Induction Of Rat Epidermal Stem Cell Differentiation Into Dental Epithelium

Background: Teeth is a specific organ, which consist of enamel, dentin and dental pulp. The outside layer of the tooth, the enamel, is the hardest tissue in the human body. The enamel surrounds another layer of the tooth called the dentin. The dental pulp lies in the middle of the tooth. The pulp contains blood vessels, nerve fibers and other connective tissue. Formation of teeth start at the early development stege of embryo. Two different types of embryonic cells -primitive oral epithelial cells and oral mesenchymal cells cooperate with each other to direct the development of tooth, its structure, size, shape. Primitive oral epithelial cells differentiate into enamel secreted cell-ameloblasts, furthermore, which form enamel organ, finally it form enamel. Otherwise, primitive oral mesenchymal cells diffetantiate into odontoblast, and continue to form dental papilla which finally form dentin and pulp,and dental follicle which finally form cemencum and other adjunctive tissue. Genes active in the embryo tightly control all these process. Tooth replacement occurs throughout life in most non-mammalian vertebrates, but only once in most mammals. Some teeth are never replaced, including part of the human dentition, and mouse dentition. Consequently, if tooth is damaged by decay or trauma, it is possible that damnification will be perpetual and unrestored.As a scientific discipline, the field of tissue engineering was occurred in 15 years ago, which dedicated to the generation of new tissue using the principles of engineering in combination with an understanding and application of the biologic sciences. More recently, the collective knowledge of tooth developmental biology, advances in stem cell research, progress in the understanding of the tooth biomineralization principles, and practice in tissue engineering approaches and biomimetic technology have provided a firm foundation for restoring the post-eruptive loss of ameloblasts and damnification of dental enamel in childrens and adults. Recent data shown tooth contains stem cells, while limited by cell source, proliferative ability and differentiation potential, it is impossible to use tooth-derived stem cell for regeneration of dental tissues until now. Therefore, the new source of plastic stem cell should be considered. It has been shown that no-dental derived stem cells, such as bone morrow derived stem cells, that can acquire characters of dental epithelia under a suitable nich . Skin tissues harbouring stem cells are of epithelial nature , and the presence of stem cells for epithelium regeneration and repair has been demonstrated for several decades. More recently, the accumulative evidences indicated that epidermal stem cells are multipotent and plasticity, which can differentiate into many kinds of cell with other tissue nature phenotypes under suitable environment. Both skin and teeth are ectoderm-derived organ, and the early development of skin and teeth are regulated by same mechanism epithelial-mesenchymal interaction mechanism. So we hypothesize that under the suitable environment skin-derived epidermal stem cell may differentiate into dental epithelium and serve as a source for ameloblasts.Objects:Establishing a effectively intercellular singal transduced induction system, which can promote skin-derived epidermal stem cells diffetentiate into a tissue with characters of dental epithelia,and successfully achieve tissue engineering enamel regeneration.Methods: Base on the isolution of epidermal stem cells, both method induction in vitro or in vivo were used in this experiment:l)Isolation,culture and identify of epidermal stem cells: Epidermal stem cells (EpiSCs) were isolated from ldpn rat by rapidly adhering to rat-tail collagen coated flasks and cultured in Kerantincyto -Suerm Free Medium (K-SFM) . Identification of EpiSCs as following described: The growth state of EpiSCs were recorded and cell grpwth curve was made; the expression of betal-integrin and cytokeratin 19 in EpiSCs were detected by immunhistochemical staining; Cell cycle are examed by Flow Cytometry ;EpiSCs is lablled by a fluorescent dye PKH26 and differentiation potential detected by transferred these cells into sub-renal capsule .2)Induction of EpiSCs diffetentiation by dental papilla and its result analysis: The tooth germs were dissociated into epithelums(Enamel Organ) and mesenchymes (Dental Papilla) after treated with trypsin and collagen(1:1). The dental papilla tissue of 1dpn rat were isolated and directly recombined with PKH26 lablled EpiSCs as experimental group 1 ;The dental epitheliums were continue to digesting to single cells and mixed with PKH26 lablled epidermal stem cells. After centrifugation, the pellets of mixture of dissociated dental epithelial cells and PKH26 lablled EpiSCs were reassociated with the dental papilla as experiment group 2. Both recombinants were co-cultured up to 48 hours in CO₂ incubator. Subsequently , the recombinants were transplaned into sub-renal capsule of adult Sprague-Dawley rat and collected at 2 weeks or 3 weeks after the grafting. Forzen sections are mounted and the histological changes of transplant were observed under fluorescent microscope and combined with H&E staining. The expression of cytokeratin -14 in recombinants were detected by immunhistochemical staining. Full tooth germ, dental epithelial tissue and dental papilla tissue as control group respectively.3) Induction of EpiSCs diffetentiation by full tooth grem cells or tooth germ cell conditioned medium and its result analysis: Full tooth germ cells of new born rat (first molar) were isolated and culture in DMEM, contain 3% Fetal bovine serum .When tooth germ cells grow to 60⁷0 percentage confluence, co-culture it with EpiSCs or directly add the tooth germ cell conditioned medium (TGC-CM) into EpiSCs in order to induce EpiaSCs differentiation toward dental epithelial cell or tissue. The morphological change of cell are observed under optical microscope. Reverse transcription-polymerase chain reaction (RT-PCR) was used to evaluate the expression of ameloblastin mRNA and amelogenin mRNA in cells obtained after co-culture. The ameloblasts and pure EpiSCs as control group 1 or control group 2 respectively.Results:1) The EpiSCs were successfully isolated by rapidly adhering to rat-tail collagen and that they can be expanded in K-SFM .There were postive expressions ofβ₁-integrin and cytokeratin 19 are positive in EpiSCs. It was indicated by cell cycle analysis that cell in G₀/G₁ stage accounted for 81.2% of the cells, suggesting slow cell cycle. Transplants were differentiated and exhibited epidermal-like structure. These data indicated the cell obtained in this experiment with character of epidermal stem cells.2) Recombinants were well developed under the environment of rat sub-renal capsule ,and with normal rigidity and color. It is indicated by H&E histological analysis that thooth like structures were formed and which contain obviously enamel-like structure.Red fluorescent emited by PKH26 were observed in enamel like structure under fluorescent microscope. The expression of cytokeratin -14 in enamel-like structure tissue are positive suggested skin derived EpiSCs participate in the formation of enamel-like structures. While the enamel-like structures were not observed in the group of direct recombinants EpiSC with dental papilla tissue.3) The morphological change of EpiSCs after co-culture with toothgrem cell are observed under optical microscope. These cell are longer than EpiSCs,part of them grow as cluster. The expression of cytokeratin -14 in these cell are positive.RT-PCR indicated the expression of ameloblastin mRNA and amelogenin mRNA in EpiSCs after co-culture are weak postive.This suggested EpiSCs incline to differentiate into dental epithelia.While in group of TGC-CM induction, a little morphological change are observed and cells are tend to death after 60 hours. Conclusions:l)Epidermal stem cells of new born rat are plastic cell, it can differentiate into enamel-like structures under certain environment.2)The dental papilla of 1dpn rat are suitable environment which contain a series of signal molecules which induce differentiation of epidermal stem cell into dental tissue.3) Co-culture epidermal stem cells with tooth grem cells can induce epidermal stem cells differentiate into dental epithelia.