Histological and immunohistochemical analyses of articular cartilage during onset and progression of pre- and early-stage osteoarthritis in a rodent model.

Early diagnosis and treatment of pre- and early-stage osteoarthritis (OA) is important. However, the cellular and cartilaginous changes occurring during these stages remain unclear. We investigated the histological and immunohistochemical changes over time between pre- and early-stage OA in a rat model of traumatic injury. Thirty-six male rats were divided into two groups, control and OA groups, based on destabilization of the medial meniscus. Histological and immunohistochemical analyses of articular cartilage were performed on days 1, 3, 7, 10, and 14 postoperatively. Cell density of proteins associated with cartilage degradation increased from postoperative day one. On postoperative day three, histological changes, including chondrocyte death, reduced matrix staining, and superficial fibrillation, were observed. Simultaneously, a compensatory increase in matrix staining was observed. The Osteoarthritis Research Society International score increased from postoperative day seven, indicating thinner cartilage. On postoperative day 10, the positive cell density decreased, whereas histological changes progressed with fissuring and matrix loss. The proteoglycan 4-positive cell density increased on postoperative day seven. These findings will help establish an experimental model and clarify the mechanism of the onset and progression of pre- and early-stage traumatic OA.

Effects of acute- and long-term aerobic exercises at different intensities on bone in mice.

INTRODUCTION: Exercise intensity determines the benefits of aerobic exercise. Our objectives were, in aerobic exercise at different intensities, to determine (1) changes in bone metabolism-related genes after acute exercise and (2) changes in bone mass, strength, remodeling, and bone formation-related proteins after long-term exercise. MATERIALS AND METHODS: Total 36 male C57BL/6J mice were divided into a control group and exercise groups at 3 different intensities: low, moderate, or high group. Each exercise group was assigned to acute- or long-term exercise groups. Tibias after acute exercise were evaluated by real-time PCR analysis. Furthermore, hindlimbs of long-term exercise were assessed by micro-CT, biomechanical, histological, and immunohistochemical analyses. RESULTS: Acute moderate-intensity exercise decreased RANKL level as bone resorption marker, whereas low- and high-intensity exercise did not alter it. Additionally, only long-term exercise at moderate intensity increased bone mass and strength. Moderate-intensity exercise promoted osteoblast activity and suppressed osteoclast activity. After low- and high-intensity exercise, osteoblast and osteoclast activity were unchanged. An increase in the number of ß-catenin-positive cells and a decrease in sclerostin-positive cells were observed in the only moderate group. CONCLUSION: These results showed that moderate-intensity exercise can inhibit bone resorption earlier, and long-term exercise can increase bone mass and strength through promoted bone formation via the Wnt/ß-catenin activation. High-intensity exercise, traditionally considered better for bone, may fail to stimulate bone remodeling, leading to no change in bone mass and strength. Our findings suggest that moderate-intensity exercise, neither too low nor high, can maintain bone health.

Analysis of Molecular Changes and Features in Rat Knee Osteoarthritis Cartilage: Progress From Cellular Changes to Structural Damage.

OBJECTIVE: Although knee osteoarthritis (KOA) is a common disease, there is a lack of specific prevention and early treatment methods. Hence, this study aimed to examine the molecular changes occurring at different stages of KOA to elucidate the dynamic nature of the disease. DESIGN: Using a low-force compression model and analyzing RNA sequencing data, we identified molecular changes in the transcriptome of knee joint cartilage, including gene expression and molecular pathways, between the cellular changes and structural damage stages of KOA progression. In addition, we validated hub genes using an external dataset. RESULTS: Gene set enrichment analysis (GSEA) identified the following pathways to be associated with KOA: "B-cell receptor signaling pathway," "cytokine-cytokine receptor interaction," and "hematopoietic cell lineage." Expression analysis revealed 585 differentially expressed genes, with 579 downregulated and 6 upregulated genes. Enrichment and clustering analyses revealed that the main molecular clusters were involved in cell cycle regulation and immune responses. Furthermore, the hub genes Csf1r, Cxcr4, Cxcl12, and Ptprc were related to immune responses. CONCLUSIONS: Our study provides insights into the dynamic nature of early-stage KOA and offers valuable information to support the development of effective intervention strategies to prevent the irreversible damage associated with KOA, thereby addressing a major clinical challenge.

Development of a novel model for intraarticular adhesion in rat knee joint.

In this study, a novel rat model of knee joint adhesion was developed, and its formation was analyzed quantitatively over time. Thirty-nine Wistar rats were randomly divided into intact control (n = 3) and experimental (n = 36) groups. The latter was equally divided into three groups according to the experimental intervention: fixed with deep bending of the knee joint (group I), fixed after incision of the capsule (group II), and fixed after exposure of the patellofemoral joint to artificial patellar subluxation (group III). All rats were subdivided according to their joint immobilization period (1, 2, or 4 weeks). Thereafter, the limited range of motion of the knee joint with (limited knee range of motion) and without (limited knee joint intrinsic range of motion) skin and muscles were measured. The lengths of adhesions of the anterior knee joint and posterior capsules were evaluated histologically. The limited intrinsic range of motion of the knee joint was found to be increased in groups II and III compared to that in group I 4 weeks after immobilization. Adhesions were confirmed within 1 week after immobilization in groups II and III. The length of the adhesions in group III was significantly longer than in other groups at 2 weeks and remained longer than in group I at 4 weeks. This model may contribute to the assessment of the adhesion process and development of new therapeutic avenues following trauma or surgical invasion.

Higher-intensity ultrasound accelerates fracture healing via mechanosensitive ion channel Piezo1.

Osteoporosis-related fractures are a major public health problem. Mechanobiological stimulation utilizing low-intensity pulsed ultrasound (LIPUS) is the most widely accepted modality for accelerating fracture healing. However, recent evidence has demonstrated the ineffectiveness of LIPUS, and the biophysical mechanisms of ultrasound-induced bone formation also remain elusive. Here, we demonstrate that ultrasound at a higher intensity than LIPUS effectively accelerates fracture healing in a mouse osteoporotic fracture model. Higher-intensity ultrasound promoted chondrogenesis and hypertrophic differentiation of chondrocytes in the fracture callus. Higher-intensity ultrasound also increased osteoblasts and newly formed bone in the callus, resulting in accelerated endochondral ossification during fracture healing. In addition, we found that accelerated fracture healing by ultrasound exposure was attenuated when the mechanosensitive ion channel Piezo1 was inhibited by GsMTx4. Ultrasound-induced new bone formation in the callus was attenuated in fractured mice treated with GsMTx4. Similar results were also confirmed in a 3D osteocyte-osteoblast co-culture system, where osteocytic Piezo1 knockdown attenuated the expression of osteoblastic genes after ultrasound exposure. Together these results demonstrate that higher-intensity ultrasound than clinically used LIPUS can accelerate endochondral ossification after fractures. Furthermore, our results suggest that mechanotransduction via Piezo1 mediates ultrasound-stimulated fracture healing and bone formation.

Ultrasound therapy for a week promotes regeneration and reduces pro-inflammatory macrophages in a rat sciatic nerve autograft model.

Peripheral nerve injury causes long-term motor dysfunction. Ultrasound (US) therapy is expected to accelerate peripheral nerve regeneration. However, its optimal usage and effects on macrophage phenotypes during peripheral nerve regeneration remain unknown. In this study, we investigated the optimal duration of US therapy and its effects on macrophage phenotype. Twenty-seven rats with autologous sciatic nerve grafting were divided into three groups: two received US therapy (1 MHz frequency, intensity of 140 mW/cm2, 20% duty cycle, 5 min/day) for one (US1) or 4 weeks (US4), and one group received sham stimulation. Immunohistochemistry was performed 3 and 7 days after injury in another set of 12 rats. Eight weeks after the injury, the compound muscle action potential amplitude of the gastrocnemius in the US1 and US4 groups was significantly higher than that in the sham group. The toe-spreading test showed functional recovery, whereas the gait pattern during treadmill walking did not recover. There were no significant differences in motor function, histomorphometry, or muscle weight between groups. Immunohistochemistry showed that US therapy decreased the number of pro-inflammatory macrophages seven days after injury. Therefore, US therapy for both one or 4 weeks can similarly promote reinnervation and reduce proinflammatory macrophages in autograft model rats.

Treadmill Exercise Suppresses Histological Progression of Disuse Atrophy in Articular Cartilage in Rat Knee Joints during Hindlimb Suspension.

OBJECTIVE: The purpose of this study was to determine the preventive effects of treadmill exercise or physiological loading on disuse atrophy in the rat knee joint cartilage and bone during hindlimb suspension. DESIGN: Twenty male rats were divided into 4 experimental groups, including the control, hindlimb suspension, physiological loading, and treadmill walking groups. Histological changes in the articular cartilage and bone of the tibia were histomorphometrically and immunohistochemically evaluated 4 weeks after the intervention. RESULTS: Compared with the control group, the hindlimb suspension group showed thinning of cartilage thickness, decreased matrix staining, and decreased proportion of noncalcified layers. Cartilage thinning, decreased matrix staining, and decreased noncalcified layers were suppressed in the treadmill walking group. The physiological loading group exhibited no significant suppression of cartilage thinning or decreased noncalcified layers, but the decreased matrix staining was significantly suppressed. No significant prevention of bone mass loss or changes in subchondral bone thickness were detected after physiological loading or treadmill walking. CONCLUSION: Disuse atrophy of the articular cartilage caused by unloading conditions could be prevented by treadmill walking in rat knee joints.

Conjoined nerve root in a patient with lumbar disc herniation accompanied by a lumbosacral spine anomaly: a case report.

BACKGROUND: A nerve root anomaly, typified by a conjoined nerve root, is a rare finding. Conjoined nerve root anomalies are easily missed even in preoperative advanced imaging modalities, which can be potentially troublesome during and after surgery. In this report, we present a case of conjoined right L5-S1 nerve root in a patient with lumbar disc herniation, accompanied by spina bifida occulta, which was undiagnosed on preoperative imaging studies. CASE REPORT: A 55-year-old Asian (Japanese) woman presented with low back pain and right leg radiating pain due to lumbar disc herniation at the right L5/S1. Physical examination revealed a positive Lasègue sign and the range of the straight leg raising test was 20° on the right side. The right patellar tendon reflex was normal; however, the right ankle jerk reflex disappeared. Although no obvious hypoesthesia was noted, mild muscle weakness (4/5) was observed in the right leg on the manual muscle test. We planned the lumbar discectomy under a microscope. During surgery, the conjoined right L5-S1 nerve root, which was compressed by herniated nucleus pulposus, was encountered. Although it was very thick and less mobile, some pieces of herniated nucleus pulposus could be removed piece by piece from the axillary part. After sequential decompressive procedures, the tightness of the conjoined right L5-S1 nerve root decreased but its mobility did not improve much. The laterality of the thickness and exit angle of the conjoined right L5-S1 nerve root was retrospectively confirmed on T2 coronal magnetic resonance images and magnetic resonance neurography. Postoperatively, right leg pain was immediately alleviated and complete improvement of muscle weakness was achieved 1 week later (5/5). CONCLUSIONS: Magnetic resonance neurography is extremely useful for the accurate diagnosis of anomalous nerve roots because of clear visualization of the neural tissue. Discectomy under a microscope, which enables magnified three-dimensional observation of the surgical field, must provide a valid and safe procedure to achieve not only secure resection of herniated discs but also adequate exposure of anomalous nerve roots.

One session of 20 ​N cyclic compression induces chronic knee osteoarthritis in rats: A long-term study.

Objective: Mechanical stimulation is a risk factor for knee osteoarthritis. Non-surgical compression has been used to study the effects of mechanical stimulation in vivo. However, the long-term effects of low-force compression on knee joint had not been studied. Therefore, we sought to identify the long-term effects of low-force cyclic compression on the rat knee joint. Design: In this study, we applied one session cyclic compression with a peak load of 20 â€‹N for 60 cycles to the rat knee joint in an approximately 140-degree flexion position (Wistar, male, 12 weeks old), followed by 1 year of observation (including data from 1 week, 2 weeks, 4 weeks, 8 weeks, 6 months, and 1 year after compression), and then performed a sub-regional analysis with hematoxylin-eosin, Safranin O and Fast Green, and MMP13 immunohistochemical staining. Results: We observed osteoarthritis-like cartilage damage, synovial inflammation, and high expression of MMP13 within 1 year after compression. However, these changes progressed slowly, with obvious matrix cracks that did not appear until 1 year after compression. In the regional analysis, we found that low-force compression caused a much slower development of injury at the compression contact site, and no significant structural cartilage damage was observed after 1 year of compression. In contrast, the non-contact site during compression at tibial cartilage in the same joint was the first to show significant structural damage. Conclusion: This study demonstrates that one session of 20 â€‹N cyclic compression induces a chronic osteoarthritis-like phenotype in the rat knee in the long term.

Ultrasound Stimulation Inhibits Morphological Degeneration of Motor Endplates in the Denervated Skeletal Muscle of Rats.

Recovery of motor function after peripheral nerve injury requires treatment of the neuromuscular junction (NMJ), as well as the injured nerve and skeletal muscle. The purpose of this study was to examine the effects of ultrasound (US) stimulation on NMJ degeneration after denervation using a rat model of peroneal nerve transection. Twelve-week-old male Wistar rats were randomly assigned to 3 groups: US stimulation, sham stimulation, and intact. US or sham stimulation was performed on the left tibialis anterior (TA) muscle starting the day after peroneal nerve transection for 5 minutes daily under anesthesia. Four weeks later, the number and morphology of the motor endplates were analyzed to assess NMJ in the TA muscle. The endplates were classified as normal, partially fragmented, or fully fragmented for morphometric analysis. In addition, the number of terminal Schwann cells (tSCs) per endplate and percentage of endplates with tSCs (tSC retention percentage) were calculated to evaluate the effect of tSCs on NMJs. Our results showed that endplates degenerated 4 weeks after transection, with a decrease in the normal type and an increase in the fully fragmented type in both the US and sham groups compared to the intact group. Furthermore, the US group showed significant suppression of the normal type decrease and a fully fragmented type increase compared to the sham group. These results suggest that US stimulation inhibits endplate degeneration in denervated TA muscles. In contrast, the number of endplates and tSC and tSC retention percentages were not significantly different between the US and sham groups. Further investigations are required to determine the molecular mechanisms by which US stimulation suppresses degeneration.

Ultrasound Therapy of Injury Site Modulates Gene and Protein Expressions in the Dorsal Root Ganglion in a Sciatic Nerve Crush Injury Rat Model.

The aim of this study was to verify the effects of ultrasound on dorsal root ganglion (DRG) neurons at the injury site in a rat model of sciatic nerve crush injury. We evaluated the mRNA expression of neurotrophic and pro-inflammatory factors by quantitative reverse transcription polymerase chain reaction 7 and 14 d post-injury. We also evaluated the protein levels of brain-derived neurotrophic factor (BDNF) 7 and 14 d post-injury. Axon regeneration and motor function analyses were performed 21 days after injury to confirm the facilitative effect of ultrasound on nerve regeneration. In the ultrasound group, BDNF and interleukin-6 mRNA expression of the DRG was significantly reduced 7 d post-injury. Compared with the sham group, the BDNF protein expression of the DRG in the ultrasound group remained at a higher level 14 d post-injury. Motor function, myelinated fiber density and myelin sheath thickness were significantly higher in the ultrasound group than in the sham group 21 d post-injury. These results indicate that ultrasound therapy at the injury site promotes nerve regeneration and modulates gene and protein expression in the DRG of a rat model of a sciatic nerve crush injury.

Improvement of an Automated Sample Injection System for Pillar Array Columns to Increase Analytical Reproducibility.

In our previous study, we developed an automatic sample injection system for pillar array columns for quantitative analysis. An autosampler was used to maintain a constant sample injection volume. However, the sample was diluted during injection using the autosampler, thus deteriorating the analytical reproducibility. In this study, we have substituted the autosampler with a syringe pump to overcome the abovementioned problem and improve the system. Sample dilution was avoided by filling the entire capillary with the sample at a constant rate. This improved system also increased the analytical reproducibility. In the previous system, the relative standard deviation (RSD) exceeded 17% of the peak height for coumarin dyes. In contrast, the improved system decreased the RSD to the range 1.2-1.8%. The analytical reproducibility was evaluated by using five types of amino acids. The RSD of each peak height was within 3.0%, confirming good reproducibility. These results indicate that the sample injection method developed in this study can be applied to biological sample analyses as a simple quantitative analysis method for pillar array columns.

Comprehensive Understanding of Inactivity-induced Gait Alteration in Rodents.

It is well known that disuse affects neural systems and that joint motions become altered; however, which outcomes properly exhibit these characteristics is still unclear. The present study describes a motion analysis approach that utilizes three-dimensional (3D) reconstruction from video captures. Using this technology, disuse-evoked alterations of walking performances were observed in rodents exposed to a simulated microgravity environment by unloading their hindlimb by their tail. After 2 weeks of unloading, the rats walked on a treadmill, and their gait motions were captured with four charge-coupled device (CCD) cameras. 3D motion profiles were reconstructed and compared to those of control subjects using the image processing software. The reconstructed outcome measures successfully portrayed distinct aspects of distorted gait motion: hyperextension of the knee and ankle joints and higher position of the hip joints during the stance phase. Motion analysis is useful for several reasons. First, it enables quantitative behavioral evaluations instead of subjective observations (e.g., pass/fail in certain tasks). Second, multiple parameters can be extracted to fit specific needs once the fundamental datasets are obtained. Despite hurdles for broader application, the disadvantages of this method, including labor intensity and cost, may be alleviated by determining comprehensive measurements and experimental procedures.

Investigating the Optimal Initiation Time of Ultrasound Therapy for Peripheral Nerve Regeneration after Axonotmesis in Rats.

This study was aimed at identifying the optimal initiation time of ultrasound (US) therapy for peripheral nerve regeneration after axonotmesis. Thirty-six rats with sciatic nerve crush injury were divided into four groups that received US irradiation initiated 1, 7 or 14 d after injury, or sham stimulation for 4 wk. Motor function analysis was conducted weekly; however, there was no significant improvement attributed to US treatment. Four weeks after injury, compound muscle action potential amplitude values of the group in which US irradiation was initiated 1 d after the injury exhibited significant improvement compared with the sham stimulation group. In addition, myelin sheath thickness was significantly greater in the 1-d group than in other groups. These results indicate that US treatment initiated 1 d after peripheral nerve injury promotes maximum regeneration.

Physiological Reloading Recovers Histologically Disuse Atrophy of the Articular Cartilage and Bone by Hindlimb Suspension in Rat Knee Joint.

OBJECTIVE: This study aimed to clarify physiological reloading on disuse atrophy of the articular cartilage and bone in the rat knee using the hindlimb suspension model. DESIGN: Thirty male rats were divided into 3 experimental groups: control group, hindlimb suspension group, and reloading after hindlimb suspension group. Histological changes in the articular cartilage and bone of the tibia were evaluated by histomorphometrical and immunohistochemical analyses at 2 and 4 weeks after reloading. RESULTS: The thinning and loss of matrix staining in the articular cartilage and the decrease in bone volume induced by hindlimb suspension recovered to the same level as the control group after 2 weeks of reloading. The proportion of the noncalcified and calcified layers of the articular cartilage and the thinning of subchondral bone recovered to the same level as the control group after 4 weeks of reloading. CONCLUSIONS: Disuse atrophy of the articular cartilage and bone induced by hindlimb suspension in the tibia of rats was improved by physiological reloading.

Intra-Articular Injections of Curcumin Monoglucuronide TBP1901 Suppresses Articular Cartilage Damage and Regulates Subchondral Bone Alteration in an Osteoarthritis Rat Model.

OBJECTIVE: Curcumin monoglucuronide (TBP1901) is highly water soluble and can convert to free form curcumin, which has pharmacological effects, on intravenous administration. This study aimed to investigate the effectiveness of TBP1901 intra-articular injections in an osteoarthritis (OA) rat model. METHODS: Sixty-four male Wistar rats (12 weeks old) who underwent destabilized medial meniscus (DMM) surgery were randomly separated into the TBP1901 injection or saline solution (control) injection group. They were sacrificed at 1, 2, 6, or 10 weeks postoperatively (weeks 1, 2, 6, and 10; n = 8 for each group). TBP1901 (30 mg/mL) or saline solution of 50 µL was injected into the knee joints twice a week during weeks 1 and 2 to investigate the effects in the acute phase of posttraumatic (PT) OA or once a week during weeks 6 and 10 to investigate it in the chronic phase of PTOA. Histology, immunohistochemistry, and micro-computed tomography were performed to evaluate the changes in OA. RESULTS: TBP1901 injections significantly reduced synovial inflammation at weeks 1 and 2, and tumor necrosis factor-α expression in the articular cartilage at week 6. The TBP1901 injections also significantly suppressed articular cartilage damage, subchondral bone (SB) plate thickening, SB plate perforation, and osteophyte formation at week 10. CONCLUSIONS: TBP1901 intra-articular injections suppressed synovial inflammation in the acute phase of PTOA in DMM rats. In the chronic phase, TBP1901 suppresses articular cartilage damage and regulates SB plate changes.

Grafting of iPS cell-derived tenocytes promotes motor function recovery after Achilles tendon rupture.

Tendon self-renewal is a rare occurrence because of the poor vascularization of this tissue; therefore, reconstructive surgery using autologous tendon is often performed in severe injury cases. However, the post-surgery re-injury rate is relatively high, and the collection of autologous tendons leads to muscle weakness, resulting in prolonged rehabilitation. Here, we introduce an induced pluripotent stem cell (iPSC)-based technology to develop a therapeutic option for tendon injury. First, we derived tenocytes from human iPSCs by recapitulating the normal progression of step-wise narrowing fate decisions in vertebrate embryos. We used single-cell RNA sequencing to analyze the developmental trajectory of iPSC-derived tenocytes. We demonstrated that iPSC-tenocyte grafting contributed to motor function recovery after Achilles tendon injury in rats via engraftment and paracrine effects. The biomechanical strength of regenerated tendons was comparable to that of healthy tendons. We suggest that iPSC-tenocytes will provide a therapeutic option for tendon injury.

Reduction of knee joint load suppresses cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis using a post-traumatic rat model.

The purpose of this study was to clarify the histological effect of reducing the loading to knee on cartilage degeneration, osteophyte formation, and synovitis in early-stage osteoarthritis (OA) using a post-traumatic rat model. Ten male rats were randomly allocated into two experimental groups: OA induction by surgical destabilization of medial meniscus (DMM, OA group) and hindlimb suspension after OA induction by DMM (OAHS group). The articular cartilage, osteophyte formation, and synovial membrane in the medial tibiofemoral joint were analyzed histologically and histomorphometrically at 2 and 4 weeks after surgery. The histological scores and changes in articular cartilage and osteophyte formation were significantly milder and slower in the OAHS group than in the OA group. At 2 and 4 weeks, there were no significant differences in cartilage thickness and matrix staining intensity between both the groups, but chondrocytes density was significantly lower in the OA group. Synovitis was milder in OAHS group than in OA group at 2 weeks. Reducing knee joint loading inhibited histological OA changes in articular cartilage, osteophyte formation, and synovial inflammation. This result supports the latest clinical guidelines for OA treatment. Further studies using biochemical and mechanical analyses are necessary to elucidate the mechanism underlying delayed OA progression caused by joint-load reduction.

A Non-Invasive Method for Generating the Cyclic Loading-Induced Intra-Articular Cartilage Lesion Model of the Rat Knee.

The pathophysiology of primary osteoarthritis (OA) remains unclear. However, a specific subclassification of OA in relatively younger age groups is likely correlated with a history of articular cartilage damage and ligament avulsion. Surgical animal models of OA of the knee play an important role in understanding the onset and progression of post-traumatic OA and aid in the development of novel therapies for this disease. However, non-surgical models have been recently considered to avoid traumatic inflammation that could affect the evaluation of the intervention. In this study, an intra-articular cartilage lesion rat model induced by in vivo cyclic compressive loading was developed, which allowed researchers to (1) determine the optimal magnitude, speed, and duration of load that could cause focal cartilage damage; (2) assess post-traumatic spatiotemporal pathological changes in chondrocyte vitality; and (3) evaluate the histological expression of destructive or protective molecules that are involved in the adaptation and repair mechanisms against joint compressive loads. This report describes the experimental protocol for this novel cartilage lesion in a rat model.