The In Vitro Study Of Cementoblasts Differentiation Induced From Placenta-derived MSCs

Periodontal disease involves three types of tissues including periodontal ligament, cementum and alveolar bone. The conformation of the three tissues is in a complex order, which requires the facilitations of various cellular components, such as osteoblasts, cementoblasts and fibroblasts. The standard of successful periodontal reconstruction is the reconstruction of the three tissues and the restoration of the periodontal structural integrity. However, due to the destruction of long-term inflammation in adult periodontal disease and the limited quantification of seed cells in the periodontal lesion area, it is difficult to effectively promote the tissue regeneration and achieve the functional reconstruction. A large-scale of seed cells are required to transplant into the periodontal lesion area in order to meet the request of periodontal tissue reconstruction. Further more, cementoblasts play an important role in the colonization of the periodontal fibers and the formation of the periodontal membrane. Therefore, it is the key of the periodontal reconstruction to select the seed cells with the potential of directional differentiation to cementoblasts.Mesenchymal stem cells, which are characterized as undifferentiated, high-efficiently self-renewal and multipotent cells with multi-directional differentiation potentials, demonstrate a good prospect in clinical treatment. Recently, the mesenchymal stem cells have been isolated in many adult tissues successfully. Mesenchymal stem cells are the main source for the reconstruction of damaged tissues and cells for gene therapy resulting from the multi-directional differentiation potential and plasticity. Of all the mesenchymal stem cells isolated, placenta-derived mesenchymal stem cells exhibit advantages of abundant sources, low immunogenicity, low rate of viral contamination and no social and ethical dispute, therefore, have better prospects for clinical application.The directional differentiation of stem cells requires the establishment of an appropriate micro-environment for the induction. In the development of periodontal tissues, enamel matrix protein derivatives play an essential role, and of which amelogenin is the main active ingredient, with the capabilities of the promotion of the cementum formation, the proliferation of periodontal ligament cells and the bone induction. The domestic and international studies have shown that amelogenin has reliable effect to induce stem cells to differentiate towards periodontal phenotypes.Therefore, the micro-environment of the induction was established in vitro on the basis of the construction of Ad-AMG in our study. Placenta-derived mesenchymal stem cells were induced to differentiate towards cementoblasts in order to provide sources of new seed cells and experimental methods for periodontal tissue engineering, especially cementum tissue engineering, and to provide the experimental reference for the reminding cementum occurrence and the reaction mechanisms under orthodontic force.Materials and Methods1. Construction of Ad-AMGHuman amelogenin X chromosome encoding cDNA sequence (GenBank Accession No. M86932) was used as the target gene, coupled with restriction enzyme sites of EcoR I and BamH I respectively at each end of the sequence, it was synthesized and inserted into an adenovirus shuttle vector by utilizing the restriction endonuclease EcoR I and BamH I, and was then homologously recombined to an adenoviral backbone vector in 293 cells.2. Isolation and identification of placenta-derived mesenchymal stem cellsThe stem cells were obtained from human’s placenta by enzymatic digestion and density gradient centrifugation, and were placed under an microscope to observe their morphologic change and growth characteristics; CCK-8 kit was used to detect cell proliferation; flow cytometry was used to detect cell cycle and the expression of surface antigen (CD73, CD90, CD105, CD29, CD31, CD34, CDllb, CD19, CD45å'ŒHLA-DR). The experiment induced placenta-derived mesenchymal stem cells to osteoblasts and adipocytes useing the osteogenic induction medium and adipogenic induction medium confirmed placenta-derived mesenchymal stem cells’ multi-differentiation ability.3. Ad-AMG induction of placenta-derived mesenchymal stem cells to differentiate towards cementoblasts in vitroPlacenta-derived mesenchymal stem cells were changed from the original fibroblast-like cells to low columnar shaped and polygonal cementoblasts under the induction of Ad-AMG overexpression. And its proliferation activity was significantly reduced, and the mineralization capability in vitro was remarkably increased. Meanwhile the expression of CAP and the expressions of related mineralized proteins, BSP, OPN, OCN, were also confirmed by RT-PCR and Immunohistochemistry. Through further study, the reaction mechanism of cementoblasts under the compressive force showed RANKL and M-CSF mRNA expression level was proportional to the pressure loading time, and the RANKL/OPG ratio increases. Under the pressure, the cementoblasts have the function to induce osteoclast formation and differentiation. After cryptiogene of FAK gene, RANKL and M-CSF mRNA expression level of cementoblasts with the pressure remained basically unchanged with the time prolonged, but OPG mRNA expression level increased and RANKL/OPG ratio decreases. Through interference experiment, it is found that FAK gene participated in the osteoclast differentiation and maturation process.The results1. The overexpression Ad-AMG vector and the contrast adenovirus vector, Ad-EGFP, were constructed. The transfection efficiency of adenovirus vector was confirmed high by the expression of Ad-EGFP. The amelogenin sequence of Ad-AMG was identified by PCR and the gene sequence was completely consistent with the gene sequence of the gene bank, and the expression of amelogenin was confirmed by Western-Blot assays.2. The placenta-derived mesenchymal stem cells grew well. The cells showed uniform spindle like, and grew in parallel or swirl shape. The immunophenotype detected by the flow cytometry showed the cells expressed the typical mesenchymal stem cells surface antigen labeling and the surface antigen of the endothelial cells and the blood cells were not expressed. Three weeks after osteogenic induction, the highlight of mineralized crystallization was obvious and showed deep red nodules by alizarin red staining. Two weeks after adipogenic induction, beaded or cribriform round vacuoles filled with whole cells and showed as orange red round bubble by oil red O staining.3. Placenta-derived mesenchymal stem cells were changed from the original fibroblast-like cells to low columnar shaped and polygonal cementoblasts under the induction of Ad-AMG overexpression. And its proliferation activity was significantly reduced, and the mineralization capability in vitro was remarkably increased. Meanwhile the expression of cementum CAP and the expressions of related mineralized proteins, BSP, OPN, OCN, were also significantly enhanced.Conclusion1. The Ad-AMG and the contrast adenovirus vector of Ad-EGFP were successfully constructed. The transfection efficiency of adenovirus vector showed high by Ad-EGFP. The amelogenin sequence of Ad-AMG was proper which identified by PCR and the expression of amelogenin was confirmed by Western-Blot assays.2. Placenta-derived mesenchymal stem cells were successfully obtained. Afer the induction by the amelogenin in vitro, they exhibited the characteristics of the cementoblasts in the cell morphology, the cell proliferation, the capabilities of the mineralization in vitro and the CAP gene expressions. It confirmed that placenta-derived mesenchymal stem cells can differentiate into cementoblasts in a suitable environment.3. Under the compressive stress, the cementoblast which induced from placenta-derived mesenchymal stem cells obtains the function to induce the osteoclast differentiation. It is proved that the periodontal tissue induced from non-odontogenic stem cells can participate in the remodeling of periodontal tissues during orthodontic treatment. At the same time, the expression of FAK gene associated with cytoskeletal reorganization involved in osteoclast related signaling factors.