Malvidin attenuates trauma-induced heterotopic ossification of tendon in rats by targeting Rheb for degradation via the ubiquitin-proteasome pathway.

The pathogenesis of trauma-induced heterotopic ossification (HO) in the tendon remains unclear, posing a challenging hurdle in treatment. Recognizing inflammation as the root cause of HO, anti-inflammatory agents hold promise for its management. Malvidin (MA), possessing anti-inflammatory properties, emerges as a potential agent to impede HO progression. This study aimed to investigate the effect of MA in treating trauma-induced HO and unravel its underlying mechanisms. Herein, the effectiveness of MA in preventing HO formation was assessed through local injection in a rat model. The potential mechanism underlying MA's treatment was investigated in the tendon-resident progenitor cells of tendon-derived stem cells (TDSCs), exploring its pathway in HO formation. The findings demonstrated that MA effectively hindered the osteogenic differentiation of TDSCs by inhibiting the mTORC1 signalling pathway, consequently impeding the progression of trauma-induced HO of Achilles tendon in rats. Specifically, MA facilitated the degradation of Rheb through the K48-linked ubiquitination-proteasome pathway by modulating USP4 and intercepted the interaction between Rheb and the mTORC1 complex, thus inhibiting the mTORC1 signalling pathway. Hence, MA presents itself as a promising candidate for treating trauma-induced HO in the Achilles tendon, acting by targeting Rheb for degradation through the ubiquitin-proteasome pathway.

Quercetin as a promising intervention for rat osteoarthritis by decreasing M1-polarized macrophages via blocking the TRPV1-mediated P2X7/NLRP3 signaling pathway.

Osteoarthritis (OA) is characterized by an imbalance between M1 and M2 polarized synovial macrophages. Quercetin has shown protective effects against OA by altering M1/M2-polarized macrophages, but the underlying mechanisms remain unclear. In this study, rat chondrocytes were treated with 10 ng/mL of IL-1ß. To create M1-polarized macrophages in vitro, rat bone marrow-derived macrophages (rBMDMs) were treated with 100 ng/mL LPS. To mimic OA conditions observed in vivo, a co-culture system of chondrocytes and macrophages was established. ATP release assays, immunofluorescence assays, Fluo-4 AM staining, Transwell assays, ELISA assays, and flow cytometry were performed. Male adult Sprague-Dawley (SD) rats were used to create an OA model. Histological analyses, including H&E, and safranin O-fast green staining were performed. Our data showed a quercetin-mediated suppression of calcium ion influx and ATP release, with concurrent downregulation of TRPV1 and P2X7 in the chondrocytes treated with IL-1ß. Activation of TRPV1 abolished the quercetin-mediated effects on calcium ion influx and ATP release in chondrocytes treated with IL-1ß. In the co-culture system, overexpression of P2X7 in macrophages attenuated the quercetin-mediated effects on M1 polarization, migration, and inflammation. Either P2X7 or NLRP3 knockdown attenuated IL-1ß-induced M1/M2 polarization, migration, and inflammation. Moreover, overexpression of TRPV1 reduced the quercetin-mediated suppressive effects on OA by promoting M1/M2-polarized macrophages in vivo. Collectively, our data showed that quercetin-induced suppression of TRPV1 leads to a delay in OA progression by shifting the macrophage polarization from M1 to M2 subtypes via modulation of the P2X7/NLRP3 pathway.

miRNA-382-5p Carried by Extracellular Vesicles in Osteoarthritis Reduces Cell Viability and Proliferation, and Promotes Cell Apoptosis by Targeting PTEN.

The objective of the study was to identify extracellular vesicle (EV) microRNAs (miRNAs) that play important roles in knee osteoarthritis (OA). Models of knee OA were surgically induced in nine male Sprague-Dawley rats. Tissue samples were collected at 0 weeks (Control), 6 weeks (6 weeks), and 12 weeks (12 weeks). The EVs were isolated and analyzed for size. Various biomarkers, including recombinant tetraspanin 30 cluster of differentiation (CD)63 and CD9 were detected. An Agilent array was used to screen for differentially expressed (DE) miRNAs. The levels of DE miRNAs and their target mRNAs were evaluated by quantitative reverse transcription-polymerase chain reaction and western blotting. The viability, proliferation, and apoptosis of lipopolysaccharide (LPS)-induced human synovial cells (HSCs) were examined by using Cell Counting Kit-8, EdU (5-ethynyl-2'-deoxyuridine), and TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling) assays, respectively. The OA model rats had significantly increased levels of inflammatory activity, damaged cells, and rough articular cartilage when compared with rats in the control group. The EVs from the model rats appeared as round vesicle-like structures with a mean diameter of ∼145 nm. Five miRNAs that showed gradual increases in the model rats were selected for further analysis; those miRNAs included miR-127-3p, miR-132-3p, miR-141-3p, miR-345-5p, and miR-382-5p. miR-382-5p was found to reduce the viability and proliferation and promote the apoptosis of LPS-induced HSCs. Moreover, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) was negatively regulated by miR-382-5p. Our findings revealed that EVs produced by the OA rats contained miR-382-5p, which might reduce cell viability and proliferation, and promote cell apoptosis by targeting PTEN.

The silencing of miR-199a-5p protects the articular cartilage through MAPK4 in osteoarthritis.

Background: Osteoarthritis (OA) is the most common joint disorder, and places a heavy burden on individuals and society. As conventional therapies, such as surgery, rarely cure the disorder, targeted therapies represent a promising alternative. This research sought to explore the potential effect of miR-199a-5p on the development of OA. Methods: Based on the OA rat model, the serum was collected at 6 and 12 weeks, and microRNA (miRNA) sequencing was performed. A bioinformatics analysis was conducted to examine the differentially expressed micro ribonucleic acids, and qRT-PCR (real-time quantitative PCR) was conducted to determine their expression in the joint tissues of rats with OA. Rats articular chondrocytes were collected and treated with a miR-199a-5p antagomir or agomir. Afterwards, cell viability, autophagy was determinated. Dual luciferase was used to verify that miR-199a-5p targets the regulation of mitogen-stimulated protein kinase 4 (MAPK4). Subsequently, in chondrocytes, MAPK was knockdown to rescue the effect of miR-199a-5p inhibition, and cell viability and autophagy were examined. Finally, the OA model was treated with miR-199a-5p antagomir to detect joint pathology, cartilage tissue and inflammatory factor and autophagy was measured. Results: MiR-199a-5p was greatly upregulated in OA, and miRNA was found to be differentially expressed in OA tissues. MAPK4 was identified to be a target gene of miR-199-5p. Inhibiting miR-199a-5p not only decreased the survival of chondrocytes and induced apoptosis, but also relieved inflammation and decreased the content of pro-inflammatory cytokines. Further, the silencing of miR-199a-5p protected the articular cartilage and improved gait abnormalities, but this effect was abrogated by the silencing of MAPK4. Conclusions: The silencing of miR-199a-5p appears to improve gait abnormalities, promote the survival of chondrocytes, and improve the condition of OA. Our findings may lead to the development of miR-199a-5p-based targeted therapy for OA.

Quercetin Alleviates Osteoarthritis Progression in Rats by Suppressing Inflammation and Apoptosis via Inhibition of IRAK1/NLRP3 Signaling.

INTRODUCTION: Quercetin was recently reported to help protect against osteoarthritis (OA) progression, but the molecular mechanism for that protective affect remains unclear. METHODS: Here, OA model rats were intraperitoneally injected with quercetin, and the severity of cartilage damage in the rats was evaluated by H&E, Safranin O, and Toluidine blue, as well as by using the Osteoarthritis Research Society International (OARSI) Scoring System. Additionally, rat chondrocytes were treated with quercetin and then stimulated with IL-1ß. The levels of pro-inflammatory cytokines (IL-1ß, IL-18, and TNF-α) were detected by ELISA.Cell apoptosis was evaluated by flow cytometry and Hoechst staining. ROS levels were measured using a DCFH-DA probe. Protein expression was evaluated by Western blotting, immunohistochemical staining, and immunofluorescence. RESULTS: Our data showed that quercetin attenuated the degeneration and erosion of articular cartilage, suppressed inflammation and apoptosis, and downregulated the levels of IRAK1, NLRP3, and caspase-3 expression. In vitro data showed that overexpression of NLRP3 could reverse the suppressive effect of quercetin on IL-1ß-induced rat chondrocyte injuries. Importantly, rescue experiments confirmed that quercetin inhibited IL-1ß-induced rat chondrocyte injuries in vitro by suppressing the IRAK1/NLRP3 signaling pathway. CONCLUSION: Our study indicated that quercetin inhibits IL-1ß-induced inflammation and cartilage degradation by suppressing the IRAK1/NLRP3 signaling pathway.

Tendon-to-bone healing after repairing full-thickness rotator cuff tear with a triple-loaded single-row method in young patients.

BACKGROUND: Arthroscopic repair is recommended for young patients with full-thickness rotator cuff tears (RCTs), but the healing rates have raised concerns. The Southern California Orthopedic Institute (SCOI) row method has been developed based on greater than 3 decades of experience with excellent clinical outcomes; however, studies with a focus on the younger patient population are limited in number. The current study assessed the short-term clinical outcome and the initial tendon-to-bone healing in a young cohort after repair of a full-thickness RCT using the SCOI row method. METHODS: A retrospective cohort study was performed. Patients < 55 years of age who had a full-thickness RCT and underwent an arthroscopic repair using the SCOI row method were reviewed. Clinical outcomes were assessed at baseline, and 3 and 6 months post-operatively. The visual analog scale (VAS), University of California at Los Angeles (UCLA) scale, and Constant-Murley score were completed to assess pain and function. Active range of motion was also examined, including abduction and flexion of the involved shoulder. A preoperative MRI was obtained to assess the condition of the torn tendon, while 3- and 6-month postoperative MRIs were obtained to assess tendon-to-bone healing. Repeated measurement ANOVA and chi-square tests were used as indicated. RESULTS: Eighty-nine patients (57 males and 32 females) with a mean age of 44.1 ± 8.6 years who met the criteria were included in the study. Compared with baseline, clinical outcomes were significantly improved 3 and 6 months postoperatively based on improvement in the VAS, UCLA score, and Constant-Murley score, as well as range of motion. Greater improvement was also noted at the 6-month postoperative assessment compared to the 3-month postoperative assessment. Three- and six-month postoperative MRIs demonstrated intact repairs in all shoulders and footprint regeneration, which supported satisfactory tendon-to-bone healing. The mean thickness of regeneration tissue was 7.35 ± 0.76 and 7.75 ± 0.79 mm as measured from the 3- and 6-month MRI (P = 0.002). The total satisfactory rate was 93.3 %. CONCLUSIONS: Arthroscopic primary rotator cuff repair of a full-thickness RCT using the SCOI row method in patients < 55 years of age yields favorable clinical outcomes and early footprint regeneration.

Less blood loss in supercapsular percutaneously assisted versus posterolateral total hip arthroplasty.

BACKGROUND: Although excellent clinical outcomes of supercapsular percutaneously assisted total hip arthroplasty (SuperPath) have been reported, the peri-operative blood loss has rarely been reported. The current study determined the blood loss during SuperPath and compared the blood loss with conventional posterolateral total hip arthroplasty (PLTH). METHODS: This retrospective study enrolled patients who underwent unilateral primary THA between January 2017 and December 2019. The demographic data, diagnoses, affected side, radiographic findings, hemoglobin concentration, hematocrit, operative time, transfusion requirements, and intra-operative blood loss were recorded. The peri-operative blood loss was calculated using the OSTHEO formula. Blood loss on the 1st, 3rd, and 5th post-operative days was calculated. Hidden blood loss (HBL) was determined by subtracting the intra-operative blood loss from the total blood loss. RESULTS: Two hundred sixty-three patients were included in the study, 85 of whom were in the SuperPath group and 178 in the posterolateral total hip arthroplasty (PLTH) group. Patient demographics, diagnoses, affected side, operative times, and pre-operative hemoglobin concentrations did not differ significantly between the two groups (all P > 0.05). Compared to the PLTH group, the SuperPath group had less blood loss, including intra-operative blood loss, 1st, 3rd, and 5th post-operative days blood loss, and HBL (all P < 0.05). Total blood loss and HBL was 790.07 ± 233.37 and 560.67 ± 195.54 mL for the SuperPath group, respectively, and 1141.26 ± 482.52 and 783.45 ± 379.24 mL for the PLTH group. PLTH led to a greater reduction in the post-operative hematocrit than SuperPath (P < 0.001). A much lower transfusion rate (P = 0.028) and transfusion volume (P = 0.019) was also noted in the SuperPath group. CONCLUSION: SuperPath resulted in less perioperative blood loss and a lower transfusion rate than conventional PLTH.

Biomechanical comparison between single-row with triple-loaded suture anchor and suture-bridge double-row rotator cuff repair.

BACKGROUND: Numerous biomechanical and clinical studies comparing different techniques for rotator cuff repair have been reported, yet universal consensus regarding the superior technique has not achieved. A medially-based single-row with triple-loaded suture anchor (also referred to as the Southern California Orthopedic Institute [SCOI] row) and a suture-bridge double-row (SB-DR) with Push-Locks have been shown to result in comparable improvement in treating rotator cuff tear, yet the biomechanical difference is unknown. The purpose of the current study was to determine whether a SCOI row repair had comparable initial biomechanical properties to a SB-DR repair. METHODS: Six matched pairs of fresh-frozen cadaveric shoulders with full-thickness supraspinatus tendon tears we created were included. Two different repairs were performed for each pair (SCOI row and SB-DR methods). Specimens were mounted on a material testing machine to undergo cyclic loading, which was cycled from 10 to 100 N at 1 Hz for 500 cycles. Construct gap formation was recorded at an interval of 50 cycles. Samples were then loaded to failure and modes of failure were recorded. Repeated-measures analysis of variance and pair-t test were used for statistical analyses. RESULTS: The construct gap formation did not differ between SCOI row and SB-DR repairs (P = 0.056). The last gap displacement was 1.93 ± 0.37 mm for SCOI row repair, and 1.49 ± 0.55 mm for SB-DR repair. The tensile load for 5 mm of elongation and ultimate failure were higher for SCOI row repair compared to SB-DR repair (P = 0.011 and 0.028, respectively). The ultimate failure load was 326.34 ± 11.52 N in the SCOI row group, and 299.82 ± 27.27 N in the SB-DR group. Rotator cuff repair with the SCOI row method failed primarily at the suture- tendon interface, whereas pullout of the lateral row anchors was the primary mechanism of failure for repair with the SB-DR method. CONCLUSION: Rotator cuff repair with the SCOI row method has superior biomechanical properties when compared with the SB-DR method. Therefore, SCOI row repair using a medially-based single-row technique with triple-loaded suture anchor is recommended to improve the initial strength in treating full-thickness rotator cuff tears.