Effect Of Bone Morphogenetic Protein-2 And Dexamethasone On Osteogensis Of Rat Dental Follicle Cells

At present, treatments on incomplete and damaged periodontal tissues caused by periodontal diseases mainly include renewing tissues and applying tissue engineering to have new periodontal adhesion and restore the structure and supporting tissues of natural periodontal tissues.The basic method of tissue engineering is to add normal tissue cells cultured in vitro on natural or synthesized extracellular matrix scaffold with good biocompatibility and biodegradation. After a period of culture, it is implanted in the body to grow into new tissues and organs with the original special function and form, aimed to repairing trauma and reconstructing function. There are studies reporting that periodontal membrane cells are used as seed cells with the method of tissue engineering to succeed in repairing periodontal tissues.Relevant studies indicate that dental follicle cells can show muliformity with specific treatment. There is no report on whether dental follicle cells as seed cells can repair incomplete and damaged periodontal tissues and combined application of BMP-2 and Dexamethasone can facilitate osteogensis in dental follicle cells.The experiment first observed the effect of BMP-2, Dexamethasone and the combined application of the two on proliferation and differentiation of rat follicle cells. Dental follicle cells andβ-tricalcium phosphate with different treatments were synthesized in vitro. The adhesion and growth of cells on the scaffold was observed. They were then implanted in a rat with immune deficiency. It was observed whether the combined effect of BMP-2 and Dexamethasone could enhance the ability of dental follicle cells to differentiate to osteo/cementoblast.Part 1. Culture and identification of rat dental follicle cells Objective: To culture the rat dental follicle cells in vitro and to identify its origin; meanwhile, to investigate it as seed cell for periodontal tissue engineering.Methods: Dental follicle and associated enamel organs were dissected from the first and second molars of 6 or 7-day-old rats, and then cultured in vitro. Vimentin and cytokeratin were examined to identify its origin. Immunocytochemistry was used to detect the expression of dentin matrix protein1, osteopontin, bone sialoprotein, type I collagen, type III collagen and fibronectin. Results: The cultured rat dental follicle cells show multiformity, elongate shape, spindle shape, and irregular triangle shape, etc. The expression of vimentin was positive while that of cytokeratin was negative. The localization of vimentin was in the cytoplasm surrounding nucleus. Osteopontin, bone sialoprotein, type I collagen, type III collagen and fibronectin were expressed in the rat dental follicle cell and protein1 negatively expressed. Conclusions: The results indicate that the rat dental follicle cells can be cultured in vitro and these cells showing multiformity originate from ectomesenchyme. Rat dental follicle cells can be used as the seeding cells for periodontal tissue engineering.Part2. Effect of Bone morphogenetic protein-2 and Dexamethasone on biological characteristics of rat dental follicle cellsObjective: The present study was conducted to investigate the combined effect of bone morphogenetic protein-2 (BMP-2) and dexamethasone (Dex) on the proliferation and differentiation of RDFCs. Methods: RDFCs of the third passage cells were induced by BMP-2 (100ng/ml), Dex (10-8mol/ml) and a mixture of BMP-2 (100ng/ml) and Dex (10-8mol/ml) were in DMEM. After 1, 3 and 5 days , proliferation of the attached cells were measured with methylthiazol tetrazolium (MTT) method. Differentiation of the attached cells were measured with alkaline phosphatase (ALP) activity measurement kit, mineralization potential was studied by Alizarin Red staining and DMP-1,DSPP,BSP,OCN,Col I synthesis were examined by RT-PCR. Results: The results showed that with treatment of BMP-2, Dex alone could promote the proliferation of RDFCs. BMP-2 had a significant effect on the 3rd day, and Dex had a significant effect on the proliferation on the 1st day. Moreover, greater proliferation of RDFCs was found in combined treatment group at different time compared with individual treatment groups. Treatment with Dex alone could also augment the expression of ALP. The above results suggested that BMP-2 and Dex could synergistically promote the proliferation of RDFCs and its differentiation to osteoblast. Conclusions: The exposure of Dex as well as BMP-2 to RDFCs with an appropriate concentration promotes osteogenic expression without reverse effects on cell proliferation, which indicates the great potential value in cell-based strategy of bone tissue engineering.Part 3. To investigate the adhesion and growth of RDFCs combined ontoβ-tricalcium phosphate (β-TCP) in vitroObjective: To construct a three-dimensional culture model of RDFCs by using porousβ-tricalcium phosphate (β-TCP) as scaffold and RDFCs as seed cells. To investigate the possibility and the biological capability of the reconstruction of the combined tissues by using RDFCs withβ-TCP in vitro. Method: RDFCs of the third passage were induced by BMP-2 (100ng/ml), Dex (10-8mol/ml) and a mixture of BMP-2 (100ng/ml) and Dex (10-8mol/ml) in DMEM. After 3 days, the control cells were collected for combination withβ-TCP scaffold and also cultured in DMEM solution. On the 3rd day and 7th day, RDFCs adhesion and growth were observed by scanning electronic microscope (SEM), the proliferation of the cells was evaluated by cell counting. Results: SEM showed that the multi-porus scaffold had 3 morphologically distinct pore types. The third passage rat dental follicle cells could be adhered and extended and proliferation on theβ-TCP by SEM. Conclusion: The pours scaffoldβ-TCP had good 3-D structure and biocompatibility, which provided safe surface for RDFCs to adhere to and proliferate. It can be used as biomaterials in bone tissue engineering.Part 4 Effect of BMP-2 and Dex on differentiation potential of rat dental follicle cells in vivoObjective: To assess the effect of BMP-2, Dex and BMP-2/Dex on differentiation potential of rat dental follicle Cells in vivo. Method: Rat dental follicle cells induced by BMP-2, Dex and BMP-2/Dex were recombined withβ-TCP scaffolds and transplanted subcutaneously into the dorsal surface of 6-week-old immunocompromised beige mice. The transplants were recovered after 8 weeks. Sections were stained with H&E and Masson. Results: Both HE and Masson staining showed that the formation of bone and osteoid and vascularization in the group of BMP-2/Dex. A small amount of new bone tissues was also observed in the group of BMP-2. However, no bone and osteoid was found in other groups. Conclusion: The exposure of BMP-2/Dex to RDFCs can exert maximum effect on promoting differentitation along the osteoblast-lineage, which provided the experiment evidence for restoration of periodontal defection using tissue engineering methods.