Effects of Calcium Hydroxide on Osteoblastic Differentiation and Mineralization

Objectives:

Calcium hydroxide has been broadly used for endodontic treatments since it exhibits antibacterial effects due to the strong alkalinity. In addition, as frequently utilized for apexification, calcium hydroxide is effective to promote mineralization. However, the detailed mechanism of calcium hydroxide to induce mineralization remains unclear. The purpose of this study was to examine the effects of calcium hydroxide on differentiation and mineralization of osteoblasts, focusing on the role of calcium ion and alkalinity.

Methods:

Murine primary osteoblasts were cultured in a-MEM containing 10% FCS under the culture conditions of different concentrations of Ca(OH)₂ and pH of the medium: 1) control without addition of Ca(OH)₂, pH 7.4, 2) 0.025 mg/ml, pH 7.5, 3) 0.25 mg/ml, pH 8.5, 4) 0.25 mg/ml, pH 7.4. Mineralization was evaluated by alizarin red staining, and the expression levels of osteoblast differentiation markers were determined using quantitative RT-PCR. The activation of ERK, p38 and JNK were examined by western blotting analysis.

Results:

For the control and cultures with 0.025 mg/ml Ca(OH)₂, no positive staining of the mineralized tissue was observed even after 21 days. The presence of 0.25 mg/ml Ca(OH)₂ induced mineralization, exhibiting greater amount of calcified tissue at pH 7.4 than 8.5. Expression levels of osteopontin, osteocalcin and alkaline phosphatase mRNA were up-regulated by 0.25 mg/ml Ca(OH)₂, and the neutral condition significantly enhanced osteopontin mRNA expression (Student t-test, p<0.05). Stimulation with 0.25 mg/ml Ca(OH)₂ strongly activated p38 and JNK at pH 7.4 but not at pH 8.5.

Conclusions:

Calcium hydroxide induces osteoblast differentiation and mineralization, and its effects are pronounced in the neutral condition based on the activation of p38 and JNK. These results suggest the importance of calcium ion on osteoblastic differentiation. (This study was supported by a Grant-in-Aid for Scientific Research No. 19659497 from the Japan Society for the Promotion of Science.)