| Objective:To explore the new ideal seed cells of dental pulp regeneration and offer the theory foundation of coculture system, the cells characteristic in Bone Marrow Stem Cells (BMMSCs) and Dental Pulp Cells (DPCs) coculture system, the optimization cells coculture environment and the condition of dental pulp regeneration with coculture system in vivo were investigated.Methods:1 Isolate, culture and identify the BMMSCs and DPCs in vitro. All cells were cocultured in the 0.4μm Transwell plate and divided into three groups:(1) DPCs group, (2) coculture group and (3) BMMSCs group. After 6 days’ coculturing, cells were extracted and detected the mRNA expression of STRO-1 and DSPP by polymerse chain reaction and the protein expression of ALP and CD 146 by western blot. In addition, the growth curves of experiment groups were obtained with the MTT assay.2 To explore the optimization coculture ratio and period, BMMSCs and DPCs were seed into 0.4μm Transwell plate in the ratio of 10:1,1:1,1:5,1:10 and cultured 3,6,9 and 12days respectively. Cells after coculture were extracted and detected the mRNA expression of STRO-1, CD146, ALP, DSPP by Real Time polymerse chain reaction and statistical analysis.3 After mechanical and chemical preparation, the root segments were divided into 4 groups randomly (1) BMMSCs group, (2) DPCs group, (3) coculture group and (4) blank peptide hydrogel group. After the cells being blended in the peptide hydrogel and perfused into the root segments accordingly, the root segments were then transplanted subcutaneously into the immune deficiency mice for 6 weeks. The root segments then were retrieved and made the histological slices and stained with hematine-eosin.Result:1 The isolated Rat BMMSCs is roundness and adherent the culture flask after 24h, showing cells morphology stability with splindle shape and colony formation which is parallel-aligned or vortex arrangement after 3 days. Flow cytometry analyses showed that both mesenchyme stem cells markers CD29, CD44 are positive and the hematopoietic stem cells markers CD45, CD 34 are negative. The mulipotential differentiation have been demonstrated by the staining of alizarin red, oil red O and alcian blue are positive, after the cells culturing in osteogenesis media, adipogenic media and chondrogenesis media for 21 days,21 days and 14days respectively. The passage 3 DPCs show cells morphology stability which is fibroblast-like with splindle shape, and the immunohistochemistry show that keratin staining is negative and vimentin staining is positive, which reveal its mesenchymal origin and can be applied to future experiments.2 Coculture of BMMSCs and DPCs can promote the expression of stem cells marker and proliferation of DPCs, which show its reprogramming tendency. Meanwhile, coculture system can restrain the proliferation and raise the expression of dental pulp cells markers in BMMSCs, which imply BMMSCs differentiation towards DPCs.3 The modifying of coculture ratio and period can affect the cells differentiation significantly. When coculture BMMSCs and DPCs at the ratio of 1:1 and 1:5, the BMMSCs can be induced differentiation and the DPCs expressed stem cells marker efficiently. The expression of dental pulp cells markers in BMMSCs and the stems cells markers in DPCs are significant increase at about 9 days in ex vivo coculture system.4 After the root segments with cells and peptide hydrogel transplanted subcutaneously into the immune deficiency mice for 6 weeks, we found that the dental-pulp like tissue with physiologic structure regenerate in the coculture group root segments, however the regeneration tissue in BMMSCs group and DPCs group is limit in both quality and quantity.Conclusion:1 BMMSCs and DPCs mutually effect in the proliferation, the expression of mRNA and protein in the coculture system. In addition, modifying the coculture condition can control the cells proliferation and differentiation to some extent. With the low proportion of DPCs which is difficult to acquire in clinical can achieve the favorable result in dental pulp regeneration with this coculture system.2 In the BMMSCs and DPCs coculture system, the expression of mRNA and protein can be effected especially when the coculture ratio is 1:1,1:5 and the coculture period is 6-9days.3 Through the result of experiment which transplante root segements subcutaneously into the immune deficiency mice, BMMSCs-DPCs coculture system can be applied as the seed cells in dental pulp regeneration. |
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