Biological Characteristic Study On The Apical Bud Cells Of Rat Incisors And Molar Hertwig's Epithelial Root Sheath Cells

Tooth development is the result of the interactions between ectoderm epithelium and mesenchyme from neural crest. Human tooth development is a reciprocal and consecutive process, which includes bud, cap, bell stages, tooth root development and tooth eruption. As human tooth and rodent molar, after completion of tooth crown, the inner and outer epithelium of enamel organ proliferate at the cervical loop and form a bilayered epithelial sheath, known as Hertwig's epithelial root sheath (HERS), which is the initiation of root development. However, the rodent incisors grow and erupt continuously the whole life. It is the adult stem cells residing on the apical bud of the incisors that leads to the continuous growth. What reasons exist in these two epithelia, the apical bud and HERS cells, resulting in the two totaly different developmental modals? The aim of this study is to explore the biological characters and differences between the two cells, in order to provide experimental evidence for the studies on the mechanism of root development.In this study, histology, immunohistochemistry, cell culture, RT-PCR, in vivo implantation and proteomics methods were used to conduct following four sections of experimental study. The major contents and results are as follows:Section 1 Study on apical bud and HERS tissuesIn experiment 1, PN0-10d SD rats'apical bud and HERS structures were systematically observed by histological method. It was found that apical bud resided on the labial tip of the mandibular incisor, which had formed at birth. Inner, outer epithelial and stellate reticulum cells comprised the apical bud and the stellate reticulum cells were pretty populous, which caused a spherical bulge between the dental papilla and follicle tissues. At PN 7d, HERS began to develop at the molar crown cervix. The inner and outer epithelia fused and developed to HERS, and the stellate reticulum cells dissappeared, leading to a continuous and flat shape.In experiment 2, the expression of some molecules, such as FGF10, Notch1 and BMPs were detected in/around the apical bud and HERS tissues by immunohistochemistry. It was found that FGF10 was possitive in the dental papilla close to the apical bud, whereas none was existed around the HERS. Notch1 were expressed in the apical bud cells. BMPs was possitive in the HERS cells. The results indicated that some certain molecules were differently expressed by the two cells, although they came from the common origin, the enamel organ.In experiment 3, the apical bud and HERS's ultrastructures were observed by transmission electron micrograph(TEM). Some apical bud cells'membrane thickened and formed brige-like cells connection. Some cells contained plenty middle fiber and presented as mucus-like epithelia phenotype. The bilayed HERS cells lined up regularly, the inner were column and the outer were flat cells. The HERS cells connected tightly by membrane insertion and fusion. Section 2 Study on the biological characters of apical bud and HERS cellsIn experiment 1, mechanical separation of the incisor germs'labial tip and molar germs'cervical portion, primary mixed culture by enzyme digestion and differential trypsinization were operated and the two epithelial cells were cultured with 10% fetal bovine serum(FBS) DMEM/F12. It was found that primary cells grew well and proliferated rapidly. Purified epithelial cells were obtained after three times of differential trypsinization, which were confirmed by immunocytochemistry.In experiment 2, the biological characters of purified apical bud and HERS cells were investigated primarily by MTT, flow cytometry (FCM), immunocyto- chemistry, alizarin red and RT-PCR. It was found that Amelogenin, Ameloblastin and Notch1 were expressed by apical bud cells, whereas BMP2,4 were expressed by HERS cells. Apical bud cells cultured in vitro had an ability to mineralization formation.Section 3 Comparative proteomics between apical bud and HERS cellsIn experiment 1, the proteins expressed by apical bud and HERS cells were investigated by two-dimensional electrophoresis. It was found that 145 differential proteins appeared. Compared to apical bud cells, 36 new spots appeared, 23 spots disappeared, 68 spots were up-regulated and 18 were down-regulated in the HERS cells.In experiment 2, differential proteins obtained from the above were investigated by peptide mass fingerprinting method. It was found that 10 proteins were identified, whose functions involved in the aspects of redox regulation of the cell, Catalysis of the NADPH-dependent reduction of 3-deoxyglucosone, and so on.Section 4 Study on the cell pellet of the apical bud or HERS cells combined by dental papilla cells implanted in vivoIn this experiment, apical bud cells and HERS cells were recombined respectively with incisor or molar dental papilla cells and implanted into the rats'renal capsules. It was found that apical bud and incisor papilla cells pellet developed into well-formed enamel and dentin tissues, whereas the combination of HERS cells and molar papilla cells produced bone-like dentin tissues. The result demonstrated that apical bud and dental papilla cells could induce each other, resulting in ameloblats and odontoblasts differentiation, and eventually form dental structures.