Effects of varied ionic calcium and phosphate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells in vitro

Background and Objective: A number of bone-filling materials containing calcium (Ca2+) and phosphate (P) ions have been used in the repair of periodontal bone defects; however, the effects that local release of Ca2+ and P ions has on biological reactions are not fully understood. In this study, we investigated the effects of various levels of Ca2+ and P ions on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells (hPDLCs). Material and Methods: The hPDLCs were obtained using an explant culture method. Defined concentrations and ratios of ionic Ca2+ to inorganic P were added to standard culture and osteogenic induction media. The ability of hPDLCs to proliferate in these growth media was assayed using the Cell Counting Kit-8. Cell apoptosis was evaluated by the fluorescein isothiocyanate-annexin V/ propidium iodide double-staining method. Osteogenic differentiation and mineralization were investigated by morphological observations, alkaline phosphatase activity and Alizarin Red S/ von Kossa staining. The mRNA expression of osteogenic related markers was analysed using RT-PCR. Results: Within the ranges ofCa(2+) andPion concentrations tested, we observed that increased concentrations of Ca2+ and P ions enhanced cell proliferation and formation of mineralized matrix nodules, whereas alkaline phosphatase activity was reduced. The RT-PCR results showed that elevated concentrations of Ca2+ and P ions led to a general increase of Runx2 mRNA expression and decreased alkaline phosphatasemRNAexpression, but gavenoclear trendonosteocalcinmRNAlevels. Conclusion: The concentrations and ratios of Ca2+ and P ions could significantly influence proliferation, differentiation and mineralization of hPDLCs. Within the range of concentrations tested, we found that the combination of 9.0 mM Ca2+ ions and 4.5 mM P ions were the optimal concentrations for proliferation, differentiation and mineralization in