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Hypertrophic differentiation is not only the terminal process of endochondral ossification in the growth plate but is also an important pathological change in osteoarthritic cartilage. Collagen type II (COL2A1) was previously considered to be only a structural component of the cartilage matrix, but recently, it has been revealed to be an extracellular signaling molecule that can significantly suppress chondrocyte hypertrophy. However, the mechanisms by which COL2A1 regulates hypertrophic differentiation remain unclear. In our study, a p.Gly1170Ser mutant mouse model was constructed, and Col2a1 loss was demonstrated in homozygotes. Loss of Col2a1 was found to accelerate chondrocyte hypertrophy through the bone morphogenetic protein (BMP)-SMAD1 pathway. Upon interacting with COL2A1, integrin β1 (ITGB1), the major receptor for COL2A1, competed with BMP receptors for binding to SMAD1 and then inhibited SMAD1 activation and nuclear import. COL2A1 could also activate ITGB1-induced ERK1/2 phosphorylation and, through ERK1/2-SMAD1 interaction, it further repressed SMAD1 activation, thus inhibiting BMP-SMAD1-mediated chondrocyte hypertrophy. Moreover, COL2A1 expression was downregulated, while chondrocyte hypertrophic markers and BMP-SMAD1 signaling activity were upregulated in degenerative human articular cartilage. Our study reveals novel mechanisms for the inhibition of chondrocyte hypertrophy by COL2A1 and suggests that the degradation and decrease in COL2A1 might initiate and promote osteoarthritis progression.

Osteoarthritis (OA) is characterized by cartilage destruction, chronic inflammation, and local pain. Evidence showed that retinoic acid receptor-related orphan receptor-α (RORα) is crucial in cartilage development and OA pathogenesis. Here, we investigated the role and molecular mechanism of RORα, an important member of the nuclear receptor family, in regulating the development of OA pathologic features. Investigation into clinical cartilage specimens showed that RORα expression level is positively correlated with the severity of OA and cartilage damage. In an in vivo OA model induced by anterior crucial ligament transaction, intra-articular injection of si-Rora adenovirus reversed the cartilage damage. The expression of cartilage matrix components type II collagen and aggrecan were elevated upon RORα blockade. RNA-seq data suggested that the IL-6/STAT3 pathway is significantly downregulated, manifesting the reduced expression level of both IL-6 and phosphorylated STAT3. RORα exerted its effect on IL-6/STAT3 signaling in two different ways, including interaction with STAT3 and IL-6 promoter. Taken together, our findings indicated the pivotal role of the RORα/IL-6/STAT3 axis in OA progression and confirmed that RORα blockade improved the matrix catabolism in OA chondrocytes. These results may provide a potential treatment target in OA therapy.

Background The protective effect of melatonin against bone metabolism imbalance in osteoporosis (OP) induced by drugs such as retinoic acid (RA) is unclear. The aim of this study was to explore the role of melatonin in bone destruction based on a mouse model. Methods RA-induced OP model mice were established. To assess the effect of melatonin on these mice, micro-CT was used to characterize the trabecular structure of normal mice and those treated with RA (model), RA + low-dose melatonin (Mlt-L), RA + high-dose melatonin (Mlt-H), and RA + alendronate sodium (positive control). The shape of the trabecular bone, the length and diameter of the femoral head and the height and diameter of vertebra(L1) of each group were also measured and the number of osteoclasts was determined by Tartrate-resistant acid phosphatase (TRACP) staining. Meanwhile, the expression of alkaline phosphatase (ALP) was evaluated by immunohistochemistry assays. The differences between groups in terms of liver and kidney oxidation–related indexes and serum and urinary indicators related to bone metabolism were also analyzed. Furthermore, qRT-PCR and western blotting were used to evaluate the effect of melatonin on osteogenic and osteoclastic differentiation in MC3T3-E1 and RAW264.7 cells, respectively. Results RA induction led to a decrease in the amount and density of trabecular bone, a decrease in the length and diameter of the femur and height, diameter of the vertebra (L1), a decrease in bone mass and density and the expression of ALP, and an increase in the number of osteoclasts. Melatonin treatment alleviated these effects induced by RA, increasing the amount of trabecular bone in OP mice, improving the microstructure of the femur and vertebra(L1) and increasing bone mass bone density and the expression of ALP, as well as decreasing the number of osteoclasts. Additionally, blood and urinary bone metabolism-related indicators showed that melatonin promoted bone formation and inhibited bone resorption. Determination of oxidant and antioxidant biomarkers in the livers and kidneys of the mice revealed that melatonin promoted the antioxidant level and suppressed the level of oxidant molecules in these organs. In vitro, RA promoted osteoclasts and inhibit osteogenesis by increasing oxidative stress levels in the RAW264.7 and MC3T3-E1 cells, but melatonin reversed this effect. Melatonin may, therefore, play a role in the ERK/SMAD and NF-κB pathways. Conclusions Melatonin can alleviate bone loss in RA-induced OP model mice, repair the trabecular microstructure, and promote bone formation. These effects may be related to reducing oxidation levels in vivo and vitro through the ERK/SMAD and NF-κB pathways.

Background Little is known about the implications of circRNAs in the effects of melatonin (MEL) on bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation and osteoporosis (OP) progression. The aim of our study was to investigate circRNAs in MEL-regulated BMSC differentiation and OP progression. Methods BMSC osteogenic differentiation was measured by qRT-PCR, western blot (WB), Alizarin Red, and alkaline phosphatase (ALP) staining. Differential circRNA and mRNA profiles of BMSCs treated by MEL were characterized by deep sequencing, followed by validation using RT-PCR, Sanger sequencing, and qRT-PCR. Silencing and overexpression of circ_0003865 were conducted for functional investigations. The sponged microRNAs and targeted mRNAs were predicted by bioinformatics and validated by qRT-PCR, RNA pull-down, and dual-luciferase reporter assay. The function of miR-3653-3p and circ_0003865/miR-3653-3p/growth arrest-specific gene 1 (GAS1) cascade was validated for the osteogenic differentiation of BMSCs by CCK-8, qRT-PCR, WB, Alizarin Red, and ALP staining. The effects of circ_0003865 on OP development were tested in murine OP model. Results MEL promoted osteogenic differentiation of BMSCs. RNA sequencing revealed significant alterations in circRNA and mRNA profiles associated with multiple biological processes and signaling pathways. Circ_0003865 expression in BMSCs was significantly decreased by MEL treatment. Silencing of circ_0003865 had no effect on proliferation while promoted osteogenic differentiation of BMSCs. Overexpression of circ_0003865 abrogated the promotion of BMSC osteogenic differentiation induced by MEL, but proliferation of BMSCs induced by MEL had no change whether circ_0003865 was overexpression or not. Furthermore, circ_0003865 sponged miR-3653-3p to promote GAS1 expression in BMSCs. BMSC osteogenic differentiation was enhanced by miR-3653-3p overexpression while BMSC proliferation was not affected. By contrast, miR-3653-3p silencing mitigated the promoted BMSC osteogenic differentiation caused by circ_0003865 silencing, but had no effect on proliferation. Finally, circ_0003865 silencing repressed OP development in mouse model. Conclusion MEL promotes BMSC osteogenic differentiation and inhibits OP pathogenesis by suppressing the expression of circ_0003865, which regulates GAS1 gene expression via sponging miR-3653-3p.

Background Mesenchymal stem cells (MSCs) possess the potential to differentiate into chondrocytes, which makes them an ideal source for healing cartilage defects. Here, we seek to identify the essential genes participating in MSCs chondrogenesis. Methods Human MSCs were induced for chondrogenesis for 7, 14, and 21 days using a high-density micromass culture system, and RNA was extracted for RNA-seq. Results A total of 6247 differentially expressed genes (DEGs) were identified on day 7, and 85 DEGs were identified on day 14. However, no significant DEGs was identified on day 21. The top 30 DEGs at day 7, including COL9A3, COL10A1, and CILP2, are closely related to extracellular matrix organization. While the top 30 DEGs at day 14 revealed that inflammation-related genes were enriched, including CXCL8, TLR2, and CCL20. We also conducted protein–protein interaction (PPI) networks analysis using the search tool for the retrieval of interacting genes (STRING) database and identified key hub genes, including CXCL8, TLR2, CCL20, and MMP3. The transcriptional factors were also analyzed, identifying the top 5 TFs: LEF1, FOXO1, RORA, BHLHE41, and SOX5. We demonstrated one particular TF, RORA, in promoting early MSCs chondrogenesis. Conclusions Taken together, our results suggested that these DEGs may have a complex effect on MSCs chondrogenesis both synergistically and solitarily.

Background Sarcopenia and intervertebral disc degeneration (IDD) are both highly prevalent among the elderly and have a substantial impact on their quality of life. However, the association between sarcopenia and IDD remains unclear. This study aimed to investigate whether sarcopenia is independently associated with an increased risk of IDD in middle‐aged and older adults, using prospective data from the UK Biobank. Methods A total of 378 773 participants from the UK Biobank were included and categorized into three groups based on the European Working Group on Sarcopenia in Older People 2 (EWGSOP2) criteria: normal, probable sarcopenia and confirmed sarcopenia. The association between sarcopenia and IDD was examined using Kaplan–Meier survival analysis and Cox proportional hazards models. Sensitivity analyses included subgroup analyses to assess the robustness of findings and interaction tests to explore potential effect modifiers. Results The median age of participants was 59 years, with females accounting for 54.8% of the cohort. Over a median follow‐up duration of 171 months, 10 585 participants developed IDD. In unadjusted Cox regression analyses, compared to the normal group, the hazard ratios (HRs) for IDD were 1.51 (95% CI: 1.41–1.61) in the probable sarcopenia group and 1.47 (95% CI: 1.14–1.90) in the confirmed sarcopenia group. After adjusting for multiple covariates, the corresponding HRs were 1.35 (95% CI: 1.26–1.44) and 1.41 (95% CI: 1.10–1.80), respectively. These associations remained consistent across subgroup analyses. Notably, in BMI‐stratified analyses, individuals with sarcopenia and a BMI > 25 had a higher risk of IDD (HR: 1.88; 95% CI: 1.31–2.71) compared to those with BMI ≤ 25 (HR: 1.51; 95% CI: 1.06–2.16), with a significant interaction (p < 0.001). Conclusions Sarcopenia is associated with an increased risk of IDD, particularly in overweight or obese individuals. Regular assessment of muscle strength and mass, along with promoting physical activity and adequate nutritional interventions in ageing populations, may help prevent sarcopenia and delay the onset of IDD.

Background Human mesenchymal stem cells (hMSCs) have been proven to have inherent chondrogenic differentiation potential, which appears to be used in cartilage regeneration. Increasing evidence suggests that irisin enhances osteoblast differentiation of MSCs, but little is known about its potential on chondrogenic differentiation. Methods In the study, we investigated the effects of irisin on chondrogenic differentiation of hMSCs using a high-density pellet culture system. The cartilage pellets were evaluated by morphology, and the metabolism of cartilage matrix was detected by qPCR, western blot and immunohistochemistry. Next, RNA-seq was performed to explore the underlying mechanism. Furthermore, using the transduction of plasmid, miRNAs mimics and inhibitor, the activation of Rap1/PI3K/AKT axis, the expression level of SIPA1L2, and the functional verification of miR-125b-5p were detected on day 7 of chondrogenic differentiation of hMSCs. Results Compared with the controls, we found that irisin treatment could significantly enhance the chondrogenic differentiation of hMSCs, enlarge the induced-cartilage tissue and up-regulate the expression levels of cartilage markers. RNA-seq indicated that irisin activated the Rap1 and PI3K/AKT signaling pathway, and the lower expression level of SIPA1L2 and the higher expression level of miR-125b-5p were found in irisin-treated group. Further, we found that irisin treatment could up-regulate the expression level of miR-125b-5p , targeting SIPA1L2 and consequently activating the Rap1/PI3K/AKT axis on the process of chondrogenic differentiation of hMSCs. Conclusions Collectively, our study reveals that irisin can enhance chondrogenic differentiation of hMSCs via the Rap1/PI3K/AKT pathway, suggesting that irisin possesses prospects in cartilage regeneration.

Background Osteoarthritis (OA) is a widespread arthritic disease and a primary cause of disability. Increasing evidence suggests that inflammation has a pivotal part in its pathogenesis. Interleukin-1β (IL-1β) is a primary mediator of local inflammatory processes in OA. Current therapies for OA mainly focus on the symptoms of the advanced stage of the disease. The possible utilization of bone marrow mesenchymal stem cells (BMSCs) to regenerate cartilage is an appealing method, but in the case of OA requires chondrogenesis to take place within an inflamed environment. Our previous study showed that melatonin (MLT) can promote chondrogenic differentiation of MSCs, but whether MLT can rescue IL-1β-impaired chondrogenesis in human BMSCs has not yet been established. MLT, which can have anti-inflammatory and prochondrogenic effects, has demonstrated potential in defeating IL-1β-induced inhibition of chondrogenesis and further study should be conducted. Methods Human bone marrow-derived MSCs were separated and cultured based on our system that was already documented. A high-density micromass culture system was used for the chondrogenic differentiation of human BMSCs, which was also described previously. Human BMSCs were induced for chondrogenesis for 7, 14, and 21 days with the treatment of IL-1β and MLT. The cultured cartilage pellets were then evaluated by morphology, extracellular matrix accumulation, and chondrogenic, metabolic, and apoptotic marker expression. Furthermore, cell apoptosis was assessed by TUNEL assay. The phosphorylation level P65 and IκBα of the NF-κB pathway activity was explored on day 21 of chondrogenic differentiation of BMSCs. Results The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs in different aspects. Firstly, MLT can restore the chondrogenic pellet size, and rescue matrix synthesis and accumulation. Secondly, MLT can upregulate chondrogenic marker COL2A1 expression at both mRNA and protein levels, and also regulate the expression levels of other chondrogenic markers like ACAN , SOX9 , and COL10A1 in the presence of IL-1β. Thirdly, MLT can maintain the metabolic balance of the chondrogenic process by suppressing expression of catabolic genes, such as MMP , MMP13 , and ADAMTS4 . Furthermore, MLT can subdue IL-1β-induced cell apoptosis of BMSCs throughout chondrogenesis. Meanwhile, MLT suppressed the phosphorylation level of P65 and IκBα, which were elevated by IL-1β treatment, indicating that MLT can attenuate the IL-1β-induced activation of NF-κB signaling. Conclusion The current evaluation showed that MLT can save IL-1β-impaired chondrogenesis of human BMSCs by restoring the pellet size and matrix accumulation, and maintaining the metabolic balance, reducing cell apoptosis. Our study also showed that MLT can attenuate the IL-1β-induced activation of the NF-κB signaling pathway, which is the most important pathway downstream of IL-1β, and plays a crucial role in inflammation, apoptosis, and metabolism. Thus, MLT has prospects for treating OA due to its multifaceted functions, such as mitigating inflammation, maintaining metabolic balance, and mitigating apoptosis.

Intervertebral disc degeneration (IDD) is a degenerative disease that is characterized by decreased matrix synthesis and extra degradation, nucleus pulposus cells (NPCs) apoptosis, and infiltration of inflammatory factors. Aloin, a colored compound from aloe plants, has been shown to be effective against skeletal degenerative diseases, but it is unclear whether it is protective against IDD. Herein, we investigated the role of aloin in NPCs. In our study, the upregulation of proinflammatory factors, apoptosis, and unbalanced matrix metabolism were observed in degenerative NP tissues. We found that aloin had a curative effect on extracellular matrix metabolism and apoptosis in TNF-alpha- (TNF-α-) treated NPCs by inhibiting oxidative stress and the proinflammatory factor expression. Further investigation revealed that aloin treatment suppressed the TAK1/NF-κB pathway. Moreover, the expression level of the NLPR3 inflammasome was downregulated after aloin treatment in TNF-α-treated NPCs. In summary, our results demonstrated that aloin treatment can reverse TNF-α-induced unbalanced matrix metabolism and apoptosis of NPCs via the TAK1/NF-κB/NLRP3 axis. This study supports that aloin can be a promising therapeutic agent for IDD.

Adolescent idiopathic scoliosis (AIS) is one of the most common spinal deformities found in adolescent populations. Recently, a genome-wide association study (GWAS) in a Japanese population indicated that three single nucleotide polymorphisms (SNPs), rs11190870, rs625039 and rs11598564, all located near the LBX1 gene, may be associated with AIS susceptibility [1]. This study suggests a novel AIS predisposition candidate gene and supports the hypothesis that somatosensory functional disorders could contribute to the pathogenesis of AIS. These findings warrant replication in other populations. First, we conducted a case-control study consisting of 953 Chinese Han individuals from southern China (513 patients and 440 healthy controls), and the three SNPs were all found to be associated with AIS predisposition. The ORs were observed as 1.49 (95% CI 1.23-1.80, P = 5.09E-5), 1.70 (95% CI 1.42-2.04, P = 1.17E-8) and 1.52 (95% CI 1.27-1.83, P = 5.54E-6) for rs625039, rs11190870 and rs11598564, respectively. Second, a case-only study including a subgroup of AIS patients (N = 234) was performed to determine the effects of these variants on the severity of the condition. However, we did not find any association between these variants and the severity of curvature. This study shows that the genetic variants near the LBX1 gene are associated with AIS susceptibility in Chinese Han population. It successfully replicates the results of the GWAS, which was performed in a Japanese population.