Fibroblast growth factor 10 delays the progression of osteoarthritis by attenuating synovial fibrosis via inhibition of IL-6/JAK2/STAT3 signaling in vivo and in vitro.

Synovial fibrosis is a driver in the progression of osteoarthritis (OA). Fibroblast growth factor 10 (FGF10) has prominent anti-fibrotic effects in many diseases. Thus, we explored the anti-fibrosis effects of FGF10 in OA synovial tissue. In vitro, fibroblast-like synoviocytes (FLSs) were isolated from OA synovial tissue and stimulated with TGF-ß to establish a cell model of fibrosis. After treatment with FGF10, we assessed the effects on FLS proliferation and migration using CCK-8, EdU, and scratch assays, and collagen production was observed using Sirius Red Stain. The JAK2/STAT3 pathway and expression of fibrotic markers were evaluated through western blotting (WB) and immunofluorescence (IF). In vivo, we treated mice with OA induced by surgical destabilization of the medial meniscus (DMM) with FGF10 and assessed the anti-OA effect using histological and immunohistochemical (IHC) staining of MMP13, and fibrosis was evaluated using HE and Masson's trichrome staining. The expression of IL-6/JAK2/STAT3 pathway components was determined using ELISA, WB, IHC, and IF. In vitro, FGF10 inhibited TGF-ß-induced FLS proliferation and migration, decreased collagen deposition, and improved synovial fibrosis. Moreover, FGF10 mitigated synovial fibrosis and improved the symptoms of OA in DMM-induced OA mice. Overall, FGF10 had promising anti-fibrotic effects on FLSs and improved OA symptoms in mice. The IL-6/STAT3/JAK2 pathway plays key roles in the anti-fibrosis effect of FGF10. This study is the first to demonstrate that FGF10 inhibited synovial fibrosis and attenuated the progression of OA by inhibiting the IL-6/JAK2/STAT3 pathway.

Knockdown of microRNA-203 reduces cisplatin chemo-sensitivity to osteosarcoma cell lines MG63 and U2OS in vitro by targeting RUNX2.

Clinical studies have reported that miRNAs abnormal expression are associated with the generation of cisplatin-resistant to osteosarcoma. Our previous research found that miR-203 is downregulated in osteosarcoma cells and overexpressed miR-203 exerts antitumor properties on osteosarcoma cells. However, the role and mechanism of miR-203 in regulating the sensitivity of cisplatin in osteosarcoma cells remains unclear. This study aimed to investigate the effects of miR-203 in cisplatin therapy for osteosarcoma cells in vitro and determined the underlying mechanism. In this study, we found that miR-203 was significantly upregulated in osteosarcoma cells after exposure to cisplatin. miR-203 knockdown reduced the sensitivity of osteosarcoma cells to cisplatin by suppressing cell apoptosis, cell cycle arrest, and inducing invasion. Meanwhile, we found that miR-203 knockdown reduces the therapeutic sensitivity of osteosarcoma cells by upregulating RUNX2. Moreover, we found that RUNX2 silencing sensitizes osteosarcoma cells to chemotherapy treatment of cisplatin. In summary, our findings demonstrated that miR-203 knockdown reduces cisplatin chemo-sensitivity to osteosarcoma cells in vitro by targeting RUNX2, and speculated that miR-203 may be a target for drug resistance of osteosarcoma to cisplatin.

Salidroside suppresses the growth and invasion of human osteosarcoma cell lines MG63 and U2OS in vitro by inhibiting the JAK2/STAT3 signaling pathway.

Previous research has reported that salidroside exerts antitumor properties on numerous types of tumor cells; however, its effect on osteosarcoma cells remains unknown. The present study aimed to investigate the effects of salidroside on the viability, apoptosis and invasion of osteosarcoma cells in vitro, and determine the underlying mechanism of action. The results of an MTT revealed that salidroside suppressed the viability of osteosarcoma cells (MG63 and U2OS cells) in a time­ and concentration­dependent manner. The results of cell morphological analysis (profile observations and Hoechst 33258 staining) and the detection of apoptosis by flow cytometry further indicated that the decrease in osteosarcoma cell viability induced by salidroside was associated with cell apoptosis. Western blot analysis not only confirmed these results but also suggested that salidroside induced the apoptosis of osteosarcoma cells by activating the caspase­9­dependent apoptotic pathway. In addition, we reported that salidroside induced G0/G1 phase arrest and suppressed the invasion of osteosarcoma cells, as measured by flow cytometric cell cycle analysis and a Transwell invasion assay, respectively. Western blot analysis confirmed the aforementioned results. Furthermore, our findings demonstrated that salidroside induced the apoptosis, G0/G1 phase arrest and suppressed the invasion of osteosarcoma cells by inhibiting the janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) signaling pathway, as determined by western blot analysis. In summary, the findings of the present study suggested that salidroside may inhibit the progression of osteosarcoma by suppressing the growth and invasion of osteosarcoma cells. Furthermore, the investigations into the underlying mechanism demonstrated that salidroside exerted notable antitumor activity in osteosarcoma cells by inhibiting the JAK2/STAT3 signaling pathway.

MicroRNA-504 modulates osteosarcoma cell chemoresistance to cisplatin by targeting p53.

Chemoresistance implicates the therapeutic value of cisplatin and remains a primary obstacle to its clinical use. MicroRNAs (miRs) negatively modulate the expression of their target genes and are associated with the occurrence and progression of various types of tumor. The abnormal expression of miR-504 has been reported in certain types of human tumor and has been associated with tumor prognosis. However, the association between miR-504 and cisplatin in human osteosarcoma remains unclear. The present study therefore aimed to assess the in vitro effects and possible mechanism of miR-504 in cell proliferation, apoptosis and cisplatin resistance in MG63 osteosarcoma cells. The results demonstrated that miR-504 was overexpressed in osteosarcoma tissues and cells. This overexpression also induced cell proliferation, as determined by MTT and EdU staining assays. Furthermore, miR-504 suppressed cisplatin-induced apoptosis, which was demonstrated via MTT, cell morphology analysis and flow cytometry. Cisplatin-induced G1 arrest was also suppressed, which was determined by flow cytometry. The potential target genes of miR-504 were predicted using bioinformatics. p53 was confirmed to be a direct target of miR-504 using a luciferase reporter assay and western blot analysis revealed that miR-504 negatively regulated p53 expression at a molecular level. These results indicate that miR-504 contributes to cisplatin resistance in MG63 osteosarcoma cells by suppressing p53. miR-504 may therefore be a potential biomarker for cisplatin resistance in patients with osteosarcoma.

Analysis of miR-148b expression differences in stage-I and II parosteal osteosarcoma.

Expression of human serum albumin-micro RNA miR-148b in patients with stage-I and II parosteal osteosarcoma and its effect on prognosis were investigated. A total of 47 cases of fresh tissues of stage-I and II parosteal osteosarcoma and the corresponding para-carcinoma normal bone tissues resected by operation were collected; the expression of miR-148b in parosteal osteosarcoma tissues and normal bone tissues was detected, and the correlations of miR-148b expression in parosteal osteosarcoma tissues with clinicopathological parameters and prognosis were analyzed. The expression level of miR-148b in parosteal osteosarcoma tissues was significantly lower than that in para-carcinoma normal tissues (P<0.05). It was found that the low expression of miR-148b was correlated with the lung metastasis (P<0.05). Moreover, Kaplan-Meier survival curve analysis showed that the overall survival rate of patients in the low-expression miR-148b group was lower than that in the high-expression group (P<0.05). Multivariate Cox regression analysis revealed that the miR-148b level (P=0.003) was an independent prognostic factor affecting the prognosis. The results of this study showed that the expression of miR-148b in stage-I and II parosteal osteosarcoma tissues declines, which is related to the poor clinical prognosis of parosteal osteosarcoma.

MicroRNA-539 promotes osteoblast proliferation and differentiation and osteoclast apoptosis through the AXNA-dependent Wnt signaling pathway in osteoporotic rats.

This study aims to explore the effects of miR-539 on osteoblast proliferation and differentiation and osteoclast apoptosis in a rat model of osteoporosis, and its mechanism involving the regulation of the AXIN1-mediated wingless-Int (Wnt) signaling pathway. A rat model of osteoporosis was successfully established by ovariectomy. With osteoblasts and osteoclasts of rats not receiving ovariectomy in the sham group as control, those of osteoporotic rats were treated with miR-539 inhibitor, miR-539 mimic, and AXIN1 shRNA. The expression of miR-53, AXIN1, the Wnt pathway related-genes, apoptosis related-genes, and osteogenic markers were measured by RT-qPCR and Western blot analysis, respectively. Alkaline phosphatase (ALP) activity in osteoblast and tartrate-resistant acid phosphatase (TRAP) activity in osteoclasts were determined after cell transfection. Osteoblast and osteoclast viability was assayed by CCK-8 assay. Cell cycle and apoptosis of osteoblasts and osteoclasts were detected by flow cytometry. Lastly, alizarin red S staining was used to detect mineralized nodules of osteoblasts. Firstly, we determined that miR-539 was down-regulated in osteoblast and osteoclast of osteoporotic rats and AXIN1 was negatively regulated by miR-539. Additionally, overexpression of miR-539 increased the expressions of ß-catenin, LEF1, c-myc, cyclin D1, RUNX2, BGP, BMP-2 in osteoblast as well as ß-catenin, RhoA, caspase-3, and Bcl-2 in osteoclasts. Finally, overexpression of miR-539 elevated ALP activity, proliferation, and mineralized nodules in osteoblast and osteoclast apoptosis, with reduced TRAP activity in osteoclasts. Our results demonstrate that miR-539 promotes osteoblast proliferation and differentiation as well as osteoclast apoptosis through the AXIN1-dependent Wnt signaling pathway in osteoporotic rats.

Glucosamine promotes osteoblast proliferation by modulating autophagy via the mammalian target of rapamycin pathway.

Glucosamine is effective in the treatment of osteoarthritis; however, its effect on osteoporosis remains unclear. Decreased activity of osteoblasts is the main cause of osteoporosis. Here, we examined the effects of glucosamine on osteoblasts. The potential underlying mechanisms were explored. The results showed that glucosamine had a biphasic effect on the viability of hFOB1.19 osteoblasts. At low concentrations (<0.6 mM), glucosamine induced hFOB1.19 cell proliferation, whereas at high concentrations (>0.8 mM) it induced apoptosis. The autophagy inhibitor 3-methyladenine (3-MA) was used to verify that glucosamine modulated hFOB1.19 cell viability via autophagy. The induction of apoptosis by high concentrations of glucosamine was significantly exacerbated by 3-MA, whereas the promotion of cell proliferation by low concentrations of glucosamine was significantly suppressed by 3-MA. Autophagy was examined by western blot detection of autophagy-related proteins including LC3, Beclin-1, and SQSTM1/p62 and by immunofluorescence analysis of autophagosomes. Glucosamine activated autophagy in a time- and concentration-dependent manner. Investigation of the underlying mechanism showed that glucosamine inhibited the phosphorylation of m-TOR in a concentration-dependent manner within 48 h, and rapamycin significantly inhibited the phosphorylation of m-TOR. These results demonstrated that glucosamine promoted hFOB1.19 cell proliferation and increased autophagy by inhibiting the m-TOR pathway, suggesting its potential as a therapeutic agent for osteoporosis.

MicroRNA-21 promotes bone mesenchymal stem cells migration in vitro by activating PI3K/Akt/MMPs pathway.

MicroRNA-21 (miR-21) contributes to anti-apoptosis in bone marrow mesenchymal stem cells (BMSC), but its role in the migration of BMSCs remains vague. The aim of this study was to determine the possible effect of miR-21 on regulating BMSCs directional migration and the expression of MMP-2/MMP-9 in BMSCs in vitro. BMSCs were successfully infected with miR-21-up lentivirus. Cell migration using Transwell assay indicated that upregulated expression of miR-21 could significantly promote BMSCs migration. Western blot analysis indicated that miR-21 significantly upregulated the expression of MMP-2 and MMP-9, which were related to metastasis-associated genes. GM6001, the specific MMPs inhibitor, abrogated the upregulated expression of MMP-2/MMP-9 and abolished the positive effect of miR-21 on promoting BMSCs migration. Meanwhile, miR-21 significantly enhanced Akt phosphorylation, as measured by Western blot analysis. LY294002, an inhibitor of Akt activation, abrogated the phosphorylation of Akt and abolished the positive effect of miR-21 on promoting BMSCs migration and upregulating MMP-2/MMP-9 expression. These results suggest that miR-21 contributes to BMSCs migration by upregulating MMP-2/MMP-9, potentially via the PI3K/Akt pathway.

MicroRNA-203 inhibits proliferation and invasion, and promotes apoptosis of osteosarcoma cells by targeting Runt-related transcription factor 2.

Accumulating evidence indicates that microRNA-203 (miR-203) is abnormally expressed in many human tumor tissues and significantly associated with the occurrence, development and clinical outcomes of human tumors. The aim of this study was to determine the target genes and functional significance of miR-203 in osteosarcoma cells. We found reduced expression of miR-203 in osteosarcoma tissues and cells (MG63 and U2-OS) compared with the adjacent normal tissues and normal osteoblastic cells (hFOB1.19), respectively. In vitro studies further demonstrated that exogenous miR-203 overexpression inhibited osteosarcoma cell proliferation and invasion, and promoted apoptosis. At the molecular level, our results confirmed that apoptosis, cell cycle and invasion-related proteins were regulated by miR-203. Our findings also revealed that Runt-related transcription factor 2 (RUNX2) was directly negatively regulated by miR-203. These results suggested that miR-203 may function as a tumor suppressor and may therefore have therapeutic potential in the treatment of human osteosarcoma.

miR-106a-5p Suppresses the Proliferation, Migration, and Invasion of Osteosarcoma Cells by Targeting HMGA2.

We aim to investigate the effect of miR-106a-5p on the proliferation, migration, and invasion of osteosarcoma (OS) cells by targeting high-mobility group AT-hook 2 (HMGA2). Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used for detecting the expressions of miR-106-5p and HMGA2 in 137 OS and adjacent normal bone tissues. Immunohistochemistry was applied for the HMGA2 protein expression detection. Luciferase reporter gene assay was conducted for verifying whether miR-106-5p targeted HMGA2. MG63 and U2SO cells were respectively divided into five groups: Blank, miR-106a-5p, scramble, HMGA2-siRNA, and miR-106a-5p+HMGA2 groups. RT-qPCR and western blot were applied for detecting the expressions of miR-106a-5p and HMGA2 in five groups. Proliferation rate, cell cycle, invasion, and migration ability of OS cells were detected using methyl thiazolyl-tetrazolium, 5-ethynyl-2'-deoxyuridine (Edu) assay, flow cytometry, and Transwell. Compared with adjacent normal tissues, OS tissues presented with decreased miR-106a-5p expressions, elevated HMGA2 mRNA, and positive expressions (all p < 0.05). The sensitivity and specificity of miR-106a-5p were 97.8%, 93.43%, and HMGA2 mRNA were 97.8%, 99.27%, separately. miR-106a-5p and HMGA2 expressions were associated with tumor size, Enneking stage, distant metastasis, and lung metastasis. Expressions of HMGA2 in OS cells in miR-106a-5p and HMGA2 siRNA groups were both significantly decreased with the same downregulation level, and the proliferation rates in both groups were obviously slowed down after 48 h (both p < 0.001). Edu positive cells, S phase cells (majority of cells blocked at G0/G1 phase), migratory and invasive cells were obviously decreased (all p < 0.05). Downregulation of miR-106a-5p was found in OS tissues, and upregulation of miR-106a-5p can inhibit the proliferation, migration, and invasion by targeting HMGA2 in OS cells.

Roles of Cell Cyle Regulators Cyclin D1, CDK4, and p53 in Knee Osteoarthritis.

OBJECTIVE: The aim of this study was to investigate the roles of cyclin D1, CDK4, and p53 in knee osteoarthritis (KOA). METHODS: A total of 76 healthy controls and 154 KOA cases (grades ranging from II to IV) were recruited. Protein expression of cyclin D1, CDK4, and p53 were detected by immunohistochemistry, and mRNA expression levels of the cyclin D1, the CDK4, and the p53 genes were measured by reverse transcription-polymerase chain reaction. RESULTS: Both protein and mRNA expression levels of cyclin D1 and CDK4 were significantly lower in KOA cases than those in healthy controls, while protein and mRNA expression of p53 was significantly higher in KOA cases than that in healthy controls (all p < 0.05). As the grades of KOA increased, Cyclin D1 and CDK4 mRNA expressions decreased, whereas p53 mRNA expression increased (all p < 0.05). In KOA cases, mRNA expression of Cyclin D1 was positively correlated to CDK4 mRNA levels (r = 0.386, p < 0.001), while negatively correlated with p53 mRNA levels (r = -0.227, p = 0.005). CONCLUSIONS: Expression of the Cyclin D1, CDK4, and p53 genes are correlated with the disease grades of KOA.

Tranexamic Acid Treatment Decreases Hidden Blood Loss in Total Knee Arthroplasty.

The aim of our meta-analysis is to investigate the effect of tranexamic acid (TXA) on hidden blood loss (HBL) in total knee arthroplasty (TKA). A literature search was undertaken to identify all cohort studies that investigated the effect of TXA on HBL in TKA. Both electronic database search and manual search were used to retrieve studies related to the topic, and the retrieved studies were screened according to our stringent inclusion and exclusion criteria. Comprehensive Meta-analysis 2.0 software (CMA 2.0) was used for statistical analysis of the data retrieved from selected case-cohort studies. A total of 480 studies were initially retrieved, and after further screening and selection, 7 studies were eventually incorporated into our meta-analysis. The 7 studies included a total of 530 osteoarthritis or rheumatic arthritis patients who had TKA, and among them, 250 patients received an intravenous injection of TXA as cases and 280 patients received an intravenous injection of sodium chloride as sterile placebo controls. Our meta-analysis revealed that the volume of HBL of cases was lower than that of controls, which was statistically significant. The ethnicity-stratified analysis suggested that the volume of HBL of cases was significantly lower than that of controls in both the Asians and whites, also at statistically significant levels. Our meta-analysis provides strong evidence that TXA significantly reduces HBL in TKA, thus TXA can be used as a standard drug to prevent/reduce HBL in TKA.

[Analysis of clinical effect of unicompartmental knee arthroplasty in treating spontaneous osteonecrosis of the knee].

OBJECTIVE: To investigate the short-term clinical outcome of unicompartmental knee arthroplasty for the treatment of spontaneous osteonecrosis of the knee. METHODS: From September 2013 to April 2014,5 patients with spontaneous osteonecrosis of the knee underwent unicompartmental knee arthroplasty, included 3 males and 2 females, aged from 65 to 80 years old with an average of 74 years. The courses of disease was from 1 to 6 years with the mean of 3 years. According to the radiographic staging criteria of Koshino, 1 case was stage II, 2 cases were stage III, 2 cases were stage IV. Clinical effects were assessed by VAS score, HSS score, and knee range of motion, tibiofemoral angle before and after operation. RESULTS: All the patients were followed up from 6 to 7 months with an average of 6.4 months. All incisions obtained primary healing, and there were no complications such as infection, thrombosis, fracture of lower limbs. All 5 patients' pain relieved and their knee function improved significantly after operation, but knee range of motion had no obviously improved. Postoperative HSS scores, VAS scores, tibiofemoral angle were significantly improved than that of preoperative. CONCLUSION: The short-term effect of unicompartmental knee arthroplasty in treating spontaneous osteonecrosis of the knee is satisfactory.

[Total hip arthroplasty for degeneration of infected hip: a report of 7 cases].

OBJECTIVE: To evaluate indications and clinical results of total hip arthroplasties for degenerative hips with history of infection. METHODS: Seven cases of degenerative hip with history of infection underwent primary total hip arthroplasties, which involved 5 males and 2 females, with an average age of 45.8 years (range, 30 to 65 years). The quiescent period of infection were more than 10 years in all hips. According to Kim classification, 3 cases were of type I, and 4 of type II. The method to exclude active infection at the site of degenerative hips preoperatively was combination of physical examination, erythrocyte sedimentation rate and C-reactive protein level. The lateral incision was adopted in all cases, and all prosthesis were cementless. The clinical results of affected hips were assessed according to Harris hip score. RESULTS: The follow-up was performed with the mean duration of 33.5 months (range, 21 to 44 months). No recurrence of infection, damage of nerve function or deep vein thrombosis of lower extremities occurred in all cases. The mean Harris hip scores improved from 44.5 points preoperatively to 84 points at the latest follow-up. No aseptic loosening of prosthesis or periprosthetic osteolysis were found at the latest follow-up. CONCLUSION: Total hip arthroplasties has good short term results for degenerative hips with history of infection. It is important to select indicated cases and rule out the possibility of active infection.