Effects of different combinations of mechanical stress intensity, duration, and frequency on the articular cartilage in mice

Effects of different combinations of mechanical stress intensity, duration, and frequency on the articular cartilage in mice | 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 Effects of different combinations of mechanical stress intensity, duration, and frequency on the articular cartilage in mice Yoshio Wakimoto, Yasushi Mimura, Shota Inoue, Masato Nomura, Hideki Moriyama This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3907866/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 29 Jul, 2024 Read the published version in Molecular Biology Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Background Understanding how healthy articular cartilage responds to mechanical stress is critical. Moderate mechanical stress has positive effects on the cartilage, such as maintaining cartilage homeostasis. The degree of mechanical stress is determined by a combination of intensity, frequency, and duration; however, the best combination of these parameters for knee cartilage remains unclear. This study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical stress on healthy knee articular cartilage in vitro and in vivo. Methods and results In this study, 33 male mice were used. Chondrocytes isolated from mouse knee joints were subjected to different cyclic tensile strains (CTSs) and assessed by measuring the expression of cartilage matrix-related genes. Furthermore, the histological characteristics of mouse tibial cartilages were quantified using different treadmill exercises. Chondrocytes and mice were divided into the control group and eight intervention groups: high-intensity, high-frequency, and long-duration; high-intensity, high-frequency, and short-duration; high-intensity, low-frequency, and long-duration; high-intensity, low-frequency, and short-duration; low-intensity, high-frequency, and long-duration; low-intensity, high-frequency, and short-duration; low-intensity, high-frequency, and short-duration; low-intensity, low-frequency, and long-duration; low-intensity, low-frequency, and short-duration. In low-intensity CTSs, chondrocytes showed anabolic responses by altering the mRNA expression of COL2A1 in short durations and SOX9 in long durations. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises minimized chondrocyte hypertrophy and enhanced aggrecan synthesis in tibial cartilages. Conclusion Low-intensity, low-frequency, and long-duration stress is the best combination for healthy knee cartilage to maintain homeostasis and activate anabolic responses. Our findings provide a significant scientific basis for exercise and lifestyle instructions. mechanical stress cyclic tensile strain treadmill exercise knee cartilage metabolism knee cartilage homeostasis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Introduction Knee joints bear a majority of the body weight; thus, the articular cartilage of the knee joints is particularly more likely to degenerate [ 1 ]. Because chondrocyte metabolism is low and intact cartilage has no blood supply [ 2 ], natural cartilage repair is limited. Mechanical stress is an essential factor for regulating cartilage homeostasis [ 3 ]; therefore, understanding how healthy articular cartilage responds to mechanical stress is critical for maintaining healthy cartilages. Moderate mechanical stress has positive effects on increasing the anabolic responses of the cartilage matrix [ 3 ], whereas excessive mechanical stress induces catabolic responses [ 4 , 5 ]. A combination of intensity, frequency, and duration determines the degree of mechanical stress [ 6 ]. The effects of these parameters on cartilage response and health have been extensively examined in vitro and in vivo [ 3 , 4 , 7 – 9 ]. Cyclic tensile strain (CTS) can be applied to chondrocytes in various strain intensities, frequencies, and durations [ 10 ]. A review showed that high-intensity, high-frequency, and long-duration CTS increases catabolic responses, and CTS of 3–10% intensity, 0.17–0.5 Hz, and 2–12 h may induce anabolic responses in healthy chondrocytes [ 8 ]. However, direct comparison is difficult because different experimental conditions (e.g., animal species, age, and cell type) were used. Running or walking is one of the most common weight-bearing activities and can simulate long-term stress on weight-bearing joints [ 11 ]. Moderate running increases cartilage thickness and promotes cartilage matrix synthesis and cartilage protection in a healthy animal model [ 12 , 13 ]. Conversely, high-intensity and long-duration or long-distance running accelerates cartilage matrix degradation and cartilage thinning [ 3 , 9 , 11 ]. In contrast, some studies have reported that the thickness and matrix of healthy cartilage do not change or conversely increase with excessive running [ 3 , 12 , 14 – 17 ]. Taken together, there is no consensus on the optimal combination of intensity, frequency, and duration for eliciting preferred responses in healthy knee articular cartilage [ 18 , 19 ]. Therefore, this study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical stress on healthy knee articular cartilages. Materials and Methods Experimental animals and animal care In this study, 33 male C57BL/6J mice (7 weeks old, with a mean body weight of 19–24 g) purchased from Japan SLC (Shizuoka, Japan) were used. The animals were housed in standard cages (3 mice/cage) under a 12-h dark/light cycle at a constant temperature of 22°C ± 1°C and allowed free access to water and standard foods. Six mice were used for CTS, and 27 mice were used for treadmill exercises. Isolation and culture of chondrocytes The mice were euthanized by exsanguination under anesthesia, and primary chondrocytes were isolated from the femoral condyles and tibial plateau according to a previous study [ 20 ]. After rinsing the cartilage pieces (excluding the subchondral bone that appears brown) with phosphate-buffered saline, chondrocytes were isolated from the cartilage using 0.4% collagenase (034-22363; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) overnight at 37°C. These chondrocytes were seeded in a 35-mm cell culture dish (353801; BD Falcon, Tokyo, Japan). They were then cultured in Dulbecco’s Modified Eagle Medium–Ham’s F12 medium (DMEN/HAM’S F-12; 042-30555; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) supplemented with 10% fetal bovine serum (Gibco 12,483,020; Thermo-Fisher Scientific, Inc., MA, USA), 50 units/mL penicillin, and 50 µg/mL streptomycin (168-23191; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) in an incubator maintained at 37°C with 5% CO 2 . The medium was changed every 3 days. At up to 80–90% confluency, the chondrocytes were harvested with a trypsin–ethylenediaminetetraacetic acid solution (209-16941; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) for passage. Chondrocytes were seeded at 2.5 × 10 5 cells in 35-mm culture dishes (353001; BD Falcon, Tokyo, Japan) (passage 1) or a 10-cm 2 chamber (STB-CH-10; Strex Inc., Osaka, Japan) coated with type I collagen (IPC-50; AteloCell, Koken, Tokyo, Japan) (passage 2). Chondrocyte cultures were continued by changing the medium every 3 days. Exposure of chondrocytes to CTS and RNA extraction After the chondrocytes of passage 2 reached 80–90% confluency, CTS was applied to the chondrocytes using the Strex device (STB-140; Strex Inc., Osaka, Japan). In the chondrocytes of several species, including mice, chondrocytes of passage 2 maintain their shape and phenotype [ 21 , 22 ]. CTS with a sinusoidal waveform was applied to chondrocytes, which were divided into the following nine groups (n = 4 chambers): control (no intervention); high-intensity, high-frequency (1.0 Hz), and long-duration (24 h) (HHL); high-intensity, high-frequency, and short-duration (12 h) (HHS); high-intensity, low-frequency (0.5 Hz), and long-duration (HLL); high-intensity, low-frequency, and short-duration (HLS); low-intensity (8%), high-frequency, and long-duration (LHL); low-intensity, high-frequency, and short-duration (LHS); low-intensity, low-frequency, and long-duration (LLL); and low-intensity, low-frequency, and short-duration (LLS) (Table 1 ). The intensity, duration, and frequency of this CTS protocol were determined based on a previous review [ 8 ]. Immediately after CTS, total RNA was extracted from chondrocytes using ISOSPIN Cell & Tissue RNA (314–08211; NIPPON GENE CO., LTD, Tokyo, Japan), according to the manufacturer’s instructions. The purity and concentration of the extracted total RNA were measured using a BioPhotometer D30 (Eppendorf, Hamburg, Germany). Table 1 Groups and stretch protocols for cyclic tensile stretch of articular chondrocytes Groups Intensity (%) Frequency (Hz) Duration (hours) Control – – – High-intensity, high-frequency, and long-duration (HHL) 15 1.0 24 High-intensity, high-frequency, and short-duration (HHS) 12 High-intensity, low-frequency, and long-duration (HLL) 0.5 24 High-intensity, low-frequency, and short-duration (HLS) 12 Low-intensity, high-frequency, and long-duration (LHL) 8 1.0 24 Low-intensity, high-frequency, and short-duration (LHS) 12 Low-intensity, low-frequency, and long-duration (LLL) 0.5 24 Low-intensity, low-frequency, and short-duration (LLS) 12 Analysis of gene expression in chondrocytes using quantitative real-time polymerase chain reaction (qRT-PCR) Reverse transcription and qRT-PCR were performed using the StepOne Real-Time PCR system (Thermo-Fisher Scientific Inc., MA, USA) with the TaqMan™ Fast Virus 1-Step Master Mix (Thermo-Fisher Scientific Inc., MA, USA) and Gene Expression Assays (Applied Biosystems, CA, USA) for collagen type II alpha1 mRNA ( COL2A1 ; Mm01309565_m1), aggrecan mRNA ( ACAN ; Mm00545794_m1), sex-determining region Y-box 9 (SOX9) mRNA ( SOX9 ; Mm00448840_m1), a disintegrin-like and metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) mRNA ( ADAMTS5 ; Mm00478620_m1), matrix metallopeptidase 13 (MMP13) mRNA ( Mmp13 ; Mm00439491_m1), and 18S ribosomal(18s) mRNA ( 18s : Mm03928990_g1). Their expression levels were analyzed using the 2 −Δ Δ CT method [ 23 , 24 ] and normalized to 18s levels [ 25 ]. Treadmill exercise protocol A treadmill device (MK-680, Muromachi Kikai Co, Ltd., Tokyo, Japan) was used to exercise the mice. Twenty-seven mice were randomly divided into the following nine groups (n = 3 mice): control (no exercise); high-intensity (18 m/min), high-frequency (every day), and long-duration (60 min/day) (HHL); high-intensity, high-frequency, and short-duration (15 min/day) (HHS); high-intensity, low-frequency (once every 3 days), and long-duration (HLL); high-intensity, low-frequency, and short-duration (HLS); low-intensity (8 m/min), high-frequency, and long-duration (LHL); low-intensity, high-frequency, and short-duration (LHS); low-intensity, low-frequency, and long-duration (LLL); and low-intensity, low-frequency, and short-duration (LLS) (Table 2 ). The gradient of the treadmill was graded 5% uphill. The speeds of 18 and 8 m/min correspond to running and walking for mice, respectively [ 26 , 27 ]. All mice were trained on the treadmill once a day for 4 weeks after a 1-week acclimation period to treadmill exercises. Table 2 Groups and exercise protocols for treadmill exercises in mice Groups Intensity (m/min) Frequency Duration (min) Total distance (km) Control – – – – High-intensity, high-frequency, and long-duration (HHL) 18 Every day 60 30.2 High-intensity, high-frequency, and short-duration (HHS) 15 7.6 High-intensity, low-frequency, and long-duration (HLL) Once every 3 days 60 10.1 High-intensity, low-frequency, and short-duration (HLS) 15 2.5 Low-intensity, high-frequency, and long-duration (LHL) 8 Every day 60 13.4 Low-intensity, high-frequency, and short-duration (LHS) 15 3.4 Low-intensity, low-frequency, and long-duration (LLL) Once every 3 days 60 4.5 Low-intensity, low-frequency, and short-duration (LLS) 15 1.1 Sampling and histological preparation We prepared undecalcified frozen sections as previously described [ 28 ]. At the end of the experimental period, all mice were euthanized by exsanguination under anesthesia, and the knee joints were harvested. The samples were immediately freeze-embedded in 5% carboxymethyl cellulose gel. Blocks were cut into slices, and 5-µm frontal sections of the proximal tibia were prepared. The right and left tibias of each animal served as different samples, and four of six tibias were randomly selected. The sample sizes were set according to Arifin et al. [ 29 ] (minimum sample size = 2.1) and confirmed by power analysis based on pilot results. Histomorphometrical analyses We measured the articular cartilage thickness and the number of chondrocytes on digitized images of histological sections stained with safranin-O/fast green. According to our previous methods [ 30 ], the articular cartilage thickness of the tibia was measured. The cartilage thickness for each specimen was determined by averaging three sections spaced 180 µm apart. The number of chondrocytes was quantified on sections stained with safranin-O/fast green [ 31 ]. The number of chondrocytes was manually counted using cells with visible nuclei [ 32 ]. Histochemical and immunohistochemical analyses Alkaline phosphatase (ALP) activity was detected according to the manufacturer’s instructions (Sigma387A-1KT, Sigma-Aldrich Japan, Tokyo, Japan), and the sections were counterstained with eosin. According to the manufacturer’s instructions for the ApopTag® Peroxidase In Situ Apoptosis Detection Kit (Chemicon S7100, Chemicon International, CA, USA), apoptosis-positive cells were detected using the TUNEL method. According to the protocols established in our laboratory [ 32 ], sections were immunostained with antibodies against type II collagen (diluted 1:150, ab21291; Abcam, Tokyo, Japan), aggrecan (diluted 1:400, AB1031; Millipore, MA, USA), SRY-Box Transcription Factor 9 (SOX9; diluted 1:500, ab3697; Abcam), matrix metalloproteinase 13 (MMP13; diluted 1:400, ab39012; Abcam), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5; diluted 1:500, ab41037; Abcam), type X collagen (diluted 1:10,000, LB-0092; LSL, Tokyo, Japan), lubricin (diluted 1:1,500, ab28484; Abcam), and proliferating cell nuclear antigen (PCNA) (diluted 1:1000, #13110, Cell Signaling Technology Inc., Tokyo, Japan). Subsequent reactions were performed using the streptavidin–biotin–peroxidase complex technique with an Elite ABC kit (diluted 1:50, PK-6100, Vector Laboratories, CA, USA). Immunoreactivity was visualized using 3,30-diaminobenzidine tetrahydrochloride (K3466; Dako Japan, Tokyo, Japan). We used hematoxylin (K3466, Dako Japan, Tokyo, Japan) as counterstaining to distinguish between calcified (deep zone of articular cartilage where calcified cartilage occupies more) and uncalcified regions, as previously described [ 33 ]. The staining intensity of type II collagen was measured as previously described [ 33 ]. For aggrecan, the immune-positive area was measured using Image J, and the ratio of the immune-positive area to the articular cartilage area was calculated. For SOX9, lubricin, MMP13, ADAMTS5, type X collagen, ALP, PCNA, and apoptosis, the number of immune-positive cells was manually quantified. Statistical analyses All statistical analyses were performed using R version 3.6.0 (R Core team). The results were compared among all groups using one-way analysis of variance (ANOVA), followed by the Tukey–Welsch test. All graphs are presented as means ± standard deviations. P -values < 0.05 were used to indicate statistical significance for all statistical analyses. A post hoc power analysis for the one-way ANOVA test using R was used to confirm that sufficient samples had been used (Supplementary Tables 1 and 2). Results Effects of CTS on chondrocytes Anabolic gene expression The relative expression of COL2A1 mRNA in the short-duration groups (i.e., HLS, LHS, and LLS groups) was significantly increased compared with that in the control group and the long-duration groups of the same intensity (Fig. 1 a). The relative expression of ACAN mRNA in the HHS group was significantly increased compared with that in the control and all long-duration (i.e., HHL, HLL, LHL, and LLL) groups (Fig. 1 b). The relative expression of SOX9 mRNA in the low-intensity and long-duration (LHL and LLL) groups was significantly decreased or tended to decrease compared with that in the control group (Fig. 1 c). Catabolic gene expression The relative expression of MMP13 mRNA showed no significant difference among the groups (Fig. 1 d). The relative expression of ADAMTS5 mRNA in the HHL group was significantly increased compared with that in all other high-intensity groups (Fig. 1 e). Effects of treadmill exercise on the tibial cartilage Cartilage thickness and number of chondrocytes The cartilage surface was intact without cartilage damage, and no obvious difference in cartilage matrix staining to safranin as a reflection of proteoglycan content was observed between all groups (Fig. 2 a). The cartilage thickness in the total layer in the HHL group was significantly decreased or tended to decrease compared with that in all groups (Fig. 2 c). Furthermore, the number of chondrocytes in the total layer in the HHL group was significantly increased compared with that in the control group, particularly in the uncalcified layer in the HHL group, which was significantly increased compared with that in all groups, except for the HHS group (Figs. 2 b and 2 d). In contrast, the cartilage thickness in the total layer in the LHL group was significantly increased compared with that in the control, HHL, and HLS groups or tended to increase compared with that in the HLL group, although no significant differences were observed among the low-intensity groups (Fig. 2 c). The number of chondrocytes in the uncalcified layer in the LHL group was significantly increased compared with that in all other low-intensity groups (Figs. 2 b and 2 d). Consistent with the increase in the number of chondrocytes in the HHL group, the percentage of PCNA-positive cells, as a marker of cell proliferation, in the total and uncalcified layers in the HHL group was significantly increased compared with that in all groups, except for the HHS group (Supplementary Fig. 2). An increase in cartilage thickness with increased matrix synthesis is beneficial to articular cartilage, whereas an increase in cartilage thickness with cellular activities, such as chondrocyte hypertrophy, may be harmful to articular cartilage [ 34 ]. Therefore, we next evaluated protein changes associated with the cartilage matrix and chondrocyte hypertrophy. Markers of inhibitory effect on chondrocyte hypertrophy SOX9-positive cells, which contribute to the suppression of chondrocyte hypertrophy and the maintenance of cartilage homeostasis [ 35 ], were observed in both the uncalcified and calcified layers (Fig. 3 a). The percentage of SOX9-positive cells in the total layer in all high-intensity groups was significantly decreased compared with that in the control and low-intensity groups, and this decrease was more pronounced in the HHL group (Figs. 3 a and 3 b). Lubricin, which maintains cartilage integrity [ 36 ] and inhibits hypertrophy and catabolism in the cartilage [ 37 ], was localized on the cartilage surface and within chondrocytes in the uncalcified layer (Fig. 3 c). The percentage of lubricin-positive cells in the high-intensity and high-frequency (HHL and HHS) and low-intensity and long-duration (LHL and LLL) groups was significantly increased or tended to increase compared with that in the control group (Figs. 3 c and 3 d). For the low-intensity groups, the percentage of lubricin-positive cells in the LHL and LLL groups was significantly increased or tended to increase compared with that in the LLS group (Figs. 3 c and 3 d). Cartilage matrix and proteases Type II collagen was evenly distributed throughout the cartilage, and staining intensity showed no significant differences among all groups (Figs. 4 a and 4 c). Aggrecan, which is the major proteoglycan in articular cartilage, was uniform across the entire cartilage and localized within or around chondrocytes (Fig. 4 b). The percentage of aggrecan-positive areas in the total layer in the HHS, LHS, and LLL groups was significantly increased compared with that in the control group and tended to increase compared with that in the HHL group (Figs. 4 b and 4 d). MMP13-positive cells, the most active in cleaving type II collagen, were mainly observed within chondrocytes in the calcified layer, whereas, in the high-intensity and high-frequency (HHL and HHS) groups, these cells were also detected in chondrocytes in the uncalcified layer (Fig. 5 a). The percentage of MMP13-positive cells in the total layer in the HHL group was significantly increased compared with that in all groups, except for the HHS group (Figs. 5 a and 5 b). ADAMTS5-positive cells, the major aggrecanase in mouse cartilage, were mainly observed within chondrocytes in the calcified layer (Fig. 5 c). The percentage of ADAMTS5-positive cells in the total layer in almost all exercise groups (except for LHL group) was significantly decreased compared with that in the control and HHL groups (Figs. 5 c and 5 d). Markers of chondrocyte hypertrophy ALP-positive cells, which are a marker of maturing chondrocytes [ 38 ], were located on the tidemark and in the calcified cartilage in all groups (Fig. 6 a). The percentage of ALP-positive cells in the uncalcified layer in all high-intensity groups was significantly increased or tended to increase compared with that in the control group (Fig. 6 c). Furthermore, the percentage of ALP-positive cells in the uncalcified layer in all high-intensity groups (except for the HLS group vs. LLS group) was significantly increased or tended to increase compared with that in the low-intensity groups (Fig. 6 c). Type X collagen-positive cells, which are a specific marker of chondrocyte hypertrophy, were observed in the calcified layer in all groups, and an increase in type X collagen-positive cells in the uncalcified layer was observed in the HHL group (Fig. 6 b). The percentage of type X collagen-positive cells in the total layer was significantly increased only in the HHL group compared with that in the control group (Fig. 6 d). The percentage of type X collagen-positive cells in the calcified layer in the high-frequency (i.e., HHL, HHS, LHL, and LHS) groups was significantly increased, regardless of the intensity, compared with that in the control group (Fig. 6 d). Furthermore, for the low-intensity groups, the percentage of type X collagen-positive cells in the calcified layer in the LHL group was significantly higher than that in the low-frequency (LLL and LLS) groups (Fig. 6 d). Furthermore, the percentage of TUNEL-positive cells, which indicates chondrocyte apoptosis [ 39 ], in the total layer in the high-intensity and high-frequency (HHL and HHS) groups was significantly increased compared with that in the control and low-intensity groups (Supplementary Figs. 5a and 5b). Discussion Our objective was to determine the combination of intensity, frequency, and duration that provides the best mechanical stress on healthy knee articular cartilage. We found that low-intensity CTS, regardless of frequency, activated anabolic gene expression in the cartilage matrix during the short duration and suppressed the downregulation of gene expression of SOX9 during the long duration. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises inhibited chondrocyte hypertrophy and increased aggrecan synthesis in the tibial cartilage. Chondrocytes adapt to changes in the mechanical environment by upregulating anabolic genes. The expression of these genes is then downregulated [ 8 , 40 ]. However, ACAN mRNA did not show time-dependent changes similar to COL2A1 mRNA. This poor response may be the reason that proteoglycan loss precedes type II collagen loss in early cartilage degeneration [ 41 ]. SOX9 mRNA, which contributes to cartilage homeostasis [ 35 ], was downregulated in the high-intensity groups, similar to the findings of previous studies [ 42 , 43 ]. However, interestingly, this downregulation also occurred in the low-intensity and short-duration groups, and the expression of SOX9 mRNA in the low-intensity and long-duration groups was maintained at the same level compared with that in the control group. TGF-β , which inhibits chondrocyte hypertrophy, contributes to the expression of SOX9 [ 44 ], and TGF-β mRNA is upregulated when CTS is applied with intensity of 5–12% and duration of 12–24 h [ 45 , 46 ]. Taken together, low-intensity CTS applied for > 12 h may be necessary to maintain cartilage homeostasis via SOX9 . Histological analysis after treadmill exercises revealed that stress in the HHL group had the most negative effects on healthy articular cartilage, and these findings are consistent with those of a previous review [ 9 ]. The increase in cartilage thickness with the increase in chondrocyte proliferation and hypertrophy, which were observed in the LHL group, have been reported as precursors to cartilage degeneration [ 34 , 47 , 48 ]. In contrast, although the cartilage thickness was similar to that in the control group, only the cartilage in the LLL group exhibited an increase in aggrecan and lubricin without an increase in chondrocyte hypertrophy compared with that in the control group, and these responses may maintain/improve the loading function of articular cartilage. Because high-frequency exercises resulted in catabolism, including chondrocyte hypertrophy, even in the low-intensity groups, daily exercises without rest may lead to chondrocyte hypertrophy [ 49 ]. In contrast, cartilage in the LLS group did not change. This is consistent with the findings of previous studies, which have reported that stress loads over a certain amount of time and frequency are necessary to maintain articular cartilage health [ 8 , 16 ]. This study has several limitations. First, the best mechanical stress on the articular cartilage presented in this study was achieved by combining large or small values for each parameter, but not specific values. Although further research is required, our findings provide useful insights for future research. Directly linking CTS to treadmill exercise results may be difficult because the load on the knee joint caused by treadmill exercises does not reflect the exact amount of stress on the cartilage or chondrocytes [ 49 ]. Interestingly, low-intensity and long-duration stress was a suitable combination for the articular cartilage in both the CTS and treadmill exercise results. Conclusions Our results show that low-intensity, low-frequency, and long-duration stress is the best combination for healthy knee articular cartilage to maintain homeostasis. Our results may provide a significant scientific basis for designing exercise programs and lifestyle instructions that consider the mechanical stress on articular cartilage. Declarations Competing interests The authors declare that they have no conflicts of interest with any financial organization regarding the materials used and discussed in the manuscript. Ethical approval All experimental procedures were approved by the Institutional Animal Care and Use Committee and performed according to the Kobe University Animal Experimentation Regulations (approval number: P140603). Funding This study was supported in part by Japan Society for the Promotion of Science KAKENHI Grant Number 25702032 and 19H04050. Author Contribution Y.W. and H.M. have given substantial contributions to the conception or the design of the manuscript; Y.W., S.I., and M.N. contributed to Collection and assembly of data; All authors contributed to analysis and interpretation of the data; Y.W., S.I., and H.M. contributed to statistical expertise; H.M. contributed to obtaining of funding; H.M. contributed to administrative, technical, or logistic support; Y.W., H.M., and Y.M. have participated to drafting the manuscript, and all authors revised it critically. All authors read and approved the final version of the manuscript. Acknowledgements We thank Toshihiro Akisue, Yuta Kohara, Eriko Mizuno, Changxin Li, Junpei Hatakeyama, and Daisuke Takamura for their assistance. Data availability All data that support the findings of this study are available from the corresponding author on reasonable request. References McGinty G, Irrgang JJ, Pezzullo D (2000) Biomechanical considerations for rehabilitation of the knee. Clin Biomech (Bristol Avon) 15:160–166. https://doi.org/10.1016/s0268-0033(99)00061-3 Chen S, Fu P, Wu H, Pei M (2017) Meniscus, articular cartilage and nucleus pulposus: a comparative review of cartilage-like tissues in anatomy, development and function. Cell Tissue Res 370:53–70. https://doi.org/10.1007/s00441-017-2613-0 Bricca A, Juhl CB, Grodzinsky AJ, Roos EM (2017) Impact of a daily exercise dose on knee joint cartilage - a systematic review and meta-analysis of randomized controlled trials in healthy animals. Osteoarthr Cartil 25:1223–1237. https://doi.org/10.1016/j.joca.2017.03.009 Ambrosio F, Tarabishy A, Kadi F, Brown EH, Sowa G (2011) Biological basis of exercise-based treatments for musculoskeletal conditions. PM R 3:S59–S63. https://doi.org/10.1016/j.pmrj.2011.05.001 Buckwalter JA, Martin JA (2006) Osteoarthritis. Adv Drug Deliv Rev 58:150–167. https://doi.org/10.1016/j.addr.2006.01.006 Mueller MJ, Maluf KS (2002) Tissue adaptation to physical stress: a proposed physical stress theory to guide physical therapist practice, education, and research. Phys Ther 82:383–403. https://doi.org/10.1093/ptj/82.4.383 Wolf A, Ackermann B, Steinmeyer J (2007) Collagen synthesis of articular cartilage explants in response to frequency of cyclic mechanical loading. Cell Tissue Res 327:155–166. https://doi.org/10.1007/s00441-006-0251-z Bleuel J, Zaucke F, Brüggemann GP, Niehoff A (2015) Effects of cyclic tensile strain on chondrocyte metabolism: A systematic review. PLoS ONE 10:e0119816. https://doi.org/10.1371/journal.pone.0119816 Mazor M, Best TM, Cesaro A, Lespessailles E, Toumi H (2019) Osteoarthritis biomarker responses and cartilage adaptation to exercise: a review of animal and human models. Scand J Med Sci Sports 29:1072–1082. https://doi.org/10.1111/sms.13435 Yang Y, Wang Y, Kong Y, Zhang X, Zhang H, Gang Y, Bai L (2019) Mechanical stress protects against osteoarthritis via regulation of the AMPK/NF-κB signaling pathway. J Cell Physiol 234:9156–9167. https://doi.org/10.1002/jcp.27592 Tang T, Muneta T, Ju YJ, Nimura A, Miyazaki K, Masuda H, Mochizuki T, Sekiya I (2008) Serum keratan sulfate transiently increases in the early stage of osteoarthritis during strenuous running of rats: protective effect of intraarticular hyaluronan injection. Arthritis Res Ther 10:R13. https://doi.org/10.1186/ar2363 Lequesne MG, Dang N, Lane NE (1997) Sport practice and osteoarthritis of the limbs. Osteoarthr Cartil 5:75–86. https://doi.org/10.1016/s1063-4584(97)80001-5 Tiderius CJ, Svensson J, Leander P, Ola T, Dahlberg L (2004) dGEMRIC (delayed gadolinium-enhanced MRI of cartilage) indicates adaptive capacity of human knee cartilage. Magn Reson Med 51:286–290. https://doi.org/10.1002/mrm.10714 Jones G, Ding C, Glisson M, Hynes K, Ma D, Cicuttini F (2003) Knee articular cartilage development in children: A longitudinal study of the effect of sex, growth, body composition, and physical activity. Pediatr Res 54:230–236. https://doi.org/10.1203/01.PDR.0000072781.93856.E6 Qi C, Changlin H (2006) Effects of moving training on histology and biomarkers levels of articular cartilage. J Surg Res 135:352–363. https://doi.org/10.1016/j.jss.2006.03.011 Racunica TL, Teichtahl AJ, Wang Y, Wluka AE, English DR, Giles GG, O’Sullivan R, Cicuttini FM (2007) Effect of physical activity on articular knee joint structures in community-based adults. Arthritis Rheum 57:1261–1268. https://doi.org/10.1002/art.22990 Changlin H, Qi C (2008) Effects of different mode high-intensity movement training on articular cartilage in histology - a randomised controlled trial on rabbit knee. Biol Sport 25:371–386 Cymet TC, Sinkov V (2006) Does long-distance running cause osteoarthritis? J Am Osteopath Assoc 106:342–345 Regnaux JP, Lefevre-Colau MM, Trinquart L, Nguyen C, Boutron I, Brosseau L, Ravaud P (2015) High-intensity versus low-intensity physical activity or exercise in people with hip or knee osteoarthritis. Cochrane Database Syst Rev 2015:CD010203. https://doi.org/10.1002/14651858.CD010203.pub2 Gosset M, Berenbaum F, Thirion S, Jacques C (2008) Primary culture and phenotyping of murine chondrocytes. Nat Protoc 3:1253–1260. https://doi.org/10.1038/nprot.2008.95 Kang SW, Yoo SP, Kim BS (2007) Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage. Bio Med Mater Eng 17:269–276 Tang Q, Zheng G, Feng Z, Tong M, Xu J, Hu Z, Shang P, Chen Y, Wang C, Lou Y, Chen D, Zhang D, Nisar M, Zhang X, Xu H, Liu H (2017) Wogonoside inhibits IL-1β induced catabolism and hypertrophy in mouse chondrocyte and ameliorates murine osteoarthritis. Oncotarget 8:61440–61456. https://doi.org/10.18632/oncotarget.18374 Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262 Schmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101–1108. https://doi.org/10.1038/nprot.2008.73 Al-Sabah A, Stadnik P, Gilbert SJ, Duance VC, Blain EJ (2016) Importance of reference gene selection for articular cartilage mechanobiology studies. Osteoarthr Cartil 24:719–730. https://doi.org/10.1016/j.joca.2015.11.007 Billat VL, Mouisel E, Roblot N, Melki J (2005) Inter- and intrastrain variation in mouse critical running speed. J Appl Physiol (1985) 98:1258–1263. https://doi.org/10.1152/japplphysiol.00991.2004 Sturgeon K, Schadler K, Muthukumaran G, Ding D, Bajulaiye A, Thomas NJ, Ferrari V, Ryeom S, Libonati JR (2014) Concomitant low-dose doxorubicin treatment and exercise. Am J Physiol Regul Integr Comp Physiol 307:R685–R692. https://doi.org/10.1152/ajpregu.00082.2014 Kawamoto T (2003) Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch Histol Cytol 66:123–143. https://doi.org/10.1679/aohc.66.123 Arifin WN, Zahiruddin WM (2017) Sample size calculation in animal studies using resource equation approach. Malays J Med Sci 24:101–105. https://doi.org/10.21315/mjms2017.24.5.11 Moriyama H, Yoshimura O, Kawamata S, Takayanagi K, Kurose T, Kubota A, Hosoda M, Tobimatsu Y (2008) Alteration in articular cartilage of rat knee joints after spinal cord injury. Osteoarthr Cartil 16:392–398. https://doi.org/10.1016/j.joca.2007.07.002 Pinamont WJ, Yoshioka NK, Young GM, Karuppagounder V, Carlson EL, Ahmad A, Elbarbary R, Kamal F (2020) Standardized histomorphometric evaluation of osteoarthritis in a surgical mouse model. J Vis Exp 159. https://doi.org/10.3791/60991 Moriyama H, Kanemura N, Brouns I, Pintelon I, Adriaensen D, Timmermans JP, Ozawa J, Kito N, Gomi T, Deie M (2012) Effects of aging and exercise training on the histological and mechanical properties of articular structures in knee joints of male rat. Biogerontology 13:369–381. https://doi.org/10.1007/s10522-012-9381-8 Nomura M, Sakitani N, Iwasawa H, Kohara Y, Takano S, Wakimoto Y, Kuroki H, Moriyama H (2017) Thinning of articular cartilage after joint unloading or immobilization. An experimental investigation of the pathogenesis in mice. Osteoarthr Cartil 25:727–736. https://doi.org/10.1016/j.joca.2016.11.013 Vignon E, Arlot M, Hartmann D, Moyen B, Ville G (1983) Hypertrophic repair of articular cartilage in experimental osteoarthrosis. Ann Rheum Dis 42:82–88. https://doi.org/10.1136/ard.42.1.82 Lefebvre V, Dvir-Ginzberg M (2017) SOX9 and the many facets of its regulation in the chondrocyte lineage. Connect Tissue Res 58:2–14. https://doi.org/10.1080/03008207.2016.1183667 Jay GD, Waller KA (2014) The biology of lubricin: near frictionless joint motion. Matrix Biol 39:17–24. https://doi.org/10.1016/j.matbio.2014.08.008 Ruan MZC, Erez A, Guse K, Dawson B, Bertin T, Chen Y, Jiang MM, Yustein J, Gannon F, Lee BH (2013) Proteoglycan 4 expression protects against the development of osteoarthritis. Sci Transl Med 5:176ra34. https://doi.org/10.1126/scitranslmed.3005409 Miao D, Scutt A (2002) Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage. J Histochem Cytochem 50:333–340. https://doi.org/10.1177/002215540205000305 Lotz M, Loeser RF (2012) Effects of aging on articular cartilage homeostasis. Bone 51:241–248. https://doi.org/10.1016/j.bone.2012.03.023 Zhu G, Qian Y, Wu W, Li R (2019) Negative effects of high mechanical tensile strain stimulation on chondrocyte injury in vitro. Biochem Biophys Res Commun 510:48–52. https://doi.org/10.1016/j.bbrc.2019.01.002 Hardingham T, Bayliss M (1990) Proteoglycans of articular cartilage: changes in aging and in joint disease. Semin Arthritis Rheum 20:12–33. https://doi.org/10.1016/0049-0172(90)90044-g Lin YY, Tanaka N, Ohkuma S, Iwabuchi Y, Tanne Y, Kamiya T, Kunimatsu R, Huang YC, Yoshioka M, Mitsuyoshi T, Tanimoto K, Tanaka E, Tanne K (2010) Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system. Eur J Oral Sci 118:151–158. https://doi.org/10.1111/j.1600-0722.2010.00710.x Sobue Y, Takahashi N, Ohashi Y, Suzuki M, Nishiume T, Kobayakawa T, Terabe K, Knudson W, Knudson C, Ishiguro N, Kojima T (2019) Inhibition of CD44 intracellular domain production suppresses bovine articular chondrocyte de-differentiation induced by excessive mechanical stress loading. Sci Rep 9:14901. https://doi.org/10.1038/s41598-019-50166-4 Tuli R, Tuli S, Nandi S, Huang X, Manner PA, Hozack WJ, Danielson KG, Hall DJ, Tuan RS (2003) Transforming growth factor-β-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk. J Biol Chem 278:41227–41236. https://doi.org/10.1074/jbc.M305312200 Ohno S, Tanaka N, Ueki M, Honda K, Tanimoto K, Yoneno K, Ohno-Nakahara M, Fujimoto K, Kato Y, Tanne K (2005) Mechanical regulation of terminal chondrocyte differentiation via RGD-CAP/beta ig-h3 induced by TGF-beta. Connect Tissue Res 46:227–234. https://doi.org/10.1080/03008200500346111 Furumatsu T, Matsumoto E, Kanazawa T, Fujii M, Lu Z, Kajiki R, Ozaki T (2013) Tensile strain increases expression of CCN2 and COL2A1 by activating TGF-β-Smad2/3 pathway in chondrocytic cells. J Biomech 46:1508–1515. https://doi.org/10.1016/j.jbiomech.2013.03.028 Calvo E, Palacios I, Delgado E, Sánchez-Pernaute O, Largo R, Egido J, Herrero-Beaumont G (2004) Histopathological correlation of cartilage swelling detected by magnetic resonance imaging in early experimental osteoarthritis. Osteoarthr Cartil 12:878–886. https://doi.org/10.1016/j.joca.2004.07.007 Dreier R (2010) Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther 12:216. https://doi.org/10.1186/ar3117 Miller RH (2017) Joint loading in runners does not initiate knee osteoarthritis. Exerc Sport Sci Rev 45:87–95. https://doi.org/10.1249/JES.0000000000000105 Additional Declarations No competing interests reported. Supplementary Files SupplementaryInformation.docx Cite Share Download PDF Status: Published Journal Publication published 29 Jul, 2024 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 02 May, 2024 Reviews received at journal 02 Mar, 2024 Reviewers agreed at journal 03 Feb, 2024 Reviewers invited by journal 01 Feb, 2024 Editor assigned by journal 01 Feb, 2024 Submission checks completed at journal 01 Feb, 2024 First submitted to journal 28 Jan, 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. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-3907866","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":270447440,"identity":"a1d16760-42a5-4454-9d90-74afb2a025fe","order_by":0,"name":"Yoshio Wakimoto","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABAklEQVRIie3PMUsDMRTA8YTA69Jwq6XgZ4gcdDrwg7jYJZ2yZ6g1U1wE1xZK/Qp1ufkdgdxy0LXgUhB0cahbR3Mn6HS9ugnmP7zh8X7DIyQW+5MBQSKycyAAhOiwYMycQLRMk4ZUNaHdJFy68cIEQm296SCi9GP8sJKuEeQLX91cJXeBHHTeTiqJxcJmTCCUKc9LNXfU0PvquZWMcGIctxIE9uyQ516ZQBi1R8jmrSau/0WWXj12kq1E16/c2cCAH3IzVesucrl9vS7mWoqEweRi6VE9BVIc+2XwINP9XmS3tmdHu/fpTK02rtgddDv5iTXTNRNPuP9u9pvjWCwW+yd9AubvYGkqKfAzAAAAAElFTkSuQmCC","orcid":"","institution":"Kobe University","correspondingAuthor":true,"prefix":"","firstName":"Yoshio","middleName":"","lastName":"Wakimoto","suffix":""},{"id":270447441,"identity":"d25a2b00-12b1-461b-8181-259ec4ebbe44","order_by":1,"name":"Yasushi Mimura","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Yasushi","middleName":"","lastName":"Mimura","suffix":""},{"id":270447442,"identity":"eb36d871-9f9a-48d0-b50d-aa6750551ba3","order_by":2,"name":"Shota Inoue","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Shota","middleName":"","lastName":"Inoue","suffix":""},{"id":270447443,"identity":"ec6536cc-3d0e-452f-af97-9ac80ed974be","order_by":3,"name":"Masato Nomura","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Masato","middleName":"","lastName":"Nomura","suffix":""},{"id":270447444,"identity":"3ea743f8-f90a-431a-864c-d57b41f5efeb","order_by":4,"name":"Hideki Moriyama","email":"","orcid":"","institution":"Kobe University","correspondingAuthor":false,"prefix":"","firstName":"Hideki","middleName":"","lastName":"Moriyama","suffix":""}],"badges":[],"createdAt":"2024-01-29 04:14:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3907866/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3907866/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-024-09762-5","type":"published","date":"2024-07-29T15:57:22+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":50556249,"identity":"9ee718b4-cfcf-43f3-a369-8b634ac36a7c","added_by":"auto","created_at":"2024-02-02 12:40:32","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":329547,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of cyclic tensile strain on gene expression in mouse articular chondrocytes. The gene expression levels of (a) \u003cem\u003eCOL2A1\u003c/em\u003e, (b) \u003cem\u003eACAN\u003c/em\u003e, (c) \u003cem\u003eSOX9\u003c/em\u003e, (d) \u003cem\u003eMMP13\u003c/em\u003e, and (e) \u003cem\u003eADAMTS5\u003c/em\u003e were analyzed using quantitative PCR, normalized to the housekeeping gene \u003cem\u003e18s\u003c/em\u003e, and relative to the control group. Untreated chondrocytes were used as the control group. Data are presented as means ± standard deviations. Statistical differences are shown as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e control; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e control; †\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HHL; ††\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHL; ‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HHS; ‡‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHS; §\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLL; ¶\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLS; ¶¶\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HLS; δδ\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e LHL; ♭\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003eLHS; ##\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e LLL. HHL: high-intensity, high-frequency, and long-duration; HHS: high-intensity, high-frequency, and short-duration; HLL: high-intensity, low-frequency, and long-duration; HLS: high-intensity, low-frequency, and short-duration; LHL: low-intensity, high-frequency, and long-duration; LHS: low-intensity, high-frequency, and short-duration; LLL: low-intensity, low-frequency, and long-duration; LLS: low-intensity, low-frequency, and short-duration; \u003cem\u003eCOL2A1\u003c/em\u003e: collagen type II alpha1; \u003cem\u003eACAN\u003c/em\u003e: aggrecan, \u003cem\u003eSOX9\u003c/em\u003e: sex-determining region Y-box 9; \u003cem\u003eMMT13\u003c/em\u003e: matrix metallopeptidase 13; \u003cem\u003eADAMTS5\u003c/em\u003e: a disintegrin-like and metallopeptidase with thrombospondin type 1 motif 5; \u003cem\u003e18s\u003c/em\u003e: 18S ribosomal mRNA\u003c/p\u003e","description":"","filename":"Fig.1.png","url":"https://assets-eu.researchsquare.com/files/rs-3907866/v1/b27ecf1eba1357aa5ab7ffd1.png"},{"id":50556263,"identity":"7d8bb167-e047-49b2-96bf-cc8b1486b0eb","added_by":"auto","created_at":"2024-02-02 12:40:32","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":3463940,"visible":true,"origin":"","legend":"\u003cp\u003eCartilage thickness and number of chondrocytes in the tibial cartilage. (a) Cartilage thickness in the tibia was measured on histological sections stained with safranin O/first green. Magnified images of the boxed area in (a) are shown in (b). Scale bars = 200 μm. (b) Histological observations of the number of chondrocytes. Scale bars = 50 μm. (c) Quantitative results of the mean cartilage thickness in the total layer. (d) Quantitative results of the mean number of chondrocytes in the total layer. Data are presented as means ± standard deviations. Statistical differences are shown as follows: *\u003cem\u003ep\u003c/em\u003e\u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e control; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e control; †\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HHL; ††\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHL; ‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HHS; ‡‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHS; §\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLL; §§\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HLL; ¶\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLS; ¶¶\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HLS; δ\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e LHL; δδ\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e LHL. HHL: high-intensity, high-frequency, and long-duration; HHS: high-intensity, high-frequency, and short-duration; HLL: high-intensity, low-frequency, and long-duration; HLS: high-intensity, low-frequency, and short-duration; LHL: low-intensity, high-frequency, and long-duration; LHS: low-intensity, high-frequency, and short-duration; LLL: low-intensity, low-frequency, and long-duration; LLS: low-intensity, low-frequency, and short-duration. All quantitative results (in the uncalcified, calcified, and total layers) are shown in Supplementary Fig. 1\u003c/p\u003e","description":"","filename":"Fig.2.png","url":"https://assets-eu.researchsquare.com/files/rs-3907866/v1/c1782fdddc7328382e789bf6.png"},{"id":50556250,"identity":"064e0055-069d-4ce5-90d7-c7fe37288109","added_by":"auto","created_at":"2024-02-02 12:40:32","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3231806,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of the tibial cartilage stained with (a) SOX9 and (c) lubricin and the quantitative results. (a) Scale bars = 40 μm. (b) Quantitative results of the percentage of SOX9-positive cells in the total layer. Data are presented as means ± standard deviations. (c) White arrowheads represented Lubricin-negative cells. Scale bars = 50 μm. (d) Quantitative results of the percentage of lubricin-negative cells in the total layer. Data are presented as means ± standard deviations. Statistical differences are shown as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e control; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e control; ††\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHL; ‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HHS; ‡‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHS; §\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLL; §§\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HLL; ¶\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLS; δ\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e LHL. HHL: high-intensity, high-frequency, and long-duration; HHS: high-intensity, high-frequency, and short-duration; HLL: high-intensity, low-frequency, and long-duration; HLS: high-intensity, low-frequency, and short-duration; LHL: low-intensity, high-frequency, and long-duration; LHS: low-intensity, high-frequency, and short-duration; LLL: low-intensity, low-frequency, and long-duration; LLS: low-intensity, low-frequency, and short-duration; SOX9: sex-determining region Y-box 9. All quantitative results of SOX9 (in the uncalcified, calcified, and total layers) are shown in Supplementary Fig. 3a\u003c/p\u003e","description":"","filename":"Fig.3.png","url":"https://assets-eu.researchsquare.com/files/rs-3907866/v1/d3fe8f4a0186e3604da1bdbc.png"},{"id":50556254,"identity":"76a4e48f-1e4c-4014-b37a-743032eea04f","added_by":"auto","created_at":"2024-02-02 12:40:32","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":3579711,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of the tibial cartilage stained with (a) type II collagen and (b) aggrecan and the quantitative results. (a) Scale bars = 100 μm. (b) Scale bars = 50 μm. (c) Quantitative results of staining intensity for type II collagen in the total layer. Data are presented as means ± standard deviations. (d) Quantitative results of the percentage of aggrecan-positive area in the total layer. Data are presented as means ± standard deviations. Statistical differences are shown as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e control; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e control; ¶\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLS. HHL: high-intensity, high-frequency, and long-duration; HHS: high-intensity, high-frequency, and short-duration; HLL: high-intensity, low-frequency, and long-duration; HLS: high-intensity, low-frequency, and short-duration; LHL: low-intensity, high-frequency, and long-duration; LHS: low-intensity, high-frequency, and short-duration; LLL: low-intensity, low-frequency, and long-duration; LLS: low-intensity, low-frequency, and short-duration. All quantitative results (in the uncalcified, calcified, and total layers) are shown in Supplementary Figs. 3b and 3c\u003c/p\u003e","description":"","filename":"Fig.4.png","url":"https://assets-eu.researchsquare.com/files/rs-3907866/v1/90f4fd43d0cf88f5f0205791.png"},{"id":50556277,"identity":"02799a72-a39c-46c7-b8ab-17fe8749efc8","added_by":"auto","created_at":"2024-02-02 12:40:32","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3137919,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of the tibial cartilage stained with (a) MMP13 and (c) ADAMTS5 and the quantitative results. (a) Black arrowheads represented MMP13-positive cells. Scale bars = 50 μm. (b) Quantitative results of the percentage of MMP13-positive cells in the total layer. Data are presented as means ± standard deviations. (c) Black arrowheads represented ADAMTS5-positive cells. Scale bars = 50 μm. (d) Quantitative results of the percentage of ADAMTS5-positive cells in the total layer. Data are presented as means ± standard deviations. Statistical differences are shown as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e control; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e control; ††\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHL; ‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HHS; ‡‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHS. HHL: high-intensity, high-frequency, and long-duration; HHS: high-intensity, high-frequency, and short-duration; HLL: high-intensity, low-frequency, and long-duration; HLS: high-intensity, low-frequency, and short-duration; LHL: low-intensity, high-frequency, and long-duration; LHS: low-intensity, high-frequency, and short-duration; LLL: low-intensity, low-frequency, and long-duration; LLS: low-intensity, low-frequency, and short-duration; MMT13: matrix metallopeptidase 13; ADAMTS5: a disintegrin-like metallopeptidase with thrombospondin type 1 motif 5. All quantitative results (in the uncalcified, calcified, and total layers) are shown in Supplementary Figs. 4a and 4b\u003c/p\u003e","description":"","filename":"Fig.5.png","url":"https://assets-eu.researchsquare.com/files/rs-3907866/v1/5f687741e1d1c0237ec53f21.png"},{"id":50556635,"identity":"01c2cc65-89de-4ff6-ae27-f43fa1041574","added_by":"auto","created_at":"2024-02-02 12:48:32","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4412450,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative images of the tibial cartilage stained with (a) ALP and (b) type X collagen and the quantitative results. (a) Scale bars = 200 μm. (b) Type X collagen-positive cells in the uncalcified layer are represented by arrowheads. Scale bars = 200 μm. (c) Quantitative results of the percentage of ALP-positive cells in the total and uncalcified layers. Data are presented as means ± standard deviations. (d) Quantitative results of the percentage of type X collagen-positive cells in the total and calcified layers. Data are presented as means ± standard deviations. Statistical differences are shown as follows: *\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs. \u003c/em\u003econtrol; **\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e control; †\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HHL; ††\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHL; ‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HHS; ‡‡\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HHS; §\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLL; §§\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HLL; ¶\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e HLS; ¶¶\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01 \u003cem\u003evs.\u003c/em\u003e HLS; δ\u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05 \u003cem\u003evs.\u003c/em\u003e LHL. HHL: high-intensity, high-frequency, and long-duration; HHS: high-intensity, high-frequency, and short-duration; HLL: high-intensity, low-frequency, and long-duration; HLS: high-intensity, low-frequency, and short-duration; LHL: low-intensity, high-frequency, and long-duration; LHS: low-intensity, high-frequency, and short-duration; LLL: low-intensity, low-frequency, and long-duration; LLS: low-intensity, low-frequency, and short-duration; ALP: alkaline phosphatase. All quantitative ALP results (in the uncalcified, calcified, and total layers) are shown in Supplementary Fig. 4c\u003c/p\u003e","description":"","filename":"Fig.6.png","url":"https://assets-eu.researchsquare.com/files/rs-3907866/v1/6dd93fa88a666f4e405996ca.png"},{"id":61793488,"identity":"e56200bd-2d39-48eb-b0be-52965da74e72","added_by":"auto","created_at":"2024-08-05 16:13:08","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":30837438,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3907866/v1/f33b95b3-e2d3-44a3-9aa5-b1101bf8a118.pdf"},{"id":50556283,"identity":"1fe27a5a-2ac4-4b71-93da-86c05739ce6e","added_by":"auto","created_at":"2024-02-02 12:40:32","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1552060,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformation.docx","url":"https://assets-eu.researchsquare.com/files/rs-3907866/v1/99300af261fd265b3ec5a7f7.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of different combinations of mechanical stress intensity, duration, and frequency on the articular cartilage in mice","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKnee joints bear a majority of the body weight; thus, the articular cartilage of the knee joints is particularly more likely to degenerate [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Because chondrocyte metabolism is low and intact cartilage has no blood supply [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], natural cartilage repair is limited. Mechanical stress is an essential factor for regulating cartilage homeostasis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]; therefore, understanding how healthy articular cartilage responds to mechanical stress is critical for maintaining healthy cartilages.\u003c/p\u003e \u003cp\u003eModerate mechanical stress has positive effects on increasing the anabolic responses of the cartilage matrix [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e], whereas excessive mechanical stress induces catabolic responses [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. A combination of intensity, frequency, and duration determines the degree of mechanical stress [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. The effects of these parameters on cartilage response and health have been extensively examined \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCyclic tensile strain (CTS) can be applied to chondrocytes in various strain intensities, frequencies, and durations [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A review showed that high-intensity, high-frequency, and long-duration CTS increases catabolic responses, and CTS of 3\u0026ndash;10% intensity, 0.17\u0026ndash;0.5 Hz, and 2\u0026ndash;12 h may induce anabolic responses in healthy chondrocytes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. However, direct comparison is difficult because different experimental conditions (e.g., animal species, age, and cell type) were used.\u003c/p\u003e \u003cp\u003eRunning or walking is one of the most common weight-bearing activities and can simulate long-term stress on weight-bearing joints [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Moderate running increases cartilage thickness and promotes cartilage matrix synthesis and cartilage protection in a healthy animal model [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. Conversely, high-intensity and long-duration or long-distance running accelerates cartilage matrix degradation and cartilage thinning [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In contrast, some studies have reported that the thickness and matrix of healthy cartilage do not change or conversely increase with excessive running [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan additionalcitationids=\"CR15 CR16\" citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTaken together, there is no consensus on the optimal combination of intensity, frequency, and duration for eliciting preferred responses in healthy knee articular cartilage [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Therefore, this study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical stress on healthy knee articular cartilages.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eExperimental animals and animal care\u003c/h2\u003e \u003cp\u003eIn this study, 33 male C57BL/6J mice (7 weeks old, with a mean body weight of 19\u0026ndash;24 g) purchased from Japan SLC (Shizuoka, Japan) were used. The animals were housed in standard cages (3 mice/cage) under a 12-h dark/light cycle at a constant temperature of 22\u0026deg;C\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C and allowed free access to water and standard foods. Six mice were used for CTS, and 27 mice were used for treadmill exercises.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eIsolation and culture of chondrocytes\u003c/h2\u003e \u003cp\u003eThe mice were euthanized by exsanguination under anesthesia, and primary chondrocytes were isolated from the femoral condyles and tibial plateau according to a previous study [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. After rinsing the cartilage pieces (excluding the subchondral bone that appears brown) with phosphate-buffered saline, chondrocytes were isolated from the cartilage using 0.4% collagenase (034-22363; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) overnight at 37\u0026deg;C. These chondrocytes were seeded in a 35-mm cell culture dish (353801; BD Falcon, Tokyo, Japan). They were then cultured in Dulbecco\u0026rsquo;s Modified Eagle Medium\u0026ndash;Ham\u0026rsquo;s F12 medium (DMEN/HAM\u0026rsquo;S F-12; 042-30555; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) supplemented with 10% fetal bovine serum (Gibco 12,483,020; Thermo-Fisher Scientific, Inc., MA, USA), 50 units/mL penicillin, and 50 \u0026micro;g/mL streptomycin (168-23191; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) in an incubator maintained at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. The medium was changed every 3 days.\u003c/p\u003e \u003cp\u003eAt up to 80\u0026ndash;90% confluency, the chondrocytes were harvested with a trypsin\u0026ndash;ethylenediaminetetraacetic acid solution (209-16941; FUJIFILM Wako Pure Chemical Co., Osaka, Japan) for passage. Chondrocytes were seeded at 2.5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e cells in 35-mm culture dishes (353001; BD Falcon, Tokyo, Japan) (passage 1) or a 10-cm\u003csup\u003e2\u003c/sup\u003e chamber (STB-CH-10; Strex Inc., Osaka, Japan) coated with type I collagen (IPC-50; AteloCell, Koken, Tokyo, Japan) (passage 2). Chondrocyte cultures were continued by changing the medium every 3 days.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eExposure of chondrocytes to CTS and RNA extraction\u003c/h2\u003e \u003cp\u003eAfter the chondrocytes of passage 2 reached 80\u0026ndash;90% confluency, CTS was applied to the chondrocytes using the Strex device (STB-140; Strex Inc., Osaka, Japan). In the chondrocytes of several species, including mice, chondrocytes of passage 2 maintain their shape and phenotype [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. CTS with a sinusoidal waveform was applied to chondrocytes, which were divided into the following nine groups (n\u0026thinsp;=\u0026thinsp;4 chambers): control (no intervention); high-intensity, high-frequency (1.0 Hz), and long-duration (24 h) (HHL); high-intensity, high-frequency, and short-duration (12 h) (HHS); high-intensity, low-frequency (0.5 Hz), and long-duration (HLL); high-intensity, low-frequency, and short-duration (HLS); low-intensity (8%), high-frequency, and long-duration (LHL); low-intensity, high-frequency, and short-duration (LHS); low-intensity, low-frequency, and long-duration (LLL); and low-intensity, low-frequency, and short-duration (LLS) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The intensity, duration, and frequency of this CTS protocol were determined based on a previous review [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Immediately after CTS, total RNA was extracted from chondrocytes using ISOSPIN Cell \u0026amp; Tissue RNA (314\u0026ndash;08211; NIPPON GENE CO., LTD, Tokyo, Japan), according to the manufacturer\u0026rsquo;s instructions. The purity and concentration of the extracted total RNA were measured using a BioPhotometer D30 (Eppendorf, Hamburg, Germany).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGroups and stretch protocols for cyclic tensile stretch of articular chondrocytes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntensity (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrequency (Hz)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDuration (hours)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-intensity, high-frequency, and long-duration (HHL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-intensity, high-frequency, and short-duration (HHS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-intensity, low-frequency, and long-duration (HLL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-intensity, low-frequency, and short-duration (HLS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-intensity, high-frequency, and long-duration (LHL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-intensity, high-frequency, and short-duration (LHS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-intensity, low-frequency, and long-duration (LLL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e24\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-intensity, low-frequency, and short-duration (LLS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eAnalysis of gene expression in chondrocytes using quantitative real-time polymerase chain reaction (qRT-PCR)\u003c/h2\u003e \u003cp\u003eReverse transcription and qRT-PCR were performed using the StepOne Real-Time PCR system (Thermo-Fisher Scientific Inc., MA, USA) with the TaqMan\u0026trade; Fast Virus 1-Step Master Mix (Thermo-Fisher Scientific Inc., MA, USA) and Gene Expression Assays (Applied Biosystems, CA, USA) for collagen type II alpha1 mRNA (\u003cem\u003eCOL2A1\u003c/em\u003e; Mm01309565_m1), aggrecan mRNA (\u003cem\u003eACAN\u003c/em\u003e; Mm00545794_m1), sex-determining region Y-box 9 (SOX9) mRNA (\u003cem\u003eSOX9\u003c/em\u003e; Mm00448840_m1), a disintegrin-like and metallopeptidase with thrombospondin type 1 motif 5 (ADAMTS5) mRNA (\u003cem\u003eADAMTS5\u003c/em\u003e; Mm00478620_m1), matrix metallopeptidase 13 (MMP13) mRNA (\u003cem\u003eMmp13\u003c/em\u003e; Mm00439491_m1), and 18S ribosomal(18s) mRNA (\u003cem\u003e18s\u003c/em\u003e: Mm03928990_g1). Their expression levels were analyzed using the 2\u003csup\u003e\u0026minus;Δ Δ CT\u003c/sup\u003e method [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] and normalized to \u003cem\u003e18s\u003c/em\u003e levels [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eTreadmill exercise protocol\u003c/h2\u003e \u003cp\u003eA treadmill device (MK-680, Muromachi Kikai Co, Ltd., Tokyo, Japan) was used to exercise the mice. Twenty-seven mice were randomly divided into the following nine groups (n\u0026thinsp;=\u0026thinsp;3 mice): control (no exercise); high-intensity (18 m/min), high-frequency (every day), and long-duration (60 min/day) (HHL); high-intensity, high-frequency, and short-duration (15 min/day) (HHS); high-intensity, low-frequency (once every 3 days), and long-duration (HLL); high-intensity, low-frequency, and short-duration (HLS); low-intensity (8 m/min), high-frequency, and long-duration (LHL); low-intensity, high-frequency, and short-duration (LHS); low-intensity, low-frequency, and long-duration (LLL); and low-intensity, low-frequency, and short-duration (LLS) (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). The gradient of the treadmill was graded 5% uphill. The speeds of 18 and 8 m/min correspond to running and walking for mice, respectively [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. All mice were trained on the treadmill once a day for 4 weeks after a 1-week acclimation period to treadmill exercises.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eGroups and exercise protocols for treadmill exercises in mice\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\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 \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eGroups\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eIntensity (m/min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrequency\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDuration (min)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTotal distance (km)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u0026ndash;\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-intensity, high-frequency, and long-duration (HHL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEvery day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e30.2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-intensity, high-frequency, and short-duration (HHS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-intensity, low-frequency, and long-duration (HLL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOnce every 3 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHigh-intensity, low-frequency, and short-duration (HLS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e2.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-intensity, high-frequency, and long-duration (LHL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003e8\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eEvery day\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-intensity, high-frequency, and short-duration (LHS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e3.4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-intensity, low-frequency, and long-duration (LLL)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eOnce every 3 days\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e4.5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLow-intensity, low-frequency, and short-duration (LLS)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSampling and histological preparation\u003c/h2\u003e \u003cp\u003eWe prepared undecalcified frozen sections as previously described [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. At the end of the experimental period, all mice were euthanized by exsanguination under anesthesia, and the knee joints were harvested. The samples were immediately freeze-embedded in 5% carboxymethyl cellulose gel. Blocks were cut into slices, and 5-\u0026micro;m frontal sections of the proximal tibia were prepared. The right and left tibias of each animal served as different samples, and four of six tibias were randomly selected. The sample sizes were set according to Arifin et al. [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] (minimum sample size\u0026thinsp;=\u0026thinsp;2.1) and confirmed by power analysis based on pilot results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eHistomorphometrical analyses\u003c/h2\u003e \u003cp\u003eWe measured the articular cartilage thickness and the number of chondrocytes on digitized images of histological sections stained with safranin-O/fast green. According to our previous methods [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], the articular cartilage thickness of the tibia was measured. The cartilage thickness for each specimen was determined by averaging three sections spaced 180 \u0026micro;m apart.\u003c/p\u003e \u003cp\u003eThe number of chondrocytes was quantified on sections stained with safranin-O/fast green [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The number of chondrocytes was manually counted using cells with visible nuclei [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eHistochemical and immunohistochemical analyses\u003c/h2\u003e \u003cp\u003eAlkaline phosphatase (ALP) activity was detected according to the manufacturer\u0026rsquo;s instructions (Sigma387A-1KT, Sigma-Aldrich Japan, Tokyo, Japan), and the sections were counterstained with eosin.\u003c/p\u003e \u003cp\u003eAccording to the manufacturer\u0026rsquo;s instructions for the ApopTag\u0026reg; Peroxidase In Situ Apoptosis Detection Kit (Chemicon S7100, Chemicon International, CA, USA), apoptosis-positive cells were detected using the TUNEL method.\u003c/p\u003e \u003cp\u003eAccording to the protocols established in our laboratory [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], sections were immunostained with antibodies against type II collagen (diluted 1:150, ab21291; Abcam, Tokyo, Japan), aggrecan (diluted 1:400, AB1031; Millipore, MA, USA), SRY-Box Transcription Factor 9 (SOX9; diluted 1:500, ab3697; Abcam), matrix metalloproteinase 13 (MMP13; diluted 1:400, ab39012; Abcam), disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5; diluted 1:500, ab41037; Abcam), type X collagen (diluted 1:10,000, LB-0092; LSL, Tokyo, Japan), lubricin (diluted 1:1,500, ab28484; Abcam), and proliferating cell nuclear antigen (PCNA) (diluted 1:1000, #13110, Cell Signaling Technology Inc., Tokyo, Japan). Subsequent reactions were performed using the streptavidin\u0026ndash;biotin\u0026ndash;peroxidase complex technique with an Elite ABC kit (diluted 1:50, PK-6100, Vector Laboratories, CA, USA). Immunoreactivity was visualized using 3,30-diaminobenzidine tetrahydrochloride (K3466; Dako Japan, Tokyo, Japan). We used hematoxylin (K3466, Dako Japan, Tokyo, Japan) as counterstaining to distinguish between calcified (deep zone of articular cartilage where calcified cartilage occupies more) and uncalcified regions, as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe staining intensity of type II collagen was measured as previously described [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eFor aggrecan, the immune-positive area was measured using Image J, and the ratio of the immune-positive area to the articular cartilage area was calculated.\u003c/p\u003e \u003cp\u003eFor SOX9, lubricin, MMP13, ADAMTS5, type X collagen, ALP, PCNA, and apoptosis, the number of immune-positive cells was manually quantified.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analyses\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed using R version 3.6.0 (R Core team). The results were compared among all groups using one-way analysis of variance (ANOVA), followed by the Tukey\u0026ndash;Welsch test. All graphs are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviations. \u003cem\u003eP\u003c/em\u003e-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05 were used to indicate statistical significance for all statistical analyses. A post hoc power analysis for the one-way ANOVA test using R was used to confirm that sufficient samples had been used (Supplementary Tables\u0026nbsp;1 and 2).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eEffects of CTS on chondrocytes\u003c/h2\u003e \u003cdiv id=\"Sec14\" class=\"Section3\"\u003e \u003ch2\u003eAnabolic gene expression\u003c/h2\u003e \u003cp\u003eThe relative expression of \u003cem\u003eCOL2A1\u003c/em\u003e mRNA in the short-duration groups (i.e., HLS, LHS, and LLS groups) was significantly increased compared with that in the control group and the long-duration groups of the same intensity (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea).\u003c/p\u003e \u003cp\u003eThe relative expression of \u003cem\u003eACAN\u003c/em\u003e mRNA in the HHS group was significantly increased compared with that in the control and all long-duration (i.e., HHL, HLL, LHL, and LLL) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003eThe relative expression of \u003cem\u003eSOX9\u003c/em\u003e mRNA in the low-intensity and long-duration (LHL and LLL) groups was significantly decreased or tended to decrease compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eCatabolic gene expression\u003c/h2\u003e \u003cp\u003eThe relative expression of \u003cem\u003eMMP13\u003c/em\u003e mRNA showed no significant difference among the groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eThe relative expression of \u003cem\u003eADAMTS5\u003c/em\u003e mRNA in the HHL group was significantly increased compared with that in all other high-intensity groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ee).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eEffects of treadmill exercise on the tibial cartilage\u003c/h2\u003e \u003cdiv id=\"Sec17\" class=\"Section3\"\u003e \u003ch2\u003eCartilage thickness and number of chondrocytes\u003c/h2\u003e \u003cp\u003eThe cartilage surface was intact without cartilage damage, and no obvious difference in cartilage matrix staining to safranin as a reflection of proteoglycan content was observed between all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea). The cartilage thickness in the total layer in the HHL group was significantly decreased or tended to decrease compared with that in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). Furthermore, the number of chondrocytes in the total layer in the HHL group was significantly increased compared with that in the control group, particularly in the uncalcified layer in the HHL group, which was significantly increased compared with that in all groups, except for the HHS group (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed). In contrast, the cartilage thickness in the total layer in the LHL group was significantly increased compared with that in the control, HHL, and HLS groups or tended to increase compared with that in the HLL group, although no significant differences were observed among the low-intensity groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec). The number of chondrocytes in the uncalcified layer in the LHL group was significantly increased compared with that in all other low-intensity groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb and \u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eConsistent with the increase in the number of chondrocytes in the HHL group, the percentage of PCNA-positive cells, as a marker of cell proliferation, in the total and uncalcified layers in the HHL group was significantly increased compared with that in all groups, except for the HHS group (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cp\u003eAn increase in cartilage thickness with increased matrix synthesis is beneficial to articular cartilage, whereas an increase in cartilage thickness with cellular activities, such as chondrocyte hypertrophy, may be harmful to articular cartilage [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Therefore, we next evaluated protein changes associated with the cartilage matrix and chondrocyte hypertrophy.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eMarkers of inhibitory effect on chondrocyte hypertrophy\u003c/h2\u003e \u003cp\u003eSOX9-positive cells, which contribute to the suppression of chondrocyte hypertrophy and the maintenance of cartilage homeostasis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], were observed in both the uncalcified and calcified layers (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea). The percentage of SOX9-positive cells in the total layer in all high-intensity groups was significantly decreased compared with that in the control and low-intensity groups, and this decrease was more pronounced in the HHL group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eLubricin, which maintains cartilage integrity [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e] and inhibits hypertrophy and catabolism in the cartilage [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], was localized on the cartilage surface and within chondrocytes in the uncalcified layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). The percentage of lubricin-positive cells in the high-intensity and high-frequency (HHL and HHS) and low-intensity and long-duration (LHL and LLL) groups was significantly increased or tended to increase compared with that in the control group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). For the low-intensity groups, the percentage of lubricin-positive cells in the LHL and LLL groups was significantly increased or tended to increase compared with that in the LLS group (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eCartilage matrix and proteases\u003c/h2\u003e \u003cp\u003eType II collagen was evenly distributed throughout the cartilage, and staining intensity showed no significant differences among all groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec). Aggrecan, which is the major proteoglycan in articular cartilage, was uniform across the entire cartilage and localized within or around chondrocytes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). The percentage of aggrecan-positive areas in the total layer in the HHS, LHS, and LLL groups was significantly increased compared with that in the control group and tended to increase compared with that in the HHL group (Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb and \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eMMP13-positive cells, the most active in cleaving type II collagen, were mainly observed within chondrocytes in the calcified layer, whereas, in the high-intensity and high-frequency (HHL and HHS) groups, these cells were also detected in chondrocytes in the uncalcified layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea). The percentage of MMP13-positive cells in the total layer in the HHL group was significantly increased compared with that in all groups, except for the HHS group (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). ADAMTS5-positive cells, the major aggrecanase in mouse cartilage, were mainly observed within chondrocytes in the calcified layer (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec). The percentage of ADAMTS5-positive cells in the total layer in almost all exercise groups (except for LHL group) was significantly decreased compared with that in the control and HHL groups (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ed).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003eMarkers of chondrocyte hypertrophy\u003c/h2\u003e \u003cp\u003eALP-positive cells, which are a marker of maturing chondrocytes [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e], were located on the tidemark and in the calcified cartilage in all groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea). The percentage of ALP-positive cells in the uncalcified layer in all high-intensity groups was significantly increased or tended to increase compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec). Furthermore, the percentage of ALP-positive cells in the uncalcified layer in all high-intensity groups (except for the HLS group \u003cem\u003evs.\u003c/em\u003e LLS group) was significantly increased or tended to increase compared with that in the low-intensity groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003eType X collagen-positive cells, which are a specific marker of chondrocyte hypertrophy, were observed in the calcified layer in all groups, and an increase in type X collagen-positive cells in the uncalcified layer was observed in the HHL group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb). The percentage of type X collagen-positive cells in the total layer was significantly increased only in the HHL group compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). The percentage of type X collagen-positive cells in the calcified layer in the high-frequency (i.e., HHL, HHS, LHL, and LHS) groups was significantly increased, regardless of the intensity, compared with that in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed). Furthermore, for the low-intensity groups, the percentage of type X collagen-positive cells in the calcified layer in the LHL group was significantly higher than that in the low-frequency (LLL and LLS) groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ed).\u003c/p\u003e \u003cp\u003eFurthermore, the percentage of TUNEL-positive cells, which indicates chondrocyte apoptosis [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], in the total layer in the high-intensity and high-frequency (HHL and HHS) groups was significantly increased compared with that in the control and low-intensity groups (Supplementary Figs.\u0026nbsp;5a and 5b).\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eOur objective was to determine the combination of intensity, frequency, and duration that provides the best mechanical stress on healthy knee articular cartilage. We found that low-intensity CTS, regardless of frequency, activated anabolic gene expression in the cartilage matrix during the short duration and suppressed the downregulation of gene expression of \u003cem\u003eSOX9\u003c/em\u003e during the long duration. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises inhibited chondrocyte hypertrophy and increased aggrecan synthesis in the tibial cartilage.\u003c/p\u003e \u003cp\u003eChondrocytes adapt to changes in the mechanical environment by upregulating anabolic genes. The expression of these genes is then downregulated [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. However, \u003cem\u003eACAN\u003c/em\u003e mRNA did not show time-dependent changes similar to \u003cem\u003eCOL2A1\u003c/em\u003e mRNA. This poor response may be the reason that proteoglycan loss precedes type II collagen loss in early cartilage degeneration [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. \u003cem\u003eSOX9\u003c/em\u003e mRNA, which contributes to cartilage homeostasis [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], was downregulated in the high-intensity groups, similar to the findings of previous studies [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. However, interestingly, this downregulation also occurred in the low-intensity and short-duration groups, and the expression of \u003cem\u003eSOX9\u003c/em\u003e mRNA in the low-intensity and long-duration groups was maintained at the same level compared with that in the control group. \u003cem\u003eTGF-β\u003c/em\u003e, which inhibits chondrocyte hypertrophy, contributes to the expression of \u003cem\u003eSOX9\u003c/em\u003e [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], and \u003cem\u003eTGF-β\u003c/em\u003e mRNA is upregulated when CTS is applied with intensity of 5\u0026ndash;12% and duration of 12\u0026ndash;24 h [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Taken together, low-intensity CTS applied for \u0026gt;\u0026thinsp;12 h may be necessary to maintain cartilage homeostasis via \u003cem\u003eSOX9\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eHistological analysis after treadmill exercises revealed that stress in the HHL group had the most negative effects on healthy articular cartilage, and these findings are consistent with those of a previous review [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The increase in cartilage thickness with the increase in chondrocyte proliferation and hypertrophy, which were observed in the LHL group, have been reported as precursors to cartilage degeneration [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. In contrast, although the cartilage thickness was similar to that in the control group, only the cartilage in the LLL group exhibited an increase in aggrecan and lubricin without an increase in chondrocyte hypertrophy compared with that in the control group, and these responses may maintain/improve the loading function of articular cartilage.\u003c/p\u003e \u003cp\u003eBecause high-frequency exercises resulted in catabolism, including chondrocyte hypertrophy, even in the low-intensity groups, daily exercises without rest may lead to chondrocyte hypertrophy [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. In contrast, cartilage in the LLS group did not change. This is consistent with the findings of previous studies, which have reported that stress loads over a certain amount of time and frequency are necessary to maintain articular cartilage health [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis study has several limitations. First, the best mechanical stress on the articular cartilage presented in this study was achieved by combining large or small values for each parameter, but not specific values. Although further research is required, our findings provide useful insights for future research. Directly linking CTS to treadmill exercise results may be difficult because the load on the knee joint caused by treadmill exercises does not reflect the exact amount of stress on the cartilage or chondrocytes [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Interestingly, low-intensity and long-duration stress was a suitable combination for the articular cartilage in both the CTS and treadmill exercise results.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eOur results show that low-intensity, low-frequency, and long-duration stress is the best combination for healthy knee articular cartilage to maintain homeostasis. Our results may provide a significant scientific basis for designing exercise programs and lifestyle instructions that consider the mechanical stress on articular cartilage.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eCompeting interests\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no conflicts of interest with any financial organization regarding the materials used and discussed in the manuscript.\u003c/p\u003e\u003ch2\u003eEthical approval\u003c/h2\u003e \u003cp\u003e All experimental procedures were approved by the Institutional Animal Care and Use Committee and performed according to the Kobe University Animal Experimentation Regulations (approval number: P140603).\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e \u003cp\u003eThis study was supported in part by Japan Society for the Promotion of Science KAKENHI Grant Number 25702032 and 19H04050.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eY.W. and H.M. have given substantial contributions to the conception or the design of the manuscript; Y.W., S.I., and M.N. contributed to Collection and assembly of data; All authors contributed to analysis and interpretation of the data; Y.W., S.I., and H.M. contributed to statistical expertise; H.M. contributed to obtaining of funding; H.M. contributed to administrative, technical, or logistic support; Y.W., H.M., and Y.M. have participated to drafting the manuscript, and all authors revised it critically. All authors read and approved the final version of the manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank Toshihiro Akisue, Yuta Kohara, Eriko Mizuno, Changxin Li, Junpei Hatakeyama, and Daisuke Takamura for their assistance.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e \u003cp\u003eAll data that support the findings of this study are available from the corresponding author on reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMcGinty G, Irrgang JJ, Pezzullo D (2000) Biomechanical considerations for rehabilitation of the knee. Clin Biomech (Bristol Avon) 15:160\u0026ndash;166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s0268-0033(99)00061-3\u003c/span\u003e\u003cspan address=\"10.1016/s0268-0033(99)00061-3\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChen S, Fu P, Wu H, Pei M (2017) Meniscus, articular cartilage and nucleus pulposus: a comparative review of cartilage-like tissues in anatomy, development and function. Cell Tissue Res 370:53\u0026ndash;70. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00441-017-2613-0\u003c/span\u003e\u003cspan address=\"10.1007/s00441-017-2613-0\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBricca A, Juhl CB, Grodzinsky AJ, Roos EM (2017) Impact of a daily exercise dose on knee joint cartilage - a systematic review and meta-analysis of randomized controlled trials in healthy animals. Osteoarthr Cartil 25:1223\u0026ndash;1237. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2017.03.009\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2017.03.009\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmbrosio F, Tarabishy A, Kadi F, Brown EH, Sowa G (2011) Biological basis of exercise-based treatments for musculoskeletal conditions. PM R 3:S59\u0026ndash;S63. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.pmrj.2011.05.001\u003c/span\u003e\u003cspan address=\"10.1016/j.pmrj.2011.05.001\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBuckwalter JA, Martin JA (2006) Osteoarthritis. Adv Drug Deliv Rev 58:150\u0026ndash;167. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.addr.2006.01.006\u003c/span\u003e\u003cspan address=\"10.1016/j.addr.2006.01.006\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMueller MJ, Maluf KS (2002) Tissue adaptation to physical stress: a proposed physical stress theory to guide physical therapist practice, education, and research. Phys Ther 82:383\u0026ndash;403. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1093/ptj/82.4.383\u003c/span\u003e\u003cspan address=\"10.1093/ptj/82.4.383\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWolf A, Ackermann B, Steinmeyer J (2007) Collagen synthesis of articular cartilage explants in response to frequency of cyclic mechanical loading. Cell Tissue Res 327:155\u0026ndash;166. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s00441-006-0251-z\u003c/span\u003e\u003cspan address=\"10.1007/s00441-006-0251-z\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBleuel J, Zaucke F, Br\u0026uuml;ggemann GP, Niehoff A (2015) Effects of cyclic tensile strain on chondrocyte metabolism: A systematic review. PLoS ONE 10:e0119816. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1371/journal.pone.0119816\u003c/span\u003e\u003cspan address=\"10.1371/journal.pone.0119816\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMazor M, Best TM, Cesaro A, Lespessailles E, Toumi H (2019) Osteoarthritis biomarker responses and cartilage adaptation to exercise: a review of animal and human models. Scand J Med Sci Sports 29:1072\u0026ndash;1082. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/sms.13435\u003c/span\u003e\u003cspan address=\"10.1111/sms.13435\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYang Y, Wang Y, Kong Y, Zhang X, Zhang H, Gang Y, Bai L (2019) Mechanical stress protects against osteoarthritis via regulation of the AMPK/NF-κB signaling pathway. J Cell Physiol 234:9156\u0026ndash;9167. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/jcp.27592\u003c/span\u003e\u003cspan address=\"10.1002/jcp.27592\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang T, Muneta T, Ju YJ, Nimura A, Miyazaki K, Masuda H, Mochizuki T, Sekiya I (2008) Serum keratan sulfate transiently increases in the early stage of osteoarthritis during strenuous running of rats: protective effect of intraarticular hyaluronan injection. Arthritis Res Ther 10:R13. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/ar2363\u003c/span\u003e\u003cspan address=\"10.1186/ar2363\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLequesne MG, Dang N, Lane NE (1997) Sport practice and osteoarthritis of the limbs. Osteoarthr Cartil 5:75\u0026ndash;86. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/s1063-4584(97)80001-5\u003c/span\u003e\u003cspan address=\"10.1016/s1063-4584(97)80001-5\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTiderius CJ, Svensson J, Leander P, Ola T, Dahlberg L (2004) dGEMRIC (delayed gadolinium-enhanced MRI of cartilage) indicates adaptive capacity of human knee cartilage. Magn Reson Med 51:286\u0026ndash;290. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/mrm.10714\u003c/span\u003e\u003cspan address=\"10.1002/mrm.10714\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJones G, Ding C, Glisson M, Hynes K, Ma D, Cicuttini F (2003) Knee articular cartilage development in children: A longitudinal study of the effect of sex, growth, body composition, and physical activity. Pediatr Res 54:230\u0026ndash;236. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1203/01.PDR.0000072781.93856.E6\u003c/span\u003e\u003cspan address=\"10.1203/01.PDR.0000072781.93856.E6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eQi C, Changlin H (2006) Effects of moving training on histology and biomarkers levels of articular cartilage. J Surg Res 135:352\u0026ndash;363. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jss.2006.03.011\u003c/span\u003e\u003cspan address=\"10.1016/j.jss.2006.03.011\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRacunica TL, Teichtahl AJ, Wang Y, Wluka AE, English DR, Giles GG, O\u0026rsquo;Sullivan R, Cicuttini FM (2007) Effect of physical activity on articular knee joint structures in community-based adults. Arthritis Rheum 57:1261\u0026ndash;1268. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/art.22990\u003c/span\u003e\u003cspan address=\"10.1002/art.22990\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eChanglin H, Qi C (2008) Effects of different mode high-intensity movement training on articular cartilage in histology - a randomised controlled trial on rabbit knee. Biol Sport 25:371\u0026ndash;386\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCymet TC, Sinkov V (2006) Does long-distance running cause osteoarthritis? J Am Osteopath Assoc 106:342\u0026ndash;345\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRegnaux JP, Lefevre-Colau MM, Trinquart L, Nguyen C, Boutron I, Brosseau L, Ravaud P (2015) High-intensity versus low-intensity physical activity or exercise in people with hip or knee osteoarthritis. Cochrane Database Syst Rev 2015:CD010203. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1002/14651858.CD010203.pub2\u003c/span\u003e\u003cspan address=\"10.1002/14651858.CD010203.pub2\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGosset M, Berenbaum F, Thirion S, Jacques C (2008) Primary culture and phenotyping of murine chondrocytes. Nat Protoc 3:1253\u0026ndash;1260. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nprot.2008.95\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2008.95\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang SW, Yoo SP, Kim BS (2007) Effect of chondrocyte passage number on histological aspects of tissue-engineered cartilage. Bio Med Mater Eng 17:269\u0026ndash;276\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTang Q, Zheng G, Feng Z, Tong M, Xu J, Hu Z, Shang P, Chen Y, Wang C, Lou Y, Chen D, Zhang D, Nisar M, Zhang X, Xu H, Liu H (2017) Wogonoside inhibits IL-1β induced catabolism and hypertrophy in mouse chondrocyte and ameliorates murine osteoarthritis. Oncotarget 8:61440\u0026ndash;61456. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.18632/oncotarget.18374\u003c/span\u003e\u003cspan address=\"10.18632/oncotarget.18374\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLivak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) method. Methods 25:402\u0026ndash;408. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1006/meth.2001.1262\u003c/span\u003e\u003cspan address=\"10.1006/meth.2001.1262\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSchmittgen TD, Livak KJ (2008) Analyzing real-time PCR data by the comparative C(T) method. Nat Protoc 3:1101\u0026ndash;1108. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/nprot.2008.73\u003c/span\u003e\u003cspan address=\"10.1038/nprot.2008.73\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAl-Sabah A, Stadnik P, Gilbert SJ, Duance VC, Blain EJ (2016) Importance of reference gene selection for articular cartilage mechanobiology studies. Osteoarthr Cartil 24:719\u0026ndash;730. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2015.11.007\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2015.11.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBillat VL, Mouisel E, Roblot N, Melki J (2005) Inter- and intrastrain variation in mouse critical running speed. J Appl Physiol (1985) 98:1258\u0026ndash;1263. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/japplphysiol.00991.2004\u003c/span\u003e\u003cspan address=\"10.1152/japplphysiol.00991.2004\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSturgeon K, Schadler K, Muthukumaran G, Ding D, Bajulaiye A, Thomas NJ, Ferrari V, Ryeom S, Libonati JR (2014) Concomitant low-dose doxorubicin treatment and exercise. Am J Physiol Regul Integr Comp Physiol 307:R685\u0026ndash;R692. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1152/ajpregu.00082.2014\u003c/span\u003e\u003cspan address=\"10.1152/ajpregu.00082.2014\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKawamoto T (2003) Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch Histol Cytol 66:123\u0026ndash;143. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1679/aohc.66.123\u003c/span\u003e\u003cspan address=\"10.1679/aohc.66.123\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eArifin WN, Zahiruddin WM (2017) Sample size calculation in animal studies using resource equation approach. Malays J Med Sci 24:101\u0026ndash;105. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.21315/mjms2017.24.5.11\u003c/span\u003e\u003cspan address=\"10.21315/mjms2017.24.5.11\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoriyama H, Yoshimura O, Kawamata S, Takayanagi K, Kurose T, Kubota A, Hosoda M, Tobimatsu Y (2008) Alteration in articular cartilage of rat knee joints after spinal cord injury. Osteoarthr Cartil 16:392\u0026ndash;398. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2007.07.002\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2007.07.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePinamont WJ, Yoshioka NK, Young GM, Karuppagounder V, Carlson EL, Ahmad A, Elbarbary R, Kamal F (2020) Standardized histomorphometric evaluation of osteoarthritis in a surgical mouse model. J Vis Exp 159. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.3791/60991\u003c/span\u003e\u003cspan address=\"10.3791/60991\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMoriyama H, Kanemura N, Brouns I, Pintelon I, Adriaensen D, Timmermans JP, Ozawa J, Kito N, Gomi T, Deie M (2012) Effects of aging and exercise training on the histological and mechanical properties of articular structures in knee joints of male rat. Biogerontology 13:369\u0026ndash;381. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s10522-012-9381-8\u003c/span\u003e\u003cspan address=\"10.1007/s10522-012-9381-8\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNomura M, Sakitani N, Iwasawa H, Kohara Y, Takano S, Wakimoto Y, Kuroki H, Moriyama H (2017) Thinning of articular cartilage after joint unloading or immobilization. An experimental investigation of the pathogenesis in mice. Osteoarthr Cartil 25:727\u0026ndash;736. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2016.11.013\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2016.11.013\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eVignon E, Arlot M, Hartmann D, Moyen B, Ville G (1983) Hypertrophic repair of articular cartilage in experimental osteoarthrosis. Ann Rheum Dis 42:82\u0026ndash;88. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1136/ard.42.1.82\u003c/span\u003e\u003cspan address=\"10.1136/ard.42.1.82\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLefebvre V, Dvir-Ginzberg M (2017) SOX9 and the many facets of its regulation in the chondrocyte lineage. Connect Tissue Res 58:2\u0026ndash;14. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/03008207.2016.1183667\u003c/span\u003e\u003cspan address=\"10.1080/03008207.2016.1183667\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJay GD, Waller KA (2014) The biology of lubricin: near frictionless joint motion. Matrix Biol 39:17\u0026ndash;24. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.matbio.2014.08.008\u003c/span\u003e\u003cspan address=\"10.1016/j.matbio.2014.08.008\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRuan MZC, Erez A, Guse K, Dawson B, Bertin T, Chen Y, Jiang MM, Yustein J, Gannon F, Lee BH (2013) Proteoglycan 4 expression protects against the development of osteoarthritis. Sci Transl Med 5:176ra34. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1126/scitranslmed.3005409\u003c/span\u003e\u003cspan address=\"10.1126/scitranslmed.3005409\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiao D, Scutt A (2002) Histochemical localization of alkaline phosphatase activity in decalcified bone and cartilage. J Histochem Cytochem 50:333\u0026ndash;340. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1177/002215540205000305\u003c/span\u003e\u003cspan address=\"10.1177/002215540205000305\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLotz M, Loeser RF (2012) Effects of aging on articular cartilage homeostasis. Bone 51:241\u0026ndash;248. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bone.2012.03.023\u003c/span\u003e\u003cspan address=\"10.1016/j.bone.2012.03.023\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu G, Qian Y, Wu W, Li R (2019) Negative effects of high mechanical tensile strain stimulation on chondrocyte injury in vitro. Biochem Biophys Res Commun 510:48\u0026ndash;52. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.bbrc.2019.01.002\u003c/span\u003e\u003cspan address=\"10.1016/j.bbrc.2019.01.002\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHardingham T, Bayliss M (1990) Proteoglycans of articular cartilage: changes in aging and in joint disease. Semin Arthritis Rheum 20:12\u0026ndash;33. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/0049-0172(90)90044-g\u003c/span\u003e\u003cspan address=\"10.1016/0049-0172(90)90044-g\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLin YY, Tanaka N, Ohkuma S, Iwabuchi Y, Tanne Y, Kamiya T, Kunimatsu R, Huang YC, Yoshioka M, Mitsuyoshi T, Tanimoto K, Tanaka E, Tanne K (2010) Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system. Eur J Oral Sci 118:151\u0026ndash;158. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1111/j.1600-0722.2010.00710.x\u003c/span\u003e\u003cspan address=\"10.1111/j.1600-0722.2010.00710.x\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSobue Y, Takahashi N, Ohashi Y, Suzuki M, Nishiume T, Kobayakawa T, Terabe K, Knudson W, Knudson C, Ishiguro N, Kojima T (2019) Inhibition of CD44 intracellular domain production suppresses bovine articular chondrocyte de-differentiation induced by excessive mechanical stress loading. Sci Rep 9:14901. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1038/s41598-019-50166-4\u003c/span\u003e\u003cspan address=\"10.1038/s41598-019-50166-4\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTuli R, Tuli S, Nandi S, Huang X, Manner PA, Hozack WJ, Danielson KG, Hall DJ, Tuan RS (2003) Transforming growth factor-β-mediated chondrogenesis of human mesenchymal progenitor cells involves N-cadherin and mitogen-activated protein kinase and Wnt signaling cross-talk. J Biol Chem 278:41227\u0026ndash;41236. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1074/jbc.M305312200\u003c/span\u003e\u003cspan address=\"10.1074/jbc.M305312200\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOhno S, Tanaka N, Ueki M, Honda K, Tanimoto K, Yoneno K, Ohno-Nakahara M, Fujimoto K, Kato Y, Tanne K (2005) Mechanical regulation of terminal chondrocyte differentiation via RGD-CAP/beta ig-h3 induced by TGF-beta. Connect Tissue Res 46:227\u0026ndash;234. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1080/03008200500346111\u003c/span\u003e\u003cspan address=\"10.1080/03008200500346111\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eFurumatsu T, Matsumoto E, Kanazawa T, Fujii M, Lu Z, Kajiki R, Ozaki T (2013) Tensile strain increases expression of CCN2 and COL2A1 by activating TGF-β-Smad2/3 pathway in chondrocytic cells. J Biomech 46:1508\u0026ndash;1515. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.jbiomech.2013.03.028\u003c/span\u003e\u003cspan address=\"10.1016/j.jbiomech.2013.03.028\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCalvo E, Palacios I, Delgado E, S\u0026aacute;nchez-Pernaute O, Largo R, Egido J, Herrero-Beaumont G (2004) Histopathological correlation of cartilage swelling detected by magnetic resonance imaging in early experimental osteoarthritis. Osteoarthr Cartil 12:878\u0026ndash;886. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.joca.2004.07.007\u003c/span\u003e\u003cspan address=\"10.1016/j.joca.2004.07.007\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDreier R (2010) Hypertrophic differentiation of chondrocytes in osteoarthritis: the developmental aspect of degenerative joint disorders. Arthritis Res Ther 12:216. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1186/ar3117\u003c/span\u003e\u003cspan address=\"10.1186/ar3117\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMiller RH (2017) Joint loading in runners does not initiate knee osteoarthritis. Exerc Sport Sci Rev 45:87\u0026ndash;95. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1249/JES.0000000000000105\u003c/span\u003e\u003cspan address=\"10.1249/JES.0000000000000105\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\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":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"mechanical stress, cyclic tensile strain, treadmill exercise, knee cartilage metabolism, knee cartilage homeostasis","lastPublishedDoi":"10.21203/rs.3.rs-3907866/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3907866/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eUnderstanding how healthy articular cartilage responds to mechanical stress is critical. Moderate mechanical stress has positive effects on the cartilage, such as maintaining cartilage homeostasis. The degree of mechanical stress is determined by a combination of intensity, frequency, and duration; however, the best combination of these parameters for knee cartilage remains unclear. This study aimed to determine which combination of intensity, frequency, and duration provides the best mechanical stress on healthy knee articular cartilage in vitro and in vivo.\u003c/p\u003e\u003ch2\u003eMethods and results\u003c/h2\u003e \u003cp\u003eIn this study, 33 male mice were used. Chondrocytes isolated from mouse knee joints were subjected to different cyclic tensile strains (CTSs) and assessed by measuring the expression of cartilage matrix-related genes. Furthermore, the histological characteristics of mouse tibial cartilages were quantified using different treadmill exercises. Chondrocytes and mice were divided into the control group and eight intervention groups: high-intensity, high-frequency, and long-duration; high-intensity, high-frequency, and short-duration; high-intensity, low-frequency, and long-duration; high-intensity, low-frequency, and short-duration; low-intensity, high-frequency, and long-duration; low-intensity, high-frequency, and short-duration; low-intensity, high-frequency, and short-duration; low-intensity, low-frequency, and long-duration; low-intensity, low-frequency, and short-duration. In low-intensity CTSs, chondrocytes showed anabolic responses by altering the mRNA expression of COL2A1 in short durations and SOX9 in long durations. Furthermore, low-intensity, low-frequency, and long-duration treadmill exercises minimized chondrocyte hypertrophy and enhanced aggrecan synthesis in tibial cartilages.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eLow-intensity, low-frequency, and long-duration stress is the best combination for healthy knee cartilage to maintain homeostasis and activate anabolic responses. Our findings provide a significant scientific basis for exercise and lifestyle instructions.\u003c/p\u003e","manuscriptTitle":"Effects of different combinations of mechanical stress intensity, duration, and frequency on the articular cartilage in mice","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-02-02 12:40:27","doi":"10.21203/rs.3.rs-3907866/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-05-02T10:52:19+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-03-03T04:17:44+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"9f623bde-1c87-4537-b62e-450425a4bc7e","date":"2024-02-03T14:30:12+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-02-01T11:47:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-02-01T08:12:52+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-02-01T08:12:52+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2024-01-29T04:12:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"01aded6a-24ea-4e74-a99e-9b6fd9deb3c5","owner":[],"postedDate":"February 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-05T16:02:13+00:00","versionOfRecord":{"articleIdentity":"rs-3907866","link":"https://doi.org/10.1007/s11033-024-09762-5","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2024-07-29 15:57:22","publishedOnDateReadable":"July 29th, 2024"},"versionCreatedAt":"2024-02-02 12:40:27","video":"","vorDoi":"10.1007/s11033-024-09762-5","vorDoiUrl":"https://doi.org/10.1007/s11033-024-09762-5","workflowStages":[]},"version":"v1","identity":"rs-3907866","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3907866","identity":"rs-3907866","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}