| Backgroud: Postmenopausal osteoporosis(PMOP) is a common frequently-occurring disease in postmenopausal women. The main reasons can be concluded as follows: the decreased ovarian function and a drop in estrogen levels which leads to metabolic imbalance of osteogenesis and bone absorption. Estrogen deficiency is an important reason of postmenopausal osteoporosis which can cause alveolar bone resorption, missing teeth, the difficulty of periodontal tissue regeneration and repair. In recent years, with the further research of bone tissue engineering, great progress has been made in repairing the defects of jaw and alveolar bone. However, the study found that the estrogen deficiency microenvironment can lead to the increase of BMSCs apoptosis and the decrease of bone differentiation, due to the lack of necessary nutrients to maintain and good local microenvironment,the implantation of BMSCs can often result in apoptosis. Therefore, how to enhance the survival rate of the BMSCs and maintain the characteristics of stem cells is the key to the success of using stem cell transplantation to repair tissue defects. Recently,the study found that EPCs have specificity of homing ability, which can form new blood vessels through its differentiation and proliferation. It has a unique advantage to improve the microenvironment. In our study, the BMSCs of osteoporotic Sprague-Dawley(SD) rats were co-cultured with EPCs in order to explore the effect of endothelial progenitor cells(EPCs) on the biological characteristics of bone marow mesenchymal stem cells(BMSCs) of osteoporosis rat in the indirect co-culture system which may lay the foundation of the repair of the periodontal tissue defect for the osteoporosis patients.Objective: This study aims to explore the effect of endothelial progenitor cells(EPCs) on proliferation, apoptosis ability, osteogenic and adipogenesis differentiation of bone marow mesenchymal stem cells(BMSCs) of osteoporosis rat in the indirect co-culture system which lies in the theoretical foundation of the repair of the periodontal tissue defect for the osteoporosis patients.Contents:(1)Establishment of osteoporosis model in rats.(2)Isolation and characterization of rat bone marrow mesenchymal stem cells and endothelial progenitor cells.(3)Effects of EPCs on proliferation, apoptosis and differentiation of BMSCs of osteoporosis rat in the indirect co-culture system.Methods:(1) To establish the model of ovariectomized SD rats Methods : Three-month-old female SD rats were selected in this study with 10% chloral hydrate intraperitoneal injection anesthesia, the bilateral dorsal incision, blunt separation into abdominal cavity, which can be removed along the fallopian tube to determine the location of ovary after ligation of retrograde stump, followed by ligation, suture. Three months after the operation, the model was evaluated by Micro-CT.(2) Isolation and cultivation of EPCs Methods: EPCs from sham group were isolated using density gradient centrifugation combined with adhesion method and identified with surface markers, cell proliferation and immunocytochemistry in vitro.(3) Isolation and cultivation of BMSCs Methods: MSCs from osteoporosis group and sham group were isolated using density gradient centrifugation combined with adhesion method and identified them by flow cytometer and multi- differentiation cultivation in vitro.(4) Establishment of indirect co-culture system of EPCs and OVX-BMSCs Methods: After indirect co-cultured with EPCs, flow cytometry were applied to detect OVX-BMSCs proliferation and apoptosis. ALP and Alizarin red S staining were used to determine ALP enzymatic activity and calcium phosphate formation ability. Oil red O staining and optical density(OD) assay were used to determine adipogenic ability. Real-time PCR was used to analyses osteogenic and adipogenic marker genes expression.Results:(1)After 3 months after either a sham surgery or bilateral ovariectomization,the average weight of the OVX rats changed significantly compared with the preoperative weight. Micro-CT evaluation of the two different groups showed that the bone marrow density,bone volume ratio, trabecular number and other aspects changed significantly, which indicating that the the osteoporotic rat model was successfully established.(2)The EPCs can be obtained by density gradient centrifugation combined with differential velocity adherent method and cultivating under special inductive culture media.The cell morphology observed under an inverted microscope showed a typical cobblestone appearance. Cell phenotype was identified by flow cytometry and the cells were stained with double fluorescence staining, which showed that the cultured cells were EPCs.(3)Cultured BMSCs showed elongated spindle or rhomboid-shaped and the cells could differentiate into adipocytes and osteoblasts when cultured in induction media. The cell surface markers were positive for mesenchymal markers and negative for hematopoietic markers by flow cytometry.(4)Under the indirect co-culture conditions, the proliferation ability and apoptosis rate of BMSCs in three groups were detected by flow cytometry, the results showed that EPCs can enhance the proliferation ability and reduce the apoptosis rate. ALP staining and alizarin red staining results showed that BMSCs of co-culture group could form more mineralized nodules than ovariectomized group and sham operation group, real-time PCR results showed that the expression levels of RUNX2?OCN?BSP were higher in co-culture group than the other groups. Oil red O staining showed that lipid droplets in co-culture group formed more than the other two groups, and real-time PCR results showed that the expression level of LPL and PPAR ? 2 increased significantly in co-culture group compared with sham operation group and ovariectomized group.Conclusions:(1)Three-months-old female SD rats were selected to be ovariectomized. Three months after the operation, the model of ovariectomized SD rats has been successfully established by micro-CT.(2)Under the indirect co-culture conditions, EPCs could reverse the proliferation ability of OVX-BMSCs and decrease the apoptosis rate.(3)Under the indirect co-culture conditions, EPCs could reverse the differentiative ability of OVX-BMSCs. |
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