In Vitro Osteoinductive Efficacy of Immortalised Human Hypertrophic Chondrocyte Matrix

INTRODUCTION: Hypertrophic cartilage is the key inducer of endochondral bone formation during embryogenesis and postnatal growth, and in fracture repair. Consequently, hypertrophic cartilage may prove to be a powerful tool for use in bone repair and regeneration, if it were freely available. To produce hypertrophic-like cartilage in vitro we have generated immortalised human hypertrophic cartilage cell lines that synthesise a complex bioactive matrix (HHCM). Our studies so far demonstrate that HHCM induces marrow stromal cells to differentiate into osteogenic cells that elaborate and mineralise an extracellular matrix. Furthermore, HHCM also induces vascularised bone formation in selected in vivo models. It is likely that numerous cytokines, matrix proteins and growth factors participate in the osteoinductive efficacy of this complex, human cell-derived material, some of which may be BMP-related. OBJECTIVES: To determine whether BMPs play a significant role in the osteoinductive activity of HHCM. METHODS: HHCM was tested in an in vitro CFU-f assay, to determine the osteoblastic differentiation of rat bone marrow stromal cells (BMSCs). Osteoblastic differentiation was quantified by measuring alkaline phosphatase activity, and the synthesis of osteocalcin. The contribution of BMPs to the osteoinductive efficacy of the HHCM was determined by co-administration of a monoclonal antibody to BMP-2/-4 (1?20 mg/ml) to neutralise any bioactivity of BMP-2 and BMP-4. RESULTS: Alkaline phosphatase activity was stimulated in rat BMSCs treated with HHCM. Similarly, osteocalcin synthesis, a later marker of osteoblastic differentiation, was increased by treatment with HHCM. Co-administration of anti?human BMP?2/?4 resulted in a significant fall in HHCM induced alkaline phosphatase activity and osteocalcin synthesis. However, in each case, a highly significant level of osteoinductive activity was maintained even at the highest BMP antibody concentrations. CONCLUSION: HHCM is able to stimulate osteoblastic differentiation of rat BMSCs through factors independent of BMP-2/-4. ACKNOWLEDGEMENTS: This research was funded by CellFactors plc, grant RF009460.