Experimental Study On Odontogenic Capability Of Developing Apical Complex

Mammalian tooth root development is a long-term process which include several stage: tooth root initiation, root elongation and periodontal attachment establishment, tooth eruption, establishment of occlusal relationship and completion of root development. However, numerous studies focus on the the stage of tooth root initiation, little information is available on the subsequent stages, refer to the stage after root intionation to root completion (further root developmental stage). During this stage, the tooth along with the periodontium forms a functional unit and is anchored in the alveolar bone. Acting as putative root growth center, apical end of developing root proliferates throughout the period of root development, promoting the elongation of root along the apical direction coincident with periodontium development. Clinically, apexification is widely utilized as an approach to induce the continued apical development of incomplete root in teeth with necrotic pulp, implying the enormous independent developmental potential of apical root tissue in situ.The development of tooth root and periodontium involves a complicated sequence of events and requires the participation of various types of cells. In a constitutive manner, the elongating apical portion of developing root contains a bilayered epithelial sheath termed Hertwig's epithelial root sheath (HERS) which subdivides the adjacent dental ectomesenchymal tissues into dental papilla and dental follicle. Despite the heterogeneity feature of this region, it exists as an inseparable integrity where the interaction and function of these components should be prerequisite for the establishment of structurally intact root/periodontal complex. Therefore, the apical portion of developing root seems to be a developmentally viable complex comprising multiple cell types closely related and indispensable of one another. The above clinical and developmental phenomena fascinate us to characterize this developing structure and uncover its possible roles in regenerating tooth root and supporting structure by tissue engineering techniques, which remains an intriguing possibility. Preliminarily we term it as developing apical complex (DAC) of tooth root and regard it as a functional integrity in continuing root and periodontium development.In this study we characterized the developing apical complex (DAC) of the rat molar root in situ, followed by evaluating the proliferation and differentiation features of the cells from this region. Using an ectopic transplantation model, we demonstrated that both isolated tissues and cultured cells of the DAC can independently generate tooth root and periodontium like tissues in vivo, mimicking normal root/periodontal complex structures in situ.Section 1 Histological observation of developing apical complex(DAC) in situIn experiment 1, the histological characteristic of apical tissue of the mandibular first molar root during the different root developmental stage of SD rat was systematically observed by histology and the biological behaviour of HERS was observed by immunohistochemistry. It was found that at PN 8d, HERS began to form and tooth root development initiated, the HERS is continuous with the dental epithelium of the crown; at PN21d, the root of the first molar had elongated further and reached approximately one-half of the complete root length. The HERS located beside the root had lost its continuity. In contrast, HERS in the apical end of the root dentin and epithelial diaphragm remained intact. Except for the apparent subdivision position of HERS, no boundary of dental papilla and follicle was observed; at PN 35d, HERS fully disrupted and tooth root development completed.In experiment 2, the histological characteristics of developing apical complex(DAC) in situ were detected by histology and immunohistochemistry. The results showed that the cells in DAC were denser than those in the pulp, especially in the sites adjacent to HERS. More numerous STRO-1 and PCNA positive cells were detected in DAC than in DP. The expression of various proteins associated with dentin, bone, or cementum formation were detected in DAC.Section 2 Study on the separation, culture and biological characteristics of DAC cellsIn experiment 1, DAC tissue was mechanically separated and the presence of HERS was confirmed by immunohistochemical stain. Primary DAC and DP cells were obtained by enzyme digestion. It was found that DAC tissue contained HERS and mesenchymal tissue. Immunocytochemistry test showed that the isolated DAC tissue contained intact HERS, suggesting that the separation method used in this experiment is available. Primary DAC cells comprised epithelial-like and fibroblast-like cells, in contrast, primary DP cells contained fibroblast-like cells exclusively.In experiment 2, proliferation activity of DAC cells were investigated by cell number, bromodeoxyuridine (BrdU) incorporation, and flow cytometry. It was found that DAC cells showed a fast proliferation stage during initial culture, with the total number of which statistically preceding that of DP cells until day 10. In addition, DAC cells showed more than 4-fold enrichment of BrdU-uptaking cells with comparison to DP cells. The cell cycle distributions analysis by the flow cytometry showed a higher percentage of cells in S (23.8%) and G2 (5.9%) phases and a lower percentage in G1 phase (70.3%) compared with DP cells (x2=20.4, P<0.01), while almost all DP cells stayed in G1 phase.In experiment 3, mineralization potential of DAC cells in culture was evaluated by alkaline phosphatase (ALP) activity, alizarin red staining and RT-PCR. It was found that DAC cells showed about 4-fold higher ALP activity than DP cells at day 14. Obvious alizarin red-positive nodules appeared in DAC cells at day 14 and increased remarkably with culture time. However, no alizarin red-positive grains were detected in DP cells at day 14. RT-PCR evaluation reflected a tendancy in accordance with above results. The DAC cells, but not DP cells, strongly express OPN, OCN, BSP and ALP, which were reported as cemento/osteoblast related markers. Both groups expressed odontoblast-specific markers DSPP and DMP-1.Section 3 Both DAC tissue and cells implantation in vivo and the effect of HERS on histological appearance of the explantsIn experiment 1, to assess the developmental capacity of DAC ex situ, we transplanted isolated DAC into the renal capsules of rat host using DP implants as a control. The results showed that after 4-week incubation in vivo, DAC grafts formed typical root like structures with supporting tissues. In histological view, most explants were highly ordered with a well-formed dentin-pulp complex containing identifiable dentinal tubules and pulp cavities. A layer of unmineralized predentine was clearly observed between mineralized dentine and odontoblasts, while newly formed cementum-like structure with fibrous tissue were lining over the dentin surface. The highly ordered structure observed in DAC explants resembled the profiles of normal root-periodontal complex formed during development. In contrast, DP implants only formed irregular dentin-pulp complex like structures with no distinct tubular structure.In experiment 2, the purified epithelial-like and fibroblast-like DAC cells were obtained by differential digestion and were identified by western blot. DACCs were produced by contact coculture of 3rd passage epithelial- and mesenchymal-like DAC cells, while DACCs-M were produced by 3rd purified mesenchymal-like cells. Both cells groups were recombined with CBB scaffolds and transplanted into the renal capsules of rat hosts. The results showed that nearly half of DACCs grafts generated root-periodontal complex like tissue which included dentin-, cementum-, periodontal ligament- and bone-like tissues. Some explants did not form such intact structures but remained cementum-like deposits with fibrous bundle embedded in them, similar to periodontal ligament in situ. However, in DACCs-M group, abundant osteodentin-like tissue can be observed and no cementum-like but only fibrous tissue formed at the surface of osteoinductive CBB.Section 4 A comparative study of DAC and apical tissue of both initial and completive stage of root development in vivo and in vitroIn experiment 1, the primary DAC cells and apical tissue cells of both initial and completive stage of root development were obtained by mechanical separation and enzyme digestion. Proliferation and mineralization potential of DAC cells and apical tissue cells of both initial and completive stage of root development were investigated by cell number, flow cytometry, ALP activity, alizarin red staining and RT-PCR. It was found that DAC and apical tissue cells of PN8d showed similary higher proliferation and mineralization potential than cells of completion stage in vitro. There is a significant difference of gene expression among DAC and other groups. The expression of genes involved in the odontoblast, cementoblast and osteoblast lineage were found to be expressed in DAC and apical cells of completion stage. In contrast, only the genes related to the osteoblast, such as OPN and BSP were detected in the apical tissue of PN8d. However, no marker of mature osteoblasts-OCN expression was detected in apical cells of this stage.In experiment 2, to compare the difference on differentiation potential and odontogenic capability of DAC cells and apical tissue cells of both initial and completive stage of root at different development stage in vivo, the primary DAC cells were recombined with CBB scaffolds and transplanted into the renal capsules of rat hosts. The results showed that both DAC and apical tissue cells of PN8d implants developed into root and periodotium like structure, containing cementum like fragments on the surface of CBB carrier with fibrous tissue adjacent or inserted into newly formed cementum and dentin like structure with identifiable dentinal tube. In contrast, no such tissues were formed in the transplants of PN35d.