Novel isolation of heparan sulfate with a specific affinity for bone morphogenetic protein 2 (2008)

As mesenchymal stem cells commit to the osteoblast lineage and become mature osteoblasts they secrete a unique and complex extracellular matrix. This matrix is rich in complex glycosaminoglycan chains that modulate a myriad of cellular responses including those involved in mesenchymal stem cell differentiation. Key to the role played by these complex sugars is the known specific interactions they display with a large number of heparin-binding growth factors. Bone morphogenetic protein 2 (BMP2) is a member of the TGF-β super-family of growth factors and is a known heparin-binding growth factor. This particular morphogen displays powerful osteoinductive properties that are thought to be modulated by the growth factor’s interaction with extracellular matrix-based glycosaminoglycans. In this study we characterised an osteoblast derived matrix (MX), obtained via the decellularisation of MC3T3-E1 monolayers, for its structural attributes via scanning electron microscopy and histology, as well as its ability to promote cell attachment, growth and viability. We further examined the capacity of MX to specifically retain BMP2 as well as identifying the critical role played by matrix-based glycosaminoglycans in mediating this effect. We proceeded to separate the glycosaminoglycan portion of MX via anion exchange chromatography and employed a novel method of affinity chromatography to isolate glycosaminoglycans with a specific affinity for BMP2 from total MX carbohydrate fractions. Our method for affinity chromatography employed the physical linking of the BMP2 heparin binding domain (BMP2-HBP) on its own to a streptavidin column, thereby abrogating the need for expensive protein column systems displaying the full BMP2 molecule. Close examination of the glycosaminoglycans separated via this methodology revealed that the MX sugars responsible for the retention of BMP2 were almost exclusively composed of heparan sulfate (HS) residues. Furthermore, analysis via heparinase III digestion indicated that full length BMP2-specific heparan sulfate chains appeared to be composed of numerous specific repeats along the length of each chain, with a virtual complete absence of non-specific regions. Furthermore, the exposure of C2C12 myoblasts to BMP2 and BMP2-specific sugar chains resulted in a significantly increased downstream induction of osteocalcin signalling. As such, this study demonstrates a novel method for the isolation of growth factor specific glycosaminoglycan chains, in particular those with an affinity for BMP2. This study represents the first time that BMP2-specifc glycosaminoglycan chains have been used in an attempt to directly modulate the efficiency of BMP2 signalling. Our results indicate that heparan sulfate appears to be almost exclusively responsible for mediating this control mechanism. Our data demonstrates a possible role for such specific HS chains in improving the efficiency of medical procedures that utilise BMP2 and help significantly refine the current understanding of how glycosaminoglycan interactions modulate BMP2-driven mesenchymal stem cell commitment to the osteoblast lineage.