Experimental Study On The Role Of Rat Molar Hertwig's Epithelial Root Sheath In Tooth Root Development

Human tooth development is a consecutive process, which includes bud stage, cap stage, bell stage and tooth root development. After completion of tooth crown, the inner and outer epithelium of enamel organ proliferate at the cervical loop, grow towards the future root tip, and form a bilayered epithelial sheath, which is called as Hertwig's epithelial root sheath (HERS). Tooth development is the result of the interactions between ectoderm epithelium and mesenchyme from neural crest, and tooth root development is regulated by the interactions between HERS and its surrounding ectoderm mesenchyme. HERS may play important role in tooth root development, for example, HERS induces dental papilla cells to differentiate into odontoblasts to form root dentin; HERS disrupts at the mineralization of dentin and contact with the dental sac cells, which are then induced to differentiate into cementoblasts to form cement; moreover, HERS may directly participate in cement formation.As HERS is a transient structure in tooth root development and comprises few epithelial cells which is surrounded by mesenchyme, it is difficult to separate and culture HERS cells in vitro, which result in little available information on the biological characteristics and precise roles of HERS in tooth root development. The aims of this study are to find suitable opportunity in tooth root development to separate and culture HERS cells, and further investigate the biological characteristics of HERS cells and the precise role of HERS in tooth root development, so as to provide experimental and theoretical bases for further investigating tooth root development and tissue engineering of tooth root.In this study, histology, immunohistochemistry, cell culture, RT-PCR, implantation in vivo and three-dimensional culture in vitro were used to conduct following four sections of experimental study. The major contents and results are as follows:Section 1 Observation of HERS in rat molar root developmentIn experiment 1, developmental time series of the mandibular first molar root of postnatal (PN) SD rat was systematically observed by histological anatomy and the biological behaviour of HERS was observed by immunohistochemistry. It was found that at PN 5d, HERS began to form and tooth root development initiated; at PN 15d, at root furcation and dental cervix, hard tissue of tooth root started to form and HERS began to disrupt; HERS cells were prolonged while disrupting and assumed round or cubical shape after disruption; at PN 35d, HERS fully disrupted and tooth root development completed.In experiment 2, proliferative ability of HERS and its surrounding mesenchyme were detected by immunohistochemistry. It was found that at PN 5d and 7-8d, HERS had powerful proliferative ability; at PN 15d, the apical end of HERS maintained powerful proliferative ability, but the proliferative ability of HERS at the cervical portion of tooth germ decreased and began to disrupt; at PN 25d, the proliferating cells in root tip were mainly mesenchymal cells; the mesenchyme surrounding HERS kept powerful proliferative ability all along tooth root development. These results demonstrate that the disruption of HERS may result from the difference in proliferative ability between HERS and the surrounding mesenchyme. In consideration of the growth, proleferative ability and the difficulty of operating, PN 7-8d is chosen as a suitable opportunity to separate and culture HERS cells.Section 2 Study on the separation, culture and biological characteristics of HERS cellsIn experiment 1, mechanical separation of the apical portion of tooth germ (APTG), primary mixed culture by enzyme digestion, differential digestion to remove fibroblast-like cells and continuous culture with keratinocyte serum-free medium (KSFM) were used to separate, culture and purify HERS cells. It was found that APTG contained dental papilla, HERS and dental sac tissue; the cells of primary culture comprised epithelial-like and fibroblast-like cells; through three differential digestions and continuous culture with KSFM, fibroblast-like cells were completely removed and the adherent cells were all epithelial-like cells; immunocytochemistry test showed that the epithelial-like cells derived from epithelium tissue and RT-PCR analyses demonstrated that the purified epithelial-like cells were not mixed with mesenchymal cells. These results demonstrate that cultured HERS cells are purified, which provides bases for further investigating the biological characteristics and function of HERS cells.In experiment 2, the biological characteristics of purified HERS cells were investigated by observing through scanning electron microscope and analyzing by MTT and RT-PCR. It was found that there were developed microvilli and cell conjunction on HERS cells surface; compared with dental mesenchymal cells, HERS cells proliferated slowly; HERS cells deposited calcium salts when cultured with mineralization inducing fluid; except for ameloblastin (AMBN) and osteocalcin (OCN) mRNA, HERS cells, being different from enamel organ cells, did not expressed the other tooth mineralization-associated protein mRNA. These results show that HERS cells assume typical surface characteristics of epithelial cells, and although deriving from enamel organ cells, HERS cells are different from enamel organ cells.Section 3 Interactions between HERS cells and mesenchymal cells of APTGIn experiment 1, conditioned media (CM) of HERS cells and mesenchymal cells of APTG were collected respectively and used to culture each other, and growth curves were examined to analysis the interactions in proliferation between these two kinds of cells. It was found that when cultured with CM of HERS cells, the growth and proliferation of mesenchymal cells of APTG had no significant change; however, when cultured with CM of mesenchymal cells of APTG, the growth and proliferation of HERS cells increased significantly. Our data show that HERS cells have no significant effect on the proliferation of mesenchymal cells of APTG; otherwise, mesenchymal cells of APTG can significantly facilitate the proliferation of HERS cells. In addition, the experimental model for investigating interactions between HERS cells and dental mesenchymal cells is successfully established in this experiment.In experiment 2, alkaline phosphatase (ALP) activity analyses, RT-PCR and implantation of cell pellet in vivo were used to evaluate the effect of HERS cell on the diffierentiation of mesenchymal cells of APTG. It was found that ALP activity and mRNA expression of dentin dentin sialophosphoprotein (DSPP), dentin matrix protein 1(DMP1), bone sialoprotein (BSP), osteopontin(OPN), OCN and ALP of mesenchymal cells of APTG, which was cultured with CM of HERS cells, increased; odontoblast differentiation and tubular dentin formation in vivo increased. These results demonstrate that HERS can facilitate cell differentiation of mesenchymal cells of APTG.Section 4 Study on the role of HERS in tooth germ and APTG implantation in vivo and culture in vitroIn experiment 1, intact mandibular first molar tooth germs of PN 7-8d SD rat were implanted into renal capsule, omentum and subcutaneous tissue of the mother rat. It was found that the implanted tooth germs formed tooth root and periodontal tissue with well structure and shape without evident immunologic rejection and inflammatory reaction in all the implantation sites, which revealed that tooth root can develop in allotopic planting enviroment, of which the key is the opportunity of tooth germs, namely the initiation of root development. It also indicates that HERS is an indispensable component in tooth root development and to culture tooth germ of the son rat in the mother rat is a good model for investigating tooth development and culturing tissue-engineered tooth in vivo.In experiment 2, the mandibular first molar germs of PN 7-8d SD rat were cut into coronal and apical portions, and each portion was analyzed with flow cytometry and implanted into renal capsule of the mother rat. It was found that most cells of APTG were arrested in G0G1 stage and many cells of the coronal portion were in S and G2 stage; APTG developed tooth root and periodontal tissue with well structure and shape, but the coronal portion of tooth germ did not. These results show that APTG of PN 7-8d rat is in the preparation period for tooth root development and is the basic tissue of tooth root development. In experiment 3, mandibular first molar tooth germs of PN 7-8d SD rat were cultured in vitro for up to 24 days. It was found that the volume and structure of tooth germs could maintained for about 12 days, and then decreased; all through the culture in vitro, tooth germs did not generate tooth root and periodontal tissue. This experiment provides a culture model in vitro for postnatal tooth germ, but owing to the complexity of tooth root development regulation, tooth germ can not continue to develop tooth root in this culture model.In experiment 4, HERS was digested into single cells or fragments by different methods, and pelleted together with dental papilla cells and dental sac cells by centrifugalization. The cell pellets were cultured in vitro for 4h and then implanted into renal capsule of the mother rat. It was found that the pellet containing fragments of HERS generated pieces of tooth root and periodontal tissue, but the pellet containing single cells of HERS just formed tubular dentin. This result indicates that the bilayered epithelial structure of HERS is important for conducting normal root development, and once missing bilayered epithelial structure, HERS just maintains limited ability to conduct tooth root formation.In experiment 5, the apical tissue of tooth root of PN 25d rat was isolated and implanted into renal capsule of the mother rat. After growing for 4w, the apical tissue of tooth root grew tooth structure like root tip, which demonstrates that the apical tissue of tooth root of PN 25d rat still has the capacbility of forming tooth root and periodontal tissue, and this capability degrade with HERS disruption.