Inamoto Takeshi, Yoshikawa Masataka, Miyamoto Ayano, Maeda Hiroshi
Department of Endodontics, School of Dentistry, Osaka Dental University, Osaka, Japan.
Dental Health Division, Health Policy Bureau, Ministry of Health, Labour and Welfare, Tokyo, Japan.
J Dent Sci. 2023 Jul;18(3):1079-1085. doi: 10.1016/j.jds.2022.11.015. Epub 2022 Nov 29.
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.
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 Ca in calcified nodules were measured. Results were analyzed using the Tukey-Kramer test.
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 Ca increased from 13.04 ± 0.44 to 20.91 ± 0.56 mg/dL ( < 0.01).
Vb12 is effective for tooth or bone regeneration by the MSCs of rats and is useful as an osteoinductive factor for MSCs.
背景/目的：牙齿或骨骼再生需要大量间充质干细胞（MSC）。虽然牙髓是合适的细胞来源，但该组织中存在的MSC数量有限，且它们需要很长时间才能再生。因此，本研究调查了维生素B12（Vb12）作为从牙髓获得的MSC的骨诱导因子。
使用牙髓锉从3只6周龄雄性Fischer 344/N Slc大鼠拔除的下颌切牙根管中取出牙髓组织，并收集全细胞。原代培养后，将细胞传代培养以在含有地塞米松（Dex）、β-甘油磷酸（β-GP）、维生素C（Vc）和Vb12的MEM中形成钙化结节。在倒置相差显微镜下确认钙化结节。测量细胞的碱性磷酸酶（ALP）活性和钙化结节中的钙含量。结果采用Tukey-Kramer检验进行分析。
用Dex、β-GP、Vc和Vb12对细胞进行传代培养后，在显微镜下观察到密集排列的钙化结节。在补充有Vb12的MEM中，ALP活性水平为0.077±0.023μmol/μg DNA，与不添加Vb12时无显著差异。在含有Dex、β-GP、Vc和Vb12的培养基中形成了大量钙结节。钙含量从13.04±0.44增加到20.91±0.56mg/dL（P<0.01）。
Vb12对大鼠MSC的牙齿或骨骼再生有效，可作为MSC的骨诱导因子。
