Long Non-coding RNA SNHG7 Suppresses Inflammation and Apoptosis of Chondrocytes Through Inactivating of p38 MAPK Signaling Pathway in Osteoarthritis.

This study aims to explore the molecular mechanism of LncRNA SNHG7 in Osteoarthritis (OA). Cartilage tissues of OA patients or patients with trauma or amputation were collected. Compared to normal cartilage tissues, SNHG7 was downregulated while miR-324-3p was upregulated in cartilage tissues of OA patients. IL-1ß was used to induce damage to chondrocytes and treatment with IL-1ß reduced SNHG7 expression in OA chondrocytes. In IL-1ß-treated OA chondrocytes, SNHG7 overexpression reduced the levels of TNF-α and IL-6, inhibited cell apoptosis, and increased cell viability. Additionally, the luciferase reporter assay proved that SNHG7 upregulated dual-specificity phosphatase 1 (DUSP1) by sponging miR-324-3p, thereby inactivating the p38 MAPK signaling pathway by regulating the miR-324-3p/DUSP1 axis. Anisomycin (a p38 MAPK activator) enhanced OA chondrocytes inflammation, promoted cell apoptosis, and reduced cell viability; however, this was reversed by SNHG7 overexpression. This study demonstrates that the SNHG7/miR-324-3p/DUSP1 axis suppresses OA chondrocytes inflammation and apoptosis by inhibiting the p38 MAPK signaling pathway. Thus, this study indicates that SNHG7 is a novel target for OA treatment.

High levels of TDO2 in relation to pro-inflammatory cytokines in synovium and synovial fluid of patients with osteoarthritis.

BACKGROUND: Tryptophan 2,3-dioxygenase (TDO2) is the primary enzyme that catabolizes tryptophan to kynurenine. Numerous studies have suggested that TDO2 is involved in inflammation-related diseases. However, its role in osteoarthritis (OA) has not yet been investigated. The aim of the present study was to explore the levels of TDO2 in the synovium and synovial fluid (SF) of patients with OA and its correlation with clinical manifestations and levels of pro-inflammatory cytokines.  METHODS : Synovium and SF samples were collected from patients with OA and patients with joint trauma (controls) during surgery. An enzyme-linked immunosorbent assay (ELISA) was used to measure TDO2, interleukin-1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) levels in the synovium and SF. Diagnostic performance of TDO2 in the synovium to discriminate between controls and OA patients was assessed using receiver operating characteristic (ROC) curve analysis. Correlations between TDO2 levels, OA clinical features, and pro-inflammatory cytokines were evaluated using Pearson correlation analysis. Effects of IL-1ß or TNF-α stimulation on TDO2 expression in OA-fibroblast-like synoviocytes (OA-FLS) were also examined. RESULTS: The levels of TDO2, IL-1ß, and TNF-α in the synovium of patients with OA were found to be significantly higher than those in controls. ROC curve analysis revealed an area under the curve (AUC) of 0.800 with 64.3% sensitivity and 85.0% specificity of TOD2 in the synovium, which enabled discriminating patients with OA from controls. Moreover, protein expression of TDO2 was upregulated to a greater extent in OA-FLS than in normal synovial fibroblasts (NSF). Furthermore, the levels of TDO2 showed significantly positive correlation with IL-1ß and TNF-α levels in the synovium and SF. TDO2 levels in the synovium were also positively correlated with the Kellgren-Lawrence score. Additionally, TDO2 protein expression was significantly increased in IL-1ß‒ or TNF-α‒stimulated OA-FLS than in control FLS. CONCLUSION: These data indicate that highTDO2 levels in the synovium can be correlated with pro-inflammatory cytokines and severity of OA.

Long noncoding RNA ZBED3-AS1 induces the differentiation of mesenchymal stem cells and enhances bone regeneration by repressing IL-1ß via Wnt/ß-catenin signaling pathway.

Bone regeneration, as a physiological process of bone formation, is regulated by multiple cytokines. Long noncoding RNAs are involved in the progress of bone formation. The present study investigated role by which ZBED3-AS1 acts to control the differentiation of mesenchymal stem cells (MSCs) and bone regeneration. Bioinformatics prediction and dual luciferase reporter gene assay identified putative ZBED3-AS1 binding sites on the 3'-untranslated region of interleukin-1ß (IL-1ß). Then, RNA immunoprecipitation and chromatin immunoprecipitation assays confirmed that ZBED3-AS1 could regulate the expression of IL-1ß by binding to the transcription factor CREB. Notably, ZBED3-AS1 was shown to negatively regulate IL-1ß expression. After model establishment in rats simulating bone injury, MSCs were isolated and delivered with ZBED3-AS1, Si-ZBED3-AS1, Si-IL-1ß, or DKK (inhibitor of Wnt/ß-catenin signaling pathway) to identify their roles in osteogenic differentiation by evaluating MSC colony formation and proliferation. Then, number of mineralized nodules, alkaline phosphatase (ALP) activity and osteocalcin (OCN) expression, and expression of osteogenesis-related genes were determined. Overexpression of ZBED3-AS1 or silencing of IL-1ß was shown to accelerate ectopic osteogenesis, as reflected by increasing the number of mineralized nodules, ALP activity, and OCN expression, and promoting MSC colony formation and proliferation. Additionally, ZBED3-AS1 activated the Wnt/ß-catenin signaling pathway by negatively regulating IL-1ß. IL-1ß inhibited osteogenic differentiation by suppressing the Wnt/ß-catenin signaling pathway. Furthermore, the effect of ZBED3-AS1 and IL-1ß on osteogenic differentiation was confirmed in vivo. Taken together, upregulation of ZBED3-AS1 could restore differentiation of MSCs and enhance bone regeneration via activation of Wnt/ß-catenin signaling pathway by repressing IL-1ß.

Interleukin-34 Synovial Fluid Was Associated with Knee Osteoarthritis Severity: A Cross-Sectional Study in Knee Osteoarthritis Patients in Different Radiographic Stages.

BACKGROUND: Inflammation might play a crucial role in the pathogenesis of osteoarthritis (OA). Interleukin-34 (IL-34) is a well-known proinflammatory cytokine. OBJECTIVE: The objective of this study was to detect IL-34 levels in serum and synovial fluid (SF) of patients with OA and to investigate their correlation with radiographic and symptomatic severity. METHODS: One hundred and eighty-two OA patients and 69 controls were recruited. IL-34 levels were measured by enzyme-linked immunosorbent assay (ELISA). Radiographic and symptomatic severity of OA was reflected by Kellgren-Lawrence (KL) grades and Western Ontario McMaster University Osteoarthritis Index (WOMAC) scores, respectively. RESULTS: SF IL-34 levels were independently associated with the KL grade (B = 0.273, 95% CI: 0.150-0.395; P < 0.001). SF IL-34 levels were significantly correlated with WOMAC scores (r = 0.265, 95% CI: 0.123-0.399; P < 0.001). The correlation between SF IL-34 levels and WOMAC scores was still significant after adjusting for confounding factors (B = 0.020, 95% CI: 0.001-0.038; P = 0.035) in OA patients. CONCLUSIONS: We found that IL-34 levels in SF were significantly associated with the radiographic and symptomatic severity of knee OA.

TRPV4 functions in flow shear stress induced early osteogenic differentiation of human bone marrow mesenchymal stem cells.

Mechanical cues have been shown to induce osteogenic differentiation of bone marrow stromal cells (MSCs). The TRPV4 channel, a Ca2+-permeable membrane ion channel, is implicated in the transduction of external mechanical stimulation into specific intracellular responses in a wide variety of bone cells. However, the role of TRPV4 in transducing and regulating the differentiation of human MSCs in response to flow shear stress (FSS) is unclear. In this study, using FSS and calcium imaging, we demonstrated that FSS activated early osteogenic differentiation, as shown by the early osteogenic differentiation marker osterix (Osx) and alkaline phosphatase (ALP) staining. Increases in intracellular Ca2+ and in the percentage of responding cells were induced by FSS. However, the late osteogenic differentiation marker Ocn and in vitro mineralization were unchanged after FSS stimulation. TRPV4 channels mediated the FSS-induced Ca2+ influx and osteogenic differentiation of MSCs, which were inhibited by a selective TRPV4 blocker HC-067047 and specific Trpv4 siRNA. Ca2+ influx through TRPV4 promoted NFATc1 nuclear localization. These results identify an essential role of TRPV4 in FSS-induced early osteogenic differentiation of human MSCs.

Gremlin-1 suppression increases BMP-2-induced osteogenesis of human mesenchymal stem cells.

Previous research focusing on rodent cells and animal models has demonstrated that gremlin-1 antagonizes bone morphogenetic proteins (BMPs) in order to suppress osteogenesis. However, the impact of gremlin­1 on osteogenesis in human bone marrow-derived mesenchymal stem cells (MSCs) remains unknown. The aim of the present study was to test the effects of gremlin-1 on viability and in vitro BMP-2-induced osteogenic differentiation of human bone marrow­derived mesenchymal stem cells (MSCs). Gremlin­1­specific small interfering RNA (siRNA) inhibited gremlin­1 mRNA and protein expression in human MSCs. The mRNA expression levels of osteoblastic genes were analyzed using reverse transcription-quantitative polymerase chain reaction, and calcification and enzymatic alkaline phosphatase (ALP) activity assessed the BMP­2­induced osteogenic differentiation of human MSCs. The results indicated that gremlin­1 suppression significantly increased human MSC metabolism and DNA content. The expression levels of osteoblastic genes were also significantly increased by gremlin­1 inhibition. In the gremlin­1­inhibited group, enzymatic ALP activity was significantly increased. In addition, due to BMP­2­inducing osteoblasts, gremlin­1 inhibition increased calcium deposits. The present study indicated that gremlin­1 inhibited the cell viability and osteogenic differentiation of human MSCs and that the suppression of gremlin­1 expression suppressed can increase the cell viability and osteogenic differentiation of human MSCs induced by BMP-2.

miR-92a promotes tumor growth of osteosarcoma by targeting PTEN/AKT signaling pathway.

MicroRNAs (miRNAs) play critical roles in human cancers including osteosarcoma (OS). miR-92a has been found to be a cancer-related miRNA in many cancer types and it is upregulated in OS cell lines. However, the expression and biological function of miR-92a in OS have not been investigated. In this study, we showed that miR-92a expression was increased in OS tissues, and its high expression was correlated with clinical stage, T classification and histological differentiation. Furthermore, patients with high expression of miR-92a had a significantly poorer survival rate. Functionally, miR-92a overexpression promoted the proliferation and cell cycle progression, and inhibited apoptosis in MG-63 cells. While inhibition of miR-92a showed contrary effects with reduced proliferation, cell cycle arrest at G1 phase and increased apoptosis in U2OS cells. Moreover, we confirmed that miR-92a inhibition reversed the tumor growth of OS cells in nude mice. Phosphatase and tensin homolog (PTEN), a well-known tumor suppressor, was confirmed to be the direct downstream target of miR-92a in OS. Notably, miR-92a consequently regulated the expression of the downstream targets of PTEN/AKT signaling pathway including p-Akt(Ser473), mTOR, p-p27(Thr157) and p-MDM2(Ser166). Furthermore, PTEN knockdown abrogated the functional effects of miR-92a silencing on the proliferation, apoptosis and cell cycle progression in OS cells. Thus, miR-92a that exerts an oncogenic role by targeting PTEN/AKT pathway in OS potentially acts as a biomarker and drug-target.

Effects of five treatment regimens on blood loss and blood transfusion in total knee arthroplasty: a preliminary study in China
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OBJECTIVE: Our study is aimed to explore effects of five treatment regimens on blood loss and blood transfusion rate in total knee arthroplasty (TKA) patients. METHODS: 191 TKA patients were divided into the rivaroxaban, nadroparin, and tranexamic acid groups (n = 37 each) as well as into the affected-limb-position and tourniquet group (n = 40 each). A 3-month follow-up after operation was needed for all patients. The total blood loss, hidden blood loss, and dominant blood loss were recorded, and hemoglobin and red blood cell changes, pain and knee swelling degrees, hospital for special surgery (HSS), and American knee society (KSS) knee scores were observed. RESULTS: When compared with the rivaroxaban, nadroparin, and tourniquet groups, TKA patients' dominant blood loss, hidden blood loss, total blood loss, rate and volume of blood transfusion in the tranexamic acid and affected-limb-position groups were significantly decreased. While 7 days after operation, the hemoglobin and red blood cells in the tranexamic acid and affected-limb-position groups were significantly increased. At 1 month and 3 months after operation, when compared with the rivaroxaban, nadroparin, and tourniquet groups, the HSS and KSS scores in the tranexamic acid and affected-limb-position groups were all increased. In comparison with the rivaroxaban, nadroparin, and tourniquet groups, the D-Dimers after operation in the tranexamic acid and affected-limb-position groups were significantly lower. CONCLUSION: These results demonstrated that for TKA patients, the tranexamic acid and affected-limb-position could obviously reduce the blood loss and blood transfusion rate.
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A predictive model to estimate the pretest probability of metastasis in patients with osteosarcoma.

Osteosarcomas (OSs) represent a huge challenge to improve the overall survival, especially in metastatic patients. Increasing evidence indicates that both tumor-associated elements but also on host-associated elements are under a remarkable effect on the prognosis of cancer patients, especially systemic inflammatory response. By analyzing a series prognosis of factors, including age, gender, primary tumor size, tumor location, tumor grade, and histological classification, monocyte ratio, and NLR ratio, a clinical predictive model was established by using stepwise logistic regression involved circulating leukocyte to compute the estimated probabilities of metastases for OS patients. The clinical predictive model was described by the following equations: probability of developing metastases = ex/(1 + ex), x = -2.150 +  (1.680 × monocyte ratio) + (1.533 × NLR ratio), where is the base of the natural logarithm, the assignment to each of the 2 variables is 1 if the ratio >1 (otherwise 0). The calculated AUC of the receiver-operating characteristic curve as 0.793 revealed well accuracy of this model (95% CI, 0.740-0.845). The predicted probabilities that we generated with the cross-validation procedure had a similar AUC (0.743; 95% CI, 0.684-0.803). The present model could be used to improve the outcomes of the metastases by developing a predictive model considering circulating leukocyte influence to estimate the pretest probability of developing metastases in patients with OS.

Inhibition of the p38-MAPK signaling pathway suppresses the apoptosis and expression of proinflammatory cytokines in human osteoarthritis chondrocytes.

This study aims to investigate the effects of p38-MAPK signaling pathway on the apoptosis and expression of proinflammatory cytokines in human osteoarthritis (OA) chondrocytes. Human articular cartilage specimens were obtained from 57 OA patients and 31 patients with lower extremity traumatic amputations. The expressions of p38-MAPK pathway-related proteins in cartilage tissue were detected by immunochemistry. Cultured chondrocytes isolated from human OA cartilage were assigned into the blank group, the IL-1ß group, the PD (PD980959, ERK pathway inhibitors)+IL-1ß group, the SB (SB203580, p38 pathway inhibitors)+IL-1ß group, and the SP (SP600125, JNK signaling pathway inhibitors)+IL-1ß group. Cell proliferation and apoptosis were detected by MTT assay and flow cytometry. Western blotting was used to detect the expressions of MAPK pathway-related proteins. The mRNA expressions of IL-1, IL-6, and TNF-α were detected by qRT-PCR. The positive rates of p-p38, p-JNK and p-ERK in OA cartilage were higher than those in normal cartilage. Compared with the blank group, cell proliferation rate was decreased, cell apoptotic rate was increased, the mRNA expressions of IL-1, IL-6, TNF-α and the expressions of p-p38, p-JNK and p-ERK were increased in the IL-1ß group, while opposite trends were observed in the PD+IL-1ß, SB+IL-1ß, and SP+IL-1ß groups. Our study provides evidence that inhibition of the p38-MAPK signaling pathway could suppress the apoptosis and expression of proinflammatory cytokines in human OA chondrocytes.

Fibroblast growth factor-21 concentration in serum and synovial fluid is associated with radiographic bone loss of knee osteoarthritis.

We aimed to evaluate whether FGF-21 concentration in serum and synovial fluid (SF) is associated with radiographic bone loss of knee osteoarthritis (OA). A total of 186 OA patients and 108 controls were recruited. The radiographic bone loss of knee OA was assessed by the Ahlbäck grading scale. FGF-21 concentration in serum and SF was measured by enzyme-linked immunosorbent assay (ELISA). We demonstrated that OA patients had significantly higher serum FGF-21 concentration compared with controls (204.30 [range 158.25-279.16] ng/L vs. 130.72 [range 94.93-218.03] ng/L, p < 0.01). FGF-21 concentration in serum was well correlated with that in paired SF samples (r = 0.668, p < 0.001). In OA patients, those with a higher Ahlbäck grade had significantly higher serum and SF FGF-21 concentration (p < 0.001 for both). FGF-21 concentration in serum and SF was significantly and independently associated with the Ahlbäck grade (r = 0.403, p < 0.001 and r = 0.410, p < 0.001; respectively). These findings indicated that FGF-21 might be a potential biomarker for predicting bone loss of OA. Therapeutic interventions by blocking FGF-21 signaling pathways to delay the degenerative process of OA warrants further investigations.

FBXW7 acts as an independent prognostic marker and inhibits tumor growth in human osteosarcoma.

F-box and WD repeat domain-containing 7 (FBXW7) is a potent tumor suppressor in human cancers including breast cancer, colorectal cancer, gastric cancer and hepatocellular carcinoma. In this study, we found that the expressions of FBXW7 protein and mRNA levels in osteosarcoma (OS) cases were significantly lower than those in normal bone tissues. Clinical analysis indicated that FBXW7 was expressed at lower levels in OS patients with advanced clinical stage, high T classification and poor histological differentiation. Furthermore, we demonstrated that high expression of FBXW7 was correlated with a better 5-year survival of OS patients. Multivariate Cox regression analysis indicated that FBXW7 was an independent prognostic marker in OS. Our in vitro studies showed that FBXW7 overexpression inhibited cell cycle transition and cell proliferation, and promoted apoptosis in both U2OS and MG-63 cells. In a nude mouse xenograft model, FBXW7 overexpression slowed down tumor growth by inducing apoptosis and growth arrest. Mechanistically, FBXW7 inversely regulated oncoprotein c-Myc and cyclin E levels in both U2OS and MG-63 cells. Together these findings suggest that FBXW7 may serve as a prognostic biomarker and inhibit tumor progression by inducing apoptosis and growth arrest in OS.

Serum fetuin-A levels are inversely associated with clinical severity in patients with primary knee osteoarthritis.

The present study aimed to detect serum fetuin-A levels in knee osteoarthritis (OA) patients and to investigate their correlation with clinical severity. We enrolled 215 knee OA patients and 76 healthy controls. We measured serum fetuin-A levels by enzyme-linked immunosorbent assay and assessed the correlation between serum fetuin-A levels and Kellgren-Lawrence grades as well as Western Ontario and McMaster Universities Arthritis Index scores in OA patients. Our results demonstrated that serum fetuin-A levels were independently and negatively correlated with greater clinical severity in OA patients.

Correlation of synovial fluid HMGB-1 levels with radiographic severity of knee osteoarthritis.

PURPOSE: This study measured high-mobility group box 1 (HMGB-1) levels in serum and synovial fluid (SF) in patients with primary knee osteoarthritis (OA) and correlated these levels with radiographic disease severity. METHODS: Seventy-eight OA patients and 30 controls were enrolled in this study. All OA patients were scored according to the Kellgren-Lawrence (KL) grading system. HMGB-1 levels were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: SF HMGB-1 levels were significantly higher in knee OA patients, compared with controls (P < 0.01). Moreover, SF HMGB-1 levels were positively associated with KL scores (P < 0.01). Multinomial logistic regression demonstrated that the SF HMGB-1 level was an independent factor for radiographic severity of OA (P=0.002); however, serum HMGB-1 levels did not differ significantly between OA patients and controls and did not correlate with KL scores (P > 0.05). CONCLUSION: These results demonstrate that HMGB-1 levels in SF of knee OA patients are independently associated with radiographic disease severity.

High pressure may inhibit periprosthetic osteogenesis.

Mechanical effects have been demonstrated to activate periprosthetic osteoclasts and hence to promote bone resorption. However, the periprosthetic mechanical effect on osteoblast function is not clearly understood. The purpose of this study was to explore whether the high pressure on bone caused by a prosthesis affects periprosthetic osteoblast function. We applied static pressure of various magnitudes to SV40-transfected human fetal osteoblast cells, then assayed bioactivities compared to cells cultured without pressure (control). The results showed that osteoblast proliferation, differentiation, apoptosis, necrosis, and mineralization were all sensitive to static pressure, and the effects were magnitude dependent. Low-level static pressure (20 kPa) enhanced osteogenesis. Under 50-100 kPa static pressure, proliferation was inhibited and apoptosis was enhanced, but the cellular phenotype could be maintained. High pressure (250-500 kPa) totally inhibited the bioactivity of the osteoblasts and induced necrosis. Mineralization nodules decreased significantly under 100 kPa pressure, while no nodules could be found under 250 and 500 kPa pressure. RUNX2, COL-1, and BGP mRNA expression was significantly downregulated under 250 and 500 kPa. SOX9 expression was significantly upregulated at 100 kPa but significantly downregulated at 250 and 500 kPa. RANKL/OPG expression was increased under pressure, and the differences were significant at 100 and 500 kPa. These results suggest that periprosthetic high pressure may inhibit osteogenesis and promote osteoclastogenesis. Countermeasures should be developed to improve periprosthetic osteogenesis.

Transforming growth factor-beta activated kinase 1 signaling pathways regulate TNF-alpha production by titanium alloy particles in RAW 264.7 cells.

Implant particles may induce inflammatory response by activating the nuclear factor kappaB (NFkappaB) and mitogen activated protein kinases (MAPK). Previous studies have shown that these signaling pathways are involved in the transforming growth factor-beta activated kinase 1 (TAK1) in signaling cascades induced by the receptor activator of NFkappaB ligand (RANKL) and tumor necrosis factor-alpha (TNF-alpha) as well as interleukin-1beta (IL-1beta). In this study, the roles of the TAK1 pathway in the production of the pro-inflammatory cytokine TNF-alpha in RAW 264.7 cells exposed to titanium alloy particles were investigated. Endogenous TAK1 was phosphorylated upon simulating RAW 264.7 cells by titanium alloy particles. The critical role for TAK1 in p38MAPK and NFkappaB activation was as well confirmed by the inhibition of p38MAPK and NFkappaB activity following 5Z-7-oxozeaenol, a selective inhibitor of TAK1. Furthermore, it was found that TNF-alpha was completely blocked by 5Z-7-oxozeaenol in RAW 264.7 cells. These results suggest that the TAK1-MAPK-NFkappaB signaling pathways may be a potential pharmacological target that can prevent instability for arthroplasty prosthesis.

The contour of cementless femoral stem has minor effect on initial periprosthetic von Mises stress distribution. A 3-dimensional finite element analysis.

OBJECTIVE: To evaluate periprosthetic von Mises stress distribution with cementless femoral stems of various contours. METHODS: The study was carried out at the Department of Orthopaedics, Shanghai 6th Hospital, Shanghai, China between May 2008 and February 2009. Finite element models of proximal femoral replacement with 4 cementless stems (Alloclassic, Ribbed Anatomic, VerSys, and Securi-fit) of various contours were set up. Under the loading conditions of walking and stair climbing, 3-dimensional periprosthetic von Mises stresses were calculated, and the stress distribution patterns were compared. RESULTS: Periprosthetic stresses were increased in level 1, 2, and 3 under the 2 loading conditions, and more considerably in level 2 and 3. The stresses were higher on the medial side in all cases. No remarkable difference was found in the patterns between the 4 stems. CONCLUSION: The contour design of femoral stem has minor effect on initial periprosthetic von Mises stress distribution.