| Osteoporosis is the most common systemic skeletal disease that affects elderly women, which is characterized by low bone mass and microstructure damage with a consequence increase in bone fragility with susceptibility to fracture. Mainly it displays in low back pain and pathological fracture in clinic, especially in postmenopausal women, and the incidence is as high as60%. With extending of the average of life span, more and more countries step into aging society. This disease threatens seriously the health and life of elderly women and results in increasing economic burden for family and society. Postmenopausal osteoporosis has become an important public health problem. Mesenchymal stem cells come from mesoderm that has self-replicated and multiple differentiation activity. They are easy to be cultured, rich in source and have differentiation of multi-potential, even low immunogenicity, portability, tissue repair ability, and they also have the capacity of cross-mesodermal differentiation, such as osteoblast, myocyte, adipocyte, and chondrocyte, etc. Recent studies have found that postmenopausal osteoporosis is associated with the increasing bone marrow fat tissue, but the specific cytology mechanism is not clear, some scholars think osteopenia and fat increase is due to the imbalance of osteoblast and adipocyte precursors-MSCs. In the basis of ovariectomied animal model, we investigate the pathogenesis of postmenopausal osteoporosis, find the changes of bone mass of the ovariectomied rats and normal rats; and the differential capacity of these BMSCs to osteoblast and adipocyte. Moreover, the study of MSCs from ovariectomied rats may provide a better understanding of the mechanisms of the postmenopausal osteoporosis.Our experiment methods:We established animal model of osteoporosis by3old-months female Sprague-Dawley rats(SD) by ovariectomy. Animal rats were randomly divided into4groups:control group (group A), model group (group B), cell treatment group (group C), drug treatment group (group D). After the successful establishment of osteoporosis model, BMSCs were transplanted via the tail vein(group C), D group received anti-osteoporosis drugs therapy(Raloxifene).Through the detection of bone mineral density, bone biochemical markers, micro-CT testing, the model was verified to be successful. Cell phenotype is tested by flow cytometry, The bone marrow mesenchymal stem cells (BMSCs) was transplanted by3×106/one rat via the tail vein.At the same time, the drug therapy group rats (group D) were given anti-osteoporosis drug, The average intake was3.4mg/day of R.Our results showed:1)12w after ovariectomy, model group compared with control group, bone mineral density decrease. Micro-CT found that bone microstructure had been damaged, the bone trabecular become slim and the quantity also had reduced (P<0.05).2) Most of BMSCs cultured had been attached after24h, until the third generation. The cells form a uniform shape, BMSCs from normal rats compared with BMSCs from the OP rats, we found the proliferation ability of osteoporosis BMSCs was lower than the normal group in vitro.3)14d after adipogenic induction, the shape of BMSCs had changed, from long shuttle to round or polygonal, we could find lipid vesicle formation by Oil Red-O staining.25d after osteogenic induction, BMSCs gradually lose their cellular structure, and the formation of calcium nodules was apparent. we could verify formation of the calcium nodes by alizarin red staining. These results showed BMSCs we cultivated could be induced in different conditions, and differentiated respectively into adipocyte and osteoblast. we confirmed BMSCs have multiple differentiation ability.4) Group A compared with group B, bone mineral density of the lumbar spine and femoral from group B rats were significantly lower than group A (P<0.01).12w after cell treatment, bone mineral density of the group C had been improved obviously, and group B also had the statistical difference (P<0.01), which was better than D group (P<0.05).5) the4w,8w,12w after different treatment, bone alkaline phosphatase and I collagen type carboxyl terminal peptide in serum were increased at different stage, their contents were higher than others(P<0.05). Cell therapy group compared with drug treatment group, the difference of the bone biological markers also was statistically significant (P<0.05).6) Results of the micro-CT detection showed that the microstructure of cell treatment group and drug treatment group rats had been improved obviously in BMD, bone trabecular number and thickness (P<0.05).Experimental results suggest that:1)12w after ovariectomy, bone mineral density of model group was significantly lower than other groups. Bone trabecular was decreased, slim, and bone biochemical markers content increased obviously.2) BMSCs from normal rats was not obviously different with BMSCs from osteoporosis rats in morphology, but the proliferation ability of them was obviously different. The phenotype of BMSCs was tested by flow cytometry. The results showed that MSCs were positive for CD44and negative for CD45by FCM. These results identify that our isolated cells are bone marrow mesenchymal stem cells, which were distinct from the hemopoietic stem cell.3) Through measuring bone mineral density, bone biochemical markers and bone tissue micro-structure, we evaluated the effect of cell treatment and drug treatment. Transplantation after12w, the BMD of the lumbar and femur, bone microstructure were significantly increased compared with model group, and the corresponding bone biochemical markers also dropped,and display balanced trend of bone metabolism. These evidences suggested BMSCs transplantation can improve bone mass and promote bone formation. |
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