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

Abstract

A number of bone-filling materials containing calcium (Ca(2+) ) and phosphate (P) ions have been used in the repair of periodontal bone defects; however, the effects that local release of Ca(2+) and P ions has on biological reactions are not fully understood. In this study, we investigated the effects of various levels of Ca(2+) and P ions on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells (hPDLCs). The hPDLCs were obtained using an explant culture method. Defined concentrations and ratios of ionic Ca(2+) 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. Within the ranges of Ca(2+) and P ion concentrations tested, we observed that increased concentrations of Ca(2+) 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 Ca(2+) and P ions led to a general increase of Runx2 mRNA expression and decreased alkaline phosphatase mRNA expression, but gave no clear trend on osteocalcin mRNA levels. The concentrations and ratios of Ca(2+) 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 Ca(2+) ions and 4.5 mm P ions were the optimal concentrations for proliferation, differentiation and mineralization in hPDLCs.