OBJECTIVES: Understanding further its mechanism of action we wished to determine whether HHCM could drive differentiation of homogeneous populations of well characterised, clonal cell lines representing pluripotential (C3H10T1/2) and myoprogenitor (C2C12) mesenchymal cell lineages. Furthermore, we wished to determine if the removal of subpopulations of cells in the marrow would influence the ability of HHCM to induce osteogenic differentiation in vitro.
METHODS: C3H10T1/2 and C2C12 cells were cultured in osteogenic medium and exposed to varying concentrations of HHCM for 14 days. Also, whole rat bone marrow, or rat bone marrow depleted of early adherent cells, was cultured in osteogenic medium and exposed to varying concentrations of HHCM for 12 days. Colonies were then stained sequentially for alkaline phosphatase, calcification, collagen synthesis and total colony formation. In addition, alkaline phosphatase activity and osteocalcin levels in the culture medium were quantified.
RESULTS: HHCM did not induce the differentiation of C3H10T1/2 or C2C12 cells. Also, the removal of early adherent cells from bone marrow reduced the osteoinductive efficacy of the HHCM by 50%.
CONCLUSIONS: The results are consistent with the hypothesis that HHCM requires the presence of accessory cells, such as monocytes, in the bone marrow to enhance/maximise its osteogenic activity.
ACKNOWLEDGEMENTS: This research was funded by CellFactors plc, grant RF009460.