title: Growth Differentiation Factor 5-Mediated Enhancement of Chondrocyte Phenotype Is Inhibited by Heparin: Implications for the Use of Heparin in the Clinic and in Tissue Engineering Applications.
abstract: The highly sulfated glycosaminoglycan (GAG) heparin is widely used in the clinic as an anticoagulant, and researchers are now using it to enhance stem cell expansion/differentiation protocols, as well as to improve the delivery of growth factors for tissue engineering (TE) strategies. Growth differentiation factor 5 (GDF5) belongs to the bone morphogenetic protein family of proteins and is vital for skeletal formation; however, its interaction with heparin and heparan sulfate (HS) has not been studied. We identify GDF5 as a novel heparin/HS binding protein and show that HS proteoglycans are vital in localizing GDF5 to the cell surface. Clinically relevant doses of heparin (≥10 nM), but not equivalent concentrations of HS, were found to inhibit GDF5's biological activity in both human mesenchymal stem/stromal cell-derived chondrocyte pellet cultures and the skeletal cell line ATDC5. We also found that heparin inhibited both GDF5 binding to cell surface HS and GDF5-induced induction of Smad 1/5/8 signaling. Furthermore, GDF5 significantly increased aggrecan gene expression in chondrocyte pellet cultures, without affecting collagen type X expression, making it a promising target for the TE of articular cartilage. Importantly, this study may explain the variable (and disappointing) results seen with heparin-loaded biomaterials for skeletal TE and the adverse skeletal effects reported in the clinic following long-term heparin treatment. Our results caution the use of heparin in the clinic and in TE applications, and prompt the transition to using more specific GAGs (e.g., HS derivatives), with better-defined structures and fewer off-target effects.
authors.affiliation: 1 Institute of Medical Biology , Agency for Science, Technology and Research (A*STAR), Singapore, Singapore .|2 Wellcome Trust Centre for Cell-Matrix Research, Division of Cell Matrix Biology and Regenerative Medicine, School of Biology, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, University of Manchester , Manchester, United Kingdom .
authors.forename: Bethanie I
authors.initials: BI
authors.lastname: Ayerst
authors.affiliation: 1 Institute of Medical Biology , Agency for Science, Technology and Research (A*STAR), Singapore, Singapore .
authors.forename: Raymond A A
authors.initials: RA
authors.lastname: Smith
authors.affiliation: 1 Institute of Medical Biology , Agency for Science, Technology and Research (A*STAR), Singapore, Singapore .
authors.forename: Victor
authors.initials: V
authors.lastname: Nurcombe
authors.affiliation: 2 Wellcome Trust Centre for Cell-Matrix Research, Division of Cell Matrix Biology and Regenerative Medicine, School of Biology, Faculty of Biology Medicine and Health, Manchester Academic Health Science Centre, University of Manchester , Manchester, United Kingdom .
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Day
authors.affiliation: 3 School of Materials, University of Manchester , Manchester, United Kingdom .|4 Wolfson Centre for Stem Cells, Tissue Engineering and Modelling, Centre for Biomolecular Sciences, University of Nottingham , Nottingham, United Kingdom .
authors.forename: Catherine L R
authors.initials: CL
authors.lastname: Merry
authors.affiliation: 1 Institute of Medical Biology , Agency for Science, Technology and Research (A*STAR), Singapore, Singapore .|5 Department of Orthopaedic Surgery, Yong Loo Lin School of Medicine, National University of Singapore , Singapore, Singapore .
authors.forename: Simon M
authors.initials: SM
authors.lastname: Cool
mesh_terms: D000925:Anticoagulants / Q000378:metabolism / Q000494:pharmacology; D002460:Cell Line; D019902:Chondrocytes / Q000187:drug effects* / Q000378:metabolism*; D055428:Growth Differentiation Factor 5 / Q000378:metabolism* / Q000494:pharmacology*; D006493:Heparin / Q000737:chemistry / Q000494:pharmacology*; D006497:Heparan Sulfate / Q000737:chemistry*; D006801:Humans; D023822:Tissue Engineering / Q000379:methods*
chemical_list: D000925:Anticoagulants; D055428:Growth Differentiation Factor 5; D006493:Heparin; D006497:Heparan Sulfate
keywords: cartilage; growth differentiation factor 5; heparan sulfate; heparin; mesenchymal stem cells; tissue engineering