MicroRNAs (miRNAs) have recently been recognized to have a role in human orthopedic disorders. The objective of our study was to explore the expression profile and biological function of miRNA-17-5p (miR-17-5p), which is well known to be related to cancer cell proliferation and invasion, in osteoblastic differentiation and in cell proliferation. The expression levels of miR-17-5p in the femoral head mesenchymal stem cells of 20 patients with non-traumatic osteonecrosis (ON) and 10 patients with osteoarthritis (OA) were examined by quantitative reverse transcription-PCR (qRT-PCR). Furthermore, the interaction between miR-17-5p and SMAD7 was observed. We found that in non-traumatic ON samples the level of mature miR-17-5p was significantly lower than that of OA samples (P=0.0002). By targeting SMAD7, miR-17-5p promoted nuclear translocation of beta-catenin, enhanced expression of COL1A1 and finally facilitated the proliferation and differentiation of HMSC-bm cells. We also demonstrated that restoring expression of SMAD7 in HMSC-bm cells partially reversed the function of miR-17-5p. Together, our data suggested a theory that dysfunction of a network containing miR-17-5p, SMAD7 and beta-catenin could contribute to ON pathogenesis. The present study prompts the potential clinical value of miR-17-5p in non-traumatic ON.
Adult ; Base Sequence ; Bone Morphogenetic Protein 2/metabolism ; Cell Differentiation ; Cell Line ; Cell Proliferation ; Female ; *Gene Expression Regulation ; Humans ; Male ; MicroRNAs/genetics/*metabolism ; Middle Aged ; Osteoarthritis/genetics/metabolism/pathology ; Osteoblasts/*cytology/metabolism/*pathology ; Osteogenesis ; Osteonecrosis/*genetics/metabolism/pathology ; Signal Transduction ; Smad7 Protein/*genetics/metabolism ; beta Catenin/metabolism

PURPOSE: To explore the value of transplanting peripheral blood-derived mesenchymal stem cells from allogenic rabbits (rPBMSCs) to treat osteonecrosis of the femoral head (ONFH). MATERIALS AND METHODS: rPBMSCs were separated/cultured from peripheral blood after granulocyte colony-stimulating factor mobilization. Afterwards, mobilized rPBMSCs from a second passage labeled with PKH26 were transplanted into rabbit ONFH models, which were established by liquid nitrogen freezing, to observe the effect of rPBMSCs on ONFH repair. Then, the mRNA expressions of BMP-2 and PPAR-γ in the femoral head were assessed by RT-PCR. RESULTS: After mobilization, the cultured rPBMSCs expressed mesenchymal markers of CD90, CD44, CD29, and CD105, but failed to express CD45, CD14, and CD34. The colony forming efficiency of mobilized rPBMSCs ranged from 2.8 to 10.8 per million peripheral mononuclear cells. After local transplantation, survival of the engrafted cells reached at least 8 weeks. Therein, BMP-2 was up-regulated, while PPAR-γ mRNA was down-regulated. Additionally, bone density and bone trabeculae tended to increase gradually. CONCLUSION: We confirmed that local transplantation of rPBMSCs benefits ONFH treatment and that the beneficial effects are related to the up-regulation of BMP-2 expression and the down-regulation of PPAR-γ expression.
Animals ; Blood Cells/*cytology ; Bone Morphogenetic Protein 2/genetics ; *Cell- and Tissue-Based Therapy ; Femur Head Necrosis/metabolism/*pathology/*therapy ; Gene Expression Regulation ; *Mesenchymal Stem Cell Transplantation ; Mesenchymal Stromal Cells/*cytology ; Osteonecrosis/*pathology/*therapy ; PPAR gamma/genetics ; Rabbits ; Transplantation, Homologous

Osteonecrosis of the femoral head is frequently observed in patients treated with excessive corticosteroids. However, the pathogenesis of corticosteroid-induced osteonecrosis remains unclear. The purpose of this study was to investigate the role of Toll-like receptor 4 (TLR4) signaling pathway in steroid-induced femoral head osteonecrosis in rats. Male Sprague-Dawley rats were injected intramuscularly with 20 mg/kg methylprednisolone (MP) for 8 weeks, twice per week. The animals were sacrificed at 2, 4 and 8 weeks after the last MP injection, respectively, and then allocated to the 2-, 4- and 8-week model groups (n=24 each). Rats in the control group (n=12) were not given any treatment. Histopathological analysis was performed and the concentration of tartrate-resistant acid phosphatase (TRAP) in plasma was determined. The activation of osteoclasts in the femoral head was assessed by TRAP staining. The expression of TLR4, MyD88, TRAF6 and NF-κB p65 that are involved in TLR4 signaling, and MCP-1 production were detected by using real-time PCR (RT-PCR) and Western blotting. The results showed that the osteonecrosis in the femoral head was clearly observed and the concentration of TRAP in the plasma was increased in the model rats. The femoral head tissues in MP-treated rats were positive for TRAP and the intensity of TRAP staining was greater in MP-treated rats than in control rats. As compared with the control group, the mRNA expression of TLR4 signaling-related factors was enhanced significantly at 4 and 8 weeks, and the protein levels of these factors increased significantly with time. It was concluded that MP could induce the femoral head osteonecrosis in rats, which was associated with osteoclast activation via the TLR4 signaling pathway. These findings suggest that TLR4 signaling pathway plays a pivotal role in the pathogenesis of steroid-induced osteonecrosis.
Acid Phosphatase ; metabolism ; Animals ; Blotting, Western ; Chemokine CCL2 ; genetics ; metabolism ; Femur Head ; metabolism ; pathology ; Gene Expression ; Immunohistochemistry ; Isoenzymes ; metabolism ; Male ; Methylprednisolone ; Myeloid Differentiation Factor 88 ; genetics ; metabolism ; Osteonecrosis ; chemically induced ; genetics ; metabolism ; Rats, Sprague-Dawley ; Reverse Transcriptase Polymerase Chain Reaction ; Signal Transduction ; TNF Receptor-Associated Factor 6 ; genetics ; metabolism ; Tartrate-Resistant Acid Phosphatase ; Time Factors ; Toll-Like Receptor 4 ; genetics ; metabolism ; Transcription Factor RelA ; genetics ; metabolism