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

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

Abstract 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

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

Abstract 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 3ʹ-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

Additional file 2 of 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

Additional file 2. The sequencing results of sample

Additional file 1 of 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

Additional file 1. The meaningful up-regulated results of sequencing