The Research Of Constructing Tooth Like Structure With Human Dental Pulp Stem Cells

Dental pulp stem cells (DPSCs) is an important candidate stem cells in tooth tissue engineering, and great progress have been made in identifying DPSC. Nevertheless, many problems have to be solved if we carry out further researches and application, for example,how to construct complex tooh like structure with DPSCs and other cells,and how to create appropriate microenvironment to guide DPSC to differentiate into specific direction, especially into odontogenic cell linage when DPSCs were cultured in vitro.This research tried to construct heterogeneous chimeric tooth structure with human DPSCs and xenogenic epithelial cells from tooth germ , to evaluate the effect of xenogenic tooth germ conditioned medium on human DPSCs, and to construct engineered bioroot by comination of human DPSC and periodontal ligament stem cells (PDLSC) .The aim of these studies was to establish experimental fondation for the further research and application of human DPSCs, to provide some useful clues for the future application of human DPSCs in tooth tissue engieering, and to explore some new methods and approaches for tooth regeneration with tooth derived stem cells.The main achivements are as follows:Section 1.Construction of heterogeneous chimeric tooth with human dental pulp stem cellsHuman DPSC were cultured with enzyme digestion solution. Results showed cultured DPSCs had typical morphology, and expressed surface markers of mesenchymal stem cells STRO-1 ,CD06 and CD29. cultured DPSCs held the potential to form colony ,and to differentiate into osteoblats or adipocytes after induction. These data indicated the DPSCs we cultured were mesenchymal stem cells. Porcine dental germ of the third molar was isolated and dental germ cells were cultured. Epithelial cells of dental germ were obtained by speed-differentiated digestion solution. Immunocytochemeical staining showed tooth germ derived epithelial cells were positive for CK14 and AMBN, but negative for Vimentin. Then human DPSCs and porcine tooth germ derived epithelial cells were combined with different methods and transplanted into subcutaneous pockets of nude mice. Implants were harvested after 6 weeks. It was found most of the implants did not form any ordinate structure, whereas some implants contained dentin-pulp like structure and enamel like structure.The probability of forming this kind of structure was just 20%. Immunohistochemistry results provided evidence that the enamel like structure was originated from porcine tooth germ derived epithelial cells while dentin like structure was originated from human DPSCs.The results of this part suggested that human DPSC and porcine tooth germ derived epithelial cells could mimic the interaction between epithelial cells and mesenchymal cells during tooth development, and induce each other and possessed the possibility to form heterogeneous chimeric tooth structure. So we could come to the conclusion that it is feasible to construct heterogeneous chimric tooth with human mesenchymal stem cells and xenogenic tooth germ derived epithelial cells, and this may be a solution to cope with ename regeneration.Section 2.Odontogenic differentiation of human dental pulp stem cells induced by swine tooth germ cell conditioned mediumPrevious studies showed that tooth germ cell conditioned medium (TGC-CM) hold the potential to induce dental pulp stem cells (DPSCs) to differentiate into odontogenic lineage. Nevertheless, human TGC-CM (hTGC-CM) is not feasible in practical application. We hypothesized that xenogenic TGC-CM may exert similar influence on DPSCs. In this study, we selected swine as a xenogenic origin and compared the effects of swine tooth germ cell conditioned medium (sTGC-CM) with hTGC-CM on human DPSCs. in vivo and in vitro. Results showed sTGC-CM, which is similar with the effect of hTGC-CM, was able to induce human DPSCs to make odontoblastic changes as indicated by remarkable morphological changes, increased proliferation ability, higher multipotential and colony forming capability, and expression of some odontogenic markers in gene and protein levels,such as DSP, DSPP, DMP1, BSP, OCN, OPN. Induced DPSC also maintained better stem-cell characteristics,. Besides, human DPSCs treated with sTGC-CM could form more regular dentine-pulp complex in vivo.The probability of forming this kind of structure was elevated from 20% to over 70%. These results was similar to DPSCs treated with hTGC-CM while quite different from that of control DPSCs.Our data provided evidence that sTGC-CM held almost the same potential as hTGC-CM in inducing DPSCs to differentiate along odontogenic direction.. The observations suggested xenogenic TGC-CM may be a useful tool in tooth regeneration and the application of xenogenic TGC-CM may facilitate generating a bioengineered tooth from tooth derived stem cells in future.Section 3 The trial of constructing engineered bioroot with human dental pulp stem cells and periodontal ligament stem cellsUndoubtedly, DPSC and PDLSC was the most promising postnatal stem cells in future tooth tissue engineering.Then, is it possible to get tooth root like structure in vivo if these two kinds of stem cells were combined and transplanted?We carried out this kind of trial. PDLSCs were cultured ex vivo and were characterizied. Results showed PDLSCs were positive for Vimentin while negative for CK , indicating they were of mesenchymal origin.They were also positive for STRO-1, which suggest cultured PDLSCs were endowed with the phenotype of mesenchymal stem cells.The experiments of calcification and adipogenesis induction confirmed the multipotentiality and plasticity of human PDLSCs. DPSCs were labled with PKH26 and then were combined with PDLSCs by different methods and transplanted into nude mice. Results revealed the implants with toot-shaped CBB as scaffold presented small tooth root like structure in gross appearance.HE staining showed all the implants formed calcified matrix, and Sharpey fiber like structure could be found in some implants. While the implants with CBB particles as scaffold formed thin and plat structure in gross appearance and HE staining demonstated calcified matrix were also formed, but less than implants with root-shaped CBB as scaffold. Most cell pellet implants did not form any corporeal structure with exception that only a few contained calcified matrix. No dentin-pulp like structure was found in all the three kinds of implants.These data suggested: When constructing engineered tooth root, preshaped scalffold was the best choice to guide related stem cells grow along the scaffold and form good-shaped structure in gross appearance; it is recommended to seed stem cells onto the scaffold respectively instead of mixing them simply if more than one kinds of stem cells were used; it is not suitable to construct tooth like structure with good–shaped gross appearance by cell pellet transplantation method.