The Research Of DPSCs And PDLSCs'Microenvirnment Construction By Cell Sheet Engineering Techniques

Dental pulp stem cells and periodontal ligament stem cells are two of the important stem cells in tissue engineering. Compared with other stem cells(SHEDs, SCAP, DFSCs et al.) in tooth tissue engineering, DPSCs and PDLSCs are superior in age limit, tissue storage and less trauma. Their high proliferation rate, multiple differentiation potential and their combination in both dental and periodontal regeneration make them two of the most promising stem cells in tissue engineering. This experiments established a periodontal ligament engineering model by cell-sheet technique and make DPSC and PDLSC'conditioned medium to simulate different extracellular microenvironment. Then ,co-cultured DPSCs/PDLSCs with different conditioned medium to find out the interaction. At last, used a new method to establish PDLSCs -DPSCs-HA/TCP models by cell sheet technique and cultured this compound in vivo to find what result would be get.Part One: Periodontal ligament stem cell sheet makingThe PDLSCs were isolated by combined tissue block and enzymatic digestion method and cell's state was observed by invert microscope. When PDLSCs reached the 4th passage, they were seeded into culture dishes. After 5 weeks culturing, single cells turned into a united cell sheet which can be operated by tools and cells in which were well stretched and multi-layered. Then this cell sheet was wrapped onto HA/TCP scaffold and transplanted subcutaneously into the side surface of immunocompromised mice. 4 weeks after transplantation , the human PDLSCs were able to form cementum on the surface of HA/TCP carrier and periodontal ligaments with micrangiums formation in it.Pare Two: The effect of DPSCs/PDLSCs cultured in their respective microenvironmentWe isolated DPSCs, PDLSCs and prepared their conditioned medium. Then we set 4 groups: DPSCs were exposed to PDLSC conditioned medium(PDL-CM)and DPSC conditioned medium(DP-CM) respectively(named group D'and D ); PDLSCs were exposed to DP-CM and PLC-CM respectively(named group P'and P).Morphological appearance, proliferation rate,alkaline phosphatase(ALP) activity, gene expression of osteoblast, fibroblast of four groups were evaluated. Realtime PCR's results showed that group D'expressed more mRNA for osteoblast, fibroblast markers, DMP,DSP,ColI,ColIII,than group D while P'expressed less osteoblast and fibroblast markers, ColI,OCN,scleraxis than P . Proliferation rate and ALP activity showed the same result between group D'vs. group D ; group P'vs. group P.From all the data above we can infer that PDL-CM can promote DPSCs'proliferation and differentiation while DP-CM can inhibit PDLSCs'proliferation and differentiation.Part Three: Establishment of PDLSC sheet-DPSC-HA/TCP complex in vivoHA/TCP carrier mixed with DPSCs were cultured in bioreactor. 10 days later, moved out HA/TCP carriers and wrapped with PDLSC sheets, then, transplanted these compounds into nude mice subcutaneously. Six weeks after transplantation, histological results showed that the human DPSCs were able to form dentin like mineralizing matrix in the outside and inside surface of HA/TCP carrier, while the mineralizing matrix in the outside surface were much more than the inside surface. Also, in the region that HA/TCP carrier attached with periodontal ligament like tissue, DPSCs showed more activity by secreting more mineralizing matrix and nucleus of DPSCs were biger and deeper dyeing than normal while the non-attached region showed little matrix. This part further confirms the fact that PDLSCs can induce and promote DPSCs.