Gene therapy by virus-like self-spooling toroidal DNA condensates for revascularization of hindlimb ischemia.

Peripheral arterial diseases (PAD) have been reported to be the leading cause for limb amputations, and the current therapeutic strategies including antiplatelet medication or intervene surgery are reported to not clinically benefit the patients with high-grade PAD. To this respect, revascularization based on angiogenetic vascular endothelial growth factor (VEGF) gene therapy was attempted for the potential treatment of critical PAD. Aiming for transcellular delivery of VEGF-encoding plasmid DNA (pDNA), we proposed to elaborate intriguing virus-like DNA condensates, wherein the supercoiled rigid micrometer-scaled plasmid DNA (pDNA) could be regulated in an orderly fashion into well-defined nano-toroids by following a self-spooling process with the aid of cationic block copolymer poly(ethylene glycol)-polylysine at an extraordinary ionic strength (NaCl: 600 mM). Moreover, reversible disulfide crosslinking was proposed between the polylysine segments with the aim of stabilizing these intriguing toroidal condensates. Pertaining to the critical hindlimb ischemia, our proposed toroidal VEGF-encoding pDNA condensates demonstrated high levels of VEGF expression at the dosage sites, which consequently contributed to the neo-vasculature (the particularly abundant formation of micro-vessels in the injected hindlimb), preventing the hindlimb ischemia from causing necrosis at the extremities. Moreover, excellent safety profiles have been demonstrated by our proposed toroidal condensates, as opposed to the apparent immunogenicity of the naked pDNA. Hence, our proposed virus-like DNA condensates herald potentials as gene therapy platform in persistent expressions of the therapeutic proteins, and might consequently be highlighted in the management of a variety of intractable diseases.

Holomycin, a novel NLRP3 inhibitor, attenuates cartilage degeneration and inflammation in osteoarthritis.

The contribution of the NLRP3 inflammasome in osteoarthritis (OA) pathogenesis has been uncovered in recent years. Holomycin (HL) has recently been identified as a novel NLRP3 inflammasome inhibitor. Herein, we aimed to explore the benefits of HL for OA. A chondrocyte-macrophage co-culture system and the destabilization of the medial meniscus (DMM) mouse model were established to study the effect of HL on OA in vitro and in vivo. ECM degradation-related proteins (MMP-13, aggrecan, and Collagen II) were detected by Western blot (WB) and immunohistochemistry (IHC). The chondrocyte senescence was determined by cell cycle, p16 and p21 expressions, and SA-ß-Gal staining. The cartilage degeneration was evaluated by OARSI score and Safranin O and H&E staining. Inflammation and NLRP3 inflammasome activation were investigated via RT-PCR, ELISA, WB, and IHC. In vitro studies showed that IL-1ß stimulation caused a significant increase of MMP13, p16, p21, and ß-galactosidase expressions, a G1-phase arrest, and a down-regulation of aggrecan and Collagen II in chondrocytes, and the increased expressions of IL-6, CXCL-1, IL-1ß, NLRP3, and Caspase 1 p20 in both chondrocyte and macrophage. Meanwhile, HL administration could partly reverse these effects induced by IL-1ß. In DMM mouse models, intra-articular administration of HL alleviated cartilage degeneration and inflammation, as evidenced by the decrease of OARSI score and MMP13, p16, p21, Collagen II, IL-6, and CXCL-1 expressions and the restoration of chondrocyte number, proteoglycan, and MMP13 expression in cartilage tissues. This study identified HL as a promising agent for OA.

[Locking loop stitch with suture-bridge technique in repair of acute closed distal Achilles tendon rupture by using suture anchors].

OBJECTIVE: To explore clinical efficacy of Locking loop stitch with suture-bridge technique in repair of acute closed distal Achilles tendon rupture by using suture anchors. METHODS: From July 2019 to March 2021, 20 patients with acute closed distal Achilles tendon rupture were treated by minimally invasive suture anchor locking suture bridging repair technique. Among them, including 18 males and 2 females, aged from 19 to 52 years old with an average of(40.0±9.0) years old. Complications were observed, and recovery of ankle function was evaluated by American Orthopaedic Foot & Ankle Society(AOFAS) ankle and hindfoot function scoring system before operation and 1 year after operation. RESULTS: All patients followed up from 6 to 18 months with an average of (12.0±3.2) months. The incisions were healed at stageⅠwithout infection and skin necrosis occurred;no gastrocnemius nerve injury and deep vein thrombosis of the lower extremities occurred;and no heel pain and Achilles tendon re-rupture occurred. AOFAS scores of ankle and hindfoot increased from(59.0±4.3) before opertaion to(95.1±2.6) at 1 year after operation (t=-32.1, P<0.05). CONCLUSION: The effect of locking suture bridging with suture anchor nails to repair acute distal Achilles tendon rupture is definite, and it could reduce incidence of complications such as Achilles tendon re-rupture, nerve injury, and skin necrosis, which has advantages of small surgical trauma, reliable anastomosis method and good functional recovery, and is an ideal method for treating acute closed distal Achilles tendon rupture.

LncRNA RGMB-AS1 up-regulates ANKRD1 Through Competitively Sponging miR-3614-5p to Promote OSA Cell Proliferation and Invasion.

BACKGROUND: Osteosarcoma (OSA) is associated with unfavorable prognosis. The overall survival rate for patients with OSA recurrence or metastasis is only about 20%. Long non-coding RNAs (lncRNAs) significantly function in gene expression and the progression of various cancers including OSA. METHODS: The expression of repulsive guidance molecule BMP co-receptor b antisense RNA 1 (RGMB-AS1) was detected in OSA cells via qRT-PCR. Western blot assay exposed the protein level of ankyrin repeat domain 1 (ANKRD1). The function assays showed the role of RGMB-AS1, miR-3614-5p, ANKRD1 on OSA cell proliferation and invasion. Subcellular Fraction assay was conducted to detect RGMB-AS1 localization. Rescue assays manifested the mechanism of RGMB-AS1/miR-3614-5p/ANKRD1 axis in OSA cells. RESULTS: FOXA1-activated RGMB-AS1 positively regulated OSA cell progression including proliferation and invasion but negatively modulated apoptosis. miR-3614-5p interacted with RGMB-AS1 and functioned as the tumor suppressor in OSA cells. ANKRD1 was targeted by miR-3614-5p and was negatively interacted by miR-3614-5p. RGMB-AS1 and ANKRD1 competitively bound with miR-3614-5p. The suppression of silencing RGMB-AS1 on OSA cell progression was rescued by ANKRD1 overexpression or miR-3614-5p down-regulation. CONCLUSIONS: FOXA1-activated RGMB-AS1 promoted cell proliferation and invasion in OSA via miR-3614-5p/ANKRD1 pathway.

Bioinformatics Analysis Reveals an Association between Autophagy, Prognosis, Tumor Microenvironment, and Immunotherapy in Osteosarcoma.

Autophagy is a catabolic pathway involved in the regulation of bone homeostasis. We explore clinical correlation of autophagy-related key molecules to establish risk signature for predicting the prognosis, tumor microenvironment (TME), and immunotherapy response of osteosarcoma. Single cell RNA sequencing data from GSE162454 dataset distinguished malignant cells from normal cells in osteosarcoma. Autophagy-related genes (ARGs) were extracted from the established risk signature of the Molecular Signatures Database of Gene Set Enrichment Analysis (GSEA) by univariate Cox and least absolute shrinkage and selection operator (LASSO) Cox regression analysis. Overall survival (OS), TME score, abundance of infiltrating immune cells, and response to immune-checkpoint blockade (ICB) treatment in patients with different risks were compared based on risk score. Nine ARGs were identified and risk signature was constructed. In all osteosarcoma datasets, the OS was significantly longer in the high-risk patients than low-risk onset. Risk signature significantly stratified clinical outcomes, including OS, metastatic status, and survival status. Risk signature was an independent variable for predicting osteosarcoma OS and showed high accuracy. A nomogram based on risk signature and metastases was developed. The calibration curve confirmed the consistency in 1-year, 3-year, and 5-year predicted OS and the actual OS. The risk score was related to 6 kinds of T cells and macrophages, myeloid-derived suppressor cell, natural killer cell, immune score, and stromal score in TME. The risk signature helped in predicting patients' response to anti-PD1/anti-PD-L1 treatment. The ARGs-led risk signature has important value for survival prediction, risk stratification, tumor microenvironment, and immune response evaluation of osteosarcoma.

Clinical Study of Autologous Cartilage Transplantation Based on Nano-Hydroxyapatite in the Treatment of Talar Osteochondral Injury.

Talus osteochondral damage is one of the common symptoms of chronic ankle pain in people's lives. The cartilage regeneration and self-repair ability are extremely limited, the joint cartilage lesions are often accompanied by the lesions of the subchondral bone, and the subchondral bone lesions can affect the metabolism of the cartilage above it, which brings certain difficulties to clinical treatment. Traditional methods of treating cartilage damage include microfractures and drilling. Due to large trauma, inconsistent clinical efficacy reports, poor tissue repair results, and limited donor sources, etc., the application of traditional treatment methods in the clinic has been largely limited. Therefore, finding an ideal treatment method for bone injury has been a hot spot in clinical research in orthopedics. Studies have shown that autologous cartilage transplantation via nano-hydroxyapatite has become a new treatment model, providing new ideas for clinical treatment of talar osteochondral damage. Nano-hydroxyapatite and its composites have good histocompatibility, biological activity, and bone conductivity. They are an ideal bone defect repair material, and have been initially applied in clinical practice. The preparation of nano-hydroxyapatite, its biological characteristics and the repairing effect on the composite defect of osteochondral bone were studied experimentally, and its feasibility for repairing osteochondral damage was discussed. In this paper, the unique structure and properties of natural cartilage layers are studied. In combination with bionics theory and methods, nano-hydroxyapatite micro-particle composite samples are prepared by the gel method, and the bone-forming properties of nano-composites are measured by in vitro drug release experiments. To establish a model of infectious bone injury in New Zealand white rabbits, and nano-hydroxyapatite composites were implanted into local lesions of New Zealand white rabbit models by autologous cartilage transplantation, and evaluated by imaging, blood biochemistry, histology, infection control and bone repair. The experimental results show that using the unique physical and chemical and biological properties of nano-hydroxyapatite materials. It is innovatively introduced into the treatment of talar osteochondral defects caused by open fractures. It has been proven in vitro and in vivo experiments that nano-hydroxyapatite materials can be used. As an ideal tissue engineering scaffold for the treatment of talar osteochondral defects, this provides a new way to solve clinical orthopedic problems using new nanomaterials.

Comprehensive biomechanical analysis of three clinically used fixation constructs for posterior malleolar fractures using cadaveric and finite element analysis.

Different fixation modalities are available for fixation of posterior malleolar fractures (PMFs), but the best method is still unclear. The purpose of this study was to carry out a comparative biomechanical analysis of three commonly used fixation constructs for PMFs using experimental and finite element analysis (FEA). 15 human cadaveric ankle specimens were randomly divided into three groups. Specimens in group-A were fixed with two anteroposterior (AP) lag screws, group-B with two posteroanterior (PA) lag screws, and for group-C, a posterior plate was used. Each model was subjected to axial load. Outcomes included loads for 0.5 mm, 1 mm, 1.5 mm, and 2 mm vertical displacements of posterior fragments were noted. 3D FE models were reconstructed from computed tomography (CT) images and subjected to vertical loads. The model's stress, fracture step-off, and resultant strains in implants were also studied in 3D FE models. Significantly higher amounts of mean compressive loads were observed to cause the same amount of vertical displacements in plate group (265 ± 60.21 N, 796 ± 57.27 N, 901.18 ± 8.88 N, 977.26 ± 13.04 N) than AP (102.7 ± 16.78 N, 169.5 ± 19.91 N, 225.32 ± 15.92 N, 269.32 ± 17.29 N) and PA (199.88 ± 31.43 N, 362.80 ± 28.46 N, 431.3 ± 28.12 N, 541.86 ± 36.05 N) lag screws respectively (P < 0.05). Simulated micro-motion analysis demonstrated that fracture step-off values in plate group (0.03 ± 0.001 mm, 0.06 ± 0.003 mm and 0.13 ± 0.010 mm) were the lowest among the three groups (P < 0.001). The cancellous bone showed the highest amount of stress in AP and PA lag groups respectively, whereas the lowest stress was noted in the plate-group. This biomechanical study concluded that posterior plating is biomechanically the most stable fixation construct for PMFs fixation. AP and PA lag screws with higher bone stress and fracture step-off values have a high tendency of bone cut-through and loss of fixation respectively.

The protective effects of exenatide against AGEs-induced articular matrix degradation in human primary chondrocytes.

Osteoarthritis (OA) presents a major global health burden and is projected to become even more prevalent in coming decades. Therefore, it is of utmost importance to uncover novel therapies for the treatment and prevention of this disease. In the present study, we investigated the effects of exenatide, a specific glucagon-like peptide (GLP) agonist, on degradation of type II collagen and aggrecan, the two main components of the articular extracellular matrix, in human primary chondrocytes. Our results reveal that exenatide could ameliorate degradation of type II collagen and aggrecan by inhibiting expression of metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) induced by advanced glycation end-products. We also found that exenatide reduces oxidative stress and inhibits activation of nuclear factor-κB through the p38 cellular signaling pathway. Taken together, the findings of this study indicate that exenatide may have potential as a novel treatment for osteoarthritis.