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

An S, Ling J, Gao Y, Xiao Y. Effects of varied ionic calcium and phosphate on the proliferation, osteogenic differentiation and mineralization of human periodontal ligament cells in vitro. J Periodont Res 2012; 47: 374–382. ©2011 John Wiley & Sons A/S 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 of Ca²⁺ and P ion 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 phosphatase mRNA expression, but gave no clear trend on osteocalcin mRNA levels. 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 hPDLCs.