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The difference in joint instability affects the onset of cartilage degeneration or subchondral bone changes

  • K. Arakawa
    Affiliations
    Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, Saitama, Japan
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  • K. Takahata
    Affiliations
    Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, Saitama, Japan
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  • S. Enomoto
    Affiliations
    Department of Physical Therapy, Health and Social Services, Saitama Prefectural University, Saitama, Japan
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  • Y. Oka
    Affiliations
    Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, Saitama, Japan
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  • K. Ozone
    Affiliations
    Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, Saitama, Japan

    Japan Society for the Promotion of Science, Tokyo, Japan
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  • S. Nakagaki
    Affiliations
    Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, Saitama, Japan
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  • K. Murata
    Affiliations
    Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, Saitama, Japan

    Department of Physical Therapy, Health and Social Services, Saitama Prefectural University, Saitama, Japan
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  • N. Kanemura
    Affiliations
    Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, Saitama, Japan

    Department of Physical Therapy, Health and Social Services, Saitama Prefectural University, Saitama, Japan
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  • T. Kokubun
    Correspondence
    Address correspondence and reprint requests to: T Kokubun, Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, 820 Sannomiya, Koshigaya-Shi, Saitama, 343-8540. Japan. Tel.: 48-973-4123.
    Affiliations
    Department of Health and Social Services, Health and Social Services, Graduate School of Saitama Prefectural University, Saitama, Japan

    Department of Physical Therapy, Health and Social Services, Saitama Prefectural University, Saitama, Japan
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Open AccessPublished:December 12, 2021DOI:https://doi.org/10.1016/j.joca.2021.12.002

      Summary

      Objective

      It has been debated whether the onset of knee osteoarthritis is initiated in cartilage or subchondral bone. The purpose of this study was to clarify the effects of increasing or decreasing joint instability on cartilage degeneration and subchondral bone changes in knee OA by comparing different models of joint instability.

      Design

      We used the anterior cruciate ligament transection (ACL-T) model and the destabilization of the medial meniscus (DMM) model. In addition, we created a controlled abnormal tibial translation (CATT) model and a controlled abnormal tibial rotation (CATR) model. We performed joint instability analysis, micro-computed tomography analysis, histological and immunohistological analysis in 4 and 6 weeks.

      Results

      The CATT group suppressed joint instability in the ACL-T group (6 weeks; P = 0.032), and the CATR group suppressed joint instability in the DMM group (6 weeks; P = 0.032). Chondrocyte hypertrophy in the ACL-T and DMM groups was increased compared to the Sham group (6 weeks; [ACL-T vs Sham], P = 0.002, 95%CI [5.983–33.025]; [DMM vs Sham], P = 0.022, 95%CI [1.691–28.733]). In the subchondral bone, the BV/TV in the DMM and CATR groups was increased compared to the ACL-T and CATT groups (6 weeks; [DMM vs ACL-T], P = 0.002, 95%CI [7.404–37.582]; [DMM vs CATT], P = 0.014, 95%CI [2.881–33.059]; [CATR vs ACL-T], P = 0.006, 95%CI [4.615–34.793]; [CATR vs CATT], P = 0.048, 95%CI [0.092–30.270]).

      Conclusions

      This study showed that joint instability promotes chondrocyte hypertrophy, but subchondral bone changes were influenced by differences in ACL and meniscus function.

      Abbreviations:

      ACL-T (Anterior Cruciate Ligament Transection), CATT (Controlled Abnormal Tibial translation), CATR (Controlled Abnormal Tibial Rotation), DMM (Destabilizaiton of the Medial Meniscus), OA (Osteoarthritis), MMTL (Medial Meniscotibial Ligament), ICR (Institute for Cancer Research), μCT (Micro-computed tomography), BV/TV (Bone Volume/Tissue Volume), Tb.Th (Trabecular Thickness), Tb.N (Trabecular Number), Tb.Sp (Trabecular Separation), OARSI (Osteoarthritis Research Society International), TRAP (Tartrate-Resistant Acid Phosphatase), Oc.S/BS (Osteoclast Surface/Bone Surface), Col X (Type X collagen), CI (Confidence intervals)

      Keywords

      Introduction

      Osteoarthritis (OA) is a degenerative joint disease characterized by articular cartilage degeneration, osteophyte formation, and subchondral bone sclerosis
      • Lohmander L.S.
      What can we do about osteoarthritis?.
      ,
      • Loeser R.F.
      • Collins J.A.
      • Diekman B.O.
      Ageing and the pathogenesis of osteoarthritis.
      . Knee OA has the highest morbidity among OA, and interferes with daily life due to pain and dysfunction of the knee joint. Radin et al., in 1986 proposed that the imbalance of subchondral bone stiffness may be the cause of articular cartilage damage. Since then, it has been debated whether the onset of knee OA is initiated in the cartilage or the subchondral bone
      • Radin E.L.
      • Rose R.M.
      Role of subchondral bone in the initiation and progression of cartilage damage.
      . Some reports have shown that articular cartilage degeneration precedes changes in subchondral bone
      • Intema F.
      • Hazewinkel H.A.W.
      • Gouwens D.
      • Bijlsma J.
      • Weinans H.
      • Lafeber F.
      • et al.
      In early OA, thinning of the subchondral plate is directly related to cartilage damage: results from a canine ACLT-meniscectomy model.
      ,
      • Panula H.E.
      • Nieminen J.
      • Parkkinen J.J.
      • Arnala I.
      • Kröger H.
      • Alhava E.
      Subchondral bone remodeling increases in early experimental osteoarthrosis in young beagle dogs.
      . However, other reports suggested that changes in subchondral bone precede articular cartilage degeneration
      • Botter S.
      • van Osch G.
      • Waarsing J.
      • van der Linden J.
      • Verhaar J.
      • van Leeuwen J.
      • et al.
      Cartilage damage pattern in relation to subchondral plate thickness in a collagenase-induced model of osteoarthritis.
      ,
      • Sniekers Y.H.
      • Intema F.
      • Lafeber F.
      • Van Osch G.
      • Van Leeuwen J.
      • Weinans H.
      • et al.
      A role for subchondral bone changes in the process of osteoarthritis; A micro-CT study of two canine models.
      . Therefore, there was no consistent view on this topic yet.
      The mechanism of knee OA onset has not been elucidated, but mechanical stress is considered to be the main factor
      • Felson D.T.
      Osteoarthritis as a disease of mechanics.
      . Although excessive mechanical stress causes articular cartilage degeneration, moderate mechanical stress contributes to articular cartilage protection
      • Nam J.
      • Aguda B.
      • Rath B.
      • Agarwal S.
      Biomechanical thresholds regulate inflammation through the NF-κB pathway: experiments and modeling.
      . In addition, a study that had used an animal model of knee OA revealed that moderate exercise with a treadmill suppressed the progression of knee OA, and, high-intensity exercise promoted the progression of knee OA
      • Iijima H.
      • Ito A.
      • Nagai M.
      • Tajino J.
      • Yamaguchi S.
      • Kiyan W.
      • et al.
      Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model.
      ,
      • Yamaguchi S.
      • Aoyama T.
      • Ito A.
      • Nagai M.
      • Iijima H.
      • Zhang X.
      • et al.
      Effects of exercise level on biomarkers in a rat knee model of osteoarthritis.
      . Our laboratory has reported that joint instability was regarded as abnormal mechanical stress and that suppression of joint instability delays cartilage degeneration
      • Murata K.
      • Kanemura N.
      • Kokubun T.
      • Fujino T.
      • Morishita Y.
      • Onitsuka K.
      • et al.
      Controlling joint instability delays the degeneration of articular cartilage in a rat model.
      ,
      • Onitsuka K.
      • Murata K.
      • Kokubun T.
      • Fujiwara S.
      • Nakajima A.
      • Morishita Y.
      • et al.
      Effects of controlling abnormal joint movement on expression of MMP13 and TIMP-1 in osteoarthritis.
      .
      Two popular rodent knee OA model includes the anterior cruciate ligament transection (ACL-T) model and the destabilization of the medial meniscus (DMM) model. The ACL-T model transects the ACL, and the DMM model transects the medial meniscotibial ligament (MMTL)
      • Glasson S.
      • Blanchet T.
      • Morris E.
      The Surgical Destabilization of the Medial Meniscus (DMM) Model of Osteoarthritis in the 129/SvEv Mouse.
      . These models cause OA by transecting the ligaments involved in knee joint stability. Therefore, these models both induce joint instability. It has been reported that articular cartilage degeneration depends on the magnitude of joint instability
      • Kamekura S.
      • Hoshi K.
      • Shimoaka T.
      • Chung U.
      • Chikuda H.
      • Yamada T.
      • et al.
      Osteoarthritis development in novel experimental mouse models induced by knee joint instability.
      , and the DMM model has a slower progression of cartilage degeneration and less joint instability than the ACL-T model
      • Glasson S.
      • Blanchet T.
      • Morris E.
      The Surgical Destabilization of the Medial Meniscus (DMM) Model of Osteoarthritis in the 129/SvEv Mouse.
      ,
      • Adebayo O.O.
      • Ko F.C.
      • Goldring S.R.
      • Goldring M.B.
      • Wright T.M.
      • van der Meulen M.C.H.
      Kinematics of meniscal- and ACL-transected mouse knees during controlled tibial compressive loading captured using roentgen stereophotogrammetry.
      . However, the DMM model showed early degeneration of the meniscus
      • Kwok J.
      • Onuma H.
      • Olmer M.
      • Lotz M.K.
      • Grogan S.P.
      • D'Lima D.D.
      Histopathological analyses of murine menisci: implications for joint aging and osteoarthritis.
      , suggesting that not only joint instability but also dysfunction of the meniscus may be involved in the development of OA. Therefore, we applied the controlled abnormal tibial translation (CATT) model
      • Kokubun T.
      • Kanemura N.
      • Murata K.
      • Moriyama H.
      • Morita S.
      • Jinno T.
      • et al.
      Effect of changing the joint kinematics of knees with a ruptured anterior cruciate ligament on the molecular biological responses and spontaneous healing in a rat model.
      , which suppresses joint instability caused by the ACL-T model, to the DMM model. Thus, we established a new controlled abnormal tibial rotation (CATR) model to suppress joint instability in the DMM model, and can evaluate the process of knee OA development with suppressed joint instability in the DMM model. Furthermore, By comparing the new model with the existing ACL-T model, DMM model, and CATT model, we can examine the effects of joint instability and the differences in the effects of ACL and meniscus dysfunction on knee OA.
      Therefore, the purpose of this study was to clarify the effects of increasing or decreasing in a different type of joint instability on cartilage degeneration and subchondral bone changes in knee OA. We compared the ACL-T model, CATT model, DMM model, and CATR model and clarified part of the knee OA development mechanism by comparing the models.

      Materials and methods

      Animals and experimental design

      This study was approved by the Animal Research Committee of Saitama Prefectural University (approval number: 2020-1), and the animals were handled in accordance with the relevant legislation and institutional guidelines for humane animal treatment. In this study, 59 adult (12-week-old) Institute for Cancer Research (ICR) male mice were randomized into one of five groups: ACL-T group (ACL-T, n = 12), CATT group (CATT, n = 12), DMM group (DMM, n = 12), CATR group (CATR, n = 12), and sham group (Sham, n = 11). We sacrificed all mice at 4 and 6 weeks after surgical intervention (4 weeks; each group: n = 6, 6 weeks; Sham group: n = 5, other groups: n = 6). All mice were housed in plastic cages, and the room had a 12-hour light/dark cycle. Mice were permitted unrestricted movement within the cage and had free access to food and water.

      Surgical procedures

      All surgical procedures were performed on the left knee joint with the mice under a combination anesthetic (medetomidine, 0.375 mg/kg; midazolam, 2.0 mg/kg; and butorphanol, 2.5 mg/kg). The medial capsule was exposed in the ACL-T group, and scissors were used to cut the ACL. CATT was performed following the same procedure as the ACL-T surgery [Fig. 1(A)]. Mice assigned to the CATT group had bone tunnels created in the distal femur and proximal tibia using a 25-gauge needle and 4-0 nylon threads threaded through them to suppress the anterior-posterior joint instability that occurs in the ACL-T model [Fig. 1(B)a,b]. Subsequently, 4-0 nylon threads were tightly tied [Fig. 1(B)c] to compensate for ACL function and suppress anterior-posterior joint instability.
      Fig. 1
      Fig. 1Surgery for the CATT and CATR model. (A) Schematic diagram of the CATT and CATR models. Nylon suture suppresses anterior-posterior joint instability caused by the ACL-T model and rotational joint instability caused by the DMM model. (B) Details of surgery. Create a bone tunnel distal to the femur and proximal to the tibia (a). Use 4-0 nylon suture to penetrate the bone tunnel (b). Tie nylon thread in a loop (d).
      DMM surgery was performed on the left knee joint of the mice as previously described
      • Glasson S.
      • Blanchet T.
      • Morris E.
      The Surgical Destabilization of the Medial Meniscus (DMM) Model of Osteoarthritis in the 129/SvEv Mouse.
      . CATR surgery was performed following the same procedure as DMM surgery. Mice assigned to the CATR group were created using the same procedure as the CATT model. The bone tunnels were positioned vertically to suppress rotational instability that occurs in the DMM model [Fig. 1(A)]. To eliminate differences between the groups, bone tunnels were created in the Sham, ACL-T, and DMM groups as well as in the CATT and CATR groups, and a nylon thread was tied loosely so as not to suppress joint instability.

      Anterior drawer test

      To assess anterior-posterior joint instability, we performed the anterior drawer test using a constant force spring (0.05 kgf; Sanko Spring Co., Ltd., Fukuoka, Japan) and a soft X-ray device (M-60; Softex Co., Ltd., Kanagawa, Japan)
      • Oka Y.
      • Murata K.
      • Kano T.
      • Ozone K.
      • Arakawa K.
      • Kokubun T.
      • et al.
      Impact of Controlling Abnormal Joint Movement on the Effectiveness of Subsequent Exercise Intervention in Mouse Models of Early Knee Osteoarthritis.
      . At 4 and 6 weeks after surgery, we collected the knee joints of mice with an intact femur, tibia, and paw. For the measurement of displacement, photographs were taken with the knee joint in 90° of flexion and the femur fixed to a clamp and with the tibia pulled forward using constant force spring. Digital images were acquired using an x-ray sensor (Naomi; RF Co. Ltd., Nagano, Japan) with a voltage of 28 kV, a current of 1.5 mA, and an exposure time of 1 s. The soft x-ray images were used to quantify the anterior displacement of the tibia using a dedicated image analysis software (ImageJ; National Institutes of Health, Bethesda, MD, USA). The anterior displacement was measured by the linear distance from the perpendicular line from the lower end of the femoral condyle to the posterior edge of the tibial joint surface.

      Tibial rotational test

      To assess rotational joint instability, we performed a tibial rotational test using a constant force spring (0.05 kgf; Sanko Spring Co., Ltd., Fukuoka, Japan) and a soft x-ray device (M-60; Softex Co., Ltd., Kanagawa, Japan). At 4 and 6 weeks after surgery, for this experiment, the femur and tibia were fixed with the knee joint at 90° of flexion. In addition, a 25-gauge needle was passed through the tibia as a measure of tibial rotation change. In the test, the proximal tibia was pulled laterally and medially with a 4-0 nylon thread. We photographed x-rays during medial and lateral traction and defined the angular change between the needle and the vertical line of the device as rotational instability. Digital images were acquired using an x-ray sensor (Naomi; RF Co. Ltd., Nagano, Japan) with the settings described above. Based on the soft X-ray images, we quantified the changes in the rotation angle of the tibia using the mentioned image analysis software.

      Analysis of the subchondral bone using micro-computed tomography (μCT)

      For analysis of the subchondral bone, each knee joint was fixed with 4% paraformaldehyde. The knee joints were scanned using a μCT system (Skyscan 1272, BRUKER, MA, USA) with the following parameters: pixel size, 6 μm; voltage, 60 kV; current, 165 μA. Subsequently, the reconstructed image was acquired using the NRecon software (BRUKER, MA, USA). We designated the region of interest on 40 slides of the medial tibial subchondral bone. We then calculated the bone volume/tissue volume fraction (BV/TV, %), trabecular thickness (Tb.Th, mm), trabecular number (Tb.N, 1/mm), and trabecular separation (Tb.Sp, mm) using CTAn software (BRUKER, MA, USA). In addition, the position of the medial meniscus was macroscopically observed from the reconstructed image.

      Histological analysis

      After evaluation of joint instability, the harvested knee joint was fixed with 4% paraformaldehyde for 1 day, decalcification in 10% ethylenediaminetetraacetic acid for 3 weeks, dehydrated and embedded in paraffin. The samples were cut in the sagittal plane (6 μm thickness) using a microtome (ROM-360; Yamato Kohki Industrial Co., Ltd., Saitama, Japan). Safranin-O/fast green staining was performed to evaluate the articular cartilage degeneration. The Osteoarthritis Research Society International (OARSI) histopathological grading system was used to assess cartilage degeneration
      • Glasson S.
      • Chambers M.
      • Van Den Berg W.
      • Little C.
      The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse.
      by two independent observers (KT and SN) who were blinded to all other sample information. We used the medial tibial plateau for analysis. And, one section without meniscal coverage was used for analysis. The average value of the scores of the two observers was used as the representative value.

      Immunohistochemical analysis and TRAP staining

      To evaluate the expression of type X collagen (Col X) and Osterix, we performed immunohistochemical staining using the avidin-biotinylated enzyme complex method and the VECTASTAIN Elite ABC Rabbit IgG Kit (Vector Laboratories, Burlingame, CA, USA). The tissue sections were deparaffinized with xylene and ethanol, and antigen activation was performed using proteinase K (Worthington Biochemical Co., Lakewood, NJ, USA) for 15 min. Endogenous peroxidase was inactivated with 0.3% H2O2/methanol for 30 min. Nonspecific binding of the primary antibody was blocked using normal goat serum for 30 min. The sections were then incubated with anti-collagen X antibody (1:300, ab58632, Abcam) and Osterix polyclonal antibody (1:250, bs-1110R, Bioss) overnight at 4°C. Afterward, the sections were incubated with biotinylated secondary anti-rabbit IgG antibody and stained using Dako Liquid DAB + Substrate Chromogen System (Dako, Glostrup, Denmark).
      Cell nuclei were stained with hematoxylin. For analysis, we calculated the ratio between the number of Col X positive cells and the number of chondrocytes in an articular cartilage area of 10,000 μm2 (100 μm × 100 μm). In the analysis of Osterix, the positive cell ratios in the medullary cavity of the subchondral bone at 2,500 μm2 (50 μm × 50 μm) were compared. We calculated Osterix positive cell ratio from the total number of bone marrow cells at two locations in the medullary cavity of 2,500 μm2 (50 μm × 50 μm) and the total number of Osterix positive cells. According to manufacturer instructions, osteoclast activity was detected by histochemical staining for tartrate-resistant acid phosphatase (TRAP) using the TRAP stain kit (Wako, Osaka, Japan). We then calculated the osteoclast surface/bone surface (Oc.S/BS, %) in the bone marrow region as an analysis of osteoclast activity.

      Statistical analysis

      All data were analyzed using R software, version 3.6.1. First, the normality of all data was verified using the Shapiro–Wilk test. Since the evaluation of joint instability and the OARSI score were nonparametric, the Kruskal–Wallis test was used. Subsequently, the Steel–Dwass test was used for post-hoc analysis. A one-way analysis of variance (ANOVA) was performed for the other data, followed by a post hoc Tukey–Kramer test. Parametric data were expressed as means with 95% confidence intervals (CI), whereas non-parametric data were expressed as medians with interquartile ranges. The statistical significance was set at P < 0.05.

      Results

      Evaluation of joint instability

      Anterior-posterior joint instability was quantified in terms of anterior displacement of the tibia using the anterior drawer test [Fig. 2(A)]. At 4 weeks, the anterior displacement of the ACL-T group increased significantly compared to that of the other groups. The anterior displacement of the CATT group significantly reduced the joint instability of the ACL-T group, but significantly increased it compared to the Sham, DMM, and CATR groups. ([ACL-T vs Sham], P = 0.031; [ACL-T vs CATT], P = 0.032; [ACL-T vs DMM], P = 0.031; [ACL-T vs CATR], P = 0.031; [CATT vs Sham], P = 0.032; [CATT vs DMM], P = 0.032; [CATT vs CATR], P = 0.032).
      Fig. 2
      Fig. 2Evaluation of joint instability using soft x-ray analysis. (A) Results of the anterior drawer test. Anterior tibial displacement was observed in the ACL-T group (a). At both 4 and 6 weeks, the amount of anterior tibial displacement in the ACL-T group was increased compared to that in other groups. The CATT group suppressed the joint instability that occurred in the ACL-T group (b). The data are presented as the median with interquartile range. (B) Results of the tibial rotational test. An increase in tibial rotation angle was observed in the DMM group (a). At both 4 and 6 weeks, the amount of change in tibial rotation angle in the CATR group was smaller than that in the DMM group. The CATR group suppressed the joint instability that occurred in the DMM group (b). The data are presented as the median with interquartile range.
      At 6 weeks, the anterior displacement of the ACL-T group increased significantly compared to that of the other groups. On the other hand, the CATT group significantly reduced the anterior displacement of the ACL-T group, and no significant difference compared to the other groups ([ACL-T vs Sham], P = 0.047; [ACL-T vs CATT], P = 0.032; [ACL-T vs DMM], P = 0.032; [ACL-T vs CATR], P = 0.032).
      Rotational joint instability was quantified in terms of change in the tibial rotation angle using a tibial rotational test [Fig. 2(B)]. At 4 weeks, the change in tibial rotation angle in the DMM group was not significantly different from those in the Sham group but was significantly increased compared to that of the other groups. In contrast, the change in the tibial rotation angle in the CATR group was not significantly different from that in the Sham, ACL-T, and CATT groups ([DMM vs ACL-T], P = 0.032; [DMM vs CATT], P = 0.032; [DMM vs CATR], P = 0.032).
      Similarly, at 6 weeks, the change in tibial rotation angle in the DMM group was not significantly different from that in the Sham group but was significantly increased compared to that of the ACL-T and CATR groups. In contrast, the change in the tibial rotation angle in the CATR group was not significantly different from that in the Sham, ACL-T, and CATT groups ([DMM vs ACL-T], P = 0.032; [DMM vs CATR], P = 0.032).

      Subchondral bone changes between models

      The 3D reconstruction images of the knee joint by μCT analysis are shown in Fig. 3(A). At 4 and 6 weeks, lateral displacement of the medial meniscus was observed in all samples in the DMM and CATR groups, but not in all other groups.
      Fig. 3
      Fig. 3Analysis result by μCT. (A) Comparison of lateral displacement of the medial meniscus. Lateral displacement of the medial meniscus was observed in the DMM and CATR groups (red triangle). No displacement of the medial meniscus was observed in the Sham, ACL-T, and CATT groups (white triangle). (B) Results of BV/TV. At 4 weeks, the DMM group showed higher values than the CATT group, and at 6 weeks, the DMM and CATR groups showed higher values than the ACL-T and CATT groups. Data are presented as the mean with 95% CI. (C) Results of Tb.Th. At 4 weeks, the ACL-T and CATT groups showed lower values than the Sham and DMM groups. At 6 weeks, the DMM group showed higher values than the Sham and ACL-T groups. Data are presented as the mean with 95% CI.
      The results of the BV/TV were shown in Fig. 3(B). At 4 weeks, the BV/TV in the DMM group was significantly higher than that in the CATT group. (P = 0.043, 95%CI [0.325–28.815]). Although there was no significant difference, the BV/TV in the ACL-T and CATT groups was lower than that in the Sham group, and the BV/TV in the DMM group was higher than that in the Sham group [Fig. 3(B)]. At 6 weeks, the BV/TV in the DMM and CATR groups was significantly higher than that in the ACL-T and CATT groups ([DMM vs ACL-T], P = 0.002, 95%CI [7.404–37.582]; [DMM vs CATT], P = 0.014, 95%CI [2.881–33.059]; [CATR vs ACL-T], P = 0.006, 95%CI [4.615–34.793]; [CATR vs CATT], P = 0.048, 95%CI [0.092–30.270]). Although there was no significant difference, the BV/TV in the ACL-T and CATT groups was lower than that in the Sham group, and the BV/TV in the DMM and CATR groups were higher than those in the Sham group.
      The Tb.Th results were shown in Fig. 3(C). At 4 weeks, Tb.Th in the ACL-T and CATT groups were significantly lower than that in the Sham and DMM groups ([ACL-T vs sham], P = 0.014, 95%CI [−0.036 to −0.003]; [ACL-T vs DMM], P = 0.037, 95%CI [0.001–0.034]; [CATT vs Sham], P = 0.002, 95%CI [−0.041 to −0.008]; [CATT vs DMM], P = 0.005, 95%CI [0.006–0.039]). At 6 weeks, Tb.Th in the DMM group was significantly higher than that in the sham and ACL-T groups ([DMM vs Sham], P = 0.011, 95%CI [0.005–0.045]; [DMM vs ACL-T], P = 0.011, 95%CI [0.004–0.043]). Although there was no significant difference, Tb.Th in the CATR group was higher than that in the Sham group.
      There was no significant difference in Tb.N and Tb.Sp between the groups (supplementary results).

      Increased joint instability promotes superficial articular cartilage degeneration

      The results of safranin-O/fast green staining were shown in Fig. 4(A). Surface fibrillation and cartilage destruction in the ACL-T group were confirmed in 5/6 samples at 4 weeks and in 5/6 samples at 6 weeks. In addition, no noticeable superficial cartilage destruction was observed in the other groups. Although there was no significant difference, the OARSI score of the ACL-T group was higher than that of the other groups at 4 weeks [Fig. 4(B)]. Similarly, there was no significant difference in OARSI score at 6 weeks [Fig. 4(B)].
      Fig. 4
      Fig. 4Comparison of articular cartilage degeneration by OARSI score. (A) Representative safranin-O/fast green-stained image of each group. The histological image shows articular cartilage of the medial tibia. In the ACL-T group, irregularity and fibrillation of articular cartilage were observed. (B) The results of OARSI score. There was no significant difference in OARSI scores. The data are presented as the median with interquartile range. Black scale bar, 200 μm; white scale bar, 100 μm.
      The results of immunohistochemical staining of Col X were shown in Fig. 5(A). At 4 weeks, the positive cell rate of Col X in the ACL-T and DMM groups was higher than that in the Sham group [Fig. 5(B)] ([ACL-T vs Sham], P < 0.001, 95%CI [8.195–37.338]; [DMM vs Sham], P = 0.002, 95%CI [6.452–35.595]). At 6 weeks, the Col X-positive cell rate in the ACL-T and DMM groups was higher than that in the Sham group, and that in the CATR group was significantly lower than that in the ACL-T group [Fig. 5(B)] ([ACL-T vs Sham], P = 0.002, 95%CI [5.983–33.025]; [DMM vs Sham], P = 0.022, 95%CI [1.691–28.733]; [CATR vs ACL-T], P = 0.008, 95%CI [−29.272 to −3.488]).
      Fig. 5
      Fig. 5Comparison of Col X positive cell rates. (A) Representative Col X-stained image of each group. (B) At 4 weeks, the positive cell rate of the ACL-T and DMM groups was higher than that of the Sham group. Similarly, at 6 weeks, the positive cell rate of the ACL-T and DMM groups was higher than that of the Sham group. The positive cell rate in the CATR group was lower than that in the ACL-T group. Data are presented as the mean with 95% CI. Scale bar: 100 μm.

      Subchondral bone remodeling differences between models

      The results of TRAP staining were shown in Fig. 6(A). Regarding the activity of osteoclasts in subchondral bone, the Oc.S/BS in the ACL-T group was higher than that in the other groups at 4 weeks [Fig. 6(B)] ([ACL-T vs Sham], P < 0.001, 95%CI [4.087–13.863]; [ACL-T vs CATT], P = 0.015, 95%CI [−10.648 to −0.872]; [ACL-T vs DMM], P = 0.001, 95%CI [−12.395 to −2.619]; [ACL-T vs CATR], P = 0.001, 95%CI [−12.433 to −2.657]). However, there was no significant difference in Oc.S/BS between the groups at 6 weeks [Fig. 6(B)].
      Fig. 6
      Fig. 6The results of TRAP staining. (A) Representative TRAP-stained image of each group. (B) The results of Oc.S/BS. At 4 weeks, Oc.S/BS in the ACL-T group was higher than that in the other groups. No significant difference was observed at 6 weeks. Data are presented as the mean with 95% CI. Scale bar: 100 μm.
      The results of immunohistochemical staining of Osterix were shown in Fig. 7(A). At 4 weeks, the positive cell rate of Osterix in the DMM and CATR groups was higher than that in the Sham group, and that in the CATR group was significantly higher than that in the ACL-T group [Fig. 7(B)] ([DMM vs Sham], P = 0.004, 95%CI [4.743–31.480]; [CATR vs Sham], P = 0.001, 95%CI [7.605–34.342]; [CATR vs ACL-T], P = 0.021, 95%CI [1.748–28.485]). However, at 6 weeks, there was no significant difference in the positive cell rate of Osterix between the groups [Fig. 7(B)].
      Fig. 7
      Fig. 7The results of immunohistochemical staining of Osterix. (A) Representative Osterix-stained image of each group. (B) Analysis results of Osterix positive cell rate. Analysis results of positive cell rate. At 4 weeks, the Osterix positive cell rate in the DMM and CATR groups was higher than the Sham group, and the positive cell rate in the CATR group was higher than that in the ACL-T group. There was no significant difference between the groups at 6 weeks. Data are presented as the mean with 95% CI. Scale bar: 100 μm.

      Discussion

      This study examined the difference between articular cartilage degeneration and subchondral bone changes in four models. CATT and CATR models have been established to control joint instability. Chondrocyte hypertrophy was observed in the ACL-T and DMM groups; however, suppressed in the CATT and CATR groups. Changes in subchondral bone were less affected by the increase or decrease in joint instability. The resorption of subchondral bone was promoted in the ACL-T and CATT groups in which ACL deficiency. On the other hand, in the DMM and CATR groups in which meniscal dysfunction occurred, bone formation of the subchondral bone was promoted.
      In this study, ACL-T and DMM models with joint instability and CATT and CATR models with suppressed joint instability were used. Soft x-ray analysis showed that the CATT group suppressed the joint instability produced by the ACL-T group (Fig. 2), and that the CATR group suppressed joint instability produced by the DMM group (Fig. 2). In addition, lateral deviation of the medial meniscus was observed in the DMM and CATR groups. These results suggest that the CATR group suppresses joint instability, but increases lateral deviation of the medial meniscus, similar to the DMM group, and the meniscus dysfunction remains.
      It has been reported that articular cartilage degeneration depends on the magnitude of joint instability
      • Kamekura S.
      • Hoshi K.
      • Shimoaka T.
      • Chung U.
      • Chikuda H.
      • Yamada T.
      • et al.
      Osteoarthritis development in novel experimental mouse models induced by knee joint instability.
      . Adebayo et al.
      • Adebayo O.O.
      • Ko F.C.
      • Goldring S.R.
      • Goldring M.B.
      • Wright T.M.
      • van der Meulen M.C.H.
      Kinematics of meniscal- and ACL-transected mouse knees during controlled tibial compressive loading captured using roentgen stereophotogrammetry.
      and Glasson et al.
      • Glasson S.
      • Blanchet T.
      • Morris E.
      The Surgical Destabilization of the Medial Meniscus (DMM) Model of Osteoarthritis in the 129/SvEv Mouse.
      had reported that the DMM model has milder joint instability and slower articular cartilage degeneration than the ACL-T model. In addition, our colleague reported that articular cartilage degeneration is delayed by suppressing joint instability that occurs in the ACL-T model
      • Murata K.
      • Kanemura N.
      • Kokubun T.
      • Fujino T.
      • Morishita Y.
      • Onitsuka K.
      • et al.
      Controlling joint instability delays the degeneration of articular cartilage in a rat model.
      • Murata K.
      • Kokubun T.
      • Onitsuka K.
      • Oka Y.
      • Kano T.
      • Mrishita Y.
      • et al.
      Controlling joint instability after anterior cruciate ligament transection inhibits transforming growth factor-beta-mediated osteophyte formation.
      . Out histological results confirmed articular cartilage degeneration in the ACL-T group at 4 and 6 weeks. There was no significant difference in the OARSI score, but the CATT, DMM, and CATR groups had mild articular cartilage degeneration. The results of the present study were milder than those of cartilage degeneration reported in previous studies
      • Glasson S.
      • Blanchet T.
      • Morris E.
      The Surgical Destabilization of the Medial Meniscus (DMM) Model of Osteoarthritis in the 129/SvEv Mouse.
      ,
      • Fang H.
      • Huang L.
      • Welch I.
      • Norley C.
      • Holdsworth D.
      • Beier F.
      • et al.
      Early changes of articular cartilage and subchondral bone in the DMM mouse model of osteoarthritis.
      ,
      • Liao L.
      • Zhang S.
      • Zhao L.
      • Chang X.
      • Han L.
      • Huang J.
      • et al.
      Acute synovitis after trauma precedes and is associated with osteoarthritis onset and progression.
      . One reason for this may be the strain of the mice. In our study using the same ICR mice as in the present study, cartilage degeneration in the ACL-T model was mild
      • Oka Y.
      • Murata K.
      • Kano T.
      • Ozone K.
      • Arakawa K.
      • Kokubun T.
      • et al.
      Impact of Controlling Abnormal Joint Movement on the Effectiveness of Subsequent Exercise Intervention in Mouse Models of Early Knee Osteoarthritis.
      , as in the present study. Therefore, the results of this study may have been influenced by the strain of the mice. The chondrocyte hypertrophy characteristic of early articular cartilage degeneration was significantly increased in the ACL-T and DMM groups, compared to the Sham group at 4 and 6 weeks. However, there was no significant difference between the CATT and CATR groups compared to the Sham group. These results support those of previous studies
      • Murata K.
      • Kanemura N.
      • Kokubun T.
      • Fujino T.
      • Morishita Y.
      • Onitsuka K.
      • et al.
      Controlling joint instability delays the degeneration of articular cartilage in a rat model.
      ,
      • Kamekura S.
      • Hoshi K.
      • Shimoaka T.
      • Chung U.
      • Chikuda H.
      • Yamada T.
      • et al.
      Osteoarthritis development in novel experimental mouse models induced by knee joint instability.
      which suggest that increased joint instability causes articular cartilage degeneration. Our comparison of different models suggested that joint instability was involved in superficial articular cartilage degeneration.
      Subchondral bone changes are important for the onset of OA. Abnormal subchondral bone remodeling is essential for OA pathology
      • Glasson S.
      • Blanchet T.
      • Morris E.
      The Surgical Destabilization of the Medial Meniscus (DMM) Model of Osteoarthritis in the 129/SvEv Mouse.
      ,
      • Nomura M.
      • Sakitani N.
      • Iwasawa H.
      • Kohara Y.
      • Takano S.
      • Wakimoto Y.
      • et al.
      Thinning of articular cartilage after joint unloading or immobilization. An experimental investigation of the pathogenesis in mice.
      ,
      • Berman A.
      • Clauser C.
      • Wunderlin C.
      • Hammond M.
      • Wallace J.
      Structural and mechanical improvements to bone are strain dependent with axial compression of the tibia in female C57BL/6 mice.
      . Previous reports that had induced subchondral bone remodeling in a single model reported that the ACL-T model causes bone loss
      • Wen C.
      • Lu W.
      • Chiu K.
      Importance of subchondral bone in the pathogenesis and management of osteoarthritis from bench to bed.
      and that the DMM model increases BV/TV early after the intervention
      • Fang H.
      • Huang L.
      • Welch I.
      • Norley C.
      • Holdsworth D.
      • Beier F.
      • et al.
      Early changes of articular cartilage and subchondral bone in the DMM mouse model of osteoarthritis.
      . Interestingly, suppression of joint instability inhibited chondrocyte hypertrophy, but had no effect on subchondral bone changes. Specifically, different changes were observed in the ACL-T and CATT groups, in which transected ACL, and in the DMM and CATR groups, in which transected MMTL. The BV/TV decreased in the ACL-T and CATT groups with ACL disconnection. Nomura et al.
      • Nomura M.
      • Sakitani N.
      • Iwasawa H.
      • Kohara Y.
      • Takano S.
      • Wakimoto Y.
      • et al.
      Thinning of articular cartilage after joint unloading or immobilization. An experimental investigation of the pathogenesis in mice.
      had reported that reducing the mechanical stress on joints reduced the BV/TV. Therefore, we speculated that in the ACL-T and CATT groups, joint instability in the anterior-posterior direction caused the decreased BV/TV. In addition, osteoclast activity by TRAP staining was increased in the Oc.S/BS in the ACL-T group. Therefore, we inferred that ACL amputation causes joint instability and distributes mechanical stress to the knee joint by dispersing the contact area, leading to osteoclast activation and BV/TV reduction. In contrast, BV/TV increased in the DMM and CATR groups in which lateral deviation of the medial meniscus was confirmed by MMTL transection. BV/TV and Tb.Th increase when an axial compression load is applied to the tibia
      • Berman A.
      • Clauser C.
      • Wunderlin C.
      • Hammond M.
      • Wallace J.
      Structural and mechanical improvements to bone are strain dependent with axial compression of the tibia in female C57BL/6 mice.
      • Willie B.
      • Birkhold A.
      • Razi H.
      • Thiele T.
      • Aido M.
      • Kruck B.
      • et al.
      Diminished response to in vivo mechanical loading in trabecular and not cortical bone in adulthood of female C57Bl/6 mice coincides with a reduction in deformation to load.
      • De Souza R.
      • Matsuura M.
      • Eckstein F.
      • Rawlinson S.
      • Lanyon L.
      • Pitsillides A.
      Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: a new model to study cortical and cancellous compartments in a single loaded element.
      . Thus, we speculated that the increased BV/TV and Tb.Th in the DMM and CATR groups may have been caused by the increased compressive load on the medial tibia due to lateral deviation of the meniscus. In addition, the Osterix-positive cell ratio was higher in the DMM and CATR groups. Therefore, we inferred that the lateral deviation of the medial meniscus promoted osteoblast differentiation and contributed to bone formation.
      Summarize, it was suggested that the suppression of joint instability inhibited articular cartilage degeneration by suppressing chondrocyte hypertrophy. However, the changes in the subchondral bone showed different characteristics in the ACL transected group and the MMTL transected group. The reason for the difference in subchondral bone changes may be the difference in the function of the ACL and the meniscus. The function of the ACL is to resist the shearing force against the knee joint. On the other hand, the main function of the meniscus is to distribute the compression stress to the knee joint and to maintain the conformity of the femur and tibia. Therefore, it can be inferred that dysfunction of the ACL leads to an increase in shear force, and dysfunction of the meniscus leads to an increase in compressive stress. Consequently, the difference in the changes of the subchondral bone may be influenced by the increase of different mechanical stresses caused by the dysfunction of the ACL and the meniscus. In future studies, we will verify whether the results obtained in this study are due to the different types of mechanical stress by reproducing the different types of mechanical stress in in vitro and ex vivo experiments.
      The main limitations of this study are the following two points. First, the joint instability was only evaluated under static conditions. Therefore, it is necessary to evaluate dynamic joint instability by analyzing the difference in joint instability during walking of mice by motion analysis. Second, the observed articular cartilage degeneration is mild, and the difference in articular cartilage degeneration and subchondral bone changes between the models may be due to the difference in progression rate. Thus, future studies should investigate the difference in articular cartilage degeneration and subchondral bone changes of each model by performing longer-term verification.
      In conclusion, the results of this study suggested that joint instability promotes chondrocyte hypertrophy and contributes to articular cartilage degeneration. The results also suggested that ACL or meniscus dysfunction may have different effects on subchondral bone changes.

      Contributions

      All authors approved the final submitted manuscript.
      Study design: KA, KM, NK, and TK.
      Data collection, Histological analysis: KA, KT, YO, KO, SN and SE.
      Manuscript composition KA, TK.

      Conflict of interest

      All authors have no conflicts of interest related to the manuscript.

      Funding sources

      This study was supported by Grant of Saitama Chapter, Japanese Physical Therapy Association for Study Promotion (Grant Number: 20-06), and Saitama Prefectural University Research Grant (Grant Number: 18009).

      Acknowledgements

      The author(s) received no financial support for the research, authorship, and/or publication of this article. And we would like to thank Editage (www.editage.com) for English language editing.

      Appendix A. Supplementary data

      The following is the Supplementary data to this article:

      References

        • Lohmander L.S.
        What can we do about osteoarthritis?.
        Arthritis Res. 2000; 2: 95-100https://doi.org/10.1186/ar74
        • Loeser R.F.
        • Collins J.A.
        • Diekman B.O.
        Ageing and the pathogenesis of osteoarthritis.
        Nat Rev Rheumatol. 2016; 12: 412-420https://doi.org/10.1038/nrrheum.2016.65
        • Radin E.L.
        • Rose R.M.
        Role of subchondral bone in the initiation and progression of cartilage damage.
        Clin Orthop Relat Res. 1986; 213: 34-40https://doi.org/10.1097/00003086-198612000-00005
        • Intema F.
        • Hazewinkel H.A.W.
        • Gouwens D.
        • Bijlsma J.
        • Weinans H.
        • Lafeber F.
        • et al.
        In early OA, thinning of the subchondral plate is directly related to cartilage damage: results from a canine ACLT-meniscectomy model.
        Osteoarthritis Cartilage. 2010; 18: 691-698https://doi.org/10.1016/j.joca.2010.01.004
        • Panula H.E.
        • Nieminen J.
        • Parkkinen J.J.
        • Arnala I.
        • Kröger H.
        • Alhava E.
        Subchondral bone remodeling increases in early experimental osteoarthrosis in young beagle dogs.
        Acta Orthop Scand. 1998; 69: 627-632https://doi.org/10.3109/17453679808999269
        • Botter S.
        • van Osch G.
        • Waarsing J.
        • van der Linden J.
        • Verhaar J.
        • van Leeuwen J.
        • et al.
        Cartilage damage pattern in relation to subchondral plate thickness in a collagenase-induced model of osteoarthritis.
        Osteoarthritis Cartilage. 2008; 16: 506-514https://doi.org/10.1016/j.joca.2007.08.005
        • Sniekers Y.H.
        • Intema F.
        • Lafeber F.
        • Van Osch G.
        • Van Leeuwen J.
        • Weinans H.
        • et al.
        A role for subchondral bone changes in the process of osteoarthritis; A micro-CT study of two canine models.
        BMC Muscoskel Disord. 2008; 9: 1-11https://doi.org/10.1186/1471-2474-9-20
        • Felson D.T.
        Osteoarthritis as a disease of mechanics.
        Osteoarthritis Cartilage. 2013; 21: 10-15https://doi.org/10.1016/j.joca.2012.09.012
        • Nam J.
        • Aguda B.
        • Rath B.
        • Agarwal S.
        Biomechanical thresholds regulate inflammation through the NF-κB pathway: experiments and modeling.
        PLoS One. 2009; 4https://doi.org/10.1371/journal.pone.0005262
        • Iijima H.
        • Ito A.
        • Nagai M.
        • Tajino J.
        • Yamaguchi S.
        • Kiyan W.
        • et al.
        Physiological exercise loading suppresses post-traumatic osteoarthritis progression via an increase in bone morphogenetic proteins expression in an experimental rat knee model.
        Osteoarthritis Cartilage. 2017; 25: 964-975https://doi.org/10.1016/j.joca.2016.12.008
        • Yamaguchi S.
        • Aoyama T.
        • Ito A.
        • Nagai M.
        • Iijima H.
        • Zhang X.
        • et al.
        Effects of exercise level on biomarkers in a rat knee model of osteoarthritis.
        J Orthop Res. 2013; 31: 1026-1031https://doi.org/10.1002/jor.22332
        • Murata K.
        • Kanemura N.
        • Kokubun T.
        • Fujino T.
        • Morishita Y.
        • Onitsuka K.
        • et al.
        Controlling joint instability delays the degeneration of articular cartilage in a rat model.
        Osteoarthritis Cartilage. 2017; 25: 297-308https://doi.org/10.1016/j.joca.2016.10.011
        • Onitsuka K.
        • Murata K.
        • Kokubun T.
        • Fujiwara S.
        • Nakajima A.
        • Morishita Y.
        • et al.
        Effects of controlling abnormal joint movement on expression of MMP13 and TIMP-1 in osteoarthritis.
        Cartilage. 2020; 11: 98-107https://doi.org/10.1177/1947603518783449
        • Glasson S.
        • Blanchet T.
        • Morris E.
        The Surgical Destabilization of the Medial Meniscus (DMM) Model of Osteoarthritis in the 129/SvEv Mouse.
        2007: 1061-1069https://doi.org/10.1016/j.joca.2007.03.006 (Published online)
        • Kamekura S.
        • Hoshi K.
        • Shimoaka T.
        • Chung U.
        • Chikuda H.
        • Yamada T.
        • et al.
        Osteoarthritis development in novel experimental mouse models induced by knee joint instability.
        Osteoarthritis Cartilage. 2005; 13: 632-641https://doi.org/10.1016/j.joca.2005.03.004
        • Adebayo O.O.
        • Ko F.C.
        • Goldring S.R.
        • Goldring M.B.
        • Wright T.M.
        • van der Meulen M.C.H.
        Kinematics of meniscal- and ACL-transected mouse knees during controlled tibial compressive loading captured using roentgen stereophotogrammetry.
        J Orthop Res. 2017; 35: 353-360https://doi.org/10.1002/jor.23285
        • Kwok J.
        • Onuma H.
        • Olmer M.
        • Lotz M.K.
        • Grogan S.P.
        • D'Lima D.D.
        Histopathological analyses of murine menisci: implications for joint aging and osteoarthritis.
        Osteoarthritis Cartilage. 2016; 24: 709-718https://doi.org/10.1016/j.joca.2015.11.006
        • Kokubun T.
        • Kanemura N.
        • Murata K.
        • Moriyama H.
        • Morita S.
        • Jinno T.
        • et al.
        Effect of changing the joint kinematics of knees with a ruptured anterior cruciate ligament on the molecular biological responses and spontaneous healing in a rat model.
        Am J Sports Med. 2016; 44: 2900-2910https://doi.org/10.1177/0363546516654687
        • Oka Y.
        • Murata K.
        • Kano T.
        • Ozone K.
        • Arakawa K.
        • Kokubun T.
        • et al.
        Impact of Controlling Abnormal Joint Movement on the Effectiveness of Subsequent Exercise Intervention in Mouse Models of Early Knee Osteoarthritis.
        Cartilage, 2019https://doi.org/10.1177/1947603519885007 (Published online)
        • Glasson S.
        • Chambers M.
        • Van Den Berg W.
        • Little C.
        The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the mouse.
        Osteoarthritis Cartilage. 2010; 18: S17-S23https://doi.org/10.1016/j.joca.2010.05.025
        • Murata K.
        • Kokubun T.
        • Onitsuka K.
        • Oka Y.
        • Kano T.
        • Mrishita Y.
        • et al.
        Controlling joint instability after anterior cruciate ligament transection inhibits transforming growth factor-beta-mediated osteophyte formation.
        Osteoarthritis Cartilage. 2019; 27: 1185-1196https://doi.org/10.1016/j.joca.2019.03.008
        • Fang H.
        • Huang L.
        • Welch I.
        • Norley C.
        • Holdsworth D.
        • Beier F.
        • et al.
        Early changes of articular cartilage and subchondral bone in the DMM mouse model of osteoarthritis.
        Sci Rep. 2018; 8: 1-9https://doi.org/10.1038/s41598-018-21184-5
        • Liao L.
        • Zhang S.
        • Zhao L.
        • Chang X.
        • Han L.
        • Huang J.
        • et al.
        Acute synovitis after trauma precedes and is associated with osteoarthritis onset and progression.
        Int J Biol Sci. 2020; 16: 970-980https://doi.org/10.7150/ijbs.39015
        • Wen C.
        • Lu W.
        • Chiu K.
        Importance of subchondral bone in the pathogenesis and management of osteoarthritis from bench to bed.
        J Orthop Transl. 2014; 2: 16-25https://doi.org/10.1016/j.jot.2013.11.004
        • Nomura M.
        • Sakitani N.
        • Iwasawa H.
        • Kohara Y.
        • Takano S.
        • Wakimoto Y.
        • et al.
        Thinning of articular cartilage after joint unloading or immobilization. An experimental investigation of the pathogenesis in mice.
        Osteoarthritis Cartilage. 2017; 25: 727-736https://doi.org/10.1016/j.joca.2016.11.013
        • Berman A.
        • Clauser C.
        • Wunderlin C.
        • Hammond M.
        • Wallace J.
        Structural and mechanical improvements to bone are strain dependent with axial compression of the tibia in female C57BL/6 mice.
        PLoS One. 2015; 10: 1-16https://doi.org/10.1371/journal.pone.0130504
        • Willie B.
        • Birkhold A.
        • Razi H.
        • Thiele T.
        • Aido M.
        • Kruck B.
        • et al.
        Diminished response to in vivo mechanical loading in trabecular and not cortical bone in adulthood of female C57Bl/6 mice coincides with a reduction in deformation to load.
        Bone. 2013; 55: 335-346https://doi.org/10.1016/j.bone.2013.04.023
        • De Souza R.
        • Matsuura M.
        • Eckstein F.
        • Rawlinson S.
        • Lanyon L.
        • Pitsillides A.
        Non-invasive axial loading of mouse tibiae increases cortical bone formation and modifies trabecular organization: a new model to study cortical and cancellous compartments in a single loaded element.
        Bone. 2005; 37: 810-818https://doi.org/10.1016/j.bone.2005.07.022