Biology subjects

Kokubun, T.

Publications and source records attributed to Kokubun, T..

10 recordsLinked to original sources

bioRxiv2026

A Novel Slow-Progressive Knee Osteoarthritis Murine Model Induced by Non-Invasive Posterior Cruciate Ligament Rupture in Mice

ObjectiveRecently, alternatives to animal testing, such as new approach methodologies, are being developed in the orthopedic research field; animal models still provide valuable insights into the pathogenesis of knee osteoarthritis (OA). However, commonly used models develop OA much more rapidly and severely than those observed in human patients. We aimed to develop a novel murine model that closely mimics the slow progression of human OA with posterior Cruciate ligament (PCL) rupture. Design12-week-old C57BL/6 mice were induced to PCL-rupture (PCL-R) by manually applying an external tibial posterior translation force. We analyzed joint kinematics, histological observations, and bone structure to confirm the absence of concurrent injury on day 0. Then, joint stability and the pathophysiological progression of knee OA were analyzed at 8, 16, and 34 weeks post-PCL-R. The destabilized medial meniscus (DMM) model was also analyzed to compare the OA progression. ResultsNon-invasive PCL-R intervention induced the complete rupture in the central region of PCL without concurrent injury. The PCL-R group showed larger posterior tibial deviation than the INTACT (P=0.008). Regarding the range of motion in the PCL-R group, there was no limitation in range of motion on day 0, but extension limitations occurred at weeks 16 and 34 weeks. Histologically, articular cartilage degeneration in PCL-R was milder than DMM. In the subchondral bone, micro-CT reconstruction images indicated that, compared with the INTACT group, the DMM group observed progressive subchondral bone formation from 16 weeks post-surgery. In contrast, the PCLR group maintained the subchondral bone structure even at 34 weeks. ConclusionsPCL-R model induced mild abnormal mechanical stress depending on posterior instability, and cartilage degeneration occurred more slowly in this model than in DMM models.

pathology↗

bioRxiv2025

Novel Biomaterial-Based Synovial Fluid Analysis Reveals Protective microRNA Signatures in a Mouse Model of Acute Synovitis-Driven Osteoarthritis

Acute synovitis and continuous mechanical stress are critical factors of Post-traumatic Osteoarthritis (PTOA); however, the effect on cartilage degeneration and its underlying mechanisms remains unclear. Here, we established a novel biomaterial-based method for synovial fluid analysis in mice to uncover the mechanisms underlying the onset of PTOA, focusing on synovitis and mechanical stress. Twelve-week-old C57BL/6J males were divided to the ACL-Transection (ACL-T), ACL-rupture (ACL-R), and Intact groups. We performed joint instability test and histological analysis for cartilage degeneration and synovitis at 2, 6, and 10 weeks. Real time PCR was performed on articular cartilage at 2 weeks and synovium at 2, 6, and 10 weeks. Tetra-slime was injected into the knee joint, and solidified slime containing synovial fluid molecules was analyzed in digital PCR at 2 weeks. Acute synovitis and cartilage degeneration were induced in the ACL-T group at 2 weeks, although no difference was observed in joint instability between both two ACL injury models. Real time PCR showed significant increase of Mmp-3, Tnf-, Ifn-{gamma}, and inos in synovium of the ACL-T group. During this tissue interaction, although there were no significant differences, miR145-5p and miR149-5p in synovial fluid were also upregulated in the ACL-T group compared to the ACL-R group. Unlike the early stage, no histological or biological differences were observed between groups at 6 and 10 weeks. In conclusion, acute synovitis caused secondary cartilage degeneration via MMP-3 and TNF- in the synovium during the early stage and may involve M1 macrophage activation. Whereas it was suggested that synovial fluid miRNA might be produced to suppress the secondary cartilage degeneration. Furthermore, mechanical stress was the dominant factor in the late stage of OA, regardless of the initial synovitis condition.

pathology↗

bioRxiv2024

The braking force to Control the Abnormal Tibial Translation Affects the Spontaneous Healing Response for Complete Ruptured Anterior Cruciate Ligament in Mice Model

Purpose of this studyThe anterior cruciate ligament (ACL) has been believed to have a low spontaneous healing capacity; growing evidence has suggested that ACL could heal spontaneously. While the healed ACL has reduced mechanical properties and incomplete tissue maturation, the mechanisms underlying these problems remain unknown. We aimed to elucidate the effect of mechanical stresses during the early phase of spontaneous ACL healing. Materials and MethodsMale and female C57BL/6 mice were subjected to ACL rupture and randomly classified into three groups: Tight-CATT; tightly controlled anterior tibial translation (ATT), Loose-CATT; loosely controlled ATT and mild increasing mechanical stress compared to Tight-CATT, and ACL-Rupture (ACL-R) group; not controlled ATT. Mice were sacrificed and analyzed immediately after injury and at 4 and 8 weeks. We evaluated the effect of controlling the braking force of the ATT of each knee, the success rate of the ACL healing, collagen maturation, COL1A1 expression in the healed ACL, and the mechanical properties of the healed ACL. ResultsThe Tight-CATT group showed a higher success rate of ACL healing than the Loose-CATT group at 4 and 8 weeks. However, collagen maturation and the mechanical properties of healed ACL did not differ between the Tight-CATT and Loose-CATT groups. ConclusionOur results suggested that loose ATT braking immediately after injury is a negative factor for the healing of the completely ruptured ACL, and that it may be necessary to apply higher mechanical stress in the later stages to achieve greater healing.

physiology↗

bioRxiv2024

Concurrent Joint Contact in Anterior Cruciate Ligament Injury induces cartilage micro-injury and subchondral bone sclerosis, resulting in knee osteoarthritis

ObjectiveAnterior Cruciate Ligament (ACL) injury initiates post-traumatic osteoarthritis (PTOA) via two distinct processes: initial direct contact injury of the cartilage surface during ACL injury, and secondary joint instability due to the ACL deficiency. Using the well-established Compression-induced ACL rupture method (ACL-R) and a novel Non-Compression ACL-R model, we aimed to reveal the individual effects of cartilage compression and joint instability on PTOA progression after ACL injury in mice. DesignTwelve-week-old C57BL/6J male were randomly divided to three experimental groups: Compression ACL-R, Non-Compression ACL-R, and Intact. Following ACL injury, we performed joint laxity testing and microscopic analysis of the articular cartilage surface at 0 days, in vivo optical imaging of matrix-metalloproteinase (MMP) activity at 3 and 7 days, and histological and microCT analysis at 0, 7, 14, and 28 days. ResultsThe Compression ACL-R group exhibited a significant increase of cartilage roughness immediately after injury compared with the Non-Compression group. At 7 days, the Compression group exhibited increased MMP-induced fluorescence intensity and MMP-13 positive cell ratio of chondrocytes. Moreover, histological cartilage degeneration was observable in the Compression group at the same time point. Sclerosis of tibial subchondral bone in the Compression group was more significantly developed than in the Non-Compression group at 28 days. ConclusionsBoth Compression and Non-Compression ACL injury initiated PTOA progression due to joint instability. However, joint contact during ACL rupture also caused initial micro-damage on the cartilage surface and initiated early MMP activity, which could accelerate PTOA progression compared to ACL injury without concurrent joint contact.

molecular biology↗

bioRxiv2024

Muscle Contraction is Essential for Tendon Healing and Muscle Function Recovery after Achilles Tendon Rupture and Surgical Repair

Incomplete tendon healing and postponed muscle weakness after Achilles tendon rupture and surgical repair lead to poor performance in patient activities. Although the effectiveness of postoperative early functional rehabilitation has been proven, the priority and each effect of specific methods in early rehabilitation remain unclear. We hypothesized early muscle contraction exercises without joint motion would promote tendon healing and prevent calf muscle atrophy; in contrast, early static stretching after surgical repair would not contribute to tendon healing and induce calf muscle atrophy. C57Bl/6 mice underwent Achilles tendon rupture and suture repair, followed by different methods of post-surgery interventions: a non-exercise group, a Static stretching group, and an Electrical muscle stimulation group. 3 and 5 weeks after surgery, we assessed ex vivo tendon mechanical properties, collagen fiber alignment, and histological muscle properties. Electrical Muscle Stimulation restored the recovery of tendon mechanical properties and muscle strength more quickly than Static stretching. Static stretching had no additional effect on them compared to the non-exercise. Our results suggested that calf muscle contraction was essential as a post-surgery early functional rehabilitation to load tensile forces on tendons and improve Achilles tendon healing. Additionally, early muscle contractions naturally promote restoring muscle function after the rupture, but further research is needed to optimize muscle contraction protocols. Statement of Clinical SignificanceThis study shows the importance of selecting appropriate exercise modalities to resolve imperfections in tendon healing and muscle recovery. The establishment of proper rehabilitation is expected to improve post-surgery outcomes. Study DesignA controlled laboratory study.

physiology↗

bioRxiv2023

The mechanical force with the physiological movement works as a biological cue in tendon development during the postnatal phase in mice

The musculoskeletal system provides structural stability and coordination to enable movement. Tendons have the essential role of efficiently transmitting force generated from muscle contraction to bone to enable ambulation. In doing so, they resist high external forces. In fact, muscle contraction during embryonic development is required to maintain tendon growth and differentiation. Nonetheless, defining the types and magnitudes of loads that act on tendons during embryonic and early postnatal periods is quite difficult. In this study, we aimed to define the physiologic limb movement and forces experienced during these stages in the murine model. We found that late-stage embryos had limited amniotic space, which attenuated limb movement. In the neonatal phase, physical ability, as measured by rollover function and locomotion, increased. These changes, which likely corresponded to increased forces applied to the tendons, corresponded with the expression of tenogenic markers during the embryo to postnatal phase. In particular, we found that the upregulation of Scx and Tnmd correlated with increased movement during the two weeks after birth. Our results help define the spatiotemporal role of mechanical force, including internal and external factors, in tendon growth and development. HighlightsO_LIAssessed limb movement in amnion. C_LIO_LISpace limitation attenuated limb movement in the late-stage embryos. C_LIO_LIDefined the mechanical force from the limbs physiological environment. C_LIO_LIScx and Tnmd were upregulated synchronically with rollover function and locomotion. C_LIO_LIMechanical forces may work as the cue of tendon development C_LI

developmental biology↗

bioRxiv2023

Characteristic differences in tibial subchondral bone changes in the post-traumatic knee osteoarthritis model

ObjectiveMeniscus degeneration and subchondral bone changes contribute to the development and progression of knee OA. The purpose of this study was to reveal the relationship between medial meniscus degeneration and characteristics of subchondral bone changes in different models DesignWe used the anterior cruciate ligament transection (ACL-T) model, destabilization of the medial meniscus (DMM) model, controlled abnormal tibial translation (CATT) model and a controlled abnormal tibial rotation (CATR) model with different mechanical stresses. We performed histological analysis and micro computed tomography analysis, at 4 and 6 weeks. In addition, we divided the tibial subchondral bone into four compartments and set the regions of interest ResultsMeniscus degeneration was observed in all groups, but there was no significant difference. The ACL-T group showed posterior displacement of the contact area, and the DMM and CATR groups showed lateral deviation of the medial meniscus. The region-specific subchondral bone changes in each model showed that changes in mechanical stress due to ACL and meniscus dysfunction, as well as changes in the contact area, affect the bony structure of the subchondral bone differently in each region. ConclusionsSubchondral bone changes in different models of mechanical stress were different in each region. In particular, changes in the contact area and increased compressive stress due to meniscus dysfunction were suggested to promote bone formation. The results of this study indicate that changes in alignment and contact area in the PTOA model may cause region-specific characteristics of the subchondral bone changes.

pathology↗

bioRxiv2022

Abnormal Tibia Translation leads Directly the Surface Cartilage Degeneration with Molecular Biological Response using a Novel Non-Invasive ACL ruptured Mice Model

ObjectiveThe ACL-deficient model helps to clarify the mechanism of knee OA; however, the conventional ACL injury model could have included concurrent onset factors such as direct compression stress to cartilage and subchondral bone. In this study, we established a novel Non-invasive ACL-Ruptured mouse model without concurrent injuries and elucidated the relationship between OA progression and joint instability. DesignWe induced the ACL-Rupture non-invasively in twelve-week-old C57BL/6 male mice and evaluated histological, macroscopical, and morphological analysis at 0 days. Next, we created the ACL-R, controlled abnormal tibial translation (CATT), and Sham groups. Then, the joint stability and OA pathophysiology were analyzed at 2, 4, and 8 weeks. ResultsNo intra-articular injuries, except for ACL rupture, were observed in the ACL-R model. ACL-R mice increased anterior tibial displacement compared to the Sham group (p<0.001, 95% CI [-1.509 to -0.966]) and CATT group (p<0.001, 95% CI [-0.841 to -0.298]) at 8 weeks. All mice in the ACL-R group caused cartilage degeneration. The degree of cartilage degeneration in the ACL-R group was higher than in the CATT group (p=0.006) at 8 weeks. The MMP-3-positive cell rate of chondrocytes increased in the ACL-R group than CATT group from 4 weeks (p=0.043; 95% CI [-28.32 to -0.364]) while that of synovial cells increased at 8 weeks (p=0.031; 95% CI [-23.398 to -1.021]). ConclusionWe successfully established a Non-invasive ACL-R model without intra-articular damage. Our model revealed that chondrocytes might react to abnormal mechanical stress prior to synovial cells while the knee OA onset.

molecular biology↗

bioRxiv2021

Effect of differences in mechanical stress in vivo on the onset and progression of knee osteoarthritis

ObjectiveThe effect of the type of mechanical stress on OA onset has not been clarified. The aim of this study was to establish a new model that reproduces the type and increase and decrease of mechanical stress in vivo and to clarify the differences in the mechanism of knee OA onset and progression among the models. DesignTo reproduce the difference in mechanical stress, 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 that suppressed the joint instability of the ACL-T and DMM model, respectively. These four models reproduced the increase and decrease in shear force due to joint instability and compressive stress due to meniscal dysfunction. We performed joint instability analysis with soft X-ray, micro computed tomography analysis, histological analysis, and immunohistological analysis in 4 and 6 weeks. ResultsJoint instability decreased in the CATT and CATR groups. The meniscus deviated in the DMM and CATR groups. Chondrocyte hypertrophy increased in the ACL-T and DMM groups with joint instability. In the subchondral bone, bone resorption was promoted in the ACL-T and CATT groups, and bone formation was promoted in the DMM and CATR groups. ConclusionsIncreased shear force causes articular cartilage degeneration and osteoclast activation in the subchondral bone. In contrast, increased compressive stress promotes bone formation in the subchondral bone earlier than articular cartilage degeneration occurs.

pathology↗

bioRxiv2021

Effect of Suppression of Rotational Joint Instability on Cartilage and Meniscus Degeneration in Mouse Osteoarthritis Model

ObjectiveJoint instability and meniscal dysfunction contribute to the onset and progression of knee osteoarthritis (OA). In the destabilization of the medial meniscus (DMM) model, secondary OA occurs due to the rotational instability and increases compressive stress resulting from the meniscal dysfunction. We created a new controlled abnormal tibial rotation (CATR) model that reduces the rotational instability that occurs in the DMM model. So, we aimed to investigate whether rotational instability affects articular cartilage degeneration using the DMM and CATR models, as confirmed using histology and immunohistochemistry. DesignTwelve-week-old male mice were randomized into 3 groups: DMM group, CATR group, and INTACT group (right knee of the DMM group). After 8 and 12 weeks, we performed the tibial rotational test, safranin-O/fast green staining, and immunohistochemical staining for TNF- and MMP-13. ResultsThe rotational instability in the DMM group was significantly higher than that of the other groups. And articular cartilage degeneration was higher in the DMM group than in the other groups. However, meniscal degeneration was observed in both DMM and CATR groups. The TNF- and MMP-13 positive cell rates in the articular cartilage of the CATR group were lower than those in the DMM group. ConclusionsWe found that the articular cartilage degeneration was effectively suppressed by controlling the rotational instability caused by meniscal dysfunction. These findings suggest that suppression of rotational instability in the knee joint is an effective therapeutic measure for preventing OA progression.

pathology↗