Background: The next generation of biomaterial surfaces for use in orthopaedic surgery will be functionalised to promote osteogenesis. This will be achieved in part by the stable addition of functional bioactive molecules onto the biomaterial surface. Heparan sulphate is a complex glycosaminoglycan (GAG) that displays cell and tissue specific differences in size and levels of sulphation. It is this heterogeneity that underlies the numerous biological roles of heparan sulphate, including binding of growth factors and proteases. Findings by others have shown that the addition of heparan sulphate proteoglycans stimulate osteoblast differentiation in vitro.
Aims: To characterise heparan sulphate structures that support and enhance osteogenesis and have the potential for tissue engineering.
Experiment 1. In order to further investigate the role of heparan-sulphate proteoglycans (HSPGs) in osteogenesis we supplemented cultures of differentiating rat osteo-blasts with sodium chlorate (an inhibitor of the enzyme that sulphates GAG chains) or 4-methylumbelliferyl-b-D-xyloside, BDX (an artificial acceptor of GAG chain synthesis). Interestingly the addition of chlorate to our culture system significantly stimulated alkaline phosphatase levels and increased the area of Von Kossa stained bone-like nodules. Whereas, when BDX was added to differentiating rat osteoblasts there was no increase in alkaline phosphatase activity or nodule area.
Experiment 2. Further characterisation of the HSPGs in chlorate treated osteoblasts showed that whilst they were less sulphated than untreated cells (as shown by low salt elution from an anion exchange chromatography column) they were much more abundant. These observations led us to hypothesise that less sulphated forms of heparan sulphate may well stimulate osteo-blast differentiation.
Experiment 3. To test this hypothesis we took the fully sulphated form of heparan sulphate, heparin and selectively desulphated it using DMSO/methanol (9:1) at 97°C and specifically N-resulphated or N-acetylated. These partially desulphated heparins were then added to osteoblasts cultured under osteogenic conditions. Quantification of bone nodule formation showed that specifically desulphated heparin significantly increased mineralised areas compared to controls whilst the addition of heparin inhibited osteogenesis. How these modified heparan sulphates exert their effect on bone cells is unknown, but a well characterised role of heparan sulphate is the support of FGF signalling. In preliminary studies we have shown through the activation of p42/44 MAPK and proliferation assays that the modified heparan sulphates are able to support FGF signalling in bone cells.
Experiment 4. Currently were are attaching our desulphated heparin to biomaterial scaffolds and examining osteoblast attachment and migration/ingrowth in cell culture.
Conclusion: We have isolated heparan sulphate chains that demonstrate osteogenic properties and have the potential for enhancing biological interactions of orthopaedic implant materials.