| Dental pulp regeneration is the focus of current research, the most important research method is based on the principle of tissue engineering, which uses the mesenchymal stem cells to conduct the heterotopic and orthotopic tissue regerenation combined with the biological scaffold material. Most of the current studies are prospective histological and animal model exploration, but until now, the dental pulp regeneration with complete histological structure and physiological function has not yet been achieved. Besides, since there are ethical restriction and biological safety risk about the application method of mesenchymal stem cells and the material properties of biological scaffold, further researches are still needed before clinical trials.To realize physiological and functional dental pulp regeneration in clinic, the source of seed cells, the selection of scaffold materials and the effective inductive microenvironment are the key points to be solved. The stem cells from human exfoliated deciduous teeth(SHED) is a kind of odontogenic stem cells in the early growth period derived from deciduous organ, it has good biological properties, including higher proliferation ability, more extensive multi-directions differentiation ability and better immune regulation ability; its clinical source is also more accessible, which is easy for the regeneration and application of autologous tissue. Besides, the cell aggregate emerging in recent years is a kind of advanced tissue engineering technology. It can not only maintain the activity of stem cell and possess the biological scaffold- autocrine intercellular matrix with good performance, but also contain many biological factors required for cell growth and differentiation. Our researches want to define the feasibility to be used for dental pulp regeneration of the full-length root, including the biological performance test on the deciduous teeth dental pulp stem cell aggregate, and the experiment of dentin pulp complex regeneration. Then the animal model treatment experiment is used to explore the therapeutic effect that SHED aggregates are applied to the treatment of young permanent teeth with pulp necrosis. Our results will be expected to provide novel treatment concepts and strategies for the endodontic treatment, and to establish the theoretical foundation and experimental basis on clinical application. Experiment one: The study on biological properties of dental pulp stem cells aggregate from human exfoliated deciduous teethObject: To define the biological behaviors of SHED and the biological properties of SHED aggregate. Method: Stem cells from human exfoliated deciduous teeth(SHED) and dental pulp stem cells(DPSC) were separated and cultivated. Then SHED and DPSC were compared by the method of cellular surface markers, colony-formation ability, proliferation level and multi-directions differentiation ability. Finally SHED aggregates were prepared to detect the biological performance by histological, immunohistochemistry method. Result: Both SHED and DPSC could positively express the surface markers STRO-1, CD90, CD105 and CD146, while the hematopoietic marker CD34 was negative. The colony-formation ability, proliferation level and osteogenic and adipogenic differentiation ability of SHED were higher than those of DPSC. SHED aggregate presented moderate thickness and tough texture; HE staining showed that SHED aggregate contained more undifferentiated cells and more extracellular matrixes; MASSON staining indicated that the cross-linked collagen fibers could form fibrillar network. Conclusion: SHED and its aggregate have good biological properties, which are the ideal seed cells and the potential application mode of dental pulp regeneration.Experiment two: The study on the ectopic reconstruction of dentin-pulp complex using SHED aggregateObject: To explore the tissue-engineering method of ectopic dentin-pulp complex reconstruction by using SHED aggregate. Method: The extracted orthodontic teeth were collected and prepared to make the scaffolds of full-length tooth root after canal disinfection; SHED aggregates were inserted into root canal in experiment group while the calcium hydroxide paste were filled in control group. Then the grafts were implanted into the subcutaneous space on the backs of nude mice. 3 months after transplantation, the mice were euthanized and the roots were removed for histological evaluations. The regeneration tissues were obtained from the root canal, and the cells were separated and cultivated. The biological performance of regeneration tissue were detected by histological and immunohistochemistry method. Meanwhile, the regeneration cells(R-SHED) and SHED were compared by the examinations of cellular surface markers, colony-formation ability, proliferation level and multi-directions differentiation ability. Result: HE staining showed that the whole root canal was almost filled with the pulp-like tissues in experiment group; while the root canal of control group were full of the admixture of calcification mass and granulation tissues. The immunohistochemistry staining showed that the regeneration tissue could positively express the makers of DSPP, CD31 and NESTIN, which indicated the formation of secondary dentin, odontoblast layer, blood vessels and nerve tissue. Moreover, Both R-SHED and SHED could positively and negatively express the same surface markers, at the same time, the clone formation ability, proliferation level and osteogenic and adipogenic differentiation ability of R-SHED are similar to those of SHED. And the immunofluorescence staining showed that most of the regenerated blood vessels and nerves were derived from SHED aggregates. Conclusion: SHED aggregate could ectopically regenerate normal dental pulp-dentin complex in root canal microenvironment.Experiment three: The study on the orthotropic pulp regeneration using dental pulp stem cells aggregate from minipig exfoliated deciduous teethObject: To detect the application effect of the orthotropic pulp regeneration using SPED aggregates in the experimental animal model. Method: The stem cells from mini-pig exfoliated deciduous teeth(SPED) were separated and cultivated. The cellular biological behaviors were observed and analyzed, then SPED aggregates were prepared. The experimental animal model of young permanent teeth with pulp necrosis was established, which were divided into SPED aggregate regeneration group, Apexification group and Healthy control Group. 4 months after transplantation, the pulp vitality of treated tooth were detected, then the mini-pigs were euthanized and the treated tooth were removed to observe the pulp regeneration and apexogenesis using the histological, immunohistochemistry and immunofluorescence examinations. Result: The radiological examination and pulp vitality test indicated the treated teeth root with SPED aggregates could allow for continued growth and present certain sensitive response to mechanical stimuli.HE staining showed that pulp-like tissues was regenerated in the entire root canal in SPED aggregate regeneration group, while immunohistochemistry and immunofluorescence staining showed the regeneration tissue could positively express the makers of CD31, NESTIN, CGRP, TRPV1, TRPM8 similar to healthy pulp. But the treated roots of apexification group were were full of the admixture of calcification mass and granulation tissues. Conclusion: SPED aggregate can realize the orthotopic dental pulp regeneration of full length root canal, and the regenerated tissue not only had the normal morphological structure, but also had the physiological functions of dentin formation, dental pulp vitality and apexogenesis. |
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