The Study Of The Application Of Dental Follicle Cells And Adipose-derived Stromal Cells In Bioengineering Periodontal Tissue-like Structure

The nature of periodontal tissue regeneration is the recapitulation of periodontal development. Therefore, it is of necessity to mimic the microenvironment of periodontal development during the construction of bioengineered periodontal tissue. The dental follicle cells (DFCs) are regarded as the precursors for periodontal tissue, which could differentiate into cementoblasts, fibroblasts and osteoblasts. However, the mechanisms regulating the differentiation of DFCs are still an open question. The possibility of the application of DFCs as seed cells for periodontal tissue engineering is uncertain as well. In addition, adipose-derived stromal cells (ADSCs) have the advantages such as being easy to obtain, sufficient sources, multidifferentiation potential, transplantation without immunological rejection and so on, which have broadly drawn many reseachers'attentions. Then, could ADSCs participate in the process of periodontal tissue regeneration after being induced by the microenvironment of periodontal development? In order to give answers to above questions, the present study is designed to compare dental follicle cells and periodontal ligament cells (PDLCs), to investigate the condition promoting DFCs differentiation, to explore the microenvironment of periodontal development, and finally, to direct the construction of bioengineered periodontal tissue by DFCs and ADSCs respectively. The results of the present study will provide theoretical foundation for the clinical practice to facilitate periodontal regeration in the future.The contents of the present study are as follows:Part One. The comparative study of dental follicle cells and periodontal ligament cellsObjective: To compare the characteristics of dental follicle cells and periodontal ligament cells. Methods: The cryopreserved human DFCs and PDLCs of the 4th passage were recovered and cultured. Cell morphological differences were observed under a phase-contrast microscope, cell surface markers were identified by flow cytometry, and these two kinds of cells were combined with CBB and transplanted subcutaneously in nude mice, the implants were examined after 4 weeks and 6 weeks. Results: Both DFCs and PDLCs displayed fibroblast-like shape. However, DFCs seemed to be shorter than PDLCs, and DFCs grew in the irregular arrangement, whereas PDLCs were paralleled with each other. Both DFCs and PDLCs expressed CD11b,CD34,CD45 negatively, and CD29,CD44,CD90,CD105 positively. The percentage of CD14+ cells in PDLCs were higher than that in DFCs(6.23% VS. 2.72%), but the percentage of STRO-1+ cells in DFCs were higher than that in PDLCs(20.99% VS. 14.1%), and the percentage of MACAM/CD146+ cells in DFCs were significantly higher than that in PDLCs(96.89% VS. 17.11%). The histological examination of implants demonstrated that DFCs and PDLCs could generate similar tissues in vivo. Conclusions: There is homology between DFCs and PDLCs since both of them come from ectomesenchymal stem cells; Both of DFCs and PDLCs contain stem cells, and the amount of undifferentiated mesenchymal stem cells in DFCs is more than that in PDLCs; The differentiations of DFCs and PDLCs need signals from the microenvironment to stimulate.Part Two: The effects of dentin non-collagenous proteins (dNCPs)on dental follicle cellsObjective: To study the effects of dentin matrix on the proliferation and differentiation of dental follicle cells. Methods: DFCs were isolated from SD rats of 6～7dpn, and cells in the experimental group were cocultured with 250μg/ml dNCPs medium. A serial of in vitro studies were performed, including cell morphological observation (phase-contrast microscopy, scanning electron microscopy and transmission electron microscopy), cell proliferation assay (MTT, BrdU incorporation and cell cycle analysis), cell mineralization assay (ALP activity, von Kossa staining ), immunocytometry analysis, RT-PCR test; In the in vivo experiment, a new model involving DFCs cell pellet and dentin carrier was applied, and the constructs were transplanted in the subrenal capsule of adult rats. Results: After dNCPs induction, DFCs presented several features of cementoblast lineages in vitro, as indicated by morphological changes, decreased proliferation, enhanced ALP (alkaline phosphatase) activity and increased matrix mineralization. Besides, the expressions of mineralization- associated proteins and genes were up-regulated after induction. Incubation of treated DFCs pellets in vivo revealed that a large amount of cementum-like tissues were formed within the novel dentin carriers. Conclusions: dNCPs can stimulate DFCs to differentiate into cementoblast lineages. The present study provides new insights into the mechanism of cementogenesis.Part Three: The effects of apical tooth germ cells-conditioned medium(ATGC-CM) on dental follicle cellsObjective: To study the effects of apical tooth germ cells-conditioned medium on the proliferation and differentiation of dental follicle cells. Methods: DFCs were isolated from SD rats of 6～7d , and cells in the experimental group were cocultured with ATGC-CM. Several in vitro experiments were carried out, including MTT assay, cell cycle analysis, ALP activity assay, immuno- cytochemistry analysis; The protocol of in vivo experiment was as same as Part Two. Results: After ATGC-CM induction, cell proliferation was inhibited, ALP activity was enhanced, mineral-forming cells and periodontal fibroblast related proteins were expressed, and bone-like tissues as well as fibers were produced in the dentin carrier. Immunohistological analysis identified the newly formed bone tissues. Conclusions: ATGC-CM contains a lot of bioactive factors responsible for tooth root and periodontal tissue development; ATGC-CM could stimulate DFCs to differentiate into osteoblasts and fibroblasts. Part Four: The application of dental follicle cells and adipose-derived stromal cells in bioengineering periodontal tissue-like structure using the ectopic modelsObjective: To explore the possibility of involving DFCs and ADSCs into the construction of bioengineered periodontal tissue. Methods: dNCPs and ATGC-CM were mixed to induce rat DFCs, then, the induced cells were combined with dentin block, encapsuled by PLGA membrane, and then transplanted in subrenal capsule of adult rats; Rat ADSCs were isolated, cultured and identified; dNCPs and ATGC-CM were mixed to induce rat ADSCs, and three constructing protocols were used as follows: the induced ADSCs were mixed with CBB; the induced ADSCs were combined with dentin block and encapsuled by PLGA membrane; the induced ADSCs were combined with dentin block within demineralized bone tube. And the constructs were transplanted into subrenal capsule of adult rats. Results: After induction, the treated DFCs formed cementum-like tissues along dentin surface, and fibers inserted into the tissue perpendicularly, which displayed cementum/PDL-like structure; The rat ADSCs had the characteristics of mesenchymal stem cells; The induced ADSCs could generate cementum/PDL-like structure along CBB and dentin surface, the structure formed in dentin group was more typical, and bone-like tissues could be produced along the demineralized bone surface. Conclusions: DFCs can be utilized to bioengineer cementum/PDL-like structure after certain induction; The ectopic model for periodontal tissue engineering is feasible; ADSCs belong to mesenchymal stem cells which possess capability of multipotent differentiation; ADSCs can produce cementum/PDL-like structure in the ectopic models after appropriate induction, and will be a kind of important seed cells for periodontal regeneration.