Bone Abstracts

Introduction: Activated protein C (APC) plays an important role in the cutaneous healing of chronic wounds arising from orthopaedic surgery and has cytoprotective and anti-inflammatory properties which may also assist bone repair. The aim of this study was to examine whether APC could directly influence osteoblasts and increase bone formation in a rodent model.

Methods: Proliferation of MG-63 osteoblast-like cells was quantified by MTT assay and direct counting. Monolayers were stained for calcium using alizarin red. ERK phosphorylation (pERK) was assayed by western blotting. Expression of APC receptors protease activated receptor 1 (PAR1) and PAR2, were immunostained on MG-63. Ectopic bone formation assay was performed in C57BL/6J mice using collagen sponge infused with rhBMP2±APC. Pellet size was assessed by X-ray and microCT. TRAP staining for osteoclast number (Oc.N) was done on sections.

Results: In vivo, APC increased BV in ectopic bone pellets by 73% (P<0.01) and TV by 104% (P<0.001) but did not alter BV/TV. APC inclusion led to 20% enhancement of Oc.N (P<0.05), suggesting that APC was pro-anabolic and not anti-resorptive. In vitro, APC increased MTT incorporation over 72 h by 15% (P<0.05) and similarly increased cell count. APC stimulated pERK activation and calcium deposition. PAR1 and PAR2 were expressed by MG-63 cells, and PAR antagonists abolished all effects of APC.

Conclusion: APC increased rhBMP2 induced ectopic bone formation, consistent with the results of increased osteoblast proliferation and matrix mineralization in cultured cells. PAR antagonists blocked the effects of APC, suggesting PAR1 and PAR2 directly mediate the effects of APC on bone.