The Study Of MicroRNA In Regulating The MSC For The Steroid Induced Osteonecrosis

BackgroundSteroid-associated femoral head necrosis is a common orthopaedic disease.There is no ideal conservative resolution and most of patients have to undergo total hip arthoplasty. One of the most recognized mechanism is that the osteogenic potential of the bone mesenchymal stem cells is suppressed by the steroid. Previously our research group screened the discrepancy of the miRNA spectrum between the steroid associated femoral head necrosis patients and the normal adult.Later,the miRNA spectrum of the normal human-derived bone mesenchymal stem cells before and after stimulated by large dose steroid were also obtained. The consistent miRNAs were found to be microRNA-23a. In vitro studies revealed that miR-23a was significantly down-regulated during osteogenic differentiation. Over-expression of miR-23a inhibited osteogenic differentiation of hBMSCs, whereas down-regulation of miR-23a enhanced the process. Target prediction analysis and dual luciferase reporter assays confirmed that low-density lipoprotein (LDL)-receptor-related protein 5 (LRP5) was a direct target of miR-23a.Furthermore, knockdown of LRP5 inhibited osteogenic differentiation of hBMSCs,similar to the effect observed in up-regulation of miR-23a.Lots of conservative therapy had been tried on the early stage femoral head necrosis, but there was still no satisfactory resolution. Teriparatide (rhPTH[l-34]) was a bone anabolic agent which was approved to treat osteoporosis both in women and men. In vitro studies had shown that teriparatide stimulated new bone formation by increasing osteoblast number, enhancing osteogenic differentiation of bone mesenchymal stem cell and improving cell survival. Besides osteoporosis, teriparatide had emerged as a potential curative drug in promoting spinal fusion,bone fracture healing and biphosphates associatedosteonecrosis of the jaw. One of the pathogenesis of femoral head necrosis was that the steroid suppressed the activity of the bone mesenchymal stem cell and decreased the osteogenic differentiation.So we hypothesized the teriparatide might treat the steroid induced femoral head necrosis.Objectives1.To analyze the osteogenic effect of the microRNA-23a in regulating the BMSC of the femoral head necrosis rearing process.2.To explore the potential effect of the rhPTH (1-34) in rat femoral head necrosis model.Methods1. Part 1:The rat bone mesenchymal stem cells were isolated by the whole bone marrow adherent culture method and were identified by induction and flow cytometry.MicroRNA-23a mimics,inhibitor and vector were transfected into the BMSC by lentivirus. The steroid-induced femoral head necrosis model was established by LPS (LPS,20μg/Kgx2) and MPS (40mg/Kgx3) and assessed by the micro CT and histopathology.After transfected by the miRNA-23a mimics, inhibitor or vector, the BMSCs were injected into the bone marrow cavity of both femurs. The femoral head of both sides were scanned by micro CT and analyzed by the software with regards of the trabeculae parameters. The histopathology was also analyzed by the HE staining. The LRP-5 were examined by the immunohistochemical method.2.Part 2:Rats in the experimental group were injected with the rhPTH (1-34) (20μg/Kg) everyday for 4 weeks while the control group received normal saline. The serum bone markers were analyzed and the incidence of osteonerosis were compared with micro CT and histopathology.Results1.The rats derived BMSCs could be isolated by the whole bone marrow adherent culture method.The BMSCs could be induced into osteogenic and adipogenic differentiation in vitro.The BMSCs were CD29(+),CD44(+),CD105(+),CD106(+)and CD31(-), CD34(-), HLA-DR(-).The microRNAs could be transfected by lentivirus and replicated after passage.The steroid-induced femoral head necrosis model could be established by LPS(LPS,20μg/Kgx2) and MPS (40mg/Kgx3),The incidence of osteonecrosis was 83%. The micro CT demonstrated cyst degeneration of the femoral head in the experimental group and the BV/TV, Tb.Th.were significantly lower in the experimental group. The histopathology demonstrated empty bone lacuna, necrosis of the bone marrow and the fibrosis of the trabeculae.The BV/TV, Tb.Th. were significantly higher in the microRNA-23a inhibitor transfected group. The Tb.N and Cor.Th.were comparable. Higher incidence of osteonecrosis and lower expression rate of LRP-5 were observed in the microRNA-23a mimics transfected group compared with the inhibitor group.2.The incidence of osteonecrosis was significantly lower (16.7% VS.75%,P<0.05) and the BV/TV, Tb.Th and the BMD were significantly higher in the experimental group.Conclusion1.Inhibiting the miRNA-23a leaded to the lower incidence of osteonecrosis, higher BV/TV and Tb.Th., higher LRP-5 expression while miRNA-23a mimics cause the opposite effect. 2.RhPTH(l-34) demonstrated therapeutic effect in steroid induced osteonerosis rat model,the optimum dose and effect in human needed to be expolored.