Construction Of EPC/BMSC Compound Cell Sheet And Its Effect On Rat Alveolar Bone Defect Repair

The repair of alveolar bone defect caused by periodontal diseases, trauma, congenital deformity, or tumor is a major issue for oral medicine. In recent years, the development of bone tissue engineering provides a new method for the repair and regeneration of alveolar bone defect. Bone marrow mesenchymal stem cells(BMSCs) are multipotent cells. They have the ability to differentiate into a wide rage of cell types such as osteoblasts, adipocytes and chondroblasts. This type of cells has been widely applied to bone tissue engineering. However, some problems, such as, inadequate neovascularization of implanted grafts and immunologic rejection or cell low availability of the scaffolds remain the main obstacles that prevent the application of bone tissue engineering. Thus, in our study, we aimed to establish a co-cultured EPC/BMSC sheet by using induction of vitamin C after directly co-cultured of EPCs and BMSCs. We try to explore a better microenvironment which is benefit for the osteogenesis. And we hope to find a more efficient strategy to repair and regenerate the alveolar bone defects by using stem cells. Objective:1.To isolate EPCs and BMSCs and to construct a compound EPC/BMSC sheet.2.To study the osteogenesis ablility of EPC/BMSC sheet and to explore the microenvironment which can improve the osteogenesis of BMSCs.3.To observe the bone repair ablility of EPC/BMSC sheet in a rat alveolar bone defect model. And to explore a new method to repair alveolar bone defect using a compound cell sheet. Methods:1.To isolate the BMSCs, the density gradient centrifugation with adherent method was used. Inverted microscope was used to observe the morphology of cultured cells. Phenotypes of cultured cells were determined by flow cytometry. The multi-differentiation potential of cultured cells was detected by osteogenic and adiopogenic differentitation.2.EPCs were isolated and separated by the density gradient centrifugation combined with adherent method and cultured with endothelial cell growth medium-2. Inverted microscope was used to observe the sequential change of cultured cells. Phenotypes of cultured cells were analyzed by flow cytometry. The cultured cells were further identified by the ability of taking up Di I-ac-LDL and binding FITC-UEA-1 simultaneously.3.EPCs and BMSCs were co-cultured directly, and then constructed the EPC/BMSC compound sheet by using the induction of vitamin C. HE staining and scanning electron microscope were carried out for morphological and microstructural observation.4.BMSCs and EPCs were stained with the lipophilic fluorescent dyes Di O and Di I respectively to observe the distribution of two types of cells in the process of contructing EPC/BMSC sheet. TUNEL assay was used to detect the apoptotic cells of cell sheets.5.ALP activity and the formation ability of calcium phosphate were detected. Real-time PCR and Western blot were used to analyze the expression of osteogenic and angiogenic markers. ELISA was used to detect the expression of VEGF secreted by cell sheets.6.Established the rat alveolar bone defect model, and implanted the cell sheets into the defect areas immediately. Gross observation, Micro-CT scanning and analysis, HE and Masson staining were used to determine the effect of bone repair ability. Results:1.Cultured BMSCs displayed a relatively homogenous confluent population that showed a rhomboid-shaped and spindle-shaped morphology. The cells were positive for the mesenchymal stem cell like markers, but were negative for the hematopoietic markers. Cultured cells were differentiated into adipogenic and osteoblastic lineages by cultured in the appropriate induction medium.2.Cultured EPCs initially appeared spindle shaped, but this kind of morphology gradually disappeared and was replaced by a cobblestone-like morphology. The cells expressed surface markers of endothelial cells and hematopoietic stem cells. Cultured cells were positive for v WF and took up Di I-ac-LDL and binded FITC-UEA-1 simultaneously.3.After 10 days of cell sheet induction, the EPC/BMSC compound cell sheet was harvested. The cell sheet showed a semi-transparent and membrane-like structure and could be detached intact from the edge of the culture dish using forcep. Histological observation showed that the EPC/BMSC compound cell sheet composed of mutiple layers of cells with abundant collagen.4.The distribution of two types of cells in EPC/BMSC compound cell sheet was even. Direct co-culture led to a close EPCs and BMSCs membrane contact. Furthermore, compared with the control groups(BMSC sheet and EPC sheet), EPC/BMSC compound cell sheet had a relatively low rate of apoptosis.5.The result of ALP activity in EPC/BMSC compound cell sheet was significantly higher compared with the other groups(BMSC sheet and EPC sheet). EPC/BMSC compound cell sheet also had a larger area of mineralized nodules than BMSC sheet and EPC sheet. The expression of osteogenic and angiogenic related markers in EPC/BMSC compound cell sheet was significantly higher than other groups.6.At different time point, EPC/BMSC compound cell sheet exhibited better osteogenic activity compared with other groups in vivo. Conclusions:1. EPC/BMSC compound cell sheet has a great potential of osteogenesis and angiogenesis in vitro study.2. There has a cell-cell communication between EPCs and BMSCs in EPC/BMSC sheet.3.EPC/BMSC compound cell sheet siginificantly improves the ability of new bone formation in vivo, which may provide a new perspective of the alveolar bone defect repair.