Odontogenic Potential Of Mesenchymal Stem Cells Derived From Rat Bone Marrow

Tooth lost and missing as a result of decay, periodontal disease, trauma, surgical operation and congenital abnormality is a prevalent disease, which generally induce the decline of the living quality. Current replacement tooth methods use synthetic materials that can elicit an immune induced host rejection response. Tooth regeneration aim at regenerating or reconstructing nature teeth-like tissue engineering tooth in tissue structure, conformation and biological function, so the rising bio-technique, a relatively new science integrating developmental, molecular/cellular biology, and genetics with the field of engineering, holds promise in this area. In the field of tooth regeneration research, the thief problem is to find seed cells that can generate dental tissue. The objective of this study was to research the odontogenic potential of bone marrow-derived mesenchymal stem cells (BM-MSCs) as seed cells in tooth regeneration, which can be derived from autologous adult tissue, and possess multi-lineage differentiation.In this study, BM-MSCs derived from rat bone marrow were cultured and proliferated in vitro, and were transferred Green Fluorescent Protein (GFP) as track. These GFP positive cells were co-cultured with oral membrane epithelium and tooth germ cells derived from fatal rat, and then were selected with flow cytometry. Theexpression of odontogenesis raletively gene Pax9, DMP1 and DSPP of these selected BM-MSCs were detected with RT-PCR technique, and the protein expression of DSPP was detected with Western Blot. To further characterize the odontogenic potential of BM-MSCs, an in vivo transplantation system, the gold standard, was executed. The BM-MSCs derived from male rat were mixed with female fatal rat oral membrane epithelium and tooth germ cells respectively. A cells mass made of BM-MSCs and oral epithelium were implanted under adult rat BM-MSCs envelop, and the mixture of BM-MSCs, tooth germ cells and alginate gel was implanted into female rat greater omentum.1-3 months later, the implants were examined with HE stain, in site hybridization. Furthermore, the mechanism of odontogenic potential on BM-MSCs was explored with the transfection with DSPP gene, and the mineralization capability of transfeted BM-MSCs was observed, the results reveal that Ad-GFP delivered GFP gene with high efficiency to rat BM-MSCs. 41.3±1.4 % of BM-MSCs infected with Ad-GFP expressed GFP gene, which is much higher than the control (12.5%). Expression of GFP gene of infected BM-MSCs maintained stabilization from 1 to 6 weeks after infection. Infected BM-MSCs possess the same alkaline phosphatase activation as non-infected BM-MSCs, and can form mineralized nodules. After co-culture of BM-MSCs and oral epithelium cells in Ell.5 rat or tooth germ cells in E17.5, the expression of Pax9, DMP1 and DSPP gene, which were related to tooth development, were find with RT-PCR. The Western blot analysis show that BM-MSCs co-cultured with oral epithelium or tooth germ cells produced gradually increasing levels of DSP protein. These results proved that BM-MSCs possess the differentiation potential in level of gene and protein. To confirm the odontogenic potential of BM-MSCs in vivo, adult female SD rats were used as hosts for mixed tooth-related tissue implantscomprising BM-MSCs. Cell mass composed of fatal rat oral epithelium cells and BM-MSCs were implanted under the renal capsule, and alginate gel scaffold mixed with BM-MSCs and tooth germ cells were embedded into omenta of syngeneic SD rat hosts. After 1-3 months, experimental implants revealed the mineralized tissues, and radiographic analyses of experimental implants revealed the presence of mineralized tissues. Histological analyses of BM-MSCs/epithelium cell mass and alginate gel cell-seeded implants demonstrated the presence of tooth-like structure. In site hybridization analysis of sry cDNA and DSPP gene to alginate gel implant containing male BM-MSCs and female tooth germ cells demonstrated that some sry-positive BM-MSCs differentiated into odontoblast-like cells expressing DSPP gene. Cell mass implant only comprised of oral epithelium and BM-MSCs, so the tooth-like mineralized implant supported the same conclusion. Moreover, to explore the feasible method of inducing BM-MSCs, we transfected DSPP gene to BM-MSCs mediated by liposome. Results show that transfected BM-MSCs exhibited changes in their morphology, tended to align themselves in straight parallel lines, and had long dendrited-like processes. Furthermore, an in vitro nodule formation assay revealed that the mineralized matrix formations in transfected BM-MSCs were enhanced according to the larger and more nodule than un-transfected BM-MSCs.Based on these findings, the study confirmed that adult BM-MSCs possess odontogenic potential. A series of experiments in vitro and vivo demonstrated that BM-MSCs can differentiate into functional odontoblast-like cells. It implies that BM-MSCs may become a seed cells source in tooth regeneration research.