Rat Knee Research Articles

Chronic synovial inflammation play important roles to establish residual pain in monoiodoacetic acid induced arthritis rat model

Purpose: Osteoarthritis (OA) is a group of diseases and mechanical abnormalities involving degradation of articular cartilage and subchondral bone. Major complaint of OA patients is persistent knee pain, which significantly decreases patients' activity of daily living (ADL). However, causal treatment has not provided for OA patients since OA is multifactorial diseases and etiology has not fully understood. Therefore, most of the current treatment strategies for OA is based on the symptom management and patients with uncontrolled knee pain usually choose knee replacement surgery. In this regard, we consider it is quite important to understand the mechanisms of persistent knee pain development during the progression of OA. We have established two distinct inflammation-induced articular cartilage degeneration models in rats. The first was to induce joint inflammation by intra-articular injection of low-dose (0.2mg) of monoiodoacetate (MIA). In this model, we observed transient synovial inflammation followed by mild articular cartilage degeneration in rat knee. The other was high-dose (1.0mg) of MIA injection method. In this model, chronic synovial inflammation and extensive articular cartilage degeneration were observed by 14 days after injection. In this study, we performed detailed histological assessments and monitored pain avoidance behavior in these rat models to understand the mechanisms how knee pain persistence established after joint inflammation. Here we showed that chronic synovial inflammation may play important roles to establish residual pain in rats. Methods: Male Wistar rats received intra-articular injection of MIA (0.2mg, or 1mg/30μl) in the right knee, and PBS (30μl) in the left knee. We performed pain behavior test (Incapacitnce Test and von Frey Test) at 0,1,3,5,7,14,21, and 28 days. At 5, 14, and 28 days, rats were sacrificed, and knee joint and dorsal root ganglion (DRG) were excised for histological assessments. Results: In the low-dose group (0.2mg), extensive inflammatory cell infiltration in the infra-patella fat pad (IFP) and synovial hyperplasia were observed by 5 days after MIA injection, then quenched by 14 days. Proteoglycan contents in both articular cartilage and meniscus gradually decreased by 28 days. Incapacitance tests indicated that weight-bearing of the MIA injected knee (right knee) was significantly decreased by 7 days after injection and returned to the even levels by 14 days when the acute synovial inflammation was quenched. Percentage of Calcitonin-Gene Related Peptide (CGRP)-positive sensory neuron in L4 DRG was significantly decreased at 28 days if compared to that in day 5. In the high-dose group (1.0mg), onset of inflammatory response was almost the same with that of low-dose group, however, it continued throughout the experimental period (28 days). Signs of articular cartilage degeneration was obviously observed as early as 5 days after injection and superficial layers were almost missing at 28 days. In contrast to the low-dose group, weight-bearing continued decreasing significantly throughout the experimental period. Immunohistochemical analyses indicated that percentage of CGRP-positive sensory neuron was much abundant and did not decrease by 28 days. Conclusions: Data suggest that persistent synovial inflammation plays important roles in establishing residual pain in the knee joint.

SM04690, a WNT pathway inhibitor: anti-inflammatory and cartilage protective effects in preclinical osteoarthritis models

Purpose: Osteoarthritis (OA) is characterized by pain, swelling, and reduced function in the knee joint. Upregulated Wnt signaling drives OA through synovial inflammation, increased subchondral bone, and thinning cartilage. SM04690, a small molecule Wnt pathway inhibitor that demonstrated chondrogenesis and anti-inflammatory properties preclinically (Deshmukh et al., OAC 2017; Deshmukh et al. Arth Rheum 2016), was further evaluated to determine its potential to reduce inflammation, protect cartilage, improve joint health and modify pain in OA. Methods: Cytokine secretion (IL-6 and TNF-α) from IL-1β stimulated- and SM04690 treated-synovial fibroblasts was measured by ELISA. A single intra-articular injection of SM04690 or vehicle was evaluated in an in vivo rat knee monosodium iodoacetate (MIA) OA model. Joint inflammation was evaluated by H&E staining, inflammatory cytokines (IL-1α, IL-1β, IL-6, TNF-α and IFN-γ) by qPCR, and cartilage protection by qPCR for matrix metalloproteinases (MMPs). Histological evaluation of cartilage health was performed using OARSI score and thickness by Safranin-O staining. Pain was measured as paw withdrawal threshold using Von Frey apparatus and weight distribution using incapacitance meter and analyzed using generalized estimating equation regression accounting for repeated measurements. Results: SM04690 dose-dependently inhibited IL-1β-induced cytokine secretion in synovial fibroblasts (EC50 ∼30nM; Fig.1). In the rat MIA OA model, compared to vehicle, SM04690 injection reduced visible knee swelling, inflammatory cells, proinflammatory cytokine and MMP production (P < 0.05). SM04690 increased (P < 0.01) paw withdrawal threshold and improved (P < 0.05) weight distribution to the affected limb in treated rats, at all post-treatment timepoints, compared to vehicle. SM04690 increased Safranin-O stained cartilage thickness and decreased OARSI score (P < 0.05) compared to vehicle (Fig.2). Conclusions: In a rat MIA OA model, SM04690 injection reduced inflammation, protease production, and pain, with improved cartilage and joint health, compared to vehicle. Previously demonstrated regenerative effects in nonclinical studies1, along with anti-inflammatory properties, show SM04690 may potentially improve symptoms and provide disease modification in OA. Clinical studies are ongoing.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Assessment on the effects of high and low in vivo cyclic compressive loading on the progression of cartilage degeneration in rat knee joint

Assessment on the effects of high and low in vivo cyclic compressive loading on the progression of cartilage degeneration in rat knee joint

The relationship between treadmill running and HIF-2α on rat knee articular cartilage

The relationship between treadmill running and HIF-2α on rat knee articular cartilage

The effect of exercise on the protein profile of rat knee joint intra- and extra-articular ligaments

Abstract Injuries to the intra-articular knee joint ligament (anterior cruciate ligament (ACL)) together with the extra-articular medial collateral ligament (MCL) result in significant joint instability, pain and immobility for the affected individual. Moderate endurance type exercise has been shown to increase ligament strength, however little is known on the effect of short-term high intensity exercise regimes such as treadmill training on the ACL and MCL and whether they may be beneficial to the extracellular matrix (ECM) structure of these ligaments. This study aimed to identify the effect of short-term high intensity exercise on the proteome of the rat ACL and MCL using mass spectrometry. Sprague Dawley male rats (n=12) were split into equal groups of control and exercise animals, which were subjected to high intensity training and followed by proteomic analysis of the ACL and MCL. Knee joint and ligament health was assessed using OARSI scoring or using a validated histological scoring system. Histopathological analyses demonstrated no significant changes in the ACL, MCL or cartilage of the knee joint, indicating that the exercise regime used in this study did not have substantial impact on tissue structure and health of several tissues within the rat knee joint. Some proteins were found to be significantly more abundant in the ACL in the exercised group than the control group. However, no proteins with a significantly different expression were identified between MCL control and MCL exercised groups. The majority of proteins expressed at higher levels in the ACL exercise group were cytoskeletal proteins, ribosomal proteins and enzymes. Several matrisomal proteins were also more abundant such as collagen proteins and proteoglycans in ACL exercise group. In conclusion, our results indicate that short-term high intensity exercise has an impact on ACL ECM protein expression, with the majority of differential expressed proteins being cellular proteins such as actins, ribosomal and heat shock proteins, indicative of metabolic and molecular responses. Further study is necessary to determine the impact of these short-term changes on ligament structure and function.

Tibial Osteotomy as a Mechanical Model of Primary Osteoarthritis in Rats

This study has presented the first purely biomechanical surgical model of osteoarthritis (OA) in rats, which could be more representative of the human primary disease than intra-articular techniques published previously. A surgical tibial osteotomy (TO) was used to induce degenerative cartilage changes in the medial knee of Sprague-Dawley rats. The presence of osteoarthritic changes in the medial knee compartment of the operated animals was evaluated histologically and through analysis of serum carboxy-terminal telepeptides of type II collagen (CTX-II). In-vivo biomechanical analyses were carried out using a musculoskeletal model of the rat hindlimb to evaluate the loading conditions in the knee pre and post-surgically. Qualitative and quantitative medial cartilage degeneration consistent with OA was found in the knees of the operated animals alongside elevated CTX-II levels and increased tibial compressive loading. The potential avoidance of joint inflammation post-surgically, the maintenance of internal joint biomechanics and the ability to quantify the alterations in joint loading should make this model of OA a better candidate for modeling primary forms of the disease in humans.

Transplantation of dedifferentiated fat cell-derived micromass pellets contributed to cartilage repair in the rat osteochondral defect model

Mature adipocyte-derived dedifferentiated fat (DFAT) cells possesses the ability to proliferate effectively and the potential to differentiate into multiple linages of mesenchymal tissue; similar to adipose-derived stem cells (ASCs). The purpose of this study is to examine the effects of DFAT cell transplantation on cartilage repair in a rat model of osteochondral defects. Full-thickness osteochondral defects were created in the knees of Sprague-Dawley rats bilaterally. Cartilage-like micromass pellets were prepared from green fluorescent protein (GFP)-labeled rat DFAT cells and subsequently transplanted into the affected right knee of these rats. Defects in the left knee were used as a control. Macroscopic and microscopic changes of treated and control defects were evaluated up to 12 weeks post-treatment with DFAT cells. To observe the transplanted cells, sectioned femurs were immunostained for GFP and type II collagen. DFAT cells formed micromass pellets expressing characteristics of immature cartilage invitro. In the DFAT cell-transplanted limbs, the defects were completely filled with white micromass pellets as early as 2 weeks post-treatment. These limbs became smooth at 4 weeks. Conversely, the defects in the control limbs were still not repaired by 4 weeks. Macroscopic ICRS scores at 2 and 4 weeks were significantly higher in the DFAT cells-transplanted limbs compared to those of the control limbs. The modified O'Driscol histological scores for the DFAT cell-transplanted limbs were significantly higher than those of the control limbs at corresponding time points. GFP-positive DAFT cells were detected in the transplanted area at 2 weeks but hardly visible at 12 weeks post-operation. Transplantation of DFAT cell-derived micromass pellets contribute to cartilage repair in a rat osteochondral defect model. DFAT cell transplantation may be a viable therapeutic strategy for the repair of osteochondral injuries.

Vasoactive intestinal peptide alleviates osteoarthritis effectively via inhibiting NF-\u03baB signaling pathway

BackgroundTo investigate the treatment effect of vasoactive intestinal peptide (VIP) on osteoarthritis (OA) and the relative mechanism.MethodThe OA model on the SD rat knee was established using the modified Hulth method, and the recombinant pcDNA3.1+/VIP plasmid was constructed. One month after the plasmids VIP were injected intra-articularly into the right knee joint of OA and sham-operated rats, the pathological changes of the OA knee joint were observed by Hematoxylin-eosin (HE) and Safranin O/fast green staining. The levels of VIP and serum inflammatory cytokines (TNF-α, IL-2 and IL-4) were measured by ELISA kits. Meanwhile, synoviocytes isolated from OA rat and sham-operated rat were cultured in vitro, and transfected with the VIP plasmid. The proliferation of synoviocytes was determined using BrdU kits. The protein expressions of TNF-α, IL-2, CollagenII, osteoprotegerin (OPG), matrix-degrading enzymes (MMP-13, ADAMTS-5), and the related protein of NF-κB signaling pathway (phosphorylated p65, phosphorylated IκBα) were evaluated by western blot.ResultsThe VIP plasmid could effectively improve the pathological state of the OA rats knee joint, significantly decrease the levels of serum TNF-α and IL-2, and clearly increase the levels of VIP and serum IL-4. At the same time, after the OA synoviocytes were treated with the VIP plasmid, the proliferation ability of OA synoviocytes was reduced, the protein expressions of Collagen II and OPG were remarkably up-regulated, and the protein expressions of TNF-α, IL-2, MMP-13 and ADAMTS-5 were significantly down-regulated. In addition, the p-p65 expression decreased and p-IκBα expression increased.ConclusionOsteoarthritis was effectively treated by VIP via inhibiting the NF-κB signaling pathway.

Can photobiomodulation associated with implantation of mesenchymal adipose-derived stem cells attenuate the expression of MMPs and decrease degradation of type II collagen in an experimental model of osteoarthritis?

This study aimed to determine whether photobiomodulation therapy (PBMT) could improve the bioavailability and chondroprotective benefits of mesenchymal stem cells injected into the knees of rats used as an experimental model of osteoarthritis (OA) as well as reduce the expression of matrix metalloproteinases (MMPs) and degradation of type II collagen (COL2-1) in the cartilage. Adipose-derived stem/stromal cells (ADSCs) were collected from three male Fischer 344 rats and characterized by flow cytometry. Fifty female Fischer 344 rats were distributed into five groups of 10 animals each. These groups were as follows: control, OA, OA PBMT, OA ADSC, and OA ADSC PBMT. OA was induced in the animals using a 4% papain solution. Animals from the OA ADSC and OA ADSC PBMT groups received an intra-articular injection of 10 × 106 ADSCs and were treated with PBMT by irradiation (wavelength: 808nm, power: 50mW, energy: 42J, energy density: 71.2J/cm2, spot size: 0.028). Euthanasia was performed 7days after the first treatment. The use of PBMT alone and the injection of ADSCs resulted in downregulation of pro-inflammatory cytokines and MPs in cartilage compared to the OA group. PBMT and ADSCs caused upregulation of tissue inhibitors of MPs 1 and 2 and mRNA and protein expression of COL2-1 in cartilage compared to the OA group. The intra-articular injection of ADSCs and PBMT prevented joint degeneration resulting from COL2-1 degradation and modulated inflammation by downregulating cytokines and MMPs in the OA group.

Co-implantation of bone marrow mesenchymal stem cells and chondrocytes increase the viability of chondrocytes in rat osteo-chondral defects.

Replacement of chondrocytes by adult stem cells was believed to improve the performance of autologous chondrocytes transplantation, since less chondrocytes were needed. Previous studies have demonstrated that the increased cartilage production in pellet co-cultures of chondrocytes and bone marrow mesenchymal stem cells (BMSCs) is due to the trophic effects of the MSC by stimulating chondrocyte proliferation and matrix production. However, the destination of MSCs or chondrocytes after implanted in osteo-chondral defects is not clear. The aim of the present study is to investigate the viability of MSCs and chondrocytes after co-implantation into a rat osteo-chondral defect model. MSCs were isolated from bone marrow and chondrocytes were extracted from knee joints of neonatal rats. Results of sulfated glycosaminoglycans (GAG) and collagen quantification demonstrated that co-culture pellets of BMSCs and chondrocytes have more GAG deposition than that of BMSCs or chondrocytes alone. Tracking cells with fluorescence protein demonstrated that MSCs disappeared following co-culture. In a rat knee injury model, co-implantation of BMSCs and chondrocytes contained more viable chondrocytes than chondrocytes implanted alone. To conclude, BMSCs were replaced by chondrocytes in pellet co-culture and BMSCs increased the viability of chondrocytes following co-implantation in a osteo-chondral defects model. Co-implantation of BMSCs and chondrocytes may be a promising approach to repairing osteo-chondral defects in the clinical setting.

Isoliquiritigenin Inhibits IL-1β-Induced Production of Matrix Metalloproteinase in Articular Chondrocytes

Osteoarthritis (OA) is a major joint disease in which inflammatory cytokine interleukin-1β (IL-1β) and matrix metalloproteinases (MMPs) play a pivotal role. Isoliquiritigenin has been reported to have anti-inflammation activity. In this study, the effect of isoliquiritigenin on IL-1β-induced production of matrix metalloproteinase and nuclear factor κB (NF-κB) activation was analyzed. We treated primary cultured articular chondrocytes with isoliquiritigenin and the expressions of MMPs were analyzed on mRNA and protein level. The phosphorylation of IκBa and p65 was analyzed to detect NF-κB activation. We also used in vivo model by treating mice with isoliquiritigenin and detecting the level of MMPs. IL-1β induced NF-κB activation and MMP-1, MMP-3, MMP-9, MMP-13, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-4 and ADAMTS-5 production on chondrocytes. A 10-μM isoliquiritigenin treatment significantly inhibited IL-1β-induced NF-κB activation and these MMPs production on chondrocytes. Injecting isoliquiritigenin into rat knee joint also inhibited IL-1β-induced NF-κB activation and MMPs production in articular cartilage. Isoliquiritigenin treatment inhibited IL-1β-induced MMPs production and NF-κB activation both in vitro and in vivo, suggesting a potential therapeutic role of isoliquiritigenin to treat osteoarthritis.

Omega-3-carboxylic acids provide efficacious anti-inflammatory activity in models of crystal-mediated inflammation

This study assesses the efficacy and exposure–response relationship of omega-3-carboxylic acids (OM-3 CA) in models of crystal-based inflammation. Human THP-1 macrophages and primary peripheral blood mononuclear cells exposed to multiple inflammatory crystal types were used to determine the anti-inflammatory potential of omega-3 (OM-3) fatty acids in vitro. Anti-inflammatory effects of OM-3 CA in vivo were tested in rat monosodium urate (MSU) crystal air pouch and rat knee intra-articular MSU injection models. Acute treatment with the OM-3 fatty acid docosahexaenoic acid suppressed MSU-, cholesterol crystal-, and calcium pyrophosphate crystal-mediated interleukin-1β (IL-1β) production in vitro. In vivo, OM-3 CA dose-dependently reduced crystal-mediated cell migration, exudate volume, and levels of IL-1β and prostaglandin E2. Following intra-articular injection of MSU, treatment with OM-3-CA (1 mL/kg) and indomethacin (1 mg/kg) resulted in similar mean reductions in pain (23% and 41%, respectively) and swelling (58% and 50%, respectively), compared with controls. Additionally, in complex formulations of OM-3 fatty acids, high levels of palmitic acid could reduce the in vivo effect on crystal-mediated IL-1β elevation. OM-3 CA has a broadly efficacious anti-inflammatory effect with a strong exposure–response relationship that could be beneficial in prevention and treatment of crystal arthritis, with potential applications in other IL-1β-mediated diseases.

Link Protein N-Terminal Peptide as a Potential Stimulating Factor for Stem Cell-Based Cartilage Regeneration.

Background Link protein N-terminal peptide (LPP) in extracellular matrix (ECM) of cartilage could induce synthesis of proteoglycans and collagen type II in cartilaginous cells. Cartilage stem/progenitor cells (CSPCs), the endogenous stem cells in cartilage, are important in cartilage degeneration and regeneration. We hypothesized that LPP could be a stimulator for stem cell-based cartilage regeneration by affecting biological behaviors of CSPC. Methods CSPCs were isolated from rat knee cartilage. We evaluated the promoting effect of LPP on proliferation, migration, and chondrogenic differentiation of CSPCs. The chondrogenic differentiation-related genes and proteins were quantitated. Three-dimensional culture of CSPC was conducted in the presence of TGF-β3 or LPP, and the harvested pellets were analyzed to assess the function of LPP on cartilage regeneration. Results LPP stimulated the proliferation of CSPC and accelerated the site-directional migration. Higher expression of SOX9, collagen II, and aggrecan were demonstrated in CSPCs treated with LPP. The pellets treated with LPP showed more distinct characteristics of chondroid differentiation than those with TGF-β3. Conclusion LPP showed application prospect in cartilage regeneration medicine by stimulating proliferation, migration, and chondrogenic differentiation of cartilage stem/progenitor cells.

Effects of autophagy on acid-sensing ion channel 1a-mediated apoptosis in rat articular chondrocytes.

Rheumatoid arthritis (RA) is a degenerative joint disease that is caused by multiple pathogenic factors. However, the precise etiology of RA is still unknown. Our previous studies demonstrated that acid-sensing ion channel 1a (ASIC1a)-mediated articular chondrocyte apoptosis played a key role in the progression of RA. In this study, we aim to explore whether ASIC1a mediates autophagy or not and the effect of autophagy on ASIC1a-mediated apoptosis. Primary articular chondrocytes, extracted from rat knee joints, were exposed to different concentrations of concentrated hydrochloric acid for different time intervals in vitro. The results indicated that extracellular acid treatment induced autophagy of rat articular chondrocytes. Moreover, inhibition of ASIC1a with either psalmotoxin 1 or ASIC1a short hairpin RNA reduced the autophagy flux. The results suggested that ASIC1a mediated acid-induced autophagy. Pretreatment with autophagy antagonist 3-methyladenine decreased the autophagy, but increased the apoptosis mediated by ASIC1a. Furthermore, knockdown of Beclin 1 by small interfering RNA attenuated autophagy but potentiated ASIC1a-mediated apoptosis of rat articular chondrocytes. Taken together, these findings suggested that both inhibition and silencing of autophagy could enhance ASIC1a-mediated apoptosis in rat articular chondrocytes, and therefore, autophagy is likely to be a new mechanism involved in ASIC1a-mediated apoptosis of articular chondrocytes during the pathogenesis of RA.

A cyclic pathway of P2 × 7, bradykinin, and dopamine receptor activation induces a sustained articular hyperalgesia in the knee joint of rats.

We investigated whether: (1) P2 × 7 receptor activation by its agonist (BzATP) induces articular hyperalgesia in the rat's knee joint via inflammatory mechanisms and (2) activation of P2 × 7 receptors by endogenous ATP contributes to the articular hyperalgesia induced by bradykinin, TNF-α, IL-1β, CINC-1, PGE2, and dopamine. The articular hyperalgesia was quantified using the rat knee joint incapacitation test. The knee joint inflammation, characterized by the concentration of pro-inflammatory cytokines and by neutrophil migration, was quantified in the synovial lavage fluid by ELISA and myeloperoxidase enzyme activity assay, respectively. BzATP induced a dose-dependent articular hyperalgesia in the rat's knee joint that was significantly reduced by the selective antagonists for P2 × 7, bradykinin B1 or B2 receptors, β1 or β2 adrenoceptors, and by pre-treatment with Indomethacin. BzATP induced a local increase of TNF-α, IL-1β, IL-6, and CINC-1 concentration and neutrophil migration into the knee joint. The co-administration of the selective P2 × 7 receptor antagonist A-740003 significantly reduced the articular hyperalgesia induced by bradykinin and dopamine, but not by TNF-α, IL-1β, CINC-1, and PGE2. P2 × 7 receptor activation induces articular hyperalgesia mediated by the previous inflammatory mediator release. P2 × 7 receptor-induced articular hyperalgesia is sustained by the involvement of this purinergic receptor in bradykinin and dopamine-induced hyperalgesia in the knee joint.

Intra-articular collagenase injection increases range of motion in a rat knee flexion contracture model

ObjectivesA knee joint contracture, a loss in passive range of motion (ROM), can be caused by prolonged immobility. In a rat knee immobilization flexion contracture model, the posterior capsule was shown to contribute to an irreversible limitation in ROM, and collagen pathways were identified as differentially expressed over the development of a contracture. Collagenases purified from Clostridium histolyticum are currently prescribed to treat Dupuytren’s and Peyronie’s contractures due to their ability to degrade collagen. The potential application of collagenases to target collagen in the posterior capsule was tested in this model.Materials and methodsRats had one hind leg immobilized, developing a knee flexion contracture. After 4 weeks, the immobilization device was removed, and the rats received one 50 µL intra-articular injection of 0.6 mg/mL purified collagenase. Control rats were injected with only the buffer. After 2 weeks of spontaneous remobilization following the injections, ROM was measured with a rat knee arthrometer, and histological sections were immunostained with antibodies against rat collagen types I and III.Results/conclusionCompared with buffer-injected control knees, collagenase-treated knees showed increased ROM in extension by 8.0°±3.8° (p-value <0.05). Immunohistochemical analysis revealed an increase in collagen type III staining (p<0.01) in the posterior capsule of collagenase-treated knees indicating an effect on the extracellular matrix due to the collagenase. Collagen I staining was unchanged (p>0.05). The current study provides experimental evidence for the pharmacological treatment of knee flexion contractures with intra-articular collagenase injection, improving the knee ROM.

Active exercise on immobilization-induced contractured rat knees develops arthrogenic joint contracture with pathological changes.

This study investigated the effects of treadmill walking during remobilization on range of motion (ROM) and histopathology in rat knee joints, which were immobilized for 3 wk in a flexed position. After fixator removal, rats were divided into a no-intervention (RM) group and a group forced to walk on a treadmill daily at 12 m/min for 60 min (WALK group). Passive knee extension ROMs were measured before (m-ROM) and after (a-ROM) knee flexor myotomy on the first and last day of a 7-day remobilization period, with m-ROM mainly reflecting myogenic factors and a-ROM reflecting arthrogenic factors. Knee joints were histologically analyzed and gene expression of inflammatory or fibrosis-related mediators in the posterior joint capsule were examined. m-ROM and a-ROM restrictions were established after immobilization. m-ROM significantly increased following the remobilization period both in RM and WALK groups compared with that of immobilized (IM) group. Conversely, a-ROM decreased following the remobilization period in both RM and WALK groups compared with that of IM group. Importantly, a-ROM was smaller in the WALK group than the RM group. Remobilization without intervention induced inflammatory and fibrotic reactions in the posterior joint capsule after 1 and 7 days. Treadmill walking promoted these reactions and also increased the expression of fibrosis-related TGF-β1 and collagen type I and III genes. While free movement after immobilization improved myogenic contracture, arthrogenic contracture worsened. Treadmill walking further aggravated arthrogenic contracture through amplified inflammatory and fibrotic reactions. Thus active exercise immediately after immobilization may not improve immobilization-induced joint contracture. NEW & NOTEWORTHY In clinical practice, it is widely accepted that facilitation of joint movements is effective in improving immobilization-induced joint contracture. However, whether active exercises improve arthrogenic contracture is not known. In this study, we revealed that treadmill walking further promoted remobilization-induced progression of arthrogenic contracture. To our knowledge, this is the first study demonstrating no favorable effect of active exercise on immobilization-induced arthrogenic contracture.

Cell-type-specific gene expression patterns in the knee cartilage in an osteoarthritis rat model

Osteoarthritis (OA) is a chronic degenerative disease that leads to joint failure, pain, and disability. Gene regulation is implicated as a driver of the imbalance between the expression of catabolic and anabolic factors that eventually leads to the degeneration of osteoarthritic cartilage. In our model, knee-joint OA was induced in male Wistar rats by intra-articular sodium monoiodoacetate (MIA) injections. Whole-genome microarrays were used to analyse the alterations in gene expression during the time-course of OA development (at 2, 14, and 28 days post-injection) in rat knee joints. The identified co-expressed groups of genes were analysed for enriched regulatory mechanisms, functional classes, and cell-type-specific expression. This analysis revealed 272 regulated transcripts (ANOVA FDR < 0.1% and fold > 2). Functionally, the five major gene expression patterns (A–E) were connected to PPAR signalling and adipogenesis (in cluster A), WNT signalling (in cluster B), endochondral ossification (in cluster C), matrix metalloproteinases and the ACE/RAGE pathway (in cluster D), and the Toll-like receptor, and IL-1 signalling pathways (in cluster E). Moreover, the dynamic profiles of these transcriptional changes were assigned to cellular compartments of the knee joint. Classifying the molecular processes associated with the development of cartilage degeneration provides novel insight into the OA disease process. Our study identified groups of co-regulated genes that share functional relationships and that may play an important role in the early and intermediate stages of OA.

Influence of cartilage interstitial fluid on gene expression in cruciate ligament fibroblasts.

Loading of articular cartilage during motion squeezes the fluid from the cartilage, termed cartilage interstitial fluid (CIF), which was found to influence gene expression in synovial membrane cells. After crucial ligaments damage, these cells are exposed to synovial fluid containing factors released from articular cartilage; the aim of the present study was to establish the influence of CIF and factors present in CIF (CIF-like cocktails) on crucial ligament fibroblasts. CIF was squeezed from articular-epiphyseal cartilage complexes of newborn rats. Fibroblasts were obtained from crucial ligaments of adult rat knee joints. Cells were cultured in control medium, CIF and CIF-like cocktails, and the expression of selected genes was evaluated using quantitative PCR. CIF stimulated the expression of HAS1, HAS2, aggrecan, lubricin, MMP3, TIMP3 and TGFβ1. Expression of collagen type I, versican, MMP2, TIMP2, TNF and IL1β was inhibited. The CIF-like cocktail stimulated HAS1, HAS2, collagen type I, versican, aggrecan, lubricin, TIMP1, TGFβ1, IL1β, IL6 and inhibited of MMP3 and TNF expression. Both agents exerted similar effects on the expression of HAS2, aggrecan, lubricin, TGFβ1 and TNF. CIF contains inhibitory and stimulatory factors affecting gene expression in crucial ligament fibroblasts and some of them were not included in the CIF-like cocktail. Due to the powerful influence of CIF on crucial ligament fibroblasts and the synovial membrane, further studies on its composition are needed. An improved CIF like-cocktail could be applied in the treatment of various joint or tendon ailments.

Bone replaces articular cartilage in the rat knee joint after prolonged immobilization

BackgroundLost joint range of motion (ROM) is common in chronic osteoarthritis, alters regional weight-bearing across the articular surfaces, and contributes to loss of cartilage and bone alterations. Limited data exist on the regional effects on joints subjected to chronic losses of ROM. ObjectiveTo characterize the regional replacement by bone as part of articular cartilage degeneration after prolonged immobilization. MethodsEleven rat knees were rigidly-immobilized in flexion for 32weeks with contralateral and sham-operated (n=6) knees as controls. Sagittal medial tibial epiphysis histological sections assessed the anterior (non-weight-bearing), middle and posterior (both weight-bearing) regions. We quantified the distribution of collagen I, collagen II, cartilage thickness, glycosaminoglycan (GAG) staining, Mankin scoring, and subchondral bone plate cross-sectional area. Using immunohistochemistry (IHC), we visualized blood vessels, osteoblasts, and mesenchymal stem cells (MSCs). ResultsImmobilized cartilage had increased collagen I content in the anterior tibial region with picrosirius red staining (immobilized=61±20%; contralateral=43±12%, p=0.033; sham=20±10%, p=0.028) and collagen I IHC (immobilized=40±10%; contralateral=11±4%, p=0.003; sham=5±3%, p=0.043). Articular cartilage was thinner anteriorly (18±30μm) in immobilized knees versus contralateral (124±40μm, p<0.001) and sham (125±43μm, p=0.043). GAG staining covered 2±4% of the anterior articular area in immobilized knees versus 28±12% contralaterally (p=0.003) and 26±7% in sham (p=0.043). Mankin scores in immobilized knees were 4.7±1.7 versus 0.2±0.4 and 0±0 for contralateral and sham (p=0.003, p=0.042), respectively. The trabecular bone plate area of anterior and posterior regions showed relative loss of cross-sectional area in immobilized knees compared to controls (immobilized/contralateral area ratios of 0.67 and 0.46 respectively, both p=0.003), while the area in the middle region was preserved. Movat's pentachrome stain and CD31 staining showed chondral vascular ingrowth from subchondral bone. Osteocalcin and CD90 MSC staining were decreased in immobilized knees versus contralateral (p=0.003, p=0.036 respectively). ConclusionsBony replacement characterizes articular cartilage degeneration of knees immobilized for 32weeks in the anterior, non-weight bearing region of the tibia. Replacement of cartilage by bone may have been mediated by chondral vascularization, suggesting irreversible changes. These findings stress the importance of weight-bearing and joint motion to maintain cartilage structure.