Pathological effects of joint stress imbalance on joint tissues

Pathological effects of joint stress imbalance on joint tissues | 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 Pathological effects of joint stress imbalance on joint tissues Jianwei Wang, Guangyan Pan, Xiaofeng Wu, Xiaojuan Gu, Yifeng Zhao, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7495716/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Objective This study aims to determine the pathological effects of joint stress imbalance on joint tissues from multiple mouse models, and provide useful information for clinicians to develop new strategies for the treatment and prevention of Osteoarthritis (OA). Methods In this study, knee joint samples were collected at 2, 4 and 8 weeks, respectively, through the established mouse knee joint long-term fixation model, weight-bearing model and excessive exercise model. The pathological changes of knee joint cartilage, synovial inflammation and osteophytes were observed by Micro-CT and histological staining. Results We found that long-term joint fixation resulted in significant pain, articular cartilage damage, synovial infiltration, and cartilaginous osteophyte of knee joints in mice, and the joint injury became more serious with the extension of fixation time. OA cartilage damage also occurs on the non-fixation weight-bearing side, and the cartilage damage becomes more severe with the extension of weight-bearing time, but it did not cause synovial inflammation and osteophyte formation. However, long-term excessive exercise in mice did not lead to obvious OA lesions such as cartilage damage, synovial inflammation and osteophyte formation, but it increases pain sensitivity. Conclusions This study can provide theoretical guidance for postoperative rehabilitation or exercise in clinical patients: the pathological process of tissue repair and OA lesions should be balanced as much as possible, and the joint fixation time should be shortened to reduce the occurrence of OA. For those who do not have meniscus lesions, moderate exercise can be performed to promote cartilage function, while for those with meniscus lesions or long-term weight bearing, joint movement should be reduced to repair meniscus lesions or reduce weight bearing to reduce the occurrence of OA. Osteoarthritis Fixation Treadmill Cartilage damage Synovium Osteophytes Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Osteoarthritis (OA) is a joint disease characterized by articular cartilage damage, osteophyte hyperplasia, and infiltration of synovitis. Its clinical manifestations include joint pain, stiffness, limited mobility, and muscle weakness, which seriously affect limb function and reduce quality of life [ 1 ] . With the aging of the population and the aggravation of obesity, the incidence and disability of OA are increasing year by year, further aggravating the social and economic burden. Although traditional therapies such as medication and surgery can partially alleviate these symptoms, their clinical application remains limited due to adverse reactions and the risk of recurrence [ 2 ] . Limited information on the etiology and pathogenesis of OA makes the development of new treatment strategies a challenging endeavor. Clinically, in addition to OA caused by injury or aging, long-term fixation, weight-bearing or excessive exercise may lead to OA injury [ 3 ] . Studies have shown that joint load is essential for maintaining the structural and functional integrity of articular cartilage, while excessive mechanical stress or excessive exercise leading to stress imbalance in articular cartilage is an important factor causing OA [ 4 ] . Articular cartilage includes a large amount of extracellular matrix and embedded chondrocytes, and the extracellular matrix is mainly composed of type II collagen and proteoglycan [ 5 ] . Pressure receptors are important regulators of chondrocyte function, and under moderate stress stimulation, the metabolic balance of chondrocytes is crucial for cell survival and the maintenance of extracellular matrix production. Mechanical stress imbalance may cause chondrocyte apoptosis, promote chondrocyte secretion of matrix decomposition proteins such as Mmp13/Adamts5, inhibit matrix synthesis proteins such as Col2a1/Aggrecan, and further promote cartilage matrix degradation, leading to a vicious cycle of cartilage damage [ 6 ] . In addition, the synovium is also an important component of the joint, and synovial inflammation is closely related to metabolic disorders and apoptosis of chondrocytes and the progression of OA [ 7 , 8 ] . There have been no systematic studies on the pathological changes of various tissues in OA caused by joint stress imbalance. We constructed mouse lower extremity weight bearing model, disuse model and excessive exercise models respectively, and comprehensively analyzed the effects of stress on joint tissue homeostasis at both the histological and animal levels, providing theoretical guidance for clinical patients' postoperative rehabilitation or exercise. Materials and Methods Animals C57BL6/J mice (12 weeks old) were purchased from the Model animal research center of Nanjing University (Nanjing, China). The mice were housed in the specific pathogen-free (SPF) facility at LENSCI Biotechnology(kunshan)Co.,LTD. All experimental protocol in this study were approved by the Institutional Animal Care and Use Committee of LENSCI Biotechnology(kunshan)Co.,LTD (20240325-0033-01). Construction of mouse model Modified the previous protocol of plaster fixation in mice [9] , specifically: Wrap the right lower limb of the mouse with gauze, then wrap and fix the right leg with soaked plaster, and then wrap the fixed outer layer of plaster with gauze and tie a knot. Finally, trim the gauze line and plaster to expose the ankle and observe blood circulation. The other leg was not treated, and bilateral knee joints were collected for analysis at the same time point. Treadmill The mice were randomly assigned to a control group (no exercise) or a treadmill exercise group. Before the experiment, the mice in the exercise group were first trained to run at a speed of 10 m/min for 1 h per day for 3 days, and then the slope was gradually increased. One week later, the treadmill model was officially started, with mice running at a slope of 5%, 10%, and 15% at 20 m/min for 1 h per day for 30 consecutive days [10] . Micro-computed tomography After sampling, the mouse knee joints were fixed with 4% paraformaldehyde for 24 hours and scanned using a high-resolution Micro-computed tomography(mCT) scanner (Skyscan 1276) at a voltage of 60kV and a current of 100uA. The segmented images were used for 3D reconstruction and data analysis [11] . Von Frey test Mechanical allodynia (Von Frey sensitivity) was tested as described previously [12] . Mice were placed on a mesh platform in isolation and allowed to acclimate in a quiet environment for 15 min, and the sensitivity of the hind PAWS to stimuli was measured by pulling them from below using calibrated von Frey wires. Histological analyses The tissue specimens were sequentially fixed with 4% paraformaldehyde, decalcified with 10% EDTA (pH 7.4), embedded in paraffin, and sliced at a thickness of 5 mm [13] . Then dried and rehydrated, and stained with hematoxylin-eosin (H/E), safranin O/fast green (SO/FG) and Masson trichrome staining. Cartilage degradation was assessed using the Osteoarthritis Research Society International (OARSI) scoring system [14] . Synovial activation was assessed using the Krenn synovitis scoring system, and osteophyte size and maturity were assessed according to relevant literature [15,16] . Each section was evaluated by two independent blinded reviewers, and the average score was used for statistical analysis. Statistical analysis GraphPad Prism 8.0 was used for analysis or plotting, and Student's t test was used to analyze the differences between the two groups. One-way analysis of variance (ANOVA) was used for multiple group comparisons. P < 0.05 was considered as significant different between groups. Results Long-term joint flexion fixation induces cartilage damage, synovial inflammation and cartilaginous osteophyte formation in mice. We first established a mouse lower limb flexion fixation model, and analyzed the samples by µCT scanning and histological staining after fixation for 2, 4 and 8 weeks, respectively. µCT 3D imaging showed that after the mice were immobilized and unable to move, there was no obvious ossification of the meniscus in the knee joints, and no obvious osteophyte formation on both sides of the tibial condyle and femoral condyle (Fig. 1 A-C). Further observation showed that the bone mass of cancellous tibia decreased significantly after joint fixation without movement, and the longer the fixation time, the more obvious the bone mass reduction was (Fig. 1 A,D,E). Further histological analysis showed that long-term joint flexion fixation could lead to the formation of cartilaginous osteophyte (Fig. 2 A), destruction of articular cartilage, thickening of the calcified area of ​​articular cartilage, and thinning of the hyaline cartilage area, resulting in OA damage (Fig. 2 B), and the longer the fixation time, the more severe OA injury (Fig. 2 C-E). Long-term fixation induces cartilage damage on the non-fixation weight-bearing knee. In clinical practice, after the joint fixation of one lower limb, the other limb will perform relevant functional compensation and increase the weight bearing. In order to further observe the histological changes of the non-fixed weight-bearing joint with compensatory motor function after joint fixation, we also analyzed the knee joint on the non-fixed weight-bearing side after 2, 4 and 8 weeks of fixation. µCT 3D imaging and sagittal section showed no obvious ossification of meniscus and no obvious osteophyte formation on tibial condyle and femoral condyle of the non-fixed weight-bearing side knee joint (Fig. 3 A-C). Further observation showed that surface articular cartilage damage could occur on the non-fixed weight-bearing side, but there was no obvious thickening in the calcified area of articular cartilage. Moreover, the longer the fixation time, the more severe the OA surface cartilage damage (Fig. 3 D,E). However, there is no obvious promoting effect on osteophyte formation and synovial inflammation (Fig. 3 F,G). Long-term treadmill exercise did not cause joint damage but caused pain sensitivity. We established a treadmill exercise model with different exercise intensities by having mice exercise on a treadmill. Samples were collected for µCT and histological analysis 8 weeks after exercise. µCT 3D reconstruction showed that, compared with the control group, after 8 weeks of exercise at different intensities, there was no obvious ossification of knee meniscus, and no obvious osteophyte formation on both sides of tibial condyle and femoral condyle in mice (Fig. 4 A-C). Further histological analysis showed that long-term exercise could enhance the pain sensitivity of mice, and the higher the slope, the more obvious the pain (Fig. 4 F). However, compared with the control group, there were no significant changes in the articular cartilage, synovium, etc. in the exercise groups of different intensities, indicating that continuous exercise would not cause OA damage (Fig. 4 D-I). Discussion This study explores the effects of different types of joint stress changes on joint homeostasis by establishing long-term joint fixation mouse models, non-fixation side weight-bearing models, and long-term exercise models. We found that long-term joint fixation resulted in significant pain, articular cartilage damage, synovial infiltration, and cartilaginous osteophyte of knee joints in mice, and the joint injury became more serious with the extension of fixation time. OA cartilage damage also occurs on the non-fixation weight-bearing side, and the cartilage damage becomes more serious with the extension of weight-bearing time, but it did not cause synovial inflammation and osteophyte formation. It was surprising to find that long-term excessive exercise in mice did not lead to obvious OA lesions such as cartilage damage, synovial inflammation and osteophyte formation, but it increases pain sensitivity. In clinical practice, after acute joint sprains, periarticular fracture surgery, arthroscopy and other surgeries, early joint fixation is a stress protection for the acute phase of tissue damage and a common treatment method for restoring joint function and relieving pain. Some studies have shown that short-term joint fixation can reduce joint pressure, stabilize joint tissue, alleviate joint inflammation, and play an important role in joint function recovery and tissue repair [ 17 , 18 ] . Chondrocytes can regulate cell function and maintain extracellular matrix homeostasis through pressure receptors on the cell surface. Sustained moderate joint stress is crucial for maintaining chondrocyte function. Long-term stress loss will weaken the biological activity and matrix formation of chondrocytes, resulting in the imbalance of cartilage homeostasis. Mutsuzaki et al. found that fixation for 4 and 8 weeks resulted in thinning of the glycosaminoglycan layer in articular cartilage and apoptosis of chondrocytes [ 19 ] . Other studies have shown that unloading conditions lead to thinning of knee cartilage after 2 weeks, and thinning of the medial tibiofemoral cartilage after 4 weeks, with a decrease in cartilage matrix [ 20 ] . Our study also showed that long-term joint fixation leads to articular cartilage damage and loss of cartilage matrix. Studies have shown that there are a large number of stem cells at the junction of the periosteum and synovium near the articular cartilage, which can cause intramembranous ossification after activation, thereby forming osteophytes in the cartilage and bone [ 21 ] . In this study, long-term joint fixation resulted in the formation of cartilaginous osteophyte on knee joints, which gradually proliferated over time, which may be related to the ossification of stem cells in the periosteum or synovium. Synovial inflammatory infiltration is an important factor in OA lesions and joint pain. Some studies have shown that moderate activity can regulate immune responses by initiating the differentiation of circulating monocytes into anti-inflammatory macrophages [ 22 ] . Moreover, collagen fragments released by articular cartilage damage caused by long-term fixation play an important role in activating synovial cells [ 23 ] . In addition, studies have also shown that long-term fixation will lead to muscle atrophy, promote the release of IL-6, and cause the up-regulation of inflammatory factors around joints, which may be the cause of OA changes after fixation [ 24 ] . We found that in addition to the fixed joint side, the other knee joint also suffered cartilage damage. Some studies have suggested that articular cartilage damage will release biological factors from the damaged joint, which will flow through the blood to the healthy joint, causing corresponding joint damage [ 25 – 27 ] , but we did not observe synovial inflammation and osteophyte formation in the other knee joint. We believe that when one leg of mice is fixed, the other leg will increase the compensatory weight during the maintenance of daily basic activities, and the knee joint stress overload will cause OA lesions. Studies have shown that the load on articular cartilage is an important factor in the occurrence and development of osteoarthritis. Moderate exercise is essential for maintaining joint stability, while excessive load will cause joint injury. Studies have shown that activation of mechanical sensing proteins in chondrocytes can increase the expression of cartilage matrix decomposition proteins and decrease the expression of cartilage matrix synthesis proteins, which can also lead to chondrocyte hypertrophy [ 28 , 29 ] . This suggests that when one side of the limb is unable to bear weight clinically, crutches, carts or other supporting aids should be used in time to reduce the load on the healthy limb. In addition, it can also be used as a mouse model to simulate OA induced by weight-bearing laborers and climbers. Sports wear and tear is an important factor in the incidence of clinical OA, and the prevalence of OA in athletes and elderly people is higher than that in normal young people [ 30 ] . However, our study shows that long-term excessive exercise does not cause obvious OA lesions. Several recent studies have reported that exercise is a form of physical therapy, such as treadmill can effectively prevent the progression of osteoarthritis [ 31 , 32 ] . However, the reasons for the exercise-induced cartilage protection have not been fully elucidated. Stable and intact meniscus is the structural basis for maintaining the function of articular cartilage. Meniscus lesions or instability may cause articular cartilage damage [ 33 ] . Some clinical studies have shown that meniscus lesions occurs before OA, and some studies believe that meniscus lesions is the result of OA lesions [ 34 ] . Studies have shown that OA progresses rapidly in DMM-induced mouse models, which is related to the stress imbalance of articular cartilage caused by meniscus injury [ 35 ] . We believe that stable mechanical load during exercise is necessary to maintain the structure and function of cartilage. Moderate exercise can slow the progression of cartilage degeneration [ 36 ] . Stress imbalance in articular cartilage is an important factor in the development of osteoarthritis. Once cartilage stress is imbalanced, even a small amount of exercise can exert a large impact on the articular cartilage, accelerating the progression of OA. This study aims to determine the pathological effects of joint stress imbalance on joint tissues at the histological and animal levels, and to provide useful information for clinicians to develop new strategies for the treatment and prevention of OA. For example, for clinical patients undergoing joint fixation after surgery, the risk of tissue repair and cartilage degeneration should be balanced. During the period of joint fixation, muscle isometric contraction exercises can be performed to increase joint load. In addition, the joint fixation time should be shortened as much as possible to reduce the occurrence of OA. Although medication and surgical therapy are effective ways to treat osteoarthritis, it is also crucial to prevent articular cartilage degeneration. For those with no meniscus lesions, moderate exercise can be carried out to promote cartilage function, while for those with meniscus lesions or weight bearing, joint movement should be reduced to repair meniscus lesions or reduce weight bearing. Conclusions Clinically, in addition to OA caused by injury or aging, long-term fixation, weight-bearing or excessive exercise may lead to OA injury. There have been no systematic studies on the pathological changes of various tissues in OA caused by joint stress imbalance. We constructed mouse lower extremity weight bearing model, disuse model and excessive exercise models respectively, and comprehensively analyzed the effects of stress on joint tissue homeostasis at both the histological and animal levels, providing theoretical guidance for clinical patients' postoperative rehabilitation or exercise. Abbreviations OA: Osteoarthritis; SPF: Specific pathogen-free; mCT: Micro-computed tomography; H/E: Hematoxylin-eosin; SO/FG: Safranin O/fast green; OARSI: Osteoarthritis Research Society International; ANOVA: Analysis of variance; Declarations Ethics approval and consent to participate All experimental protocol in this study were approved by the Institutional Animal Care and Use Committee of LENSCI Biotechnology(kunshan)Co.,LTD (20240325-0033-01). Consent for publication Not applicable. Availability of data and material The datasets analyzed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests. Funding This work was supported by the Suzhou Clinical Key Disease Diagnosis and Treatment Technology Special Project (LCZX202127). Authors' contributions Study design: XJ and JW. Study conduct and data collection: JW, XW, XG, and YZ. Data analysis: JW, GP, XW, XG, and YZ. Data interpretation: XJ and JW. Drafting the manuscript: XJ, GP, and JW. XJ and JW take the responsibility for the integrity of the data analysis. References Sharma L. Osteoarthritis of the Knee. N Engl J Med 2021;384(1):51-59. [eng]. Katz JN, Arant KR, Loeser RF. Diagnosis and Treatment of Hip and Knee Osteoarthritis: A Review. Jama 2021;325(6):568-78. [eng]. Abramoff B, Caldera FE. Osteoarthritis: Pathology, Diagnosis, and Treatment Options. Med Clin North Am 2020;104(2):293-311. [eng]. Hodgkinson T, Kelly DC, Curtin CM, O'Brien FJ. Mechanosignalling in cartilage: an emerging target for the treatment of osteoarthritis. 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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-7495716","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":512714482,"identity":"aa0603a4-aa92-421c-9cbc-6fa02a413473","order_by":0,"name":"Jianwei Wang","email":"","orcid":"","institution":"Kunshan Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Jianwei","middleName":"","lastName":"Wang","suffix":""},{"id":512714483,"identity":"5c98de39-6183-4c56-8915-7f2f8b295c25","order_by":1,"name":"Guangyan Pan","email":"","orcid":"","institution":"Tongji University","correspondingAuthor":false,"prefix":"","firstName":"Guangyan","middleName":"","lastName":"Pan","suffix":""},{"id":512714484,"identity":"965e92c0-175b-4868-a9bf-18677095d0fa","order_by":2,"name":"Xiaofeng Wu","email":"","orcid":"","institution":"Kunshan Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaofeng","middleName":"","lastName":"Wu","suffix":""},{"id":512714485,"identity":"61b00eb4-825e-4329-86c3-397ec2759997","order_by":3,"name":"Xiaojuan Gu","email":"","orcid":"","institution":"Kunshan Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Xiaojuan","middleName":"","lastName":"Gu","suffix":""},{"id":512714486,"identity":"1217e487-04c0-40ff-8e26-b7e2b4372855","order_by":4,"name":"Yifeng Zhao","email":"","orcid":"","institution":"Kunshan Hospital of Traditional Chinese Medicine","correspondingAuthor":false,"prefix":"","firstName":"Yifeng","middleName":"","lastName":"Zhao","suffix":""},{"id":512714487,"identity":"5a957992-d8d1-4e31-8bd2-28e204ddc5bc","order_by":5,"name":"Xinghua Jiang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwUlEQVRIiWNgGAWjYBACxgYehgMSBhJyDMwkaGF8YFFgY0y8FgYGHmaDig9piQ1Ea2CekXtM4obB4fT57bwHPzDU2EQTdljPuTTJGQaHczcc5kuWYDiWlkvQOsb2HjNpCZAWZh4DCcaGw0RoaeYxk/4DdJh8M4/xD+K0tPcYG0gYpCUwHOYxI9KWnjOGDyQMbAw3ALVYJBDjF8MZOQYHJP5IyMv3nzG+8aHGhggtKCoSCCkHAXliFI2CUTAKRsEIBwDX+TujmItvuQAAAABJRU5ErkJggg==","orcid":"","institution":"Kunshan Hospital of Traditional Chinese Medicine","correspondingAuthor":true,"prefix":"","firstName":"Xinghua","middleName":"","lastName":"Jiang","suffix":""}],"badges":[],"createdAt":"2025-08-30 13:53:24","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7495716/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7495716/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91072505,"identity":"9cc279d2-3e7c-4ec4-b79f-bb85d015bcf9","added_by":"auto","created_at":"2025-09-11 10:54:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":596445,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term joint flexion fixation stimulates a decrease in long bone mass, but has no effect on meniscal ossification in mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 3D and 2D reconstruction images of the knee joint at 0, 2, 4, and 8 weeks after flexion fixation. The first row is the anterior view of the 3D reconstruction of the knee joint; the second row is the lateral view of the 3D reconstruction of the knee joint; the third row is the 2D coronal view of the knee joint. The red arrow shows the distribution of trabeculae in the proximal tibia. (B,C) Quantitative μCT analyses of bone volume (BV) and bone volume fraction (BV/TV) of meniscus. (D,E) Quantitative μCT analyses of trabecular separation (Tb.Sp), trabecular number(Tb.N) of tibia. \u003cem\u003eN\u003c/em\u003e=6. Results are expressed as mean \u003cu\u003e+\u003c/u\u003estandard deviation (s.d.). **\u003cem\u003eP\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-7495716/v1/625da1a8adae327b5d3fe98e.png"},{"id":91072507,"identity":"7ae18d41-4278-412b-b392-d18a55fcac1b","added_by":"auto","created_at":"2025-09-11 10:54:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1426792,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term joint flexion fixation induces cartilage damage, synovial inflammation and cartilaginous osteophyte at the joint margins of mice.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) H/E staining of the knee joints at 0, 2, 4, and 8 weeks after fixation. The first row is a 4×image, and the second row is a local magnification. The green arrow indicates cartilaginous osteophyte. (B) Safranin O/fast green (SO/FG) staining of the knee joints at 0, 2, 4, and 8 weeks after fixation. The first row is a 10×image, and the second row is a local magnification. The green arrow indicates cartilaginous osteophyte. The red arrow indicates the damage of the articular cartilage, and the black arrow indicates the calcified cartilage area. (C-E) Quantitative data of OARSI score (C), Osteophyte score (D) and Synovitis score(E) based on staining results in (A, B). \u003cem\u003eN\u003c/em\u003e=6. Results are expressed as mean + standard deviation (s.d.). \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-7495716/v1/6df914194ffb0ef516dfdeae.png"},{"id":91072508,"identity":"49d0af2d-75d4-4225-a47c-930efec5ee55","added_by":"auto","created_at":"2025-09-11 10:54:51","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1747020,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eCartilage damage occurred on the non-fixation weight-bearing knee after Long-term joint fixation.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 3D reconstruction and sagittal section of μCT scan of the non-fixation weight-bearing knee joint at 0, 2, 4 and 8 weeks after fixation. (B,C) Quantitative μCT analyses of BV、BV/TV of meniscus based on (A). (D) SO/FG staining of the non-fixation weight-bearing side knee joint at 0, 2, 4, and 8 weeks after fixation. The first row is a 10×image, and the second row is a local magnification. The red arrow indicates the damage of the articular cartilage, and the black arrow indicates the calcified cartilage area. (E-G) Quantitative data of OARSI score (E), Osteophyte score (F) and Synovitis score(G) based on staining results in (D). \u003cem\u003eN\u003c/em\u003e=6. Results are expressed as mean + standard deviation (s.d.). \u003cem\u003e**P\u003c/em\u003e \u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-7495716/v1/c2628214df25a611ccaba22f.png"},{"id":91072506,"identity":"48cbe1fb-fc66-4e77-ad7b-9e8f209df2f6","added_by":"auto","created_at":"2025-09-11 10:54:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1148262,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term treadmill exercise did not cause joint damage but caused pain sensitivity.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(A) 3D reconstruction of knee joint with μCT scan after 8 weeks of treadmill exercise in different groups. The first row is a 3D reconstruction of the knee joint, and the second row is a partial magnification of the joint. (B,C) Quantitative μCT analyses of BV、BV/TV of meniscus based on (A). (D) H/E staining of the knee joints in different groups after 8 weeks of treadmill exercise. (E) SO/FG staining of the knee joints in different groups after 8 weeks of treadmill exercise. (E) Von Frey scores of different groups after 8 weeks of treadmill exercise. (G-I) Quantitative data of OARSI score (G), Osteophyte score (H) and Synovitis score(I) based on staining results in (D,E). \u003cem\u003eN\u003c/em\u003e=6. Results are expressed as mean + standard deviation (s.d.). \u003cem\u003e**P \u003c/em\u003e\u0026lt; 0.01.\u003c/p\u003e","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-7495716/v1/e89561233bae19a458f9987e.png"},{"id":91331289,"identity":"a73731d9-8c0d-4141-85cd-dc3f0b29c7fc","added_by":"auto","created_at":"2025-09-15 11:02:03","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6201852,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7495716/v1/f942db3e-b2e7-4fbb-99c8-5a915c04ee2c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pathological effects of joint stress imbalance on joint tissues","fulltext":[{"header":"Introduction","content":"\u003cp\u003eOsteoarthritis (OA) is a joint disease characterized by articular cartilage damage, osteophyte hyperplasia, and infiltration of synovitis. Its clinical manifestations include joint pain, stiffness, limited mobility, and muscle weakness, which seriously affect limb function and reduce quality of life\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. With the aging of the population and the aggravation of obesity, the incidence and disability of OA are increasing year by year, further aggravating the social and economic burden. Although traditional therapies such as medication and surgery can partially alleviate these symptoms, their clinical application remains limited due to adverse reactions and the risk of recurrence\u003csup\u003e[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]\u003c/sup\u003e. Limited information on the etiology and pathogenesis of OA makes the development of new treatment strategies a challenging endeavor.\u003c/p\u003e\u003cp\u003eClinically, in addition to OA caused by injury or aging, long-term fixation, weight-bearing or excessive exercise may lead to OA injury\u003csup\u003e[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that joint load is essential for maintaining the structural and functional integrity of articular cartilage, while excessive mechanical stress or excessive exercise leading to stress imbalance in articular cartilage is an important factor causing OA \u003csup\u003e[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]\u003c/sup\u003e. Articular cartilage includes a large amount of extracellular matrix and embedded chondrocytes, and the extracellular matrix is mainly composed of type II collagen and proteoglycan \u003csup\u003e[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]\u003c/sup\u003e. Pressure receptors are important regulators of chondrocyte function, and under moderate stress stimulation, the metabolic balance of chondrocytes is crucial for cell survival and the maintenance of extracellular matrix production. Mechanical stress imbalance may cause chondrocyte apoptosis, promote chondrocyte secretion of matrix decomposition proteins such as Mmp13/Adamts5, inhibit matrix synthesis proteins such as Col2a1/Aggrecan, and further promote cartilage matrix degradation, leading to a vicious cycle of cartilage damage\u003csup\u003e[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]\u003c/sup\u003e. In addition, the synovium is also an important component of the joint, and synovial inflammation is closely related to metabolic disorders and apoptosis of chondrocytes and the progression of OA \u003csup\u003e[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e. There have been no systematic studies on the pathological changes of various tissues in OA caused by joint stress imbalance.\u003c/p\u003e\u003cp\u003eWe constructed mouse lower extremity weight bearing model, disuse model and excessive exercise models respectively, and comprehensively analyzed the effects of stress on joint tissue homeostasis at both the histological and animal levels, providing theoretical guidance for clinical patients' postoperative rehabilitation or exercise.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003eAnimals\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eC57BL6/J mice (12 weeks old) were purchased from the Model animal research center of Nanjing University (Nanjing, China). The mice were housed in the specific pathogen-free (SPF) facility at LENSCI Biotechnology(kunshan)Co.,LTD. All experimental protocol in this study were approved by the Institutional Animal Care and Use Committee of LENSCI Biotechnology(kunshan)Co.,LTD (20240325-0033-01).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConstruction of mouse model\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eModified the previous protocol of plaster fixation in mice\u003csup\u003e[9]\u003c/sup\u003e, specifically: Wrap the right lower limb of the mouse with gauze, then wrap and fix the right leg with soaked plaster, and then wrap the fixed outer layer of plaster with gauze and tie a knot. Finally, trim the gauze line and plaster to expose the ankle and observe blood circulation. The other leg was not treated, and bilateral knee joints were collected for analysis at the same time point.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTreadmill\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe mice were randomly assigned to a control group (no exercise) or a treadmill exercise group. Before the experiment, the mice in the exercise group were first trained to run at a speed of 10 m/min for 1 h per day for 3 days, and then the slope was gradually increased. One week later, the treadmill model was officially started, with mice running at a slope of 5%, 10%, and 15% at 20 m/min for 1 h per day for 30 consecutive days\u003csup\u003e[10]\u003c/sup\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMicro-computed tomography\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter sampling, the mouse knee joints were fixed with 4% paraformaldehyde for 24 hours and scanned using a high-resolution Micro-computed tomography(mCT) scanner (Skyscan 1276) at a voltage of 60kV and a current of 100uA. The segmented images were used for 3D reconstruction and data analysis \u003csup\u003e[11]\u003c/sup\u003e. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eVon Frey test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMechanical allodynia (Von Frey sensitivity) was tested as described previously\u003csup\u003e\u0026nbsp;\u003c/sup\u003e\u003csup\u003e[12]\u003c/sup\u003e. Mice were placed on a mesh platform in isolation and allowed to acclimate in a quiet environment for 15 min, and the sensitivity of the hind PAWS to stimuli was measured by pulling them from below using calibrated von Frey wires.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analyses\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe tissue specimens were sequentially fixed with 4% paraformaldehyde, decalcified with 10% EDTA (pH 7.4), embedded in paraffin, and sliced at a thickness of 5\u0026nbsp;mm\u003csup\u003e[13]\u003c/sup\u003e.\u0026nbsp;Then dried and rehydrated, and stained with hematoxylin-eosin (H/E), safranin O/fast green (SO/FG) and Masson trichrome staining. Cartilage degradation was assessed using the Osteoarthritis Research Society International (OARSI) scoring system\u003csup\u003e[14]\u003c/sup\u003e. Synovial activation was assessed using the Krenn synovitis scoring system, and osteophyte size and maturity were assessed according to relevant literature\u003csup\u003e[15,16]\u003c/sup\u003e. Each section was evaluated by two independent blinded reviewers, and the average score was used for statistical analysis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStatistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism 8.0 was used for analysis or plotting, and Student\u0026apos;s t test was used to analyze the differences between the two groups. One-way analysis of variance (ANOVA) was used for multiple group comparisons. \u003cem\u003eP\u003c/em\u003e \u0026lt; 0.05 was considered as significant different between groups.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eLong-term joint flexion fixation induces cartilage damage, synovial inflammation and cartilaginous osteophyte formation in mice.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe first established a mouse lower limb flexion fixation model, and analyzed the samples by \u0026micro;CT scanning and histological staining after fixation for 2, 4 and 8 weeks, respectively. \u0026micro;CT 3D imaging showed that after the mice were immobilized and unable to move, there was no obvious ossification of the meniscus in the knee joints, and no obvious osteophyte formation on both sides of the tibial condyle and femoral condyle (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-C). Further observation showed that the bone mass of cancellous tibia decreased significantly after joint fixation without movement, and the longer the fixation time, the more obvious the bone mass reduction was (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA,D,E). Further histological analysis showed that long-term joint flexion fixation could lead to the formation of cartilaginous osteophyte (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA), destruction of articular cartilage, thickening of the calcified area of ​​articular cartilage, and thinning of the hyaline cartilage area, resulting in OA damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB), and the longer the fixation time, the more severe OA injury (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-E).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLong-term fixation induces cartilage damage on the non-fixation weight-bearing knee.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn clinical practice, after the joint fixation of one lower limb, the other limb will perform relevant functional compensation and increase the weight bearing. In order to further observe the histological changes of the non-fixed weight-bearing joint with compensatory motor function after joint fixation, we also analyzed the knee joint on the non-fixed weight-bearing side after 2, 4 and 8 weeks of fixation. \u0026micro;CT 3D imaging and sagittal section showed no obvious ossification of meniscus and no obvious osteophyte formation on tibial condyle and femoral condyle of the non-fixed weight-bearing side knee joint (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-C). Further observation showed that surface articular cartilage damage could occur on the non-fixed weight-bearing side, but there was no obvious thickening in the calcified area of articular cartilage. Moreover, the longer the fixation time, the more severe the OA surface cartilage damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD,E). However, there is no obvious promoting effect on osteophyte formation and synovial inflammation (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF,G).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eLong-term treadmill exercise did not cause joint damage but caused pain sensitivity.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eWe established a treadmill exercise model with different exercise intensities by having mice exercise on a treadmill. Samples were collected for \u0026micro;CT and histological analysis 8 weeks after exercise. \u0026micro;CT 3D reconstruction showed that, compared with the control group, after 8 weeks of exercise at different intensities, there was no obvious ossification of knee meniscus, and no obvious osteophyte formation on both sides of tibial condyle and femoral condyle in mice (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). Further histological analysis showed that long-term exercise could enhance the pain sensitivity of mice, and the higher the slope, the more obvious the pain (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF). However, compared with the control group, there were no significant changes in the articular cartilage, synovium, etc. in the exercise groups of different intensities, indicating that continuous exercise would not cause OA damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD-I).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study explores the effects of different types of joint stress changes on joint homeostasis by establishing long-term joint fixation mouse models, non-fixation side weight-bearing models, and long-term exercise models. We found that long-term joint fixation resulted in significant pain, articular cartilage damage, synovial infiltration, and cartilaginous osteophyte of knee joints in mice, and the joint injury became more serious with the extension of fixation time. OA cartilage damage also occurs on the non-fixation weight-bearing side, and the cartilage damage becomes more serious with the extension of weight-bearing time, but it did not cause synovial inflammation and osteophyte formation. It was surprising to find that long-term excessive exercise in mice did not lead to obvious OA lesions such as cartilage damage, synovial inflammation and osteophyte formation, but it increases pain sensitivity.\u003c/p\u003e\u003cp\u003eIn clinical practice, after acute joint sprains, periarticular fracture surgery, arthroscopy and other surgeries, early joint fixation is a stress protection for the acute phase of tissue damage and a common treatment method for restoring joint function and relieving pain. Some studies have shown that short-term joint fixation can reduce joint pressure, stabilize joint tissue, alleviate joint inflammation, and play an important role in joint function recovery and tissue repair \u003csup\u003e[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]\u003c/sup\u003e. Chondrocytes can regulate cell function and maintain extracellular matrix homeostasis through pressure receptors on the cell surface. Sustained moderate joint stress is crucial for maintaining chondrocyte function. Long-term stress loss will weaken the biological activity and matrix formation of chondrocytes, resulting in the imbalance of cartilage homeostasis. Mutsuzaki et al. found that fixation for 4 and 8 weeks resulted in thinning of the glycosaminoglycan layer in articular cartilage and apoptosis of chondrocytes \u003csup\u003e[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]\u003c/sup\u003e. Other studies have shown that unloading conditions lead to thinning of knee cartilage after 2 weeks, and thinning of the medial tibiofemoral cartilage after 4 weeks, with a decrease in cartilage matrix \u003csup\u003e[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/sup\u003e. Our study also showed that long-term joint fixation leads to articular cartilage damage and loss of cartilage matrix. Studies have shown that there are a large number of stem cells at the junction of the periosteum and synovium near the articular cartilage, which can cause intramembranous ossification after activation, thereby forming osteophytes in the cartilage and bone \u003csup\u003e[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/sup\u003e. In this study, long-term joint fixation resulted in the formation of cartilaginous osteophyte on knee joints, which gradually proliferated over time, which may be related to the ossification of stem cells in the periosteum or synovium. Synovial inflammatory infiltration is an important factor in OA lesions and joint pain. Some studies have shown that moderate activity can regulate immune responses by initiating the differentiation of circulating monocytes into anti-inflammatory macrophages \u003csup\u003e[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e. Moreover, collagen fragments released by articular cartilage damage caused by long-term fixation play an important role in activating synovial cells \u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. In addition, studies have also shown that long-term fixation will lead to muscle atrophy, promote the release of IL-6, and cause the up-regulation of inflammatory factors around joints, which may be the cause of OA changes after fixation \u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWe found that in addition to the fixed joint side, the other knee joint also suffered cartilage damage. Some studies have suggested that articular cartilage damage will release biological factors from the damaged joint, which will flow through the blood to the healthy joint, causing corresponding joint damage \u003csup\u003e[\u003cspan additionalcitationids=\"CR26\" citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e, but we did not observe synovial inflammation and osteophyte formation in the other knee joint. We believe that when one leg of mice is fixed, the other leg will increase the compensatory weight during the maintenance of daily basic activities, and the knee joint stress overload will cause OA lesions. Studies have shown that the load on articular cartilage is an important factor in the occurrence and development of osteoarthritis. Moderate exercise is essential for maintaining joint stability, while excessive load will cause joint injury. Studies have shown that activation of mechanical sensing proteins in chondrocytes can increase the expression of cartilage matrix decomposition proteins and decrease the expression of cartilage matrix synthesis proteins, which can also lead to chondrocyte hypertrophy \u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. This suggests that when one side of the limb is unable to bear weight clinically, crutches, carts or other supporting aids should be used in time to reduce the load on the healthy limb. In addition, it can also be used as a mouse model to simulate OA induced by weight-bearing laborers and climbers.\u003c/p\u003e\u003cp\u003eSports wear and tear is an important factor in the incidence of clinical OA, and the prevalence of OA in athletes and elderly people is higher than that in normal young people \u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. However, our study shows that long-term excessive exercise does not cause obvious OA lesions. Several recent studies have reported that exercise is a form of physical therapy, such as treadmill can effectively prevent the progression of osteoarthritis \u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. However, the reasons for the exercise-induced cartilage protection have not been fully elucidated. Stable and intact meniscus is the structural basis for maintaining the function of articular cartilage. Meniscus lesions or instability may cause articular cartilage damage\u003csup\u003e[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Some clinical studies have shown that meniscus lesions occurs before OA, and some studies believe that meniscus lesions is the result of OA lesions \u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]\u003c/sup\u003e. Studies have shown that OA progresses rapidly in DMM-induced mouse models, which is related to the stress imbalance of articular cartilage caused by meniscus injury \u003csup\u003e[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. We believe that stable mechanical load during exercise is necessary to maintain the structure and function of cartilage. Moderate exercise can slow the progression of cartilage degeneration \u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Stress imbalance in articular cartilage is an important factor in the development of osteoarthritis. Once cartilage stress is imbalanced, even a small amount of exercise can exert a large impact on the articular cartilage, accelerating the progression of OA.\u003c/p\u003e\u003cp\u003eThis study aims to determine the pathological effects of joint stress imbalance on joint tissues at the histological and animal levels, and to provide useful information for clinicians to develop new strategies for the treatment and prevention of OA. For example, for clinical patients undergoing joint fixation after surgery, the risk of tissue repair and cartilage degeneration should be balanced. During the period of joint fixation, muscle isometric contraction exercises can be performed to increase joint load. In addition, the joint fixation time should be shortened as much as possible to reduce the occurrence of OA. Although medication and surgical therapy are effective ways to treat osteoarthritis, it is also crucial to prevent articular cartilage degeneration. For those with no meniscus lesions, moderate exercise can be carried out to promote cartilage function, while for those with meniscus lesions or weight bearing, joint movement should be reduced to repair meniscus lesions or reduce weight bearing.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eClinically, in addition to OA caused by injury or aging, long-term fixation, weight-bearing or excessive exercise may lead to OA injury. There have been no systematic studies on the pathological changes of various tissues in OA caused by joint stress imbalance. We constructed mouse lower extremity weight bearing model, disuse model and excessive exercise models respectively, and comprehensively analyzed the effects of stress on joint tissue homeostasis at both the histological and animal levels, providing theoretical guidance for clinical patients' postoperative rehabilitation or exercise.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eOA: Osteoarthritis;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSPF: Specific pathogen-free;\u003c/p\u003e\n\u003cp\u003emCT: Micro-computed tomography; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eH/E: Hematoxylin-eosin;\u003c/p\u003e\n\u003cp\u003eSO/FG: Safranin O/fast green;\u003c/p\u003e\n\u003cp\u003eOARSI: Osteoarthritis Research Society International;\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eANOVA: Analysis of variance;\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll experimental protocol in this study were approved by the Institutional Animal Care and Use Committee of LENSCI Biotechnology(kunshan)Co.,LTD (20240325-0033-01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis work was supported by the Suzhou Clinical Key Disease Diagnosis and Treatment Technology Special Project (LCZX202127).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eStudy design: XJ and JW. Study conduct and data collection: JW, XW, XG, and YZ. Data analysis: JW, GP, XW, XG, and YZ. Data interpretation: XJ and JW. Drafting the manuscript: XJ, GP, and JW. XJ and JW take the responsibility for the integrity of the data analysis. \u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eSharma L. Osteoarthritis of the Knee. N Engl J Med 2021;384(1):51-59. [eng].\u003c/li\u003e\n\u003cli\u003eKatz JN, Arant KR, Loeser RF. Diagnosis and Treatment of Hip and Knee Osteoarthritis: A Review. Jama 2021;325(6):568-78. [eng].\u003c/li\u003e\n\u003cli\u003eAbramoff B, Caldera FE. Osteoarthritis: Pathology, Diagnosis, and Treatment Options. Med Clin North Am 2020;104(2):293-311. [eng].\u003c/li\u003e\n\u003cli\u003eHodgkinson T, Kelly DC, Curtin CM, O\u0026apos;Brien FJ. Mechanosignalling in cartilage: an emerging target for the treatment of osteoarthritis. Nat Rev Rheumatol 2022;18(2):67-84. 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Repurposing of Pirfenidone (Anti-Pulmonary Fibrosis Drug) for Treatment of Rheumatoid Arthritis. Front Pharmacol 2021;12:631891. [eng].\u003c/li\u003e\n\u003cli\u003eGlasson SS, Chambers MG, Van Den Berg WB, Little CB. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse. Osteoarthritis Cartilage 2010;18 Suppl 3:S17-23. [eng].\u003c/li\u003e\n\u003cli\u003eZhang H, Shao Y, Yao Z, Liu L, Zhang H, Yin J, et al. Mechanical overloading promotes chondrocyte senescence and osteoarthritis development through downregulating FBXW7. Ann Rheum Dis 2022;81(5):676-86. [eng].\u003c/li\u003e\n\u003cli\u003eZhou F, Mei J, Han X, Li H, Yang S, Wang M, et al. Kinsenoside attenuates osteoarthritis by repolarizing macrophages through inactivating NF-\u0026kappa;B/MAPK signaling and protecting chondrocytes. Acta Pharm Sin B 2019;9(5):973-85. 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[eng].\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Osteoarthritis, Fixation, Treadmill, Cartilage damage, Synovium, Osteophytes","lastPublishedDoi":"10.21203/rs.3.rs-7495716/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7495716/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e\u003cp\u003eThis study aims to determine the pathological effects of joint stress imbalance on joint tissues from multiple mouse models, and provide useful information for clinicians to develop new strategies for the treatment and prevention of Osteoarthritis (OA).\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eIn this study, knee joint samples were collected at 2, 4 and 8 weeks, respectively, through the established mouse knee joint long-term fixation model, weight-bearing model and excessive exercise model. The pathological changes of knee joint cartilage, synovial inflammation and osteophytes were observed by Micro-CT and histological staining.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eWe found that long-term joint fixation resulted in significant pain, articular cartilage damage, synovial infiltration, and cartilaginous osteophyte of knee joints in mice, and the joint injury became more serious with the extension of fixation time. OA cartilage damage also occurs on the non-fixation weight-bearing side, and the cartilage damage becomes more severe with the extension of weight-bearing time, but it did not cause synovial inflammation and osteophyte formation. However, long-term excessive exercise in mice did not lead to obvious OA lesions such as cartilage damage, synovial inflammation and osteophyte formation, but it increases pain sensitivity.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eThis study can provide theoretical guidance for postoperative rehabilitation or exercise in clinical patients: the pathological process of tissue repair and OA lesions should be balanced as much as possible, and the joint fixation time should be shortened to reduce the occurrence of OA. For those who do not have meniscus lesions, moderate exercise can be performed to promote cartilage function, while for those with meniscus lesions or long-term weight bearing, joint movement should be reduced to repair meniscus lesions or reduce weight bearing to reduce the occurrence of OA.\u003c/p\u003e","manuscriptTitle":"Pathological effects of joint stress imbalance on joint tissues","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-11 10:54:46","doi":"10.21203/rs.3.rs-7495716/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"23143945-a1d5-491b-a2b3-65f79025d0a1","owner":[],"postedDate":"September 11th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-15T10:53:53+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-11 10:54:46","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7495716","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7495716","identity":"rs-7495716","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}