Hypertrophic Cartilage Requires Accessory Cells to Trigger Osteogenic Activity

Hypertrophic chondrocytes orchestrate normal bone formation via the process of endochondral ossification. This process is recapitulated in fracture repair, whereby cartilage formed at the fracture site signals new bone formation. The process is highly regulated, relying on a multiplicity of growth factors, cytokines and matrix proteins to act in concert, and may also require activity from accessory cells within the local milieu for bone formation to occur. Previously, we have demonstrated that human hypertrophic cartilage matrix (HHCM) harvested from immortalised hypertrophic chondrocytes is osteoinductive, but requires the presence of accessory cells to induce the differentiation of mesenchymal progenitor cells to osteoblasts. OBJECTIVES: This study seeks to elucidate further the mechanism of HHCM osteoactivity and determine whether accessory cells are required to promote HHCM osteogenic activity in addition to osteoinductive activity. METHODS: MC3T3-E1 (osteoblastic), C3H10T1/2 (mesenchymal/pluripotential), C2C12 (myoprogenitor), NIH3T3 (fibroblast) cell lines and primary rat bone marrow cells were cultured in the presence of varying concentrations of HHCM for periods up to 14 days. CFU-F formation, alkaline phosphatase activity, osteocalcin expression, and the ability to elaborate and calcify an extracellular matrix in response to increasing concentrations of HHCM, were measured. RESULTS: HHCM induced CFU-F formation, extracellular matrix elaboration, and increased alkaline phosphatase activity and osteocalcin expression of primary rat marrow cultures. However, no osteoinductive or osteogenic effect of HHCM was seen for any of the pluripotential, myoprogenitor, fibroblastic, or osteoblastic clonal cell lines. Conversely, HHCM was shown to inhibit alkaline phosphatase activity in the osteoblastic MC3T3-E1 cell line at concentrations above 0.33ug/ml. CONCLUSIONS: The results are consistent with the hypothesis that accessory cells are required to activate HHCM osteogenic activity, in addition to their requirement for HHCM osteoinductive activation. Further studies are needed to elucidate how accessory cells trigger the osteoactivity of hypertrophic cartilage matrix.