Odontoblast-like Cell Differentiation Of Cranial Neural Crest Stem Cell In Vitro Culture And In Vivo Implants With Scaffolds

Tooth loss due to periodontal disease, dental caries, trauma, or other disorders continues to affect most adults adversely at some time in their lives. Courrent therapies mostly rely on artificial material to reconstruct tooth structure, but its function could not be restorated completely. With the rapid development of life science and tissue engineering, A biological tooth substitute that could replace lost teeth would provide a vital alternative to currently available clinical treatments. In this study, depending on the courrent study results of tooth development, stem cell biology and tissue engineering, cranial neural crest stem cells (CNCSCs) were cultured, and odontoblast-like cell differentiation of CNCSC were induced in vitro and in vivo, in order to investigate the feasibility of regenerating tooth tissue by seeding CNCSC onto biodegradable collagen-chitosan polymeric scaffolds, providing the basis of theoretics and approaches for engineering tooth furthely.Cranial neural tubes, dissected from mouse E9d, were explanted onto fibronectin- coated dishes. CNCSCs emigrated from the explanted neural tubes, cultured in a free-serum modified DMEM/F12 medium. Biological characteristics of CNCSC were detected by morphology, nuclear labeled with BurdU and immunocytochemistry. Fibroblast-like CNCSCs demonstrated the characteristics of stem cell, such as clonality, self-renewal, and multipotentiality. The result of immunocytochemical stain showed that CNCSC expressed HNK-1 antigen. CNCSC were cultured successfully. Providing a experimental basis for study on tooth/jaw-like differention of CNCSC in vitro, especially, providing cell source for investigating tooth/jaw regeneration.Cranial neural crest explants were cultured in free-serum medium containing modified DMEM/F12 and different doses of FGF8.The differentiation type of CNCSC were determined by In situ hybridization for Hoxa2 and immunocytochemistry for vimentin. Pre-emigrating CNCSC demonstrated the negative Hoxa2 stain and positive vimentin stain after treated by lOOng/ml FGF8. On the early stage of CNCSC emigration, the first branchial arch phenotype of CNCSC could be induced by FGF8.This experiment could provide in vitro model for study on the mechanisam of tooth/jaw regeneration.To study the effects of TGFP l,BMP2,dentin matrix non-collagen protein(DMNCP) on CNCSC differentiating into odontoblast-like cell phenotype.The expression of collagen I/dentin sialophosphoprotein (DSPP), the activating of alkaline phosphatase (ALP), and the forming of calcium accumulation were analyzed by morphology, immunocytochemistry, Alizarin Red staining. The results showed that DMNCP could induce odontoblast-like cell differentiation of CNCSC, demonstrating positive expression of collagen type I / DSPP, increasing activating of ALP, and forming calcium nodules. TGF ï¿¡ 1,BMP2 could enforce the effects of induction .In this experiment ,we built in vitro model odontoblast-like cell phenotypy differentiation of CNCSC induced by DMNCP/ TGF P l/BMP2,providing experiment approaches for engineering tooth by seeding CNCSC onto the collagen-chitosan polymeric scaffolds .In vitro cell cultures in combination with histological assays in vivo implants into immunodeficiency mouse model, characteristics of CNCSC , seeded onto the scaffolds ,were detected by morphology, nuclear labeled with BurdU ,histology,and immunohistochemical staining.The results showed that CNCSC have excellent adhension and proliferation capability on chitosan scaffolds,collagen could improve the adhension and proliferation of CNCSC on scaffolds. 1 or 2months later,implants with CNCSC induced by DMNCP/ TGFP 1/BMP2 ,were analyzed by HE and immunohistochemical staining. While scaffolds were destroyed, columnarcells possessing polarized nuclei and matrix produced by cells were seen in some regions. Immunohistochemical staining demonstrated that collagen type I andDSPP were expressed throughout the cytoplasm and matrix produced by cells. According to tissue engineering approaches,this experiment furtherly verified odontoblast-like cell phenotypy differentiation ofCNCSC.lt is feasibility investigating tooth regeneration with CNCSC.In this Study, after CNCSC were successfully obtained , odontoblast-like cell phenotypy differentiation of CNCSC were induced by FGF8/ DMNCP/ TGF 3 1/BMP2 in vitro culture and in vivo implants. Investigation into CNCSC contribution to bioengineered tooth structures is a hopeful direction.