Artemisinin relieves osteoarthritis by activating mitochondrial autophagy through reducing TNFSF11 expression and inhibiting PI3K/AKT/mTOR signaling in cartilage.

Osteoarthritis (OA) is a widespread chronic degenerative joint disease characterized by the degeneration of articular cartilage or inflamed joints. Our findings indicated that treatment with artemisinin (AT) downregulates the protein levels of MMP3, MMP13, and ADAMTS5, which are cartilage degradation-related proteins in OA, and inhibits the expression of inflammatory factors in interleukin-1ß (IL-1ß)-stimulated chondrocytes. However, the mechanism of the role of AT in OA remains unclear. Here, we performed gene sequencing and bioinformatics analysis in control, OA, and OA + AT groups to demonstrate that several mRNA candidates were enriched in the PI3K/AKT/mTOR signaling pathway, and TNFSF11 was significantly downregulated after AT treatment. TNFSF11 was downregulated in the OA + AT group, whereas it was upregulated in rat OA tissues and OA chondrocytes. Therefore, we confirmed that TNFSF11 was the target gene of AT. In addition, our study revealed that AT relieved cartilage degradation and defection by activating mitochondrial autophagy via inhibiting the PI3K/AKT/mTOR signaling pathway in IL-1ß-induced chondrocytes. Furthermore, an OA model was established in rats with medial meniscus destabilization. Injecting AT into the knee joints of OA rat alleviated surgical resection-induced cartilage destruction. Thus, these findings revealed that AT relieves OA by activating mitochondrial autophagy by reducing TNFSF11 expression and inhibiting PI3K/AKT/mTOR signaling.

Effect of kartogenin-loaded gelatin methacryloyl hydrogel scaffold with bone marrow stimulation for enthesis healing in rotator cuff repair.

BACKGROUND: Strategies involving microfracture, biomaterials, growth factors, and chemical agents have been evaluated for improving enthesis healing. Kartogenin (KGN) promotes selective differentiation of bone marrow mesenchymal stem cells (BMSCs) into chondrocytes. Gelatin methacryloyl (GelMA) is a promising biomaterial for engineering scaffolds and drug carriers. Herein, we investigated KGN-loaded GelMA hydrogel scaffolds with a bone marrow-stimulating technique for the repair of rotator cuff tear. METHODS: KGN-loaded GelMA hydrogel scaffolds were obtained by ultraviolet GelMA crosslinking and vacuum freeze-drying. Fifty-four New Zealand rabbits were randomly divided into (1) repair only (control), (2) microfracture + repair (BMS), and (3) microfracture + repair augmentation with a KGN-loaded GelMA hydrogel scaffold (combined) groups. Tendons were repaired by transosseous sutures. The structure, degradation, and in vitro KGN release of the scaffolds were characterized. Animals were euthanized 4, 8, and 12 weeks after repair. Enthesis healing was evaluated by macroscopy, microcomputed tomography, histology, and biomechanical tests. RESULTS: The KGN-loaded GelMA hydrogel scaffolds are porous with a 60.4 ± 28.2-µm average pore size, and they degrade quickly in 2.5 units/mL collagenase solution. Nearly 81% of KGN was released into phosphate-buffered saline within 12 hours, whereas the remaining KGN was released in 7 days. Macroscopically, the repaired tendons were attached to the footprint. No differences were detected postoperatively in microcomputed tomography analysis among groups. Fibrous scar tissue was the main component at the tendon-to-bone interface in the control group. Disorderly arranged cartilage formation was observed at the tendon-to-bone interface in the BMS and combined groups 4 weeks after repair; the combined group exhibited relatively more cartilage. The combined group showed improved cartilage regeneration 8 and 12 weeks after repair. Similar results were found in tendon maturation scores. The ultimate load to failure and stiffness of the repaired tendon increased in all 3 groups. At 4 weeks after repair, the BMS and combined groups exhibited greater ultimate load to failure than the control group, although there was no difference in stiffness among groups. The BMS and combined groups exhibited greater ultimate load to failure and stiffness than the control group, and the combined group exhibited better values than the BMS group at 8 and 12 weeks after repair. CONCLUSION: Compared with the bone marrow-stimulating technique, the KGN-loaded GelMA hydrogel scaffold with bone marrow stimulation improved enthesis healing by promoting fibrocartilage formation and improving the mechanical properties.

[Finite element analysis of arthroscopic repair of rotator cuff injury with different transosseous techniques].

OBJECTIVE: To compare and analyze the biomechanical differences between different transosseous techniques in arthroscopic repairment of rotator cuff injuries by finite element analysis. METHODS: Finite element models of traditional arthroscopic transosseous(ATO) technique, giant needle technique, and ArthroTunneler(AT) technique were established based on the shoulder CT data of a healthy adult. Then, loads of 10 N and 20 N were applied to the sutures on the different technical models, respectively. Compare and analyze the stress changes of the bone tunnels and sutures of the three models were compared and analyzed. RESULTS: Under the same condition of loading, the stress on the lateral bone tunnels and sutures of the traditional ATO technology model was the largest, followed by the giant needle technology model. The stress on the mid-section bone tunnels and sutures of the AT technology model was the largest, followed by the giant needle technology model. Under the different conditions of loading, the high-stress areas of the three models were mainly concentrated on the contact area between the sutures and the bone tunnels. Besides, compared with the traditional ATO technology model, the stress distribution of the lateral bone tunnels and sutures of the giant needle technology and AT technology model were more dispersed, but there was obvious stress concentration phenomena in the stress distribution in the mid-section bone tunnels and sutures in the AT technology model. CONCLUSION: Compared with the traditional ATO technique, both the giant needle technique and the AT technique can reduce the risk of cutout between the bone tunnel and suture, and may be better treatments for rotator cuff tear. However, compared with the giant needle technique, the application of AT technique in patients with osteoporosis may be limited.

[Effect of microfracture combined with biomimetic hydrogel scaffold on rotator cuff tendon-to-bone healing in rabbits].

OBJECTIVE: To assess the effect of microfracture and biomimetic hydrogel scaffold on tendon-to-bone healing in a rabbit rotator cuff tear model. METHODS: Gelatin and methacrylic anhydride were used to synthesize gelatin methacryloyl (GelMA). Then the GelMA were treated with ultraviolet rays and vacuum freeze-drying method to obtain a biomimetic hydrogel scaffold. The morphology of the scaffold was observed by gross observation and scanning electron microscope. Degradation of the scaffold was determined at different time points. Twenty-four adult New Zealand rabbits, weighting 2.8-3.5 kg and male or female, were surgically created the bilateral acute rotator cuff tear models. One shoulder was treated with microfractures on the footprint and transosseous suture (control group, n=24). The other shoulder was treated with the same way, except for putting the scaffold on the footprint before transosseous suture (experimental group, n=24). The general conditions of rabbits were observed postoperatively. Tendon-to-bone healing was evaluated by gross observation, Micro-CT, HE staining, and bio-mechanical testing at 4 and 8 weeks after operation. RESULTS: The scaffold was white and has a porous structure with pore size of 31.7-89.9 µm, which degraded slowly in PBS solution. The degradation rate was about 95% at 18 days. All the rabbits survived to the completion of the experiment. Micro-CT showed that there was no obvious defect and re-tear at the tendon-to-bone interface in both groups. No difference was found in bone mineral density (BMD), tissue mineral density (TMD), and bone volume/total volume (BV/TV) between the two groups at 4 and 8 weeks postoperatively ( P>0.05). HE staining showed that the fibrous scar tissue was the main component at the tendon-to-bone interface in the control group at 4 and 8 weeks postoperatively; the disorderly arranged mineralized cartilage and fibrocartilage formation were observed at the tendon-to-bone interface in the experimental group at 4 weeks, and the orderly arranged cartilage formation was observed at 8 weeks. Besides, the tendon maturation scores of the experimental group were significantly higher than those of the control group at 4 and 8 weeks ( P<0.05). There was no significant difference in the ultimate load to failure and stiffness between the two groups at 4 weeks ( P>0.05); the ultimate load to failure at 8 weeks was significantly higher in the experiment group than in the control group ( t=4.162, P=0.009), and no significant difference was found in stiffness between the two groups at 8 weeks ( t=2.286, P=0.071). CONCLUSION: Compared with microfracture alone, microfracture combined with biomimetic hydrogel scaffold can enhance tendon-to-bone healing and improve the ultimate load to failure in rabbits.

MicroRNA-451 blockade promotes osteoblastic differentiation and skeletal anabolic effects by promoting YWHAZ-mediated RUNX2 protein stabilization.

Background: senile osteoporosis researchers are now seeking to promote osteoblastogenesis and resultant bone formation to directly counteract age-related bone loss. Targeting microRNA (miRNA) activity in adult osteoblasts may be a successful therapeutic strategy for age-related bone loss. We investigated the mechanism(s) by which miRNAs negatively regulate osteoblastogenesis and bone formation in vitro and in vivo. Methods: we performed a miRNA microarray screen followed by PCR validation in adult bone marrow-derived mesenchymal stem cells during the proliferation-to-mineralization transition to identify downregulated miRNAs, most notably miR-451. Primary human calvarial pre-osteoblasts were isolated and transfected with miR-451's agomir or antagomir for in vitro assays. Bioinformatics analysis and in vitro experiments verified YWHAZ as a miR-451 target gene. We next investigated the effects of YWHAZ knockdown on osteoblastic differentiation. To examine the effects of miR-451's antagomir in vivo, we injected ovariectomized (OVX) or sham-operated mice with miR-451's antagomir over a period of six weeks. We isolated stromal cells from murine bone marrow on week six for further ex vivo experimentation. Results: miR-451's antagomir stimulated pre-osteoblast differentiation into a more differentiated, mineralized phenotype. This phenotype was associated with upregulated RUNX2, ALP, and COL1A1 protein expression. miR-451's antagomir derepresses YWHAZ expression, thereby enhancing RUNX2 protein stability and promoting osteoblastic differentiation. When injected in vivo, miR-451's antagomir promotes osteoblastogenesis and mineralization, reversed OVX-induced bone loss, and increased bone strength in OVX and sham-operated mice. Conclusions: miR-451 suppresses osteoblastogenesis in vitro and in vivo. miR-451 inhibition may serve as an effective anabolic therapeutic strategy in senile osteoporosis patients.

Aquaporin-1 expression as an indicator in evaluating the efficacy of meloxicam in the treatment of ankylosing spondylitis: A comparative study.

OBJECTIVE: The key objective of the study was to investigate the correlation between the expression of aquaporin-1 (AQP1) and the efficacy of meloxicam and expressions of pro-inflammatory cytokines in ankylosing spondylitis (AS). METHODS: 40 AS patients whom had received meloxicam were recruited and subsequently placed into the experiment, while 40 healthy individuals were recruited as control group. Clinical indicators were detected before treatment (0 week), and at 2, 4, 6, 8, 10 and 12 week intervals after treatment, which included various assessments including Ankylosing Spondylitis 20% (ASAS20) response, Bath ankylosing spondylitis disease activity index (BASDAI), visual analog scale (VAS) for back pain, duration of morning stiffness, Bath ankylosing spondylitis functional index (BASFI), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) levels. Healthy volunteers were examined for ESR and CRP levels. The mRNA and protein expressions of AQP1 and pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2), in peripheral blood mononuclear cells (PBMCs) were detected 6 and 12 weeks after treatment using reverse transcription quantitative polymerase chain reaction (RT-qPCR) and Western blotting. Correlation of expressions of AQP1, efficacy of meloxicam and expression of pro-inflammatory cytokines were determined via Pearson correlation analysis. RESULTS: Following 12 weeks of meloxicam treatment, the ASAS20 response reached 93.7±3.61%. 6 weeks after treatment, BASDAI, VAS for back pain, duration of morning stiffness, BASFI, ESR, and CRP levels all exhibited considerably reduced levels compared to the initial levels observed prior to the commencement of treatment. Compared with before treatment, the expressions of TNF-α, IL-2 and AQP1 mRNA and protein all displayed decreases in the experiment group after both 6 and 12-week periods of treatment. Pre and post treatment levels of TNF-α, IL-2 and AQP1 mRNA and protein expressions were higher than those in the control group. The expressions of AQP1 mRNA and protein in the experiment group were positively correlated with clinical indicators and expressions of pro-inflammatory cytokines. CONCLUSION: Our findings indicated that AQP1 was both highly expressed and positively correlated with the efficacy of meloxicam and expressions of pro-inflammatory cytokines in AS patients, thereby highlighting the promise of meloxicam as a potential indicator in predicting the efficacy in the treatment of AS.