Calcium ions released from mineral trioxide aggregate convert the differentiation pathway of C2C12 cells into osteoblast lineage.

INTRODUCTION: The purpose of this study was to examine the effect of mineral trioxide aggregate (MTA) on pluripotent-mesenchymal cell differentiation. METHODS: The pluripotent-mesenchymal cell line C2C12 was cultured in a 5% serum medium to induce cell differentiation with or without MTA. The differentiation to myoblasts was analyzed by the immunocytochemical staining of myosin heavy chains. The cellular phenotype-specific markers characterizing the osteoblasts (Runx2 and osterix), chondroblasts (Sox9), myoblasts (MyoD), and adipocytes (LPL) were estimated with mRNA and protein levels by using real-time polymerase chain reaction and Western blot analysis, respectively. To verify that the effect of MTA was caused by the released calcium ions, the mRNA levels were analyzed in the presence or absence of MTA with ethylene glycol tetraacetic acid, calcium chloride, or verapamil. RESULTS: C2C12 cells cultured without MTA altered their phenotype to myoblasts, exhibiting positive reactions to myosin heavy chains. However, the cells cultured with MTA were strongly inhibited from developing into myoblasts. The mRNA and protein expressions of Runx2, osterix, and Sox9 significantly increased with MTA; the expressions of MyoD and LPL decreased significantly. Calcium chloride addition without MTA presented a significant increase of mRNA levels of Runx2, osterix, and Sox9; ethylene glycol tetraacetic acid addition with MTA presented a significant increase of mRNA levels of MyoD and LPL. Verapamil blocked the stimulating or suppressing effect of MTA on these transcription factors. CONCLUSIONS: Our study showed that MTA converted the differentiation pathway of C2C12 cells into osteoblast and/or chondroblast lineages as a result of elution components such as calcium ions from MTA.

Nicotine affects bone resorption and suppresses the expression of cathepsin K, MMP-9 and vacuolar-type H(+)-ATPase d2 and actin organization in osteoclasts.

Tobacco smoking is an important risk factor for the development of several cancers, osteoporosis, and inflammatory diseases such as periodontitis. Nicotine is one of the major components of tobacco. In previous study, we showed that nicotine inhibits mineralized nodule formation by osteoblasts, and the culture medium from osteoblasts containing nicotine and lipopolysaccharide increases osteoclast differentiation. However, the direct effect of nicotine on the differentiation and function of osteoclasts is poorly understood. Thus, we examined the direct effects of nicotine on the expression of nicotine receptors and bone resorption-related enzymes, mineral resorption, actin organization, and bone resorption using RAW264.7 cells and bone marrow cells as osteoclast precursors. Cells were cultured with 10(-5), 10(-4), or 10(-3) M nicotine and/or 50 µM α-bungarotoxin (btx), an 7 nicotine receptor antagonist, in differentiation medium containing the soluble RANKL for up 7 days. 1-5, 7, 9, and 10 nicotine receptors were expressed on RAW264.7 cells. The expression of 7 nicotine receptor was increased by the addition of nicotine. Nicotine suppressed the number of tartrate-resistant acid phosphatase positive multinuclear osteoclasts with large nuclei(≥10 nuclei), and decreased the planar area of each cell. Nicotine decreased expression of cathepsin K, MMP-9, and V-ATPase d2. Btx inhibited nicotine effects. Nicotine increased CA II expression although decreased the expression of V-ATPase d2 and the distribution of F-actin. Nicotine suppressed the planar area of resorption pit by osteoclasts, but did not affect mineral resorption. These results suggest that nicotine increased the number of osteoclasts with small nuclei, but suppressed the number of osteoclasts with large nuclei. Moreover, nicotine reduced the planar area of resorption pit by suppressing the number of osteoclasts with large nuclei, V-ATPase d2, cathepsin K and MMP-9 expression and actin organization.

Esthetic and endodontic management of a deep crown-root fracture of a maxillary central incisor.

Treatment of trauma to anterior teeth should aim at preserving the affected teeth so as to restore function and esthetic appearance. Recently, patients have come to expect adequate esthetics immediately after trauma. In the present case, a deep crown-root fracture compromised the pulp and extended subgingivally on the palatal aspect. After using the fractured fragment as a provisional crown, the patient received conventional root canal treatment, which provided immediately satisfactory esthetic results and reliable short-term restoration of the crown-root fractured tooth. Rehabilitation of the fractured central incisor was performed with a post-core-supported prosthetic restoration.