Humoral Immune Response To Transplantation Of Allogeneic Periodontal Ligament Stem Cells

Restoration of lost teeth and regeneration of periodontal tissue are one of the key issues in dental clinic and research. At present, the treatment of bone defect of periodontitis is still difficult. Artificial teeth including fixed teeth, removal teeth and dental implant are prosthetic appliances, which are not truly biological restoration. Recently, the new prospect on bio-regeneration of teeth and periodontal tissue is brought about by the progress of dental related stem cells and tissue engineering technology. Currently, many studies present that dental stem cells are regarded as the best seed cells resource for the regeneration of teeth and periodontal tissue with a booming clinical application. However, autologous dental stem cells are limited in the patients. If dental stem cells can be used in allogeneic individuals, the source of seed cells can be expanded greatly which will greatly promote the development on restoration of lost teeth and regeneration of periodontal tissue. Adaptive immune divides into two parts including cellar immune and humoral immune. Our previous studies have proved that PDLSCs possessed low immunogenecity and immunosuppressive function through secretion of PGE2. But there is no study on humoral immune response to transplantation of allogeneic periodontal ligament stem cells. The aim of this study is to address the immunological characteristics of PDLSCs which effect on B lymphocyte functions, the mechanisms underlying the PDLSCs-mediated immunological suppression, observe the ability of regenerating bone defect of periodontitis by using allogeneic PDLSCs in the miniature pig model and the present of humoral immune rejection in vivo. Part1Culture and identification of the periodontal ligament stem cells of human in vitroObjective:To culture and identify the periodontal ligament stem cells of human in vitro for providing the necessary foundation and prerequisite for the next step to study other characteristics of the periodontal ligament stem cells.Methods:Normal impacted third molars of healthy individuals were extracted, and periodontal ligament stem cells were separated and cultured referring to the methods of human’s related stem cells. The growth characteristics of these stem cells were observed, the surface markers were detected and the abilities of trans-differentiation were investigated.Results:The cultured human periodontal ligament stem cells shaped in typical fibroblast cells under light microscope. The colony forming units-fibroblasts were from15to28units/105in periodontal ligament cells. These cells were positive for STRO-1, CD146, CD90, SSEA-4and OCT-4,5MSCs markers, identifying them as PDLSCs. Moreover, the cultured human periodontal ligament stem cells had the abilities to be induced to form calcium and adipose materials in vitro.Conclusion:The human periodontal ligament stem cells, which can be cultured successfully in vitro showed good growth and could undergo multilineage differentiation. Part2Modulation of B lymphocyte function in vitro by hPDLSCsObjective:To study hPDLSCs modulate B lymphocyte function in vitro.Methods:B lymphocyte were extracted from healthy human and cultured with hPDLSCs in the absence or presence of the following stimuli:the CpG synthetic oligonucleotide, recombinant CD40L, anti-human immunoglobulin goat antibodies, interleukin2and IL-4, co-cultured with hBMSCs as a positive control. Proliferation, apoptosis, differentiation, chemotatic function, expression of costimulatory molecules and secretion of B lymphocyte were detected by flow cytometry to study hPDLSCs modulate B lymphocyte function. Furthermore, transwell culture experiments, quantification of soluble factors, neutralization experiments, and detection of apoptotic B cells were performed to identify the possible mechanisms of the immunosuppressive and apoptosis function of hPDLSCs on B lymphocyte.Results:HPDLSCs could not initiate B lymphocyte proliferative responses. B lymphocyte proliferation was significantly inhibited by hPDLSCs in a dose dependent manner in the presence of CpG ODN, rCD40L, anti-immunoglobulin, IL-2, and IL-4. Moreover, proliferative of B lymphocyte was inhibited by delaying to co-cultured with hPDLSCs. The experiment also proved that the expression of costimulatory molecules(CD40, CD80, CD86, HLA DR) and secretion of B lymphocyte was not impacted by hPDLSCs, but the differentiation and chemotactic function of B cell was inhibited. By using transwell chambers, we found that the inhibition of B cells proliferation was mediated by cell-to-cell contact and soluble factors. By using flow cytometry, we found that the expression of PD-1, PD-L1and PD-L2on the surface of hPDLSCs was changed. Furthermore, neutralization experiments showed that anti-PD-1antibody, anti-PD-L1antibody and anti-PD-L2antibody partially restored B cells proliferation inhibited by hPDLSCs. Apoptosis showed that inhibition B cells proliferation by hPDLSCs was not mediated by inducing apoptosis of B cells. On the contrary, hPDLSCs inhibited apoptosis of B cells by IL-6secretion. HBMSCs as a positive control, the results on the effect on B cells were the same to hPDLSCs.Conclusion:HPDLSCs, similar to hBMSCs, can inhibit the proliferation of B lymphocyte in a dose dependent manner partially by PD-land PD-L1and have no relationship with B cells apoptosis. They also can inhibit apoptosis, differentiation and chemotatic function of B cells. But they cannot modulate the expression of costimulatory molecules(CD40, CD80, CD86, HLA DR) and secretion of B lymphocyte.Part3Humoral immune response to transplantation of allogeneic periodontal ligament stem cells of minipig in vivoObjective:To study humoral immune response to transplantation of allogeneic periodontal ligament stem cells of minipig in vivo.Methods:We generated periodontitis-lesions in12female Wuzhishan inbred minipigs, totally24defects. Then the miniature pig’s periodontal ligament stem cells were separated and cultured in vitro. The24defects were randomly assigned to four different groups:(1) Control group (6defects in3minipigs):no treatment;(2) HA/TCP group (6defects in3minipigs):flap surgery, transplantation of HA/TCP scaffolds, and covering of the defects with gelatin membranes;(3)HA/TCP scaffolds+autologous PDLSCs group (6defects in3minipigs): transplantation of autologous PDLSCs combined with HA/TCP scaffolds, and covering of the defects with gelatin membranes;(4) HA/TCP scaffolds+allogeneic Guizhou minipig PDLSCs group (6defects in3minipigs):transplantation of allogeneic Guizhou minipig PDLSCs combined with HA/TCP scaffolds, and covering of the defects with gelatin membranes. The regeneration of bone defect was observed by clinical assessments including probing depth (PD), gingival recession (GR) and attachment loss (AL), CT scanning, and histopathological study. Blood routine test, biochemical routine test, immunoglobulin in serum, gingival crevicular fluid and periodontal tissues test and B cells related markers, i.e. percentage of CD20+cells, CD25+cells, B220+cells, and the expression of activated B cells marker.Results:We successfully established periodontitis model and there was comparable between them. At12weeks post-transplantation, either autologous or allogeneic PDLSCs treatment significantly improved periodontal tissue regeneration in comparison with HA/TCP and the control groups, and there was no difference between autologous and allogeneic PDLSCs groups. CT scanning showed that the height of alveolar bone in autologous and allogeneic PDLSCs groups recovered to approximately the normal levels. In contrast, HA/TCP groups and the control groups showed very limited or no bone regeneration. In addition, histopathological photomicrographs showed increased new bone and periodontal tissues, including cementum and periodontal ligament, were regenerated in the periodontal defect area in autologous and allogeneic PDLSCs groups. However, typical periodontitis status, including deep periodontal pocket and shortage of new bone and periodontal ligament fibers, was still found in the HA/TCP and control groups. Furthermore, the data of blood routine tests, biochemical routine test, immunoglobulin test and B cells related immunological markers(CD20,CD25and B220) were almost the same as those of4weeks pre-transplantation, suggesting there were no marked immunological rejections in the animals received allogeneic PDLSCs transplantation. By using ELISA, we found that there was no different on immunoglobulin in serum, gingival crevicular fluid and periodontal tissues between autologous and allogeneic PDLSCs groups. Moreover, by using real time-PCR, we detected the mRNA of IL-6, PD-1, PD-L1and PD-L2in autologous and allogeneic PDLSCs groups at1week and2week after transplantation. We found there was no difference between them on the expression of IL-6at different time. However, there was significantly difference of the mRNA expression of PD-1, PD-L1and PD-L2between autologous and allogeneic PDLSCs groups at1week and2week after transplantation.Conclusion:Allogeneic PDLSCs can effectively repair bone defect of periodontitis in minipig and do not induce humoral immunological rejections in which PD-1and PD-L1maybe play an important role.