Effect of CTGF on the Undifferentiated Dental Mesenchymal Cells

Objectives: A recent finding, utilizing a novel bioengineering technique, demonstrates the regeneration of mammalian dental tissues from tooth derived cells. CTGF (CCN2) was found to regulate chondrocyte and osteoblast function. However, the effect of CTGF on the undifferentiated dental mesenchymal cells remain largely unknown. Here we provide experimental evidence that CTGF induce undifferentiated dental mesenchymal cells into odontoblasts. Methods: Molar tooth germs from 3-month-old calves were obtained from a local slaughterhouse. Samples were rinsed in cold Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% fetal calf serum for 5 min, and dental epithelial and mesenchymal tissue layers were then separated by microsurgical procedures. In situ hybridization, ALPase activity, alizarin red staining, calcium analysis and Northern blotting were used to evaluate the effects of CTGF in undifferentiated mesenchymal cells differentiation and mineralization. Human recombinant CTGF was prepared by baculovirus expression system. Results: CTGF mRNA was detected in preodontoblasts and the gene expression patterns were maintained in mature odontoblasts. We first established a novel culture system with undifferentiated dental mesenchymal cells from bovine bell staged tooth germs and promoted their differentiation into odontoblasts. CTGF, in the presence of 5μg/ml ascorbic acid and 1mM β-glycerophosphate, stimulated the expressions of early odontoblast differentiation markers; ALP and collagen type I mRNA, additionally, maturation markers osteopontin and dentin sialophosphoprotein (DSPP) mRNA during the nine-day-culture period. Histological and histochemical analyses confirmed that CTGF treatment stimulated high alkaline phosphatase activity, calcium incorporation and hydroxyapatite deposition. Conclusion: These findings demonstrated for the first time that the novel culture system can maintain the osteogenic potential of undifferentiated dental mesenchymal cells and promote their differentiation into odontoblasts. Furthermore, CTGF is capable of promoting the differentiation of tooth germ mesenchymal cells into odontoblasts, suggesting that CTGF may be useful for future therapeutic treatment for tooth regeneration. Supported by NIDR grant RO1DE13206.