Mesenchymal stem cells improve osteoarthritis by secreting superoxide dismutase to regulate oxidative stress response

Mesenchymal stem cells improve osteoarthritis by secreting superoxide dismutase to regulate oxidative stress response | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Mesenchymal stem cells improve osteoarthritis by secreting superoxide dismutase to regulate oxidative stress response Yao Yao, Juan Cao, Congzhu Ding This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4235031/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Apr, 2025 Read the published version in BMC Musculoskeletal Disorders → Version 1 posted 16 You are reading this latest preprint version Abstract Background To investigate the therapeutic effect of intraarticular injection of mesenchymal stem cells (MSC) in a rabbit osteoarthritis (OA) model. And to suppose whether MSC play a pivotal role in OA therapy by improving oxidative stress through secreting superoxide dismutase (SOD). Methods MSC were isolated and cultured in vitro. SOD gene of MSC was silenced by siRNA technology to prepare the SOD-siRNA-MSC for in-vivo study. Twenty healthy adult New Zealand white rabbits underwent papain injection to induce OA and then received intra-articular injection with MSC, siRAN-MSC, or normal saline. The rabbits were divided into 4 groups (n = 5), such as the control group, the model group, the MSC group, the siRNA-MSC group. Cytokines determination was performed 2 and 4 weeks after treatment. Magnetic resonance imaging (MRI) and histopathology and immunohistochemistry determination were performed 4 weeks after treatment. Results COMP, TNF-α, MMP-2 and MMP-13 in the MSC group were significantly decreased compared to those in model group (P < 0.05). However, MMP2 and MMP13 in the siRNA-MSC group were not significantly decreased compared to the model group (P < 0.05). Magnetic resonance results revealed a significant improvement in cartilage and synovial membrane 4 weeks after MSC injection. Histopathology determination showed that cartilage structure was also significantly improved in MSC group. Immunohistochemical analysis revealed amelioration in the expression levels of proteoglycan, COL-2, P21 and P53 in MSC group. On the other hand, MRI, histopathologic and immunohistochemical analysis also indicated a decreased therapeutic effect with SOD-siRNA -MSC. Conclusion Our study demonstrated for the first time that MSC might be a promising therapy in OA through anti-apoptosis and regeneration in chondrocyte by secreting SOD and improving oxidative stress. might play a pivotal role in the treatment of OA through anti-apoptosis and regeneration in chondrocyte by secreting SOD and improving oxidative stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Key Points 1. It is the first time to be reported that MSC may be a promising therapy in OA. 2. MSC might play a pivotal role in the treatment of OA through anti-apoptosis and regeneration in chondrocyte by secreting SOD and improving oxidative stress. 1. Introduction Osteoarthritis (OA) is a highly prevalent degenerative condition of the joints, most commonly the knee and hip, which affects more than 300 million people in the world [ 1 ]. Patients with OA suffer from mobility problems and chronic pain, resulting in a lifelong disability and reduced quality of life quality [ 2 ]. In addition, chronic OA management imposes a heavy burden on the family and medical resources. The pathogenesis of OA is complex and is not fully understood, but it is characterized by skeletal inflammation and cartilage degeneration and subchondrical bone sclerosis [ 3 ]. In addition to physical therapy and weight loss program, paracetamol or non-steroidal anti-inflammatory drugs (NSAIDs), and intraarticular injections of corticosteroids or hyaluronic acid may be the main current non-surgical treatments for OA [ 4 ]. Patients with no response to the treatment mentioned above, would consider other pain relief treatment such as tramadol or amitriptyline. The final option for end-stage knee OA is joint replacement. However, this surgery is not suitable for all patients for the risk of complications [ 5 ]. Furthermore, all these treatments have limited effects on the healing potential of damaged cartilage. Although several types of cells are involved in pathogeny of OA, chondrocytes are primarily considered to play an important role in OA induction by cellular senescence [ 6 ]. Normal chondrocytes usually show moderate metabolic and proliferative activity under normal conditions. However, some senescence chondrocytes lose their differentiated phenotype under inflammation conditions. Recent studies have identified that OA pathology is obviously related to oxidative stress and reactive oxygen species (ROS), which would regulate chondrocyte apoptosis and senescence, matrix metalloproteinase (MMP) production, and extracellular matrix synthesis and degradation [ 7 ]. MSC play an important role in repairing cartilage by tissue regeneration, producing factors that promote resident chondrocytes in the joint to form articular cartilage, and inhibiting inflammation [ 8 ]. A summary paper reviewed MSC therapy for OA in preclinical and clinical studies in the last 5 years, which certified the benefits and safety of intra-articular injections of MSC. Nevertheless, the effects and mechanisms of intra-articular injections of MSC in OA are not well defined due to the low-quality evidence. Furthermore, in vivo MSC differentiation is relatively rare, indicating that MSC differentiation in chondrocytes could not explain the therapeutic effect [ 9 ]. Klein D et al. verified that the antioxidant enzyme superoxide dismutase 1 (SOD1) could be an MSC secreted factor, which could mediate ROS [ 10 ]. The aim of our study was to observe whether intra-articular injection of MSC could change knee joint morphology using magnetic resonance imaging (MRI). Furthermore, inflammation determination, histopathological examination and immunohistochemical analysis were also carried out to establish cartilage injury and senescence improvement of intra-articular MSC injection. In addition, SOD gene was silenced to suppose whether MSC play a pivotal role in OA treatment through oxidative stress pathway. 2. Materials and methods 2.1 Culture and Characterization of UCB-MSC hUCB-MSC (passage 4–6, Jiangsu Keygen Biotech Corp, China) were cultured in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) at 37°C with 5% CO2. Characterization of hUCB-MSC were performed according to published procedures through a flow cytometer [ 11 ]. 2.2 SOD siRNA construction and transfection The siRNA primer sequence of SOD1 was designed following GENE BANK. There were three sequences listed as below. Gene Sequence SOD1siRNA-1 forward 5’-GGG CAA AGG UGG AAA UGA ATT-3’ Reverse 5’-UUC AUU UCC ACC UUU GCC CTT-3’ SOD1 siRNA-2 Forward 5’-GUU CAU GAG UUU GGA GAU ATT-3’ Reverse 5’-UAU CUC CAA ACU CAU GAA CTT-3’ SOD1 siRNA-3 Forward 5’-CAA UGU GAC UGC UGA CAA ATT-3’ Reverse 5’-UUU GUC AGC AGU CAC AUU GTT-3’ One day before transfection, an appropriate number of MSC cells were inoculated into 6-well cell culture plates, so that the cell density at transfection could reach 80–90%. siRNA was diluted to 20uM with 125µL serum-free Opti-MEM medium (Gibco, Gaithersburg, MD, USA), mixed gently, and incubated at room temperature for 5 min. Five µL lipo3000 was also diluted with 125µL serum-free Opti-MEM medium, mixed gently, and incubated at room temperature for 5 min. Then siRNA and lipo3000 (Invitrogen, thermos, USA) were gently mixed and incubated for 20 min at room temperature. The mixture was added to the cell in 750µL complete medium and cultured in CO 2 incubator at 37℃ for 48h. qPCR was used for identification the best silencing sequence. And the follow-up experiment was conducted with the best silencing SOD-siRNA-MSC. In addition, cell viability of MSC and siRNA-MSC was both verified through Trypan blue (Jiangsu Keygen Biotech Corp, China) dye exclusion test after transfection for 24h. 2.3 Experimental Animals and groups This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Drum Tower Hospital affiliated with the Nanjing University School of Medicine and was in accordance with ARRIVE guidelines. Twenty healthy adult New Zealand white rabbits with body weight between 2.5 and 3.0 kg (provided by the Experimental Animal Centre of Drum Tower Hospital affiliated to Nanjing University School of Medicine) were selected and divided into 4 groups (n = 5), control group, model group, MSC group and SOD-siRNA-MSC group. The environment of the animal laboratory was controlled at a temperature of 23 ± 2°C, a relative humidity of 55 ± 10%, a ventilation frequency of 12 times/h, and a lighting cycle of 12 h. And a normal diet and sterile distilled water was provided without restriction. 2.4 Establishment of osteoarthritis model and experimental procedures Four percent papain injection (Sigma, USA) 0.5mL was locally articular injected into the posterior knee cavity of the model group, MSC group and siRNA-MSC group on day 1, 4 and 7 for three times in total. The control group was injected with normal saline. Two weeks later, the experimental joints were observed by magnetic resonance imaging (MRI) to verify whether the OA model had been established successfully. And serum was collected through the marginal ear vein for determination of cytokines. Subsequently, the MSC group was injected with 1×10 6 /mL 0.5mL MSC, and the siRNA-MSC group was injected with 1×10 6 /mL 0.5mL siRNA-MSC, while the model group and control group were injected with 0.5ml normal saline. After 4 weeks of treatment, the morphology of the joints was observed by MRI. After observation, the experimental animals were sacrificed and serum, synovial membrane and articular cartilage were collected for further study. 2.5 MRI evaluation MRI was performed 4 weeks after treatment. In this study, the Philips 3.0T magnetic resonance imaging system (Ingenia, Philips, The Netherlands) was used. After anesthesia through propofol (5mg/kg) injection,, rabbits were fixed in the supine position on the experimental plate. Coronal and sagittal scans were performed with 8-channel orthogonal magnetic resonance coils of the head to obtain PDW-SPAIR sequence images. The following sequences were utilized: TR3000 MS, TE30 MS, turn angle 90°, field of vision 10 cm, layer thickness 1.5 mm, acquisition time 2 minutes 30 seconds. 2.6 Detection of cytokines in serum Cytokine determination was performed 2 and 4 weeks after treatment. Rabbit serum was collected through marginal ear vein. Serum levels of IL-1β, L-6, TNFα, MMP2, 13 and COMP were determined by enzyme-linked immunosorbent assay (ELISA lab, Hubei, China) according to each instruction, respectively. 2.7 Histopathologic examination and immunohistochemistry Rabbits were sacrificed after MRI evaluation at week 4. The supracondylar portion of the femur was collected, including the arthritis-induced site. The joint tissue specimens were fixed with 10% formalin solution, embedded in paraffin, sectioned into 4µm sections and stained with HE. The articular cartilage was fixed in 10% formalin solution, decalcified with EDTA, rinsed with water, and then treated with conventional embedding, sectioning and HE staining. All tissues were observed under a light microscope. Expression of P21, P53, COL-2 and proteoglycan in articular cartilage tissues was detected by Streptomyces antibiotin protein-peroxidase immunohistochemical method. The percentage of microscopically positive cells and staining intensity were evaluated by semiquantitative interpretation. Percentages of positive staining cell scores were calculated as follows: 0 for positive cells < 5%, 1 for 5%-25%, 2 for 26%-50%, 3 for 51%-75%, and 4 for 76%-100%. Positive stain intensity scores were calculated as follows: 0 for colorless, 1 for light yellow, and 2 for brownish yellow, 3 for tan. The multiplication of 2 scores can be considered a positive rating: 0 for negative (-), 1–4 for weakly positive (+), 5–8 for positive (+ +), 9–12 for strong positive (+ + +). 2.8 Statistical analysis Multiple comparison tests were conducted to compare different groups. For parametric multiple comparisons, one-way analysis of variance (ANOVA) was used to analyze the data. The T test was used to compare the mean data between the two groups. SPSS 22.0 statistical software was used for analysis. 3. Results 3.1 MSC identification Flow cytometry was used to monitor the proportion of CD29 and CD90 expression cells. It was indicated that the number of CD29 positive expression cells was 99.76%, and the number of CD90 positive expression cells was 94.87%, both of which were more than 90%. The number of CD29 positive cells was 0.13% and CD90 positive cells was 0.28% in the homotype control group (Fig.1) 3.2 SOD1 SiRNA Sequence Design and the best choice The siRNA primer sequence of SOD1 was designed by GENE BANK, and the synthesized primer sequence was transfected into MSC by liposome. qPCR was used to verify whether the sequence could block SOD1 expression in MSC. The results suggested that the expression of SOD1 in MSC was significantly decreased, either transfected with sequence 1, 2 or 3 (P 0.05) (Tab.1). Sequence 3 was applied as the SOD-siRNA sequence in subsequent experiments. In addition, the cell viability assay indicated that more than 95% cell survivor after transfection (Tab.2), which was suitable for intraarticular injection. 3.3 Cytokines outcomes The results of serum cytokines and cartilage damage markers are shown in Fig.2. COMP, IL-1β, IL-6, TNF-α, MMP-2 and MMP-13 levels in model group were significantly higher than those in control group (P <0.05), which indicated OA model was established successfully. Two weeks after intra-articular injection, the levels of COMP, IL-1β, IL-6, TNF-α, MMP-2 and MMP-13 in MSC group were significantly decreased compared with those in model group (P ≤0.05). Moreover, the levels of COMP, TNF-α, MMP-2 and MMP-13 in the MSC group were still lower than those in model group at 4 weeks after intra-articular injection (P ≤0.05). Two weeks after intra-articular injection, IL-1β, MMP-2, MMP-13 and TNF-α in the SOD- siRNA-MSC group was decreased compared with model group (P0.05). Moreover, IL-1β, IL-6 and MMP2 in SOD-siRNA-MSC group were elevated compared with MSC group (P<0.05). Four weeks after intraarticular injection, IL-1β, IL-6 and MMP-13 in the siRNA-MSC group did not significantly decrease compared to the model group (P<0.05). 3.4 MRI outcomes All rabbits were performed MRI (Fig. 3). Images showed that the cartilage surface of the femoral condyle joint was smooth, no abnormal signal was found in the subchondral bone, the synovial signal was normal, and a small amount of synovial fluid was found in the joint cavity on T2WI fat saturation sequence in control group. However, rough cartilage surface of femoral condylar article, poor meniscus polish, slightly increased signal of anterior cruciate ligament, slight thickening of synovium, slightly increased signal of subchondral bone, and moderate effusion in the joint cavity were showed in model group. The MRI images on T2WI fat saturation sequence of the joint after 4w treatment in the siRNA-MSC group showed that the cartilage surface of the femoral condyle joint was rough, the meniscus was not polished, the anterior cruciate ligament signal was increased, the synovium was slightly thickened, the subchondral bone signal was slightly increased, and there was fluid in the joint cavity, which did not change much compared with the model group. In MSC group, MRI findings showed that the cartilage signal of femoral condyle joint and tibial plateau was uneven, the meniscus was normal and intact, the anterior cruciate ligament was normal, the synovial signal was slightly increased, which was improved compared with the model group. In addition, the subchondral bone signal was normal, and the joint effusion was significantly reduced compared with the model group. 3.5 Histopathologic outcomes HE staining was performed on articular cartilage and synovial membrane of rabbits in all experimental groups (Fig.4). The results suggested that the posterior articular cartilage of rabbits in the control group was continuous and smooth. There was no obvious infiltration of cartilage, inflammatory cells, infiltration of synovial tissue inflammatory cells, or thickening of the synovial membrane. In the model group, the articular surface of the posterior knee cartilage was not smooth, the cartilage became thin, the synovial membrane was infiltrated by a large number of inflammatory cells and the synovial membrane was hyperplasia and edema. Compared with the model group, the infiltration of inflammatory cells in MSC group was obviously reduced, indicating that MSC treatment of OA could improve the inflammation of articular cartilage and have a protective effect on articular cartilage. Four weeks after intra-articular injection with siRNA-MSC, the articular surface of the posterior knee cartilage was not smooth, the articular cartilage was fractured, the synovial membrane was infiltrated by a large number of inflammatory cells, and the synovial membrane was hyperplastic and edema, and the inflammatory infiltration was worse than that of the model group, suggesting that siRNA-MSC had no protective effect on the joint, and the inflammatory injury was aggravated. 3.6 Immunohistochemistry outcomes Immunohistochemical results of expression of P21, P53, proteoglycan and COL-2 in articular cartilage of experimental rabbit joints are shown in Fig5. Semiquantitative results showed that proteoglycan expression was 3+, col-2 expression was 2+, P21 expression was +, P53 expression was + in the control group. Additionally, in the model group, proteoglycan expression was +, col-2 expression was 2+, P21 expression was 3+ and P53 expression was 3+. Moreover, in MSC group, proteoglycan expression was 2+, col-2 expression was 2+, P21 expression was + and P53 expression was +. However, in the siRNA group, proteoglycan expression was 2+, col-2 expression was 2+, P21 expression was 3+ and P53 expression was 2 + (Tab.3). 4. Discussion In this study, we confirmed that intra-articular injection with MSC could improve OA and the mechanism could be anti-chondrocyte senescence by secreting SOD. To our knowledge, several papers have demonstrated the therapeutic effects of MSC in either rabbit, beagles or sheep. However, the mechanism is still under debate. The ability of MSC to differentiate along a mesodermal lineage, such as chondrocytes, has suggested its intrinsic potential for tissue repair and regeneration [ 12 – 14 ]. However, it is likely that MSC manipulate the OA local environment via paracrine signaling, rather than direct differentiation, which leads to the disease modification [ 15 ]. MSC transferred by siRNA SOD was applied to the model rabbit to verify the mechanism of MSC through SOD secretion. Furthermore, the therapy effect was also contrasted between two different treatment courses. Thus, we believe that our study was valuable for assessing the effect and mechanism of MSC as an OA treatment strategy. Due to large and thick joints for cartilage, there are rabbit, beagle, and sheep suitable for cartilage repair experiments. Rabbits offer advantages as they are cost effective, easy to handle, and to house. Rabbits have been widely applied to in vivo OA research in previous studies [ 16 ]. Papain is a proteolytic enzyme which could induce OA through releasing chondroitin sulfate from a protein-polysaccharide complex of the articular cartilage matrix and producing inflammatory cytokines (such as TNF-α and IL-1β) [ 17 ]. Along with inflammatory cytokine secretion, MMPs levels and free radical products increase, which could promote the pathology of OA [ 18 ]. Therefore, rabbits were selected as our model animals in this study and induced OA via papain injection. It is common sense that defect articular cartilage in OA limits self-healing capability. Originally, destructive processes limited the supply of nutrients and oxygen. Then, it will reduce the synthesis of extracellular matrix (ECM) components and increase the synthesis of tissue-destructive proteinases. Ultimately, there will be general apoptosis of chondrocytes and synovial inflammation [ 19 ]. In addition, apoptosis chondrocytes have little self-repairing abilities, resulting in further progressive degradation of articular cartilage, which is a major challenge for OA therapy [ 20 ]. As a result, OA therapy should aim to repair cartilage that recapitulates the native properties of healthy cartilage. MSC might be a potential cell-based therapies in OA for its excellent tissue repair ability [ 21 ]. According to our study, intra-articular injection of MSC could reduce the secretion of cytokine factors associated with OA, even after 4 weeks. The MRI and HE results showed that MSC therapeutic effect might due to chondrocyte regeneration. Recent studies had concluded that intra-articular MSC injection was effective in OA through inhibiting cartilage degradation [ 22 – 24 ]. Furthermore, Gupta et al. [ 25 ] indicated that MSC were a potential treatment strategy of OA and contributed cartilage regeneration via 3 distinct pathways: differentiating to chondrocytes, suppressing inflammatory and secreting trophic factors that modulate ECM synthesis. The injection of MSC weekly was found to provide a more significant chondroprotective effects compared to a single injection [ 26 ]. In our study, the amelioration effect of MSC was relatively decreased at 4 weeks in both anti-inflammation and anti-cartilage degradation. Our findings were consistent with this hypothesis. In previous studies, it was demonstrated that oxidative stress and reactive oxygen species (ROS) was critical in pathogenesis of OA through regulating chondrocyte apoptosis and senescence, MMPs production and extracellular matrix degradation [ 7 ]. Among multiple types of endogenous and exogenous antioxidants, SOD is a major catalytic antioxidant in articular cartilage [ 27 ]. However, both preclinical and clinical studies confirmed that direct use of free SOD only provided a slight protective effect against oxidative damage due to its inadequate retention and rapid inactivation at the site of the disease [ 28 ]. Gui T’s work demonstrated the therapeutic potential of SOD-loaded porous polymersome nanoparticles (SOD-NP) and inferred that targeting SOD could be a great promise for OA therapy [ 29 ]. In addition, MSC secreted SOD would be a cellular based antioxidant therapy in OA [ 30 ]. In our study, SOD-siRNA MSC was prepared and injected to another group of rabbits. The results showed that SOD inhibition in MSC would decrease the therapeutic effect of MSC in cytokines and tissue structure. Furthermore, the p53/p21 protein was determined through immunohistochemistry assay. In addition to tumor suppressant function, p53 is also involved in a serious number of pathologies associated with ageing [ 31 ]. P53/P21 signaling is associated with pathogenesis of OA. And p53 expression is obviously increased in OA patients than in healthy volunteers [ 32 ]. Loss of Sirt1 in cartilage led to accelerate OA pathogenesis through aberrant activation of p53/p21, which mediated senescence associated secretory phenotype, hypertrophy and apoptosis [ 33 ]. The results in current study showed that the expression of p53 and p21 in articular cartilage were decreased by MSC treatment, which was reversed by SOD-siRNA MSC. Watanabe K et al also verified that SOD loss decreased the mitochondrial membrane potential of chondrocytes and led to p53 activation, which contributed to growth arrest and apoptosis of chondrocyte [ 34 ]. Therefore, we have reasons to believe that MSC might be a promising therapy in OA through anti-apoptosis and promoting regeneration in chondrocyte by secreting SOD and improving oxidative stress. However, there was still some residue improvement in MMP2 and TNF-α levels even if SOD expression is blocked in MSC either in 2 weeks or 4 weeks. We assumed that MSC can still secrete anti-inflammatory factors in addition to SOD silencing. Conclusion In conclusion, our study demonstrated that intra-articular MSC injection inhibited the progression of OA through cartilage regeneration. Furthermore, we first discussed that the mechanism might be that SOD secreted by MSC played an important role in improving oxidative stress and anti-apoptosis chondrocyte. To adequately evaluate the effectiveness and mechanism of MSC therapy in preclinical studies, a reliable animal model with appropriate doses and injection frequency should be considered in future. Abbreviations Full name abbreviation extracellular matrix ECM magnetic resonance imaging MRI matrix metalloproteinase MMP mesenchymal stem cells MSC non-steroidal anti-inflammatory drugs NSAIDs osteoarthritis OA reactive oxygen species ROS superoxide dismutase SOD Declarations Ethics approval and consent to participate This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Drum Tower Hospital affiliated with the Nanjing University School of Medicine. Consent for publication Not applicable. Availability of data and materials The datasets used during the current study are available from the corresponding author on reasonable request Conflicts of Interest The authors declare that they have no conflicts of interest. Fundings: This work was supported by Nanjing Health Bureau Medical Science and technology development project (No. ZKX17020 and No. YKK15067). Authors’ Contributions Congzhu Ding was responsible for the overall research work arrangement. 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Gao X, Ma YQ, Zhang GJ, Tang FY, Zhang JJ, Cao JC, Liu CH. Targeted elimination of intracellular reactive oxygen species using nanoparticle-like chitosan- superoxide dismutase conjugate for treatment of monoiodoacetateinduced osteoarthritis. Int J Pharm. 2020;590:119947. Gui T, Luo L, Chhay B, Zhong LL, Wei YL, Yao LT, Yu W, Li J, Nelson CL, Tsourkas A, Qin L, Cheng ZL. Superoxide dismutase-loaded porous polymersomes as highly efficient antioxidant nanoparticles targeting synovium for osteoarthritis therapy. Biomaterials. 2022;283:121437. Nightingale H, Kemp K, Gray E, Hares K, Mallam E, Scolding N, Wilkins A. Changes in expression of the antioxidant enzyme SOD3 occur upon differentiation of human bone marrow-derived mesenchymal stem cells in vitro. STEM CELLS DEV. 2012;21(11):2026–35. Vigneron A, Vousden KH. P53, ROS and senescence in the control of aging. Aging. 2010;2(8):471–4. Zhu XB, Yang SW, Lin WJ, Wang L, Ying JW, Ding YW, Chen X. Roles of cell cyle regulators cyclin D1, CDK4, and p53 in knee osteoarthritis. Genet Test Mol Biomarkers. 2016;20(9):529–34. Xu M, Feng M, Peng H, Qian Z, Zhao LT, Wu SF. Epigenetic regulation of chondrocyte hypertrophy and apoptosis through Sirt1/P53/P21 pathway in surgery-induced osteoarthritis. BIOCHEM BIOPH RES CO. 2020;528(1):179–85. Watanabe K, Shibuya S, Koyama H, Ozawa Y, Toda T, Yokote K, Shimizu T. Sod1 loss induces intrinsic superoxide accumulation leading to p53-mediated growth arrest and apoptosis. Int J Mol Sci. 2013;14(6):10998–1010. Tables Tab.1 The relative expression level of SOD1 after MSC transfection Groups SOD1 relative expression MSC 1.002±0.083 MSC-NC 0.963±0.023 MSC-siRNA-1 0.025±0.001 ** MSC-siRNA-2 0.038±0.001 ** MSC-siRNA-3 0.022±0.002 ** ** P < 0.01, significant difference compared with MSC-NC. Each experiment was repeated three times. Tab.2 Cell viability of MSC and siRNA-MSC after transferring for 24h Groups Cell viability(%) MSC 97±0.4 MSC-NC 96±1.0 MSC-siRNA 96±0.7 Cell viability was calculated through normal cell counts/total cell counts. Each experiment was repeated three times. Tab.3 Intensity scores of Intensity scores of 4 proteins in articular cartilage Proetins intensity scores control model MSC siRNA-MSC proteoglycan 3+ 1+ 2+ 2+ Col-2 2+ 2+ 2+ 2+ P21 1+ 3+ 1+ 3+ P53 1+ 3+ 1+ 2+ Protein expression scores of proteoglycans, Col-2, P21 and P53 in control, model, MSC and siRNA-MSC groups were calculated through method described above in 2.7. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Apr, 2025 Read the published version in BMC Musculoskeletal Disorders → Version 1 posted Editorial decision: Revision requested 28 Aug, 2024 Reviews received at journal 27 Aug, 2024 Reviews received at journal 24 Aug, 2024 Reviews received at journal 19 Aug, 2024 Reviewers agreed at journal 19 Aug, 2024 Reviewers agreed at journal 17 Aug, 2024 Reviewers agreed at journal 15 Aug, 2024 Reviewers agreed at journal 14 Aug, 2024 Reviews received at journal 13 Aug, 2024 Reviewers agreed at journal 11 Aug, 2024 Reviewers agreed at journal 08 Aug, 2024 Reviewers invited by journal 08 Aug, 2024 Editor assigned by journal 02 Jul, 2024 Editor invited by journal 14 Apr, 2024 Submission checks completed at journal 14 Apr, 2024 First submitted to journal 08 Apr, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4235031","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":291144887,"identity":"f1083412-1a9c-4854-959b-ddff9cb5f1a4","order_by":0,"name":"Yao Yao","email":"","orcid":"","institution":"The Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Yao","middleName":"","lastName":"Yao","suffix":""},{"id":291144888,"identity":"495109e8-08d6-48a8-8aa5-207c185c85a4","order_by":1,"name":"Juan Cao","email":"","orcid":"","institution":"The Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"","lastName":"Cao","suffix":""},{"id":291144889,"identity":"da75bbcc-5c36-4d78-88d3-8c7a721c05bf","order_by":2,"name":"Congzhu Ding","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1klEQVRIiWNgGAWjYDCCwzCGBAPjg4SKGtK0MBs8OHOMCC0HEFrYJB+2MBPWwXec9/CLHxV37ObP7jGrSGxgY+Bv707Aq0XyMF+aZc+ZZ8mNc86Y3UjcIcMgcebsBrxaDA7zmBkzth1OZpbIAWo5w8ZgIJFLpBY2oJaCxDZmorQYPwZqseMBamEgSosk0BbGnjOHEyQk0oolEs4c4yHoF77zZ4w//Kg4bC8/I3njxx8VNXL87b34tQABmwSQSGyA8ngIKQcB5g9Awp4YlaNgFIyCUTBCAQD3YElQKJxkEAAAAABJRU5ErkJggg==","orcid":"","institution":"The Affiliated Drum Tower Hospital of Nanjing University Medical School","correspondingAuthor":true,"prefix":"","firstName":"Congzhu","middleName":"","lastName":"Ding","suffix":""}],"badges":[],"createdAt":"2024-04-08 08:27:38","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4235031/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4235031/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12891-025-08670-4","type":"published","date":"2025-04-24T15:57:14+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":55061786,"identity":"9ed8d48e-9763-4433-97ec-b82f5ebf232e","added_by":"auto","created_at":"2024-04-22 02:51:37","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":56104,"visible":true,"origin":"","legend":"\u003cp\u003eIdentification of MSC through flow cytometry\u003c/p\u003e\n\u003cp\u003eA and C present homo-type control cells. B and D present MSC. The cells were identified CD29 and CD90 positive expression, which Consistent with the performance of MSC.\u003c/p\u003e","description":"","filename":"FIG.1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4235031/v1/01e8d2988dd51ed75784159c.jpg"},{"id":55061790,"identity":"09cd18d7-0b9a-4e28-a600-876ad0523393","added_by":"auto","created_at":"2024-04-22 02:51:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":500827,"visible":true,"origin":"","legend":"\u003cp\u003eInflammatory index results of each group after 2w and 4w treatment\u003c/p\u003e\n\u003cp\u003eDifferent bars represent control, model, MSC and siRNA-MSC for 2weeks (A) and 4weeks (B). The horizontal line shows the comparison between the two groups. * P\u0026lt;0.05, significant difference between groups.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4235031/v1/1aee33830bc2eed77994f2ac.png"},{"id":55061789,"identity":"5a2f6897-ef54-44c1-8e93-06537501c0dd","added_by":"auto","created_at":"2024-04-22 02:51:37","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":180970,"visible":true,"origin":"","legend":"\u003cp\u003eMagnetic resonance of knee joint of rabbits in each group after 4w treatment\u003c/p\u003e\n\u003cp\u003eA represent control group, B represent OA group, C represent MSC group. D represent siRNA-MSC group. 1 and 2 represent sagittal and coronal magnetic resonance images in each group. The arrows pointed to the fluid in the joint cavity in each group.\u003c/p\u003e","description":"","filename":"FIG.3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4235031/v1/6f3f1f80abfd99804b0b0b54.jpg"},{"id":55062793,"identity":"ed5218f4-1f9b-4585-b85b-e61544a1b753","added_by":"auto","created_at":"2024-04-22 02:59:37","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":344035,"visible":true,"origin":"","legend":"\u003cp\u003eHistological findings of each group\u003c/p\u003e\n\u003cp\u003eA: synovial membrane of control group; B: Cartilage of control group; C: synovial membrane in the model group; D: Cartilage of Model group; E: synovial membrane in MSC group; F: Cartilage of MSC group; G: synovial membrane of siRNA-MSC group; H: Cartilage of siRNA-MSC group. (HE staining ×100) . The arrows in the left column pointed to lymphocyte, while the arrows in the right column pointed to the cartilage surface.\u003c/p\u003e","description":"","filename":"FIG.4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4235031/v1/161630189e7fe11bbb416f87.jpg"},{"id":55061787,"identity":"10954aeb-9be2-4016-9e3a-844d6bde452c","added_by":"auto","created_at":"2024-04-22 02:51:37","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":278098,"visible":true,"origin":"","legend":"\u003cp\u003eImmunohistochemical results of each group\u003c/p\u003e\n\u003cp\u003eA represent control group, B represent OA group, C represent MSC group. D represent siRNA-MSC group. 1 to 4 represent proteoglycan, COL-2, P21 and P53 in each group, respectively (×200).\u003c/p\u003e","description":"","filename":"FIG.5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4235031/v1/6ecb22cc95f30790dc5c057f.jpg"},{"id":81569562,"identity":"2a47a535-643e-4334-828f-6e5b96290cbc","added_by":"auto","created_at":"2025-04-28 16:07:18","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2141913,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4235031/v1/e0f40dcf-17f4-4a58-a6b9-685e0c141fc4.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Mesenchymal stem cells improve osteoarthritis by secreting superoxide dismutase to regulate oxidative stress response","fulltext":[{"header":"Key Points","content":"\u003cp\u003e1.\u0026nbsp;It is the first time to be reported that MSC may be a promising therapy in OA.\u003c/p\u003e\n\u003cp\u003e2. MSC might play a pivotal role in the treatment of OA through anti-apoptosis and regeneration in chondrocyte by secreting SOD and improving oxidative stress.\u0026nbsp;\u003c/p\u003e"},{"header":"1. Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a highly prevalent degenerative condition of the joints, most commonly the knee and hip, which affects more than 300\u0026nbsp;million people in the world [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Patients with OA suffer from mobility problems and chronic pain, resulting in a lifelong disability and reduced quality of life quality [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. In addition, chronic OA management imposes a heavy burden on the family and medical resources. The pathogenesis of OA is complex and is not fully understood, but it is characterized by skeletal inflammation and cartilage degeneration and subchondrical bone sclerosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. In addition to physical therapy and weight loss program, paracetamol or non-steroidal anti-inflammatory drugs (NSAIDs), and intraarticular injections of corticosteroids or hyaluronic acid may be the main current non-surgical treatments for OA [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Patients with no response to the treatment mentioned above, would consider other pain relief treatment such as tramadol or amitriptyline. The final option for end-stage knee OA is joint replacement. However, this surgery is not suitable for all patients for the risk of complications [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, all these treatments have limited effects on the healing potential of damaged cartilage.\u003c/p\u003e \u003cp\u003eAlthough several types of cells are involved in pathogeny of OA, chondrocytes are primarily considered to play an important role in OA induction by cellular senescence [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Normal chondrocytes usually show moderate metabolic and proliferative activity under normal conditions. However, some senescence chondrocytes lose their differentiated phenotype under inflammation conditions. Recent studies have identified that OA pathology is obviously related to oxidative stress and reactive oxygen species (ROS), which would regulate chondrocyte apoptosis and senescence, matrix metalloproteinase (MMP) production, and extracellular matrix synthesis and degradation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMSC play an important role in repairing cartilage by tissue regeneration, producing factors that promote resident chondrocytes in the joint to form articular cartilage, and inhibiting inflammation [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. A summary paper reviewed MSC therapy for OA in preclinical and clinical studies in the last 5 years, which certified the benefits and safety of intra-articular injections of MSC. Nevertheless, the effects and mechanisms of intra-articular injections of MSC in OA are not well defined due to the low-quality evidence. Furthermore, in vivo MSC differentiation is relatively rare, indicating that MSC differentiation in chondrocytes could not explain the therapeutic effect [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Klein D et al. verified that the antioxidant enzyme superoxide dismutase 1 (SOD1) could be an MSC secreted factor, which could mediate ROS [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe aim of our study was to observe whether intra-articular injection of MSC could change knee joint morphology using magnetic resonance imaging (MRI). Furthermore, inflammation determination, histopathological examination and immunohistochemical analysis were also carried out to establish cartilage injury and senescence improvement of intra-articular MSC injection. In addition, SOD gene was silenced to suppose whether MSC play a pivotal role in OA treatment through oxidative stress pathway.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Culture and Characterization of UCB-MSC\u003c/h2\u003e \u003cp\u003ehUCB-MSC (passage 4\u0026ndash;6, Jiangsu Keygen Biotech Corp, China) were cultured in RPMI-1640 medium (Gibco, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS, Gibco, Gaithersburg, MD, USA) at 37\u0026deg;C with 5% CO2. Characterization of hUCB-MSC were performed according to published procedures through a flow cytometer [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 SOD siRNA construction and transfection\u003c/h2\u003e \u003cp\u003eThe siRNA primer sequence of SOD1 was designed following GENE BANK. There were three sequences listed as below.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGene\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSequence\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD1siRNA-1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eforward 5\u0026rsquo;-GGG CAA AGG UGG AAA UGA ATT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse 5\u0026rsquo;-UUC AUU UCC ACC UUU GCC CTT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD1 siRNA-2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-GUU CAU GAG UUU GGA GAU ATT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse 5\u0026rsquo;-UAU CUC CAA ACU CAU GAA CTT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSOD1 siRNA-3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eForward 5\u0026rsquo;-CAA UGU GAC UGC UGA CAA ATT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eReverse 5\u0026rsquo;-UUU GUC AGC AGU CAC AUU GTT-3\u0026rsquo;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOne day before transfection, an appropriate number of MSC cells were inoculated into 6-well cell culture plates, so that the cell density at transfection could reach 80\u0026ndash;90%. siRNA was diluted to 20uM with 125\u0026micro;L serum-free Opti-MEM medium (Gibco, Gaithersburg, MD, USA), mixed gently, and incubated at room temperature for 5 min. Five \u0026micro;L lipo3000 was also diluted with 125\u0026micro;L serum-free Opti-MEM medium, mixed gently, and incubated at room temperature for 5 min. Then siRNA and lipo3000 (Invitrogen, thermos, USA) were gently mixed and incubated for 20 min at room temperature. The mixture was added to the cell in 750\u0026micro;L complete medium and cultured in CO\u003csub\u003e2\u003c/sub\u003e incubator at 37℃ for 48h. qPCR was used for identification the best silencing sequence. And the follow-up experiment was conducted with the best silencing SOD-siRNA-MSC. In addition, cell viability of MSC and siRNA-MSC was both verified through Trypan blue (Jiangsu Keygen Biotech Corp, China) dye exclusion test after transfection for 24h.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental Animals and groups\u003c/h2\u003e \u003cp\u003e This experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Drum Tower Hospital affiliated with the Nanjing University School of Medicine and was in accordance with ARRIVE guidelines. Twenty healthy adult New Zealand white rabbits with body weight between 2.5 and 3.0 kg (provided by the Experimental Animal Centre of Drum Tower Hospital affiliated to Nanjing University School of Medicine) were selected and divided into 4 groups (n\u0026thinsp;=\u0026thinsp;5), control group, model group, MSC group and SOD-siRNA-MSC group. The environment of the animal laboratory was controlled at a temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C, a relative humidity of 55\u0026thinsp;\u0026plusmn;\u0026thinsp;10%, a ventilation frequency of 12 times/h, and a lighting cycle of 12 h. And a normal diet and sterile distilled water was provided without restriction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Establishment of osteoarthritis model and experimental procedures\u003c/h2\u003e \u003cp\u003eFour percent papain injection (Sigma, USA) 0.5mL was locally articular injected into the posterior knee cavity of the model group, MSC group and siRNA-MSC group on day 1, 4 and 7 for three times in total. The control group was injected with normal saline. Two weeks later, the experimental joints were observed by magnetic resonance imaging (MRI) to verify whether the OA model had been established successfully. And serum was collected through the marginal ear vein for determination of cytokines. Subsequently, the MSC group was injected with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/mL 0.5mL MSC, and the siRNA-MSC group was injected with 1\u0026times;10\u003csup\u003e6\u003c/sup\u003e/mL 0.5mL siRNA-MSC, while the model group and control group were injected with 0.5ml normal saline.\u003c/p\u003e \u003cp\u003eAfter 4 weeks of treatment, the morphology of the joints was observed by MRI. After observation, the experimental animals were sacrificed and serum, synovial membrane and articular cartilage were collected for further study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 MRI evaluation\u003c/h2\u003e \u003cp\u003eMRI was performed 4 weeks after treatment. In this study, the Philips 3.0T magnetic resonance imaging system (Ingenia, Philips, The Netherlands) was used. After anesthesia through propofol (5mg/kg) injection,, rabbits were fixed in the supine position on the experimental plate. Coronal and sagittal scans were performed with 8-channel orthogonal magnetic resonance coils of the head to obtain PDW-SPAIR sequence images. The following sequences were utilized: TR3000 MS, TE30 MS, turn angle 90\u0026deg;, field of vision 10 cm, layer thickness 1.5 mm, acquisition time 2 minutes 30 seconds.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Detection of cytokines in serum\u003c/h2\u003e \u003cp\u003eCytokine determination was performed 2 and 4 weeks after treatment. Rabbit serum was collected through marginal ear vein. Serum levels of IL-1β, L-6, TNFα, MMP2, 13 and COMP were determined by enzyme-linked immunosorbent assay (ELISA lab, Hubei, China) according to each instruction, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Histopathologic examination and immunohistochemistry\u003c/h2\u003e \u003cp\u003eRabbits were sacrificed after MRI evaluation at week 4. The supracondylar portion of the femur was collected, including the arthritis-induced site. The joint tissue specimens were fixed with 10% formalin solution, embedded in paraffin, sectioned into 4\u0026micro;m sections and stained with HE. The articular cartilage was fixed in 10% formalin solution, decalcified with EDTA, rinsed with water, and then treated with conventional embedding, sectioning and HE staining. All tissues were observed under a light microscope. Expression of P21, P53, COL-2 and proteoglycan in articular cartilage tissues was detected by Streptomyces antibiotin protein-peroxidase immunohistochemical method. The percentage of microscopically positive cells and staining intensity were evaluated by semiquantitative interpretation. Percentages of positive staining cell scores were calculated as follows: 0 for positive cells\u0026thinsp;\u0026lt;\u0026thinsp;5%, 1 for 5%-25%, 2 for 26%-50%, 3 for 51%-75%, and 4 for 76%-100%. Positive stain intensity scores were calculated as follows: 0 for colorless, 1 for light yellow, and 2 for brownish yellow, 3 for tan. The multiplication of 2 scores can be considered a positive rating: 0 for negative (-), 1\u0026ndash;4 for weakly positive (+), 5\u0026ndash;8 for positive (+ +), 9\u0026ndash;12 for strong positive (+ + +).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Statistical analysis\u003c/h2\u003e \u003cp\u003eMultiple comparison tests were conducted to compare different groups. For parametric multiple comparisons, one-way analysis of variance (ANOVA) was used to analyze the data. The T test was used to compare the mean data between the two groups. SPSS 22.0 statistical software was used for analysis.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMSC\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eidentification\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFlow cytometry\u0026nbsp;was used to monitor the proportion of CD29 and CD90 expression cells. It was indicated that the number of CD29 positive expression cells was 99.76%, and the number of CD90 positive expression cells was 94.87%, both of which were more than 90%. The number of CD29 positive cells was 0.13% and CD90 positive cells was 0.28% in the homotype control group (Fig.1)\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 SOD1\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;SiRNA Sequence Design and the best choice\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe siRNA primer sequence of SOD1 was designed by GENE BANK, and the synthesized primer sequence was transfected into MSC by liposome. qPCR was used to verify whether the sequence could block SOD1 expression in MSC. The results suggested that the expression of SOD1 in MSC was significantly decreased, either transfected with sequence 1, 2 or 3 (P \u0026lt; 0.05). MSC transfected with sequence 3 had a stronger inhibition effect on SOD1 expression than those transfected with sequence 1 or 2, but there was no statistical difference (P \u0026gt; 0.05) (Tab.1). Sequence 3 was applied as the SOD-siRNA sequence in subsequent experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn addition, the cell viability assay indicated that more than 95% cell survivor after transfection (Tab.2), which was suitable for intraarticular injection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Cytokines outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe results of serum cytokines and cartilage damage markers are shown in Fig.2. \u0026nbsp;COMP, IL-1\u0026beta;, IL-6, TNF-\u0026alpha;, MMP-2 and MMP-13 levels in model group were significantly higher than those in control group (P \u0026lt;0.05), which indicated OA model was established successfully. Two weeks after intra-articular injection, the levels of COMP, IL-1\u0026beta;, IL-6, TNF-\u0026alpha;, MMP-2 and MMP-13 in MSC group were significantly decreased compared with those in model group (P \u0026le;0.05). Moreover, the levels of COMP, TNF-\u0026alpha;, MMP-2 and MMP-13 in the MSC group were still lower than those in model group at 4 weeks after intra-articular injection (P \u0026le;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTwo weeks after intra-articular injection, IL-1\u0026beta;, MMP-2, MMP-13 and TNF-\u0026alpha; in the SOD- siRNA-MSC group was decreased compared with model group (P\u0026lt;0.05), while COMP, and IL-6 did not significantly change (P\u0026gt;0.05). Moreover, IL-1\u0026beta;, IL-6 and MMP2 in SOD-siRNA-MSC group were elevated compared with MSC group (P\u0026lt;0.05). Four weeks after intraarticular injection, IL-1\u0026beta;, IL-6 and MMP-13 in the siRNA-MSC group did not significantly decrease compared to the model group (P\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 MRI outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll rabbits were performed MRI (Fig. 3). Images showed that the cartilage surface of the femoral condyle joint was smooth, no abnormal signal was found in the subchondral bone, the synovial signal was normal, and a small amount of synovial fluid was found in the joint cavity on T2WI fat saturation sequence in control group. However, rough cartilage surface of femoral condylar article, poor meniscus polish, slightly increased signal of anterior cruciate ligament, slight thickening of synovium, slightly increased signal of subchondral bone, and moderate effusion in the joint cavity were showed in model group. The MRI images on T2WI fat saturation sequence of the joint after 4w treatment in the siRNA-MSC group showed that the cartilage surface of the femoral condyle joint was rough, the meniscus was not polished, the anterior cruciate ligament signal was increased, the synovium was slightly thickened, the subchondral bone signal was slightly increased, and there was fluid in the joint cavity, which did not change much compared with the model group. In MSC group, MRI findings showed that the cartilage signal of femoral condyle joint and tibial plateau was uneven, the meniscus was normal and intact, the anterior cruciate ligament was normal, the synovial signal was slightly increased, which was improved compared with the model group. In addition, the subchondral bone signal was normal, and the joint effusion was significantly reduced compared with the model group.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Histopathologic outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHE staining was performed on articular cartilage and synovial membrane of rabbits in all experimental groups (Fig.4). The results suggested that the posterior articular cartilage of rabbits in the control group was continuous and smooth. There was no obvious infiltration of cartilage, inflammatory cells, infiltration of synovial tissue inflammatory cells, or thickening of the synovial membrane. In the model group, the articular surface of the posterior knee cartilage was not smooth, the cartilage became thin, the synovial membrane was infiltrated by a large number of inflammatory cells and the synovial membrane was hyperplasia and edema. Compared with the model group, the infiltration of inflammatory cells in MSC group was obviously reduced, indicating that MSC treatment of OA could improve the inflammation of articular cartilage and have a protective effect on articular cartilage. Four weeks after intra-articular injection with siRNA-MSC, the articular surface of the posterior knee cartilage was not smooth, the articular cartilage was fractured, the synovial membrane was infiltrated by a large number of inflammatory cells, and the synovial membrane was hyperplastic and edema, and the inflammatory infiltration was worse than that of the model group, suggesting that siRNA-MSC had no protective effect on the joint, and the inflammatory injury was aggravated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.6 Immunohistochemistry outcomes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eImmunohistochemical results of expression of P21, P53, proteoglycan and COL-2 in articular cartilage of experimental rabbit joints are shown in Fig5. Semiquantitative results showed that proteoglycan expression was 3+, col-2 expression was 2+, P21 expression was +, P53 expression was + in the control group. Additionally, in the model group, proteoglycan expression was +, col-2 expression was 2+, P21 expression was 3+ and P53 expression was 3+. Moreover, in MSC group, proteoglycan expression was 2+, col-2 expression was 2+, P21 expression was + and P53 expression was +. However, in the siRNA group, proteoglycan expression was 2+, col-2 expression was 2+, P21 expression was 3+ and P53 expression was 2 + (Tab.3).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn this study, we confirmed that intra-articular injection with MSC could improve OA and the mechanism could be anti-chondrocyte senescence by secreting SOD. To our knowledge, several papers have demonstrated the therapeutic effects of MSC in either rabbit, beagles or sheep. However, the mechanism is still under debate. The ability of MSC to differentiate along a mesodermal lineage, such as chondrocytes, has suggested its intrinsic potential for tissue repair and regeneration [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e–\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, it is likely that MSC manipulate the OA local environment via paracrine signaling, rather than direct differentiation, which leads to the disease modification [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. MSC transferred by siRNA SOD was applied to the model rabbit to verify the mechanism of MSC through SOD secretion. Furthermore, the therapy effect was also contrasted between two different treatment courses. Thus, we believe that our study was valuable for assessing the effect and mechanism of MSC as an OA treatment strategy.\u003c/p\u003e \u003cp\u003eDue to large and thick joints for cartilage, there are rabbit, beagle, and sheep suitable for cartilage repair experiments. Rabbits offer advantages as they are cost effective, easy to handle, and to house. Rabbits have been widely applied to in vivo OA research in previous studies [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Papain is a proteolytic enzyme which could induce OA through releasing chondroitin sulfate from a protein-polysaccharide complex of the articular cartilage matrix and producing inflammatory cytokines (such as TNF-α and IL-1β) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Along with inflammatory cytokine secretion, MMPs levels and free radical products increase, which could promote the pathology of OA [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. Therefore, rabbits were selected as our model animals in this study and induced OA via papain injection.\u003c/p\u003e \u003cp\u003eIt is common sense that defect articular cartilage in OA limits self-healing capability. Originally, destructive processes limited the supply of nutrients and oxygen. Then, it will reduce the synthesis of extracellular matrix (ECM) components and increase the synthesis of tissue-destructive proteinases. Ultimately, there will be general apoptosis of chondrocytes and synovial inflammation [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. In addition, apoptosis chondrocytes have little self-repairing abilities, resulting in further progressive degradation of articular cartilage, which is a major challenge for OA therapy [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. As a result, OA therapy should aim to repair cartilage that recapitulates the native properties of healthy cartilage. MSC might be a potential cell-based therapies in OA for its excellent tissue repair ability [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAccording to our study, intra-articular injection of MSC could reduce the secretion of cytokine factors associated with OA, even after 4 weeks. The MRI and HE results showed that MSC therapeutic effect might due to chondrocyte regeneration. Recent studies had concluded that intra-articular MSC injection was effective in OA through inhibiting cartilage degradation [\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e–\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Furthermore, Gupta et al. [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] indicated that MSC were a potential treatment strategy of OA and contributed cartilage regeneration via 3 distinct pathways: differentiating to chondrocytes, suppressing inflammatory and secreting trophic factors that modulate ECM synthesis. The injection of MSC weekly was found to provide a more significant chondroprotective effects compared to a single injection [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. In our study, the amelioration effect of MSC was relatively decreased at 4 weeks in both anti-inflammation and anti-cartilage degradation. Our findings were consistent with this hypothesis.\u003c/p\u003e \u003cp\u003eIn previous studies, it was demonstrated that oxidative stress and reactive oxygen species (ROS) was critical in pathogenesis of OA through regulating chondrocyte apoptosis and senescence, MMPs production and extracellular matrix degradation [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Among multiple types of endogenous and exogenous antioxidants, SOD is a major catalytic antioxidant in articular cartilage [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, both preclinical and clinical studies confirmed that direct use of free SOD only provided a slight protective effect against oxidative damage due to its inadequate retention and rapid inactivation at the site of the disease [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Gui T’s work demonstrated the therapeutic potential of SOD-loaded porous polymersome nanoparticles (SOD-NP) and inferred that targeting SOD could be a great promise for OA therapy [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In addition, MSC secreted SOD would be a cellular based antioxidant therapy in OA [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. In our study, SOD-siRNA MSC was prepared and injected to another group of rabbits. The results showed that SOD inhibition in MSC would decrease the therapeutic effect of MSC in cytokines and tissue structure. Furthermore, the p53/p21 protein was determined through immunohistochemistry assay. In addition to tumor suppressant function, p53 is also involved in a serious number of pathologies associated with ageing [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. P53/P21 signaling is associated with pathogenesis of OA. And p53 expression is obviously increased in OA patients than in healthy volunteers [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Loss of Sirt1 in cartilage led to accelerate OA pathogenesis through aberrant activation of p53/p21, which mediated senescence associated secretory phenotype, hypertrophy and apoptosis [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The results in current study showed that the expression of p53 and p21 in articular cartilage were decreased by MSC treatment, which was reversed by SOD-siRNA MSC. Watanabe K et al also verified that SOD loss decreased the mitochondrial membrane potential of chondrocytes and led to p53 activation, which contributed to growth arrest and apoptosis of chondrocyte [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, we have reasons to believe that MSC might be a promising therapy in OA through anti-apoptosis and promoting regeneration in chondrocyte by secreting SOD and improving oxidative stress. However, there was still some residue improvement in MMP2 and TNF-α levels even if SOD expression is blocked in MSC either in 2 weeks or 4 weeks. We assumed that MSC can still secrete anti-inflammatory factors in addition to SOD silencing.\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, our study demonstrated that intra-articular MSC injection inhibited the progression of OA through cartilage regeneration. Furthermore, we first discussed that the mechanism might be that SOD secreted by MSC played an important role in improving oxidative stress and anti-apoptosis chondrocyte. To adequately evaluate the effectiveness and mechanism of MSC therapy in preclinical studies, a reliable animal model with appropriate doses and injection frequency should be considered in future.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFull name\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eabbreviation\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003eextracellular matrix\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003eECM\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003emagnetic resonance imaging\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003eMRI\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003ematrix metalloproteinase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003eMMP\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003emesenchymal stem cells\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003eMSC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003enon-steroidal anti-inflammatory drugs\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003eNSAIDs\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003eosteoarthritis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003eOA\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003ereactive oxygen species\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003eROS\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"50.287907869481764%\" valign=\"top\"\u003e\n \u003cp\u003esuperoxide dismutase\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"49.712092130518236%\" valign=\"top\"\u003e\n \u003cp\u003eSOD\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment was approved by the Institutional Animal Care and Use Committee (IACUC) of Drum Tower Hospital affiliated with the Nanjing University School of Medicine.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used during the current study are available from the corresponding author on reasonable request \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFundings:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by Nanjing Health Bureau Medical Science and technology development project (No. ZKX17020 and No. YKK15067).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eCongzhu Ding was responsible for the overall research work arrangement. Yao Yao was responsible for animal research. Juan Cao was responsible for Statistic work and . Yao Yao wrote the first draft of the manuscript. Yao Yao and Juan Cao revised the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eKloppenburg M, Berenbaum F. Osteoarthritis year in review 2019: epidemiology and therapy. Osteoarthritis Cartilage. 2020;28(3):242\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLohan C, Coates G, Clewes P, Stevenson H, Wood R, Tritton T, Massey L, Knaggs R, Dickson AJ, Walsh D. Estimating the cost and epidemiology of mild to severe chronic pain associated with osteoarthritis in England: a retrospective analysis of linked primary and secondary care data. 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Foot Ankle Clin. 2015;10(4):651\u0026ndash;65.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWright A, Arthaud-Day ML, Weiss ML. Therapeutic Use of Mesenchymal Stromal Cells: The Need for Inclusive Characterization Guidelines to Accommodate All Tissue Sources and Species. Front Cell Dev Biol. 2021;9:632717.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXie RH, Gong SG, Song J, Wu PP, Hu WL. Effect of mesenchymal stromal cells transplantation on the outcomes of patients with knee osteoarthritis: A systematic review and meta-analysis. J ORTHOPAED RES. 2023; 2023: 1\u0026ndash;16.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCaplan A. What are MSC therapeutic? New data: new insight. J Pathol. 2009;217:318\u0026ndash;24.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoran CJ, Ramesh A, Brama PA. The benefits and limitations of animal models for translational research in cartilage repair. J Exp Orthop. 2016;3(1):1.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLampropoulou-Adamidou K, Lelovas P, Karadimas EV, Liakou C, Triantafillopoulos IK, Dontas I, Papaioannou NA. Useful animal models for the research of osteoarthritis. Eur J Orthop Surg Traumatol. 2014;24(3):263\u0026ndash;71.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHorv\u0026aacute;th E, S\u0026oacute;lyom \u0026Aacute;, Sz\u0026eacute;kely J, Nagy EE, Popoviciu H. Inflammatory and Metabolic Signaling Interfaces of the Hypertrophic and Senescent Chondrocyte Phenotypes Associated with Osteoarthritis. Int J Mol Sci. 2023;24(22):16468.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBijlsma JW, Berenbaum F, Lafeber FP. Osteoarthritis: an update with relevance for clinical practice. Lancet. 2011;377(9783):2115\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiekman BO, Guilak F. Stem cell-based therapies for osteoarthritis: challenges and opportunities. Curr Opin Rheumatol. 2013;25(1):119\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHarrell CR, Markovic BS, Fellabaum C, Arsenijevic A, Volarevic V. Mesenchymal stem cell-based therapy of osteoarthritis: current knowledge and future perspectives. Biomed Pharmacother. 2019;109:2318\u0026ndash;26.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSaulnier N, Viguier E, Perrier-Groult E, Chenu C, Pillet E, Roger T, Maddens S, Boulocher C. Intra-articular administration of xenogeneic neonatal Mesenchymal Stromal Cells early after meniscal injury down-regulates metalloproteinase gene expression in synovium and prevents cartilage degradation in a rabbit model of osteoarthritis. Osteoarthritis Cartilage. 2015;23(1):122\u0026ndash;33.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSato M, Uchida K, Nakajima H, Miyazaki T, Guerrero AR, Watanabe S, Roberts S, Baba H. Direct transplantation of mesenchymal stem cells into the knee joints of Hartley strain guinea pigs with spontaneous osteoarthritis. Arthritis Res Ther. 2012;14(1):R31.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLi M, Luo X, Lv X, Liu V, Zhao G, Zhang X, Cao W, Wang R, Wang W. In vivo human adipose-derived mesenchymal stem cell tracking after intra-articular delivery in a rat osteoarthritis model. Stem Cell Res Ther. 2016;7(1):160.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta PK, Das AK, Chullikana A, Majumdar AS. Mesenchymal stem cells for cartilage repair in osteoarthritis. Stem Cell Res Ther. 2012;3(4):25.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOzeki N, Muneta T, Koga H, Nakagawa Y, Mizuno M, Tsuji K, Mabuchi Y, Akazawa C, Kobayashi E, Matsumoto K, Futamura K, Saito T, Sekiya I. Not single but periodic injections of synovial mesenchymal stem cells maintain viable cells in knees and inhibit osteoarthritis progression in rats. Osteoarthritis Cartilage. 2016;24(6):1061\u0026ndash;70.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKoike M, Nojiri H, Kanazawa H, Yamaguchi H, Miyagawa K, Nagura N, Banno S, Iwase Y, Kurosawa H, Kaneko K. Superoxide dismutase activity is significantly lower in end-stage osteoarthritic cartilage than non-osteoarthritic cartilage. PLoS ONE. 2018;13(9):e0203944.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGao X, Ma YQ, Zhang GJ, Tang FY, Zhang JJ, Cao JC, Liu CH. Targeted elimination of intracellular reactive oxygen species using nanoparticle-like chitosan- superoxide dismutase conjugate for treatment of monoiodoacetateinduced osteoarthritis. Int J Pharm. 2020;590:119947.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGui T, Luo L, Chhay B, Zhong LL, Wei YL, Yao LT, Yu W, Li J, Nelson CL, Tsourkas A, Qin L, Cheng ZL. Superoxide dismutase-loaded porous polymersomes as highly efficient antioxidant nanoparticles targeting synovium for osteoarthritis therapy. Biomaterials. 2022;283:121437.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNightingale H, Kemp K, Gray E, Hares K, Mallam E, Scolding N, Wilkins A. Changes in expression of the antioxidant enzyme SOD3 occur upon differentiation of human bone marrow-derived mesenchymal stem cells in vitro. STEM CELLS DEV. 2012;21(11):2026\u0026ndash;35.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVigneron A, Vousden KH. P53, ROS and senescence in the control of aging. Aging. 2010;2(8):471\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu XB, Yang SW, Lin WJ, Wang L, Ying JW, Ding YW, Chen X. Roles of cell cyle regulators cyclin D1, CDK4, and p53 in knee osteoarthritis. Genet Test Mol Biomarkers. 2016;20(9):529\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu M, Feng M, Peng H, Qian Z, Zhao LT, Wu SF. Epigenetic regulation of chondrocyte hypertrophy and apoptosis through Sirt1/P53/P21 pathway in surgery-induced osteoarthritis. BIOCHEM BIOPH RES CO. 2020;528(1):179\u0026ndash;85.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWatanabe K, Shibuya S, Koyama H, Ozawa Y, Toda T, Yokote K, Shimizu T. Sod1 loss induces intrinsic superoxide accumulation leading to p53-mediated growth arrest and apoptosis. Int J Mol Sci. 2013;14(6):10998\u0026ndash;1010.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTab.1 The relative expression level of SOD1 after MSC transfection\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003eSOD1 relative expression\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eMSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003e1.002\u0026plusmn;0.083\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eMSC-NC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003e0.963\u0026plusmn;0.023\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eMSC-siRNA-1\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003e0.025\u0026plusmn;0.001\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eMSC-siRNA-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003e0.038\u0026plusmn;0.001\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eMSC-siRNA-3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003e0.022\u0026plusmn;0.002\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003e** P \u0026lt; 0.01, significant difference compared with MSC-NC. Each experiment was repeated three times.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTab.2\u0026nbsp;Cell viability of MSC and siRNA-MSC after transferring for 24h\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eGroups\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003eCell viability(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eMSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003e97\u0026plusmn;0.4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eMSC-NC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003e96\u0026plusmn;1.0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"47.712418300653596%\" valign=\"top\"\u003e\n \u003cp\u003eMSC-siRNA\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"52.287581699346404%\" valign=\"top\"\u003e\n \u003cp\u003e96\u0026plusmn;0.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eCell viability was calculated through normal cell counts/total cell counts. Each experiment was repeated three times.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eTab.3 Intensity scores of Intensity scores of 4 proteins in articular cartilage\u003c/p\u003e\n\u003cdiv align=\"\"\u003e\n \u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"510\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.88235294117647%\" rowspan=\"2\"\u003e\n \u003cp\u003eProetins\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"74.11764705882354%\" colspan=\"4\"\u003e\n \u003cp\u003eintensity scores\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"27.440633245382585%\"\u003e\n \u003cp\u003econtrol\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.065963060686016%\"\u003e\n \u003cp\u003emodel\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"22.427440633245382%\"\u003e\n \u003cp\u003eMSC\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"25.065963060686016%\"\u003e\n \u003cp\u003esiRNA-MSC\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.831702544031312%\"\u003e\n \u003cp\u003eproteoglycan\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.35225048923679%\"\u003e\n \u003cp\u003e3+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.59099804305284%\"\u003e\n \u003cp\u003e1+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.634050880626223%\"\u003e\n \u003cp\u003e2+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.59099804305284%\"\u003e\n \u003cp\u003e2+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.831702544031312%\"\u003e\n \u003cp\u003eCol-2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.35225048923679%\"\u003e\n \u003cp\u003e2+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.59099804305284%\"\u003e\n \u003cp\u003e2+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.634050880626223%\"\u003e\n \u003cp\u003e2+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.59099804305284%\"\u003e\n \u003cp\u003e2+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.831702544031312%\"\u003e\n \u003cp\u003eP21\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.35225048923679%\"\u003e\n \u003cp\u003e1+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.59099804305284%\"\u003e\n \u003cp\u003e3+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.634050880626223%\"\u003e\n \u003cp\u003e1+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.59099804305284%\"\u003e\n \u003cp\u003e3+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"25.831702544031312%\"\u003e\n \u003cp\u003eP53\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"20.35225048923679%\"\u003e\n \u003cp\u003e1+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.59099804305284%\"\u003e\n \u003cp\u003e3+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.634050880626223%\"\u003e\n \u003cp\u003e1+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.59099804305284%\"\u003e\n \u003cp\u003e2+\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n\u003c/div\u003e\n\u003cp\u003eProtein expression scores of proteoglycans, Col-2, P21 and P53 in control, model, MSC and siRNA-MSC groups were calculated through method described above in 2.7.\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-musculoskeletal-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmsd","sideBox":"Learn more about [BMC Musculoskeletal Disorders](http://bmcmusculoskeletdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://author-welcome.nature.com/12891","title":"BMC Musculoskeletal Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"might play a pivotal role in the treatment of OA through anti-apoptosis and regeneration in chondrocyte by secreting SOD and improving oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-4235031/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4235031/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eTo investigate the therapeutic effect of intraarticular injection of mesenchymal stem cells (MSC) in a rabbit osteoarthritis (OA) model. And to suppose whether MSC play a pivotal role in OA therapy by improving oxidative stress through secreting superoxide dismutase (SOD).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eMSC were isolated and cultured in vitro. SOD gene of MSC was silenced by siRNA technology to prepare the SOD-siRNA-MSC for in-vivo study. Twenty healthy adult New Zealand white rabbits underwent papain injection to induce OA and then received intra-articular injection with MSC, siRAN-MSC, or normal saline. The rabbits were divided into 4 groups (n\u0026thinsp;=\u0026thinsp;5), such as the control group, the model group, the MSC group, the siRNA-MSC group. Cytokines determination was performed 2 and 4 weeks after treatment. Magnetic resonance imaging (MRI) and histopathology and immunohistochemistry determination were performed 4 weeks after treatment.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eCOMP, TNF-α, MMP-2 and MMP-13 in the MSC group were significantly decreased compared to those in model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). However, MMP2 and MMP13 in the siRNA-MSC group were not significantly decreased compared to the model group (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Magnetic resonance results revealed a significant improvement in cartilage and synovial membrane 4 weeks after MSC injection. Histopathology determination showed that cartilage structure was also significantly improved in MSC group. Immunohistochemical analysis revealed amelioration in the expression levels of proteoglycan, COL-2, P21 and P53 in MSC group. On the other hand, MRI, histopathologic and immunohistochemical analysis also indicated a decreased therapeutic effect with SOD-siRNA -MSC.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur study demonstrated for the first time that MSC might be a promising therapy in OA through anti-apoptosis and regeneration in chondrocyte by secreting SOD and improving oxidative stress.\u003c/p\u003e","manuscriptTitle":"Mesenchymal stem cells improve osteoarthritis by secreting superoxide dismutase to regulate oxidative stress response","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-22 02:51:31","doi":"10.21203/rs.3.rs-4235031/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-28T07:46:47+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-27T23:43:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-24T08:58:50+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-19T20:58:46+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"54698824259136316093434064551851971640","date":"2024-08-19T18:16:01+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171626690151707311535625056642019508139","date":"2024-08-18T02:05:14+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"101729930145921345145802832361861652203","date":"2024-08-15T23:06:41+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"11068667018269736531696438959795470419","date":"2024-08-14T23:42:44+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-08-13T08:52:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"288810435456701616560798971969376951077","date":"2024-08-12T02:29:21+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"44586588763826947272551266359490874046","date":"2024-08-09T01:47:13+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-08-08T16:49:08+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-03T02:36:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-04-15T03:07:17+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-04-15T03:04:23+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Musculoskeletal Disorders","date":"2024-04-08T08:26:22+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-musculoskeletal-disorders","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmsd","sideBox":"Learn more about [BMC Musculoskeletal Disorders](http://bmcmusculoskeletdisord.biomedcentral.com/)","snPcode":"","submissionUrl":"https://author-welcome.nature.com/12891","title":"BMC Musculoskeletal Disorders","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"32b82e4c-c7ba-4856-9412-2497059ca9ee","owner":[],"postedDate":"April 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-04-28T16:00:05+00:00","versionOfRecord":{"articleIdentity":"rs-4235031","link":"https://doi.org/10.1186/s12891-025-08670-4","journal":{"identity":"bmc-musculoskeletal-disorders","isVorOnly":false,"title":"BMC Musculoskeletal Disorders"},"publishedOn":"2025-04-24 15:57:14","publishedOnDateReadable":"April 24th, 2025"},"versionCreatedAt":"2024-04-22 02:51:31","video":"","vorDoi":"10.1186/s12891-025-08670-4","vorDoiUrl":"https://doi.org/10.1186/s12891-025-08670-4","workflowStages":[]},"version":"v1","identity":"rs-4235031","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4235031","identity":"rs-4235031","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}