Effects of vitamin B12 in culture medium for calcified nodule formation by rat dental pulp cells

Abstract

Background/purpose: Tooth or bone regeneration requires large numbers of mesenchymal stem cells (MSCs). Although dental pulp is a suitable cell source, the number of MSCs present in this tissue is limited and they require a long period for regeneration. Therefore, the present study investigated vitamin B12 (Vb12) as an osteoinductive factor for MSCs obtained from dental pulp. Materials and methods: Dental pulp tissue was removed from the root canals of the extracted mandibular incisors of three 6-week-old male Fischer 344/N Slc rats using an endodontic file and whole cells were harvested. After the primary culture, cells were sub-cultured for calcified nodule formation in MEM containing dexamethasone (Dex), β-glycerophosphate (β-GP), vitamin C (Vc), and Vb12. Calcified nodules were confirmed under an inverted phase-contrast microscope. The alkaline phosphatase (ALP) activity of cells and quantity of Ca2+ in calcified nodules were measured. Results were analyzed using the Tukey-Kramer test. Results: Densely arranged calcified nodules were microscopically observed after the subculture of cells with Dex, β-GP, Vc, and Vb12. The ALP activity level was 0.077 ± 0.023 μmol/μg DNA in MEM supplemented with Vb12, which did not significantly differ from that without Vb12. A mass of calcium nodules formed in culture medium containing Dex, β-GP, Vc, and Vb12. The quantity of Ca2+ increased from 13.04 ± 0.44 to 20.91 ± 0.56 mg/dL (P < 0.01). Conclusion: Vb12 is effective for in vitro tooth or bone regeneration by the MSCs of rats and is useful as an osteoinductive factor for MSCs.

Keywords

• Calcified nodules
• Dental pulp stem cells
• In vitro
• Vitamin B12