The effect of extracellular calcium and inorganic phosphate on the growth and osteogenic differentiation of mesenchymal stem cells in vitro: implication for bone tissue engineering.

The aim of this study is to demonstrate the effect of extracellular calcium ion (Ca2+) and inorganic phosphate (Pi) concentrations on the growth and differentiation of bone-marrow-derived mesenchymal stem cells (MSCs), which is essential to understand the interaction between calcium phosphate ceramic (CPC) scaffolds and seeded cells during the construction of tissue-engineered bones. MSCs were separated from rabbits and cultured in media with different concentrations of Ca2+ and Pi supplements. Their proliferation, apoptosis, mineralization and osteogenic differentiation were determined by the MTT assay, TUNEL assay, Vonkossa stain and RT-PCR examination. A two-way ANOVA calculation with comparisons of estimated marginal means by LSD was used for statistical analysis. Results showed that the optimal extracellular Ca2+ and Pi concentrations for the cells to proliferate and differentiate were 1.8 mM and 0.09 mM, respectively, which are the concentrations supplied in many commonly used culture media such as DMEM and alpha-MEM. Cell proliferation and differentiation decreased significantly with greater or lower concentrations of the Pi supplement. Greater Pi concentrations also led to significant cell apoptosis. Greater Ca2+ concentrations did not change cell proliferation but significantly inhibited cell differentiation. In addition, greater Ca2+ concentrations could significantly enhance cell mineralization. In conclusion, extracellular Ca2+ and Pi significantly influence the growth and osteogenic differentiation of MSCs. It is important to take the cellular effect of Ca2+ and Pi into consideration when designing or constructing scaffolds for bone tissue engineering with CPC.