Human Bone Marrow Mesenchymal Stromal Cell-Derived Extracellular Vesicles Promote Proliferation of Degenerated Nucleus Pulposus Cells and the Synthesis of Extracellular Matrix Through the SOX4/Wnt/ß-Catenin Axis.

Objective: Intervertebral disk degeneration (IDD) is a major cause of pain in the back, neck, and radiculus. Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are therapeutic in musculoskeletal degenerative diseases such as IDD. This study explored the effect and functional mechanism of human bone MSCs (hBMSCs)-derived EVs in proliferation and apoptosis of degenerated nucleus pulposus cells (DNPCs) and extracellular matrix (ECM) synthesis. Methods: Extracellular vesicles were isolated from hBMSCs and identified. DNPCs were induced by TNF-α. EVs were incubated with DNPCs for 24h. Internalization of EVs by DNPCs, DNPCs proliferation, apoptosis, and expressions of ECM synthetic genes, degrading genes and miR-129-5p were assessed. Downstream target genes of miR-129-5p were predicted. Target relation between miR-129-5p and SRY-box transcription factor 4 (SOX4) was verified. DNPCs proliferation, apoptosis, and ECM synthesis were measured after treatment with EVs and miR-129-5p inhibitor or SOX4 overexpression. Expressions of SOX4 and Wnt/ß-catenin pathway-related proteins were determined. Results: hBMSC-EVs promoted DNPCs proliferation, inhibited apoptosis, increased expressions of ECM synthetic genes, and reduced expressions of ECM degrading genes. hBMSC-EVs carried miR-129-5p into DNPCs. Silencing miR-129-5p in EVs partially inverted the effect of EVs on DNPCs proliferation and ECM synthesis. miR-129-5p targeted SOX4. SOX4 overexpression annulled the effect of EVs on DNPCs proliferation and ECM synthesis. Expressions of Wnt1 and ß-catenin were decreased in EVs-treated DNPCs, while silencing miR-129-5p in EVs promoted expressions of Wnt1 and ß-catenin. Conclusion: hBMSC-EVs promoted DNPCs proliferation and ECM synthesis by carrying miR-129-5p into DNPCs to target SOX4 and deactivating the Wnt/ß-catenin axis.

Mettl14-mediated m6A modification modulates neuron apoptosis during the repair of spinal cord injury by regulating the transformation from pri-mir-375 to miR-375.

BACKGROUND: Spinal cord injury (SCI) is a disabling disorder, resulting in neurological impairments. This study investigated the mechanism of methyltransferase-like 14 (Mettl14) on apoptosis of spinal cord neurons during SCI repair by mediating pri-microRNA (miR) dependent N6-methyladenosine (m6A) methylation. METHODS: The m6A content in total RNA and Mettl14 levels in spinal cord tissues of SCI rats were detected. Mettl14 expression was intervened in SCI rats to examine motor function, neuron apoptosis, and recovery of neurites. The cell model of SCI was established and intervened with Mettl14. miR-375, related to SCI and positively related to Mettl14, was screened out. The expression of miR-375 and pri-miR-375 after Mettl14 intervention was detected. The expression of pri-miR-375 combined with DiGeorge critical region 8 (DGCR8) and that modified by m6A was detected. Furthermore, the possible downstream gene and pathway of miR-375 were analysed. SCI cell model with Mettl14 intervention was combined with Ras-related dexamethasone-induced 1 (RASD1)/miR-375 intervention to observe the apoptosis. RESULTS: Mettl14 level and m6A content in spinal cord tissue were significantly increased. After Mettl14 knockdown, the injured motor function was restored and neuron apoptosis was reduced. In vitro, Mettl14 silencing reduced the apoptosis of SCI cells; miR-375 was reduced and pri-miR-375 was increased; miR-375 targeted RASD1. Silencing Mettl14 inactivated the mTOR pathway. The apoptosis in cells treated with silencing Mettl14 + RASD1/miR-375 was inhibited. CONCLUSIONS: Mettl14-mediated m6A modification inhibited RASD1 and induced the apoptosis of spinal cord neurons in SCI by promoting the transformation of pri-miR-375 to mature miR-375.

Identification of Hub Genes in Duchenne Muscular Dystrophy: Evidence from Bioinformatic Analysis.

The hub genes and signaling pathways associated with Duchenne muscular dystrophy (DMD) were predicted by bioinformatic methods to improve the therapeutic effect and quality of life of patients. Microarray data sets GSE465, GSE1004, and GSE1007 were downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were identified by GEO2R, and function enrichment analyses were performed by DAVID. The protein-protein interaction (PPI) network was constructed and the module analysis was performed using STRING and Cytoscape. A total of 195 DEGs were identified. The enriched functions and pathways of the DEGs include extracellular exosome, focal adhesion, extracellular matrix (ECM), focal adhesion, PI3K-Akt signaling pathway, calcium signaling pathway, and ECM-receptor interaction. Fifteen hub genes were identified. DEGs and hub genes identified in the present study help us understand the molecular mechanisms underlying the pathogenesis and progression of DMD, and provide candidate targets for treatment of DMD.

Two-Dimensional Ultrasound and Shear Wave Elastography in Infants With Late-Referral Congenital Muscular Torticollis.

OBJECTIVES: To investigate the feasibility of using 2-dimensional ultrasound (US) and shear wave elastography (SWE) to evaluate infants with late-referral congenital muscular torticollis (CMT). METHODS: A total of 46 infants with late-referral CMT were enrolled and divided into 4 groups according to the degree of the passive range of motion (PROM) deficit in neck rotation. We introduced 6 metrics to represent the US features of the sternocleidomastoid muscle detected by 2-dimensional US and SWE studies. RESULTS: There were no significant differences in the age at referral, sex, weight, or side of involvement between the 4 groups (P > .05). The thickness and shear modulus of the involved sternocleidomastoid muscle were positively correlated with the degree of the PROM deficit in neck rotation in late-referral infants with CMT (r = 0.71 and 0.82, respectively; P < .01). However, the difference in the shear modulus between adjacent PROM-limited groups of late-referral infants was not as significant as that of early-referral infants. CONCLUSIONS: The shear modulus ratio may be one of the best diagnostic indicators of CMT available. However, more effective differential diagnostic SWE indicators late-referral infants with CMT need to be further explored.

[Effect and mechanisms of vitamin E on early steroid-induced avascular necrosis of femoral head in rats].

OBJECTIVE: To investigate the possibility of mitochondria-dependent apoptosis as a mechanism of early steroid-induced avascular necrosis of femoral head (SANFH) in rats and vitamin E as a possible prevention strategy. METHODS: Seventy-two male Sprague Dawley rats were randomly divided into control group, model group, and intervention group, with 24 rats in each group. The rats in control group were not treated as normal control. The rats in model group and intervention group were established early SANFH models by lipopolysaccharide combined with methylprednisolone injection. At the same time, the rats in intervention group were injected with vitamin E (40 mg/kg) every day for 7 days. At 2, 4, and 8 weeks after the final injection, the bilateral femoral heads were harvested and observed by HE staining, TUNEL assay, immunohistochemical staining, and Western blot. The rate of empty lacunae, apoptotic index, and the expressions of Caspase-9, Caspase-3, and cytochrome-c (Cyt-c) proteins were calculated. RESULTS: According to histological staining, there were significant differences in the rate of empty lacunae between intervention group and control group at 8 weeks ( P<0.05) and between intervention group and model group at 4 and 8 weeks ( P<0.05). The apoptotic index of intervention group was significantly lower than that of model group at each time point ( P<0.05). And there was significant difference between the intervention group and the control group at 8 weeks ( P<0.05). According to immunohistochemistry staining and Western blot, the expressions of Cyt-c, Caspase-9, and Caspase-3 all significantly decreased in intervention group than those in model group at each time point ( P<0.05); and the differences were significant between intervention group and control group at 8 weeks ( P<0.05). CONCLUSION: Vitamin E can delay the progression of early SANFH by reducing mitochondrial dependent osteocyte apoptosis.

Carbon dots intensified poly (ethylene glycol)/chitosan/sodium glycerophosphate hydrogel as artificial synovium tissue with slow-release lubricant.

The ultra-high molecular weight polyethylene (UHMWPE) and metal artificial joint pair is limited by wear debris and short service life. Here we report the development of a hydrogel which exhibits lubricant release to intensify the lubrication effect of artificial joints.This study adopted an injectable method to prepare carbon dots/poly (ethylene glycol)/chitosan/sodium glycerophosphate (CDs/PEG/CS/GP) composite hydrogel, and the carbon dots were used to intensify the rheological and mechanical properties. In addition, the composite hydrogel had slow-release properties, and the release solution contained CDs, PEG and GP has excellent lubrication effect. At last, the MTT assay, LIVE/DEAD staining, H&E staining results and safety evaluation in BALC/c mice proved that the hydrogels had good biocompatibilility and were safety for application in vivo.

Effects of periarticular injection on analgesic effects and NSAID use in total knee arthroplasty and total hip arthroplasty

OBJECTIVES: This study examined periarticular multimodal drug injection and the use of nonsteroidal anti-inflammatory drugs for an early analgesic effect after total knee arthroplasty and total hip arthroplasty. Patient satisfaction and benefits from the treatment were also assessed. METHODS: A total of 110 patients who were scheduled to undergo total knee arthroplasty and 86 patients who were scheduled to undergo total hip arthroplasty were divided into two groups, the study group and the control group. The study group received a periarticular multimodal drug injection during surgery. The control group received an equal volume of normal saline. All patients received an analgesia pump and a moderate dose of nonsteroidal anti-inflammatory drugs. Resting and motion Numeric Rating Scale scores, the Western Ontario and McMaster Universities Arthritis Index, knee or hip joint range of motion, length of postoperative hospital stay, patient satisfaction, total nonsteroidal anti-inflammatory drug consumption and side effects were recorded. RESULTS: Both study groups exhibited significant improvement in pain Numeric Rating Scale scores during rest and exercise several days after the surgery. The range of joint motion was greater in the study group, and the length of postoperative hospital stay was shorter than that in the control group. Patients in the study group consumed fewer nonsteroidal anti-inflammatory drugs and reported greater satisfaction with surgery. CONCLUSION: Intraoperative periarticular multimodal drug injection significantly relieved pain after surgery and reduced nonsteroidal anti-inflammatory drug consumption. These patient had a better postoperative experience, including satisfaction and rehabilitation.

miR-20b/106a modulate Ngn2 gene expression during neural differentiation of human umbilical cord mesenchymal stem cells.

The aim of this study was to perform neural differentiation of mesenchymal stem cells derived from Wharton's jelly of human umbilical cord (HUMSCs) and explore the role of miR-20b and miR-106a, which may regulate Neurogenin-2 (Ngn2) expression during the neural differentiation. HUMSCs were cultured and induced in vitro. The cells were stained for nestin and microtubule-associated protein 2 (MAP2) by immunofluorescence. The interactional binding sites between the 3'-untranslated region (3'-UTR) of Ngn2 mRNA and miR-20b or miR-106a were predicted by bioinformatics and identified by a dual-luciferase assay. The expressions of Ngn2, miR-20b, and miR-106a were determined by real-time PCR and western blot before and after the neural differentiation. After infection of miR-20b or miR-106a, the expressions of Ngn2, MAP2, and ß III-tubulin (TUBB3) were measured. HUMSCs showed a uniform pattern with a typical short spindle-shaped morphology. Fourteen days after neural differentiation, HUMSCs showed neuronal traits of pyramidal appearance. TargetScan and miRanda showed that miR-20b and miR-106a were well complementary with Ngn2 3'-UTR. Identified by the dual-luciferase assay, we found that miR-20b and miR-106a inhibit Ngn2 expression by binding to its 3'-UTR. Furthermore, the expression of Ngn2 mRNA was almost reciprocal to that of miR-20b and miR-106a by real-time PCR during the neural differentiation of HUMSCs. Overexpression of miR-20b and miR-106a downregulated the expressions of Ngn2, MAP2, and TUBB3. miR-20b and miR-106a may directly or indirectly regulate neuronal genes expression to modulate the neural differentiation of HUMSCs.

Diagnostic Value of Virtual Touch Tissue Imaging Quantification for Evaluating Median Nerve Stiffness in Carpal Tunnel Syndrome.

OBJECTIVES: To measure the shear wave velocity (SWV) of the median nerve by Virtual Touch tissue imaging quantification (VTIQ; Siemens AG, Erlangen, Germany) through the beginning of the carpal tunnel and to determine whether VTIQ could be used to diagnose carpal tunnel syndrome. METHODS: This study recruited 49 consecutive patients (72 wrists) with a definitive diagnosis of carpal tunnel syndrome and 23 healthy volunteers (46 wrists). We measured the median nerve diameter and cross-sectional area by 2-dimensional sonography and the SWV by VTIQ. The interobserver variability was analyzed, and diagnostic values were evaluated by drawing a receiver operating characteristic curve. RESULTS: The median nerve SWV was significantly higher in the carpal tunnel syndrome group (3.857 m/s) than the control group (2.542 m/s; P < .05). A 3.0-m/s SWV cutoff value revealed sensitivity, specificity, positive predictive value, negative predictive value, and accuracy of 83.3%, 91.3%, 93.8%, 77.8%, and 86.4%, respectively. The interobserver agreement was excellent for the SWV measurements. CONCLUSIONS: The median nerve SWV at the carpal tunnel inlet is significantly higher in patients with carpal tunnel syndrome, for whom VTIQ appears to be a highly reproducible diagnostic technique.

Effects of periarticular injection on analgesic effects and NSAID use in total knee arthroplasty and total hip arthroplasty.

OBJECTIVES: This study examined periarticular multimodal drug injection and the use of nonsteroidal anti-inflammatory drugs for an early analgesic effect after total knee arthroplasty and total hip arthroplasty. Patient satisfaction and benefits from the treatment were also assessed. METHODS: A total of 110 patients who were scheduled to undergo total knee arthroplasty and 86 patients who were scheduled to undergo total hip arthroplasty were divided into two groups, the study group and the control group. The study group received a periarticular multimodal drug injection during surgery. The control group received an equal volume of normal saline. All patients received an analgesia pump and a moderate dose of nonsteroidal anti-inflammatory drugs. Resting and motion Numeric Rating Scale scores, the Western Ontario and McMaster Universities Arthritis Index, knee or hip joint range of motion, length of postoperative hospital stay, patient satisfaction, total nonsteroidal anti-inflammatory drug consumption and side effects were recorded. RESULTS: Both study groups exhibited significant improvement in pain Numeric Rating Scale scores during rest and exercise several days after the surgery. The range of joint motion was greater in the study group, and the length of postoperative hospital stay was shorter than that in the control group. Patients in the study group consumed fewer nonsteroidal anti-inflammatory drugs and reported greater satisfaction with surgery. CONCLUSION: Intraoperative periarticular multimodal drug injection significantly relieved pain after surgery and reduced nonsteroidal anti-inflammatory drug consumption. These patient had a better postoperative experience, including satisfaction and rehabilitation.

[EFFECT OF MACROPHAGE MIGRATION INHIBITORY FACTOR ON VASCULAR REPAIR OF STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN VITRO].

OBJECTIVE: To interpret the mechanisms of vascular repair disorders in steroid-induced avascular necrosis of the femoral head (SANFH) via detection of the changes of proliferation, migration, and macrophage migration inhibitory factor (MIF)/vascular endothelial growth factor (VEGF) expressions of endothelial cells (ECs) under hypoxia/glucocorticoid. METHODS: According to culture conditions, human umbilical vein ECs (HUVECs) at passage 3 were divided into group A (normal), group B (1.0×10-6 mol/L dexamethasone), group C (hypoxia), and group D (hypoxia+1.0×10-6 mol/L dexamethasone). The cell activity was detected by AlamarBlue; the number of viable cells was detected in live/dead cell staining; the cell morphology was observed after cytoskeleton staining; cell migration ability was compared by scratch test; and the levels of MIF and VEGF expressions were detected by ELISA. RESULTS: At 24 hours after culture, the cell activity and the number of living cells in group C were significantly higher than those in the other 3 groups, showing significant difference between groups (P<0.05), and group D had the worst cell activity and least living cells. Cytoskeleton staining showed that cells had normal morphology in groups A and B; cells had rich cytoskeleton and secretion granules in group C; cytoskeleton form disorder and nucleus pyknosis were observed in group D. Scratch test showed that the cell migration ability of group C was strongest while cell migration ability of group D was weakest. Accumulated concentration of MIF and VEGF in 4 groups significantly increased with time extending. Accumulated concentration of MIF in group C were significantly higher than that in other 3 groups at each time point (P<0.05). Within 24 hours after intervention, stage concentration of MIF during 1-8 hours was significantly lower than that during 0-1 hour and 8-24 hours in every group (P<0.05). Stage concentration of MIF in group C was significantly higher than other groups during 0-1 hour and 8-24 hours (P<0.05). Within 2 hours after intervention, stage concentration of MIF in 4 groups during 0.5-1 hour was significantly higher than that during other stages (P<0.05). Accumulated concentration of VEGF in group C was significantly higher than that in other groups at 8 and 24 hours (P<0.05). The stage concentration of VEGF in groups C and D during 8-24 hours was significantly higher than that during 0-1 hour and 1-8 hours (P<0.05). There was no significant difference in the stage concentration of VEGF within and among group A, B, C, and D at every stage within 2 hours after intervention (P>0.05). CONCLUSIONS: In hypoxia environment, the proliferation and migration of ECs is enhanced, and the secretion of VEGF and MIF is increased. High concentration of dexamethasone will suppress the process above, which induces vascular repair disorders and aggravating SANFH.

[EFFECT OF Wnt/ß-catenin SIGNAL PATHWAY ON APOPTOSIS IN STEROID-INDUCED AVASCULAR NECROSIS OF FEMORAL HEAD IN RATS].

OBJECTIVE: To investigate the effect of Wnt/ß-catenin signal pathway on the apoptosis in steroid-induced avascular necrosis of femoral head (SANFH) in rats. METHODS: Seventy-two male Sprague Dawley rats (weighing, 200-230 g) were randomly divided into the control group (group A, n=24), the model group (group B, n=24), and the intervening group (group C, n=24). The rats in groups B and C were injected with lipopolysaccharide and methylprednisolone (MPS) to establish the SANFH model. The rats in group C were injected intramuscularly with human recombinant secreted frizzled related protein 1 (SFRP1) [1 µg/(kg·d)] at the first time of MPS administration for 30 days. The rats in group A received saline injection at the same injection time of group B. The general condition of rats in groups B and C was observed during modeling and after modeling. At 2, 4, and 8 weeks after last injection of MPS, 8?rats were sacrificed to harvest the femoral head. Histological staining was performed to evaluate osteonecrosis. Apoptosis was detected via TUNEL staining. The expressions of Wnt/ß-cate nin pathway signaling molecules (activated ß-catenin and c-Myc) were detected by immunohistochemistry and Western blot. RESULTS: Six rats were added in groups B and C because of 6 deaths. The other rats survived to the end of experiment. Normal bone structure was observed in group A; osteonecrosis of bone structure disturbance and disruption of the trabecula were found with time in groups B and C. Group C had the highest empty lacuna rate and apoptosis rate, followed by groups B and A, showing significant difference between groups (P < 0.05). The expression levels of activated ß-catenin and c-Myc were significantly lower in group C than groups A and B (P < 0.05), and in group B than group A (P < 0.05). CONCLUSIONS: Wnt/ß-catenin signal pathway is involved in the pathogenesis in early SANFH model and its?possible mechanism?is to affect the cell cycle and cell apoptosis by the regulation of c-Myc expression.