  doi = {10.3390/ijms150814427},\\n  issn = {14220067},\\n  journal = {International Journal of Molecular Sciences},\\n  keywords = {Articular cartilage,Inflammation,Mechanical stretch,Nerve growth factor,Osteoarthritis,Pain,p38 MAPK},\\n  number = {8},\\n  pages = {14427--14441},\\n  title = {{Low-frequency high-magnitude mechanical strain of articular chondrocytes activates p38 MAPK and induces phenotypic changes associated with osteoarthritis and pain}},\\n  volume = {15},\\n  year = {2014}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Quinn, T. M.\",\"Haglun, L.\"],\"key\":\"Rosenzweig201414427\",\"id\":\"Rosenzweig201414427\",\"bibbaseid\":\"rosenzweig-quinn-haglun-lowfrequencyhighmagnitudemechanicalstrainofarticularchondrocytesactivatesp38mapkandinducesphenotypicchangesassociatedwithosteoarthritisandpain-2014\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Articular cartilage\",\"Inflammation\",\"Mechanical stretch\",\"Nerve growth factor\",\"Osteoarthritis\",\"Pain\",\"p38 MAPK\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Introduction:Excessive mechanical loading of intervertebral discs (IVDs) is thought to alter matrix properties and influence disc cell metabolism, contributing to degenerative disc disease and development of discogenic pain. However, little is known about how mechanical strain induces these changes. This study investigated the cellular and molecular changes as well as which inflammatory receptors and cytokines were upregulated in human intervertebral disc cells exposed to high mechanical strain (HMS) at low frequency. The impact of these metabolic changes on neuronal differentiation was also explored to determine a role in the development of disc degeneration and discogenic pain.Methods:Isolated human annulus fibrosus (AF) and nucleus pulposus (NP) cells were exposed to HMS (20 % cyclical stretch at 0.001 Hz) on high-extension silicone rubber dishes coupled to a mechanical stretching apparatus and compared to static control cultures. Gene expression of Toll-like receptors (TLRs), neuronal growth factor (NGF) and tumour necrosis factor alpha (TNFalpha) was assessed. Collected conditioned media were analysed for cytokine content and applied to rat pheocromocytoma PC12 cells for neuronal differentiation assessment.Results:HMS caused upregulation of TLR2, TLR4, NGF and TNFalpha gene expression in IVD cells. Medium from HMS cultures contained elevated levels of growth-related oncogene, interleukin 6 (IL-6), IL-8, IL-15, monocyte chemoattractant protein 1 (MCP-1), MCP-3, monokine induced by gamma interferon, transforming growth factor beta1, TNFalpha and NGF. Exposure of PC12 cells to HMS-conditioned media resulted in both increased neurite sprouting and cell death.Conclusions:HMS culture of IVD cells in vitro drives cytokine and inflammatory responses associated with degenerative disc disease and low-back pain. This study provides evidence for a direct link between cellular strain, secretory factors, neoinnervation and potential degeneration and discogenic pain in vivo. (C) 2014 Gawri et al.; licensee BioMed Central Ltd.\",\"annote\":\"cited By 58\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Gawri\"],\"firstnames\":[\"Rahul\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Krock\"],\"firstnames\":[\"Emerson\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ouellet\"],\"firstnames\":[\"Jean\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Stone\"],\"firstnames\":[\"Laura\",\"S.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.1186/ar4449\",\"issn\":\"14786362\",\"journal\":\"Arthritis Research and Therapy\",\"number\":\"1\",\"title\":\"High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain\",\"volume\":\"16\",\"year\":\"2014\",\"bibtex\":\"@article{Gawri2014,\\n  abstract = {Introduction:Excessive mechanical loading of intervertebral discs (IVDs) is thought to alter matrix properties and influence disc cell metabolism, contributing to degenerative disc disease and development of discogenic pain. However, little is known about how mechanical strain induces these changes. This study investigated the cellular and molecular changes as well as which inflammatory receptors and cytokines were upregulated in human intervertebral disc cells exposed to high mechanical strain (HMS) at low frequency. The impact of these metabolic changes on neuronal differentiation was also explored to determine a role in the development of disc degeneration and discogenic pain.Methods:Isolated human annulus fibrosus (AF) and nucleus pulposus (NP) cells were exposed to HMS (20 % cyclical stretch at 0.001 Hz) on high-extension silicone rubber dishes coupled to a mechanical stretching apparatus and compared to static control cultures. Gene expression of Toll-like receptors (TLRs), neuronal growth factor (NGF) and tumour necrosis factor alpha (TNFalpha) was assessed. Collected conditioned media were analysed for cytokine content and applied to rat pheocromocytoma PC12 cells for neuronal differentiation assessment.Results:HMS caused upregulation of TLR2, TLR4, NGF and TNFalpha gene expression in IVD cells. Medium from HMS cultures contained elevated levels of growth-related oncogene, interleukin 6 (IL-6), IL-8, IL-15, monocyte chemoattractant protein 1 (MCP-1), MCP-3, monokine induced by gamma interferon, transforming growth factor beta1, TNFalpha and NGF. Exposure of PC12 cells to HMS-conditioned media resulted in both increased neurite sprouting and cell death.Conclusions:HMS culture of IVD cells in vitro drives cytokine and inflammatory responses associated with degenerative disc disease and low-back pain. This study provides evidence for a direct link between cellular strain, secretory factors, neoinnervation and potential degeneration and discogenic pain in vivo. (C) 2014 Gawri et al.; licensee BioMed Central Ltd.},\\n  annote = {cited By 58},\\n  author = {Gawri, Rahul and Rosenzweig, Derek H. and Krock, Emerson and Ouellet, Jean A. and Stone, Laura S. and Quinn, Thomas M. and Haglund, Lisbet},\\n  doi = {10.1186/ar4449},\\n  issn = {14786362},\\n  journal = {Arthritis Research and Therapy},\\n  number = {1},\\n  title = {{High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain}},\\n  volume = {16},\\n  year = {2014}\\n  }\\n  \",\"author_short\":[\"Gawri, R.\",\"Rosenzweig, D. H.\",\"Krock, E.\",\"Ouellet, J. A.\",\"Stone, L. S.\",\"Quinn, T. M.\",\"Haglund, L.\"],\"key\":\"Gawri2014\",\"id\":\"Gawri2014\",\"bibbaseid\":\"gawri-rosenzweig-krock-ouellet-stone-quinn-haglund-highmechanicalstrainofprimaryintervertebraldisccellspromotessecretionofinflammatoryfactorsassociatedwithdiscdegenerationandpain-2014\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Background: Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur. Methods: Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone. Results: Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde-dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes. Conclusions and general significance: Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents. (C) 2013 Elsevier B.V. All rights reserved.\",\"annote\":\"cited By 6\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Moeini\"],\"firstnames\":[\"Mohammad\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Decker\"],\"firstnames\":[\"Sarah\",\"G.A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Chin\"],\"firstnames\":[\"Hooi\",\"Chuan\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Shafieyan\"],\"firstnames\":[\"Yousef\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1016/j.bbagen.2013.10.022\",\"issn\":\"03044165\",\"journal\":\"Biochimica et Biophysica Acta - General Subjects\",\"keywords\":\"Articular cartilage,Contrast agent,Dextrans,FITC,Injury,Solute adsorption\",\"number\":\"1\",\"pages\":\"605–614\",\"title\":\"Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents\",\"volume\":\"1840\",\"year\":\"2014\",\"bibtex\":\"@article{Moeini2014605,\\n  abstract = {Background: Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur. Methods: Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone. Results: Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde-dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes. Conclusions and general significance: Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents. (C) 2013 Elsevier B.V. All rights reserved.},\\n  annote = {cited By 6},\\n  author = {Moeini, Mohammad and Decker, Sarah G.A. and Chin, Hooi Chuan and Shafieyan, Yousef and Rosenzweig, Derek H. and Quinn, Thomas M.},\\n  doi = {10.1016/j.bbagen.2013.10.022},\\n  issn = {03044165},\\n  journal = {Biochimica et Biophysica Acta - General Subjects},\\n  keywords = {Articular cartilage,Contrast agent,Dextrans,FITC,Injury,Solute adsorption},\\n  number = {1},\\n  pages = {605--614},\\n  title = {{Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents}},\\n  volume = {1840},\\n  year = {2014}\\n  }\\n  \",\"author_short\":[\"Moeini, M.\",\"Decker, S. G.\",\"Chin, H. C.\",\"Shafieyan, Y.\",\"Rosenzweig, D. H.\",\"Quinn, T. M.\"],\"key\":\"Moeini2014605\",\"id\":\"Moeini2014605\",\"bibbaseid\":\"moeini-decker-chin-shafieyan-rosenzweig-quinn-decreasedsoluteadsorptionontocrackedsurfacesofmechanicallyinjuredarticularcartilagetowardsthedesignofcartilagespecificfunctionalcontrastagents-2014\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Articular cartilage\",\"Contrast agent\",\"Dextrans\",\"FITC\",\"Injury\",\"Solute adsorption\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-alpha, interleukin-1beta, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo. (C) 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.\",\"annote\":\"cited By 60\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Krock\"],\"firstnames\":[\"Emerson\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Chabot-Dore\"],\"firstnames\":[\"Anne\",\"Julie\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Jarzem\"],\"firstnames\":[\"Peter\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ouellet\"],\"firstnames\":[\"Jean\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Stone\"],\"firstnames\":[\"Laura\",\"S.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"Lisbet\"],\"suffixes\":[]}],\"doi\":\"10.1111/jcmm.12268\",\"issn\":\"15824934\",\"journal\":\"Journal of Cellular and Molecular Medicine\",\"keywords\":\"CGRP,Discogenic pain,Human,Inflammatory cytokines,Intervertebral disc degeneration,Nerve growth factor\",\"number\":\"6\",\"pages\":\"1213–1225\",\"title\":\"Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors\",\"volume\":\"18\",\"year\":\"2014\",\"bibtex\":\"@article{Krock20141213,\\n  abstract = {Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-alpha, interleukin-1beta, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo. (C) 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley {&} Sons Ltd and Foundation for Cellular and Molecular Medicine.},\\n  annote = {cited By 60},\\n  author = {Krock, Emerson and Rosenzweig, Derek H. and Chabot-Dore, Anne Julie and Jarzem, Peter and Weber, Michael H. and Ouellet, Jean A. and Stone, Laura S. and Haglund, Lisbet},\\n  doi = {10.1111/jcmm.12268},\\n  issn = {15824934},\\n  journal = {Journal of Cellular and Molecular Medicine},\\n  keywords = {CGRP,Discogenic pain,Human,Inflammatory cytokines,Intervertebral disc degeneration,Nerve growth factor},\\n  number = {6},\\n  pages = {1213--1225},\\n  title = {{Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors}},\\n  volume = {18},\\n  year = {2014}\\n  }\\n  \",\"author_short\":[\"Krock, E.\",\"Rosenzweig, D. H.\",\"Chabot-Dore, A. J.\",\"Jarzem, P.\",\"Weber, M. H.\",\"Ouellet, J. A.\",\"Stone, L. S.\",\"Haglund, L.\"],\"key\":\"Krock20141213\",\"id\":\"Krock20141213\",\"bibbaseid\":\"krock-rosenzweig-chabotdore-jarzem-weber-ouellet-stone-haglund-painfuldegeneratingintervertebraldiscsupregulateneuritesproutingandcgrpthroughnociceptivefactors-2014\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"CGRP\",\"Discogenic pain\",\"Human\",\"Inflammatory cytokines\",\"Intervertebral disc degeneration\",\"Nerve growth factor\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5 % (non-injured) or 30 % (injured) of disc height. 30 % strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured- IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROalpha, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.\",\"annote\":\"cited By 45\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Alkhatib\"],\"firstnames\":[\"B.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"D.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Krock\"],\"firstnames\":[\"E.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Roughley\"],\"firstnames\":[\"P.\",\"J.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Beckman\"],\"firstnames\":[\"L.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Steffen\"],\"firstnames\":[\"T.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"M.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ouellet\"],\"firstnames\":[\"J.\",\"A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Haglund\"],\"firstnames\":[\"L.\"],\"suffixes\":[]}],\"doi\":\"10.22203/eCM.v028a08\",\"issn\":\"14732262\",\"journal\":\"European Cells and Materials\",\"keywords\":\"Degeneration,Extracellular matrix,Inflammation,Intervertebral disc,Mechanical injury,Nerve growth factor\",\"pages\":\"98–111\",\"title\":\"Acute mechanical injury of the human intervertebral disc: link to degeneration and pain\",\"volume\":\"28\",\"year\":\"2014\",\"bibtex\":\"@article{Alkhatib201498,\\n  abstract = {Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5 % (non-injured) or 30 % (injured) of disc height. 30 % strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured- IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROalpha, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.},\\n  annote = {cited By 45},\\n  author = {Alkhatib, B. and Rosenzweig, D. H. and Krock, E. and Roughley, P. J. and Beckman, L. and Steffen, T. and Weber, M. H. and Ouellet, J. A. and Haglund, L.},\\n  doi = {10.22203/eCM.v028a08},\\n  issn = {14732262},\\n  journal = {European Cells and Materials},\\n  keywords = {Degeneration,Extracellular matrix,Inflammation,Intervertebral disc,Mechanical injury,Nerve growth factor},\\n  pages = {98--111},\\n  title = {{Acute mechanical injury of the human intervertebral disc: link to degeneration and pain}},\\n  volume = {28},\\n  year = {2014}\\n  }\\n  \",\"author_short\":[\"Alkhatib, B.\",\"Rosenzweig, D. H.\",\"Krock, E.\",\"Roughley, P. J.\",\"Beckman, L.\",\"Steffen, T.\",\"Weber, M. H.\",\"Ouellet, J. A.\",\"Haglund, L.\"],\"key\":\"Alkhatib201498\",\"id\":\"Alkhatib201498\",\"bibbaseid\":\"alkhatib-rosenzweig-krock-roughley-beckman-steffen-weber-ouellet-etal-acutemechanicalinjuryofthehumanintervertebraldisclinktodegenerationandpain-2014\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Degeneration\",\"Extracellular matrix\",\"Inflammation\",\"Intervertebral disc\",\"Mechanical injury\",\"Nerve growth factor\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.\",\"annote\":\"cited By 14\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"D.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Chicatun\"],\"firstnames\":[\"F.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Nazhat\"],\"firstnames\":[\"S.\",\"N.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"T.\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1016/j.actbio.2013.07.024\",\"issn\":\"17427061\",\"journal\":\"Acta Biomaterialia\",\"keywords\":\"Articular chondrocytes,Dense collagen,Hydrogel,Plastic compression,Tissue engineering\",\"number\":\"12\",\"pages\":\"9360–9369\",\"title\":\"Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels\",\"volume\":\"9\",\"year\":\"2013\",\"bibtex\":\"@article{Rosenzweig20139360,\\n  abstract = {Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.},\\n  annote = {cited By 14},\\n  author = {Rosenzweig, D. H. and Chicatun, F. and Nazhat, S. N. and Quinn, T. M.},\\n  doi = {10.1016/j.actbio.2013.07.024},\\n  issn = {17427061},\\n  journal = {Acta Biomaterialia},\\n  keywords = {Articular chondrocytes,Dense collagen,Hydrogel,Plastic compression,Tissue engineering},\\n  number = {12},\\n  pages = {9360--9369},\\n  title = {{Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels}},\\n  volume = {9},\\n  year = {2013}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Chicatun, F.\",\"Nazhat, S. N.\",\"Quinn, T. M.\"],\"key\":\"Rosenzweig20139360\",\"id\":\"Rosenzweig20139360\",\"bibbaseid\":\"rosenzweig-chicatun-nazhat-quinn-cartilaginousconstructsusingprimarychondrocytesfromcontinuousexpansioncultureseededindensecollagengels-2013\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Articular chondrocytes\",\"Dense collagen\",\"Hydrogel\",\"Plastic compression\",\"Tissue engineering\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"The development of cartilage-specific imaging agents supports the improvement of tissue assessment by minimally invasive means. Techniques for highlighting cartilage surface damage in clinical images could provide for sensitive indications of posttraumatic injury and early stage osteoarthritis. Previous studies in our laboratory have demonstrated that fluorescent solutes interact with cartilage surfaces strongly enough to affect measurement of their partition coefficients within the tissue bulk. In this study, these findings were extended by examining solute adsorption and distribution near the articular surface of mechanically injured cartilage. Using viable cartilage explants injured by an established protocol, solute distributions near the articular surface of three commonly used fluorophores (fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and carboxytetramethylrhodamine (TAMRA)) were observed after absorption and subsequent desorption to assess solute-specific matrix interactions and reversibility. Both absorption and desorption processes demonstrated a trend of significantly less solute adsorption at surfaces of fissures compared to adjacent intact surfaces of damaged explants or surfaces of uninjured explants. After adsorption, normalized mean surface intensities of fissured surfaces of injured explants were 6%, 40%, and 32% for FITC, TRITC, and TAMRA, respectively, compared to uninjured surfaces. Similar values were found for sliced explants and after a desorption process. After desorption, a trend of increased solute adsorption at the site of intact damaged surfaces was noted (316% and 238% for injured and sliced explants exposed to FITC). Surface adsorption of solute was strongest for FITC and weakest for TAMRA; no solutes negatively affected cell viability. Results support the development of imaging agents that highlight distinct differences between fissured and intact cartilage surfaces. (C) 2013 Biophysical Society.\",\"annote\":\"cited By 9\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Decker\"],\"firstnames\":[\"Sarah\",\"G.A.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Moeini\"],\"firstnames\":[\"Mohammad\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Chin\"],\"firstnames\":[\"Hooi\",\"Chuan\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1016/j.bpj.2013.09.037\",\"issn\":\"00063495\",\"journal\":\"Biophysical Journal\",\"number\":\"10\",\"pages\":\"2427–2436\",\"title\":\"Adsorption and distribution of fluorescent solutes near the articular surface of mechanically injured cartilage\",\"volume\":\"105\",\"year\":\"2013\",\"bibtex\":\"@article{Decker20132427,\\n  abstract = {The development of cartilage-specific imaging agents supports the improvement of tissue assessment by minimally invasive means. Techniques for highlighting cartilage surface damage in clinical images could provide for sensitive indications of posttraumatic injury and early stage osteoarthritis. Previous studies in our laboratory have demonstrated that fluorescent solutes interact with cartilage surfaces strongly enough to affect measurement of their partition coefficients within the tissue bulk. In this study, these findings were extended by examining solute adsorption and distribution near the articular surface of mechanically injured cartilage. Using viable cartilage explants injured by an established protocol, solute distributions near the articular surface of three commonly used fluorophores (fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and carboxytetramethylrhodamine (TAMRA)) were observed after absorption and subsequent desorption to assess solute-specific matrix interactions and reversibility. Both absorption and desorption processes demonstrated a trend of significantly less solute adsorption at surfaces of fissures compared to adjacent intact surfaces of damaged explants or surfaces of uninjured explants. After adsorption, normalized mean surface intensities of fissured surfaces of injured explants were 6%, 40%, and 32% for FITC, TRITC, and TAMRA, respectively, compared to uninjured surfaces. Similar values were found for sliced explants and after a desorption process. After desorption, a trend of increased solute adsorption at the site of intact damaged surfaces was noted (316% and 238% for injured and sliced explants exposed to FITC). Surface adsorption of solute was strongest for FITC and weakest for TAMRA; no solutes negatively affected cell viability. Results support the development of imaging agents that highlight distinct differences between fissured and intact cartilage surfaces. (C) 2013 Biophysical Society.},\\n  annote = {cited By 9},\\n  author = {Decker, Sarah G.A. and Moeini, Mohammad and Chin, Hooi Chuan and Rosenzweig, Derek H. and Quinn, Thomas M.},\\n  doi = {10.1016/j.bpj.2013.09.037},\\n  issn = {00063495},\\n  journal = {Biophysical Journal},\\n  number = {10},\\n  pages = {2427--2436},\\n  title = {{Adsorption and distribution of fluorescent solutes near the articular surface of mechanically injured cartilage}},\\n  volume = {105},\\n  year = {2013}\\n  }\\n  \",\"author_short\":[\"Decker, S. G.\",\"Moeini, M.\",\"Chin, H. C.\",\"Rosenzweig, D. H.\",\"Quinn, T. M.\"],\"key\":\"Decker20132427\",\"id\":\"Decker20132427\",\"bibbaseid\":\"decker-moeini-chin-rosenzweig-quinn-adsorptionanddistributionoffluorescentsolutesnearthearticularsurfaceofmechanicallyinjuredcartilage-2013\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Cartilage defects can be addressed with replacement strategies such as autologous chondrocyte implantation (ACI). Expansion of autologous chondrocytes in vitro is an essential step to obtain the necessary cell numbers required for ACI. A major problem with this approach is dedifferentiation of chondrocytes during expansion, resulting in cells with fibroblast-like features. These cells generate cartilage tissue with fibrotic instead of hyaline characteristics. The use of serum is a common feature in most expansion protocols and a potential factor contributing to the dedifferentiation process. The aim of this study was to assess if heat inactivation of serum used in the expansion medium might be a valid approach to generate cells with an improved phenotype and in relevant numbers. We used bovine chondrocyte expansion cultures incubated with heat inactivated allogeneic serum (HIFBS) as a model system. We here show that heat inactivation protects the differentiated phenotype of chondrocytes compared to cultures with regular serum. This is not only true for primary cultures but holds up after two passages. Moreover, using relatively low cell seeding densities, clinically relevant cell numbers can already be reached after the first passage in cultures with HIFBS. In short we here introduce a simple way to improve cell quality while generating relevant amounts of cells during monolayer expansion of bovine chondrocytes in a relative short time period. Our results could have wider implications when translated to the expansion of human chondrocytes. (C) 2012 Biomedical Engineering Society.\",\"annote\":\"cited By 10\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Matmati\"],\"firstnames\":[\"Mourad\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ng\"],\"firstnames\":[\"Tat\",\"Fong\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1007/s10439-012-0732-z\",\"issn\":\"00906964\",\"journal\":\"Annals of Biomedical Engineering\",\"keywords\":\"Chondrocyte,Expansion culture,Heat inactivation,Serum\",\"number\":\"5\",\"pages\":\"894–903\",\"title\":\"Protection of bovine chondrocyte phenotype by heat inactivation of allogeneic serum in monolayer expansion cultures\",\"volume\":\"41\",\"year\":\"2013\",\"bibtex\":\"@article{Matmati2013894,\\n  abstract = {Cartilage defects can be addressed with replacement strategies such as autologous chondrocyte implantation (ACI). Expansion of autologous chondrocytes in vitro is an essential step to obtain the necessary cell numbers required for ACI. A major problem with this approach is dedifferentiation of chondrocytes during expansion, resulting in cells with fibroblast-like features. These cells generate cartilage tissue with fibrotic instead of hyaline characteristics. The use of serum is a common feature in most expansion protocols and a potential factor contributing to the dedifferentiation process. The aim of this study was to assess if heat inactivation of serum used in the expansion medium might be a valid approach to generate cells with an improved phenotype and in relevant numbers. We used bovine chondrocyte expansion cultures incubated with heat inactivated allogeneic serum (HIFBS) as a model system. We here show that heat inactivation protects the differentiated phenotype of chondrocytes compared to cultures with regular serum. This is not only true for primary cultures but holds up after two passages. Moreover, using relatively low cell seeding densities, clinically relevant cell numbers can already be reached after the first passage in cultures with HIFBS. In short we here introduce a simple way to improve cell quality while generating relevant amounts of cells during monolayer expansion of bovine chondrocytes in a relative short time period. Our results could have wider implications when translated to the expansion of human chondrocytes. (C) 2012 Biomedical Engineering Society.},\\n  annote = {cited By 10},\\n  author = {Matmati, Mourad and Ng, Tat Fong and Rosenzweig, Derek H. and Quinn, Thomas M.},\\n  doi = {10.1007/s10439-012-0732-z},\\n  issn = {00906964},\\n  journal = {Annals of Biomedical Engineering},\\n  keywords = {Chondrocyte,Expansion culture,Heat inactivation,Serum},\\n  number = {5},\\n  pages = {894--903},\\n  title = {{Protection of bovine chondrocyte phenotype by heat inactivation of allogeneic serum in monolayer expansion cultures}},\\n  volume = {41},\\n  year = {2013}\\n  }\\n  \",\"author_short\":[\"Matmati, M.\",\"Ng, T. F.\",\"Rosenzweig, D. H.\",\"Quinn, T. M.\"],\"key\":\"Matmati2013894\",\"id\":\"Matmati2013894\",\"bibbaseid\":\"matmati-ng-rosenzweig-quinn-protectionofbovinechondrocytephenotypebyheatinactivationofallogeneicseruminmonolayerexpansioncultures-2013\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Chondrocyte\",\"Expansion culture\",\"Heat inactivation\",\"Serum\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell-based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes 'dedifferentiation' into fibroblastic cells. Mitogen-activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up-regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow-on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell-based therapies. (C) 2013.\",\"annote\":\"cited By 21\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ou\"],\"firstnames\":[\"Sing\",\"J.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1111/jcmm.12034\",\"issn\":\"15821838\",\"journal\":\"Journal of Cellular and Molecular Medicine\",\"keywords\":\"Chondrocyte,Dedifferentiation,Extra Cellular Matrix,Gene Expression,MAPK,Signal Transduction\",\"number\":\"4\",\"pages\":\"508–517\",\"pmid\":\"23480786\",\"title\":\"P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture\",\"volume\":\"17\",\"year\":\"2013\",\"bibtex\":\"@article{Rosenzweig2013508,\\n  abstract = {Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell-based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes 'dedifferentiation' into fibroblastic cells. Mitogen-activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up-regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow-on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell-based therapies. (C) 2013.},\\n  annote = {cited By 21},\\n  author = {Rosenzweig, Derek H. and Ou, Sing J. and Quinn, Thomas M.},\\n  doi = {10.1111/jcmm.12034},\\n  issn = {15821838},\\n  journal = {Journal of Cellular and Molecular Medicine},\\n  keywords = {Chondrocyte,Dedifferentiation,Extra Cellular Matrix,Gene Expression,MAPK,Signal Transduction},\\n  number = {4},\\n  pages = {508--517},\\n  pmid = {23480786},\\n  title = {{P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture}},\\n  volume = {17},\\n  year = {2013}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Ou, S. J.\",\"Quinn, T. M.\"],\"key\":\"Rosenzweig2013508\",\"id\":\"Rosenzweig2013508\",\"bibbaseid\":\"rosenzweig-ou-quinn-p38mitogenactivatedproteinkinasepromotesdedifferentiationofprimaryarticularchondrocytesinmonolayerculture-2013\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Chondrocyte\",\"Dedifferentiation\",\"Extra Cellular Matrix\",\"Gene Expression\",\"MAPK\",\"Signal Transduction\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Objective: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. Design: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. Results: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. Conclusions: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries. (C) 2012 Osteoarthritis Research Society International.\",\"annote\":\"cited By 19\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"D.\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Djap\"],\"firstnames\":[\"M.\",\"J.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ou\"],\"firstnames\":[\"S.\",\"J.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"T.\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1016/j.joca.2012.08.012\",\"issn\":\"10634584\",\"journal\":\"Osteoarthritis and Cartilage\",\"keywords\":\"Chondrocyte,Compression,Gene expression,Injury,MAP kinase,Signalling\",\"number\":\"12\",\"pages\":\"1591–1602\",\"pmid\":\"22935788\",\"title\":\"Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis\",\"volume\":\"20\",\"year\":\"2012\",\"bibtex\":\"@article{Rosenzweig20121591,\\n  abstract = {Objective: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. Design: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. Results: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. Conclusions: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries. (C) 2012 Osteoarthritis Research Society International.},\\n  annote = {cited By 19},\\n  author = {Rosenzweig, D. H. and Djap, M. J. and Ou, S. J. and Quinn, T. M.},\\n  doi = {10.1016/j.joca.2012.08.012},\\n  issn = {10634584},\\n  journal = {Osteoarthritis and Cartilage},\\n  keywords = {Chondrocyte,Compression,Gene expression,Injury,MAP kinase,Signalling},\\n  number = {12},\\n  pages = {1591--1602},\\n  pmid = {22935788},\\n  title = {{Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis}},\\n  volume = {20},\\n  year = {2012}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Djap, M. J.\",\"Ou, S. J.\",\"Quinn, T. M.\"],\"key\":\"Rosenzweig20121591\",\"id\":\"Rosenzweig20121591\",\"bibbaseid\":\"rosenzweig-djap-ou-quinn-mechanicalinjuryofbovinecartilageexplantsinducesdepthdependenttransientchangesinmapkinaseactivityassociatedwithapoptosis-2012\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Chondrocyte\",\"Compression\",\"Gene expression\",\"Injury\",\"MAP kinase\",\"Signalling\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and alpha-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies. (C) Copyright 2012, Mary Ann Liebert, Inc.\",\"annote\":\"cited By 32\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Matmati\"],\"firstnames\":[\"Mourad\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Khayat\"],\"firstnames\":[\"Ghazaleh\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Chaudhry\"],\"firstnames\":[\"Sidharth\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Hinz\"],\"firstnames\":[\"Boris\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1089/ten.tea.2012.0215\",\"issn\":\"19373341\",\"journal\":\"Tissue Engineering - Part A\",\"number\":\"23-24\",\"pages\":\"2466–2476\",\"title\":\"Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation\",\"volume\":\"18\",\"year\":\"2012\",\"bibtex\":\"@article{Rosenzweig20122466,\\n  abstract = {Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and alpha-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies. (C) Copyright 2012, Mary Ann Liebert, Inc.},\\n  annote = {cited By 32},\\n  author = {Rosenzweig, Derek H. and Matmati, Mourad and Khayat, Ghazaleh and Chaudhry, Sidharth and Hinz, Boris and Quinn, Thomas M.},\\n  doi = {10.1089/ten.tea.2012.0215},\\n  issn = {19373341},\\n  journal = {Tissue Engineering - Part A},\\n  number = {23-24},\\n  pages = {2466--2476},\\n  title = {{Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation}},\\n  volume = {18},\\n  year = {2012}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Matmati, M.\",\"Khayat, G.\",\"Chaudhry, S.\",\"Hinz, B.\",\"Quinn, T. M.\"],\"key\":\"Rosenzweig20122466\",\"id\":\"Rosenzweig20122466\",\"bibbaseid\":\"rosenzweig-matmati-khayat-chaudhry-hinz-quinn-cultureofprimarybovinechondrocytesonacontinuouslyexpandingsurfaceinhibitsdedifferentiation-2012\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Culture on silicone rubber surfaces has been shown to partially overcome the chondrocyte dedifferentiation characteristic of standard culture on rigid polystyrene. These methods typically involve functionalization of culture surfaces with proteins. Collagen type I is often used, but more cartilage-specific proteins may be more appropriate for chondrocytes. To explore this hypothesis, a twofold experimental design was applied. First, chondrocytes were cultured in rigid Petri dishes coated with silicone rubber (\\\"static silicone\\\" or SS culture) functionalized with either cartilage extracellular matrix (ECM) extract or collagen type I. Second, chondrocytes were cultured on monotonically expanded high extension silicone rubber dishes (\\\"continuous expansion\\\" or CE culture) functionalized with ECM extract and compared to cells grown in SS culture. There were no differential effects of surface functionalization with the ECM extract vs. collagen type I on chondrocyte morphology, viability, proliferation or apoptosis in SS culture. However, chondrocyte growth on the ECM extract was associated with significantly reduced collagen types I and X gene expression and significantly increased glycosaminoglycan (GAG) secretion. After 3 passages (P3) on ECM-coated SS culture, chondrocyte phenotype and GAG secretion was enhanced compared to cells passaged on collagen type I. Pellet cultures from P3 SS culture displayed enhanced collagen type II content when ECM extract was used for functionalization rather than collagen type I. In CE culture with ECM functionalization, chondrocyte dedifferentiation was significantly inhibited vs. SS cultures, as evidenced by both gene expression and pellet cultures. Functionalization of extendable culture surfaces with cartilage ECM extract therefore supports enhanced preservation of chondrocyte phenotype. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.\",\"annote\":\"cited By 22\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Solar-Cafaggi\"],\"firstnames\":[\"Sofia\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1016/j.actbio.2012.05.032\",\"issn\":\"17427061\",\"journal\":\"Acta Biomaterialia\",\"keywords\":\"Chondrocyte,Collagen,Dedifferentiation,Extendable surfaces,Extracellular matrix\",\"number\":\"9\",\"pages\":\"3333–3341\",\"title\":\"Functionalization of dynamic culture surfaces with a cartilage extracellular matrix extract enhances chondrocyte phenotype against dedifferentiation\",\"volume\":\"8\",\"year\":\"2012\",\"bibtex\":\"@article{Rosenzweig20123333,\\n  abstract = {Culture on silicone rubber surfaces has been shown to partially overcome the chondrocyte dedifferentiation characteristic of standard culture on rigid polystyrene. These methods typically involve functionalization of culture surfaces with proteins. Collagen type I is often used, but more cartilage-specific proteins may be more appropriate for chondrocytes. To explore this hypothesis, a twofold experimental design was applied. First, chondrocytes were cultured in rigid Petri dishes coated with silicone rubber (\\\"static silicone\\\" or SS culture) functionalized with either cartilage extracellular matrix (ECM) extract or collagen type I. Second, chondrocytes were cultured on monotonically expanded high extension silicone rubber dishes (\\\"continuous expansion\\\" or CE culture) functionalized with ECM extract and compared to cells grown in SS culture. There were no differential effects of surface functionalization with the ECM extract vs. collagen type I on chondrocyte morphology, viability, proliferation or apoptosis in SS culture. However, chondrocyte growth on the ECM extract was associated with significantly reduced collagen types I and X gene expression and significantly increased glycosaminoglycan (GAG) secretion. After 3 passages (P3) on ECM-coated SS culture, chondrocyte phenotype and GAG secretion was enhanced compared to cells passaged on collagen type I. Pellet cultures from P3 SS culture displayed enhanced collagen type II content when ECM extract was used for functionalization rather than collagen type I. In CE culture with ECM functionalization, chondrocyte dedifferentiation was significantly inhibited vs. SS cultures, as evidenced by both gene expression and pellet cultures. Functionalization of extendable culture surfaces with cartilage ECM extract therefore supports enhanced preservation of chondrocyte phenotype. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.},\\n  annote = {cited By 22},\\n  author = {Rosenzweig, Derek H. and Solar-Cafaggi, Sofia and Quinn, Thomas M.},\\n  doi = {10.1016/j.actbio.2012.05.032},\\n  issn = {17427061},\\n  journal = {Acta Biomaterialia},\\n  keywords = {Chondrocyte,Collagen,Dedifferentiation,Extendable surfaces,Extracellular matrix},\\n  number = {9},\\n  pages = {3333--3341},\\n  title = {{Functionalization of dynamic culture surfaces with a cartilage extracellular matrix extract enhances chondrocyte phenotype against dedifferentiation}},\\n  volume = {8},\\n  year = {2012}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Solar-Cafaggi, S.\",\"Quinn, T. M.\"],\"key\":\"Rosenzweig20123333\",\"id\":\"Rosenzweig20123333\",\"bibbaseid\":\"rosenzweig-solarcafaggi-quinn-functionalizationofdynamicculturesurfaceswithacartilageextracellularmatrixextractenhanceschondrocytephenotypeagainstdedifferentiation-2012\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Chondrocyte\",\"Collagen\",\"Dedifferentiation\",\"Extendable surfaces\",\"Extracellular matrix\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Oscillatory mechanical stimulation at relatively high frequencies (0.1. Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01. Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01. Hz frequency for 6. h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARgamma) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARgamma after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages. (C) 2012 International Society of Differentiation.\",\"annote\":\"cited By 25\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Khayat\"],\"firstnames\":[\"Ghazaleh\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Quinn\"],\"firstnames\":[\"Thomas\",\"M.\"],\"suffixes\":[]}],\"doi\":\"10.1016/j.diff.2011.12.004\",\"issn\":\"03014681\",\"journal\":\"Differentiation\",\"keywords\":\"Adipogenesis,C3H10T1/2,Differentiation,Gene expression,Low frequency,Mechanical stimulation\",\"number\":\"4\",\"pages\":\"179–184\",\"title\":\"Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells\",\"volume\":\"83\",\"year\":\"2012\",\"bibtex\":\"@article{Khayat2012179,\\n  abstract = {Oscillatory mechanical stimulation at relatively high frequencies (0.1. Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01. Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01. Hz frequency for 6. h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARgamma) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARgamma after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages. (C) 2012 International Society of Differentiation.},\\n  annote = {cited By 25},\\n  author = {Khayat, Ghazaleh and Rosenzweig, Derek H. and Quinn, Thomas M.},\\n  doi = {10.1016/j.diff.2011.12.004},\\n  issn = {03014681},\\n  journal = {Differentiation},\\n  keywords = {Adipogenesis,C3H10T1/2,Differentiation,Gene expression,Low frequency,Mechanical stimulation},\\n  number = {4},\\n  pages = {179--184},\\n  title = {{Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells}},\\n  volume = {83},\\n  year = {2012}\\n  }\\n  \",\"author_short\":[\"Khayat, G.\",\"Rosenzweig, D. H.\",\"Quinn, T. M.\"],\"key\":\"Khayat2012179\",\"id\":\"Khayat2012179\",\"bibbaseid\":\"khayat-rosenzweig-quinn-lowfrequencymechanicalstimulationinhibitsadipogenicdifferentiationofc3h10t12mesenchymalstemcells-2012\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Adipogenesis\",\"C3H10T1/2\",\"Differentiation\",\"Gene expression\",\"Low frequency\",\"Mechanical stimulation\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between Galphat (the transducin alpha subunit) and retGC. Galphat co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-Galphat complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with bothGalphat and retGC. The Galphat-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound Galphat stronger than the GTP[S] (GTPgammaS; guanosine 5′-[gamma-thio]triphosphate) form. Neither Galphat nor Gbetagamma. affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between Galphat and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors. (C) The Authors Journal compilation (C) 2009 Biochemical Society.\",\"annote\":\"cited By 3\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Nair\"],\"firstnames\":[\"Saidas\",\"K.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Levay\"],\"firstnames\":[\"Konstantin\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Peshenko\"],\"firstnames\":[\"Igor\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Crabb\"],\"firstnames\":[\"John\",\"W.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Dizhoor\"],\"firstnames\":[\"Alexander\",\"M.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Slepak\"],\"firstnames\":[\"Vladlen\",\"Z.\"],\"suffixes\":[]}],\"doi\":\"10.1042/BJ20081513\",\"issn\":\"02646021\",\"journal\":\"Biochemical Journal\",\"keywords\":\"CGMP,G-protein,Photoreceptor,Subcellular localization\",\"number\":\"3\",\"pages\":\"803–812\",\"title\":\"Interaction of retinal guanylate cyclase with the alpha subunit of transducin: Potential role in transducin localization\",\"volume\":\"417\",\"year\":\"2009\",\"bibtex\":\"@article{Rosenweig2009803,\\n  abstract = {Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between Galphat (the transducin alpha subunit) and retGC. Galphat co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-Galphat complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with bothGalphat and retGC. The Galphat-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound Galphat stronger than the GTP[S] (GTPgammaS; guanosine 5′-[gamma-thio]triphosphate) form. Neither Galphat nor Gbetagamma. affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between Galphat and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors. (C) The Authors Journal compilation (C) 2009 Biochemical Society.},\\n  annote = {cited By 3},\\n  author = {Rosenweig, Derek H. and Nair, Saidas K. and Levay, Konstantin and Peshenko, Igor and Crabb, John W. and Dizhoor, Alexander M. and Slepak, Vladlen Z.},\\n  doi = {10.1042/BJ20081513},\\n  issn = {02646021},\\n  journal = {Biochemical Journal},\\n  keywords = {CGMP,G-protein,Photoreceptor,Subcellular localization},\\n  number = {3},\\n  pages = {803--812},\\n  title = {{Interaction of retinal guanylate cyclase with the alpha subunit of transducin: Potential role in transducin localization}},\\n  volume = {417},\\n  year = {2009}\\n  }\\n  \",\"author_short\":[\"Rosenweig, D. H.\",\"Nair, S. K.\",\"Levay, K.\",\"Peshenko, I.\",\"Crabb, J. W.\",\"Dizhoor, A. M.\",\"Slepak, V. Z.\"],\"key\":\"Rosenweig2009803\",\"id\":\"Rosenweig2009803\",\"bibbaseid\":\"rosenweig-nair-levay-peshenko-crabb-dizhoor-slepak-interactionofretinalguanylatecyclasewiththealphasubunitoftransducinpotentialroleintransducinlocalization-2009\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"CGMP\",\"G-protein\",\"Photoreceptor\",\"Subcellular localization\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments. Copyright (C) 2007 Society for Neuroscience.\",\"annote\":\"cited By 51\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Saidas Nair\"],\"firstnames\":[\"K.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Wei\"],\"firstnames\":[\"Junhua\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Wang\"],\"firstnames\":[\"Qiang\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Garwin\"],\"firstnames\":[\"Greg\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Saari\"],\"firstnames\":[\"John\",\"C.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Chen\"],\"firstnames\":[\"Ching\",\"Kang\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Smrcka\"],\"firstnames\":[\"Alan\",\"V.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Swaroop\"],\"firstnames\":[\"Anand\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Lem\"],\"firstnames\":[\"Janis\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Hurley\"],\"firstnames\":[\"James\",\"B.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Slepak\"],\"firstnames\":[\"Vladlen\",\"Z.\"],\"suffixes\":[]}],\"doi\":\"10.1523/JNEUROSCI.1421-07.2007\",\"issn\":\"02706474\",\"journal\":\"Journal of Neuroscience\",\"keywords\":\"Cone,Diffusion,Retina,Rod,Signal transduction,Transducin\",\"number\":\"20\",\"pages\":\"5484–5494\",\"pmid\":\"17507570\",\"title\":\"Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins\",\"volume\":\"27\",\"year\":\"2007\",\"bibtex\":\"@article{Rosenzweig20075484,\\n  abstract = {Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments. Copyright (C) 2007 Society for Neuroscience.},\\n  annote = {cited By 51},\\n  author = {Rosenzweig, Derek H. and {Saidas Nair}, K. and Wei, Junhua and Wang, Qiang and Garwin, Greg and Saari, John C. and Chen, Ching Kang and Smrcka, Alan V. and Swaroop, Anand and Lem, Janis and Hurley, James B. and Slepak, Vladlen Z.},\\n  doi = {10.1523/JNEUROSCI.1421-07.2007},\\n  issn = {02706474},\\n  journal = {Journal of Neuroscience},\\n  keywords = {Cone,Diffusion,Retina,Rod,Signal transduction,Transducin},\\n  number = {20},\\n  pages = {5484--5494},\\n  pmid = {17507570},\\n  title = {{Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins}},\\n  volume = {27},\\n  year = {2007}\\n  }\\n  \",\"author_short\":[\"Rosenzweig, D. H.\",\"Saidas Nair, K.\",\"Wei, J.\",\"Wang, Q.\",\"Garwin, G.\",\"Saari, J. C.\",\"Chen, C. K.\",\"Smrcka, A. V.\",\"Swaroop, A.\",\"Lem, J.\",\"Hurley, J. B.\",\"Slepak, V. Z.\"],\"key\":\"Rosenzweig20075484\",\"id\":\"Rosenzweig20075484\",\"bibbaseid\":\"rosenzweig-saidasnair-wei-wang-garwin-saari-chen-smrcka-etal-subunitdissociationanddiffusiondeterminethesubcellularlocalizationofrodandconetransducins-2007\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"Cone\",\"Diffusion\",\"Retina\",\"Rod\",\"Signal transduction\",\"Transducin\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"PURPOSE. Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells. METHODS. Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding. RESULTS. LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange. CONCLUSIONS. Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.\",\"annote\":\"cited By 13\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Nair\"],\"firstnames\":[\"K.\",\"Saidas\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Mendez\"],\"firstnames\":[\"Ana\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Blumer\"],\"firstnames\":[\"Joe\",\"B.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H.\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Slepak\"],\"firstnames\":[\"Vladlen\",\"Z.\"],\"suffixes\":[]}],\"doi\":\"10.1167/iovs.04-1006\",\"issn\":\"01460404\",\"journal\":\"Investigative Ophthalmology and Visual Science\",\"number\":\"1\",\"pages\":\"383–389\",\"title\":\"The presence of a Leu-Gly-Asn repeat-enriched protein (LGN), a putative binding partner of transducin, in ROD photoreceptors\",\"volume\":\"46\",\"year\":\"2005\",\"bibtex\":\"@article{Nair2005383,\\n  abstract = {PURPOSE. Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells. METHODS. Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding. RESULTS. LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange. CONCLUSIONS. Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.},\\n  annote = {cited By 13},\\n  author = {Nair, K. Saidas and Mendez, Ana and Blumer, Joe B. and Rosenzweig, Derek H. and Slepak, Vladlen Z.},\\n  doi = {10.1167/iovs.04-1006},\\n  issn = {01460404},\\n  journal = {Investigative Ophthalmology and Visual Science},\\n  number = {1},\\n  pages = {383--389},\\n  title = {{The presence of a Leu-Gly-Asn repeat-enriched protein (LGN), a putative binding partner of transducin, in ROD photoreceptors}},\\n  volume = {46},\\n  year = {2005}\\n  }\\n  \",\"author_short\":[\"Nair, K. S.\",\"Mendez, A.\",\"Blumer, J. B.\",\"Rosenzweig, D. H.\",\"Slepak, V. Z.\"],\"key\":\"Nair2005383\",\"id\":\"Nair2005383\",\"bibbaseid\":\"nair-mendez-blumer-rosenzweig-slepak-thepresenceofaleuglyasnrepeatenrichedproteinlgnaputativebindingpartneroftransducininrodphotoreceptors-2005\",\"role\":\"author\",\"urls\":{},\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":0,\"html\":\"\"},{\"bibtype\":\"article\",\"type\":\"article\",\"abstract\":\"Critical-size bone defects are those that will not heal without intervention and can arise secondary to trauma, infection and surgical resection of tumours. Treatment options are currently limited to filling the defect with autologous bone, of which there is not always an abundant supply, or ceramic pastes that only allow for limited osteo-inductive and -conductive capacity. In this study we investigate the repair of bone defects using a 3D printed LayFomm scaffold. LayFomm is a polymer blend of polyvinyl alcohol (PVA) and polyurethane (PU). It can be printed using the most common method of 3D printing, fused deposition modelling, before being washed in water-based solutions to remove the PVA. This leaves a more compliant, micro-porous PU elastomer. In vitro analysis of dental pulp stem cells seeded onto macro-porous scaffolds showed their ability to adhere, proliferate and form mineralised matrix on the scaffold in the presence of osteogenic media. Subcutaneous implantation of LayFomm in a rat model showed the formation of a vascularised fibrous capsule, but without a chronic inflammatory response. Implantation into a mandibular defect showed significantly increased mineralised tissue production when compared to a currently approved bone putty. While their mechanical properties are insufficient for use in load-bearing defects, these findings are promising for the use of polyurethane scaffolds in craniofacial bone regeneration.\",\"author\":[{\"propositions\":[],\"lastnames\":[\"Cooke\"],\"firstnames\":[\"Megan\",\"Elin\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Ramirez-GarciaLuna\"],\"firstnames\":[\"Jose\",\"L\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rangel-Berridi\"],\"firstnames\":[\"Karla\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Park\"],\"firstnames\":[\"Hyeree\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Nazhat\"],\"firstnames\":[\"Showan\",\"N\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Weber\"],\"firstnames\":[\"Michael\",\"H\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Henderson\"],\"firstnames\":[\"Janet\",\"E\"],\"suffixes\":[]},{\"propositions\":[],\"lastnames\":[\"Rosenzweig\"],\"firstnames\":[\"Derek\",\"H\"],\"suffixes\":[]}],\"doi\":\"10.3389/FBIOE.2020.557215\",\"issn\":\"2296-4185\",\"journal\":\"Frontiers in Bioengineering and Biotechnology\",\"keywords\":\"3D printing,Bone Regeneration,Fused depositing modeling (FDM),Mandibular defect,Poly-urethane,layfomm\",\"pages\":\"1190\",\"publisher\":\"Frontiers\",\"title\":\"3D printed polyurethane scaffolds for the repair of bone defects\",\"volume\":\"8\",\"year\":\"2020\",\"bibtex\":\"@article{Cooke2020,\\n  abstract = {Critical-size bone defects are those that will not heal without intervention and can arise secondary to trauma, infection and surgical resection of tumours. Treatment options are currently limited to filling the defect with autologous bone, of which there is not always an abundant supply, or ceramic pastes that only allow for limited osteo-inductive and -conductive capacity. In this study we investigate the repair of bone defects using a 3D printed LayFomm scaffold. LayFomm is a polymer blend of polyvinyl alcohol (PVA) and polyurethane (PU). It can be printed using the most common method of 3D printing, fused deposition modelling, before being washed in water-based solutions to remove the PVA. This leaves a more compliant, micro-porous PU elastomer. In vitro analysis of dental pulp stem cells seeded onto macro-porous scaffolds showed their ability to adhere, proliferate and form mineralised matrix on the scaffold in the presence of osteogenic media. Subcutaneous implantation of LayFomm in a rat model showed the formation of a vascularised fibrous capsule, but without a chronic inflammatory response. Implantation into a mandibular defect showed significantly increased mineralised tissue production when compared to a currently approved bone putty. While their mechanical properties are insufficient for use in load-bearing defects, these findings are promising for the use of polyurethane scaffolds in craniofacial bone regeneration.},\\n  author = {Cooke, Megan Elin and Ramirez-GarciaLuna, Jose L and Rangel-Berridi, Karla and Park, Hyeree and Nazhat, Showan N and Weber, Michael H and Henderson, Janet E and Rosenzweig, Derek H},\\n  doi = {10.3389/FBIOE.2020.557215},\\n  issn = {2296-4185},\\n  journal = {Frontiers in Bioengineering and Biotechnology},\\n  keywords = {3D printing,Bone Regeneration,Fused depositing modeling (FDM),Mandibular defect,Poly-urethane,layfomm},\\n  pages = {1190},\\n  publisher = {Frontiers},\\n  title = {{3D printed polyurethane scaffolds for the repair of bone defects}},\\n  volume = {8},\\n  year = {2020}\\n  }\\n\",\"author_short\":[\"Cooke, M. E.\",\"Ramirez-GarciaLuna, J. L\",\"Rangel-Berridi, K.\",\"Park, H.\",\"Nazhat, S. N\",\"Weber, M. H\",\"Henderson, J. E\",\"Rosenzweig, D. H\"],\"key\":\"Cooke2020\",\"id\":\"Cooke2020\",\"bibbaseid\":\"cooke-ramirezgarcialuna-rangelberridi-park-nazhat-weber-henderson-rosenzweig-3dprintedpolyurethanescaffoldsfortherepairofbonedefects-2020\",\"role\":\"author\",\"urls\":{},\"keyword\":[\"3D printing\",\"Bone Regeneration\",\"Fused depositing modeling (FDM)\",\"Mandibular defect\",\"Poly-urethane\",\"layfomm\"],\"metadata\":{\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\"downloads\":1,\"html\":\"\"}],\n      metadata: {\"owner\":\"rosenzweig, d\",\"authorlinks\":{\"rosenzweig, d\":\"https://rosenzweiglab.com/publication/\"}},\n      ownerID: \"QftFYMfuBhvSrqkqw\"\n    };\n  </script>\n\n  <script type=\"text/javascript\">\n  var site$;\n  if (typeof $ != 'undefined') {\n    console.log('existing jquery: storing and then restoring');\n    site$ = $;\n    $ = null;\n  }\n  </script>\n\n  <script src=\"https://ajax.googleapis.com/ajax/libs/jquery/2.2.4/jquery.min.js\">\n  </script>\n  <script type=\"text/javascript\">\n  if (site$) {\n    console.log('existing jquery: avoiding conflict and restoring');\n    bb$ = $.noConflict(true);\n    $ = site$;\n  } else {\n    bb$ = $;\n  }\n  </script>\n\n  <script src=\"//bibbase.org/js/bibbase.min.js\"\n          type=\"text/javascript\">\n  </script>\n\n  <script type=\"text/javascript\"\n          src=\"https://cdnjs.cloudflare.com/ajax/libs/mathjax/2.7.1/MathJax.js?config=TeX-AMS-MML_HTMLorMML\">\n  </script>\n  <script type=\"text/x-mathjax-config\">\n    MathJax.Hub.Config({tex2jax: {inlineMath: [['$','$'], ['\\\\(','\\\\)']]},\n                        skipTags: [\"body\"],\n                        processClass: \"bibbase_paper\"});\n  </script>\n\n  <style>\n  @media print {\n    .dontprint {\n        display: none !important;\n    }\n\n  .bibbase_group {\n    background-color: #E0E0E0;\n    font-style: italic;\n    font-size: larger;\n    text-shadow: 0px 0px 3px #FFFFFF;\n    border-radius: 4px 4px 4px 4px;\n    -moz-border-radius: 4px 4px 4px 4px;\n    -webkit-border-radius: 4px;\n    padding: 5px 40px 5px;\n    margin-top: 10px;\n    margin-bottom: 5px;\n    cursor: pointer;\n  }\n\n  .bibbase_paper {\n    margin-bottom: 5px;\n  }\n\n  }\n  </style>\n\n  \n  <div style=\"float: right; margin-right: 10px; margin-top: 10px; text-align: right; font-size: 12px\">\n    generated by\n    <a href=\"//bibbase.org\"\n       onclick=\"trackOutboundLink('bibbase', 'logo clicked', window.location.href, '//bibbase.org'); return false;\">\n      <img src=\"//bibbase.org/img/logo.svg\"\n           style=\"vertical-align: middle; margin-left: 3px; height: 16px;\"/\n           alt=\"bibbase.org\"\n           title=\"BibBase – The easiest way to maintain your publications page.\"\n        ></a>\n    \n  </div>\n  \n\n  <div id=\"bibbase_header\" class=\"dontprint\" style=\"\">\n\n    <ul class=\"nav nav-pills\" style=\"margin: 0px;\">\n\n      <li class=\"dropdown\" id=\"groupby_dropdown\">\n      <a class=\"dropdown-toggle\"\n         data-toggle=\"dropdown\"\n         href=\"#\">\n          Group by\n          <b class=\"caret\"></b>\n      </a>\n      <ul class=\"dropdown-menu\">\n        <li class=\"groupby year\"><a onclick=\"groupby('year')\">\n            Year</a>\n        </li>\n        <li class=\"groupby author\"><a onclick=\"groupby('author_short')\">\n            Author</a>\n        </li>\n        <li class=\"groupby type\"><a onclick=\"groupby('type')\">\n            Type</a>\n        </li>\n        <li class=\"groupby keyword\"><a onclick=\"groupby('keyword')\">\n            Keyword</a>\n        </li>\n        <li class=\"groupby downloads\"><a onclick=\"groupby('downloads')\">\n            Downloads</a>\n        </li>\n      </ul>\n      </li>\n\n\n      <li class=\"dropdown\" id=\"menu_dropdown\">\n      <a class=\"dropdown-toggle\"\n         data-toggle=\"dropdown\"\n         href=\"#\" alt=\"controls\">\n          <i class=\"fa fa-reorder\" alt=\"controls\"></i>\n          <b class=\"caret\"></b>\n      </a>\n      <ul class=\"dropdown-menu\">\n        <li>\n          <a onclick=\"toggleAll()\">\n            <i class=\"fa fa-chevron-down fa-fw\"></i> Expand/Collapse All\n          </a>\n        </li>\n        <li>\n          <a download=\"references.bib\" href=\"data:text/bibtex;base64,@article {Fattah2020.09.22.308411,
	author = {Fattah, Abdel Rahman Abdel and Daza, Brian and Rustandi, Gregorius and Berrocal-Rubio, Miguel Angel and Gorissen, Benjamin and Poovathingal, Suresh and Davie, Kristofer and Cao, Xuanye and Rosenzweig, Derek Hadar and Lei, Yunping and Finnell, Richard and Verfaillie, Catherine and Sampaolesi, Maurilio and Dedecker, Peter and Van Oosterwyck, Hans and Aerts, Stein and Ranga, Adrian},
	title = {Actuation Enhances Patterning in Human Neural Tube Organoids},
	elocation-id = {2020.09.22.308411},
	year = {2020},
	doi = {10.1101/2020.09.22.308411},
	publisher = {Cold Spring Harbor Laboratory},
	abstract = {Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.Competing Interest StatementRF was CEO of Teratomic Consulting, LLC, which has been dissolved. He is also an associate editor of the journal Reproductive and Developmental Medicine, which provides his travel expenses.},
	URL = {https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411},
	eprint = {https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411.full.pdf},
	journal = {bioRxiv}
}



@article{Azizipour2020,
abstract = {Following the advancements in microfluidics and lab-on-a-chip (LOC) technologies, a novel biomedical application for microfluidic based devices has emerged in recent years and microengineered cell culture platforms have been created. These micro-devices, known as organ-on-a-chip (OOC) platforms mimic the in vivo like microenvironment of living organs and offer more physiologically relevant in vitro models of human organs. Consequently, the concept of OOC has gained great attention from researchers in the field worldwide to offer powerful tools for biomedical researches including disease modeling, drug development, etc. This review highlights the background of biochip development. Herein, we focus on applications of LOC devices as a versatile tool for POC applications. We also review current progress in OOC platforms towards body-on-a-chip, and we provide concluding remarks and future perspectives for OOC platforms for POC applications.},
author = {Azizipour, Neda and Avazpour, Rahi and Rosenzweig, Derek H. and Sawan, Mohamad and Ajji, Abdellah},
doi = {10.3390/mi11060599},
issn = {2072666X},
journal = {Micromachines},
keywords = {BioMEMS,Lab-on-a-chip,Microengineering,Microfluidics,Organ-on-a-chip,Personalized medicine,Point-of-care},
number = {6},
pages = {1--15},
title = {{Evolution of biochip technology: A review from lab-on-a-chip to organ-on-a-chip}},
volume = {11},
year = {2020}
}


@article{Pitaru2020,
abstract = {The current gold standard technique for treatment of anterior cruciate ligament (ACL) injury is reconstruction with autograft. These treatments have a relatively high failure and re-tear rate. To overcome this, tissue engineering and additive manufacturing are being used to explore the potential of 3D scaffolds as autograft substitutes. However, mechanically optimal polymers for this have yet to be identified. Here, we use 3D printing technology and various materials with the aim of fabricating constructs better matching the mechanical properties of the native ACL. A fused deposition modeling (FDM) 3D printer was used to microfabricate dog bone-shaped specimens from six different polymers-PLA, PETG, Lay FOMM 60, NinjaFlex, NinjaFlex-SemiFlex, and FlexiFil-at three different raster angles. The tensile mechanical properties of these polymers were determined from stress-strain curves. Our results indicate that no single material came close enough to successfully match reported mechanical properties of the native ACL. However, PLA and PETG had similar ultimate tensile strengths. Lay FOMM 60 displayed a percentage strain at failure similar to reported values for native ACL. Furthermore, raster angle had a significant impact on some mechanical properties for all of the materials except for FlexiFil. We therefore conclude that while none of these materials alone is optimal for mimicking ACL mechanical properties, there may be potential for creating a 3D-printed composite constructs to match ACL mechanical properties. Further investigations involving co-printing of stiff and elastomeric materials must be explored.},
author = {Pitaru, Audrey A. and Lacombe, Jean Gabriel and Cooke, Megan E. and Beckman, Lorne and Steffen, Thomas and Weber, Michael H. and Martineau, Paul A. and Rosenzweig, Derek H.},
doi = {10.3390/MI11090846},
issn = {2072666X},
journal = {Micromachines},
keywords = {3D printing,Elastic,Ligament,Mechanical strain,Polymers,Scaffolds,Tissue engineering},
number = {9},
pages = {1--15},
title = {{Investigating commercial filaments for 3D printing of stiff and elastic constructs with ligament-like mechanics}},
volume = {11},
year = {2020}
}

  

@article{Haglund2019142,
  abstract = {With the expiration of key patents on additive manufacturing, 3D printing devices have become extremely inexpensive. Therefore, the use of 3D printing in biomedical applications has blossomed in the past decade. Low-cost, high-quality instruments have become widely used in both industrial and academic settings. Fused deposition modeling using polymers such as polylactic acid, polycaprolactone, or other composites are now combined with bioprinting to open new avenues to cutting-edge research toward musculoskeletal repair and regeneration such as prevascularized bone or soft-tissue constructs. Most importantly, these tools are being widely used to generate composite scaffolds representing matrices on which to culture cells of various tissue types such as bone, cartilage, cardiac, and nervous tissue. 3D printed composite matrix scaffolds are being tested in sophisticated in vitro and in vivo preclinical models, paving the way for future clinical translation where the ultimate goal is to generate functional replacement tissues.},
  annote = {cited By 0},
  author = {Haglund, Lisbet and Ahangar, Pouyan and Rosenzweig, Derek H.},
  doi = {10.1016/j.cobme.2019.06.002},
  issn = {24684511},
  journal = {Current Opinion in Biomedical Engineering},
  keywords = {3D printed,Bioprinted,Composite,Hydrogels,Matrix scaffold,Tissue engineering},
  pages = {142--148},
  title = {{Advancements in 3D printed scaffolds to mimic matrix complexities for musculoskeletal repair}},
  volume = {10},
  year = {2019}
  }
  

@article{Fairag201915306,
  abstract = {Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 mum), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 mum pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 mum pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 mum pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.},
  annote = {cited By 7},
  author = {Fairag, Rayan and Rosenzweig, Derek H. and Ramirez-Garcialuna, Jose L. and Weber, Michael H. and Haglund, Lisbet},
  doi = {10.1021/acsami.9b02502},
  issn = {19448252},
  journal = {ACS Applied Materials and Interfaces},
  keywords = {3D printing,PLA,bone defect,bone repair,human osteoblasts,low-cost,mesenchymal stem cells,tissue engineering},
  number = {17},
  pages = {15306--15315},
  title = {{Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bone-like Matrix Deposition in Vitro}},
  volume = {11},
  year = {2019}
  }
  

@article{Ahangar2019,
  abstract = {Additive manufacturing (AM) has emerged over the past four decades as a cost-effective, on-demand modality for fabrication of geometrically complex objects. The ability to design and print virtually any object shape using a diverse array of materials, such as metals, polymers, ceramics and bioinks, has allowed for the adoption of this technology for biomedical applications in both research and clinical settings. Current advancements in tissue engineering and regeneration, therapeutic delivery, medical device fabrication and operative management planning ensure that AM will continue to play an increasingly important role in the future of healthcare. In this review, we outline current biomedical applications of common AM techniques and materials.},
  annote = {cited By 11},
  author = {Ahangar, Pouyan and Cooke, Megan E. and Weber, Michael H. and Rosenzweig, Derek H.},
  doi = {10.3390/app9081713},
  issn = {20763417},
  journal = {Applied Sciences (Switzerland)},
  keywords = {3D printing,Additive manufacturing,Biomedical devices,Bioprinting,Rapid prototyping,Regenerative medicine,Tissue engineering},
  number = {8},
  title = {{Current biomedical applications of 3D printing and additive manufacturing}},
  volume = {9},
  year = {2019}
  }
  

@article{Akoury2019,
  abstract = {Background: Bisphosphonates (BPs) including zoledronate (zol) have become standard care for bone metastases as they effectively inhibit tumor-induced osteolysis and associated pain. Several studies have also suggested that zol has direct anti-tumor activity. Systemic administration at high doses is the current approach to deliver zol, yet it has been associated with debilitating side effects. Local therapeutic delivery offers the ability to administer much lower total dosage, while at the same time maintaining sustained high-local drug concentration directly at the target treatment site. Here, we aimed to assess effects of lower doses of zol on bone metastases over a longer time. Methods: Prostate cancer cell line LAPC4 and prostate-induced bone metastasis cells were treated with zol at 1, 3 and 10 muM for 7 days. Following treatment, cell proliferation was assessed using Almarblue{(R)}, Vybrant MTT{(R)}, and Live/Dead{(R)} viability/cytotoxicity assays. Additionally, cell migration and invasion were carried out using Falcon™ cell culture inserts and Cultrex{(R)} 3D spheroid cell invasion assays respectively. Results: We show that treatment with 3-10 muM zol over 7-days significantly decreased cell proliferation in both the prostate cancer cell line LAPC4 and cells from spine metastases secondary to prostate cancer. Using the same low-dose and longer time course for treatment, we demonstrate that 10 muM zol also significantly inhibits tumor cell migration and 3D-cell growth/invasion. Conclusions: This project harnesses the potential of using zol at low doses for longer treatment periods, which may be a viable treatment modality when coupled with biomaterials or biodevices for local delivery.},
  annote = {cited By 1},
  author = {Akoury, Elie and Ahangar, Pouyan and Nour, Antone and Lapointe, Jacques and Gu{'{e}}rard, Karl Philippe and Haglund, Lisbet and Rosenzweig, Derek H. and Weber, Michael H.},
  doi = {10.1186/s12935-019-0745-x},
  issn = {14752867},
  journal = {Cancer Cell International},
  keywords = {Bone metastases secondary to prostate,Cellular assays,Direct in vitro treatment,Low doses,Zoledronate},
  number = {1},
  title = {{Low-dose zoledronate for the treatment of bone metastasis secondary to prostate cancer}},
  volume = {19},
  year = {2019}
  }
  

@conference{Akoury20191245,
  abstract = {Zoledronate (Zol) is a bone-preserving/ anti-Tumoral drug that is widely used for the treatment of many cancers including spinal bone metastases. High systemic Zol doses required to elicit an adequate effect in the spine often lead to significant side effects, limiting its prolonged use and effectiveness. Here, we aim to provide an alternative strategy to locally deliver Zol at the tumor site. We show that nanoporous 3D-printed scaffolds can be loaded with Zol and possess the ability to release Zol (10-28%) over a sustained period. Additionally, we demonstrate that Zol-impregnated scaffolds, mostly Gel Lay, impair the proliferation of the prostate cancer cell line LAPC4 and the prostate-induced bone metastasis cells in vitro. 3D-printed nanoporous polymers offer a novel and versatile opportunity for potential local delivery of drugs in future clinical settings. These polymers can decrease systemic exposure and related side effects of Zol while at the same time concentrating the drug effect at the tumor site thereby inhibiting tumor proliferation. Also, these scaffolds could be co-printed or coupled with other materials to produce custom implants that offer better structural support for bone growth at the tumor site following resection.},
  annote = {cited By 1},
  author = {Akoury, Elie and Weber, Michael H. and Rosenzweig, Derek H.},
  booktitle = {MRS Advances},
  doi = {10.1557/adv.2019.156},
  issn = {20598521},
  keywords = {3D printing,nanostructure},
  number = {21},
  pages = {1245--1251},
  title = {{3D-Printed Nanoporous Scaffolds Impregnated with Zoledronate for the Treatment of Spinal Bone Metastases}},
  volume = {4},
  year = {2019}
  }
  

@article{Ahangar2018,
  abstract = {The spine is the most common site of bone metastasis, often originating from prostate, lung, and breast cancers. High systemic doses of chemotherapeutics such as doxorubicin (DOX), cisplatin, or paclitaxel often have severe side effects. Surgical removal of spine metastases also leaves large defects which cannot spontaneously heal and require bone grafting. To circumvent these issues, we designed an approach for local chemotherapeutic delivery within 3D-printed scaffolds which could also potentially serve as a bone substitute. Direct treatment of prostate cancer cell line LAPC4 and patient derived spine metastases cells with 0.01 muM DOX significantly reduced metabolic activity, proliferation, migration, and spheroid growth. We then assessed uptake and release of DOX in a series of porous 3D-printed scaffolds on LAPC4 cells as well as patient-derived spine metastases cells. Over seven days, 60-75% of DOX loaded onto scaffolds could be released, which significantly reduced metabolic activity and proliferation of both LAPC4 and patient derived cells, while unloaded scaffolds had no effect. Porous 3D-printed scaffolds may provide a novel and inexpensive approach to locally deliver chemotherapeutics in a patient-specific manner at tumor resection sites. With a composite design to enhance strength and promote sustained drug release, the scaffolds could reduce systemic negative effects, enhance bone repair, and improve patient outcomes.},
  annote = {cited By 5},
  author = {Ahangar, Pouyan and Akoury, Elie and Luna, Ana Sofia Ramirez Garcia and Nour, Antone and Weber, Michael H. and Rosenzweig, Derek H.},
  doi = {10.3390/ma11091485},
  issn = {19961944},
  journal = {Materials},
  keywords = {Bone metastases,Bone substitute,Doxorubicin,Local delivery,Low-cost 3D printing,Nanoporous filament,Prostate cancer},
  number = {9},
  title = {{Nanoporous 3D-printed scaffolds for local doxorubicin delivery in bone metastases secondary to prostate cancer}},
  volume = {11},
  year = {2018}
  }
  

@article{Gutman2018E365,
  abstract = {Study Design.Meta-analysis of published randomized controlled trials (RCTs).Objective.To determine whether anterior cervical discectomy and fusion (ACDF), cervical disc replacement (CDR), or minimally invasive posterior cervical foraminotomy (MI-PCF) provides the best outcomes for patients with symptomatic single-level, single-side, and cervical radiculopathy.Summary of Background Data.The surgical treatment of cervical radiculopathy is still controversial. ACDF has been widely used as a "gold standard." CDR has evolved and become a motion-preserving alternative with a potentially lower incidence of adjacent segment disease. However, both techniques require anterior neck dissection that carries a potential for serious morbidity. MI-PCF is a motion-preserving technique that can be performed with minimal invasiveness but has not gained universal acceptance.Methods.Electronic database search for RCTs comparing the efficacy and effectiveness of ACDF, CDR, and MI-PCF was performed. Meta-analysis was done for secondary surgical procedures and adverse events.Results.A total of 358 studies were retrieved, of which four RCT reports met the inclusion criteria for this study. Three studies present clinical data comparing ACDF and CDR, and one study presents data comparing ACDF and MI-PCF. Available data from the RCTs analyzed concluded that ACDF, CDR, and MI-PCF result in significant improvements in relevant symptoms, clinical, and functional outcomes in patients with single-level, single side cervical radiculopathy refractory to nonoperative treatment. CDR had the lowest percentage of secondary surgical procedures (P=0.0178) whereas MICPF had the lowest percentage of adverse events (P<0.0001).Conclusion.All three techniques are effective in treating cervical radicular symptoms. MI-PCF has the lowest rate of adverse events whereas CDR has the lowest rate of secondary procedures. There is insufficient evidence to show which technique is the most effective and provides the longest-lasting symptom relief.Level of Evidence.},
  annote = {cited By 14},
  author = {Gutman, Gabriel and Rosenzweig, Derek H. and Golan, Jeff D.},
  doi = {10.1097/BRS.0000000000002324},
  issn = {15281159},
  journal = {Spine},
  keywords = {anterior cervical discectomy and fusion,cervical disc arthroplasty,cervical radiculopathy,minimally invasive posterior cervical foraminotomy,spine surgery},
  number = {6},
  pages = {E365--E372},
  title = {{Surgical Treatment of Cervical Radiculopathy}},
  volume = {43},
  year = {2018}
  }
  

@article{Rosenzweig2018200,
  abstract = {Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1rho magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1rho values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture setup, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.},
  annote = {cited By 4},
  author = {Rosenzweig, D. H. and Fairag, R. and Mathieu, A. P. and Li, L. and Eglin, D. and D'este, M. and Steffen, T. and Weber, M. H. and Ouellet, J. A. and Haglund, Lisbet},
  doi = {10.22203/eCM.v036a15},
  issn = {14732262},
  journal = {European Cells and Materials},
  keywords = {Autologous cell implantation,Bioreactors,Human intervertebral disc,Hydrogel,Nucleus pulposus,T1rho magnetic resonance imaging,Tissue engineering},
  pages = {200--217},
  pmid = {30370912},
  title = {{Thermoreversi ble hyaluronan-hydrogel and autologous nucleus pulposus cell deli very regenerates human i ntervertebral di scs i n an ex vi vo, physi ologi cal organ culture model}},
  volume = {36},
  year = {2018}
  }
  

@article{Krock2017,
  abstract = {Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1beta and nerve growth factor gene expression following treatment with Pam2CSK4 (TLR2/6 agonist) but not Pam3CSK4 (TLR1/2 agonist). Challenging NP cells with Pam2CSK4 or 30 kDa fibronectin fragments (FN-f, an endogenous TLR2 and TLR4 alarmin) increased secretion of proinflammatory cytokines. We then investigated the effect of TLR activation in intact, non-degenerate, ex vivo human discs. Discs were injected with PBS, Pam2CSK4 and FN-f, and cultured for 28 days. TLR activation increased proteoglycan and ECM protein release into the culture media and decreased proteoglycan content in the NP. Proteases, including MMP3, 13 and HTRA1, are secreted at higher levels following TLR activation. In addition, proinflammatory cytokine levels, including IL-6, TNFalpha and IFNgamma, increased following TLR activation. These results indicate that TLR activation induces degeneration in human discs. Therefore, TLRs are potential disease-modifying therapeutic targets to slow disc degeneration.},
  annote = {cited By 7},
  author = {Krock, Emerson and Rosenzweig, Derek H. and Currie, J. Brooke and Bisson, Daniel G. and Ouellet, Jean A. and Haglund, Lisbet},
  doi = {10.1038/s41598-017-17472-1},
  issn = {20452322},
  journal = {Scientific Reports},
  number = {1},
  title = {{Toll-like receptor activation induces degeneration of human intervertebral discs}},
  volume = {7},
  year = {2017}
  }
  

@article{Nooh20178,
  abstract = {Bisphosphonates (BPs) have recently been shown to have direct anti-tumor properties. Systemic treatment with BPs can have multiple adverse effects such as osteonecrosis of the jaw and BP induced bone fracturing and spine instability. While benefits of systemic BP treatments may outweigh risks, local treatment with BPs has been explored as an alternate strategy to reduce unwarranted risk. In the present study, we examined whether local delivery of BPs inhibits tumor-induced osteolysis and tumor growth more effectively than systemic treatment in an animal model of tumor-induced bone disease. Following establishment of an intra-tibial model of bone metastases in athymic mice, the experimental group was treated by local administration of zoledronate into the tibial lesion. A comparison of the effect of local versus systemic delivery of zoledronate on the formation of tumor-induced osteolysis was also carried out. A significant increase in mean bone volume/tissue volume % (BV/TV) of the locally treated group (12.30±2.80%) compared to the control group (7.13±1.22%) (P<0.001). Additionally, there was a significant increase in the BV/TV (10.90±1.25%) in the locally treated group compared to the systemically treated group (7.53±0.75%) (P=0.005). These preliminary results suggest that local delivery of BPs outperforms both systemic and control treatments to inhibit tumor-induced osteolysis.},
  annote = {cited By 3},
  author = {Nooh, Anas and Zhang, Yu Ling and Sato, Daisuke and Rosenzweig, Derek H. and Tabari{`{e}}s, S{'{e}}bastien and Siegel, Peter and Barralet, Jake E. and Weber, Michael H.},
  doi = {10.1016/j.jbo.2017.01.001},
  issn = {22121374},
  journal = {Journal of Bone Oncology},
  keywords = {Bisphosphonates,Bone metastases,Cancer pain,Local treatment,Osteolysis,Xenograft},
  pages = {8--15},
  title = {{Intra-tumor delivery of zoledronate mitigates metastasis-induced osteolysis superior to systemic administration}},
  volume = {6},
  year = {2017}
  }
  

@article{Rosenzweig2017240,
  abstract = {Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD) degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP) phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD) gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.},
  annote = {cited By 7},
  author = {Rosenzweig, D. H. and {Tremblay Gravel}, J. and Bisson, D. and Ouellet, J. A. and Weber, M. H. and Haglund, Lisbet},
  doi = {10.22203/eCM.v033a18},
  issn = {14732262},
  journal = {European Cells and Materials},
  keywords = {Cell culture,Elastic culture surfaces,Intervertebral disc,Nucleus pulposus cells,Tissue engineering},
  pages = {240--251},
  title = {{Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications}},
  volume = {33},
  year = {2017}
  }
  

@article{Krock20163541,
  abstract = {Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity but more recently were found to be activated by endogenous alarmins such as fragmented extracellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1beta (control) treatment increased NGF, brain-derived neurotrophic factor (BDNF), and IL-1beta gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1beta treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2, and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-kappaB, and to a lesser extent p38, but not ERK1/2 activity, blocked TLR2-driven NGF up-regulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-kappaB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-kappaB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.},
  annote = {cited By 26},
  author = {Krock, Emerson and Currie, J. Brooke and Weber, Michael H. and Ouellet, Jean A. and Stone, Laura S. and Rosenzweig, Derek H. and Haglund, Lisbet},
  doi = {10.1074/jbc.M115.675900},
  issn = {1083351X},
  journal = {Journal of Biological Chemistry},
  number = {7},
  pages = {3541--3551},
  title = {{Nerve growth factor is regulated by toll-like receptor 2 in human intervertebral discs}},
  volume = {291},
  year = {2016}
  }
  

@article{Jalani20161078,
  abstract = {Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent nanotransducers for converting longer wavelength near-infrared (NIR) light to shorter wavelengths spanning the ultraviolet (UV) to the visible (Vis) regions of the spectrum via a multiphoton absorption process, known as upconversion. Here, we report the development of NIR to UV-Vis-NIR UCNPs consisting of LiYF4:Yb3+/Tm3+@SiO2 individually coated with a 10 ± 2 nm layer of chitosan (CH) hydrogel cross-linked with a photocleavable cross-linker (PhL). We encapsulated fluorescent-bovine serum albumin (FITC-BSA) inside the gel. Under 980 nm excitation, the upconverted UV emission cleaves the PhL cross-links and instantaneously liberates the FITC-BSA under 2 cm thick tissue. The release is immediately arrested if the excitation source is switched off. The upconverted NIR light allows for the tracking of particles under the tissue. Nucleus pulposus (NP) cells cultured with UCNPs are viable both in the presence and in the absence of laser irradiation. Controlled drug delivery of large biomolecules and deep tissue imaging make this system an excellent theranostic platform for tissue engineering, biomapping, and cellular imaging applications.},
  annote = {cited By 104},
  author = {Jalani, Ghulam and Naccache, Rafik and Rosenzweig, Derek H. and Haglund, Lisbet and Vetrone, Fiorenzo and Cerruti, Marta},
  doi = {10.1021/jacs.5b12357},
  issn = {15205126},
  journal = {Journal of the American Chemical Society},
  number = {3},
  pages = {1078--1083},
  title = {{Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery}},
  volume = {138},
  year = {2016}
  }
  

@article{Rosenzweig201626,
  abstract = {Low back pain originating from intervertebral disc (IVD) degenerationaffectsthequalityoflifeformillionsof people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivomodels of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. Abioreactor wasdevelopedthat facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.6 and high 0.1-1.2 MPa. Cell viability was maintained > 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility oftissuerepairstrategieswithcellsupplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.},
  annote = {cited By 10},
  author = {Rosenzweig, D. H. and Gawri, R. and Moir, J. and Beckman, L. and Eglin, D. and Steffen, T. and Roughley, P. J. and Ouellet, J. A. and Haglund, Lisbet},
  doi = {10.22203/eCM.v031a03},
  issn = {14732262},
  journal = {European Cells and Materials},
  keywords = {Bioreactor,Cell therapy,Dynamic loading,Hydrogel,Intervertebral discs,Organ culture,Tissue regeneration},
  pages = {26--39},
  pmid = {26728497},
  title = {{Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor}},
  volume = {31},
  year = {2016}
  }
  

@article{Jalani201511255,
  abstract = {To design a biodegradable hydrogel as cell support, one should know its in vivo degradation rate. A technique commonly used to track gel degradation is fluorescence spectroscopy. However, the fluorescence from conventional fluorophores quickly decays, and the fluorophores are often moderately cytotoxic. Most importantly, they require ultraviolet or visible (UV-Vis) light as the excitation source, which cannot penetrate deeply through biological tissues. Lanthanide-doped upconverting nanoparticles (UCNPs) are exciting alternatives to conventional fluorophores because they can convert near-infrared (NIR) to UV-Vis-NIR light via a sequential multiphoton absorption process referred to as upconversion. NIR light can penetrate up to few cm inside tissues, thus making these UCNPs much better probes than conventional fluorophores for in vivo monitoring. Also, UCNPs have narrow emission bands, high photoluminescence (PL) signal-to-noise ratio, low cytotoxicity and good physical and chemical stability. Here, we show a nanocomposite system consisting of a biodegradable, in situ thermogelling injectable hydrogel made of chitosan and hyaluronic acid encapsulating silica-coated LiYF4:Yb3+/Tm3+ UCNPs. We use these UCNPs as photoluminescent tags to monitor the gel degradation inside live, cultured intervertebral discs (IVDs) over a period of 3 weeks. PL spectroscopy and NIR imaging show that NIR-to-NIR upconversion of LiYF4:Yb3+/Tm3+@SiO2 UCNPs allows for tracking of the gel degradation in living tissues. Both in vitro and ex vivo release of UCNPs follow a similar trend during the first 5 days; after this time, ex vivo release becomes faster than in vitro, indicating a faster gel degradation ex vivo. Also, the amount of released UCNPs in vitro increases continuously up to 3 weeks, while it plateaus after 15 days inside the IVDs showing a homogenous distribution of UCNPs throughout the IVD tissue. This non-invasive optical method for real time, live tissue imaging holds great potential for tissue analysis, biomapping and bioimaging applications.},
  annote = {cited By 33},
  author = {Jalani, Ghulam and Naccache, Rafik and Rosenzweig, Derek H. and Lerouge, Sophie and Haglund, Lisbet and Vetrone, Fiorenzo and Cerruti, Marta},
  doi = {10.1039/c5nr02482j},
  issn = {20403372},
  journal = {Nanoscale},
  number = {26},
  pages = {11255--11262},
  title = {{Real-time, non-invasive monitoring of hydrogel degradation using LiYF4:Yb3+/Tm3+ NIR-to-NIR upconverting nanoparticles}},
  volume = {7},
  year = {2015}
  }
  

@article{Rosenzweig201515118,
  abstract = {Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (NP) cells were cultured on ABS and PLA scaffolds for three weeks. Both cell types proliferated well, showed high viability, and produced ample amounts of proteoglycan and collagen type II on both scaffolds. NP generated more matrix than chondrocytes; however, no difference was observed between scaffold types. Mechanical testing revealed sustained scaffold stability. This study demonstrates that chondrocytes and NP cells can proliferate on both ABS and PLA scaffolds printed with a simplistic, inexpensive desktop 3D printer. Moreover, NP cells produced more proteoglycan than chondrocytes, irrespective of thermoplastic type, indicating that cells maintain individual phenotype over the three-week culture period. Future scaffold designs covering larger pore sizes and better mimicking native tissue structure combined with more flexible or resorbable materials may provide implantable constructs with the proper structure, function, and cellularity necessary for potential cartilage and disc tissue repair in vivo.},
  annote = {cited By 128},
  author = {Rosenzweig, Derek H. and Carelli, Eric and Steffen, Thomas and Jarzem, Peter and Haglund, Lisbet},
  doi = {10.3390/ijms160715118},
  issn = {14220067},
  journal = {International Journal of Molecular Sciences},
  keywords = {3D printing,ABS,Chondrocyte,Intervertebral disc,Nucleus pulposus,PLA,Tissue engineering},
  number = {7},
  pages = {15118--15135},
  title = {{3D-printed ABS and PLA scaffolds for cartilage and nucleus pulposustissue regeneration}},
  volume = {16},
  year = {2015}
  }
  

@article{Krock2015317,
  abstract = { 2015 Bentham Science Publishers. Intervertebral disc degeneration is directly linked to chronic low back pain, a condition that affects multitudes of people world-wide and presents tremendous direct and indirect health costs. Water- loss, inflammation and disruption of the extracellular matrix ultimately result in loss of tissue function and associated pain. Cytokines present in degenerate tissue can upregulate protease activity and directly causes pain. Non-invasive therapies provide limited efficacy for pain management, and surgical intervention is therefore often required to treat chronic low back pain. Disc removal can offer immediate pain-relief, however degeneration of adjacent segments can occur and pain can return. To circumvent the caveats of recurring pain and invasive surgeries, stem cell therapy is currently being investigated as a promising means to repair degenerating discs. However, while initial studies have shown promise, few studies have addressed whether stem cell therapies can modulate the inflammatory microenvironment or whether cytokines can affect the ability of the implanted cells to repair damaged tissue. This review focuses briefly on mechanisms of disc degeneration, with more attention given to the role of inflammatory milieu in this process. Cytokine upregulation in disc degeneration, the potential role of tolllike receptor signaling, and effects of these inflammatory factors on stem cells will be discussed. We find that while stem cell differentiation can be negatively influenced by inflammatory cytokines, stem cells can potentially have anti-inflammatory effects. We conclude that further investigation of stem cell interactions with the inflammatory microenvironment is required, and that priming of stem cells under various conditions may be necessary for optimal therapeutic value for intervertebral disc repair and pain reduction.},
  annote = {cited By 20},
  author = {Krock, Emerson and Rosenzweig, Derek and Haglund, Lisbet},
  doi = {10.2174/1574888x10666150211161956},
  issn = {1574888X},
  journal = {Current Stem Cell Research & Therapy},
  number = {4},
  pages = {317--328},
  title = {{The Inflammatory Milieu of the Degenerate Disc: Is Mesenchymal Stem Cell-based Therapy for Intervertebral Disc Repair a Feasible Approach?}},
  volume = {10},
  year = {2015}
  }
  

@article{Jalani2015473,
  abstract = {Injectable hydrogels are extensively used in drug delivery and tissue engineering to administer drugs, genes, growth factors and live cells. We report a method to produce tough, in-situ thermogelling, non-toxic, injectable hydrogels made of chitosan and hyaluronic acid co-crosslinked with carboxytetramethylrhodamine-glycerophophate and genipin. The gels are highly homogeneous and form within 32 min, i.e., faster than gels crosslinked with either genipin or beta-glycerophophate. The shear strength of co-crosslinked hydrogels is 3.5 kPa, higher than any chitosan-based gel reported. Chondrocytes and nucleus pulposus cells thrive inside the gels and produce large amounts of collagen II. Injection in rats shows that the gels form in-vivo within a short time and remain well localized for more than one week while the rats remain healthy and active. The excellent mechanical properties, fast in-situ gelation, good biocompatibility and the ability to encapsulate live cells at physiological conditions make these hydrogels ideal for tissue engineering, especially cartilage regeneration. Homogenous, insitu thermogelling, highly tough, non-toxic injectable hydrogels from chitosan and hyaluronic acid co-crosslinked with beta-glycerophophate and genipin are produced. The co-crosslinked gels form faster than genipin-only crosslinked gels and are 2.5 times stronger than ionically crosslinked gels. Homogenously dispersed cells express enhanced amount of collagen II, showing the great potential of these gels for cartilage tissue engineering.},
  annote = {cited By 15},
  author = {Jalani, Ghulam and Rosenzweig, Derek H. and Makhoul, Georges and Abdalla, Sherif and Cecere, Renzo and Vetrone, Fiorenzo and Haglund, Lisbet and Cerruti, Marta},
  doi = {10.1002/mabi.201400406},
  issn = {16165195},
  journal = {Macromolecular Bioscience},
  keywords = {crosslinking,genipin,hydrogels,in-situ thermogelling,injectable},
  number = {4},
  pages = {473--480},
  title = {{Tough, in-situ thermogelling, injectable hydrogels for biomedical applications}},
  volume = {15},
  year = {2015}
  }
  

@article{Rosenzweig201414427,
  abstract = {Osteoarthritis (OA) is a debilitating joint disorder resulting from an incompletely understood combination of mechanical, biological, and biochemical processes. OA is often accompanied by inflammation and pain, whereby cytokines associated with chronic OA can up-regulate expression of neurotrophic factors such as nerve growth factor (NGF). Several studies suggest a role for cytokines and NGF in OA pain, however the effects of changing mechanical properties in OA tissue on chondrocyte metabolism remain unclear. Here, we used high-extension silicone rubber membranes to examine if high mechanical strain (HMS) of primary articular chondrocytes increases inflammatory gene expression and promotes neurotrophic factor release. HMS cultured chondrocytes displayed up-regulated NGF, TNFalpha and ADAMTS4 gene expression while decreasing TLR2 expression, as compared to static controls. HMS culture increased p38 MAPK activity compared to static controls. Conditioned medium from HMS dynamic cultures, but not static cultures, induced significant neurite sprouting in PC12 cells. The increased neurite sprouting was accompanied by consistent increases in PC12 cell death. Low-frequency high-magnitude mechanical strain of primary articular chondrocytes in vitro drives factor secretion associated with degenerative joint disease and joint pain. This study provides evidence for a direct link between cellular strain, secretory factors, neo-innervation, and pain in OA pathology. (C) 2014 by the authors; licensee MDPI, Basel, Switzerland.},
  annote = {cited By 13},
  author = {Rosenzweig, Derek H. and Quinn, Thomas M. and Haglun, Lisbet},
  doi = {10.3390/ijms150814427},
  issn = {14220067},
  journal = {International Journal of Molecular Sciences},
  keywords = {Articular cartilage,Inflammation,Mechanical stretch,Nerve growth factor,Osteoarthritis,Pain,p38 MAPK},
  number = {8},
  pages = {14427--14441},
  title = {{Low-frequency high-magnitude mechanical strain of articular chondrocytes activates p38 MAPK and induces phenotypic changes associated with osteoarthritis and pain}},
  volume = {15},
  year = {2014}
  }
  

@article{Gawri2014,
  abstract = {Introduction:Excessive mechanical loading of intervertebral discs (IVDs) is thought to alter matrix properties and influence disc cell metabolism, contributing to degenerative disc disease and development of discogenic pain. However, little is known about how mechanical strain induces these changes. This study investigated the cellular and molecular changes as well as which inflammatory receptors and cytokines were upregulated in human intervertebral disc cells exposed to high mechanical strain (HMS) at low frequency. The impact of these metabolic changes on neuronal differentiation was also explored to determine a role in the development of disc degeneration and discogenic pain.Methods:Isolated human annulus fibrosus (AF) and nucleus pulposus (NP) cells were exposed to HMS (20 % cyclical stretch at 0.001 Hz) on high-extension silicone rubber dishes coupled to a mechanical stretching apparatus and compared to static control cultures. Gene expression of Toll-like receptors (TLRs), neuronal growth factor (NGF) and tumour necrosis factor alpha (TNFalpha) was assessed. Collected conditioned media were analysed for cytokine content and applied to rat pheocromocytoma PC12 cells for neuronal differentiation assessment.Results:HMS caused upregulation of TLR2, TLR4, NGF and TNFalpha gene expression in IVD cells. Medium from HMS cultures contained elevated levels of growth-related oncogene, interleukin 6 (IL-6), IL-8, IL-15, monocyte chemoattractant protein 1 (MCP-1), MCP-3, monokine induced by gamma interferon, transforming growth factor beta1, TNFalpha and NGF. Exposure of PC12 cells to HMS-conditioned media resulted in both increased neurite sprouting and cell death.Conclusions:HMS culture of IVD cells in vitro drives cytokine and inflammatory responses associated with degenerative disc disease and low-back pain. This study provides evidence for a direct link between cellular strain, secretory factors, neoinnervation and potential degeneration and discogenic pain in vivo. (C) 2014 Gawri et al.; licensee BioMed Central Ltd.},
  annote = {cited By 58},
  author = {Gawri, Rahul and Rosenzweig, Derek H. and Krock, Emerson and Ouellet, Jean A. and Stone, Laura S. and Quinn, Thomas M. and Haglund, Lisbet},
  doi = {10.1186/ar4449},
  issn = {14786362},
  journal = {Arthritis Research and Therapy},
  number = {1},
  title = {{High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain}},
  volume = {16},
  year = {2014}
  }
  

@article{Moeini2014605,
  abstract = {Background: Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur. Methods: Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone. Results: Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde-dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes. Conclusions and general significance: Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents. (C) 2013 Elsevier B.V. All rights reserved.},
  annote = {cited By 6},
  author = {Moeini, Mohammad and Decker, Sarah G.A. and Chin, Hooi Chuan and Shafieyan, Yousef and Rosenzweig, Derek H. and Quinn, Thomas M.},
  doi = {10.1016/j.bbagen.2013.10.022},
  issn = {03044165},
  journal = {Biochimica et Biophysica Acta - General Subjects},
  keywords = {Articular cartilage,Contrast agent,Dextrans,FITC,Injury,Solute adsorption},
  number = {1},
  pages = {605--614},
  title = {{Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents}},
  volume = {1840},
  year = {2014}
  }
  

@article{Krock20141213,
  abstract = {Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-alpha, interleukin-1beta, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo. (C) 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley {&} Sons Ltd and Foundation for Cellular and Molecular Medicine.},
  annote = {cited By 60},
  author = {Krock, Emerson and Rosenzweig, Derek H. and Chabot-Dore, Anne Julie and Jarzem, Peter and Weber, Michael H. and Ouellet, Jean A. and Stone, Laura S. and Haglund, Lisbet},
  doi = {10.1111/jcmm.12268},
  issn = {15824934},
  journal = {Journal of Cellular and Molecular Medicine},
  keywords = {CGRP,Discogenic pain,Human,Inflammatory cytokines,Intervertebral disc degeneration,Nerve growth factor},
  number = {6},
  pages = {1213--1225},
  title = {{Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors}},
  volume = {18},
  year = {2014}
  }
  

@article{Alkhatib201498,
  abstract = {Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5 % (non-injured) or 30 % (injured) of disc height. 30 % strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured- IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROalpha, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.},
  annote = {cited By 45},
  author = {Alkhatib, B. and Rosenzweig, D. H. and Krock, E. and Roughley, P. J. and Beckman, L. and Steffen, T. and Weber, M. H. and Ouellet, J. A. and Haglund, L.},
  doi = {10.22203/eCM.v028a08},
  issn = {14732262},
  journal = {European Cells and Materials},
  keywords = {Degeneration,Extracellular matrix,Inflammation,Intervertebral disc,Mechanical injury,Nerve growth factor},
  pages = {98--111},
  title = {{Acute mechanical injury of the human intervertebral disc: link to degeneration and pain}},
  volume = {28},
  year = {2014}
  }
  

@article{Rosenzweig20139360,
  abstract = {Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.},
  annote = {cited By 14},
  author = {Rosenzweig, D. H. and Chicatun, F. and Nazhat, S. N. and Quinn, T. M.},
  doi = {10.1016/j.actbio.2013.07.024},
  issn = {17427061},
  journal = {Acta Biomaterialia},
  keywords = {Articular chondrocytes,Dense collagen,Hydrogel,Plastic compression,Tissue engineering},
  number = {12},
  pages = {9360--9369},
  title = {{Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels}},
  volume = {9},
  year = {2013}
  }
  

@article{Decker20132427,
  abstract = {The development of cartilage-specific imaging agents supports the improvement of tissue assessment by minimally invasive means. Techniques for highlighting cartilage surface damage in clinical images could provide for sensitive indications of posttraumatic injury and early stage osteoarthritis. Previous studies in our laboratory have demonstrated that fluorescent solutes interact with cartilage surfaces strongly enough to affect measurement of their partition coefficients within the tissue bulk. In this study, these findings were extended by examining solute adsorption and distribution near the articular surface of mechanically injured cartilage. Using viable cartilage explants injured by an established protocol, solute distributions near the articular surface of three commonly used fluorophores (fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and carboxytetramethylrhodamine (TAMRA)) were observed after absorption and subsequent desorption to assess solute-specific matrix interactions and reversibility. Both absorption and desorption processes demonstrated a trend of significantly less solute adsorption at surfaces of fissures compared to adjacent intact surfaces of damaged explants or surfaces of uninjured explants. After adsorption, normalized mean surface intensities of fissured surfaces of injured explants were 6%, 40%, and 32% for FITC, TRITC, and TAMRA, respectively, compared to uninjured surfaces. Similar values were found for sliced explants and after a desorption process. After desorption, a trend of increased solute adsorption at the site of intact damaged surfaces was noted (316% and 238% for injured and sliced explants exposed to FITC). Surface adsorption of solute was strongest for FITC and weakest for TAMRA; no solutes negatively affected cell viability. Results support the development of imaging agents that highlight distinct differences between fissured and intact cartilage surfaces. (C) 2013 Biophysical Society.},
  annote = {cited By 9},
  author = {Decker, Sarah G.A. and Moeini, Mohammad and Chin, Hooi Chuan and Rosenzweig, Derek H. and Quinn, Thomas M.},
  doi = {10.1016/j.bpj.2013.09.037},
  issn = {00063495},
  journal = {Biophysical Journal},
  number = {10},
  pages = {2427--2436},
  title = {{Adsorption and distribution of fluorescent solutes near the articular surface of mechanically injured cartilage}},
  volume = {105},
  year = {2013}
  }
  

@article{Matmati2013894,
  abstract = {Cartilage defects can be addressed with replacement strategies such as autologous chondrocyte implantation (ACI). Expansion of autologous chondrocytes in vitro is an essential step to obtain the necessary cell numbers required for ACI. A major problem with this approach is dedifferentiation of chondrocytes during expansion, resulting in cells with fibroblast-like features. These cells generate cartilage tissue with fibrotic instead of hyaline characteristics. The use of serum is a common feature in most expansion protocols and a potential factor contributing to the dedifferentiation process. The aim of this study was to assess if heat inactivation of serum used in the expansion medium might be a valid approach to generate cells with an improved phenotype and in relevant numbers. We used bovine chondrocyte expansion cultures incubated with heat inactivated allogeneic serum (HIFBS) as a model system. We here show that heat inactivation protects the differentiated phenotype of chondrocytes compared to cultures with regular serum. This is not only true for primary cultures but holds up after two passages. Moreover, using relatively low cell seeding densities, clinically relevant cell numbers can already be reached after the first passage in cultures with HIFBS. In short we here introduce a simple way to improve cell quality while generating relevant amounts of cells during monolayer expansion of bovine chondrocytes in a relative short time period. Our results could have wider implications when translated to the expansion of human chondrocytes. (C) 2012 Biomedical Engineering Society.},
  annote = {cited By 10},
  author = {Matmati, Mourad and Ng, Tat Fong and Rosenzweig, Derek H. and Quinn, Thomas M.},
  doi = {10.1007/s10439-012-0732-z},
  issn = {00906964},
  journal = {Annals of Biomedical Engineering},
  keywords = {Chondrocyte,Expansion culture,Heat inactivation,Serum},
  number = {5},
  pages = {894--903},
  title = {{Protection of bovine chondrocyte phenotype by heat inactivation of allogeneic serum in monolayer expansion cultures}},
  volume = {41},
  year = {2013}
  }
  

@article{Rosenzweig2013508,
  abstract = {Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell-based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes 'dedifferentiation' into fibroblastic cells. Mitogen-activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up-regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow-on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell-based therapies. (C) 2013.},
  annote = {cited By 21},
  author = {Rosenzweig, Derek H. and Ou, Sing J. and Quinn, Thomas M.},
  doi = {10.1111/jcmm.12034},
  issn = {15821838},
  journal = {Journal of Cellular and Molecular Medicine},
  keywords = {Chondrocyte,Dedifferentiation,Extra Cellular Matrix,Gene Expression,MAPK,Signal Transduction},
  number = {4},
  pages = {508--517},
  pmid = {23480786},
  title = {{P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture}},
  volume = {17},
  year = {2013}
  }
  

@article{Rosenzweig20121591,
  abstract = {Objective: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. Design: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. Results: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. Conclusions: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries. (C) 2012 Osteoarthritis Research Society International.},
  annote = {cited By 19},
  author = {Rosenzweig, D. H. and Djap, M. J. and Ou, S. J. and Quinn, T. M.},
  doi = {10.1016/j.joca.2012.08.012},
  issn = {10634584},
  journal = {Osteoarthritis and Cartilage},
  keywords = {Chondrocyte,Compression,Gene expression,Injury,MAP kinase,Signalling},
  number = {12},
  pages = {1591--1602},
  pmid = {22935788},
  title = {{Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis}},
  volume = {20},
  year = {2012}
  }
  

@article{Rosenzweig20122466,
  abstract = {Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and alpha-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies. (C) Copyright 2012, Mary Ann Liebert, Inc.},
  annote = {cited By 32},
  author = {Rosenzweig, Derek H. and Matmati, Mourad and Khayat, Ghazaleh and Chaudhry, Sidharth and Hinz, Boris and Quinn, Thomas M.},
  doi = {10.1089/ten.tea.2012.0215},
  issn = {19373341},
  journal = {Tissue Engineering - Part A},
  number = {23-24},
  pages = {2466--2476},
  title = {{Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation}},
  volume = {18},
  year = {2012}
  }
  

@article{Rosenzweig20123333,
  abstract = {Culture on silicone rubber surfaces has been shown to partially overcome the chondrocyte dedifferentiation characteristic of standard culture on rigid polystyrene. These methods typically involve functionalization of culture surfaces with proteins. Collagen type I is often used, but more cartilage-specific proteins may be more appropriate for chondrocytes. To explore this hypothesis, a twofold experimental design was applied. First, chondrocytes were cultured in rigid Petri dishes coated with silicone rubber ("static silicone" or SS culture) functionalized with either cartilage extracellular matrix (ECM) extract or collagen type I. Second, chondrocytes were cultured on monotonically expanded high extension silicone rubber dishes ("continuous expansion" or CE culture) functionalized with ECM extract and compared to cells grown in SS culture. There were no differential effects of surface functionalization with the ECM extract vs. collagen type I on chondrocyte morphology, viability, proliferation or apoptosis in SS culture. However, chondrocyte growth on the ECM extract was associated with significantly reduced collagen types I and X gene expression and significantly increased glycosaminoglycan (GAG) secretion. After 3 passages (P3) on ECM-coated SS culture, chondrocyte phenotype and GAG secretion was enhanced compared to cells passaged on collagen type I. Pellet cultures from P3 SS culture displayed enhanced collagen type II content when ECM extract was used for functionalization rather than collagen type I. In CE culture with ECM functionalization, chondrocyte dedifferentiation was significantly inhibited vs. SS cultures, as evidenced by both gene expression and pellet cultures. Functionalization of extendable culture surfaces with cartilage ECM extract therefore supports enhanced preservation of chondrocyte phenotype. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.},
  annote = {cited By 22},
  author = {Rosenzweig, Derek H. and Solar-Cafaggi, Sofia and Quinn, Thomas M.},
  doi = {10.1016/j.actbio.2012.05.032},
  issn = {17427061},
  journal = {Acta Biomaterialia},
  keywords = {Chondrocyte,Collagen,Dedifferentiation,Extendable surfaces,Extracellular matrix},
  number = {9},
  pages = {3333--3341},
  title = {{Functionalization of dynamic culture surfaces with a cartilage extracellular matrix extract enhances chondrocyte phenotype against dedifferentiation}},
  volume = {8},
  year = {2012}
  }
  

@article{Khayat2012179,
  abstract = {Oscillatory mechanical stimulation at relatively high frequencies (0.1. Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01. Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01. Hz frequency for 6. h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARgamma) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARgamma after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages. (C) 2012 International Society of Differentiation.},
  annote = {cited By 25},
  author = {Khayat, Ghazaleh and Rosenzweig, Derek H. and Quinn, Thomas M.},
  doi = {10.1016/j.diff.2011.12.004},
  issn = {03014681},
  journal = {Differentiation},
  keywords = {Adipogenesis,C3H10T1/2,Differentiation,Gene expression,Low frequency,Mechanical stimulation},
  number = {4},
  pages = {179--184},
  title = {{Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells}},
  volume = {83},
  year = {2012}
  }
  

@article{Rosenweig2009803,
  abstract = {Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between Galphat (the transducin alpha subunit) and retGC. Galphat co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-Galphat complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with bothGalphat and retGC. The Galphat-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound Galphat stronger than the GTP[S] (GTPgammaS; guanosine 5′-[gamma-thio]triphosphate) form. Neither Galphat nor Gbetagamma. affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between Galphat and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors. (C) The Authors Journal compilation (C) 2009 Biochemical Society.},
  annote = {cited By 3},
  author = {Rosenweig, Derek H. and Nair, Saidas K. and Levay, Konstantin and Peshenko, Igor and Crabb, John W. and Dizhoor, Alexander M. and Slepak, Vladlen Z.},
  doi = {10.1042/BJ20081513},
  issn = {02646021},
  journal = {Biochemical Journal},
  keywords = {CGMP,G-protein,Photoreceptor,Subcellular localization},
  number = {3},
  pages = {803--812},
  title = {{Interaction of retinal guanylate cyclase with the alpha subunit of transducin: Potential role in transducin localization}},
  volume = {417},
  year = {2009}
  }
  

@article{Rosenzweig20075484,
  abstract = {Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments. Copyright (C) 2007 Society for Neuroscience.},
  annote = {cited By 51},
  author = {Rosenzweig, Derek H. and {Saidas Nair}, K. and Wei, Junhua and Wang, Qiang and Garwin, Greg and Saari, John C. and Chen, Ching Kang and Smrcka, Alan V. and Swaroop, Anand and Lem, Janis and Hurley, James B. and Slepak, Vladlen Z.},
  doi = {10.1523/JNEUROSCI.1421-07.2007},
  issn = {02706474},
  journal = {Journal of Neuroscience},
  keywords = {Cone,Diffusion,Retina,Rod,Signal transduction,Transducin},
  number = {20},
  pages = {5484--5494},
  pmid = {17507570},
  title = {{Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins}},
  volume = {27},
  year = {2007}
  }
  

@article{Nair2005383,
  abstract = {PURPOSE. Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells. METHODS. Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding. RESULTS. LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange. CONCLUSIONS. Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.},
  annote = {cited By 13},
  author = {Nair, K. Saidas and Mendez, Ana and Blumer, Joe B. and Rosenzweig, Derek H. and Slepak, Vladlen Z.},
  doi = {10.1167/iovs.04-1006},
  issn = {01460404},
  journal = {Investigative Ophthalmology and Visual Science},
  number = {1},
  pages = {383--389},
  title = {{The presence of a Leu-Gly-Asn repeat-enriched protein (LGN), a putative binding partner of transducin, in ROD photoreceptors}},
  volume = {46},
  year = {2005}
  }
  

@article{Cooke2020,
  abstract = {Critical-size bone defects are those that will not heal without intervention and can arise secondary to trauma, infection and surgical resection of tumours. Treatment options are currently limited to filling the defect with autologous bone, of which there is not always an abundant supply, or ceramic pastes that only allow for limited osteo-inductive and -conductive capacity. In this study we investigate the repair of bone defects using a 3D printed LayFomm scaffold. LayFomm is a polymer blend of polyvinyl alcohol (PVA) and polyurethane (PU). It can be printed using the most common method of 3D printing, fused deposition modelling, before being washed in water-based solutions to remove the PVA. This leaves a more compliant, micro-porous PU elastomer. In vitro analysis of dental pulp stem cells seeded onto macro-porous scaffolds showed their ability to adhere, proliferate and form mineralised matrix on the scaffold in the presence of osteogenic media. Subcutaneous implantation of LayFomm in a rat model showed the formation of a vascularised fibrous capsule, but without a chronic inflammatory response. Implantation into a mandibular defect showed significantly increased mineralised tissue production when compared to a currently approved bone putty. While their mechanical properties are insufficient for use in load-bearing defects, these findings are promising for the use of polyurethane scaffolds in craniofacial bone regeneration.},
  author = {Cooke, Megan Elin and Ramirez-GarciaLuna, Jose L and Rangel-Berridi, Karla and Park, Hyeree and Nazhat, Showan N and Weber, Michael H and Henderson, Janet E and Rosenzweig, Derek H},
  doi = {10.3389/FBIOE.2020.557215},
  issn = {2296-4185},
  journal = {Frontiers in Bioengineering and Biotechnology},
  keywords = {3D printing,Bone Regeneration,Fused depositing modeling (FDM),Mandibular defect,Poly-urethane,layfomm},
  pages = {1190},
  publisher = {Frontiers},
  title = {{3D printed polyurethane scaffolds for the repair of bone defects}},
  volume = {8},
  year = {2020}
  }
\">\n            <i class=\"fa fa-download fa-fw\"></i> Download BibTeX\n          </a>\n        </li>\n        <li>\n          <a href=\"//bibbase.org/rss?bib=https://raw.githubusercontent.com/rosenzweiglab/rosenzweiglab.github.io/master/docs/references.bib\">\n            <i class=\"fa fa-rss fa-fw\"></i> RSS Feed\n          </a>\n          </li>\n      </ul>\n      </li>\n\n      \n\n\n      \n    </ul>\n\n\n    <div class=\"alert alert-success bibbase_msg\" id=\"bibbase_msg_embed\"\n         style=\"display: none;\">\n\n      Excellent! Next you can\n      <a href=\"/network/register?next=/network/newSiteFromTemplate%3Fbiburl=https://raw.githubusercontent.com/rosenzweiglab/rosenzweiglab.github.io/master/docs/references.bib\"\n      >create a new website with this list</a>, or\n      embed it in an existing web page by copying &amp; pasting\n      any of the following snippets.\n\n      <div style=\"text-align: left; margin-top: 1em;\">\n        <strong>JavaScript</strong>\n        <span class=\"comment recommended\">(easiest)</span>\n        <div class=\"well well-small\">\n          <code>\n            &lt;script src=\"https://bibbase.org/show?bib=https%3A%2F%2Fraw.githubusercontent.com%2Frosenzweiglab%2Frosenzweiglab.github.io%2Fmaster%2Fdocs%2Freferences.bib&amp;authorfirst=1&amp;theme=simple&amp;jsonp=1&amp;jsonp=1\"&gt;&lt;/script&gt;\n          </code>\n        </div>\n\n        <strong>PHP</strong>\n        <div class=\"well well-small\">\n          <code>\n            &lt;?php\n            $contents = file_get_contents(\"https://bibbase.org/show?bib=https%3A%2F%2Fraw.githubusercontent.com%2Frosenzweiglab%2Frosenzweiglab.github.io%2Fmaster%2Fdocs%2Freferences.bib&amp;authorfirst=1&amp;theme=simple&amp;jsonp=1\");\n            print_r($contents);\n            ?&gt;\n          </code>\n        </div>\n\n        <strong>iFrame</strong>\n        <span class=\"comment\">(not recommended)</span>\n        <div class=\"well well-small\">\n          <code>\n            &lt;iframe src=\"https://bibbase.org/show?bib=https%3A%2F%2Fraw.githubusercontent.com%2Frosenzweiglab%2Frosenzweiglab.github.io%2Fmaster%2Fdocs%2Freferences.bib&amp;authorfirst=1&amp;theme=simple&amp;jsonp=1\"&gt;&lt;/iframe&gt;\n          </code>\n        </div>\n\n        <p>\n          For more details see the <a href=\"//bibbase.org/help\">documention</a>.\n        </p>\n      </div>\n    </div>\n\n    <div class=\"alert alert-success bibbase_msg\" id=\"bibbase_msg_preview\"\n         style=\"display: none;\">\n\n      This is a preview! To use this list on your own web site\n      or create a new web site from it,\n      <a href=\"/network/register?next=/network/newAccountWithFile%3FfileId=\"\n      >create a free account</a>. The file will be added\n      and you will be able to edit it in the File Manager.\n      We will show you instructions once you've created your account.\n    </div>\n\n    <div class=\"alert alert-warning bibbase_msg\" id=\"bibbase_msg_mendeley1\"\n         style=\"display: none;\">\n\n      <p>To the site owner:</p>\n\n      <p><strong>Action required!</strong> Mendeley is changing its\n        API. In order to keep using Mendeley with BibBase past April\n        14th, you need to:\n        <ol>\n          <li>renew the authorization for BibBase on Mendeley, and</li>\n          <li>update the BibBase URL\n            in your page the same way you did when you initially set up\n            this page.\n          </li>\n        </ol>\n      </p>\n\n      <p>\n        <a href=\"http://bibbase.org/cgi-bin/mendeley2/get.py\">\n          <i class=\"fa fa-angle-double-right\"></i>\n          Fix it now</a>\n      </p>\n    </div>\n\n  </div>\n\n\n  <div id=\"bibbase_body\">\n    \n  \n  <div class=\"bibbase_group_whole\" id=\"group_2020\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2020')\"\n      onkeypress=\"toggleGroup('group_2020')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2020</span>\n      <span class=\"bibbase_group_count\">\n        \n        (4)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"fattah-daza-rustandi-berrocalrubio-gorissen-poovathingal-davie-cao-etal-actuationenhancespatterninginhumanneuraltubeorganoids-2020\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  <a href=\"https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411\"\n     onclick=\"return trackOutboundLink('publications', 'click', 'fattah-daza-rustandi-berrocalrubio-gorissen-poovathingal-davie-cao-etal-actuationenhancespatterninginhumanneuraltubeorganoids-2020', 'https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411', event);\">\n    Actuation Enhances Patterning in Human Neural Tube Organoids.\n  </a>\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Fattah, A. R. A.; Daza, B.; Rustandi, G.; Berrocal-Rubio, M. A.; Gorissen, B.; Poovathingal, S.; Davie, K.; Cao, X.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Lei, Y.; Finnell, R.; Verfaillie, C.; Sampaolesi, M.; Dedecker, P.; Van Oosterwyck, H.; Aerts, S.;  and Ranga, A.\n</span>\n\n  \n\n</span>\n\n  <i>bioRxiv</i>.  2020.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n  <a href=\"https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411\"\n     onclick=\"log_download('fattah-daza-rustandi-berrocalrubio-gorissen-poovathingal-davie-cao-etal-actuationenhancespatterninginhumanneuraltubeorganoids-2020', 'https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411', event.ctrlKey, event)\">\n    <img src=\"//bibbase.org/img/filetypes/link.svg\"\n\t     alt=\"Actuation Enhances Patterning in Human Neural Tube Organoids [link]\"\n       title=\"Paper\"\n\t     class=\"bibbase_icon\"\n         ><span class=\"bibbase_icon_text\">Paper</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"http://doi.org/10.1101/2020.09.22.308411\"\n     onclick=\"log_download('fattah-daza-rustandi-berrocalrubio-gorissen-poovathingal-davie-cao-etal-actuationenhancespatterninginhumanneuraltubeorganoids-2020', 'http://doi.org/10.1101/2020.09.22.308411', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/fattah-daza-rustandi-berrocalrubio-gorissen-poovathingal-davie-cao-etal-actuationenhancespatterninginhumanneuraltubeorganoids-2020\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n  &nbsp;\n  <span class=\"bibbase_stats_paper\" style=\"color: #777;\">\n    <span>4 downloads</span>\n  </span>\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('fattah-daza-rustandi-berrocalrubio-gorissen-poovathingal-davie-cao-etal-actuationenhancespatterninginhumanneuraltubeorganoids-2020')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article {Fattah2020.09.22.308411,\n\tauthor = {Fattah, Abdel Rahman Abdel and Daza, Brian and Rustandi, Gregorius and Berrocal-Rubio, Miguel Angel and Gorissen, Benjamin and Poovathingal, Suresh and Davie, Kristofer and Cao, Xuanye and Rosenzweig, Derek Hadar and Lei, Yunping and Finnell, Richard and Verfaillie, Catherine and Sampaolesi, Maurilio and Dedecker, Peter and Van Oosterwyck, Hans and Aerts, Stein and Ranga, Adrian},\n\ttitle = {Actuation Enhances Patterning in Human Neural Tube Organoids},\n\telocation-id = {2020.09.22.308411},\n\tyear = {2020},\n\tdoi = {10.1101/2020.09.22.308411},\n\tpublisher = {Cold Spring Harbor Laboratory},\n\tabstract = {Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.Competing Interest StatementRF was CEO of Teratomic Consulting, LLC, which has been dissolved. He is also an associate editor of the journal Reproductive and Developmental Medicine, which provides his travel expenses.},\n\tURL = {https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411},\n\teprint = {https://www.biorxiv.org/content/early/2020/09/22/2020.09.22.308411.full.pdf},\n\tjournal = {bioRxiv}\n}\n\n</pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Tissues achieve their complex spatial organization through an interplay between gene regulatory networks, cell-cell communication, and physical interactions mediated by mechanical forces. Current strategies to generate in-vitro tissues have largely failed to implement such active, dynamically coordinated mechanical manipulations, relying instead on extracellular matrices which respond to, rather than impose mechanical forces. Here we develop devices that enable the actuation of organoids. We show that active mechanical forces increase growth and lead to enhanced patterning in an organoid model of the neural tube derived from single human pluripotent stem cells (hPSC). Using a combination of single-cell transcriptomics and immunohistochemistry, we demonstrate that organoid mechanoregulation due to actuation operates in a temporally restricted competence window, and that organoid response to stretch is mediated extracellularly by matrix stiffness and intracellularly by cytoskeleton contractility and planar cell polarity. Exerting active mechanical forces on organoids using the approaches developed here is widely applicable and should enable the generation of more reproducible, programmable organoid shape, identity and patterns, opening avenues for the use of these tools in regenerative medicine and disease modelling applications.Competing Interest StatementRF was CEO of Teratomic Consulting, LLC, which has been dissolved. He is also an associate editor of the journal Reproductive and Developmental Medicine, which provides his travel expenses.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"azizipour-avazpour-rosenzweig-sawan-ajji-evolutionofbiochiptechnologyareviewfromlabonachiptoorganonachip-2020\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Evolution of biochip technology: A review from lab-on-a-chip to organ-on-a-chip.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Azizipour, N.; Avazpour, R.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Sawan, M.;  and Ajji, A.\n</span>\n\n  \n\n</span>\n\n  <i>Micromachines</i>, 11(6): 1–15.  2020.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.3390/mi11060599\"\n     onclick=\"log_download('azizipour-avazpour-rosenzweig-sawan-ajji-evolutionofbiochiptechnologyareviewfromlabonachiptoorganonachip-2020', 'http://doi.org/10.3390/mi11060599', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/azizipour-avazpour-rosenzweig-sawan-ajji-evolutionofbiochiptechnologyareviewfromlabonachiptoorganonachip-2020\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('azizipour-avazpour-rosenzweig-sawan-ajji-evolutionofbiochiptechnologyareviewfromlabonachiptoorganonachip-2020')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Azizipour2020,\nabstract = {Following the advancements in microfluidics and lab-on-a-chip (LOC) technologies, a novel biomedical application for microfluidic based devices has emerged in recent years and microengineered cell culture platforms have been created. These micro-devices, known as organ-on-a-chip (OOC) platforms mimic the in vivo like microenvironment of living organs and offer more physiologically relevant in vitro models of human organs. Consequently, the concept of OOC has gained great attention from researchers in the field worldwide to offer powerful tools for biomedical researches including disease modeling, drug development, etc. This review highlights the background of biochip development. Herein, we focus on applications of LOC devices as a versatile tool for POC applications. We also review current progress in OOC platforms towards body-on-a-chip, and we provide concluding remarks and future perspectives for OOC platforms for POC applications.},\nauthor = {Azizipour, Neda and Avazpour, Rahi and Rosenzweig, Derek H. and Sawan, Mohamad and Ajji, Abdellah},\ndoi = {10.3390/mi11060599},\nissn = {2072666X},\njournal = {Micromachines},\nkeywords = {BioMEMS,Lab-on-a-chip,Microengineering,Microfluidics,Organ-on-a-chip,Personalized medicine,Point-of-care},\nnumber = {6},\npages = {1--15},\ntitle = {{Evolution of biochip technology: A review from lab-on-a-chip to organ-on-a-chip}},\nvolume = {11},\nyear = {2020}\n}\n</pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Following the advancements in microfluidics and lab-on-a-chip (LOC) technologies, a novel biomedical application for microfluidic based devices has emerged in recent years and microengineered cell culture platforms have been created. These micro-devices, known as organ-on-a-chip (OOC) platforms mimic the in vivo like microenvironment of living organs and offer more physiologically relevant in vitro models of human organs. Consequently, the concept of OOC has gained great attention from researchers in the field worldwide to offer powerful tools for biomedical researches including disease modeling, drug development, etc. This review highlights the background of biochip development. Herein, we focus on applications of LOC devices as a versatile tool for POC applications. We also review current progress in OOC platforms towards body-on-a-chip, and we provide concluding remarks and future perspectives for OOC platforms for POC applications.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"pitaru-lacombe-cooke-beckman-steffen-weber-martineau-rosenzweig-investigatingcommercialfilamentsfor3dprintingofstiffandelasticconstructswithligamentlikemechanics-2020\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Investigating commercial filaments for 3D printing of stiff and elastic constructs with ligament-like mechanics.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Pitaru, A. A.; Lacombe, J. G.; Cooke, M. E.; Beckman, L.; Steffen, T.; Weber, M. H.; Martineau, P. A.;  and <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>\n</span>\n\n  \n\n</span>\n\n  <i>Micromachines</i>, 11(9): 1–15.  2020.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.3390/MI11090846\"\n     onclick=\"log_download('pitaru-lacombe-cooke-beckman-steffen-weber-martineau-rosenzweig-investigatingcommercialfilamentsfor3dprintingofstiffandelasticconstructswithligamentlikemechanics-2020', 'http://doi.org/10.3390/MI11090846', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/pitaru-lacombe-cooke-beckman-steffen-weber-martineau-rosenzweig-investigatingcommercialfilamentsfor3dprintingofstiffandelasticconstructswithligamentlikemechanics-2020\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n  &nbsp;\n  <span class=\"bibbase_stats_paper\" style=\"color: #777;\">\n    <span>2 downloads</span>\n  </span>\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('pitaru-lacombe-cooke-beckman-steffen-weber-martineau-rosenzweig-investigatingcommercialfilamentsfor3dprintingofstiffandelasticconstructswithligamentlikemechanics-2020')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Pitaru2020,\nabstract = {The current gold standard technique for treatment of anterior cruciate ligament (ACL) injury is reconstruction with autograft. These treatments have a relatively high failure and re-tear rate. To overcome this, tissue engineering and additive manufacturing are being used to explore the potential of 3D scaffolds as autograft substitutes. However, mechanically optimal polymers for this have yet to be identified. Here, we use 3D printing technology and various materials with the aim of fabricating constructs better matching the mechanical properties of the native ACL. A fused deposition modeling (FDM) 3D printer was used to microfabricate dog bone-shaped specimens from six different polymers-PLA, PETG, Lay FOMM 60, NinjaFlex, NinjaFlex-SemiFlex, and FlexiFil-at three different raster angles. The tensile mechanical properties of these polymers were determined from stress-strain curves. Our results indicate that no single material came close enough to successfully match reported mechanical properties of the native ACL. However, PLA and PETG had similar ultimate tensile strengths. Lay FOMM 60 displayed a percentage strain at failure similar to reported values for native ACL. Furthermore, raster angle had a significant impact on some mechanical properties for all of the materials except for FlexiFil. We therefore conclude that while none of these materials alone is optimal for mimicking ACL mechanical properties, there may be potential for creating a 3D-printed composite constructs to match ACL mechanical properties. Further investigations involving co-printing of stiff and elastomeric materials must be explored.},\nauthor = {Pitaru, Audrey A. and Lacombe, Jean Gabriel and Cooke, Megan E. and Beckman, Lorne and Steffen, Thomas and Weber, Michael H. and Martineau, Paul A. and Rosenzweig, Derek H.},\ndoi = {10.3390/MI11090846},\nissn = {2072666X},\njournal = {Micromachines},\nkeywords = {3D printing,Elastic,Ligament,Mechanical strain,Polymers,Scaffolds,Tissue engineering},\nnumber = {9},\npages = {1--15},\ntitle = {{Investigating commercial filaments for 3D printing of stiff and elastic constructs with ligament-like mechanics}},\nvolume = {11},\nyear = {2020}\n}\n\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  The current gold standard technique for treatment of anterior cruciate ligament (ACL) injury is reconstruction with autograft. These treatments have a relatively high failure and re-tear rate. To overcome this, tissue engineering and additive manufacturing are being used to explore the potential of 3D scaffolds as autograft substitutes. However, mechanically optimal polymers for this have yet to be identified. Here, we use 3D printing technology and various materials with the aim of fabricating constructs better matching the mechanical properties of the native ACL. A fused deposition modeling (FDM) 3D printer was used to microfabricate dog bone-shaped specimens from six different polymers-PLA, PETG, Lay FOMM 60, NinjaFlex, NinjaFlex-SemiFlex, and FlexiFil-at three different raster angles. The tensile mechanical properties of these polymers were determined from stress-strain curves. Our results indicate that no single material came close enough to successfully match reported mechanical properties of the native ACL. However, PLA and PETG had similar ultimate tensile strengths. Lay FOMM 60 displayed a percentage strain at failure similar to reported values for native ACL. Furthermore, raster angle had a significant impact on some mechanical properties for all of the materials except for FlexiFil. We therefore conclude that while none of these materials alone is optimal for mimicking ACL mechanical properties, there may be potential for creating a 3D-printed composite constructs to match ACL mechanical properties. Further investigations involving co-printing of stiff and elastomeric materials must be explored.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"cooke-ramirezgarcialuna-rangelberridi-park-nazhat-weber-henderson-rosenzweig-3dprintedpolyurethanescaffoldsfortherepairofbonedefects-2020\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  3D printed polyurethane scaffolds for the repair of bone defects.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Cooke, M. E.; Ramirez-GarciaLuna, J. L; Rangel-Berridi, K.; Park, H.; Nazhat, S. N; Weber, M. H; Henderson, J. E;  and <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H</a>\n</span>\n\n  \n\n</span>\n\n  <i>Frontiers in Bioengineering and Biotechnology</i>, 8: 1190.  2020.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.3389/FBIOE.2020.557215\"\n     onclick=\"log_download('cooke-ramirezgarcialuna-rangelberridi-park-nazhat-weber-henderson-rosenzweig-3dprintedpolyurethanescaffoldsfortherepairofbonedefects-2020', 'http://doi.org/10.3389/FBIOE.2020.557215', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/cooke-ramirezgarcialuna-rangelberridi-park-nazhat-weber-henderson-rosenzweig-3dprintedpolyurethanescaffoldsfortherepairofbonedefects-2020\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n  &nbsp;\n  <span class=\"bibbase_stats_paper\" style=\"color: #777;\">\n    <span>1 download</span>\n  </span>\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('cooke-ramirezgarcialuna-rangelberridi-park-nazhat-weber-henderson-rosenzweig-3dprintedpolyurethanescaffoldsfortherepairofbonedefects-2020')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Cooke2020,\n  abstract = {Critical-size bone defects are those that will not heal without intervention and can arise secondary to trauma, infection and surgical resection of tumours. Treatment options are currently limited to filling the defect with autologous bone, of which there is not always an abundant supply, or ceramic pastes that only allow for limited osteo-inductive and -conductive capacity. In this study we investigate the repair of bone defects using a 3D printed LayFomm scaffold. LayFomm is a polymer blend of polyvinyl alcohol (PVA) and polyurethane (PU). It can be printed using the most common method of 3D printing, fused deposition modelling, before being washed in water-based solutions to remove the PVA. This leaves a more compliant, micro-porous PU elastomer. In vitro analysis of dental pulp stem cells seeded onto macro-porous scaffolds showed their ability to adhere, proliferate and form mineralised matrix on the scaffold in the presence of osteogenic media. Subcutaneous implantation of LayFomm in a rat model showed the formation of a vascularised fibrous capsule, but without a chronic inflammatory response. Implantation into a mandibular defect showed significantly increased mineralised tissue production when compared to a currently approved bone putty. While their mechanical properties are insufficient for use in load-bearing defects, these findings are promising for the use of polyurethane scaffolds in craniofacial bone regeneration.},\n  author = {Cooke, Megan Elin and Ramirez-GarciaLuna, Jose L and Rangel-Berridi, Karla and Park, Hyeree and Nazhat, Showan N and Weber, Michael H and Henderson, Janet E and Rosenzweig, Derek H},\n  doi = {10.3389/FBIOE.2020.557215},\n  issn = {2296-4185},\n  journal = {Frontiers in Bioengineering and Biotechnology},\n  keywords = {3D printing,Bone Regeneration,Fused depositing modeling (FDM),Mandibular defect,Poly-urethane,layfomm},\n  pages = {1190},\n  publisher = {Frontiers},\n  title = {{3D printed polyurethane scaffolds for the repair of bone defects}},\n  volume = {8},\n  year = {2020}\n  }\n</pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Critical-size bone defects are those that will not heal without intervention and can arise secondary to trauma, infection and surgical resection of tumours. Treatment options are currently limited to filling the defect with autologous bone, of which there is not always an abundant supply, or ceramic pastes that only allow for limited osteo-inductive and -conductive capacity. In this study we investigate the repair of bone defects using a 3D printed LayFomm scaffold. LayFomm is a polymer blend of polyvinyl alcohol (PVA) and polyurethane (PU). It can be printed using the most common method of 3D printing, fused deposition modelling, before being washed in water-based solutions to remove the PVA. This leaves a more compliant, micro-porous PU elastomer. In vitro analysis of dental pulp stem cells seeded onto macro-porous scaffolds showed their ability to adhere, proliferate and form mineralised matrix on the scaffold in the presence of osteogenic media. Subcutaneous implantation of LayFomm in a rat model showed the formation of a vascularised fibrous capsule, but without a chronic inflammatory response. Implantation into a mandibular defect showed significantly increased mineralised tissue production when compared to a currently approved bone putty. While their mechanical properties are insufficient for use in load-bearing defects, these findings are promising for the use of polyurethane scaffolds in craniofacial bone regeneration.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2019\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2019')\"\n      onkeypress=\"toggleGroup('group_2019')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2019</span>\n      <span class=\"bibbase_group_count\">\n        \n        (5)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"haglund-ahangar-rosenzweig-advancementsin3dprintedscaffoldstomimicmatrixcomplexitiesformusculoskeletalrepair-2019\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Advancements in 3D printed scaffolds to mimic matrix complexities for musculoskeletal repair.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Haglund, L.; Ahangar, P.;  and <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>\n</span>\n\n  \n\n</span>\n\n  <i>Current Opinion in Biomedical Engineering</i>, 10: 142–148.  2019.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1016/j.cobme.2019.06.002\"\n     onclick=\"log_download('haglund-ahangar-rosenzweig-advancementsin3dprintedscaffoldstomimicmatrixcomplexitiesformusculoskeletalrepair-2019', 'http://doi.org/10.1016/j.cobme.2019.06.002', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/haglund-ahangar-rosenzweig-advancementsin3dprintedscaffoldstomimicmatrixcomplexitiesformusculoskeletalrepair-2019\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('haglund-ahangar-rosenzweig-advancementsin3dprintedscaffoldstomimicmatrixcomplexitiesformusculoskeletalrepair-2019')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Haglund2019142,\n  abstract = {With the expiration of key patents on additive manufacturing, 3D printing devices have become extremely inexpensive. Therefore, the use of 3D printing in biomedical applications has blossomed in the past decade. Low-cost, high-quality instruments have become widely used in both industrial and academic settings. Fused deposition modeling using polymers such as polylactic acid, polycaprolactone, or other composites are now combined with bioprinting to open new avenues to cutting-edge research toward musculoskeletal repair and regeneration such as prevascularized bone or soft-tissue constructs. Most importantly, these tools are being widely used to generate composite scaffolds representing matrices on which to culture cells of various tissue types such as bone, cartilage, cardiac, and nervous tissue. 3D printed composite matrix scaffolds are being tested in sophisticated in vitro and in vivo preclinical models, paving the way for future clinical translation where the ultimate goal is to generate functional replacement tissues.},\n  annote = {cited By 0},\n  author = {Haglund, Lisbet and Ahangar, Pouyan and Rosenzweig, Derek H.},\n  doi = {10.1016/j.cobme.2019.06.002},\n  issn = {24684511},\n  journal = {Current Opinion in Biomedical Engineering},\n  keywords = {3D printed,Bioprinted,Composite,Hydrogels,Matrix scaffold,Tissue engineering},\n  pages = {142--148},\n  title = {{Advancements in 3D printed scaffolds to mimic matrix complexities for musculoskeletal repair}},\n  volume = {10},\n  year = {2019}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  With the expiration of key patents on additive manufacturing, 3D printing devices have become extremely inexpensive. Therefore, the use of 3D printing in biomedical applications has blossomed in the past decade. Low-cost, high-quality instruments have become widely used in both industrial and academic settings. Fused deposition modeling using polymers such as polylactic acid, polycaprolactone, or other composites are now combined with bioprinting to open new avenues to cutting-edge research toward musculoskeletal repair and regeneration such as prevascularized bone or soft-tissue constructs. Most importantly, these tools are being widely used to generate composite scaffolds representing matrices on which to culture cells of various tissue types such as bone, cartilage, cardiac, and nervous tissue. 3D printed composite matrix scaffolds are being tested in sophisticated in vitro and in vivo preclinical models, paving the way for future clinical translation where the ultimate goal is to generate functional replacement tissues.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"fairag-rosenzweig-ramirezgarcialuna-weber-haglund-threedimensionalprintedpolylacticacidscaffoldspromotebonelikematrixdepositioninvitro-2019\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bone-like Matrix Deposition in Vitro.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Fairag, R.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Ramirez-Garcialuna, J. L.; Weber, M. H.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>ACS Applied Materials and Interfaces</i>, 11(17): 15306–15315.  2019.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1021/acsami.9b02502\"\n     onclick=\"log_download('fairag-rosenzweig-ramirezgarcialuna-weber-haglund-threedimensionalprintedpolylacticacidscaffoldspromotebonelikematrixdepositioninvitro-2019', 'http://doi.org/10.1021/acsami.9b02502', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/fairag-rosenzweig-ramirezgarcialuna-weber-haglund-threedimensionalprintedpolylacticacidscaffoldspromotebonelikematrixdepositioninvitro-2019\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('fairag-rosenzweig-ramirezgarcialuna-weber-haglund-threedimensionalprintedpolylacticacidscaffoldspromotebonelikematrixdepositioninvitro-2019')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Fairag201915306,\n  abstract = {Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 mum), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 mum pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 mum pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 mum pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.},\n  annote = {cited By 7},\n  author = {Fairag, Rayan and Rosenzweig, Derek H. and Ramirez-Garcialuna, Jose L. and Weber, Michael H. and Haglund, Lisbet},\n  doi = {10.1021/acsami.9b02502},\n  issn = {19448252},\n  journal = {ACS Applied Materials and Interfaces},\n  keywords = {3D printing,PLA,bone defect,bone repair,human osteoblasts,low-cost,mesenchymal stem cells,tissue engineering},\n  number = {17},\n  pages = {15306--15315},\n  title = {{Three-Dimensional Printed Polylactic Acid Scaffolds Promote Bone-like Matrix Deposition in Vitro}},\n  volume = {11},\n  year = {2019}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Large bone defects represent a significant challenge for clinicians and surgeons. Tissue engineering for bone regeneration represents an innovative solution for this dilemma and may yield attractive alternate bone substitutes. Three-dimensional (3D) printing with inexpensive desktop printers shows promise in generating high-resolution structures mimicking native tissues using biocompatible, biodegradable, and cost-effective thermoplastics, which are already FDA-approved for food use, drug delivery, and many medical devices. Microporous 3D-printed polylactic acid scaffolds, with different pore sizes (500, 750, and 1000 mum), were designed and manufactured using an inexpensive desktop 3D printer, and the mechanical properties were assessed. The scaffolds were compared for cell growth, activity, and bone-like tissue formation using primary human osteoblasts. Osteoblasts showed high proliferation, metabolic activity, and osteogenic matrix protein production, in which 750 mum pore-size scaffolds showed superiority. Further experimentation using human mesenchymal stem cells on 750 mum pore scaffolds showed their ability in supporting osteogenic differentiation. These findings suggest that even in the absence of any surface modifications, low-cost 750 mum pore-size 3D-printed scaffolds may be suitable as a bone substitute for repair of large bone defects.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"ahangar-cooke-weber-rosenzweig-currentbiomedicalapplicationsof3dprintingandadditivemanufacturing-2019\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Current biomedical applications of 3D printing and additive manufacturing.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Ahangar, P.; Cooke, M. E.; Weber, M. H.;  and <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>\n</span>\n\n  \n\n</span>\n\n  <i>Applied Sciences (Switzerland)</i>, 9(8).  2019.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.3390/app9081713\"\n     onclick=\"log_download('ahangar-cooke-weber-rosenzweig-currentbiomedicalapplicationsof3dprintingandadditivemanufacturing-2019', 'http://doi.org/10.3390/app9081713', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/ahangar-cooke-weber-rosenzweig-currentbiomedicalapplicationsof3dprintingandadditivemanufacturing-2019\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('ahangar-cooke-weber-rosenzweig-currentbiomedicalapplicationsof3dprintingandadditivemanufacturing-2019')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Ahangar2019,\n  abstract = {Additive manufacturing (AM) has emerged over the past four decades as a cost-effective, on-demand modality for fabrication of geometrically complex objects. The ability to design and print virtually any object shape using a diverse array of materials, such as metals, polymers, ceramics and bioinks, has allowed for the adoption of this technology for biomedical applications in both research and clinical settings. Current advancements in tissue engineering and regeneration, therapeutic delivery, medical device fabrication and operative management planning ensure that AM will continue to play an increasingly important role in the future of healthcare. In this review, we outline current biomedical applications of common AM techniques and materials.},\n  annote = {cited By 11},\n  author = {Ahangar, Pouyan and Cooke, Megan E. and Weber, Michael H. and Rosenzweig, Derek H.},\n  doi = {10.3390/app9081713},\n  issn = {20763417},\n  journal = {Applied Sciences (Switzerland)},\n  keywords = {3D printing,Additive manufacturing,Biomedical devices,Bioprinting,Rapid prototyping,Regenerative medicine,Tissue engineering},\n  number = {8},\n  title = {{Current biomedical applications of 3D printing and additive manufacturing}},\n  volume = {9},\n  year = {2019}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Additive manufacturing (AM) has emerged over the past four decades as a cost-effective, on-demand modality for fabrication of geometrically complex objects. The ability to design and print virtually any object shape using a diverse array of materials, such as metals, polymers, ceramics and bioinks, has allowed for the adoption of this technology for biomedical applications in both research and clinical settings. Current advancements in tissue engineering and regeneration, therapeutic delivery, medical device fabrication and operative management planning ensure that AM will continue to play an increasingly important role in the future of healthcare. In this review, we outline current biomedical applications of common AM techniques and materials.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"akoury-ahangar-nour-lapointe-guerard-haglund-rosenzweig-weber-lowdosezoledronateforthetreatmentofbonemetastasissecondarytoprostatecancer-2019\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Low-dose zoledronate for the treatment of bone metastasis secondary to prostate cancer.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Akoury, E.; Ahangar, P.; Nour, A.; Lapointe, J.; Gu'erard, K. P.; Haglund, L.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>;  and Weber, M. H.\n</span>\n\n  \n\n</span>\n\n  <i>Cancer Cell International</i>, 19(1).  2019.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1186/s12935-019-0745-x\"\n     onclick=\"log_download('akoury-ahangar-nour-lapointe-guerard-haglund-rosenzweig-weber-lowdosezoledronateforthetreatmentofbonemetastasissecondarytoprostatecancer-2019', 'http://doi.org/10.1186/s12935-019-0745-x', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/akoury-ahangar-nour-lapointe-guerard-haglund-rosenzweig-weber-lowdosezoledronateforthetreatmentofbonemetastasissecondarytoprostatecancer-2019\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('akoury-ahangar-nour-lapointe-guerard-haglund-rosenzweig-weber-lowdosezoledronateforthetreatmentofbonemetastasissecondarytoprostatecancer-2019')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Akoury2019,\n  abstract = {Background: Bisphosphonates (BPs) including zoledronate (zol) have become standard care for bone metastases as they effectively inhibit tumor-induced osteolysis and associated pain. Several studies have also suggested that zol has direct anti-tumor activity. Systemic administration at high doses is the current approach to deliver zol, yet it has been associated with debilitating side effects. Local therapeutic delivery offers the ability to administer much lower total dosage, while at the same time maintaining sustained high-local drug concentration directly at the target treatment site. Here, we aimed to assess effects of lower doses of zol on bone metastases over a longer time. Methods: Prostate cancer cell line LAPC4 and prostate-induced bone metastasis cells were treated with zol at 1, 3 and 10 muM for 7 days. Following treatment, cell proliferation was assessed using Almarblue{(R)}, Vybrant MTT{(R)}, and Live/Dead{(R)} viability/cytotoxicity assays. Additionally, cell migration and invasion were carried out using Falcon™ cell culture inserts and Cultrex{(R)} 3D spheroid cell invasion assays respectively. Results: We show that treatment with 3-10 muM zol over 7-days significantly decreased cell proliferation in both the prostate cancer cell line LAPC4 and cells from spine metastases secondary to prostate cancer. Using the same low-dose and longer time course for treatment, we demonstrate that 10 muM zol also significantly inhibits tumor cell migration and 3D-cell growth/invasion. Conclusions: This project harnesses the potential of using zol at low doses for longer treatment periods, which may be a viable treatment modality when coupled with biomaterials or biodevices for local delivery.},\n  annote = {cited By 1},\n  author = {Akoury, Elie and Ahangar, Pouyan and Nour, Antone and Lapointe, Jacques and Gu{&#x27;{e}}rard, Karl Philippe and Haglund, Lisbet and Rosenzweig, Derek H. and Weber, Michael H.},\n  doi = {10.1186/s12935-019-0745-x},\n  issn = {14752867},\n  journal = {Cancer Cell International},\n  keywords = {Bone metastases secondary to prostate,Cellular assays,Direct in vitro treatment,Low doses,Zoledronate},\n  number = {1},\n  title = {{Low-dose zoledronate for the treatment of bone metastasis secondary to prostate cancer}},\n  volume = {19},\n  year = {2019}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Background: Bisphosphonates (BPs) including zoledronate (zol) have become standard care for bone metastases as they effectively inhibit tumor-induced osteolysis and associated pain. Several studies have also suggested that zol has direct anti-tumor activity. Systemic administration at high doses is the current approach to deliver zol, yet it has been associated with debilitating side effects. Local therapeutic delivery offers the ability to administer much lower total dosage, while at the same time maintaining sustained high-local drug concentration directly at the target treatment site. Here, we aimed to assess effects of lower doses of zol on bone metastases over a longer time. Methods: Prostate cancer cell line LAPC4 and prostate-induced bone metastasis cells were treated with zol at 1, 3 and 10 muM for 7 days. Following treatment, cell proliferation was assessed using Almarblue(R), Vybrant MTT(R), and Live/Dead(R) viability/cytotoxicity assays. Additionally, cell migration and invasion were carried out using Falcon™ cell culture inserts and Cultrex(R) 3D spheroid cell invasion assays respectively. Results: We show that treatment with 3-10 muM zol over 7-days significantly decreased cell proliferation in both the prostate cancer cell line LAPC4 and cells from spine metastases secondary to prostate cancer. Using the same low-dose and longer time course for treatment, we demonstrate that 10 muM zol also significantly inhibits tumor cell migration and 3D-cell growth/invasion. Conclusions: This project harnesses the potential of using zol at low doses for longer treatment periods, which may be a viable treatment modality when coupled with biomaterials or biodevices for local delivery.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"akoury-weber-rosenzweig-3dprintednanoporousscaffoldsimpregnatedwithzoledronateforthetreatmentofspinalbonemetastases-2019\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  3D-Printed Nanoporous Scaffolds Impregnated with Zoledronate for the Treatment of Spinal Bone Metastases.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Akoury, E.; Weber, M. H.;  and <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>\n</span>\n\n  \n\n</span>\n\n   2019.<!-- NOTE FROM BIBBASE: This entry has an invalid bibtex entry type: conference. Therefore, we don't know how to render it properly. Please consider fixing this. -->\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1557/adv.2019.156\"\n     onclick=\"log_download('akoury-weber-rosenzweig-3dprintednanoporousscaffoldsimpregnatedwithzoledronateforthetreatmentofspinalbonemetastases-2019', 'http://doi.org/10.1557/adv.2019.156', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/akoury-weber-rosenzweig-3dprintednanoporousscaffoldsimpregnatedwithzoledronateforthetreatmentofspinalbonemetastases-2019\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('akoury-weber-rosenzweig-3dprintednanoporousscaffoldsimpregnatedwithzoledronateforthetreatmentofspinalbonemetastases-2019')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@conference{Akoury20191245,\n  abstract = {Zoledronate (Zol) is a bone-preserving/ anti-Tumoral drug that is widely used for the treatment of many cancers including spinal bone metastases. High systemic Zol doses required to elicit an adequate effect in the spine often lead to significant side effects, limiting its prolonged use and effectiveness. Here, we aim to provide an alternative strategy to locally deliver Zol at the tumor site. We show that nanoporous 3D-printed scaffolds can be loaded with Zol and possess the ability to release Zol (10-28%) over a sustained period. Additionally, we demonstrate that Zol-impregnated scaffolds, mostly Gel Lay, impair the proliferation of the prostate cancer cell line LAPC4 and the prostate-induced bone metastasis cells in vitro. 3D-printed nanoporous polymers offer a novel and versatile opportunity for potential local delivery of drugs in future clinical settings. These polymers can decrease systemic exposure and related side effects of Zol while at the same time concentrating the drug effect at the tumor site thereby inhibiting tumor proliferation. Also, these scaffolds could be co-printed or coupled with other materials to produce custom implants that offer better structural support for bone growth at the tumor site following resection.},\n  annote = {cited By 1},\n  author = {Akoury, Elie and Weber, Michael H. and Rosenzweig, Derek H.},\n  booktitle = {MRS Advances},\n  doi = {10.1557/adv.2019.156},\n  issn = {20598521},\n  keywords = {3D printing,nanostructure},\n  number = {21},\n  pages = {1245--1251},\n  title = {{3D-Printed Nanoporous Scaffolds Impregnated with Zoledronate for the Treatment of Spinal Bone Metastases}},\n  volume = {4},\n  year = {2019}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Zoledronate (Zol) is a bone-preserving/ anti-Tumoral drug that is widely used for the treatment of many cancers including spinal bone metastases. High systemic Zol doses required to elicit an adequate effect in the spine often lead to significant side effects, limiting its prolonged use and effectiveness. Here, we aim to provide an alternative strategy to locally deliver Zol at the tumor site. We show that nanoporous 3D-printed scaffolds can be loaded with Zol and possess the ability to release Zol (10-28%) over a sustained period. Additionally, we demonstrate that Zol-impregnated scaffolds, mostly Gel Lay, impair the proliferation of the prostate cancer cell line LAPC4 and the prostate-induced bone metastasis cells in vitro. 3D-printed nanoporous polymers offer a novel and versatile opportunity for potential local delivery of drugs in future clinical settings. These polymers can decrease systemic exposure and related side effects of Zol while at the same time concentrating the drug effect at the tumor site thereby inhibiting tumor proliferation. Also, these scaffolds could be co-printed or coupled with other materials to produce custom implants that offer better structural support for bone growth at the tumor site following resection.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2018\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2018')\"\n      onkeypress=\"toggleGroup('group_2018')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2018</span>\n      <span class=\"bibbase_group_count\">\n        \n        (3)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"ahangar-akoury-luna-nour-weber-rosenzweig-nanoporous3dprintedscaffoldsforlocaldoxorubicindeliveryinbonemetastasessecondarytoprostatecancer-2018\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Nanoporous 3D-printed scaffolds for local doxorubicin delivery in bone metastases secondary to prostate cancer.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Ahangar, P.; Akoury, E.; Luna, A. S. R. G.; Nour, A.; Weber, M. H.;  and <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>\n</span>\n\n  \n\n</span>\n\n  <i>Materials</i>, 11(9).  2018.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.3390/ma11091485\"\n     onclick=\"log_download('ahangar-akoury-luna-nour-weber-rosenzweig-nanoporous3dprintedscaffoldsforlocaldoxorubicindeliveryinbonemetastasessecondarytoprostatecancer-2018', 'http://doi.org/10.3390/ma11091485', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/ahangar-akoury-luna-nour-weber-rosenzweig-nanoporous3dprintedscaffoldsforlocaldoxorubicindeliveryinbonemetastasessecondarytoprostatecancer-2018\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n  &nbsp;\n  <span class=\"bibbase_stats_paper\" style=\"color: #777;\">\n    <span>1 download</span>\n  </span>\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('ahangar-akoury-luna-nour-weber-rosenzweig-nanoporous3dprintedscaffoldsforlocaldoxorubicindeliveryinbonemetastasessecondarytoprostatecancer-2018')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Ahangar2018,\n  abstract = {The spine is the most common site of bone metastasis, often originating from prostate, lung, and breast cancers. High systemic doses of chemotherapeutics such as doxorubicin (DOX), cisplatin, or paclitaxel often have severe side effects. Surgical removal of spine metastases also leaves large defects which cannot spontaneously heal and require bone grafting. To circumvent these issues, we designed an approach for local chemotherapeutic delivery within 3D-printed scaffolds which could also potentially serve as a bone substitute. Direct treatment of prostate cancer cell line LAPC4 and patient derived spine metastases cells with 0.01 muM DOX significantly reduced metabolic activity, proliferation, migration, and spheroid growth. We then assessed uptake and release of DOX in a series of porous 3D-printed scaffolds on LAPC4 cells as well as patient-derived spine metastases cells. Over seven days, 60-75% of DOX loaded onto scaffolds could be released, which significantly reduced metabolic activity and proliferation of both LAPC4 and patient derived cells, while unloaded scaffolds had no effect. Porous 3D-printed scaffolds may provide a novel and inexpensive approach to locally deliver chemotherapeutics in a patient-specific manner at tumor resection sites. With a composite design to enhance strength and promote sustained drug release, the scaffolds could reduce systemic negative effects, enhance bone repair, and improve patient outcomes.},\n  annote = {cited By 5},\n  author = {Ahangar, Pouyan and Akoury, Elie and Luna, Ana Sofia Ramirez Garcia and Nour, Antone and Weber, Michael H. and Rosenzweig, Derek H.},\n  doi = {10.3390/ma11091485},\n  issn = {19961944},\n  journal = {Materials},\n  keywords = {Bone metastases,Bone substitute,Doxorubicin,Local delivery,Low-cost 3D printing,Nanoporous filament,Prostate cancer},\n  number = {9},\n  title = {{Nanoporous 3D-printed scaffolds for local doxorubicin delivery in bone metastases secondary to prostate cancer}},\n  volume = {11},\n  year = {2018}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  The spine is the most common site of bone metastasis, often originating from prostate, lung, and breast cancers. High systemic doses of chemotherapeutics such as doxorubicin (DOX), cisplatin, or paclitaxel often have severe side effects. Surgical removal of spine metastases also leaves large defects which cannot spontaneously heal and require bone grafting. To circumvent these issues, we designed an approach for local chemotherapeutic delivery within 3D-printed scaffolds which could also potentially serve as a bone substitute. Direct treatment of prostate cancer cell line LAPC4 and patient derived spine metastases cells with 0.01 muM DOX significantly reduced metabolic activity, proliferation, migration, and spheroid growth. We then assessed uptake and release of DOX in a series of porous 3D-printed scaffolds on LAPC4 cells as well as patient-derived spine metastases cells. Over seven days, 60-75% of DOX loaded onto scaffolds could be released, which significantly reduced metabolic activity and proliferation of both LAPC4 and patient derived cells, while unloaded scaffolds had no effect. Porous 3D-printed scaffolds may provide a novel and inexpensive approach to locally deliver chemotherapeutics in a patient-specific manner at tumor resection sites. With a composite design to enhance strength and promote sustained drug release, the scaffolds could reduce systemic negative effects, enhance bone repair, and improve patient outcomes.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"gutman-rosenzweig-golan-surgicaltreatmentofcervicalradiculopathy-2018\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Surgical Treatment of Cervical Radiculopathy.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Gutman, G.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>;  and Golan, J. D.\n</span>\n\n  \n\n</span>\n\n  <i>Spine</i>, 43(6): E365–E372.  2018.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1097/BRS.0000000000002324\"\n     onclick=\"log_download('gutman-rosenzweig-golan-surgicaltreatmentofcervicalradiculopathy-2018', 'http://doi.org/10.1097/BRS.0000000000002324', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/gutman-rosenzweig-golan-surgicaltreatmentofcervicalradiculopathy-2018\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('gutman-rosenzweig-golan-surgicaltreatmentofcervicalradiculopathy-2018')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Gutman2018E365,\n  abstract = {Study Design.Meta-analysis of published randomized controlled trials (RCTs).Objective.To determine whether anterior cervical discectomy and fusion (ACDF), cervical disc replacement (CDR), or minimally invasive posterior cervical foraminotomy (MI-PCF) provides the best outcomes for patients with symptomatic single-level, single-side, and cervical radiculopathy.Summary of Background Data.The surgical treatment of cervical radiculopathy is still controversial. ACDF has been widely used as a &quot;gold standard.&quot; CDR has evolved and become a motion-preserving alternative with a potentially lower incidence of adjacent segment disease. However, both techniques require anterior neck dissection that carries a potential for serious morbidity. MI-PCF is a motion-preserving technique that can be performed with minimal invasiveness but has not gained universal acceptance.Methods.Electronic database search for RCTs comparing the efficacy and effectiveness of ACDF, CDR, and MI-PCF was performed. Meta-analysis was done for secondary surgical procedures and adverse events.Results.A total of 358 studies were retrieved, of which four RCT reports met the inclusion criteria for this study. Three studies present clinical data comparing ACDF and CDR, and one study presents data comparing ACDF and MI-PCF. Available data from the RCTs analyzed concluded that ACDF, CDR, and MI-PCF result in significant improvements in relevant symptoms, clinical, and functional outcomes in patients with single-level, single side cervical radiculopathy refractory to nonoperative treatment. CDR had the lowest percentage of secondary surgical procedures (P=0.0178) whereas MICPF had the lowest percentage of adverse events (P&lt;0.0001).Conclusion.All three techniques are effective in treating cervical radicular symptoms. MI-PCF has the lowest rate of adverse events whereas CDR has the lowest rate of secondary procedures. There is insufficient evidence to show which technique is the most effective and provides the longest-lasting symptom relief.Level of Evidence.},\n  annote = {cited By 14},\n  author = {Gutman, Gabriel and Rosenzweig, Derek H. and Golan, Jeff D.},\n  doi = {10.1097/BRS.0000000000002324},\n  issn = {15281159},\n  journal = {Spine},\n  keywords = {anterior cervical discectomy and fusion,cervical disc arthroplasty,cervical radiculopathy,minimally invasive posterior cervical foraminotomy,spine surgery},\n  number = {6},\n  pages = {E365--E372},\n  title = {{Surgical Treatment of Cervical Radiculopathy}},\n  volume = {43},\n  year = {2018}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Study Design.Meta-analysis of published randomized controlled trials (RCTs).Objective.To determine whether anterior cervical discectomy and fusion (ACDF), cervical disc replacement (CDR), or minimally invasive posterior cervical foraminotomy (MI-PCF) provides the best outcomes for patients with symptomatic single-level, single-side, and cervical radiculopathy.Summary of Background Data.The surgical treatment of cervical radiculopathy is still controversial. ACDF has been widely used as a \"gold standard.\" CDR has evolved and become a motion-preserving alternative with a potentially lower incidence of adjacent segment disease. However, both techniques require anterior neck dissection that carries a potential for serious morbidity. MI-PCF is a motion-preserving technique that can be performed with minimal invasiveness but has not gained universal acceptance.Methods.Electronic database search for RCTs comparing the efficacy and effectiveness of ACDF, CDR, and MI-PCF was performed. Meta-analysis was done for secondary surgical procedures and adverse events.Results.A total of 358 studies were retrieved, of which four RCT reports met the inclusion criteria for this study. Three studies present clinical data comparing ACDF and CDR, and one study presents data comparing ACDF and MI-PCF. Available data from the RCTs analyzed concluded that ACDF, CDR, and MI-PCF result in significant improvements in relevant symptoms, clinical, and functional outcomes in patients with single-level, single side cervical radiculopathy refractory to nonoperative treatment. CDR had the lowest percentage of secondary surgical procedures (P=0.0178) whereas MICPF had the lowest percentage of adverse events (P<0.0001).Conclusion.All three techniques are effective in treating cervical radicular symptoms. MI-PCF has the lowest rate of adverse events whereas CDR has the lowest rate of secondary procedures. There is insufficient evidence to show which technique is the most effective and provides the longest-lasting symptom relief.Level of Evidence.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"rosenzweig-fairag-mathieu-li-eglin-deste-steffen-weber-etal-thermoreversiblehyaluronanhydrogelandautologousnucleuspulposuscelldeliveryregenerateshumanintervertebraldiscsinanexvivophysiologicalorganculturemodel-2018\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Thermoreversi ble hyaluronan-hydrogel and autologous nucleus pulposus cell deli very regenerates human i ntervertebral di scs i n an ex vi vo, physi ologi cal organ culture model.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Fairag, R.; Mathieu, A. P.; Li, L.; Eglin, D.; D'este, M.; Steffen, T.; Weber, M. H.; Ouellet, J. A.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>European Cells and Materials</i>, 36: 200–217.  2018.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.22203/eCM.v036a15\"\n     onclick=\"log_download('rosenzweig-fairag-mathieu-li-eglin-deste-steffen-weber-etal-thermoreversiblehyaluronanhydrogelandautologousnucleuspulposuscelldeliveryregenerateshumanintervertebraldiscsinanexvivophysiologicalorganculturemodel-2018', 'http://doi.org/10.22203/eCM.v036a15', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-fairag-mathieu-li-eglin-deste-steffen-weber-etal-thermoreversiblehyaluronanhydrogelandautologousnucleuspulposuscelldeliveryregenerateshumanintervertebraldiscsinanexvivophysiologicalorganculturemodel-2018\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-fairag-mathieu-li-eglin-deste-steffen-weber-etal-thermoreversiblehyaluronanhydrogelandautologousnucleuspulposuscelldeliveryregenerateshumanintervertebraldiscsinanexvivophysiologicalorganculturemodel-2018')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig2018200,\n  abstract = {Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1rho magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1rho values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture setup, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.},\n  annote = {cited By 4},\n  author = {Rosenzweig, D. H. and Fairag, R. and Mathieu, A. P. and Li, L. and Eglin, D. and D&#x27;este, M. and Steffen, T. and Weber, M. H. and Ouellet, J. A. and Haglund, Lisbet},\n  doi = {10.22203/eCM.v036a15},\n  issn = {14732262},\n  journal = {European Cells and Materials},\n  keywords = {Autologous cell implantation,Bioreactors,Human intervertebral disc,Hydrogel,Nucleus pulposus,T1rho magnetic resonance imaging,Tissue engineering},\n  pages = {200--217},\n  pmid = {30370912},\n  title = {{Thermoreversi ble hyaluronan-hydrogel and autologous nucleus pulposus cell deli very regenerates human i ntervertebral di scs i n an ex vi vo, physi ologi cal organ culture model}},\n  volume = {36},\n  year = {2018}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Numerous studies show promise for cell-based tissue engineering strategies aiming to repair painful intervertebral disc (IVD) degeneration. However, clinical translation to human IVD repair is slow. In the present study, the regenerative potential of an autologous nucleus pulposus (NP)-cell-seeded thermoresponsive hyaluronic acid hydrogel in human lumbar IVDs was assessed under physiological conditions. First, agarose-encased in vitro constructs were developed, showing greater than 90 % NP cell viability and high proteoglycan deposition within HA-pNIPAM hydrogels following 3 weeks of dynamic loading. Second, a bovine-induced IVD degeneration model was used to optimise and validate T1rho magnetic resonance imaging (MRI) for detection of changes in proteoglycan content in isolated intact IVDs. Finally, isolated intact human lumbar IVDs were pre-scanned using the established MRI sequence. Then, IVDs were injected with HA-pNIPAM hydrogel alone or autologous NP-cell-seeded. Next, the treated IVDs were cultured under cyclic dynamic loading for 5 weeks. Post-treatment T1rho values were significantly higher as compared to pre-treatment scans within the same IVD and region of interest. Histological evaluation of treated human IVDs showed that the implanted hydrogel alone accumulated proteoglycans, while those that contained NP cells also displayed neo-matrix-surrounded cells within the gel. The study indicated a clinical potential for repairing early degenerative human IVDs using autologous cells/hydrogel suspensions. This unique IVD culture setup, combined with the long-term physiological culture of intact human IVDs, allowed for a more clinically relevant evaluation of human tissue repair and regeneration, which otherwise could not be replicated using the available in vitro and in vivo models.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2017\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2017')\"\n      onkeypress=\"toggleGroup('group_2017')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2017</span>\n      <span class=\"bibbase_group_count\">\n        \n        (3)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"krock-rosenzweig-currie-bisson-ouellet-haglund-tolllikereceptoractivationinducesdegenerationofhumanintervertebraldiscs-2017\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Toll-like receptor activation induces degeneration of human intervertebral discs.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Krock, E.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Currie, J. B.; Bisson, D. G.; Ouellet, J. A.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>Scientific Reports</i>, 7(1).  2017.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1038/s41598-017-17472-1\"\n     onclick=\"log_download('krock-rosenzweig-currie-bisson-ouellet-haglund-tolllikereceptoractivationinducesdegenerationofhumanintervertebraldiscs-2017', 'http://doi.org/10.1038/s41598-017-17472-1', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/krock-rosenzweig-currie-bisson-ouellet-haglund-tolllikereceptoractivationinducesdegenerationofhumanintervertebraldiscs-2017\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('krock-rosenzweig-currie-bisson-ouellet-haglund-tolllikereceptoractivationinducesdegenerationofhumanintervertebraldiscs-2017')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Krock2017,\n  abstract = {Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1beta and nerve growth factor gene expression following treatment with Pam2CSK4 (TLR2/6 agonist) but not Pam3CSK4 (TLR1/2 agonist). Challenging NP cells with Pam2CSK4 or 30 kDa fibronectin fragments (FN-f, an endogenous TLR2 and TLR4 alarmin) increased secretion of proinflammatory cytokines. We then investigated the effect of TLR activation in intact, non-degenerate, ex vivo human discs. Discs were injected with PBS, Pam2CSK4 and FN-f, and cultured for 28 days. TLR activation increased proteoglycan and ECM protein release into the culture media and decreased proteoglycan content in the NP. Proteases, including MMP3, 13 and HTRA1, are secreted at higher levels following TLR activation. In addition, proinflammatory cytokine levels, including IL-6, TNFalpha and IFNgamma, increased following TLR activation. These results indicate that TLR activation induces degeneration in human discs. Therefore, TLRs are potential disease-modifying therapeutic targets to slow disc degeneration.},\n  annote = {cited By 7},\n  author = {Krock, Emerson and Rosenzweig, Derek H. and Currie, J. Brooke and Bisson, Daniel G. and Ouellet, Jean A. and Haglund, Lisbet},\n  doi = {10.1038/s41598-017-17472-1},\n  issn = {20452322},\n  journal = {Scientific Reports},\n  number = {1},\n  title = {{Toll-like receptor activation induces degeneration of human intervertebral discs}},\n  volume = {7},\n  year = {2017}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Toll-like receptors (TLR) are activated by endogenous alarmins such as fragmented extracellular matrix compounds found in the degenerating disc. TLRs regulate cytokine, neurotrophin, and protease expression in human disc cells in vitro, and thus control key factors in disc degeneration. However, whether TLR activation leads to degenerative changes in intact human discs is unclear. Nucleus pulposus (NP) cells isolated from non-degenerating discs increase IL-1beta and nerve growth factor gene expression following treatment with Pam2CSK4 (TLR2/6 agonist) but not Pam3CSK4 (TLR1/2 agonist). Challenging NP cells with Pam2CSK4 or 30 kDa fibronectin fragments (FN-f, an endogenous TLR2 and TLR4 alarmin) increased secretion of proinflammatory cytokines. We then investigated the effect of TLR activation in intact, non-degenerate, ex vivo human discs. Discs were injected with PBS, Pam2CSK4 and FN-f, and cultured for 28 days. TLR activation increased proteoglycan and ECM protein release into the culture media and decreased proteoglycan content in the NP. Proteases, including MMP3, 13 and HTRA1, are secreted at higher levels following TLR activation. In addition, proinflammatory cytokine levels, including IL-6, TNFalpha and IFNgamma, increased following TLR activation. These results indicate that TLR activation induces degeneration in human discs. Therefore, TLRs are potential disease-modifying therapeutic targets to slow disc degeneration.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"nooh-zhang-sato-rosenzweig-tabaries-siegel-barralet-weber-intratumordeliveryofzoledronatemitigatesmetastasisinducedosteolysissuperiortosystemicadministration-2017\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Intra-tumor delivery of zoledronate mitigates metastasis-induced osteolysis superior to systemic administration.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Nooh, A.; Zhang, Y. L.; Sato, D.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Tabari`es, S.; Siegel, P.; Barralet, J. E.;  and Weber, M. H.\n</span>\n\n  \n\n</span>\n\n  <i>Journal of Bone Oncology</i>, 6: 8–15.  2017.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1016/j.jbo.2017.01.001\"\n     onclick=\"log_download('nooh-zhang-sato-rosenzweig-tabaries-siegel-barralet-weber-intratumordeliveryofzoledronatemitigatesmetastasisinducedosteolysissuperiortosystemicadministration-2017', 'http://doi.org/10.1016/j.jbo.2017.01.001', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/nooh-zhang-sato-rosenzweig-tabaries-siegel-barralet-weber-intratumordeliveryofzoledronatemitigatesmetastasisinducedosteolysissuperiortosystemicadministration-2017\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('nooh-zhang-sato-rosenzweig-tabaries-siegel-barralet-weber-intratumordeliveryofzoledronatemitigatesmetastasisinducedosteolysissuperiortosystemicadministration-2017')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Nooh20178,\n  abstract = {Bisphosphonates (BPs) have recently been shown to have direct anti-tumor properties. Systemic treatment with BPs can have multiple adverse effects such as osteonecrosis of the jaw and BP induced bone fracturing and spine instability. While benefits of systemic BP treatments may outweigh risks, local treatment with BPs has been explored as an alternate strategy to reduce unwarranted risk. In the present study, we examined whether local delivery of BPs inhibits tumor-induced osteolysis and tumor growth more effectively than systemic treatment in an animal model of tumor-induced bone disease. Following establishment of an intra-tibial model of bone metastases in athymic mice, the experimental group was treated by local administration of zoledronate into the tibial lesion. A comparison of the effect of local versus systemic delivery of zoledronate on the formation of tumor-induced osteolysis was also carried out. A significant increase in mean bone volume/tissue volume % (BV/TV) of the locally treated group (12.30±2.80%) compared to the control group (7.13±1.22%) (P&lt;0.001). Additionally, there was a significant increase in the BV/TV (10.90±1.25%) in the locally treated group compared to the systemically treated group (7.53±0.75%) (P=0.005). These preliminary results suggest that local delivery of BPs outperforms both systemic and control treatments to inhibit tumor-induced osteolysis.},\n  annote = {cited By 3},\n  author = {Nooh, Anas and Zhang, Yu Ling and Sato, Daisuke and Rosenzweig, Derek H. and Tabari{&#x60;{e}}s, S{&#x27;{e}}bastien and Siegel, Peter and Barralet, Jake E. and Weber, Michael H.},\n  doi = {10.1016/j.jbo.2017.01.001},\n  issn = {22121374},\n  journal = {Journal of Bone Oncology},\n  keywords = {Bisphosphonates,Bone metastases,Cancer pain,Local treatment,Osteolysis,Xenograft},\n  pages = {8--15},\n  title = {{Intra-tumor delivery of zoledronate mitigates metastasis-induced osteolysis superior to systemic administration}},\n  volume = {6},\n  year = {2017}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Bisphosphonates (BPs) have recently been shown to have direct anti-tumor properties. Systemic treatment with BPs can have multiple adverse effects such as osteonecrosis of the jaw and BP induced bone fracturing and spine instability. While benefits of systemic BP treatments may outweigh risks, local treatment with BPs has been explored as an alternate strategy to reduce unwarranted risk. In the present study, we examined whether local delivery of BPs inhibits tumor-induced osteolysis and tumor growth more effectively than systemic treatment in an animal model of tumor-induced bone disease. Following establishment of an intra-tibial model of bone metastases in athymic mice, the experimental group was treated by local administration of zoledronate into the tibial lesion. A comparison of the effect of local versus systemic delivery of zoledronate on the formation of tumor-induced osteolysis was also carried out. A significant increase in mean bone volume/tissue volume % (BV/TV) of the locally treated group (12.30±2.80%) compared to the control group (7.13±1.22%) (P<0.001). Additionally, there was a significant increase in the BV/TV (10.90±1.25%) in the locally treated group compared to the systemically treated group (7.53±0.75%) (P=0.005). These preliminary results suggest that local delivery of BPs outperforms both systemic and control treatments to inhibit tumor-induced osteolysis.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"rosenzweig-tremblaygravel-bisson-ouellet-weber-haglund-comparativeanalysisincontinuousexpansionofbovineandhumanprimarynucleuspulposuscellsfortissuerepairapplications-2017\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Tremblay Gravel, J.; Bisson, D.; Ouellet, J. A.; Weber, M. H.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>European Cells and Materials</i>, 33: 240–251.  2017.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.22203/eCM.v033a18\"\n     onclick=\"log_download('rosenzweig-tremblaygravel-bisson-ouellet-weber-haglund-comparativeanalysisincontinuousexpansionofbovineandhumanprimarynucleuspulposuscellsfortissuerepairapplications-2017', 'http://doi.org/10.22203/eCM.v033a18', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-tremblaygravel-bisson-ouellet-weber-haglund-comparativeanalysisincontinuousexpansionofbovineandhumanprimarynucleuspulposuscellsfortissuerepairapplications-2017\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-tremblaygravel-bisson-ouellet-weber-haglund-comparativeanalysisincontinuousexpansionofbovineandhumanprimarynucleuspulposuscellsfortissuerepairapplications-2017')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig2017240,\n  abstract = {Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD) degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP) phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD) gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.},\n  annote = {cited By 7},\n  author = {Rosenzweig, D. H. and {Tremblay Gravel}, J. and Bisson, D. and Ouellet, J. A. and Weber, M. H. and Haglund, Lisbet},\n  doi = {10.22203/eCM.v033a18},\n  issn = {14732262},\n  journal = {European Cells and Materials},\n  keywords = {Cell culture,Elastic culture surfaces,Intervertebral disc,Nucleus pulposus cells,Tissue engineering},\n  pages = {240--251},\n  title = {{Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications}},\n  volume = {33},\n  year = {2017}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD) degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP) phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD) gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells – isolated from acute herniation tissue – produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2016\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2016')\"\n      onkeypress=\"toggleGroup('group_2016')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2016</span>\n      <span class=\"bibbase_group_count\">\n        \n        (3)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"krock-currie-weber-ouellet-stone-rosenzweig-haglund-nervegrowthfactorisregulatedbytolllikereceptor2inhumanintervertebraldiscs-2016\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Nerve growth factor is regulated by toll-like receptor 2 in human intervertebral discs.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Krock, E.; Currie, J. B.; Weber, M. H.; Ouellet, J. A.; Stone, L. S.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>Journal of Biological Chemistry</i>, 291(7): 3541–3551.  2016.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1074/jbc.M115.675900\"\n     onclick=\"log_download('krock-currie-weber-ouellet-stone-rosenzweig-haglund-nervegrowthfactorisregulatedbytolllikereceptor2inhumanintervertebraldiscs-2016', 'http://doi.org/10.1074/jbc.M115.675900', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/krock-currie-weber-ouellet-stone-rosenzweig-haglund-nervegrowthfactorisregulatedbytolllikereceptor2inhumanintervertebraldiscs-2016\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('krock-currie-weber-ouellet-stone-rosenzweig-haglund-nervegrowthfactorisregulatedbytolllikereceptor2inhumanintervertebraldiscs-2016')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Krock20163541,\n  abstract = {Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity but more recently were found to be activated by endogenous alarmins such as fragmented extracellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1beta (control) treatment increased NGF, brain-derived neurotrophic factor (BDNF), and IL-1beta gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1beta treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2, and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-kappaB, and to a lesser extent p38, but not ERK1/2 activity, blocked TLR2-driven NGF up-regulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-kappaB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-kappaB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.},\n  annote = {cited By 26},\n  author = {Krock, Emerson and Currie, J. Brooke and Weber, Michael H. and Ouellet, Jean A. and Stone, Laura S. and Rosenzweig, Derek H. and Haglund, Lisbet},\n  doi = {10.1074/jbc.M115.675900},\n  issn = {1083351X},\n  journal = {Journal of Biological Chemistry},\n  number = {7},\n  pages = {3541--3551},\n  title = {{Nerve growth factor is regulated by toll-like receptor 2 in human intervertebral discs}},\n  volume = {291},\n  year = {2016}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Nerve growth factor (NGF) contributes to the development of chronic pain associated with degenerative connective tissue pathologies, such as intervertebral disc degeneration and osteoarthritis. However, surprisingly little is known about the regulation of NGF in these conditions. Toll-like receptors (TLR) are pattern recognition receptors classically associated with innate immunity but more recently were found to be activated by endogenous alarmins such as fragmented extracellular matrix proteins found in degenerating discs or cartilage. In this study we investigated if TLR activation regulates NGF and which signaling mechanisms control this response in intervertebral discs. TLR2 agonists, TLR4 agonists, or IL-1beta (control) treatment increased NGF, brain-derived neurotrophic factor (BDNF), and IL-1beta gene expression in human disc cells isolated from healthy, pain-free organ donors. However, only TLR2 activation or IL-1beta treatment increased NGF protein secretion. TLR2 activation increased p38, ERK1/2, and p65 activity and increased p65 translocation to the cell nucleus. JNK activity was not affected by TLR2 activation. Inhibition of NF-kappaB, and to a lesser extent p38, but not ERK1/2 activity, blocked TLR2-driven NGF up-regulation at both the transcript and protein levels. These results provide a novel mechanism of NGF regulation in the intervertebral disc and potentially other pathogenic connective tissues. TLR2 and NF-kappaB signaling are known to increase cytokines and proteases, which accelerate matrix degradation. Therefore, TLR2 or NF-kappaB inhibition may both attenuate chronic pain and slow the degenerative progress in vivo.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"jalani-naccache-rosenzweig-haglund-vetrone-cerruti-photocleavablehydrogelcoatedupconvertingnanoparticlesamultifunctionaltheranosticplatformfornirimagingandondemandmacromoleculardelivery-2016\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Jalani, G.; Naccache, R.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Haglund, L.; Vetrone, F.;  and Cerruti, M.\n</span>\n\n  \n\n</span>\n\n  <i>Journal of the American Chemical Society</i>, 138(3): 1078–1083.  2016.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1021/jacs.5b12357\"\n     onclick=\"log_download('jalani-naccache-rosenzweig-haglund-vetrone-cerruti-photocleavablehydrogelcoatedupconvertingnanoparticlesamultifunctionaltheranosticplatformfornirimagingandondemandmacromoleculardelivery-2016', 'http://doi.org/10.1021/jacs.5b12357', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/jalani-naccache-rosenzweig-haglund-vetrone-cerruti-photocleavablehydrogelcoatedupconvertingnanoparticlesamultifunctionaltheranosticplatformfornirimagingandondemandmacromoleculardelivery-2016\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('jalani-naccache-rosenzweig-haglund-vetrone-cerruti-photocleavablehydrogelcoatedupconvertingnanoparticlesamultifunctionaltheranosticplatformfornirimagingandondemandmacromoleculardelivery-2016')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Jalani20161078,\n  abstract = {Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent nanotransducers for converting longer wavelength near-infrared (NIR) light to shorter wavelengths spanning the ultraviolet (UV) to the visible (Vis) regions of the spectrum via a multiphoton absorption process, known as upconversion. Here, we report the development of NIR to UV-Vis-NIR UCNPs consisting of LiYF4:Yb3+/Tm3+@SiO2 individually coated with a 10 ± 2 nm layer of chitosan (CH) hydrogel cross-linked with a photocleavable cross-linker (PhL). We encapsulated fluorescent-bovine serum albumin (FITC-BSA) inside the gel. Under 980 nm excitation, the upconverted UV emission cleaves the PhL cross-links and instantaneously liberates the FITC-BSA under 2 cm thick tissue. The release is immediately arrested if the excitation source is switched off. The upconverted NIR light allows for the tracking of particles under the tissue. Nucleus pulposus (NP) cells cultured with UCNPs are viable both in the presence and in the absence of laser irradiation. Controlled drug delivery of large biomolecules and deep tissue imaging make this system an excellent theranostic platform for tissue engineering, biomapping, and cellular imaging applications.},\n  annote = {cited By 104},\n  author = {Jalani, Ghulam and Naccache, Rafik and Rosenzweig, Derek H. and Haglund, Lisbet and Vetrone, Fiorenzo and Cerruti, Marta},\n  doi = {10.1021/jacs.5b12357},\n  issn = {15205126},\n  journal = {Journal of the American Chemical Society},\n  number = {3},\n  pages = {1078--1083},\n  title = {{Photocleavable Hydrogel-Coated Upconverting Nanoparticles: A Multifunctional Theranostic Platform for NIR Imaging and On-Demand Macromolecular Delivery}},\n  volume = {138},\n  year = {2016}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Lanthanide-doped upconverting nanoparticles (UCNPs) have emerged as excellent nanotransducers for converting longer wavelength near-infrared (NIR) light to shorter wavelengths spanning the ultraviolet (UV) to the visible (Vis) regions of the spectrum via a multiphoton absorption process, known as upconversion. Here, we report the development of NIR to UV-Vis-NIR UCNPs consisting of LiYF4:Yb3+/Tm3+@SiO2 individually coated with a 10 ± 2 nm layer of chitosan (CH) hydrogel cross-linked with a photocleavable cross-linker (PhL). We encapsulated fluorescent-bovine serum albumin (FITC-BSA) inside the gel. Under 980 nm excitation, the upconverted UV emission cleaves the PhL cross-links and instantaneously liberates the FITC-BSA under 2 cm thick tissue. The release is immediately arrested if the excitation source is switched off. The upconverted NIR light allows for the tracking of particles under the tissue. Nucleus pulposus (NP) cells cultured with UCNPs are viable both in the presence and in the absence of laser irradiation. Controlled drug delivery of large biomolecules and deep tissue imaging make this system an excellent theranostic platform for tissue engineering, biomapping, and cellular imaging applications.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"rosenzweig-gawri-moir-beckman-eglin-steffen-roughley-ouellet-etal-dynamicloadingmatrixmaintenanceandcellinjectiontherapyofhumanintervertebraldiscsculturedinabioreactor-2016\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Gawri, R.; Moir, J.; Beckman, L.; Eglin, D.; Steffen, T.; Roughley, P. J.; Ouellet, J. A.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>European Cells and Materials</i>, 31: 26–39.  2016.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.22203/eCM.v031a03\"\n     onclick=\"log_download('rosenzweig-gawri-moir-beckman-eglin-steffen-roughley-ouellet-etal-dynamicloadingmatrixmaintenanceandcellinjectiontherapyofhumanintervertebraldiscsculturedinabioreactor-2016', 'http://doi.org/10.22203/eCM.v031a03', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-gawri-moir-beckman-eglin-steffen-roughley-ouellet-etal-dynamicloadingmatrixmaintenanceandcellinjectiontherapyofhumanintervertebraldiscsculturedinabioreactor-2016\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-gawri-moir-beckman-eglin-steffen-roughley-ouellet-etal-dynamicloadingmatrixmaintenanceandcellinjectiontherapyofhumanintervertebraldiscsculturedinabioreactor-2016')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig201626,\n  abstract = {Low back pain originating from intervertebral disc (IVD) degenerationaffectsthequalityoflifeformillionsof people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivomodels of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. Abioreactor wasdevelopedthat facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.6 and high 0.1-1.2 MPa. Cell viability was maintained &gt; 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility oftissuerepairstrategieswithcellsupplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.},\n  annote = {cited By 10},\n  author = {Rosenzweig, D. H. and Gawri, R. and Moir, J. and Beckman, L. and Eglin, D. and Steffen, T. and Roughley, P. J. and Ouellet, J. A. and Haglund, Lisbet},\n  doi = {10.22203/eCM.v031a03},\n  issn = {14732262},\n  journal = {European Cells and Materials},\n  keywords = {Bioreactor,Cell therapy,Dynamic loading,Hydrogel,Intervertebral discs,Organ culture,Tissue regeneration},\n  pages = {26--39},\n  pmid = {26728497},\n  title = {{Dynamic loading, matrix maintenance and cell injection therapy of human intervertebral discs cultured in a bioreactor}},\n  volume = {31},\n  year = {2016}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Low back pain originating from intervertebral disc (IVD) degenerationaffectsthequalityoflifeformillionsof people, and it is a major contributor to global healthcare costs. Long-term culture of intact IVDs is necessary to develop ex vivomodels of human IVD degeneration and repair, where the relationship between mechanobiology, disc matrix composition and metabolism can be better understood. Abioreactor wasdevelopedthat facilitates culture of intact human IVDs in a controlled, dynamically loaded environment. Tissue integrity and cell viability was evaluated under 3 different loading conditions: low 0.1-0.3, medium 0.1-0.6 and high 0.1-1.2 MPa. Cell viability was maintained > 80 % throughout the disc at low and medium loads, whereas it dropped to approximately 70 % (NP) and 50 % (AF) under high loads. Although cell viability was affected at high loads, there was no evidence of sGAG loss, changes in newly synthesised collagen type II or chondroadherin fragmentation. Sulphated GAG content remained at a stable level of approximately 50 µg sGAG/mg tissue in all loading protocols. To evaluate the feasibility oftissuerepairstrategieswithcellsupplementation, human NP cells were transplanted into discs within a thermoreversible hyaluronan hydrogel. The discs were loaded under medium loads, and the injected cells remained largely localised to the NP region. This study demonstrates the feasibility of culturing human IVDs for 14 days under cyclic dynamic loading conditions. The system allows the determination a safe range-of-loading and presents a platform to evaluate cell therapies and help to elucidate the effect of load following cell-based therapies.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2015\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2015')\"\n      onkeypress=\"toggleGroup('group_2015')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2015</span>\n      <span class=\"bibbase_group_count\">\n        \n        (4)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"jalani-naccache-rosenzweig-lerouge-haglund-vetrone-cerruti-realtimenoninvasivemonitoringofhydrogeldegradationusingliyf4yb3tm3nirtonirupconvertingnanoparticles-2015\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Real-time, non-invasive monitoring of hydrogel degradation using LiYF4:Yb3+/Tm3+ NIR-to-NIR upconverting nanoparticles.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Jalani, G.; Naccache, R.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Lerouge, S.; Haglund, L.; Vetrone, F.;  and Cerruti, M.\n</span>\n\n  \n\n</span>\n\n  <i>Nanoscale</i>, 7(26): 11255–11262.  2015.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1039/c5nr02482j\"\n     onclick=\"log_download('jalani-naccache-rosenzweig-lerouge-haglund-vetrone-cerruti-realtimenoninvasivemonitoringofhydrogeldegradationusingliyf4yb3tm3nirtonirupconvertingnanoparticles-2015', 'http://doi.org/10.1039/c5nr02482j', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/jalani-naccache-rosenzweig-lerouge-haglund-vetrone-cerruti-realtimenoninvasivemonitoringofhydrogeldegradationusingliyf4yb3tm3nirtonirupconvertingnanoparticles-2015\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('jalani-naccache-rosenzweig-lerouge-haglund-vetrone-cerruti-realtimenoninvasivemonitoringofhydrogeldegradationusingliyf4yb3tm3nirtonirupconvertingnanoparticles-2015')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Jalani201511255,\n  abstract = {To design a biodegradable hydrogel as cell support, one should know its in vivo degradation rate. A technique commonly used to track gel degradation is fluorescence spectroscopy. However, the fluorescence from conventional fluorophores quickly decays, and the fluorophores are often moderately cytotoxic. Most importantly, they require ultraviolet or visible (UV-Vis) light as the excitation source, which cannot penetrate deeply through biological tissues. Lanthanide-doped upconverting nanoparticles (UCNPs) are exciting alternatives to conventional fluorophores because they can convert near-infrared (NIR) to UV-Vis-NIR light via a sequential multiphoton absorption process referred to as upconversion. NIR light can penetrate up to few cm inside tissues, thus making these UCNPs much better probes than conventional fluorophores for in vivo monitoring. Also, UCNPs have narrow emission bands, high photoluminescence (PL) signal-to-noise ratio, low cytotoxicity and good physical and chemical stability. Here, we show a nanocomposite system consisting of a biodegradable, in situ thermogelling injectable hydrogel made of chitosan and hyaluronic acid encapsulating silica-coated LiYF4:Yb3+/Tm3+ UCNPs. We use these UCNPs as photoluminescent tags to monitor the gel degradation inside live, cultured intervertebral discs (IVDs) over a period of 3 weeks. PL spectroscopy and NIR imaging show that NIR-to-NIR upconversion of LiYF4:Yb3+/Tm3+@SiO2 UCNPs allows for tracking of the gel degradation in living tissues. Both in vitro and ex vivo release of UCNPs follow a similar trend during the first 5 days; after this time, ex vivo release becomes faster than in vitro, indicating a faster gel degradation ex vivo. Also, the amount of released UCNPs in vitro increases continuously up to 3 weeks, while it plateaus after 15 days inside the IVDs showing a homogenous distribution of UCNPs throughout the IVD tissue. This non-invasive optical method for real time, live tissue imaging holds great potential for tissue analysis, biomapping and bioimaging applications.},\n  annote = {cited By 33},\n  author = {Jalani, Ghulam and Naccache, Rafik and Rosenzweig, Derek H. and Lerouge, Sophie and Haglund, Lisbet and Vetrone, Fiorenzo and Cerruti, Marta},\n  doi = {10.1039/c5nr02482j},\n  issn = {20403372},\n  journal = {Nanoscale},\n  number = {26},\n  pages = {11255--11262},\n  title = {{Real-time, non-invasive monitoring of hydrogel degradation using LiYF4:Yb3+/Tm3+ NIR-to-NIR upconverting nanoparticles}},\n  volume = {7},\n  year = {2015}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  To design a biodegradable hydrogel as cell support, one should know its in vivo degradation rate. A technique commonly used to track gel degradation is fluorescence spectroscopy. However, the fluorescence from conventional fluorophores quickly decays, and the fluorophores are often moderately cytotoxic. Most importantly, they require ultraviolet or visible (UV-Vis) light as the excitation source, which cannot penetrate deeply through biological tissues. Lanthanide-doped upconverting nanoparticles (UCNPs) are exciting alternatives to conventional fluorophores because they can convert near-infrared (NIR) to UV-Vis-NIR light via a sequential multiphoton absorption process referred to as upconversion. NIR light can penetrate up to few cm inside tissues, thus making these UCNPs much better probes than conventional fluorophores for in vivo monitoring. Also, UCNPs have narrow emission bands, high photoluminescence (PL) signal-to-noise ratio, low cytotoxicity and good physical and chemical stability. Here, we show a nanocomposite system consisting of a biodegradable, in situ thermogelling injectable hydrogel made of chitosan and hyaluronic acid encapsulating silica-coated LiYF4:Yb3+/Tm3+ UCNPs. We use these UCNPs as photoluminescent tags to monitor the gel degradation inside live, cultured intervertebral discs (IVDs) over a period of 3 weeks. PL spectroscopy and NIR imaging show that NIR-to-NIR upconversion of LiYF4:Yb3+/Tm3+@SiO2 UCNPs allows for tracking of the gel degradation in living tissues. Both in vitro and ex vivo release of UCNPs follow a similar trend during the first 5 days; after this time, ex vivo release becomes faster than in vitro, indicating a faster gel degradation ex vivo. Also, the amount of released UCNPs in vitro increases continuously up to 3 weeks, while it plateaus after 15 days inside the IVDs showing a homogenous distribution of UCNPs throughout the IVD tissue. This non-invasive optical method for real time, live tissue imaging holds great potential for tissue analysis, biomapping and bioimaging applications.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"rosenzweig-carelli-steffen-jarzem-haglund-3dprintedabsandplascaffoldsforcartilageandnucleuspulposustissueregeneration-2015\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  3D-printed ABS and PLA scaffolds for cartilage and nucleus pulposustissue regeneration.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Carelli, E.; Steffen, T.; Jarzem, P.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>International Journal of Molecular Sciences</i>, 16(7): 15118–15135.  2015.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.3390/ijms160715118\"\n     onclick=\"log_download('rosenzweig-carelli-steffen-jarzem-haglund-3dprintedabsandplascaffoldsforcartilageandnucleuspulposustissueregeneration-2015', 'http://doi.org/10.3390/ijms160715118', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-carelli-steffen-jarzem-haglund-3dprintedabsandplascaffoldsforcartilageandnucleuspulposustissueregeneration-2015\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-carelli-steffen-jarzem-haglund-3dprintedabsandplascaffoldsforcartilageandnucleuspulposustissueregeneration-2015')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig201515118,\n  abstract = {Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (NP) cells were cultured on ABS and PLA scaffolds for three weeks. Both cell types proliferated well, showed high viability, and produced ample amounts of proteoglycan and collagen type II on both scaffolds. NP generated more matrix than chondrocytes; however, no difference was observed between scaffold types. Mechanical testing revealed sustained scaffold stability. This study demonstrates that chondrocytes and NP cells can proliferate on both ABS and PLA scaffolds printed with a simplistic, inexpensive desktop 3D printer. Moreover, NP cells produced more proteoglycan than chondrocytes, irrespective of thermoplastic type, indicating that cells maintain individual phenotype over the three-week culture period. Future scaffold designs covering larger pore sizes and better mimicking native tissue structure combined with more flexible or resorbable materials may provide implantable constructs with the proper structure, function, and cellularity necessary for potential cartilage and disc tissue repair in vivo.},\n  annote = {cited By 128},\n  author = {Rosenzweig, Derek H. and Carelli, Eric and Steffen, Thomas and Jarzem, Peter and Haglund, Lisbet},\n  doi = {10.3390/ijms160715118},\n  issn = {14220067},\n  journal = {International Journal of Molecular Sciences},\n  keywords = {3D printing,ABS,Chondrocyte,Intervertebral disc,Nucleus pulposus,PLA,Tissue engineering},\n  number = {7},\n  pages = {15118--15135},\n  title = {{3D-printed ABS and PLA scaffolds for cartilage and nucleus pulposustissue regeneration}},\n  volume = {16},\n  year = {2015}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Painful degeneration of soft tissues accounts for high socioeconomic costs. Tissue engineering aims to provide biomimetics recapitulating native tissues. Biocompatible thermoplastics for 3D printing can generate high-resolution structures resembling tissue extracellular matrix. Large-pore 3D-printed acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA) scaffolds were compared for cell ingrowth, viability, and tissue generation. Primary articular chondrocytes and nucleus pulposus (NP) cells were cultured on ABS and PLA scaffolds for three weeks. Both cell types proliferated well, showed high viability, and produced ample amounts of proteoglycan and collagen type II on both scaffolds. NP generated more matrix than chondrocytes; however, no difference was observed between scaffold types. Mechanical testing revealed sustained scaffold stability. This study demonstrates that chondrocytes and NP cells can proliferate on both ABS and PLA scaffolds printed with a simplistic, inexpensive desktop 3D printer. Moreover, NP cells produced more proteoglycan than chondrocytes, irrespective of thermoplastic type, indicating that cells maintain individual phenotype over the three-week culture period. Future scaffold designs covering larger pore sizes and better mimicking native tissue structure combined with more flexible or resorbable materials may provide implantable constructs with the proper structure, function, and cellularity necessary for potential cartilage and disc tissue repair in vivo.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"krock-rosenzweig-haglund-theinflammatorymilieuofthedegeneratediscismesenchymalstemcellbasedtherapyforintervertebraldiscrepairafeasibleapproach-2015\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  The Inflammatory Milieu of the Degenerate Disc: Is Mesenchymal Stem Cell-based Therapy for Intervertebral Disc Repair a Feasible Approach?.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Krock, E.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D.</a>;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>Current Stem Cell Research & Therapy</i>, 10(4): 317–328.  2015.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.2174/1574888x10666150211161956\"\n     onclick=\"log_download('krock-rosenzweig-haglund-theinflammatorymilieuofthedegeneratediscismesenchymalstemcellbasedtherapyforintervertebraldiscrepairafeasibleapproach-2015', 'http://doi.org/10.2174/1574888x10666150211161956', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/krock-rosenzweig-haglund-theinflammatorymilieuofthedegeneratediscismesenchymalstemcellbasedtherapyforintervertebraldiscrepairafeasibleapproach-2015\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('krock-rosenzweig-haglund-theinflammatorymilieuofthedegeneratediscismesenchymalstemcellbasedtherapyforintervertebraldiscrepairafeasibleapproach-2015')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Krock2015317,\n  abstract = { 2015 Bentham Science Publishers. Intervertebral disc degeneration is directly linked to chronic low back pain, a condition that affects multitudes of people world-wide and presents tremendous direct and indirect health costs. Water- loss, inflammation and disruption of the extracellular matrix ultimately result in loss of tissue function and associated pain. Cytokines present in degenerate tissue can upregulate protease activity and directly causes pain. Non-invasive therapies provide limited efficacy for pain management, and surgical intervention is therefore often required to treat chronic low back pain. Disc removal can offer immediate pain-relief, however degeneration of adjacent segments can occur and pain can return. To circumvent the caveats of recurring pain and invasive surgeries, stem cell therapy is currently being investigated as a promising means to repair degenerating discs. However, while initial studies have shown promise, few studies have addressed whether stem cell therapies can modulate the inflammatory microenvironment or whether cytokines can affect the ability of the implanted cells to repair damaged tissue. This review focuses briefly on mechanisms of disc degeneration, with more attention given to the role of inflammatory milieu in this process. Cytokine upregulation in disc degeneration, the potential role of tolllike receptor signaling, and effects of these inflammatory factors on stem cells will be discussed. We find that while stem cell differentiation can be negatively influenced by inflammatory cytokines, stem cells can potentially have anti-inflammatory effects. We conclude that further investigation of stem cell interactions with the inflammatory microenvironment is required, and that priming of stem cells under various conditions may be necessary for optimal therapeutic value for intervertebral disc repair and pain reduction.},\n  annote = {cited By 20},\n  author = {Krock, Emerson and Rosenzweig, Derek and Haglund, Lisbet},\n  doi = {10.2174/1574888x10666150211161956},\n  issn = {1574888X},\n  journal = {Current Stem Cell Research &amp; Therapy},\n  number = {4},\n  pages = {317--328},\n  title = {{The Inflammatory Milieu of the Degenerate Disc: Is Mesenchymal Stem Cell-based Therapy for Intervertebral Disc Repair a Feasible Approach?}},\n  volume = {10},\n  year = {2015}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  2015 Bentham Science Publishers. Intervertebral disc degeneration is directly linked to chronic low back pain, a condition that affects multitudes of people world-wide and presents tremendous direct and indirect health costs. Water- loss, inflammation and disruption of the extracellular matrix ultimately result in loss of tissue function and associated pain. Cytokines present in degenerate tissue can upregulate protease activity and directly causes pain. Non-invasive therapies provide limited efficacy for pain management, and surgical intervention is therefore often required to treat chronic low back pain. Disc removal can offer immediate pain-relief, however degeneration of adjacent segments can occur and pain can return. To circumvent the caveats of recurring pain and invasive surgeries, stem cell therapy is currently being investigated as a promising means to repair degenerating discs. However, while initial studies have shown promise, few studies have addressed whether stem cell therapies can modulate the inflammatory microenvironment or whether cytokines can affect the ability of the implanted cells to repair damaged tissue. This review focuses briefly on mechanisms of disc degeneration, with more attention given to the role of inflammatory milieu in this process. Cytokine upregulation in disc degeneration, the potential role of tolllike receptor signaling, and effects of these inflammatory factors on stem cells will be discussed. We find that while stem cell differentiation can be negatively influenced by inflammatory cytokines, stem cells can potentially have anti-inflammatory effects. We conclude that further investigation of stem cell interactions with the inflammatory microenvironment is required, and that priming of stem cells under various conditions may be necessary for optimal therapeutic value for intervertebral disc repair and pain reduction.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"jalani-rosenzweig-makhoul-abdalla-cecere-vetrone-haglund-cerruti-toughinsituthermogellinginjectablehydrogelsforbiomedicalapplications-2015\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Tough, in-situ thermogelling, injectable hydrogels for biomedical applications.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Jalani, G.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Makhoul, G.; Abdalla, S.; Cecere, R.; Vetrone, F.; Haglund, L.;  and Cerruti, M.\n</span>\n\n  \n\n</span>\n\n  <i>Macromolecular Bioscience</i>, 15(4): 473–480.  2015.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1002/mabi.201400406\"\n     onclick=\"log_download('jalani-rosenzweig-makhoul-abdalla-cecere-vetrone-haglund-cerruti-toughinsituthermogellinginjectablehydrogelsforbiomedicalapplications-2015', 'http://doi.org/10.1002/mabi.201400406', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/jalani-rosenzweig-makhoul-abdalla-cecere-vetrone-haglund-cerruti-toughinsituthermogellinginjectablehydrogelsforbiomedicalapplications-2015\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('jalani-rosenzweig-makhoul-abdalla-cecere-vetrone-haglund-cerruti-toughinsituthermogellinginjectablehydrogelsforbiomedicalapplications-2015')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Jalani2015473,\n  abstract = {Injectable hydrogels are extensively used in drug delivery and tissue engineering to administer drugs, genes, growth factors and live cells. We report a method to produce tough, in-situ thermogelling, non-toxic, injectable hydrogels made of chitosan and hyaluronic acid co-crosslinked with carboxytetramethylrhodamine-glycerophophate and genipin. The gels are highly homogeneous and form within 32 min, i.e., faster than gels crosslinked with either genipin or beta-glycerophophate. The shear strength of co-crosslinked hydrogels is 3.5 kPa, higher than any chitosan-based gel reported. Chondrocytes and nucleus pulposus cells thrive inside the gels and produce large amounts of collagen II. Injection in rats shows that the gels form in-vivo within a short time and remain well localized for more than one week while the rats remain healthy and active. The excellent mechanical properties, fast in-situ gelation, good biocompatibility and the ability to encapsulate live cells at physiological conditions make these hydrogels ideal for tissue engineering, especially cartilage regeneration. Homogenous, insitu thermogelling, highly tough, non-toxic injectable hydrogels from chitosan and hyaluronic acid co-crosslinked with beta-glycerophophate and genipin are produced. The co-crosslinked gels form faster than genipin-only crosslinked gels and are 2.5 times stronger than ionically crosslinked gels. Homogenously dispersed cells express enhanced amount of collagen II, showing the great potential of these gels for cartilage tissue engineering.},\n  annote = {cited By 15},\n  author = {Jalani, Ghulam and Rosenzweig, Derek H. and Makhoul, Georges and Abdalla, Sherif and Cecere, Renzo and Vetrone, Fiorenzo and Haglund, Lisbet and Cerruti, Marta},\n  doi = {10.1002/mabi.201400406},\n  issn = {16165195},\n  journal = {Macromolecular Bioscience},\n  keywords = {crosslinking,genipin,hydrogels,in-situ thermogelling,injectable},\n  number = {4},\n  pages = {473--480},\n  title = {{Tough, in-situ thermogelling, injectable hydrogels for biomedical applications}},\n  volume = {15},\n  year = {2015}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Injectable hydrogels are extensively used in drug delivery and tissue engineering to administer drugs, genes, growth factors and live cells. We report a method to produce tough, in-situ thermogelling, non-toxic, injectable hydrogels made of chitosan and hyaluronic acid co-crosslinked with carboxytetramethylrhodamine-glycerophophate and genipin. The gels are highly homogeneous and form within 32 min, i.e., faster than gels crosslinked with either genipin or beta-glycerophophate. The shear strength of co-crosslinked hydrogels is 3.5 kPa, higher than any chitosan-based gel reported. Chondrocytes and nucleus pulposus cells thrive inside the gels and produce large amounts of collagen II. Injection in rats shows that the gels form in-vivo within a short time and remain well localized for more than one week while the rats remain healthy and active. The excellent mechanical properties, fast in-situ gelation, good biocompatibility and the ability to encapsulate live cells at physiological conditions make these hydrogels ideal for tissue engineering, especially cartilage regeneration. Homogenous, insitu thermogelling, highly tough, non-toxic injectable hydrogels from chitosan and hyaluronic acid co-crosslinked with beta-glycerophophate and genipin are produced. The co-crosslinked gels form faster than genipin-only crosslinked gels and are 2.5 times stronger than ionically crosslinked gels. Homogenously dispersed cells express enhanced amount of collagen II, showing the great potential of these gels for cartilage tissue engineering.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2014\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2014')\"\n      onkeypress=\"toggleGroup('group_2014')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2014</span>\n      <span class=\"bibbase_group_count\">\n        \n        (5)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"rosenzweig-quinn-haglun-lowfrequencyhighmagnitudemechanicalstrainofarticularchondrocytesactivatesp38mapkandinducesphenotypicchangesassociatedwithosteoarthritisandpain-2014\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Low-frequency high-magnitude mechanical strain of articular chondrocytes activates p38 MAPK and induces phenotypic changes associated with osteoarthritis and pain.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Quinn, T. M.;  and Haglun, L.\n</span>\n\n  \n\n</span>\n\n  <i>International Journal of Molecular Sciences</i>, 15(8): 14427–14441.  2014.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.3390/ijms150814427\"\n     onclick=\"log_download('rosenzweig-quinn-haglun-lowfrequencyhighmagnitudemechanicalstrainofarticularchondrocytesactivatesp38mapkandinducesphenotypicchangesassociatedwithosteoarthritisandpain-2014', 'http://doi.org/10.3390/ijms150814427', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-quinn-haglun-lowfrequencyhighmagnitudemechanicalstrainofarticularchondrocytesactivatesp38mapkandinducesphenotypicchangesassociatedwithosteoarthritisandpain-2014\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-quinn-haglun-lowfrequencyhighmagnitudemechanicalstrainofarticularchondrocytesactivatesp38mapkandinducesphenotypicchangesassociatedwithosteoarthritisandpain-2014')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig201414427,\n  abstract = {Osteoarthritis (OA) is a debilitating joint disorder resulting from an incompletely understood combination of mechanical, biological, and biochemical processes. OA is often accompanied by inflammation and pain, whereby cytokines associated with chronic OA can up-regulate expression of neurotrophic factors such as nerve growth factor (NGF). Several studies suggest a role for cytokines and NGF in OA pain, however the effects of changing mechanical properties in OA tissue on chondrocyte metabolism remain unclear. Here, we used high-extension silicone rubber membranes to examine if high mechanical strain (HMS) of primary articular chondrocytes increases inflammatory gene expression and promotes neurotrophic factor release. HMS cultured chondrocytes displayed up-regulated NGF, TNFalpha and ADAMTS4 gene expression while decreasing TLR2 expression, as compared to static controls. HMS culture increased p38 MAPK activity compared to static controls. Conditioned medium from HMS dynamic cultures, but not static cultures, induced significant neurite sprouting in PC12 cells. The increased neurite sprouting was accompanied by consistent increases in PC12 cell death. Low-frequency high-magnitude mechanical strain of primary articular chondrocytes in vitro drives factor secretion associated with degenerative joint disease and joint pain. This study provides evidence for a direct link between cellular strain, secretory factors, neo-innervation, and pain in OA pathology. (C) 2014 by the authors; licensee MDPI, Basel, Switzerland.},\n  annote = {cited By 13},\n  author = {Rosenzweig, Derek H. and Quinn, Thomas M. and Haglun, Lisbet},\n  doi = {10.3390/ijms150814427},\n  issn = {14220067},\n  journal = {International Journal of Molecular Sciences},\n  keywords = {Articular cartilage,Inflammation,Mechanical stretch,Nerve growth factor,Osteoarthritis,Pain,p38 MAPK},\n  number = {8},\n  pages = {14427--14441},\n  title = {{Low-frequency high-magnitude mechanical strain of articular chondrocytes activates p38 MAPK and induces phenotypic changes associated with osteoarthritis and pain}},\n  volume = {15},\n  year = {2014}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Osteoarthritis (OA) is a debilitating joint disorder resulting from an incompletely understood combination of mechanical, biological, and biochemical processes. OA is often accompanied by inflammation and pain, whereby cytokines associated with chronic OA can up-regulate expression of neurotrophic factors such as nerve growth factor (NGF). Several studies suggest a role for cytokines and NGF in OA pain, however the effects of changing mechanical properties in OA tissue on chondrocyte metabolism remain unclear. Here, we used high-extension silicone rubber membranes to examine if high mechanical strain (HMS) of primary articular chondrocytes increases inflammatory gene expression and promotes neurotrophic factor release. HMS cultured chondrocytes displayed up-regulated NGF, TNFalpha and ADAMTS4 gene expression while decreasing TLR2 expression, as compared to static controls. HMS culture increased p38 MAPK activity compared to static controls. Conditioned medium from HMS dynamic cultures, but not static cultures, induced significant neurite sprouting in PC12 cells. The increased neurite sprouting was accompanied by consistent increases in PC12 cell death. Low-frequency high-magnitude mechanical strain of primary articular chondrocytes in vitro drives factor secretion associated with degenerative joint disease and joint pain. This study provides evidence for a direct link between cellular strain, secretory factors, neo-innervation, and pain in OA pathology. (C) 2014 by the authors; licensee MDPI, Basel, Switzerland.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"gawri-rosenzweig-krock-ouellet-stone-quinn-haglund-highmechanicalstrainofprimaryintervertebraldisccellspromotessecretionofinflammatoryfactorsassociatedwithdiscdegenerationandpain-2014\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Gawri, R.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Krock, E.; Ouellet, J. A.; Stone, L. S.; Quinn, T. M.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>Arthritis Research and Therapy</i>, 16(1).  2014.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1186/ar4449\"\n     onclick=\"log_download('gawri-rosenzweig-krock-ouellet-stone-quinn-haglund-highmechanicalstrainofprimaryintervertebraldisccellspromotessecretionofinflammatoryfactorsassociatedwithdiscdegenerationandpain-2014', 'http://doi.org/10.1186/ar4449', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/gawri-rosenzweig-krock-ouellet-stone-quinn-haglund-highmechanicalstrainofprimaryintervertebraldisccellspromotessecretionofinflammatoryfactorsassociatedwithdiscdegenerationandpain-2014\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('gawri-rosenzweig-krock-ouellet-stone-quinn-haglund-highmechanicalstrainofprimaryintervertebraldisccellspromotessecretionofinflammatoryfactorsassociatedwithdiscdegenerationandpain-2014')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Gawri2014,\n  abstract = {Introduction:Excessive mechanical loading of intervertebral discs (IVDs) is thought to alter matrix properties and influence disc cell metabolism, contributing to degenerative disc disease and development of discogenic pain. However, little is known about how mechanical strain induces these changes. This study investigated the cellular and molecular changes as well as which inflammatory receptors and cytokines were upregulated in human intervertebral disc cells exposed to high mechanical strain (HMS) at low frequency. The impact of these metabolic changes on neuronal differentiation was also explored to determine a role in the development of disc degeneration and discogenic pain.Methods:Isolated human annulus fibrosus (AF) and nucleus pulposus (NP) cells were exposed to HMS (20 % cyclical stretch at 0.001 Hz) on high-extension silicone rubber dishes coupled to a mechanical stretching apparatus and compared to static control cultures. Gene expression of Toll-like receptors (TLRs), neuronal growth factor (NGF) and tumour necrosis factor alpha (TNFalpha) was assessed. Collected conditioned media were analysed for cytokine content and applied to rat pheocromocytoma PC12 cells for neuronal differentiation assessment.Results:HMS caused upregulation of TLR2, TLR4, NGF and TNFalpha gene expression in IVD cells. Medium from HMS cultures contained elevated levels of growth-related oncogene, interleukin 6 (IL-6), IL-8, IL-15, monocyte chemoattractant protein 1 (MCP-1), MCP-3, monokine induced by gamma interferon, transforming growth factor beta1, TNFalpha and NGF. Exposure of PC12 cells to HMS-conditioned media resulted in both increased neurite sprouting and cell death.Conclusions:HMS culture of IVD cells in vitro drives cytokine and inflammatory responses associated with degenerative disc disease and low-back pain. This study provides evidence for a direct link between cellular strain, secretory factors, neoinnervation and potential degeneration and discogenic pain in vivo. (C) 2014 Gawri et al.; licensee BioMed Central Ltd.},\n  annote = {cited By 58},\n  author = {Gawri, Rahul and Rosenzweig, Derek H. and Krock, Emerson and Ouellet, Jean A. and Stone, Laura S. and Quinn, Thomas M. and Haglund, Lisbet},\n  doi = {10.1186/ar4449},\n  issn = {14786362},\n  journal = {Arthritis Research and Therapy},\n  number = {1},\n  title = {{High mechanical strain of primary intervertebral disc cells promotes secretion of inflammatory factors associated with disc degeneration and pain}},\n  volume = {16},\n  year = {2014}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Introduction:Excessive mechanical loading of intervertebral discs (IVDs) is thought to alter matrix properties and influence disc cell metabolism, contributing to degenerative disc disease and development of discogenic pain. However, little is known about how mechanical strain induces these changes. This study investigated the cellular and molecular changes as well as which inflammatory receptors and cytokines were upregulated in human intervertebral disc cells exposed to high mechanical strain (HMS) at low frequency. The impact of these metabolic changes on neuronal differentiation was also explored to determine a role in the development of disc degeneration and discogenic pain.Methods:Isolated human annulus fibrosus (AF) and nucleus pulposus (NP) cells were exposed to HMS (20 % cyclical stretch at 0.001 Hz) on high-extension silicone rubber dishes coupled to a mechanical stretching apparatus and compared to static control cultures. Gene expression of Toll-like receptors (TLRs), neuronal growth factor (NGF) and tumour necrosis factor alpha (TNFalpha) was assessed. Collected conditioned media were analysed for cytokine content and applied to rat pheocromocytoma PC12 cells for neuronal differentiation assessment.Results:HMS caused upregulation of TLR2, TLR4, NGF and TNFalpha gene expression in IVD cells. Medium from HMS cultures contained elevated levels of growth-related oncogene, interleukin 6 (IL-6), IL-8, IL-15, monocyte chemoattractant protein 1 (MCP-1), MCP-3, monokine induced by gamma interferon, transforming growth factor beta1, TNFalpha and NGF. Exposure of PC12 cells to HMS-conditioned media resulted in both increased neurite sprouting and cell death.Conclusions:HMS culture of IVD cells in vitro drives cytokine and inflammatory responses associated with degenerative disc disease and low-back pain. This study provides evidence for a direct link between cellular strain, secretory factors, neoinnervation and potential degeneration and discogenic pain in vivo. (C) 2014 Gawri et al.; licensee BioMed Central Ltd.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"moeini-decker-chin-shafieyan-rosenzweig-quinn-decreasedsoluteadsorptionontocrackedsurfacesofmechanicallyinjuredarticularcartilagetowardsthedesignofcartilagespecificfunctionalcontrastagents-2014\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Moeini, M.; Decker, S. G.; Chin, H. C.; Shafieyan, Y.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Biochimica et Biophysica Acta - General Subjects</i>, 1840(1): 605–614.  2014.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1016/j.bbagen.2013.10.022\"\n     onclick=\"log_download('moeini-decker-chin-shafieyan-rosenzweig-quinn-decreasedsoluteadsorptionontocrackedsurfacesofmechanicallyinjuredarticularcartilagetowardsthedesignofcartilagespecificfunctionalcontrastagents-2014', 'http://doi.org/10.1016/j.bbagen.2013.10.022', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/moeini-decker-chin-shafieyan-rosenzweig-quinn-decreasedsoluteadsorptionontocrackedsurfacesofmechanicallyinjuredarticularcartilagetowardsthedesignofcartilagespecificfunctionalcontrastagents-2014\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('moeini-decker-chin-shafieyan-rosenzweig-quinn-decreasedsoluteadsorptionontocrackedsurfacesofmechanicallyinjuredarticularcartilagetowardsthedesignofcartilagespecificfunctionalcontrastagents-2014')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Moeini2014605,\n  abstract = {Background: Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur. Methods: Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone. Results: Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde-dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes. Conclusions and general significance: Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents. (C) 2013 Elsevier B.V. All rights reserved.},\n  annote = {cited By 6},\n  author = {Moeini, Mohammad and Decker, Sarah G.A. and Chin, Hooi Chuan and Shafieyan, Yousef and Rosenzweig, Derek H. and Quinn, Thomas M.},\n  doi = {10.1016/j.bbagen.2013.10.022},\n  issn = {03044165},\n  journal = {Biochimica et Biophysica Acta - General Subjects},\n  keywords = {Articular cartilage,Contrast agent,Dextrans,FITC,Injury,Solute adsorption},\n  number = {1},\n  pages = {605--614},\n  title = {{Decreased solute adsorption onto cracked surfaces of mechanically injured articular cartilage: Towards the design of cartilage-specific functional contrast agents}},\n  volume = {1840},\n  year = {2014}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Background: Currently available methods for contrast agent-based magnetic resonance imaging (MRI) and computed tomography (CT) of articular cartilage can only detect cartilage degradation after biochemical changes have occurred within the tissue volume. Differential adsorption of solutes to damaged and intact surfaces of cartilage may be used as a potential mechanism for detection of injuries before biochemical changes in the tissue volume occur. Methods: Adsorption of four fluorescent macromolecules to surfaces of injured and sliced cartilage explants was studied. Solutes included native dextran, dextrans modified with aldehyde groups or a chondroitin sulfate (CS)-binding peptide and the peptide alone. Results: Adsorption of solutes to fissures was significantly less than to intact surfaces of injured and sliced explants. Moreover, solute adsorption at intact surfaces of injured and sliced explants was less reversible than at surfaces of uninjured explants. Modification of dextrans with aldehyde or the peptide enhanced adsorption with the same level of differential adsorption to cracked and intact surfaces. However, aldehyde-dextran exhibited irreversible adsorption. Equilibration of explants in solutes did not decrease the viability of chondrocytes. Conclusions and general significance: Studied solutes showed promising potential for detection of surface injuries based on differential interactions with cracked and intact surfaces. Additionally, altered adsorption properties at surfaces of damaged cartilage which visually look healthy can be used to detect micro-damage or biochemical changes in these regions. Studied solutes can be used in in vivo fluorescence imaging methods or conjugated with MRI or CT contrast agents to develop functional imaging agents. (C) 2013 Elsevier B.V. All rights reserved.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"krock-rosenzweig-chabotdore-jarzem-weber-ouellet-stone-haglund-painfuldegeneratingintervertebraldiscsupregulateneuritesproutingandcgrpthroughnociceptivefactors-2014\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Krock, E.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Chabot-Dore, A. J.; Jarzem, P.; Weber, M. H.; Ouellet, J. A.; Stone, L. S.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>Journal of Cellular and Molecular Medicine</i>, 18(6): 1213–1225.  2014.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1111/jcmm.12268\"\n     onclick=\"log_download('krock-rosenzweig-chabotdore-jarzem-weber-ouellet-stone-haglund-painfuldegeneratingintervertebraldiscsupregulateneuritesproutingandcgrpthroughnociceptivefactors-2014', 'http://doi.org/10.1111/jcmm.12268', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/krock-rosenzweig-chabotdore-jarzem-weber-ouellet-stone-haglund-painfuldegeneratingintervertebraldiscsupregulateneuritesproutingandcgrpthroughnociceptivefactors-2014\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('krock-rosenzweig-chabotdore-jarzem-weber-ouellet-stone-haglund-painfuldegeneratingintervertebraldiscsupregulateneuritesproutingandcgrpthroughnociceptivefactors-2014')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Krock20141213,\n  abstract = {Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-alpha, interleukin-1beta, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo. (C) 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley {&amp;} Sons Ltd and Foundation for Cellular and Molecular Medicine.},\n  annote = {cited By 60},\n  author = {Krock, Emerson and Rosenzweig, Derek H. and Chabot-Dore, Anne Julie and Jarzem, Peter and Weber, Michael H. and Ouellet, Jean A. and Stone, Laura S. and Haglund, Lisbet},\n  doi = {10.1111/jcmm.12268},\n  issn = {15824934},\n  journal = {Journal of Cellular and Molecular Medicine},\n  keywords = {CGRP,Discogenic pain,Human,Inflammatory cytokines,Intervertebral disc degeneration,Nerve growth factor},\n  number = {6},\n  pages = {1213--1225},\n  title = {{Painful, degenerating intervertebral discs up-regulate neurite sprouting and CGRP through nociceptive factors}},\n  volume = {18},\n  year = {2014}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Intervertebral disc degeneration (IVD) can result in chronic low back pain, a common cause of morbidity and disability. Inflammation has been associated with IVD degeneration, however the relationship between inflammatory factors and chronic low back pain remains unclear. Furthermore, increased levels of nerve growth factor (NGF) and brain derived neurotrophic factor (BDNF) are both associated with inflammation and chronic low back pain, but whether degenerating discs release sufficient concentrations of factors that induce nociceptor plasticity remains unclear. Degenerating IVDs from low back pain patients and healthy, painless IVDs from human organ donors were cultured ex vivo. Inflammatory and nociceptive factors released by IVDs into culture media were quantified by enzyme-linked immunosorbent assays and protein arrays. The ability of factors released to induce neurite growth and nociceptive neuropeptide production was investigated. Degenerating discs release increased levels of tumour necrosis factor-alpha, interleukin-1beta, NGF and BDNF. Factors released by degenerating IVDs increased neurite growth and calcitonin gene-related peptide expression, both of which were blocked by anti-NGF treatment. Furthermore, protein arrays found increased levels of 20 inflammatory factors, many of which have nociceptive effects. Our results demonstrate that degenerating and painful human IVDs release increased levels of NGF, inflammatory and nociceptive factors ex vivo that induce neuronal plasticity and may actively diffuse to induce neo-innervation and pain in vivo. (C) 2014 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"alkhatib-rosenzweig-krock-roughley-beckman-steffen-weber-ouellet-etal-acutemechanicalinjuryofthehumanintervertebraldisclinktodegenerationandpain-2014\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Acute mechanical injury of the human intervertebral disc: link to degeneration and pain.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Alkhatib, B.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Krock, E.; Roughley, P. J.; Beckman, L.; Steffen, T.; Weber, M. H.; Ouellet, J. A.;  and Haglund, L.\n</span>\n\n  \n\n</span>\n\n  <i>European Cells and Materials</i>, 28: 98–111.  2014.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.22203/eCM.v028a08\"\n     onclick=\"log_download('alkhatib-rosenzweig-krock-roughley-beckman-steffen-weber-ouellet-etal-acutemechanicalinjuryofthehumanintervertebraldisclinktodegenerationandpain-2014', 'http://doi.org/10.22203/eCM.v028a08', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/alkhatib-rosenzweig-krock-roughley-beckman-steffen-weber-ouellet-etal-acutemechanicalinjuryofthehumanintervertebraldisclinktodegenerationandpain-2014\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('alkhatib-rosenzweig-krock-roughley-beckman-steffen-weber-ouellet-etal-acutemechanicalinjuryofthehumanintervertebraldisclinktodegenerationandpain-2014')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Alkhatib201498,\n  abstract = {Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5 % (non-injured) or 30 % (injured) of disc height. 30 % strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured- IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROalpha, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.},\n  annote = {cited By 45},\n  author = {Alkhatib, B. and Rosenzweig, D. H. and Krock, E. and Roughley, P. J. and Beckman, L. and Steffen, T. and Weber, M. H. and Ouellet, J. A. and Haglund, L.},\n  doi = {10.22203/eCM.v028a08},\n  issn = {14732262},\n  journal = {European Cells and Materials},\n  keywords = {Degeneration,Extracellular matrix,Inflammation,Intervertebral disc,Mechanical injury,Nerve growth factor},\n  pages = {98--111},\n  title = {{Acute mechanical injury of the human intervertebral disc: link to degeneration and pain}},\n  volume = {28},\n  year = {2014}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Excessive mechanical loading or acute trauma to intervertebral discs (IVDs) is thought to contribute to degeneration and pain. However, the exact mechanisms by which mechanical injury initiates and promotes degeneration remain unclear. This study investigates biochemical changes and extracellular matrix disruption in whole-organ human IVD cultures following acute mechanical injury. Isolated healthy human IVDs were rapidly compressed by 5 % (non-injured) or 30 % (injured) of disc height. 30 % strain consistently cracked cartilage endplates, confirming disc trauma. Three days post-loading, conditioned media were assessed for proteoglycan content and released cytokines. Tissue extracts were assessed for proteoglycan content and for aggrecan integrity. Conditioned media were applied to PC12 cells to evaluate if factors inducing neurite growth were released. Compared to controls, IVD injury caused significant cell death. Injury also caused significantly reduced tissue proteoglycan content with a reciprocal increase of proteoglycan content in culture media. Increased aggrecan fragmentation was observed in injured tissue due to increased matrix metalloproteinase and aggrecanase activity. Injured- IVD conditioned media contained significantly elevated interleukin (IL)-5, IL-6, IL-7, IL-8, MCP-2, GROalpha, and MIG, and ELISA analysis showed significantly increased nerve growth factor levels compared to non-injured media. Injured-disc media caused significant neurite sprouting in PC12 cells compared to non-injured media. Acute mechanical injury of human IVDs ex vivo initiates release of factors and enzyme activity associated with degeneration and back pain. This work provides direct evidence linking acute trauma, inflammatory factors, neo-innervation and potential degeneration and discogenic pain in vivo.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2013\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2013')\"\n      onkeypress=\"toggleGroup('group_2013')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2013</span>\n      <span class=\"bibbase_group_count\">\n        \n        (4)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"rosenzweig-chicatun-nazhat-quinn-cartilaginousconstructsusingprimarychondrocytesfromcontinuousexpansioncultureseededindensecollagengels-2013\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Chicatun, F.; Nazhat, S. N.;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Acta Biomaterialia</i>, 9(12): 9360–9369.  2013.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1016/j.actbio.2013.07.024\"\n     onclick=\"log_download('rosenzweig-chicatun-nazhat-quinn-cartilaginousconstructsusingprimarychondrocytesfromcontinuousexpansioncultureseededindensecollagengels-2013', 'http://doi.org/10.1016/j.actbio.2013.07.024', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-chicatun-nazhat-quinn-cartilaginousconstructsusingprimarychondrocytesfromcontinuousexpansioncultureseededindensecollagengels-2013\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-chicatun-nazhat-quinn-cartilaginousconstructsusingprimarychondrocytesfromcontinuousexpansioncultureseededindensecollagengels-2013')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig20139360,\n  abstract = {Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.},\n  annote = {cited By 14},\n  author = {Rosenzweig, D. H. and Chicatun, F. and Nazhat, S. N. and Quinn, T. M.},\n  doi = {10.1016/j.actbio.2013.07.024},\n  issn = {17427061},\n  journal = {Acta Biomaterialia},\n  keywords = {Articular chondrocytes,Dense collagen,Hydrogel,Plastic compression,Tissue engineering},\n  number = {12},\n  pages = {9360--9369},\n  title = {{Cartilaginous constructs using primary chondrocytes from continuous expansion culture seeded in dense collagen gels}},\n  volume = {9},\n  year = {2013}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Cell-based therapies such as autologous chondrocyte implantation require in vitro cell expansion. However, standard culture techniques require cell passaging, leading to dedifferentiation into a fibroblast-like cell type. Primary chondrocytes grown on continuously expanding culture dishes (CE culture) limits passaging and protects against dedifferentiation. The authors tested whether CE culture chondrocytes were advantageous for producing mechanically competent cartilage matrix when three-dimensionally seeded in dense collagen gels. Primary chondrocytes, grown either in CE culture or passaged twice on static silicone dishes (SS culture; comparable to standard methods), were seeded in dense collagen gels and cultured for 3 weeks in the absence of exogenous chondrogenic growth factors. Compared with gels seeded with SS culture chondrocytes, CE chondrocyte-seeded gels had significantly higher chondrogenic gene expression after 2 and 3 weeks in culture, correlating with significantly higher aggrecan and type II collagen protein accumulation. There was no obvious difference in glycosaminoglycan content from either culture condition, yet CE chondrocyte-seeded gels were significantly thicker and had a significantly higher dynamic compressive modulus than SS chondrocyte-seeded gels after 3 weeks. Chondrocytes grown in CE culture and seeded in dense collagen gels produce more cartilaginous matrix with superior mechanical properties, making them more suitable than SS cultured cells for tissue engineering applications. (C) 2013 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"decker-moeini-chin-rosenzweig-quinn-adsorptionanddistributionoffluorescentsolutesnearthearticularsurfaceofmechanicallyinjuredcartilage-2013\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Adsorption and distribution of fluorescent solutes near the articular surface of mechanically injured cartilage.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Decker, S. G.; Moeini, M.; Chin, H. C.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Biophysical Journal</i>, 105(10): 2427–2436.  2013.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1016/j.bpj.2013.09.037\"\n     onclick=\"log_download('decker-moeini-chin-rosenzweig-quinn-adsorptionanddistributionoffluorescentsolutesnearthearticularsurfaceofmechanicallyinjuredcartilage-2013', 'http://doi.org/10.1016/j.bpj.2013.09.037', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/decker-moeini-chin-rosenzweig-quinn-adsorptionanddistributionoffluorescentsolutesnearthearticularsurfaceofmechanicallyinjuredcartilage-2013\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('decker-moeini-chin-rosenzweig-quinn-adsorptionanddistributionoffluorescentsolutesnearthearticularsurfaceofmechanicallyinjuredcartilage-2013')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Decker20132427,\n  abstract = {The development of cartilage-specific imaging agents supports the improvement of tissue assessment by minimally invasive means. Techniques for highlighting cartilage surface damage in clinical images could provide for sensitive indications of posttraumatic injury and early stage osteoarthritis. Previous studies in our laboratory have demonstrated that fluorescent solutes interact with cartilage surfaces strongly enough to affect measurement of their partition coefficients within the tissue bulk. In this study, these findings were extended by examining solute adsorption and distribution near the articular surface of mechanically injured cartilage. Using viable cartilage explants injured by an established protocol, solute distributions near the articular surface of three commonly used fluorophores (fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and carboxytetramethylrhodamine (TAMRA)) were observed after absorption and subsequent desorption to assess solute-specific matrix interactions and reversibility. Both absorption and desorption processes demonstrated a trend of significantly less solute adsorption at surfaces of fissures compared to adjacent intact surfaces of damaged explants or surfaces of uninjured explants. After adsorption, normalized mean surface intensities of fissured surfaces of injured explants were 6%, 40%, and 32% for FITC, TRITC, and TAMRA, respectively, compared to uninjured surfaces. Similar values were found for sliced explants and after a desorption process. After desorption, a trend of increased solute adsorption at the site of intact damaged surfaces was noted (316% and 238% for injured and sliced explants exposed to FITC). Surface adsorption of solute was strongest for FITC and weakest for TAMRA; no solutes negatively affected cell viability. Results support the development of imaging agents that highlight distinct differences between fissured and intact cartilage surfaces. (C) 2013 Biophysical Society.},\n  annote = {cited By 9},\n  author = {Decker, Sarah G.A. and Moeini, Mohammad and Chin, Hooi Chuan and Rosenzweig, Derek H. and Quinn, Thomas M.},\n  doi = {10.1016/j.bpj.2013.09.037},\n  issn = {00063495},\n  journal = {Biophysical Journal},\n  number = {10},\n  pages = {2427--2436},\n  title = {{Adsorption and distribution of fluorescent solutes near the articular surface of mechanically injured cartilage}},\n  volume = {105},\n  year = {2013}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  The development of cartilage-specific imaging agents supports the improvement of tissue assessment by minimally invasive means. Techniques for highlighting cartilage surface damage in clinical images could provide for sensitive indications of posttraumatic injury and early stage osteoarthritis. Previous studies in our laboratory have demonstrated that fluorescent solutes interact with cartilage surfaces strongly enough to affect measurement of their partition coefficients within the tissue bulk. In this study, these findings were extended by examining solute adsorption and distribution near the articular surface of mechanically injured cartilage. Using viable cartilage explants injured by an established protocol, solute distributions near the articular surface of three commonly used fluorophores (fluorescein isothiocyanate (FITC), tetramethylrhodamine isothiocyanate (TRITC), and carboxytetramethylrhodamine (TAMRA)) were observed after absorption and subsequent desorption to assess solute-specific matrix interactions and reversibility. Both absorption and desorption processes demonstrated a trend of significantly less solute adsorption at surfaces of fissures compared to adjacent intact surfaces of damaged explants or surfaces of uninjured explants. After adsorption, normalized mean surface intensities of fissured surfaces of injured explants were 6%, 40%, and 32% for FITC, TRITC, and TAMRA, respectively, compared to uninjured surfaces. Similar values were found for sliced explants and after a desorption process. After desorption, a trend of increased solute adsorption at the site of intact damaged surfaces was noted (316% and 238% for injured and sliced explants exposed to FITC). Surface adsorption of solute was strongest for FITC and weakest for TAMRA; no solutes negatively affected cell viability. Results support the development of imaging agents that highlight distinct differences between fissured and intact cartilage surfaces. (C) 2013 Biophysical Society.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"matmati-ng-rosenzweig-quinn-protectionofbovinechondrocytephenotypebyheatinactivationofallogeneicseruminmonolayerexpansioncultures-2013\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Protection of bovine chondrocyte phenotype by heat inactivation of allogeneic serum in monolayer expansion cultures.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Matmati, M.; Ng, T. F.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Annals of Biomedical Engineering</i>, 41(5): 894–903.  2013.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1007/s10439-012-0732-z\"\n     onclick=\"log_download('matmati-ng-rosenzweig-quinn-protectionofbovinechondrocytephenotypebyheatinactivationofallogeneicseruminmonolayerexpansioncultures-2013', 'http://doi.org/10.1007/s10439-012-0732-z', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/matmati-ng-rosenzweig-quinn-protectionofbovinechondrocytephenotypebyheatinactivationofallogeneicseruminmonolayerexpansioncultures-2013\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('matmati-ng-rosenzweig-quinn-protectionofbovinechondrocytephenotypebyheatinactivationofallogeneicseruminmonolayerexpansioncultures-2013')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Matmati2013894,\n  abstract = {Cartilage defects can be addressed with replacement strategies such as autologous chondrocyte implantation (ACI). Expansion of autologous chondrocytes in vitro is an essential step to obtain the necessary cell numbers required for ACI. A major problem with this approach is dedifferentiation of chondrocytes during expansion, resulting in cells with fibroblast-like features. These cells generate cartilage tissue with fibrotic instead of hyaline characteristics. The use of serum is a common feature in most expansion protocols and a potential factor contributing to the dedifferentiation process. The aim of this study was to assess if heat inactivation of serum used in the expansion medium might be a valid approach to generate cells with an improved phenotype and in relevant numbers. We used bovine chondrocyte expansion cultures incubated with heat inactivated allogeneic serum (HIFBS) as a model system. We here show that heat inactivation protects the differentiated phenotype of chondrocytes compared to cultures with regular serum. This is not only true for primary cultures but holds up after two passages. Moreover, using relatively low cell seeding densities, clinically relevant cell numbers can already be reached after the first passage in cultures with HIFBS. In short we here introduce a simple way to improve cell quality while generating relevant amounts of cells during monolayer expansion of bovine chondrocytes in a relative short time period. Our results could have wider implications when translated to the expansion of human chondrocytes. (C) 2012 Biomedical Engineering Society.},\n  annote = {cited By 10},\n  author = {Matmati, Mourad and Ng, Tat Fong and Rosenzweig, Derek H. and Quinn, Thomas M.},\n  doi = {10.1007/s10439-012-0732-z},\n  issn = {00906964},\n  journal = {Annals of Biomedical Engineering},\n  keywords = {Chondrocyte,Expansion culture,Heat inactivation,Serum},\n  number = {5},\n  pages = {894--903},\n  title = {{Protection of bovine chondrocyte phenotype by heat inactivation of allogeneic serum in monolayer expansion cultures}},\n  volume = {41},\n  year = {2013}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Cartilage defects can be addressed with replacement strategies such as autologous chondrocyte implantation (ACI). Expansion of autologous chondrocytes in vitro is an essential step to obtain the necessary cell numbers required for ACI. A major problem with this approach is dedifferentiation of chondrocytes during expansion, resulting in cells with fibroblast-like features. These cells generate cartilage tissue with fibrotic instead of hyaline characteristics. The use of serum is a common feature in most expansion protocols and a potential factor contributing to the dedifferentiation process. The aim of this study was to assess if heat inactivation of serum used in the expansion medium might be a valid approach to generate cells with an improved phenotype and in relevant numbers. We used bovine chondrocyte expansion cultures incubated with heat inactivated allogeneic serum (HIFBS) as a model system. We here show that heat inactivation protects the differentiated phenotype of chondrocytes compared to cultures with regular serum. This is not only true for primary cultures but holds up after two passages. Moreover, using relatively low cell seeding densities, clinically relevant cell numbers can already be reached after the first passage in cultures with HIFBS. In short we here introduce a simple way to improve cell quality while generating relevant amounts of cells during monolayer expansion of bovine chondrocytes in a relative short time period. Our results could have wider implications when translated to the expansion of human chondrocytes. (C) 2012 Biomedical Engineering Society.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"rosenzweig-ou-quinn-p38mitogenactivatedproteinkinasepromotesdedifferentiationofprimaryarticularchondrocytesinmonolayerculture-2013\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Ou, S. J.;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Journal of Cellular and Molecular Medicine</i>, 17(4): 508–517.  2013.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1111/jcmm.12034\"\n     onclick=\"log_download('rosenzweig-ou-quinn-p38mitogenactivatedproteinkinasepromotesdedifferentiationofprimaryarticularchondrocytesinmonolayerculture-2013', 'http://doi.org/10.1111/jcmm.12034', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-ou-quinn-p38mitogenactivatedproteinkinasepromotesdedifferentiationofprimaryarticularchondrocytesinmonolayerculture-2013\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-ou-quinn-p38mitogenactivatedproteinkinasepromotesdedifferentiationofprimaryarticularchondrocytesinmonolayerculture-2013')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig2013508,\n  abstract = {Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell-based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes &#x27;dedifferentiation&#x27; into fibroblastic cells. Mitogen-activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up-regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow-on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell-based therapies. (C) 2013.},\n  annote = {cited By 21},\n  author = {Rosenzweig, Derek H. and Ou, Sing J. and Quinn, Thomas M.},\n  doi = {10.1111/jcmm.12034},\n  issn = {15821838},\n  journal = {Journal of Cellular and Molecular Medicine},\n  keywords = {Chondrocyte,Dedifferentiation,Extra Cellular Matrix,Gene Expression,MAPK,Signal Transduction},\n  number = {4},\n  pages = {508--517},\n  pmid = {23480786},\n  title = {{P38 mitogen-activated protein kinase promotes dedifferentiation of primary articular chondrocytes in monolayer culture}},\n  volume = {17},\n  year = {2013}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Articular cartilage is an avascular tissue with poor regenerative capacity following injury, a contributing factor to joint degenerative disease. Cell-based therapies for cartilage tissue regeneration have rapidly advanced; however, expansion of autologous chondrocytes in vitro using standard methods causes 'dedifferentiation' into fibroblastic cells. Mitogen-activated protein kinase (MAPK) signalling is crucial for chondrocyte metabolism and matrix production, and changes in MAPK signals can affect the phenotype of cultured cells. We investigated the effects of inhibition of MAPK signalling on chondrocyte dedifferentiation during monolayer culture. Blockade of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) signalling caused a significant increase in cartilage gene expression, however, also caused up-regulation of fibrotic gene expression. Inhibition of p38 MAPK (p38) caused a significant up-regulation of collagen type II while suppressing collagen type I expression. P38 inhibition also resulted in consistently more organized secretion of collagen type II protein deposits on cell culture surfaces. Follow-on pellet culture of treated cells revealed that MAPK inhibition reduced cell migration from the pellet. ERK and JNK inhibition caused more collagen type I accumulation in pellets versus controls while p38 inhibition strongly promoted collagen type II accumulation with no effect on collagen type I. Blockade of all three MAPKs caused increased GAG content in pellets. These results indicate a role for MAPK signalling in chondrocyte phenotype loss during monolayer culture, with a strong contribution from p38 signalling. Thus, blockade of p38 enhances chondrocyte phenotype in monolayer culture and may promote more efficient cartilage tissue regeneration for cell-based therapies. (C) 2013.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2012\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2012')\"\n      onkeypress=\"toggleGroup('group_2012')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2012</span>\n      <span class=\"bibbase_group_count\">\n        \n        (4)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"rosenzweig-djap-ou-quinn-mechanicalinjuryofbovinecartilageexplantsinducesdepthdependenttransientchangesinmapkinaseactivityassociatedwithapoptosis-2012\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Djap, M. J.; Ou, S. J.;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Osteoarthritis and Cartilage</i>, 20(12): 1591–1602.  2012.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1016/j.joca.2012.08.012\"\n     onclick=\"log_download('rosenzweig-djap-ou-quinn-mechanicalinjuryofbovinecartilageexplantsinducesdepthdependenttransientchangesinmapkinaseactivityassociatedwithapoptosis-2012', 'http://doi.org/10.1016/j.joca.2012.08.012', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-djap-ou-quinn-mechanicalinjuryofbovinecartilageexplantsinducesdepthdependenttransientchangesinmapkinaseactivityassociatedwithapoptosis-2012\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-djap-ou-quinn-mechanicalinjuryofbovinecartilageexplantsinducesdepthdependenttransientchangesinmapkinaseactivityassociatedwithapoptosis-2012')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig20121591,\n  abstract = {Objective: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. Design: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. Results: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. Conclusions: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries. (C) 2012 Osteoarthritis Research Society International.},\n  annote = {cited By 19},\n  author = {Rosenzweig, D. H. and Djap, M. J. and Ou, S. J. and Quinn, T. M.},\n  doi = {10.1016/j.joca.2012.08.012},\n  issn = {10634584},\n  journal = {Osteoarthritis and Cartilage},\n  keywords = {Chondrocyte,Compression,Gene expression,Injury,MAP kinase,Signalling},\n  number = {12},\n  pages = {1591--1602},\n  pmid = {22935788},\n  title = {{Mechanical injury of bovine cartilage explants induces depth-dependent, transient changes in MAP kinase activity associated with apoptosis}},\n  volume = {20},\n  year = {2012}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Objective: To characterize mitogen activated protein (MAP) kinase activity and chondrocyte apoptosis in an in vitro model of cartilage mechanical injury as a function of tissue depth and time post-injury. Design: Mechanically injured osteochondral explants were assessed for cell viability, MAP kinase and caspase-3 activity over 15 days using immunofluorescence microscopy and Western blot. Zonal distributions of cell viability and apoptosis were quantified in the presence of specific mitogen activated protein kinase inhibitors. Results: Viability rapidly decreased post-injury, most significantly in the superficial zone, with some involvement of the middle and deep zones, which correlated with increased caspase-3 activity. Transient and significant increases in extracellular-regulated protein kinase (ERK) activity were observed in middle and deep zones at 1 and 6 days post-injury, while c-Jun-amino terminal protein kinase activity increased in the deep zone at 1 and 6 days compared to uninjured controls. Changes in p38 activity were particularly pronounced, with significant increases in all three zones 30 min post-injury, but only in the middle and deep zones after 1 and 6 days. Inhibition of ERK and p38 increased chondrocyte viability which correlated with decreased apoptosis. Conclusions: Spatiotemporal patterns of MAP kinase signalling in cartilage after mechanical injury strongly correlate with changes in cell viability and chondrocyte apoptosis. Importantly, these signals may be pro-survival or pro-apoptotic depending on zonal location and time post-injury. These data yield mechanistic insights which may improve the diagnosis and treatment of cartilage injuries. (C) 2012 Osteoarthritis Research Society International.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"rosenzweig-matmati-khayat-chaudhry-hinz-quinn-cultureofprimarybovinechondrocytesonacontinuouslyexpandingsurfaceinhibitsdedifferentiation-2012\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Matmati, M.; Khayat, G.; Chaudhry, S.; Hinz, B.;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Tissue Engineering - Part A</i>, 18(23-24): 2466–2476.  2012.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1089/ten.tea.2012.0215\"\n     onclick=\"log_download('rosenzweig-matmati-khayat-chaudhry-hinz-quinn-cultureofprimarybovinechondrocytesonacontinuouslyexpandingsurfaceinhibitsdedifferentiation-2012', 'http://doi.org/10.1089/ten.tea.2012.0215', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-matmati-khayat-chaudhry-hinz-quinn-cultureofprimarybovinechondrocytesonacontinuouslyexpandingsurfaceinhibitsdedifferentiation-2012\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-matmati-khayat-chaudhry-hinz-quinn-cultureofprimarybovinechondrocytesonacontinuouslyexpandingsurfaceinhibitsdedifferentiation-2012')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig20122466,\n  abstract = {Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and alpha-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies. (C) Copyright 2012, Mary Ann Liebert, Inc.},\n  annote = {cited By 32},\n  author = {Rosenzweig, Derek H. and Matmati, Mourad and Khayat, Ghazaleh and Chaudhry, Sidharth and Hinz, Boris and Quinn, Thomas M.},\n  doi = {10.1089/ten.tea.2012.0215},\n  issn = {19373341},\n  journal = {Tissue Engineering - Part A},\n  number = {23-24},\n  pages = {2466--2476},\n  title = {{Culture of primary bovine chondrocytes on a continuously expanding surface inhibits dedifferentiation}},\n  volume = {18},\n  year = {2012}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Expansion of autologous chondrocytes in vitro is used to generate adequate populations for cell-based therapies. However, standard (SD) culture methods cause loss of chondrocyte phenotype and dedifferentiation to fibroblast-like cells. Here, we use a novel surface expansion culture system in an effort to inhibit chondrocyte dedifferentiation. A highly elastic silicone rubber culture surface was continuously stretched over a 13-day period to 600% of its initial surface area. This maintained cells at a high density while limiting contact inhibition and reducing the need for passaging. Gene expression analysis, biochemical assays, and immunofluorescence microscopy of follow-on pellet cultures were used to characterize the results of continuous expansion (CE) culture versus SD cultures on rigid polystyrene. CE culture yielded cells with a more chondrocyte-like morphology and higher RNA-level expression of the chondrogenic markers collagen type II, aggrecan, and cartilage oligomeric matrix protein. Furthermore, the expression of collagen type I RNA and alpha-smooth muscle actin protein were significantly reduced, indicating suppression of fibroblastic features. Pellet cultures from CE chondrocytes contained more sulphated glycosaminoglycan and collagen type II than pellets from SD culture. Additional control cultures on static (unexpanded) silicone (SS culture) indicated that benefits of CE culture were partially due to features of the culture surface itself and partially due to the reduced passaging which that surface enabled through CE. Chondrocytes grown in CE culture may, therefore, be a superior source for cell-based therapies. (C) Copyright 2012, Mary Ann Liebert, Inc.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"rosenzweig-solarcafaggi-quinn-functionalizationofdynamicculturesurfaceswithacartilageextracellularmatrixextractenhanceschondrocytephenotypeagainstdedifferentiation-2012\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Functionalization of dynamic culture surfaces with a cartilage extracellular matrix extract enhances chondrocyte phenotype against dedifferentiation.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Solar-Cafaggi, S.;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Acta Biomaterialia</i>, 8(9): 3333–3341.  2012.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1016/j.actbio.2012.05.032\"\n     onclick=\"log_download('rosenzweig-solarcafaggi-quinn-functionalizationofdynamicculturesurfaceswithacartilageextracellularmatrixextractenhanceschondrocytephenotypeagainstdedifferentiation-2012', 'http://doi.org/10.1016/j.actbio.2012.05.032', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-solarcafaggi-quinn-functionalizationofdynamicculturesurfaceswithacartilageextracellularmatrixextractenhanceschondrocytephenotypeagainstdedifferentiation-2012\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-solarcafaggi-quinn-functionalizationofdynamicculturesurfaceswithacartilageextracellularmatrixextractenhanceschondrocytephenotypeagainstdedifferentiation-2012')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig20123333,\n  abstract = {Culture on silicone rubber surfaces has been shown to partially overcome the chondrocyte dedifferentiation characteristic of standard culture on rigid polystyrene. These methods typically involve functionalization of culture surfaces with proteins. Collagen type I is often used, but more cartilage-specific proteins may be more appropriate for chondrocytes. To explore this hypothesis, a twofold experimental design was applied. First, chondrocytes were cultured in rigid Petri dishes coated with silicone rubber (&quot;static silicone&quot; or SS culture) functionalized with either cartilage extracellular matrix (ECM) extract or collagen type I. Second, chondrocytes were cultured on monotonically expanded high extension silicone rubber dishes (&quot;continuous expansion&quot; or CE culture) functionalized with ECM extract and compared to cells grown in SS culture. There were no differential effects of surface functionalization with the ECM extract vs. collagen type I on chondrocyte morphology, viability, proliferation or apoptosis in SS culture. However, chondrocyte growth on the ECM extract was associated with significantly reduced collagen types I and X gene expression and significantly increased glycosaminoglycan (GAG) secretion. After 3 passages (P3) on ECM-coated SS culture, chondrocyte phenotype and GAG secretion was enhanced compared to cells passaged on collagen type I. Pellet cultures from P3 SS culture displayed enhanced collagen type II content when ECM extract was used for functionalization rather than collagen type I. In CE culture with ECM functionalization, chondrocyte dedifferentiation was significantly inhibited vs. SS cultures, as evidenced by both gene expression and pellet cultures. Functionalization of extendable culture surfaces with cartilage ECM extract therefore supports enhanced preservation of chondrocyte phenotype. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.},\n  annote = {cited By 22},\n  author = {Rosenzweig, Derek H. and Solar-Cafaggi, Sofia and Quinn, Thomas M.},\n  doi = {10.1016/j.actbio.2012.05.032},\n  issn = {17427061},\n  journal = {Acta Biomaterialia},\n  keywords = {Chondrocyte,Collagen,Dedifferentiation,Extendable surfaces,Extracellular matrix},\n  number = {9},\n  pages = {3333--3341},\n  title = {{Functionalization of dynamic culture surfaces with a cartilage extracellular matrix extract enhances chondrocyte phenotype against dedifferentiation}},\n  volume = {8},\n  year = {2012}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Culture on silicone rubber surfaces has been shown to partially overcome the chondrocyte dedifferentiation characteristic of standard culture on rigid polystyrene. These methods typically involve functionalization of culture surfaces with proteins. Collagen type I is often used, but more cartilage-specific proteins may be more appropriate for chondrocytes. To explore this hypothesis, a twofold experimental design was applied. First, chondrocytes were cultured in rigid Petri dishes coated with silicone rubber (\"static silicone\" or SS culture) functionalized with either cartilage extracellular matrix (ECM) extract or collagen type I. Second, chondrocytes were cultured on monotonically expanded high extension silicone rubber dishes (\"continuous expansion\" or CE culture) functionalized with ECM extract and compared to cells grown in SS culture. There were no differential effects of surface functionalization with the ECM extract vs. collagen type I on chondrocyte morphology, viability, proliferation or apoptosis in SS culture. However, chondrocyte growth on the ECM extract was associated with significantly reduced collagen types I and X gene expression and significantly increased glycosaminoglycan (GAG) secretion. After 3 passages (P3) on ECM-coated SS culture, chondrocyte phenotype and GAG secretion was enhanced compared to cells passaged on collagen type I. Pellet cultures from P3 SS culture displayed enhanced collagen type II content when ECM extract was used for functionalization rather than collagen type I. In CE culture with ECM functionalization, chondrocyte dedifferentiation was significantly inhibited vs. SS cultures, as evidenced by both gene expression and pellet cultures. Functionalization of extendable culture surfaces with cartilage ECM extract therefore supports enhanced preservation of chondrocyte phenotype. (C) 2012 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.\n</div>\n\n\n</div>\n\n\n  <div class=\"bibbase_paper\" id=\"khayat-rosenzweig-quinn-lowfrequencymechanicalstimulationinhibitsadipogenicdifferentiationofc3h10t12mesenchymalstemcells-2012\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Khayat, G.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>;  and Quinn, T. M.\n</span>\n\n  \n\n</span>\n\n  <i>Differentiation</i>, 83(4): 179–184.  2012.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1016/j.diff.2011.12.004\"\n     onclick=\"log_download('khayat-rosenzweig-quinn-lowfrequencymechanicalstimulationinhibitsadipogenicdifferentiationofc3h10t12mesenchymalstemcells-2012', 'http://doi.org/10.1016/j.diff.2011.12.004', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/khayat-rosenzweig-quinn-lowfrequencymechanicalstimulationinhibitsadipogenicdifferentiationofc3h10t12mesenchymalstemcells-2012\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('khayat-rosenzweig-quinn-lowfrequencymechanicalstimulationinhibitsadipogenicdifferentiationofc3h10t12mesenchymalstemcells-2012')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Khayat2012179,\n  abstract = {Oscillatory mechanical stimulation at relatively high frequencies (0.1. Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01. Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01. Hz frequency for 6. h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARgamma) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARgamma after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages. (C) 2012 International Society of Differentiation.},\n  annote = {cited By 25},\n  author = {Khayat, Ghazaleh and Rosenzweig, Derek H. and Quinn, Thomas M.},\n  doi = {10.1016/j.diff.2011.12.004},\n  issn = {03014681},\n  journal = {Differentiation},\n  keywords = {Adipogenesis,C3H10T1/2,Differentiation,Gene expression,Low frequency,Mechanical stimulation},\n  number = {4},\n  pages = {179--184},\n  title = {{Low frequency mechanical stimulation inhibits adipogenic differentiation of C3H10T1/2 mesenchymal stem cells}},\n  volume = {83},\n  year = {2012}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Oscillatory mechanical stimulation at relatively high frequencies (0.1. Hz) has been shown to inhibit adipogenic and promote osteogenic differentiation of mesenchymal stem cells. However, for physiological interpretations and ease of implementation it is of interest to know whether different rates of mechanical stimulation can produce similar results. We hypothesized that relatively low frequency mechanical stimulation (0.01. Hz) can inhibit adipogenic differentiation of C3H10T1/2 mouse mesenchymal stem cells, even in a potent adipogenic differentiation medium. C3H10T1/2 cells were cultured in adipogenic medium under control (non-mechanically stimulated) conditions and under oscillatory surface stretch with 10% amplitude and 0.01. Hz frequency for 6. h per day for up to 5 days. Cell population was assessed by counting and adipogenic differentiation was assessed by real-time quantitative PCR (qPCR) analysis of peroxisome proliferator-activated receptor gamma (PPARgamma) and fatty acid binding protein 4 (FABP4) after 3 and 5 days. Involvement of the ERK signaling pathway was assessed by Western blot. Low frequency mechanical stimulation significantly decreased expression of PPARgamma after 3 days and FABP4 after 3 and 5 days versus non-stimulated culture. ERK signaling was decreased in mechanically-stimulated culture, indicating a role in the inhibition of adipogenic differentiation. Application of this study: Low frequency mechanical stimulation may provide a technically simple means for control of mesenchymal stem cell differentiation in cell-based therapies, particularly for inhibition of differentiation toward undesired adipogenic lineages. (C) 2012 International Society of Differentiation.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2009\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2009')\"\n      onkeypress=\"toggleGroup('group_2009')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2009</span>\n      <span class=\"bibbase_group_count\">\n        \n        (1)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"rosenweig-nair-levay-peshenko-crabb-dizhoor-slepak-interactionofretinalguanylatecyclasewiththealphasubunitoftransducinpotentialroleintransducinlocalization-2009\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Interaction of retinal guanylate cyclase with the alpha subunit of transducin: Potential role in transducin localization.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Rosenweig, D. H.; Nair, S. K.; Levay, K.; Peshenko, I.; Crabb, J. W.; Dizhoor, A. M.;  and Slepak, V. Z.\n</span>\n\n  \n\n</span>\n\n  <i>Biochemical Journal</i>, 417(3): 803–812.  2009.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1042/BJ20081513\"\n     onclick=\"log_download('rosenweig-nair-levay-peshenko-crabb-dizhoor-slepak-interactionofretinalguanylatecyclasewiththealphasubunitoftransducinpotentialroleintransducinlocalization-2009', 'http://doi.org/10.1042/BJ20081513', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenweig-nair-levay-peshenko-crabb-dizhoor-slepak-interactionofretinalguanylatecyclasewiththealphasubunitoftransducinpotentialroleintransducinlocalization-2009\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenweig-nair-levay-peshenko-crabb-dizhoor-slepak-interactionofretinalguanylatecyclasewiththealphasubunitoftransducinpotentialroleintransducinlocalization-2009')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenweig2009803,\n  abstract = {Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between Galphat (the transducin alpha subunit) and retGC. Galphat co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-Galphat complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with bothGalphat and retGC. The Galphat-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound Galphat stronger than the GTP[S] (GTPgammaS; guanosine 5′-[gamma-thio]triphosphate) form. Neither Galphat nor Gbetagamma. affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between Galphat and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors. (C) The Authors Journal compilation (C) 2009 Biochemical Society.},\n  annote = {cited By 3},\n  author = {Rosenweig, Derek H. and Nair, Saidas K. and Levay, Konstantin and Peshenko, Igor and Crabb, John W. and Dizhoor, Alexander M. and Slepak, Vladlen Z.},\n  doi = {10.1042/BJ20081513},\n  issn = {02646021},\n  journal = {Biochemical Journal},\n  keywords = {CGMP,G-protein,Photoreceptor,Subcellular localization},\n  number = {3},\n  pages = {803--812},\n  title = {{Interaction of retinal guanylate cyclase with the alpha subunit of transducin: Potential role in transducin localization}},\n  volume = {417},\n  year = {2009}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between Galphat (the transducin alpha subunit) and retGC. Galphat co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-Galphat complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with bothGalphat and retGC. The Galphat-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound Galphat stronger than the GTP[S] (GTPgammaS; guanosine 5′-[gamma-thio]triphosphate) form. Neither Galphat nor Gbetagamma. affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between Galphat and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors. (C) The Authors Journal compilation (C) 2009 Biochemical Society.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2007\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2007')\"\n      onkeypress=\"toggleGroup('group_2007')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2007</span>\n      <span class=\"bibbase_group_count\">\n        \n        (1)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"rosenzweig-saidasnair-wei-wang-garwin-saari-chen-smrcka-etal-subunitdissociationanddiffusiondeterminethesubcellularlocalizationofrodandconetransducins-2007\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>; Saidas Nair, K.; Wei, J.; Wang, Q.; Garwin, G.; Saari, J. C.; Chen, C. K.; Smrcka, A. V.; Swaroop, A.; Lem, J.; Hurley, J. B.;  and Slepak, V. Z.\n</span>\n\n  \n\n</span>\n\n  <i>Journal of Neuroscience</i>, 27(20): 5484–5494.  2007.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1523/JNEUROSCI.1421-07.2007\"\n     onclick=\"log_download('rosenzweig-saidasnair-wei-wang-garwin-saari-chen-smrcka-etal-subunitdissociationanddiffusiondeterminethesubcellularlocalizationofrodandconetransducins-2007', 'http://doi.org/10.1523/JNEUROSCI.1421-07.2007', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/rosenzweig-saidasnair-wei-wang-garwin-saari-chen-smrcka-etal-subunitdissociationanddiffusiondeterminethesubcellularlocalizationofrodandconetransducins-2007\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('rosenzweig-saidasnair-wei-wang-garwin-saari-chen-smrcka-etal-subunitdissociationanddiffusiondeterminethesubcellularlocalizationofrodandconetransducins-2007')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Rosenzweig20075484,\n  abstract = {Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments. Copyright (C) 2007 Society for Neuroscience.},\n  annote = {cited By 51},\n  author = {Rosenzweig, Derek H. and {Saidas Nair}, K. and Wei, Junhua and Wang, Qiang and Garwin, Greg and Saari, John C. and Chen, Ching Kang and Smrcka, Alan V. and Swaroop, Anand and Lem, Janis and Hurley, James B. and Slepak, Vladlen Z.},\n  doi = {10.1523/JNEUROSCI.1421-07.2007},\n  issn = {02706474},\n  journal = {Journal of Neuroscience},\n  keywords = {Cone,Diffusion,Retina,Rod,Signal transduction,Transducin},\n  number = {20},\n  pages = {5484--5494},\n  pmid = {17507570},\n  title = {{Subunit dissociation and diffusion determine the subcellular localization of rod and cone transducins}},\n  volume = {27},\n  year = {2007}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  Activation of rod photoreceptors by light induces a massive redistribution of the heterotrimeric G-protein transducin. In darkness, transducin is sequestered within the membrane-enriched outer segments of the rod cell. In light, it disperses throughout the entire neuron. We show here that redistribution of rod transducin by light requires activation, but it does not require ATP. This observation rules out participation of molecular motors in the redistribution process. In contrast to the light-stimulated redistribution of rod transducin in rods, cone transducin in cones does not redistribute during activation. Remarkably, when cone transducin is expressed in rods, it does undergo light-stimulated redistribution. We show here that the difference in subcellular localization of activated rod and cone G-proteins correlates with their affinity for membranes. Activated rod transducin releases from membranes, whereas activated cone transducin remains bound to membranes. A synthetic peptide that dissociates G-protein complexes independently of activation facilitates dispersion of both rod and cone transducins within the cells. This peptide also facilitates detachment of both G-proteins from the membranes. Together, these results show that it is the dissociation state of transducin that determines its localization in photoreceptors. When rod transducin is stimulated, its subunits dissociate, leave outer segment membranes, and equilibrate throughout the cell. Cone transducin subunits do not dissociate during activation and remain sequestered within the outer segment. These findings indicate that the subunits of some heterotrimeric G-proteins remain associated during activation in their native environments. Copyright (C) 2007 Society for Neuroscience.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n  <div class=\"bibbase_group_whole\" id=\"group_2005\">\n    <div class=\"bibbase_group\"\n      onclick=\"toggleGroup('group_2005')\"\n      onkeypress=\"toggleGroup('group_2005')\"\n      tabindex=\"0\">\n      <i class=\"fa fa-angle-down bibbase_group_head_icon\"></i>&nbsp;\n      <span>2005</span>\n      <span class=\"bibbase_group_count\">\n        \n        (1)\n        \n      </span>\n    </div>\n    <div class=\"bibbase_group_body\">\n      \n  \n  <div class=\"bibbase_paper\" id=\"nair-mendez-blumer-rosenzweig-slepak-thepresenceofaleuglyasnrepeatenrichedproteinlgnaputativebindingpartneroftransducininrodphotoreceptors-2005\">\n  <span class=\"bibbase_paper_titleauthoryear\">\n\n  \n  <span class=\"bibbase_paper_title\">\n  \n  \n  \n  The presence of a Leu-Gly-Asn repeat-enriched protein (LGN), a putative binding partner of transducin, in ROD photoreceptors.\n  \n  \n  \n</span>\n\n  <span class=\"bibbase_paper_author\">\n  Nair, K. S.; Mendez, A.; Blumer, J. B.; <a class=\"bibbase author link\" href=\"https://rosenzweiglab.com/publication/\">Rosenzweig, D. H.</a>;  and Slepak, V. Z.\n</span>\n\n  \n\n</span>\n\n  <i>Investigative Ophthalmology and Visual Science</i>, 46(1): 383–389.  2005.\n  <span class=\"note\"></span>\n\n<span class=\"bibbase_note\"></span>\n\n<br class=\"bibbase_paper_content\"/>\n\n<span class=\"bibbase_paper_content dontprint\">\n\n  \n\n  \n  <a href=\"http://doi.org/10.1167/iovs.04-1006\"\n     onclick=\"log_download('nair-mendez-blumer-rosenzweig-slepak-thepresenceofaleuglyasnrepeatenrichedproteinlgnaputativebindingpartneroftransducininrodphotoreceptors-2005', 'http://doi.org/10.1167/iovs.04-1006', event.ctrlKey)\"\n     class=\"bibbase doi link\">\n    <span>doi</span></a>\n  &nbsp;\n  \n\n  \n  <a href=\"//bibbase.org/network/publication/nair-mendez-blumer-rosenzweig-slepak-thepresenceofaleuglyasnrepeatenrichedproteinlgnaputativebindingpartneroftransducininrodphotoreceptors-2005\"\n     class=\"bibbase bibbase_page link\">link</a>\n  &nbsp;\n  \n\n  <a href=\"#\"\n     onclick=\"showBib(event); return false;\"\n     class=\"bibbase bibtex link\">bibtex\n    <i class=\"fa fa-caret-down\"></i></a>\n\n  \n  &nbsp;\n  <a class=\"bibbase_abstract_link bibbase link\"\n     href=\"#\"\n     onclick=\"showAbstract(event); return false;\">\n    abstract <i class=\"fa fa-caret-down\"></i></a>\n  \n\n  \n\n  <!-- <a class=\"bibbase read link hidden\" -->\n  <!--    href=\"javascript:toggleRead('nair-mendez-blumer-rosenzweig-slepak-thepresenceofaleuglyasnrepeatenrichedproteinlgnaputativebindingpartneroftransducininrodphotoreceptors-2005')\" -->\n  <!--    title=\"Marking this paper as read adds it to your library on BibBase.\" -->\n  <!--    > -->\n  <!--   <i class=\"fa fa-check\"></i>&nbsp; -->\n  <!--   <span>mark as read</span> -->\n  <!-- </a> -->\n\n  &nbsp;\n  \n  \n\n</span>\n\n<div class=\"well well-small bibbase\" data-type=\"bibtex\"\n     style=\"display:none\">\n  <pre style=\"white-space: pre-wrap;\">@article{Nair2005383,\n  abstract = {PURPOSE. Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells. METHODS. Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding. RESULTS. LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange. CONCLUSIONS. Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.},\n  annote = {cited By 13},\n  author = {Nair, K. Saidas and Mendez, Ana and Blumer, Joe B. and Rosenzweig, Derek H. and Slepak, Vladlen Z.},\n  doi = {10.1167/iovs.04-1006},\n  issn = {01460404},\n  journal = {Investigative Ophthalmology and Visual Science},\n  number = {1},\n  pages = {383--389},\n  title = {{The presence of a Leu-Gly-Asn repeat-enriched protein (LGN), a putative binding partner of transducin, in ROD photoreceptors}},\n  volume = {46},\n  year = {2005}\n  }\n  </pre>\n</div>\n\n\n<div class=\"well well-small bibbase\" data-type=\"abstract\"\n     style=\"display:none\">\n  PURPOSE. Heterotrimeric G proteins are regulated by receptors that act as guanine nucleotide exchange factors (GEFs) and by RGS proteins, which act as guanosine triphosphatase (GTPase) activating proteins (GAPs). Guanosine diphosphate (GDP) dissociation inhibitors (GDIs), such as activators of G protein signaling (AGS)-1 and -3 and Leu-Gly-Asn repeat-enriched (LGN) proteins regulate the Gi family of G proteins. AGS3 and LGN contain four characteristic G protein regulator (GPR) domains that are responsible for its GDI function. This study investigates the presence of a GDI for transducin in photoreceptor cells. METHODS. Western blot analysis of bovine and mouse retina was performed using specific antibodies to AGS and LGN proteins. The subcellular localization of LGN in retina was studied by immunofluorescence microscopy of mouse retinal sections and fractionation of retinal lysates, using sucrose density gradients. The interaction of LGN with transducin was studied using pull-down assays with GST-fused LGN constructs, co-immunoprecipitation and assays for GTPgammaS binding. RESULTS. LGN, but not AGS3 and AGS1, was present in the retina, where it was localized mostly in the inner segments and outer plexiform layer of photoreceptor cells in both light and dark conditions. LGN was present in the cytosol, membrane, and the detergent-resistant cytoskeletal fraction. The amount of LGN relative to transducin was at least 1:15. The alpha subunit of transducin in its GDP-bound state interacted with endogenous and recombinant LGN, and the recombinant GPR domain of LGN reduced the rate of GTP exchange. CONCLUSIONS. Photoreceptor inner segments contain LGN, which can bind to the alpha subunit of transducin and potentially regulate its function.\n</div>\n\n\n</div>\n\n\n\n\n\n    </div>\n  </div>\n\n\n\n\n  </div>\n\n\n  <!-- Modal -->\n\n  <!-- <iframe id=\"bibbase_network_frame\" -->\n  <!--         src=\"//bibbase.org/network/control\" -->\n  <!--         style=\"display: none;\"> -->\n  <!-- </iframe> -->\n\n</div>\n"}; document.write(bibbase_data.data);