{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,16]],"date-time":"2026-10-16T17:58:09Z","timestamp":1792173489921,"version":"4.4.0"},"reference-count":68,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2022,1,15]],"date-time":"2022-01-15T00:00:00Z","timestamp":1642204800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"Deutsche Forschungsgemeinschaft","doi-asserted-by":"publisher","award":["326998133"],"award-info":[{"award-number":["326998133"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["IJMS"],"abstract":"<jats:p>In 3D bioprinting for cartilage regeneration, bioinks that support chondrogenic development are of key importance. Growth factors covalently bound in non-printable hydrogels have been shown to effectively promote chondrogenesis. However, studies that investigate the functionality of tethered growth factors within 3D printable bioinks are still lacking. Therefore, in this study, we established a dual-stage crosslinked hyaluronic acid-based bioink that enabled covalent tethering of transforming growth factor-beta 1 (TGF-\u03b21). Bone marrow-derived mesenchymal stromal cells (MSCs) were cultured over three weeks in vitro, and chondrogenic differentiation of MSCs within bioink constructs with tethered TGF-\u03b21 was markedly enhanced, as compared to constructs with non-covalently incorporated TGF-\u03b21. This was substantiated with regard to early TGF-\u03b21 signaling, chondrogenic gene expression, qualitative and quantitative ECM deposition and distribution, and resulting construct stiffness. Furthermore, it was successfully demonstrated, in a comparative analysis of cast and printed bioinks, that covalently tethered TGF-\u03b21 maintained its functionality after 3D printing. Taken together, the presented ink composition enabled the generation of high-quality cartilaginous tissues without the need for continuous exogenous growth factor supply and, thus, bears great potential for future investigation towards cartilage regeneration. Furthermore, growth factor tethering within bioinks, potentially leading to superior tissue development, may also be explored for other biofabrication applications.<\/jats:p>","DOI":"10.3390\/ijms23020924","type":"journal-article","created":{"date-parts":[[2022,1,16]],"date-time":"2022-01-16T20:44:00Z","timestamp":1642365840000},"page":"924","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":52,"title":["Tethered TGF-\u03b21 in a Hyaluronic Acid-Based Bioink for Bioprinting Cartilaginous Tissues"],"prefix":"10.3390","volume":"23","author":[{"given":"Julia","family":"Hauptstein","sequence":"first","affiliation":[{"name":"Department of Trauma, Hand, Plastic and Reconstructive Surgery, University of W\u00fcrzburg, 97080 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leonard","family":"Forster","sequence":"additional","affiliation":[{"name":"Department for Functional Materials in Medicine and Dentistry, Bavarian Polymer Institute (BPI), University of W\u00fcrzburg, 97070 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ali","family":"Nadernezhad","sequence":"additional","affiliation":[{"name":"Department for Functional Materials in Medicine and Dentistry, Bavarian Polymer Institute (BPI), University of W\u00fcrzburg, 97070 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3167-8466","authenticated-orcid":false,"given":"J\u00fcrgen","family":"Groll","sequence":"additional","affiliation":[{"name":"Department for Functional Materials in Medicine and Dentistry, Bavarian Polymer Institute (BPI), University of W\u00fcrzburg, 97070 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7057-5369","authenticated-orcid":false,"given":"J\u00f6rg","family":"Te\u00dfmar","sequence":"additional","affiliation":[{"name":"Department for Functional Materials in Medicine and Dentistry, Bavarian Polymer Institute (BPI), University of W\u00fcrzburg, 97070 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Torsten","family":"Blunk","sequence":"additional","affiliation":[{"name":"Department of Trauma, Hand, Plastic and Reconstructive Surgery, University of W\u00fcrzburg, 97080 W\u00fcrzburg, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,1,15]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"013001","DOI":"10.1088\/1758-5090\/8\/1\/013001","article-title":"Biofabrication: Reappraising the definition of an evolving field","volume":"8","author":"Groll","year":"2016","journal-title":"Biofabrication"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"384","DOI":"10.1016\/j.tibtech.2017.10.015","article-title":"Biofabrication: A Guide to Technology and Terminology","volume":"36","author":"Moroni","year":"2018","journal-title":"Trends Biotechnol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"e1906423","DOI":"10.1002\/adma.201906423","article-title":"From Shape to Function: The Next Step in Bioprinting","volume":"32","author":"Levato","year":"2020","journal-title":"Adv. Mater."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"327","DOI":"10.1177\/1947603516665445","article-title":"Three-Dimensional Bioprinting and Its Potential in the Field of Articular Cartilage Regeneration","volume":"8","author":"Mouser","year":"2017","journal-title":"Cartilage"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"76","DOI":"10.1177\/1947603518809410","article-title":"Three-Dimensional Bioprinting of Articular Cartilage: A Systematic Review","volume":"12","author":"Wu","year":"2021","journal-title":"Cartilage"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"21","DOI":"10.1038\/s41578-018-0006-y","article-title":"Biofabrication strategies for 3D in vitro models and regenerative medicine","volume":"3","author":"Moroni","year":"2018","journal-title":"Nat. Rev. Mater."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"045006","DOI":"10.1088\/1758-5090\/aad8d9","article-title":"Biofabrication of human articular cartilage: A path towards the development of a clinical treatment","volume":"10","author":"Onofrillo","year":"2018","journal-title":"Biofabrication"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"032001","DOI":"10.1088\/1758-5090\/ab8752","article-title":"Hyaluronic acid as a bioink for extrusion-based 3D printing","volume":"12","author":"Petta","year":"2020","journal-title":"Biofabrication"},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"H41","DOI":"10.1002\/adma.201003963","article-title":"Hyaluronic acid hydrogels for biomedical applications","volume":"23","author":"Burdick","year":"2011","journal-title":"Adv. Mater."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"35","DOI":"10.1016\/j.copbio.2016.02.008","article-title":"Recent advances in hyaluronic acid hydrogels for biomedical applications","volume":"40","author":"Highley","year":"2016","journal-title":"Curr. Opin. Biotechnol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"044108","DOI":"10.1088\/1758-5090\/aa8cb7","article-title":"Double printing of hyaluronic acid\/poly(glycidol) hybrid hydrogels with poly(\u03b5-caprolactone) for MSC chondrogenesis","volume":"9","author":"Stichler","year":"2017","journal-title":"Biofabrication"},{"key":"ref_12","doi-asserted-by":"crossref","unstructured":"Poldervaart, M.T., Goversen, B., de Ruijter, M., Abbadessa, A., Melchels, F.P.W., \u00d6ner, F.C., Dhert, W.J.A., Vermonden, T., and Alblas, J. (2017). 3D bioprinting of methacrylated hyaluronic acid (MeHA) hydrogel with intrinsic osteogenicity. PLoS ONE, 12.","DOI":"10.1371\/journal.pone.0177628"},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"025008","DOI":"10.1088\/1758-5090\/ab03ed","article-title":"Plant seed-inspired cell protection, dormancy, and growth for large-scale biofabrication","volume":"11","author":"Pan","year":"2019","journal-title":"Biofabrication"},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"3088","DOI":"10.1021\/acsbiomaterials.8b00416","article-title":"Three-Dimensional Printing of a Tyramine Hyaluronan Derivative with Double Gelation Mechanism for Independent Tuning of Shear Thinning and Postprinting Curing","volume":"4","author":"Petta","year":"2018","journal-title":"ACS Biomater. Sci. Eng."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"014106","DOI":"10.1088\/1758-5090\/ac3acb","article-title":"Fabrication of MSC-laden composites of hyaluronic acid hydrogels reinforced with MEW scaffolds for cartilage repair","volume":"14","author":"Galarraga","year":"2021","journal-title":"Biofabrication"},{"key":"ref_16","doi-asserted-by":"crossref","unstructured":"Hauptstein, J., Forster, L., Nadernezhad, A., Horder, H., Stahlhut, P., Groll, J., Blunk, T., and Te\u00dfmar, J. (2021). Bioink Platform Utilizing Dual-stage Crosslinking of Hyaluronic Acid Tailored for Chondrogenic Differentiation of Mesenchymal Stromal Cells. Macromol. Biosci., e2100331.","DOI":"10.1002\/mabi.202100331"},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"196","DOI":"10.1016\/0167-4889(91)90123-F","article-title":"Characterization of proteoglycans synthesized by rabbit articular chondrocytes in response to transforming growth factor-beta (TGF-beta)","volume":"1093","author":"Redini","year":"1991","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"1414","DOI":"10.1136\/ard.2005.045971","article-title":"Expression of transforming growth factor-beta (TGFbeta) and the TGFbeta signalling molecule SMAD-2P in spontaneous and instability-induced osteoarthritis: Role in cartilage degradation, chondrogenesis and osteophyte formation","volume":"65","author":"Vitters","year":"2006","journal-title":"Ann. Rheum. Dis."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"704","DOI":"10.3349\/ymj.2000.41.6.704","article-title":"Cytokine delivery and tissue engineering","volume":"41","author":"Lee","year":"2000","journal-title":"Yonsei Med. J."},{"key":"ref_20","first-page":"279","article-title":"Transforming growth factor-beta 1 stimulates articular chondrocyte proteoglycan synthesis and induces osteophyte formation in the murine knee joint","volume":"71","author":"Arntz","year":"1994","journal-title":"Lab. Invest."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"597","DOI":"10.1016\/j.joca.2007.02.005","article-title":"TGF-beta and osteoarthritis","volume":"15","year":"2007","journal-title":"Osteoarthr. Cartil."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"106","DOI":"10.1089\/ten.teb.2013.0271","article-title":"Transforming growth factor Beta-releasing scaffolds for cartilage tissue engineering","volume":"20","author":"Madry","year":"2014","journal-title":"Tissue Eng. Part B"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"607","DOI":"10.1586\/erd.11.27","article-title":"Design properties of hydrogel tissue-engineering scaffolds","volume":"8","author":"Zhu","year":"2011","journal-title":"Expert Rev. Med. Devices"},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"1700390","DOI":"10.1002\/mabi.201700390","article-title":"TGF-\u03b21-Modified Hyaluronic Acid\/Poly(glycidol) Hydrogels for Chondrogenic Differentiation of Human Mesenchymal Stromal Cells","volume":"18","author":"Schill","year":"2018","journal-title":"Macromol. Biosci."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"4464","DOI":"10.1002\/jbm.a.35115","article-title":"Covalently tethered TGF-\u03b21 with encapsulated chondrocytes in a PEG hydrogel system enhances extracellular matrix production","volume":"102","author":"Sridhar","year":"2014","journal-title":"J. Biomed. Mater. Res. Part A"},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"305","DOI":"10.1007\/s13346-012-0090-2","article-title":"Covalently tethered transforming growth factor beta in PEG hydrogels promotes chondrogenic differentiation of encapsulated human mesenchymal stem cells","volume":"2","author":"McCall","year":"2012","journal-title":"Drug Deliv. Transl. Res."},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"326","DOI":"10.1002\/adhm.201500677","article-title":"Controlling Shear Stress in 3D Bioprinting is a Key Factor to Balance Printing Resolution and Stem Cell Integrity","volume":"5","author":"Blaeser","year":"2016","journal-title":"Adv. Healthcare Mater."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"e00034","DOI":"10.1016\/j.bprint.2018.e00034","article-title":"Towards preserving post-printing cell viability and improving the resolution: Past, present, and future of 3D bioprinting theory","volume":"11","author":"Lepowsky","year":"2018","journal-title":"Bioprinting"},{"key":"ref_29","first-page":"81","article-title":"Shear Stress in 3D-Bioprinting strongly impacts Human MSC Survival and Proliferation Potential","volume":"16","author":"Blaeser","year":"2015","journal-title":"BioNanoMaterials"},{"key":"ref_30","doi-asserted-by":"crossref","unstructured":"M\u00fcller, S.J., Mirzahossein, E., Iftekhar, E.N., B\u00e4cher, C., Schr\u00fcfer, S., Schubert, D.W., Fabry, B., and Gekle, S. (2020). Flow and hydrodynamic shear stress inside a printing needle during biofabrication. PLoS ONE, 15.","DOI":"10.1371\/journal.pone.0236371"},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"733","DOI":"10.1002\/bip.21646","article-title":"The effects of shear flow on protein structure and function","volume":"95","author":"Bekard","year":"2011","journal-title":"Biopolymers"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"443","DOI":"10.1007\/s10529-010-0469-4","article-title":"Effects of shear on proteins in solution","volume":"33","author":"Thomas","year":"2011","journal-title":"Biotechnol. Lett."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"97","DOI":"10.1016\/j.actbio.2019.03.046","article-title":"An in vitro and in vivo comparison of cartilage growth in chondrocyte-laden matrix metalloproteinase-sensitive poly(ethylene glycol) hydrogels with localized transforming growth factor \u03b23","volume":"93","author":"Schneider","year":"2019","journal-title":"Acta Biomater."},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"83","DOI":"10.1089\/ten.tea.2010.0198","article-title":"Controlled delivery of transforming growth factor \u03b21 by self-assembling peptide hydrogels induces chondrogenesis of bone marrow stromal cells and modulates Smad2\/3 signaling","volume":"17","author":"Kopesky","year":"2011","journal-title":"Tissue Eng. Part A"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"742","DOI":"10.1039\/C4BM00431K","article-title":"Covalently conjugated transforming growth factor-\u03b21 in modular chitosan hydrogels for the effective treatment of articular cartilage defects","volume":"3","author":"Choi","year":"2015","journal-title":"Biomater. Sci."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1639","DOI":"10.1016\/j.joca.2009.07.003","article-title":"Macromer density influences mesenchymal stem cell chondrogenesis and maturation in photocrosslinked hyaluronic acid hydrogels","volume":"17","author":"Erickson","year":"2009","journal-title":"Osteoarthr. Cartil."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"413","DOI":"10.1016\/j.biomaterials.2012.09.052","article-title":"The influence of hyaluronic acid hydrogel crosslinking density and macromolecular diffusivity on human MSC chondrogenesis and hypertrophy","volume":"34","author":"Bian","year":"2013","journal-title":"Biomaterials"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"2000737","DOI":"10.1002\/adhm.202000737","article-title":"Hyaluronic Acid-Based Bioink Composition Enabling 3D Bioprinting and Improving Quality of Deposited Cartilaginous Extracellular Matrix","volume":"9","author":"Hauptstein","year":"2020","journal-title":"Adv. Healthc. Mater."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"167","DOI":"10.1016\/j.actbio.2018.11.022","article-title":"Enhancing chondrogenesis and mechanical strength retention in physiologically relevant hydrogels with incorporation of hyaluronic acid and direct loading of TGF-\u03b2","volume":"83","author":"Deng","year":"2019","journal-title":"Acta Biomater."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"1571","DOI":"10.1016\/j.actbio.2013.07.025","article-title":"Design of cell-matrix interactions in hyaluronic acid hydrogel scaffolds","volume":"10","author":"Lam","year":"2014","journal-title":"Acta Biomater."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"573","DOI":"10.1002\/jbm.10011","article-title":"Retention and activity of BMP-2 in hyaluronic acid-based scaffolds in vitro","volume":"59","author":"Kim","year":"2002","journal-title":"J. Biomed. Mater. Res."},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"6054","DOI":"10.1016\/j.biomaterials.2005.03.012","article-title":"Injectable glycosaminoglycan hydrogels for controlled release of human basic fibroblast growth factor","volume":"26","author":"Cai","year":"2005","journal-title":"Biomaterials"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"5011","DOI":"10.1002\/adma.201302042","article-title":"25th Anniversary Article: Engineering Hydrogels for Biofabrication","volume":"25","author":"Malda","year":"2013","journal-title":"Adv. Mater."},{"key":"ref_44","doi-asserted-by":"crossref","unstructured":"Tzavlaki, K., and Moustakas, A. (2020). TGF-\u03b2 Signaling. Biomolecules, 10.","DOI":"10.3390\/biom10030487"},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1186\/2045-3701-2-9","article-title":"Regulation of TGF-\u03b2 receptor activity","volume":"2","author":"Huang","year":"2012","journal-title":"Cell Biosci."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"e00119","DOI":"10.1016\/j.bprint.2020.e00119","article-title":"Rheology, simulation and data analysis toward bioprinting cell viability awareness","volume":"21","author":"Lucas","year":"2021","journal-title":"Bioprinting"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"535","DOI":"10.1039\/D0BM00973C","article-title":"Recent progress in extrusion 3D bioprinting of hydrogel biomaterials for tissue regeneration: A comprehensive review with focus on advanced fabrication techniques","volume":"9","author":"Askari","year":"2020","journal-title":"Biomater. Sci."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"221","DOI":"10.1089\/ten.tec.2015.0375","article-title":"Analytic Models of Oxygen and Nutrient Diffusion, Metabolism Dynamics, and Architecture Optimization in Three-Dimensional Tissue Constructs with Applications and Insights in Cerebral Organoids","volume":"22","author":"McMurtrey","year":"2016","journal-title":"Tissue Eng. Part C"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"1293","DOI":"10.1089\/scd.2017.0066","article-title":"Roles of Diffusion Dynamics in Stem Cell Signaling and Three-Dimensional Tissue Development","volume":"26","author":"McMurtrey","year":"2017","journal-title":"Stem Cells Dev."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"47","DOI":"10.1002\/wdev.86","article-title":"The TGFbeta superfamily signaling pathway","volume":"2","author":"Weiss","year":"2013","journal-title":"Wiley Interdiscip. Rev. Dev. Biol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"379","DOI":"10.1016\/j.cytogfr.2009.10.010","article-title":"Smad signaling in skeletal development and regeneration","volume":"20","author":"Song","year":"2009","journal-title":"Cytokine Growth Factor Rev."},{"key":"ref_52","doi-asserted-by":"crossref","first-page":"37","DOI":"10.1002\/bdrc.21058","article-title":"TGF\u03b2 signaling in cartilage development and maintenance","volume":"102","author":"Wang","year":"2014","journal-title":"Birth Defects Res. Part C"},{"key":"ref_53","doi-asserted-by":"crossref","first-page":"111388","DOI":"10.1016\/j.msec.2020.111388","article-title":"Crosslinker-free silk\/decellularized extracellular matrix porous bioink for 3D bioprinting-based cartilage tissue engineering","volume":"118","author":"Zhang","year":"2021","journal-title":"Mater. Sci. Eng. C"},{"key":"ref_54","doi-asserted-by":"crossref","first-page":"185101","DOI":"10.1088\/1361-6528\/aaafa1","article-title":"3D bioprinting mesenchymal stem cell-laden construct with core-shell nanospheres for cartilage tissue engineering","volume":"29","author":"Zhu","year":"2018","journal-title":"Nanotechnology"},{"key":"ref_55","doi-asserted-by":"crossref","first-page":"130","DOI":"10.1016\/j.actbio.2021.04.016","article-title":"Affinity-bound growth factor within sulfated interpenetrating network bioinks for bioprinting cartilaginous tissues","volume":"128","author":"Wang","year":"2021","journal-title":"Acta Biomater."},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"044104","DOI":"10.1088\/1758-5090\/7\/4\/044104","article-title":"3D bioprinting of BMSC-laden methacrylamide gelatin scaffolds with CBD-BMP2-collagen microfibers","volume":"7","author":"Du","year":"2015","journal-title":"Biofabrication"},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"035029","DOI":"10.1088\/1758-5090\/ab9492","article-title":"The effect of BMP-mimetic peptide tethering bioinks on the differentiation of dental pulp stem cells (DPSCs) in 3D bioprinted dental constructs","volume":"12","author":"Park","year":"2020","journal-title":"Biofabrication"},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"283","DOI":"10.1007\/BF00184030","article-title":"An efficient method for lipase-catalysed preparation of acrylic and methacrylic acid esters","volume":"10","author":"Warwel","year":"1996","journal-title":"Biotechnol. Tech."},{"key":"ref_59","doi-asserted-by":"crossref","first-page":"162","DOI":"10.1016\/j.bej.2018.11.011","article-title":"Solvent-free lipase-catalyzed production of (meth)acrylate monomers: Experimental results and kinetic modeling","volume":"142","author":"Heeres","year":"2019","journal-title":"Biochem. Eng. J."},{"key":"ref_60","doi-asserted-by":"crossref","first-page":"S113","DOI":"10.1016\/j.joca.2010.05.026","article-title":"Basic methods in histopathology of joint tissues","volume":"18","author":"Schmitz","year":"2010","journal-title":"Osteoarthr. Cartil."},{"key":"ref_61","doi-asserted-by":"crossref","first-page":"656","DOI":"10.1114\/1.205","article-title":"Method for quantitative analysis of glycosaminoglycan distribution in cultured natural and engineered cartilage","volume":"27","author":"Martin","year":"1999","journal-title":"Ann. Biomed. Eng."},{"key":"ref_62","doi-asserted-by":"crossref","first-page":"168","DOI":"10.1016\/0003-2697(88)90532-5","article-title":"Fluorometric assay of DNA in cartilage explants using Hoechst 33258","volume":"174","author":"Kim","year":"1988","journal-title":"Anal. Biochem."},{"key":"ref_63","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1016\/0304-4165(86)90306-5","article-title":"Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue","volume":"883","author":"Farndale","year":"1986","journal-title":"Biochim. Biophys. Acta"},{"key":"ref_64","doi-asserted-by":"crossref","first-page":"440","DOI":"10.1016\/0003-9861(61)90291-0","article-title":"The determination of hydroxyproline in tissue and protein samples containing small proportions of this imino acid","volume":"93","author":"Woessner","year":"1961","journal-title":"Arch. Biochem. Biophys."},{"key":"ref_65","doi-asserted-by":"crossref","first-page":"1722","DOI":"10.1172\/JCI117156","article-title":"Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay","volume":"93","author":"Hollander","year":"1994","journal-title":"J. Clin. Investig."},{"key":"ref_66","doi-asserted-by":"crossref","first-page":"402","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method","volume":"25","author":"Livak","year":"2001","journal-title":"Methods"},{"key":"ref_67","doi-asserted-by":"crossref","first-page":"686","DOI":"10.1021\/bc9701095","article-title":"Synthesis and in Vitro Degradation of New Polyvalent Hydrazide Cross-Linked Hydrogels of Hyaluronic Acid","volume":"8","author":"Vercruysse","year":"1997","journal-title":"Bioconjugate Chem."},{"key":"ref_68","doi-asserted-by":"crossref","first-page":"1132","DOI":"10.1002\/pi.5159","article-title":"Synthesis of PEGylated poly(amino acid) pentablock copolymers and their self-assembly","volume":"65","author":"Iijima","year":"2016","journal-title":"Polym. 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