LncRNA MSTRG.22719.16 mediates the reduction of enoxaparin sodium high-viscosity bone cement-induced thrombosis by targeting the ocu-miR-326-5p/CD40 axis.

OBJECTIVE: Polymethylmethacrylate (PMMA) bone cement promotes the development of local thrombi. Our study found that a novel material, ES-PMMA bone cement, can reduce local thrombosis. We used a simple and reproducible animal model to confirm the reduction in local thrombosis and explored the associated molecular mechanism. METHODS: New Zealand rabbits, which were used to model thrombosis using extracorporeal carotid artery shunts, were divided into the following two groups, with 3 rabbits in each group: the PMMA bone cement group and the ES-PMMA bone cement group. Four hours after modelling, experimental samples, including thrombotic and vascular tissues, were collected. Thrombotic samples from the PMMA group and ES-PMMA group were subjected to lncRNA sequencing, and a lncRNA microarray was used to screen the differentially expressed lncRNAs. The expression of thrombomodulin in endothelial cells was quantified in vascular tissue samples. Differences in the lncRNA expression profiles between the thrombotic samples of the PMMA group and ES-PMMA group were assessed by base-to-base alignment in the intergenic regions of genomes. The lncRNA-miRNA-mRNA competitive endogenous RNA (ceRNA) network was established in light of ceRNA theory. Thrombosis was observed in the PMMA group and ES-PMMA group. RESULTS: The thrombotic weight was 0.00706 ± 0.00136 g/cm in the PMMA group and 0.00551 ± 0.00115 g/cm in the ES-PMMA group. Quantitative real-time polymerase chain reaction (RT-q-CR) and Western blotting revealed that the expression of CD40, which can regulate thrombosis in vascular endothelial cells, was significantly lower in the ES-PMMA group than in the PMMA group. High-throughput sequencing was used to identify 111 lncRNAs with lower expression in the ES-PMMA group than in the PMMA group. Through bioinformatics investigation, lncRNA MSTRG22719.16/ocu-miR-326-5p/CD40 binding sites were selected. Fluorescent in situ RNA hybridization (FISH) was performed to verify the lower expression of lncRNA MSTRG.22719.16 in vascular tissues from the ES-PMMA group. A dual-luciferase reporter gene assay was applied to verify that ocu-miR-326-5p binds the CD40 3'-UTR and targets lncRNA MSTRG.22719.16. CONCLUSION: Compared with PMMA bone cement, ES-PMMA bone cement can reduce thrombosis through the lncRNA MSTRG.22719.16/ocu-miR-326-5p/CD40 axis.

ATF2-driven osteogenic activity of enoxaparin sodium-loaded polymethylmethacrylate bone cement in femoral defect regeneration.

BACKGROUND: Polymethylmethacrylate (PMMA) bone cement loaded with enoxaparin sodium (PMMA@ES) has been increasingly highlighted to affect the bone repair of bone defects, but the molecular mechanisms remain unclear. We addressed this issue by identifying possible molecular mechanisms of PMMA@ES involved in femoral defect regeneration based on bioinformatics analysis and network pharmacology analysis. METHODS: The upregulated genes affecting the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) were selected through bioinformatics analysis, followed by intersection with the genes of ES-induced differentiation of BMSCs identified by network pharmacology analysis. PMMA@ES was constructed. Rat primary BMSCs were isolated and cultured in vitro in the proliferation medium (PM) and osteogenic medium (OM) to measure alkaline phosphatase (ALP) activity, mineralization of the extracellular matrix, and the expression of RUNX2 and OCN using gain- or loss-of-function experiments. A rat femoral bone defect model was constructed to detect the new bone formation in rats. RESULTS: ATF2 may be a key gene in differentiating BMSCs into osteoblasts. In vitro cell assays showed that PMMA@ES promoted the osteogenic differentiation of BMSCs by increasing ALP activity, extracellular matrix mineralization, and RUNX2 and OCN expression in PM and OM. In addition, ATF2 activated the transcription of miR-335-5p to target ERK1/2 and downregulate the expression of ERK1/2. PMMA@ES induced femoral defect regeneration and the repair of femoral defects in rats by regulating the ATF2/miR-335-5p/ERK1/2 axis. CONCLUSION: The evidence provided by our study highlighted the ATF2-mediated mechanism of PMMA@ES in the facilitation of the osteogenic differentiation of BMSCs and femoral defect regeneration.

Enoxaparin sodium bone cement displays local anti-inflammatory effects by regulating the expression of IL-6 and TNF-α.

Objective: To explore the roles of Enoxaparin Sodium-Polymethyl methacrylate bone cement on inflammatory factors Interleukin-6 and Tumour Necrosis Factor-α in a rabbit knee replacement model. As well as the mechanisms underlying its potential effects on lipopolysaccharide-induced endothelial cell injury. Methods: A knee replacement model was established using New Zealand rabbits. Forty rabbits were randomly divided into four groups: PMMA, ES-PMMA, sham-operated, and blank control groups (n = 10 in each group). Local tissues around the incision were taken at the 30th, 60th, and 90th minute after the surgical implantation of the corresponding bone cement. Immunohistochemistry in the surgical field was used to measure the expression of local inflammatory factors IL-6 and TNF-α. In the in vitro experiments, 1 cm3 of bone cement was immersed in 3 mL of the medium for 24 h. The bone cement was discarded and diluted to 25% with normal medium. Pre-experiments were screened for the best LPS-inducing concentration of 100 mg/mL, and the most compatible LPS concentration was used for subsequent experiments simulating the primary cultures of rats' Inferior Vena Cava Endothelial Cells. The experiments were divided into four groups: blank control group, LPS induction group, PMMA + LPS group, and ES-PMMA + LPS group. The apoptosis rate was detected by flow cytometry, and the expression levels of TNF-α and IL-6 in the cells and supernatant were measured by ELISA, western blotting, and immunofluorescence. Results: According to immunohistochemical results, IL-6-positive cells were concentrated in the tissue interstitial space. In the PMMA and sham-operated groups, the number of IL-6-positive cells gradually increased over time. At all time points, IL-6 expression in the ES-PMMA group was much lower than in the PMMA and sham-operated groups. At 30 min, TNF-α positive cells in the ES-PMMA group expressed less than those in the PMMA and sham-operated groups, with no discernible difference between the PMMA and ES-PMMA groups at 60 or 90 min. Using ELISA and flow cytometry, the expression levels of IL-6 and TNF-α were improved and the apoptosis rate was magnified in the LPS-induced group (***P < 0.001) in contrast with the blank control group. Additionally, the expression levels of IL-6 and TNF-α were reduced in the ES-PMMA + LPS group compared with the LPS-induced group (*P < 0.05) and the apoptosis rate was reduced (***P < 0.001), with statistically significant variations. Western blotting and immunofluorescence analysis confirmed that IL-6 and TNF-α protein expression in cells was upregulated in the LPS-induced group compared to the blank control group (***P < 0.001), and the mean fluorescence intensity was enlarged (***P < 0.001). Meanwhile, IL-6 and TNF-α expression in the ES-PMMA + LPS group were down-regulated (**P < 0.01 or *P < 0.05) compared with the LPS-induced group and PMMA + LPS crew protein expression, and the average fluorescence intensity of IL-6 and TNF-α was lowered in the ES-PMMA + LPS group compared to the LPS-induced group (***P < 0.001). Conclusions: ES-PMMA bone cement reduced the expression levels of local inflammatory factors IL-6 and TNF-α in a rabbit knee model. ES-PMMA bone cement reduced the rate of LPS-induced endothelial cell apoptosis and diminished local inflammatory damage by regulating the secretion of inflammatory factors TNF-α and IL-6.

The mechanism by which enoxaparin sodium-high-viscosity bone cement reduces thrombosis by regulating CD40 expression in endothelial cells.

OBJECTIVE: PMMA bone cement leads to the development of local thrombi. Our study found that ES-PMMA bone cement, a novel material, can reduce local thrombosis. We used a simple and reproducible animal model to confirm the reduction in local thrombosis and preliminarily explored the associated molecular mechanism. METHODS: New Zealand rabbits, which were used to model thrombosis using extracorporeal carotid artery shunts, were divided into the following three groups, with 10 rabbits in each group: the sham group, PMMA group and ES-PMMA group. Four hours after modelling, experimental samples were collected, and the degree of thrombosis was compared between the groups. The expression of thrombomodulin in endothelial cells was quantified in vascular tissues samples. RESULTS: Thrombosis was observed in the PMMA group and ES-PMMA group but not in the sham group. The thrombosis weight was 0.00732 ± 0.00089 g/cm in the PMMA group and 0.00554 ± 0.00077 g/cm in the ES-PMMA group (P < 0.001). Quantitative real-time polymerase chain reaction (RT-qPCR) and Western blotting revealed that the expression of CD40, which can regulate thrombosis in vascular endothelial cells, was significantly lower in the ES-PMMA group than in the PMMA group. CONCLUSION: Compared with PMMA bone cement, ES-PMMA bone cement can reduce local thrombosis by decreasing the expression of the thrombus-associated regulatory protein CD40 in vascular endothelial cells.

LncPVT1 promotes cartilage degradation in diabetic OA mice by downregulating miR-146a and activating TGF-ß/SMAD4 signaling.

INTRODUCTION: To investigate the role of LncRNA PVT1 (plasmacytoma variant translocation 1) in hyperglycemia-triggered cartilage damage using the diabetic osteoarthritis (OA) mice model. MATERIALS AND METHODS: Streptozotocin (STZ) was used to induce mouse diabetes. Knee OA model was induced through transection of anterior cruciate ligament (ACLT). Severity of arthritis was assessed histologically by Safranin O-Fast Green Staining using Mankin Scores. LncRNA PVT1 and miR-146a were detected by real-time polymerase chain reaction (PCR) in cartilage tissue. Moreover, the interaction among PVT1, miR-146a, and SMAD4 was examined by luciferase reporter assays. Mice were injected intra-articularly with ad-siRNA-PVT1 and ad-siRNA scramble control. Articular concentrations of TNF-α, IL-1, IL-6 and TGF-ß1 were determined using enzyme-linked immunosorbent assay. Levels of type II Collagen (COL2A1), TGF-ß1, p-SMAD2, SMAD2, p-SMAD3, SMAD3, SMAD4 and nuclear SMAD4 were detected by western blot analysis. RESULTS: PVT1 expression was significantly increased, whereas miR-146a was markedly decreased in diabetic OA mice than in non-diabetic OA and control. Increased PVT1 expression in diabetic OA mice was significantly associated with Mankin score and reduced miR-146a as well as Collagen alpha-1(II) (COL2A1) expressions. In vivo, intra-articular injection of ad-siRNA-PVT1 efficiently increased miR-146a and COL2A1 expressions, alleviated joint inflammation, decreased the expression of pro-inflammatory mediators, and suppressed TGF-ß/SMAD4 pathway in diabetic OA mice. CONCLUSIONS: Our results demonstrate LncRNA PVT1 is involved in cartilage degradation in diabetic OA and correlated with disease severity. Efficiency of ad-siRNA-PVT1 in controlling joint inflammation in diabetic OA mice is associated with the suppression of the expression of miR-146a, pro-inflammatory cytokines and activation of TGF-ß/SMAD4 pathway.

Decreased Synovial Fluid Biomarkers Levels Are Associated with Rehabilitation of Function and Pain in Rotator Cuff Tear Patients Following Electroacupuncture Therapy.

BACKGROUND The aim of this study was to assess inflammatory cytokines levels in synovial fluid (SF) before and after electroacupuncture (EA) treatment and to explore whether these biomarkers are associated with function of rotator cuff tear (RCT) patients. MATERIAL AND METHODS We recruited 54 patients with RCT and separated them into an EA group and a control group. The SF biomarker levels were detected at baseline and at 6-week and 6-month follow-up. The symptomatic severity was evaluated by visual analog scale (VAS), Constant-Murley score, and American Shoulder and Elbow Surgeons score (ASES). We also investigated the correlation between symptomatic severity and biomarker levels in SF of the shoulder joint. RESULTS The reductions in VAS and improved functional score (ASES and Constant-Murley score) were significantly different between the 2 groups, and SF biomarker concentrations were significantly lower in the EA group. IL-1ß levels were significantly negatively correlated with Constant-Murley score (r=-0.73, P=0.04) and ASES score (r=-0.59, P<0.001) and positively correlated with VAS scores (r=0.81, P=0.004). IL-6 levels were significantly negatively correlated with Constant-Murley score (r=-0.67, P=0.03) and positively correlated with VAS score (r=0.7, P=0.01). MMP-1 levels were significantly negatively correlated with ASES score (r=-0.57, P<0.001). CONCLUSIONS The biomarkers in SF were directly associated with shoulder pain and shoulder function in rotator cuff tear. EA, as a safe and effective conservative therapy, obviously decreased the level of inflammatory cytokines in RCT patients, accompanied by a reduction in shoulder pain and improved function.

[Effect of electroacupuncture on rehabilitation of knee joint movement after anterior cruciate ligament reconstruction].

OBJECTIVE: To observe the effect of electroacupuncture (EA) on the rehabilitation of knee joint function after anterior cruciate ligament (ACL) reconstruction. METHODS: A total of 140 patients with ACL reconstruction were randomly divided into an observation group (58 cases recruited, 12 cases dropped out) and a control group (65 cases recruited, 5 cases dropped out). The patients in the control group were treated with routine rehabilitation treatment. The patients in the observation group, on the basis of the treatment in the control group, were treated with EA at Fengshi (GB 31), Futu (ST 32), Zusanli (ST 36), Shangjuxu (ST 37), Fenglong (ST 40), Xuanzhong (GB 39), Diji (SP 8) and Sanyinjiao (SP 6) on the affected side (2 Hz/100 Hz of dilatational wave, 2-5 mA). Each EA treatment lasted 20-30 min, twice a day for 7 days. The swelling degree (d), pain visual analogue scale (VAS), knee joint range of motion (ROM), scores of International Knee Documentation Committee (IKDC) subjective short form and scores of Lysholm were observed in the two groups 1 day, 1 month, 3 months, 6 months and 1 year after operation. RESULTS: One month and 3 months after operation, the swelling degree (d) and VAS scores in the observation group were lower than those in the control group (P<0.05); 6 months and 1 year after operation, there was no significant difference between the two groups on the swelling degree (d) and VAS scores (P>0.05). One month, 3 months, 6 months and 1 year after operation, the ROM of the knee joint in the observation group was higher than that in the control group (P<0.05), the IKDC score and Lysholm score were higher than those in the control group (P<0.05). Within one year, there were no relaxations, fractures and other related complications in the two groups. The pivot shift test, anterior drawer test and the Lachman test were all negative. CONCLUSION: EA combined with routine rehabilitation training could obviously reduce the pain of knee joint, improve the swelling degree, increase the ROM of knee joint, promote the functional recovery in patients with ACL reconstruction, which are superior to rehabilitation training alone.

Relationship between serum and synovial fluid CCL20 concentrations with disease severity in primary knee osteoarthritis.

OBJECTIVES: The current study was performed to examine serum and synovial fluid (SF) CCL20 levels and their correlations with disease severity in primary knee osteoarthritis patients. METHODS: A total of 99 patients diagnosed with primary knee OA were enrolled in the study, and 95 healthy individuals receiving regular medical examination were recruited as controls. Serum and SF CCL20 concentrations were determined using an enzyme-linked immunosorbent assay. The radiographic severity of OA was assessed by the Kellgren-Lawrence (K-L) classification system. The Lequesne algofunctional index and a visual analogue scale (VAS) were used to evaluate the clinical severity of knee OA in patients. RESULTS: The serum CCL20 levels were not significantly different between patients with knee OA and controls. Patients with a K-L grade of 4 had significantly higher SF CCL20 levels than those with K-L grades of 2 and 3. Knee OA patients with a K-L grade of 3 showed significantly higher levels of CCL20 in SF than those with a K-L grade of 2. In addition, SF CCL20 levels were significantly related to the Lequesne algofunctional index and VAS score. CONCLUSIONS: These findings suggest that local CCL20 levels may reflect the disease severity of knee OA.

Synovial fluid alpha-melanocyte-stimulating hormone may act as a protective biomarker for primary knee osteoarthritis.

OBJECTIVE: The current study was carried out to investigate the serum and synovial fluid (SF) alpha-melanocyte-stimulating hormone (α-MSH) levels in correlation with disease severity in primary knee osteoarthritis (OA). METHODS: This study comprised of 105 primary knee OA patients and 98 healthy controls. The radiographic severity was verified according to the Kellgren-Lawrence radiographic grading criteria. The symptomatic severity of knee OA was assessed by the Western Ontario and McMaster Universities (WOMAC) Osteoarthritis Index. Serum α-MSH concentrations were measured by ELISA. The inflammation markers IL-6 and TNF-α, as well as cartilage damage markers MMP-3 (matrix metalloproteinase 3) and COMP (cartilage oligomeric matrix protein), were also measured. The receiver operating characteristic (ROC) curve was used to evaluate the diagnostic value between α-MSH and other four markers with regard to the radiographic progression. RESULTS: SF α-MSH concentrations were negatively related to Kellgren-Lawrence grades and WOMAC index. SF α-MSH levels in knee OA patients were negatively associated with inflammation markers IL-6, TNF-α, and cartilage damage factors COMP and MMP-3. In addition, ROC analysis implied that attenuated α-MSH levels may serve as a favorable diagnostic marker for the radiographic progression. The difference of serum α-MSH concentration was not significant between knee OA patients and healthy controls. CONCLUSIONS: Reduced SF α-MSH expression may be a characteristic of OA patients. Attenuated α-MSH level in SF may serve as a potential biomarker for disease severity of knee OA, and further studies are needed to identify its potential application for monitoring the course of the disease and the efficacy of therapies in OA patients.

[TREATMENT OF PRONATION EXTERNAL ROTATION ANKLE FRACTURE COMBINED WITH SEPARATION OF DISTAL TIBIOFIBULAR SYNDESMOSIS].

OBJECTIVE: To evaluate the difference between using and not using syndesmotic screw to treat pronation external rotation (PER) ankle fracture combined with separation of distal tibiofibular syndesmosis. METHODS: Between April 2011 and October 2014, 46 cases of PER ankle fracture combined with separation of distal tibiofibular syndesmosis were treated, and syndesmotic screw was used in 24 cases (fixation group) and syndesmotic screw was not used in 22 cases (non-fixation group). There was no significant difference in gender, age, weight, cause of injury, side, injury to operation time, and fracture type between 2 groups (P>0.05). The time for full weight-bearing, fracture healing time, and complications were recorded after operation. Anteroposterior and lateral X-ray films were taken to measure the tibiofibular overlap (TBOL) and tibiofibular clear space (TBCS). Baird-Jackson score was used to evaluate functional recovery of the ankle. RESULTS: All incision healed by first intention without complications. The cases were followed up 13-18 months (mean, 15.2 months) in 2 groups. The time for full weight-bearing was 8-12 weeks (median, 11 weeks) in fixation group, which was significantly later than that in non-fixation group (range, 6-10 weeks; median, 8 weeks) (Z=-5.049, P=0.000). X-ray examination showed reduction of separation of distal tibiofibular syndesmosis. All fractures healed. The fracture healing time was (13.83±1.37) weeks in fixation group, and was (13.91±1.31) weeks in non-fixation group, showing no significant difference (t=-0.191, P=0.945). No separation of distal tibiofibular syndesmosis, delayed union, nonunion, loosening, or breakage of fixation devices was observed in 2 groups. There was no significant difference in TBOL, TBCS, Baird-Jackson score and the excellent and good rate between 2 groups (P>0.05). CONCLUSIONS: If the medial, lateral, and posterior structures of the ankle could be repaired according to injury, no significant influence on functional outcome of ankle or radiologic findings could be detected whether syndesmotic fixation is given or not in treating PER ankle fracture (exclude Maisonneuve fracture) combined with separation of distal tibiofibular syndesmosis.

Possible Involvement of Serum and Synovial Fluid Resistin in Knee Osteoarthritis: Cartilage Damage, Clinical, and Radiological Links.

BACKGROUND: Resistin is an adipocytokine associated with inflammation and insulin resistance. Recent studies have shown that resistin plays an important role in the pathogenesis and progression in osteoarthritis (OA) patients. The current study was aimed at investigating the relationship between resistin in serum and synovial fluid (SF) and disease severity in patients with knee osteoarthritis. METHOD: Seventy-four patients diagnosed with knee OA and 79 healthy controls receiving regular body check in our hospital were recruited in the study. The Noyes score method was used to assess articular cartilage damage arthroscopically. The symptomatic severity was evaluated according to the Western Ontario McMaster University Osteoarthritis (WOMAC) scores. The radiographic disease severity of OA was assessed by the Kellgren-Lawrence (K-L) grading system. The resistin levels in serum and SF were determined by enzyme-linked immunosorbent assay. Cartilage degradation marker CTX-II in SF was also examined. RESULTS: SF but not serum resistin levels are positively associated with Noyes scores, K-L grading scores WOMAC pain scores, physical functional scores and WOMAC total scores. In addition, SF resistin correlated positively with CTX-II. CONCLUSION: Resistin in SF might serve as a potential biomarker for reflecting the disease severity and cartilage degenerative extent of knee OA.

Increased Synovial Fluid YKL-40 Levels are Linked with Symptomatic Severity in Knee Osteoarthritis Patients.

BACKGROUND: Elevated serum and synovial fluid (SF) YKL-40 levels have been detected in knee osteoarthritis (OA) patients. The current study was focused on the correlation between YKL-40 levels in serum or SF and symptomatic severity in patients with knee osteoarthritis. METHODS: 144 patients with knee OA and 151 healthy individuals were recruited into this study. Symptomatic severity was determined using Western Ontario and McMaster Universities Arthritis Index (WOMAC) scores from OA patients. Serum and SF levels of YKL-40 were explored by enzyme-linked immunosorbent assay. RESULTS: We found that YKL-40 levels in SF but not serum were independently and positively related to WOMAC pain (r = 0.531, p = 0.001), physical disability (r = 0.380, p = 0.025), and total scores (r = 0.407, p = 0.01) in knee OA patients. CONCLUSIONS: YKL-40 in SF could represent a potential biomarker for assessing the symptomatic severity of OA.