| Osteoporosis is a systemic skeletal disease characterized by low bone mass and deterioration of bone microarchitecture, with a consequent increased fragility of bone and susceptibility to fracture. The incidence of osteoporosis is rising up as longevity being increased. The osteoporotic fracture, as the most dangerous complication of osteoporosis, is more difficult to treat for the poor tissue basic, coupled with higher activity of osteoclast and the insufficient ability of bone formation, which undoubtedly threatens the elders'health and normal life. Effective medicine treatment that could decrease the incidence of and promote the healing process of osteoporotic fracture, therefore improve the elders'life quanlity, is being expected.Simvastatin, an inhibitor of hydroxymethylglutaryl-coenzyme A(HMG-CoA) reductase,has been widely used for the treatment of hyperlipidemia through lowering cholesterol levels by blocking enzymes that are essential for cholesterol production.Since the statins were reported to be potentially effective for bone formation firstly in 1999, more and more studies were performed to investigate the bone-formation- promotion ability of statins, including simvastatin, while the results were contradictory for the different study desigh.The inconsistent conclusions exist mainly in the clinical and in-vivo animal studies, though it was reported that simvastatin stimulated bone turnover and showed the tendency of decreasing bone mass in normal rats, another study observed the beneficial effect of simvastatin on osteoporosis rat, while further study was needed to investigate the effect of simvastatin on osteoporotic fracture repair.In vitro studies, unlike those in vivo, had a consistent conclusion that simvastatin stimulated the osteogenic differentiation of bone marrow stem cells or osteoprogenitor cell, but the molecular mechanism is still unclear.The present study aimed to investigate the effect and related mechanism of simvastatin on bone formation from treating rat osteoporotic fracture model in vivo and bone marrow stromal cells cultured in vitro.Part 1 Effect of simvastatin on fracture healing in osteoporotic ratsThe impact of osteoporosis on fracture healing process was demonstrated by most basic and clinical studies, though few of those reported no significant delaying of osteoprotic fracture compared to normal bone.Simvastatin has been demonstrated of its potential benefit for bone formation, but few study was focused on the effect of simvastatin on osteoporotic fracture repair. Bilateral ovariectomy has been widely used to establish an animal model of post-menopause osteoporosis in human being, which had been used in the present study to mimic osteoporotic fracture with a femur fracture operation 4 weeks after bilateral ovariectomy.Objective:The present study aimed to verify the delayed process of fracture healing in osteoporisis rat, as well as the effect and related mechanism of simvastatin on osteoprotic fracture healing.Methods:Fouty 12-week old female Sprague-Dawley rats were randomly divided into 5 groups of 8 animals in each group:Sham group, OVX group, N+Fx group, OVX+Fx+V group and OVX+Fx+SIM group.All but Sham and N+Fx rats received bilateral ovariectomy. Rats in N+Fx, OVX+Fx+V and OVX+Fx+ SIM group underwent an operation to establish the midshaft femur fracture model 4 weeks after ovariectomy and intramedullary stabilization was achieved with titanium Kirschner wire,all fractured rats were treated with simvastatin(OVX+Fx+SIM group,20mg/kg/d) or vehicle(N+Fx,OVX+Fx+V) as control.Femurs from the Sham and OVX group rats sacrificed 4 weeks after Sham/ovariectomy operation were harvested for the bone mineral density assessment by dual-energy X-ray absorptiometry (DEXA) to confirm the successful establishment of osteoporosis model.The fractured rats were sacrificed 6 weeks after fracture.A radiographic evaluation(CR film) were taken to observe the fracture healing, a scoring system for CR film, bone mineral density were used to evaluate of fracture healing quantitatively. After fixation for 2 days in 10% normal buffered formaldehyde, dehydration, undecalcitied embedding in paraffin followed, longitudinal sections in a sagittal plane were cut at 5um for the HE staining and subsequent histological observation.Results:1 Four weeks after the ovariectomy operation, the BMD of OVX rats were significantly lower than those of Sham rats;while the body weights of OVX rats were markedly higher than those of Sham group (P<0.05).2 BMD assessment of fractured groups:In contrast to the N+Fx rats,a significant decreased BMD of were observed in OVX+Fx+V and OVX+Fx +SIM group(P<0.05), while a slight increase of BMD was observed in OVX+Fx+SIM compared to that of OVX+Fx+V group, with no statistical significance (P>0.05).3 CR film:rats in OVX+Fx+SIM and OVX+Fx+V group showed more clear fracture gap in most examples, indicated delayed fracture healing process compared to N+Fx group, in which rats showed more progressed callus consolidation, accordingly, the scores for evaluation of fracture healing in N+Fx group was significantly higher than those of OVX+Fx+SIM and OVX+Fx+V group(P<0.05). Though slight higher scores in OVX+Fx+SIM group comparing to OVX+Fx+V group were observed, no statistical significance was found.4 Histomorphological observation of callus by HE staining:rats in N+Fx group showed more mature callus with partially lamellar bone formation, while a delayed fracture healing process were observed in OVX+Fx+V and OVX+Fx+SIM rats charactered by more cartilage callus but no lamellar bone formation.Conclusions:1 Osteoporotic fracture model could be successfully achieved by performing fracture operation on rats with markedly decreased BMD compared to normal rats 4 weeks after bilateral ovariectomy.2 Comparing to normal rats, osteoporotic rats showed delayed process of fracture rapair.Though simvastatin showed the tendency of potential ability to prevent bone loss and thereby to promote fracture healing, the contribution is so limited that more study of simvastatin on osteoporotic fracture healing is required.Part 2 Simvastatin promotes the osteoblast differentiation of rat bone marrow stromal cellsBMSCs can differentiate along multiple lineages such as osteoblasts in the specific induction medium. The ease of harvest from a donor bone marrow together with the ability to form bone in vivo make BMSCs ideal for clinical applications in tissue engineering, cell transplantation and gene therapy. Expression and functional activity changes of specific proteins were considered the markers of differentiating from BMSCs to any lineage respectively. Increased expression of alkaline Phosphatase during the process of osteogenic differentiation of BMSCs was a specific indication, bBone morphogenetic proteins 2, BMP-2 is one of the most widely accepted factors with strong activity of inducing bone formation. The mineralization of extracellular matrix is also a specific biological behaviour of mature osteoblast.Simvastatin has been widely used for the treatment of hyperlipidemia through lowering cholesterol levels by blocking enzymes that are essential for cholesterol production and was first reported to be a potent stimulator of bone formation in 1999, which has been attracting much more attention gradually.Objective:To investigate the effect of simvastatin on the osteogenic differentiation and on extracellular matrix mineralization in rat bone marrow stromal cells, cultured and stimulated by simvastatin in vitro.Methods:Bone marrow stromal cells from the femurs and tibias of 6-week old rats were cultured in vitro, three days later the cells were cultured in osteogenic medium (50μg/ml ascorbic acid and lOmMβ-glycerophosphate sodium) treated with simvastatin(1×10-7mol/L) (SIM group) or vehicle(V group). Alkaline phosphatase staining and activity was performed at 14th day to observe the osteoblastic differentiation of BMSCs, at the same time point,the expressions of CD45,CD11b were assessed by flow cytometry, Von Kossa staining for observing extracellular matrix mineralization was performed at 21th day.Results:1 Flow cytometry showed the positive expression rates for CD45 and CD11b were 6.75±1.26% and 7.91±0.96% separately.2 Cells in SIM showed significantly higher ALP activity and positive cell rate at 14th day.3 SIM could markedly promote the ability of extracellular matrix mineralization compared with those of V group at 21th day.Conclusions:1 The method for culturing BMSCs used in this study had been proved successful, as the cells cultured in the inducing medium showed the abilty of osteogenic differentiation and extracellular matrix mineralization.2 Simvastatin promoted the osteogenic differentiation of BMSCs indicated by the higher ALP activity and enhanced ability of extracellular matrix mineralization.Part 3 Gene expresssion profiles of simvastatin stimulated rat bone marrow stromal cellsSimvastatin was demonstrated to be effective in promoting osteogenic differentiation of bone marrow stromal cells, but the molecular mechanism was unclear, only a few genes were focused on in previous studies, such as BMP-2,cbfa1(key genes for osteogenesis) and LPL,PPARγ2(markers of adipogenic differentiation),the development of microarray gene chip make it possible to learn much more genes expression simultaneously, the establish of gene expression profile of osteogenic BMSCs with or without simvastatin stimulation would undoubtedly supply a profound reference to verify the possible genes that participate in this process.Objective:To establish the gene expresssion profiles of simvastatin stimulated rat bone marrow stromal cells in osteogenic differentiation by rats olige microarray gene chip, and to provide a theory base and reference for the subsequent studies and the possible use of simvastatin in bone disease, as well as to further understand the mechanism of simvastatin stimulation of bone formation at least.Methods:Bone marrow stromal cells from the femurs and tibias of 6-week old rats were cultured in vitro, three days later the cells were treated with simvastatin(1×10-7mol/L)(SIM group) or vehicle(V group). At 21th day total RNA was extracted for detecting the gene expression profiles by Oligonucleotides microarray chip and were confirmed by real-time quantitative RT-PCR.Results:1 The D260/ D280 value of total RNA in V group and SIM group were 2.14 and 2.15,analyzing of RNA using 1.0% agarose gel electrophoresis showed that 18S rRNA and 28S rRNA straps were clear, suggested that we have acquired the highly purified total RNA.2 Oligonucleotides microarray chip analysis showed that 502 genes out of 22,575 rat genes had differential expression (≥1.5 fold or≤0.67 fold), including genes known to be related to osteogenesis, such as, ALP1,TGFβ1, OCN,BMP-2,IBSP,MMP13,as well as some involved in lipid metabolism, cytokine, transcription factors and cell signal pathway.3 Validation of gene expression profiling results by real-time PCR:TGF-β1, BMP-2, MMP-13, OCN and ALP differentially expressed between V group and SIM group,indicating congruity results between microarray and real-time PCR assessment. Conclusions:Simvastatin could regulate lots of genes expression during the osteogenic differentiation of BMSCs in vitro, many genes associated with osteogenesis may participate in the process of simvastin promoting BMSCs osteogenic differentiation.
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