Effect of enamel matrix derivative on the differentiation of C2C12 cells

Background: Although enamel matrix derivative (EMD) can initiate de novo cementum and bone formation by stimulating and inducing differentiation of mesenchymal cells in the periodontal ligament, the molecular mechanism of this phenomenon is not fully understood. The purpose of this study was to determine the effect of EMD on the differentiation of pluripotential mesenchymal cells. Methods: A typical pluripotential mesenchymal cell line, C2C12, was used to clarify the effect of EMD on cell differentiation. The cells were cultured in 5% serum-containing medium to induce cell differentiation, either with or without the addition of EMD. Differentiation to myoblasts was analyzed by immunostaining of desmin and type II myosin heavy chains. Osteoblast differentiation was evaluated by measuring alkaline phosphatase (ALPase) activity. Furthermore, to verify the cell lineage after culture with EMD, mRNA expression of cellular phenotype-specific markers characterizing osteoblasts (ALPase and osteocalcin), chondroblasts (type X collagen), myoblasts (desmin and MyoD), and adipocytes (lipoprotein lipase) was studied using semiquantitative reverse transcription-polymerase chain reaction. Results: C2C12 cells cultured in differentiation medium without EMD altered their phenotype to myoblasts, exhibiting positive reactions to desmin and myosin heavy chains by immunological analysis. However, the cells cultured in the presence of EMD were strongly inhibited from developing into myoblasts, and showed high ALPase activity that was approximately 2 to 4 times greater than that of the vehicle. The mRNA expression of ALPase, osteocalcin, and type X collagen was increased markedly by the EMD-stimulated medium, whereas the expression of desmin, MyoD, and lipoprotein lipase was drastically decreased. Conclusions: Our study provides clear evidence that EMD converts the differentiation pathway of C2C12 cells into the osteoblast and/or chondroblast lineage.