| Dental papilla is a temporal tissue in tooth development. It consists of undifferentiated mesenchymal cells which are distributed below the enamel organ. The formation of Primary dentin is before the completion of tooth development. And the formation of primary dentin is controlled by the interactions of dental epithelial cells and mesenchymal cells. The regulation is mediated by the basement membrane. The basement membrane consists of a thin layer of extracellular matrix which is between dental epithelial and dental mesenchymal. It participates in the differentiation of variety kinds of cells and the forming of cell function.Basement membrane is the nature scaffold of inducing odontoblast differentiation and dentin formation. It is a fiber network structure which is formed by dental epithelial and dental mesenchymal inducing by the signal of dental epithelial organ. But dental epithelial organ gradually shrinks away as the complete maturation of tooth in adult body. It is very difficult to find dental epithelial components in adult body. This is bad for the formation of basement membrane. It is so crucial to find a way to imitate the function which is induced by dental epithelial organ that can be beneficial for the formation of basement membrane and dentin. The previous study shows that millipore filter combined with dentin matrix protein or TGF-βcan induce odontoblast differentiation and dentin formation on the model of dental pulp exposure of dog. It promotes the defense of reparative dentin on the lesion site. Can we use the similar method to study for tissue engineering of dentin. We suppose that a membrane material can be found to play the part of functions of the basement membrane that can induce the formation of tubular dentin in the dentin bio-regeneration. In order to achieve our purpose, we design our experiments as follows:1. Dental papilla tissue and TGF-β1 combined with the microporous membrane after the induction of dentin-like structure combined with in vitro regeneration4d after birth the young mice were killed after 75% ethanol, immersed in the 15s. Separation of upper and lower under sterile conditions, under a stereomicroscope PBS solution will pick open jaw plate, separating the first and second molars, the teeth and soft tissue to embryos outside the net, the mechanical separation, and tooth enamel nipple, the isolated dental papilla group and TGF-βcombined with the porous membrane combination, placed on Millicell culture insert dish. Remove the culture week, 4% paraformaldehyde for 24 hours for scanning electron microscopy, HE and immunohistochemical staining. The results show that the dental papilla cells attached at the membrane surface, the growth of cell processes inserted into the membrane pore size in combination with a solid membrane; HE staining showed that dental papilla tissue combined with the microporous membrane in close along the porous membrane observed were layered arrangement of cells, cell height of about the same, some cells were polarized state perpendicular to the membrane surface. Immunofluorescence staining and DMP-1,DSP visible surface of the cell membrane adhesion positive2. Dental papilla tissue and TGF-β1 combined with the microporous membrane after the induction of dentin-like structure combined with in vivo regeneration4d after birth the young mice were killed after 75% ethanol, immersed in the 15s. Separation of upper and lower under sterile conditions, under a stereomicroscope PBS solution will pick open jaw plate, separating the first and second molars, the teeth and soft tissue to embryos outside the net, the mechanical separation, and tooth enamel nipple. Cut under a stereomicroscope dental papilla, the prepared microporous membrane was inserted, and placed in DMEM were incubated at 37℃in an incubator 24 hours of prepared recombinant was used for transplantation. 300g SD rats exposed to the kidney, the re-implantation under the kidney capsule. Carotid 14 days after feeding rats were killed off, the grafts used for detection. The results shows that mineralization in the porous membrane layer is formed above the surface, uniform thickness, mineralization density structure. HE staining showed a large number of graft mineralized matrix deposition along the porous membrane, the thickness of mineralization than the same polarization in the substrate surface odontoblast-like cell distribution. DSP and DMP-1 immunofluorescence staining showed mineralization in the matrix near the odontoblast-like cells positive zonal distribution.3. Dental papilla cells with TGF-β1 combined with the microporous membrane after the induction of dentin-like structure combined with in vitro regeneration4d after birth the pups were isolated dental papilla tissue, it will cut into 1mm3 dental papilla tissue the size of tissue, digestion of primary cultured dental papilla cells. Covered after the passage. When the second generation of cells to 80 ~ 90% of the shop, digestion centrifuge into college, moved to the prepared microporous filters placed together in DMEM containing serum, 37℃, 5% CO2 incubator 7d. Remove the culture, and 4% paraformaldehyde 48h, for scanning electron microscopy, HE and immunohistochemical staining. The results showed that cell clusters grew well in the microporous membrane, part of the cell processes in the deep pore size membrane, HE staining showed cell clusters with clear boundaries porous membrane, microporous membrane of the cells close to the polarization, the cell perpendicular to the filter membrane arrangement of tall columnar cell shape changes occur. Immunofluorescence staining DSP and DMP-1 distribution along the porous membrane of high columnar cells were weakly positive differentiation.4. Dental papilla cells with TGF-β1 combined with the microporous membrane after the induction of dentin-like structure combined with in vivo regeneration4d after birth the pups were isolated dental papilla tissue, it will cut into 1mm3 dental papilla tissue the size of tissue, digestion of primary cultured dental papilla cells. Covered after the passage. When the second generation of cells to 80 ~ 90% of the shop, digestion centrifuge into college, moved to the prepared microporous filters, placed in DMEM were incubated at 37℃in an incubator 24 hours of prepared recombinant was used for transplantation . W 300g SD rats exposed to the kidney, the re-implantation under the kidney capsule. Carotid 14 days after feeding rats were killed off, the grafts used for detection. The results detected a lot of mineralized matrix deposition along the porous membrane, a more consistent substrate thickness has been mineralized part of the present tubular dentin-like structure. Neatly arranged in the substrate surface of the odontoblast-like cell distribution. DSP and DMP-1 immunofluorescence staining of columnar cells near the substrate with high expression in soft tissue around the occasionally expressed. |
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