| Tooth loss is a common and frequently occurring disease that adversely affects masticatory efficiency, language function, facial esthetics, and psychological health. With the emergence of tissue engineering techniques, the development of natural tooth substitutes may be a prospect, especially the recent isolation of postnatal dental pulp stem cells (DPSCs) in humans,because dental pulp stem cells were able to differentiate toward odontoblast-like cells in under specific culture conditions and could give rise to a regular-shaped dentin–pulp complex-like structures in vivo. Equally as important as the cells in the classic dentin tissue engineering paradigm is the scaffold as a temporary extracellular matrix. An ideal scaffold should provide chemical stability and physical properties matching the surrounding tissue to provide excellent cytocompatibility, and to support cell adhesion, proliferation and differentiation. In addition, the cell proliferation and differentiation are influenced by many factors, including the local microenvironment of the cell, the interaction among cells and intra/extra-cellular signaling molecules.In this paper,we investigated the effect of extracellular matrix(cell sheet constructed by cell-synthesized matrixes, and the scaffold materials) on dental pulp stem cell proliferation and odontogenic differentiation and the role of MAPK signaling pathway mechanism in dental pulp stem cell differentiation and MAPK signaling pathway in dental pulp stem cell responding to different scaffold materials to provide the theory basis for dentin regeneration research.Part 1. Dental pulp stem cells for odontogenic differentiation based on cell sheet teehniquesIn order to verify the role of ascorbic acid on dentin pulp stem cells, we analyzed the proliferation and mineralization activity of dental pulp stem cells, which exposure to conditioned medium of ascorbic acid in culture.The results showed that ascorbic acid treatment promoted the proliferation,differentiation and extracellular matrix synthesis in rat dental pulp stem cells significantly. Then, we used ascorbic acid to fabricate a monolayer cell-matrix membrane (cell sheet) of dental pulp stem cells. To further examine dental pulp stem cells for odontogenic differentiation based on cell sheet teehniques,We compared the ability of dental pulp stem cells for odontogenic differentiation based on cell sheet technology and the current cell seeding technology.The results showed that dental pulp stem cells exhibited higher odontogenic differentiation ability based on cell sheet seeded on CBB than the cell seeded on CBB. It was suggested that the extracellular matrix and interaction among cells in cell sheet played very important roles in dental pulp stem cell odontogenic differerntion and which provided a novel strategy for dental pulp stem cells in dentin tissue engineering.Part 2. Comparison of different three-dimensional scaffold materials for dental pulp stem cells based on dentin tissue engineeringThe present study was designed to select the most appropriate scaffold for dental pulp stem cells based on dentin tissue engineering, through comparing the in vitro and in vivo behavior of dental pulp stem cells seeded on different scaffold materials: i.e. a demineralized dentin matrix (DDM), a ceramic bvine bone (CBB), a acellular small intestinal submucosa (SIS), a poly lactic-glycotic acid (PLGA), and a collagen-hyaluronic acid-chondroitin sulfate (Col-HA-CS). It was found that dental pulp stem cells exhibiting more high levels of adhesion, proliferation, alkaline phosphatase (ALP) activity and mRNA expression of bone sialoprotein (BSP), osteocalcin (OCN), dentin sialophosphoprotein (DSPP) and dentin matrix protein -1 (DMP-1) in natural scaffold biomaterials(DDM,CBB and SIS)than in the composite materials(PLGA and Col-HA-CS). Importantly, a large amount of dentin-like tissue including pulp, odontoblasts predentin and dentin was found in natural DDM and CBB derived from mineralized tissues in vivo, which demonstrated that DDM and CBB derived from mineralized tissues as attractive biomaterials for dentin tissue engineering promoted dental pulp stem cell odontogenic differentiation.It was indicated that the extracellular matrix in the dentin should be taken consideration when designing scaffold material for dentin tissue engineering. Meanwhile, the results present evidences of sorting bimatrials for dentin regeneration research.Part 3. MAPK molecular mechanism in the differentiation of dental pulp stem cellsIn this study, we investigated the contributions of MAPK pathway on the osteogenic differentiation of dental pulp stem cells in differentiation medium by assessing an ALP activity and mineral deposition through inhibition of MAPK pathway. It was demonstrated that the inhibition of ERK1/2 and p38 MAPK pathways resulted lower levels of ALP activity and mineralization in PD98059 and SB203580 groups than in untreated groups significantly.We further investigated the role of MAPK pathway in differentiation and mineralization of dental pulp stem cells on different biomaterials and whether there might be a link between the signaling pathway and the scaffolds.The date indicated the activation of MAPK pathway were regulated by the various scaffolds during the dental pulp stem cell differentiation. In the case of the MAPK, we found that DDM, CBB and SIS could promoted ERK1/2 and p-38 activation more efficiently than PLGA and Co-CS-HA could. Moreover, it was demonstrated that the inhibition of ERK1/2 and p38 MAPK pathways resulted in significantly lower levels of ALP activity of dental pulp stem cells seeded on natural biomaterials in PD98059 and SB203580 groups than in untreated controls.These results indicated that ERK1/2 and p38 MAPK activation regulated the differentiation of dental pulp stem cells and the scaffold-induced increase of dental pulp stem cell ostogenic differentiation is correlated with ERK1/2 and p38 MAPK activation, which might provide a novel strategy that the regulation of cell function could be regulated through MAPK mechanism activation for dentin regeneration and reparation. |
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