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.

The Good the Bad and the Ugly of Glycosaminoglycans in Tissue Engineering Applications

“…Our data suggests that GDF5 could be used to generate a clinically useful cartilage matrix with a high PG content, while maintaining the chondrocytes in a mature articular cartilage phenotype. As well as building on the results of Zhang et al [177], our research [266] also complements several other studies published over the past few years [175,179,181,269]. For example, work in an OA rat model has demonstrated that GDF5 is expressed in healthy pre-hypertrophic cartilage, but is not evident as OA develops [175].…”

“…These results therefore suggest a positive role for HSPGs in BMP signalling, perhaps by acting as a co-receptor for BMPs (as with FGF signalling [467,468]), or by modulating the bioavailability of BMPs at the cell surface. In favour of the latter hypothesis, our data [266] demonstrated that pericellular HSPGs play a key role in localising GDF5 to the cell surface, with a positive correlation between HSPG and GDF5 concentration being observed (Figure 4D). This is in good agreement with results reported for BMP2 [190,469], and further emphasises the importance of HSPGs in regulating the activity of many TGFβ superfamily members.…”

“…In contrast to TGFβ1, we found that GDF5 induced aggrecan and Sox9 expression (both markers associated with chondrogenesis and ECM production [267]), without increasing the expression of collagen X (the major marker of chondrocyte hypertrophy [268]); see Figure 4A–C [266]. Our data suggests that GDF5 could be used to generate a clinically useful cartilage matrix with a high PG content, while maintaining the chondrocytes in a mature articular cartilage phenotype.…”

Fully Synthetic Heparan Sulfate-Based Neural Tissue Construct That Maintains the Undifferentiated State of Neural Stem Cells

“…For example, growth differentiation factor 5 (GDF5), which is involved in cartilage/ skeletal formation and homeostasis, is inhibited by heparin. 46 In addition, heparin can cause hemorrhagic side effects and induce thrombocytopenia. It is derived from animal products exhibiting batch to batch differences and has been contaminated with other sulfated molecules.…”

Heparin, Heparan Sulphate and the TGF-β Cytokine Superfamily

“…The binding sites within these two BMPs are yet to be established, but their aminoterminal sequences are quite distinctive from those of BMP-2 and -4, being nearly three times longer. GDF-9 (BMP-15) [ 17 ] and most recently GDF-5 (BMP-14) [ 16 ], each representing a further subfamily, have also been shown to bind to heparin and HS. In neither case has the binding site been established.…”