A synthetic heparan sulphate mimetic for enhancing BMP-2-mediated osteogenesis and bone regeneration

Background & Aim The bone morphogenetic proteins (BMPs) are a family of morphogens belonging to the TGF-b superfamily. BMP-2, a potent inducer of osteogenesis in vitro and in vivo, has been subject to extensive research for use in bone repair applications. Previously our lab has studied the effects of heparan sulphate (HS), a highly sulphated linear polysaccharides with a complex and heterogeneous structure, on the bioactivity of BMP-2 in vitro and in vivo. Whilst HS displays great potential for therapeutic applications in regenerative medicine and bone repair, animal-derived HS species are less desirable when compared to recombinant or synthetically-derived molecules. In the present study, a panel of 15 fully synthetic HS mimetics were screened against BMP-2 in an effort to identify potential targets for therapeutic applications in bone repair. Methods, Results & Conclusion Of the 15 candidates, a four-armed dendrimer functionalized with oversulphated maltose residues and tetrahedral geometry (termed ‘HS20’) displayed the highest affinity toward BMP-2 via SPR completion and greatest enhancement BMP-2 bioactivity in vitro. Interestingly, the monomeric equivalent displayed little ability to bind, stabilize and enhance the bioactivity of BMP-2. Further analysis by fluorescence anisotropy and differential scanning fluorimetry confirmed HS20 is capable of avidly binding BMP-2. Moreover, HS20 was able to enhance association between BMP-2 and BMPRII in a dose-dependent manner. In vitro assays indicated that HS20 is capable of enhancing BMP-2-mediated Smad 1/5/9 phosphorylation, osteogenic gene transcription and alkaline phosphatase activity in murine C2C12 cells. HS20 was also able to enhance calcium deposition in Human MSCs during osteogenic differentiation. Additionally, inhibition assays indicated that the pro-osteogenic effects of HS20 can be inhibited through small molecule-mediated BMPR-I antagonism. Finally, HS20 was able to enhance in vivo bone formation in a critical-sized rabbit tibia defect over scaffold alone. These data suggest that a fully synthetic short-armed tetramer bearing oversulphated maltose residues could function as a HS mimetic in bone regeneration applications. The synthesis of this compound is cost-effective and scalable, giving it great potential for translation of this fully synthetic HS mimetic into a clinical setting for bone regeneration applications.