Anterior Cruciate Ligament Transection Knees Research Articles

Wnt5a deficiency in osteocalcin-expressing cells could not alleviate the osteoarthritic phenotype in a mouse model of post-traumatic osteoarthritis.

Wnt5a, which regulates the activities of osteoblasts and osteoclasts, is reportedly overexpressed in osteoarthritis (OA) tissues. The purpose of this study was to elucidate its role in the development of OA by deleting Wnt5a in osteocalcin (OCN)-expressing cells. Knee OA was induced by anterior cruciate ligament transection (ACLT) in OCN-Cre;Wnt5afl/fl knockout (Wnt5a-cKO) mice and control littermates. Eight weeks after surgery, histological changes, cell apoptosis, and matrix metabolism of cartilage were evaluated by toluidine blue, TUNEL staining, and im-immunohistochemistry analyses, respectively. In addition, the subchondral bone microarchitecture of mice was examined by micro-computed tomography (micro-CT). Histological scores show substantial cartilage degeneration occurred in ACLT knees, coupled with decreased collagen type II expression and enhanced matrix metalloproteinase 13 expression, as well as higher proportions of apoptotic cells. Micro-CT results show that ACLT resulted in decreased bone mineral density, bone volume/trabecular volume, trabecular number, and structure model index of subchondral bones in both Wnt5a-cKO and control littermates; although Wnt5a-cKO mice display lower BMD and BV/TV values, no significant difference was observed between Wnt5a-cKO and control mice for any of these values. Our findings indicate that Wnt5a deficiency in OCN-expressing cells could not prevent an osteoarthritic phenotype in a mouse model of post-traumatic OA.

Quantitative high-resolution 7T MRI to assess longitudinal changes in articular cartilage after anterior cruciate ligament injury in a rabbit model of post-traumatic osteoarthritis

ObjectiveTo demonstrate an ultra-high field (UHF) 7 ​T delayed gadolinium-enhanced MRI of cartilage (dGEMRIC) protocol for quantitative post-traumatic osteoarthritis (PTOA) detection and monitoring in a rabbit anterior cruciate ligament transection (ACLT) model. DesignACL transections were performed unilaterally in 5 rabbits (33-weeks-old, 3.5 ​± ​0.5 ​kg) to induce PTOA. MRI exams were performed at 7 ​T prior to and 2, 4, 7 and 10-weeks after ACLT using a modified dGEMRIC protocol. Voxel-based T1 and T2 maps were created over manually drawn femoral cartilage ROIs from the center of the tibial plateau to the posterior meniscus. Femoral, tibial, and patellar epiphyses were harvested 10-weeks post-surgery and processed for μCT imaging and histology. ResultsQuantitative analysis revealed a 35% and 39% decrease in dGEMRIC index in the medial ACLT knee compartment 7- and 10-weeks post-surgery, respectively (p ​= ​0.009 and p ​= ​0.006) when compared to baseline. There was no significant change in the lateral ACLT compartment or in either compartment of the control knees. Visual inspection of histology confirmed PTOA in the ACLT knees. Osteophytes were found only in ACLT knees (osteophyte volume in femur: 94.53 ​± ​44.08 ​mm3, tibia: 29.35 ​± ​13.79 ​mm3, and patella: 3.84 ​± ​0.92 ​mm3) and were significantly larger in the medial compartments of the femur than lateral (p ​= ​0.0312). ConclusionThe dGEMRIC technique quantitatively applied at 7 ​T UHF-MRI demonstrates site-specific cartilage degeneration in a large animal PTOA model. This should encourage further investigation, with potential applications in drug and therapeutic animal trials as well as human studies.

An Anterior Cruciate Ligament In Vitro Rupture Model Based on Clinical Imaging

Background: Biomechanical studies on anterior cruciate ligament (ACL) injuries and reconstructions are based on ACL transection instead of realistic injury trauma. Purpose: To replicate an ACL injury in vitro and compare the laxity that occurs with that after an isolated ACL transection injury before and after ACL reconstruction. Study Design: Controlled laboratory study. Methods: Nine paired knees were ACL injured or ACL transected. For ACL injury, knees were mounted in a rig that imposed tibial anterior translation at 1000 mm/min to rupture the ACL at 22.5° of flexion, 5° of internal rotation, and 710 N of joint compressive force, replicating data published on clinical bone bruise locations. In contralateral knees, the ACL was transected arthroscopically at midsubstance. Both groups had ACL reconstruction with bone–patellar tendon–bone graft. Native, ACL-deficient, and reconstructed knee laxities were measured in a kinematics rig from 0° to 100° of flexion with optical tracking: anterior tibial translation (ATT), internal rotation (IR), anterolateral (ATT + IR), and pivot shift (IR + valgus). Results: The ACL ruptured at 26 ± 5 mm of ATT and 1550 ± 620 N of force (mean ± SD) with an audible spring-back tibiofemoral impact with 5o of valgus. ACL injury and transection increased ATT (P < .001). ACL injury caused greater ATT than ACL transection by 1.4 mm (range, 0.4-2.2 mm; P = .033). IR increased significantly in ACL-injured knees between 0° and 30° of flexion and in ACL transection knees from 0° to 20° of flexion. ATT during the ATT + IR maneuver was increased by ACL injury between 0° and 80° and after ACL transection between 0° and 60°. Residual laxity persisted after ACL reconstruction from 0° to 40° after ACL injury and from 0° to 20° in the ACL transection knees. ACL deficiency increased ATT and IR in the pivot-shift test (P < .001). The ATT in the pivot-shift increased significantly at 0° to 20° after ACL transection and 0° to 50° after ACL injury, and this persisted across 0° to 20° and 0° to 40° after ACL reconstruction. Conclusion: This study developed an ACL injury model in vitro that replicated clinical ACL injury as evidenced by bone bruise patterns. ACL injury caused larger increases of laxity than ACL transection, likely because of damage to adjacent tissues; these differences often persisted after ACL reconstruction. Clinical Relevance: This in vitro model created more realistic ACL injuries than surgical transection, facilitating future evaluation of ACL reconstruction techniques.

Detection of Osteoarthritis Inflammation by Single-Photon Emission Computed Tomography Based on an Inflammation-Targeting Peptide cFLFLF.

Although inflammation has been recognized as a key process in the pathogenesis of osteoarthritis (OA), there remains no clinical noninvasive imaging modality that can specifically diagnose inflammatory activity of OA. In this study, a formyl peptide receptor 1 (Fpr1) targeting probe cFLFLF-PEG-HYNIC-99mTc and single-photon emission computed tomography (SPECT) imaging was used to detect inflammatory activity by targeting macrophages involved in the pathogenesis of OA. In vitro experiments were performed to evaluate Fpr1 expression during macrophage inflammatory response. In the in vivo studies, anterior cruciate ligament transection (ACLT) surgery was performed, and magnetic resonance imaging (MRI) and histological data were assessed to analyze the OA model in both mice and rats. The radioactive probe cFLFLF-PEG-HYNIC-99mTc and SPECT imaging were used to corroborate OA-related inflammation and compare ACLT vs sham knees. In vitro macrophage activation resulted in a remarkable increase in Fpr1 expression. In vivo experiments in mice and rats produced similar results. MRI and histological analysis demonstrated significant joint degeneration in the ACLT knee. The ACLT knee produced a much stronger signal from the probe when compared to the sham knee. It is important to note that the ratio of ACLT/sham knee signal intensity decreased with OA progression, indicating greater differences earlier in the progression of OA. The radioactive probe cFLFLF-PEG-HYNIC-99mTc and SPECT imaging are effective for detecting and monitoring inflammation during OA progression by targeting Fpr1 expression in the knee joint.

Effects of ultrasound on vascular endothelial growth factor in cartilage, synovial fluid, and synovium in rabbit knee osteoarthritis.

Ultrasound is commonly used to treat knee osteoarthritis (KOA), which has unique advantages with regard to relieving pain and inflammation as well as delaying cartilage degeneration, but the underlying mechanisms are less clear. The study aimed to investigate the therapeutic effects of ultrasound on vascular endothelial growth factor (VEGF) expression in cartilage, the synovium, and synovial fluid (SF) in a rabbit model of KOA. Twenty-four New Zealand rabbits were randomly divided into ultrasound (group A), sham ultrasound (group B) and no-ACLT control groups (group C). Six weeks after undergoing anterior cruciate ligament transection (ACLT), group A was treated with ultrasound and group B was treated with sham ultrasound. Two weeks thereafter, the morphology of the synovium and cartilage were observed. Cartilage and synovium were scored using the Mankin scale and Krenn V scores, respectively. VEGF expression in the cartilage, SF, and synovium of ACLT knee joints was analyzed via immunohistochemistry, western blotting, and RT-PCR. Cartilage degeneration and synovitis were the most severe in group B and the least severe in group C. Similarly, Mankin scores and Krenn V scores were highest in group B and lowest in group C (p<0.05). There were also significant differences in the VEGF IOD of cartilage or synovium, VEGF protein content in SF, and VEGF mRNA expression in cartilage or SF (p<0.05). Ultrasound can relieve synovitis and delay cartilage degradation, and the mechanisms of ultrasound for the treatment of KOA may involve inhibition of the expression of VEGF in the synovium, SF, and cartilage.

Disturbances in Metabolic Pathways and the Identification of a Potential Biomarker Panel for Early Cartilage Degeneration in a Rabbit Anterior Cruciate Ligament Transection Model.

This study aimed to assess the association between synovial fluid (SF) metabolites and magnetic resonance imaging (MRI) measurements of cartilage biochemical composition to identify potential SF biomarkers for detecting the early onset of cartilage degeneration in a rabbit model. Both knees of 12 New Zealand White rabbits were used. The anterior cruciate ligament transection (ACLT) model was performed on right knees, and the sham surgery on left knees. MRI UTE-T2* scanning and SF sample collection were performed on ACLT knees at 4 and 8 weeks postsurgery and on sham surgery knees at 4 weeks postsurgery. Ultra-performance liquid chromatography-mass spectrometry and multivariate statistical analysis were used to distinguish samples in three groups. Pathway and receiver operating characteristic analyses were utilized to identify potential metabolite biomarkers. There were 12 knees in sham surgery models, 11 in ACLT models at 4 weeks postsurgery, and 10 in ACLT models at 8 weeks postsurgery. UTE-T2* values for the lateral tibia cartilage showed significant decreases over the study period. Levels of 103 identified metabolites in SF were markedly different among three groups. Furthermore, 24 metabolites were inversely correlated with UTE-T2* values of the lateral tibia cartilage, while hippuric acid was positively correlated with UTE-T2* values of the lateral tibia cartilage. Among 25 potential markers, N1-acetylspermidine, 2-amino-1,3,4-octadecanetriol, l-phenylalanine, 5-hydroxy-l-tryptophan, and l-tryptophan were identified as potential biomarkers with high area under the curve values and Pearson correlation coefficients. Five differential metabolites in SF were found as potential biomarkers for the early detection of cartilage degeneration in the rabbit ACLT model.

Clinical-like cryotherapy improves gait function and reduces synovial inflammation in rats with knee osteoarthritis

Purpose: To evaluate the effects of clinical-like cryotherapy on gait function and synovial inflammation in rats with knee osteoarthritis (KOA) induced by anterior cruciate ligament transection (ACLT). Methods: The experimental protocol was in accordance with the National Guide for the Care and Use of Laboratory Animals (National Research Council, 1996). All procedures were approved by the institutional Ethics Committee and conducted by trained professionals blinded to the identity of the experimental groups. Two-month-old male Wistarrats (n=32; 297±25 g) were studied. The animals were housed in pathogen-free conditions at 24ºC±1°C under a reverse light cycle (12/12 light/dark) with free access to standard rat chow and water. Animals were randomly allocated into four groups (n=8 per group): Control (without surgery and intervention); ACLT knee surgery (KOA); ACLT knee surgery submitted to ice pack (KOA+Cryotherapy), and ACLT knee surgery submitted to a sand pack (KOA+Placebo). An adapted ACLT-induced KOA model was used. The groups were analyzed one day before and 60 days after ACLT surgery from the least to the most stressful tests: skin temperature (thermography), gait test (paw print), thermal response threshold (hotplate) and swelling (digital caliper). Next, both the KOA+Cryotherapy and KOA+Placebo groups underwent the interventions (ice pack or sand pack) twice a day, for 20 min each, over five consecutive days (from the 61st to the 65th days). The four groups were then assessed on the 66th day, and after euthanasia, the synovial fluid (account of leukocytes and cytokine levels) and synovial membrane (histopathological analysis) were collected (Figure 1). Results: Among the KOA groups, only the Cryotherapy group increased their paw contact area (P=0.004; 14%) after interventions, with no difference in relation to the Control group [Figure 2(A)]. Cryotherapy decreased the number of leukocytes (P<0.001; ≥95.0%; Figure 3) and cytokine levels (P<0.001; ≥55%; Figure 4) in synovial fluid in relation to the KOA and Placebo groups. There were no differences in synovial score and fibrosis in the synovial membrane of KOA groups. Conclusions: Clinical-like cryotherapy improves the gait function and reduces the number of leukocytes and inflammatory cytokines in synovial fluid of rats with ACLT-induced KOA. These results provide new evidence of the beneficial effects of cryotherapy and suggest it can be used as a non-pharmacological and complementary treatment to control joint inflammation of KOA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Anterior cruciate ligament transection alters the n-3/n-6 fatty acid balance in the lapine infrapatellar fat pad

BackgroundThe infrapatellar fat pad (IFP) of the knee joint has received lots of attention recently due to its emerging role in the pathogenesis of osteoarthritis (OA), where it displays an inflammatory phenotype. The aim of the present study was to examine the infrapatellar fatty acid (FA) composition in a rabbit (Oryctolagus cuniculus) model of early OA created by anterior cruciate ligament transection (ACLT).MethodsOA was induced randomly in the left or right knee joint of skeletally mature New Zealand White rabbits by ACLT, while the contralateral knee was left intact. A separate group of unoperated rabbits served as controls. The IFP of the ACLT, contralateral, and control knees were harvested following euthanasia 2 or 8 weeks post-ACLT and their FA composition was determined with gas chromatography–mass spectrometry.ResultsThe n-3/n-6 polyunsaturated FA (PUFA) ratio shifted in a pro-inflammatory direction after ACLT, already observed 2 weeks after the operation (0.20 ± 0.008 vs. 0.18 ± 0.009). At 8 weeks, the FA profile of the ACLT group was characterized with increased percentages of 20:4n-6 (0.44 ± 0.064 vs. 0.98 ± 0.339 mol-%) and 22:6n-3 (0.03 ± 0.014 vs. 0.07 ± 0.015 mol-%) and with decreased monounsaturated FA (MUFA) sums (37.19 ± 1.586 vs. 33.20 ± 1.068 mol-%) and n-3/n-6 PUFA ratios (0.20 ± 0.008 vs. 0.17 ± 0.008). The FA signature of the contralateral knees resembled that of the unoperated controls in most aspects, but had increased proportions of total n-3 PUFA and reduced MUFA sums.ConclusionsThese findings provide novel information on the effects of early OA on the infrapatellar FA profile in the rabbit ACLT model. The reduction in the n-3/n-6 PUFA ratio of the IFP is in concordance with the inflammation and cartilage degradation in early OA and could contribute to disease pathogenesis.

Site-specific glycosaminoglycan content is better maintained in the pericellular matrix than the extracellular matrix in early post-traumatic osteoarthritis.

IntroductionOne of the characteristics of early osteoarthritis (OA) is the loss of fixed charged density (FCD) of glycosaminoglycans in the superficial zone of articular cartilage. However, possible local changes in the FCD content of the pericellular matrix (PCM) are not fully understood. Hence, our aim was to investigate the effect of unilateral anterior cruciate ligament transection (ACLT) in rabbit knees on estimated FCD in the PCM compared to that in the ECM, and relate these results with cell morphology.MethodsArticular cartilage samples were collected from ACLT, contralateral and intact control knee joints from lateral and medial femoral condyles and tibial plateaus, and from the femoral groove and patella. Histological samples were prepared and stained with Safranin-O to estimate the FCD content around the chondrocytes in the PCM and the ECM with digital densitometry.ResultsAs a result of ACLT, the greatest decreases in the FCD content of the PCM were observed in the superficial zone of the lateral femoral condyle (p = 0.02), medial tibial plateau (p = 0.002) and patellar (p < 0.001) cartilage. The normalized FCD content of the PCM compared to the surrounding ECM was increased most in the femoral condyles (p < 0.01) and medial tibial plateau (p = 0.02) cartilage. The high normalized FCD content of the PCM in the superficial zone of lateral femoral condyle cartilage was consistent with the round cell morphology in that location.ConclusionsIn conclusion, we suggest that certain sites in the knee joint, particularly the lateral femoral condyle cartilage, experience less FCD loss in the PCM than in the ECM in early post-traumatic OA, which could lead to altered cell shape.

Protective effect of zoledronic acid on articular cartilage and subchondral bone of rabbits with experimental knee osteoarthritis.

Subchondral bone reabsorption and remodeling are responsible for the initiation and progression of osteoarthritis (OA). Zoledronic acid (ZOL), a third-generation bisphosphonate (BIS), is an inhibitor of bone reabsorption. However, the intervention effect of ZOL on OA has not been fully characterized and remains to be directly demonstrated in animal experiments. The present study examined the microscopic and macroscopic changes in the anterior cruciate ligament transection (ACLT) model of OA in rabbits and evaluated the effects of ZOL on cartilage degeneration and subchondral bone loss. A total of 32 New Zealand white rabbits were randomly divided into four groups: High-, medium- and low-dose ZOL groups, which received an intravenous injection of 250, 50 and 10 µg/kg ZOL, respectively, after modeling, as well as an untreated group. The bone mineral density (BMD) of the knee joint was evaluated by dual-energy X-ray absorptiometry scanning immediately after modeling and at 4 and 8 weeks. At week 8, quantitative measurement of cartilage was performed by a specialized magnetic resonance imaging (MRI) technique, including three-dimensional fat-suppressed spoil gradient-recalled sequence and T2 mapping. The rabbits were sacrificed by air embolism after anesthesia and both knee joints were harvested and evaluated by general and histological observation. Toluidine blue and hematoxylin and eosin staining were used to assess histological changes in the articular cartilage. Quantitative analysis of cartilage histopathology was performed according to the Mankin scoring system. The BMD of ACLT joints dropped after modeling, which was effectively suppressed by ZOL at the high and medium dose but not the low dose. MRI scans demonstrated that in the untreated group, articular cartilages on ACLT knees were thinner than those on normal knees. The high dose of ZOL preserved the cartilage tissue thickness more efficiently than the medium and low doses. Observation of specimens and pathological slices revealed that the articular cartilage degeneration in the high-dose ZOL group was lightest, while that in the medium- and low-dose ZOL group was moderate, and the untreated group exhibited the most severe defect. The untreated group had the highest Mankin score, whereas the high-dose ZOL group had the lowest score. In conclusion, ZOL increased the subchondral bone density, improved the microstructure and reduced the degeneration of articular cartilage in OA according to morphological as well as quantitative observation. ZOL exerted significant chondroprotective effects in a dose-dependent manner. A favorable chondroprotective effect was induced at the dose of 250 µg/kg. ZOL may represent a novel promising drug to complement the treatment of OA.

Feasibility of Na18F PET/CT and MRI for Noninvasive In Vivo Quantification of Knee Pathophysiological Bone Metabolism in a Canine Model of Post-traumatic Osteoarthritis.

Purpose:To assess and quantify by molecular imaging knee osseous metabolic changes serially in an in vivo canine model of posttraumatic osteoarthritis (PTOA) of the knee utilizing sodium fluoride (Na18F) positron emission tomography (PET)/computed tomography (CT) coregistered with magnetic resonance imaging (MRI).Materials and Methods:Sodium fluoride PET imaging of 5 canines was performed prior to anterior cruciate ligament transection (ACLT) and 2 times post-ACLT (3 and 12 weeks). The PET/CT was coregistered with MRI, enabling serial anatomically guided visual and quantitative three-dimensional (3D) region of interest (ROI) assessment by maximum standardized uptake value.Results:Prior to ACLT, every 3D ROI assessed in both knees showed no Na18F uptake above background. The uptake of Na18F in the bone of the ACLT knees increased exponentially, presenting significantly higher uptake at 12 weeks in every region compared to the ACLT knees at baseline. Furthermore, the uninjured contralateral limb and the ipsilateral distal bones and joints presented Na18F uptake at 3 and 12 weeks post-ACLT.Conclusion:This study demonstrated that Na18F PET/CT coregistered with MRI is a feasible molecular imaging biomarker to assess knee osseous metabolic changes serially in an in vivo canine model of knee PTOA. Moreover, it brings a novel musculoskeletal preclinical imaging methodology that can provide unique insights into PTOA pathophysiology.

Preclinical Multimodal Molecular Imaging Using 18F-FDG PET/CT and MRI in a Phase I Study of a Knee Osteoarthritis in In Vivo Canine Model.

The aim of this study was to use a multimodal molecular imaging approach to serially assess regional metabolic changes in the knee in an in vivo anterior cruciate ligament transection (ACLT) canine model of osteoarthritis (OA). Five canine underwent ACLT in one knee and the contralateral knee served as uninjured control. Prior, 3, 6, and 12 weeks post-ACLT, the dogs underwent 18F-fluoro-d-glucose (18F-FDG) positron emission tomography (PET)/computed tomography (CT) and magnetic resonance imaging (MRI). The MRI was coregistered with the PET/CT, and 3-dimensional regions of interest (ROIs) were traced manually and maximum standardized uptake values (SUVmax) were evaluated. 18F-fluoro-d-glucose SUVmax in the ACLT knee ROIs was significantly higher compared to the uninjured contralateral knees at 3, 6, and 12 weeks. Higher 18F-FDG uptake observed in ACLT knees compared to the uninjured knees reflects greater metabolic changes in the injured knees over time. Knee 18F-FDG uptake in an in vivo ACLT canine model using combined PET/CT and MRI demonstrated to be highly sensitive in the detection of metabolic alterations in osseous and nonosteochondral structures comprising the knee joint. 18F-fluoro-d-glucose appeared to be a capable potential imaging biomarker for early human knee OA diagnosis, prognosis, and management.

Mapping articular cartilage biomechanical properties of normal and osteoarthritis mice using indentation

s / Osteoarthritis and Cartilage 23 (2015) A82eA416 A254 could be a relative increase in trabecular bone volume, which decrease in advance OA. For the tibias, only the SMI was increased in the ACLT knees versus the controls. It might be that 12 weeks is not sufficient time to detect OA trabecular bone microarchitecture changes in the tibia after the Pond-Nuki surgically induced secondary OA in the canine model. This study quantitatively analyzed the trabecular bone in the anterior cruciate ligament transection (ACLT) Pond-Nuki dog model. The results may help establish the standard characteristics of the knee joint trabecular bone microarchitecture upon development of OA, and to provide a greater understanding of OA from the clinical viewpoint and to aid in its treatment. Figure 1. showing 3D BMD color maps for the control femur (A) and tibia (B) and the ACLT femur (B) and tibia (D). The ACLT femur (B) shows lower BMD than the control (A). 391 MAPPING ARTICULAR CARTILAGE BIOMECHANICAL PROPERTIES OF NORMAL AND OSTEOARTHRITIS MICE USING INDENTATION J.-F. Lavoie yz, S. Sim yx, E. Quenneville x, M. Garon x, A. Moreau kz, M.D. Bushmann y, C.E. Aubin yz. y Polytechnique Montreal, Montreal, QC, Canada; z Sainte-Justine Univ. Hosp. Ctr., Montreal, QC, Canada; xBiomomentum, Laval, QC, Canada; k Faculty of Med., Dept of Biochemistry and Dept of Stomatology, Univ. of Montreal, Montreal, QC,

Intra-articular injection of the selective cyclooxygenase-2 inhibitor meloxicam (Mobic) reduces experimental osteoarthritis and nociception in rats

To study the effect of intra-articular injection of meloxicam (Mobic) on the development of osteoarthritis (OA) in rats and examine concomitant changes in nociceptive behavior and the expression of mitogen-activated protein kinases (MAPKs) in articular cartilage chondrocytes. OA was induced in Wistar rats by right anterior cruciate ligament transection (ACLT); the left knee was not treated. The OA + meloxicam (1.0 mg) group was injected intra-articularly in the ACLT knee with 1.0 mg of meloxicam once a week for 5 consecutive weeks starting 5 weeks after ACLT. The OA + meloxicam (0.25 mg) group was treated similarly with 0.25 mg meloxicam. The sham group underwent arthrotomy only and received vehicle of 0.1 mL sterile 0.9% saline injections, whereas the naive rats in meloxicam-only groups were treated similarly with 1.0- and 0.25-mg meloxicam. Nociception was measured as secondary mechanical allodynia and hind paw weight-bearing distribution at before (pre-) and 5, 10, 15, and 20 weeks post-ACLT. Histopathology of the cartilage and synovia was examined 20 weeks after ACLT. Immunohistochemical analysis was performed to examine the effect of meloxicam on MAPKs (p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK)) expression in the articular cartilage chondrocytes. OA rats receiving intra-articular meloxicam treatment showed significantly less cartilage degeneration and synovitis than saline-treated controls. Nociception were improved in the OA + meloxicam groups compared with the OA group. Moreover, meloxicam attenuated p38 and JNK but enhanced ERK expression in OA-affected cartilage. Intra-articular injection of meloxicam (1) attenuates the development of OA, (2) concomitantly reduces nociception, and (3) modulates chondrocyte metabolism, possibly through inhibition of cellular p38 and JNK, but enhances ERK expression.

Regulation of the inflammatory response by tin protoporphyrin IX in the rat anterior cruciate ligament transection model of osteoarthritis

The purpose of this study was to investigate several inflammatory mediators and cartilage degradation molecules as possible biomarkers of joint lesion in the anterior cruciate ligament transection (ACLT) model of osteoarthritis in rats. We also assessed whether the treatment with the anti-inflammatory agent tin protoporphyrin IX (SnPP) reduces the progression of disease. Our results indicate that serum levels of interleukin (IL)-6 and PGE(2) are significantly increased in ACLT rats 10 weeks after surgery, whereas the increases in IL-1β and tumor necrosis-α were not significant. In addition, our data suggest that IL-17 is the main pro-inflammatory cytokine in the ACLT joint. We have shown that serum C-telopeptide of type II collagen (CTX-II) is a biomarker better than cartilage oligomeric matrix protein, whereas hyaluronic acid showed no correlation with disease progression. Administration of SnPP (i.p. 12 mg/kg BW/day for 10 weeks) reduces the severity of structural changes and the levels of CTX-II, IL-6, and PGE(2) in serum and IL-17 and PGE(2) in ACLT knees. These effects on PGE(2) are dependent on the down-regulation of cyclooxygenase-2. Our findings indicate that systemic and local inflammatory mediators participate in the rat ACLT model and anti-inflammatory strategies may help to reduce the progression of joint lesion.

Imaging of a rat osteoarthritis model using 18F-fluoride positron emission tomography

Currently, conventional radiography is the standard method for the diagnosis and evaluation of the severity of osteoarthritis (OA), but it takes a couple of years to detect cartilage loss. Magnetic resonance imaging can delineate articular cartilage and accurately assess cartilage volume and thickness, but its reliability for very early diagnosis of OA is still controversial. The purpose of this study was to confirm the potential of (18)F-fluoride PET for the early diagnosis of OA by using a surgically induced rat OA model. Seventeen 16-week-old male Sprague-Dawley rats underwent anterior cruciate ligament transection (ACLT) in the right knee to induce OA. The left knee underwent sham operation. At 2, 4, and 8 weeks after operation, the rats were injected with 2.5 MBq/kg of (18)F-fluoride, and 30 min after injection, each rat was killed and the bilateral knees were resected. The femur and tibia were cut horizontally, approximately 2 mm from the joint surface excluding the growth plate, and were cut into the medial and lateral condyles. The patella was also resected and blood samples were collected. The radioactivity of each sample was measured by gamma counting. Assays for serum cartilage oligomeric matrix protein and serum C-telopeptide of type II collagen were performed. Histopathological grading was performed according to a modified Mankin's scoring system. Two rats underwent PET scans at 2, 4, and 8 weeks after operation. The rats were injected with 30 MBq of (18)F-fluoride, and 30 min after injection, bilateral knee images with a 30-min acquisition time were obtained with an animal PET system. The uptake of (18)F-fluoride was significantly higher in ACLT knees than sham-operated knees in the medial femur and medial tibia at 2 weeks after operation. At 4 weeks after operation, the medial femur, medial tibia, and lateral tibia of OA knees showed significantly higher uptake of (18)F-fluoride compared with sham-operated knees. At 8 weeks, all sections showed significant differences. The uptake of (18)F-fluoride significantly increased as time elapsed in all sections. Uptake showed a significant correlation with histological scores. Our results suggest that (18)F-fluoride is potentially useful for the early detection of osteoarthritic changes.

Degeneration of normal articular cartilage induced by late phase osteoarthritic synovial fluid in beagle dogs

Objective To investigate the pathogenesis of late phase osteoarthritic (OA) synovial fluid (SF) on normal articular cartilage in vivo and provide an understanding of degenerative cartilage extending in OA joint. Methods A random knee, each of 8 beagle dogs, received anterior cruciate ligament transection (ACLT) and was confirmed to have late phase OA degenerative changes at 24 weeks after operation. Thereafter, one random elbow of each canine was injected with autologous late phase OA knee SF. The contralateral elbow was injected with normal saline (NS) of the same volume as SF aspirated from ACLT knee. These two groups of elbows were labeled “SF” and “NS”. 8 other beagle dogs were left intact and placed in Group Control. After aseptic arthrocentesis was performed weekly on both elbows for 24 weeks, morphological changes were observed in the cartilage of the elbows, and expressions of 7 biological etiological factors of chondrocytes of the elbows were determined in Group SF, Group NS and Group Control, respectively. Results Morphological changes were observed in articular cartilage of the elbows in Group SF. Levels of unit area of collagen type I in the noncalcified, calcified and full zones of articular cartilage of the elbows in Group SF increased significantly. Level of unit area of collagen type III in the calcified zone of articular cartilage of the elbows in Group SF remained unchanged. Meanwhile, expressions of MMP-1 and MMP-3 of chondrocytes of the elbows in Group SF increased significantly. There was almost no difference between articular cartilage in Group NS and Group Control. Conclusion Based on these results, we conclude that OA degeneration of normal articular cartilage can be independently induced by late phase OA SF. Endogenous OA biological etiological factor may be one of the reasons causing degenerative cartilage extending in OA joint.

Increase in excitatory amino acid concentration and transporters expression in osteoarthritic knees of anterior cruciate ligament transected rabbits

The present study aimed to determine the role of excitatory amino acids (EAAs) and EAA transporters (EAATs) in an osteoarthritis (OA) model of rabbit knees. OA was induced in New Zealand white male rabbits by anterior cruciate ligament transection (ACLT) in one knee of one hind limb; the other knee left unoperated. Rabbits that received ACLT of knee were assigned to the ACLT group (n=6), while a sham-operated group (n=6) underwent arthrotomy with no ACLT. Six naïve rabbits that received no surgery were used as normal control. The width of the knee joint was measured to determine the severity of joint inflammation. Before operation and at 10, 20, and 30 weeks after operation, knee joint dialysates were collected by microdialysis and assayed for EAAs by high-performance liquid chromatography. Gross morphology and histopathology and EAATs glutamate/aspartate transporter (GLAST) and glutamate transporter-1 (GLT-1) expression in the articular cartilage of the knees were evaluated by immunohistochemistry and western blot analysis. In the ACLT knees, a significant increase in the joint width was observed (5.3+/-0.9 mm, P<0.05) at 30 weeks after operation, while the sham-operated and naïve knees showed no difference as compared with the basal values. The concentrations (microM) of aspartate and glutamate in knee dialysates at 30 weeks after ACLT in naïve, sham, and ACLT were 0.36+/-0.07 and 4.5+/-1.10; 0.38+/-0.09 and 4.61+/-1.11; 0.67+/-0.18 and 9.71+/-2.89, respectively. Levels of glutamate and aspartate in the dialysates obtained from the ACLT knees increased by 213.3+/-29.6% and 187.5+/-33.8% (P<0.05) when compared to those in the sham-operated knees. Both naïve and ACLT chondrocytes were positively stained by antibodies against GLAST and GLT-1. GLAST and GLT-1 protein expressions were significantly increased in the ACLT knees (P<0.05). Our findings indicate an involvement of EAAs and EAATs in the pathogenesis of OA in ACLT rabbits.

Knee joint ultrasonography of the ACLT rabbit experimental model of osteoarthritis: relevance and effectiveness in detecting meniscal lesions

To develop a protocol for rabbit knee joint ultrasonography (US); to grade ultrasonographically the meniscal injuries of the anterior cruciate ligament transection (ACLT) rabbit model of osteoarthritis (OA); to assess with US the effectiveness of the ACLT; to compare final US with macroscopy for the evaluation of medial and lateral meniscal injuries depending on the age and weight when ACLT is performed. Twenty-two skeletally mature and adolescent New Zealand white rabbits were housed during the same period at the Institut Claude-Bourgelat, Lyon, France. Surgical ACLT was performed in the left knee of nine adolescent and five adult rabbits. Final US and macroscopic semi-quantitative grading of the meniscal injuries were compared 5 months after ACLT. A standardised protocol was developed to evaluate the rabbit knee joint. US was performed in both control and ACLT knees. Normal and abnormal meniscal US appearances were described. A semi-quantitative scale to grade US meniscal injuries was created. Macroscopic and US total meniscal scores were significantly positively correlated (P<0.001, r=0.70). US detection of meniscal injuries was 92% sensitive and 87.5% specific compared to macroscopy. Positive and negative predictive values of US were, respectively, 92% and 87.5%. US detection of the ACLT effectiveness was 100% specific and 78.5% sensitive. A significant relationship was found between ultrasonographic and macroscopic grading of meniscal injuries. US was both specific and sensitive in detecting meniscal lesions. We propose US as a non-invasive, non-expensive, in vivo imaging technique for preclinical studies in the ACLT rabbit OA model.

Intra-articular injection of the cyclooxygenase-2 inhibitor parecoxib attenuates osteoarthritis progression in anterior cruciate ligament-transected knee in rats: role of excitatory amino acids

Our present study examined the effect of intra-articular cyclooxygenase-2 (COX-2) inhibitor parecoxib on osteoarthritis (OA) progression and the concomitant changes in excitatory amino acids' (EAAs) levels of the anterior cruciate ligament-transected (ACLT) knee joint dialysates. OA was induced in Wistar rats by anterior cruciate ligament transection of the knee of one hindlimb, the other was left unoperated and untreated. Rats were placed into four groups: Group ACLT/P received intra-articular parecoxib injection (100 microg) in the ACLT knee once a week for 5 consecutive weeks starting at 8 weeks after surgery. Group ACLT/S received the same procedure as group ACLT/P with saline injection instead. Naïve (Naïve/P) rats received only intra-articular parecoxib injection in one knee once a week for 5 consecutive weeks without surgery. The sham-operated rats underwent arthrotomy only without treatment. Twenty weeks after surgery, knee joint dialysates were collected and EAAs' concentration was assayed by high-performance liquid chromatography, and gross morphology and histopathology (Mankin and synovitis grading) were examined on the medial femoral condyles and synovia. Parecoxib alone had no effect on cartilage and synovium of normal knees in Naïve/P rats. In ACLT/P rats, parecoxib treatment showed a significant inhibition of cartilage degeneration of the medial femoral condyle at both the macroscopic level (1.15+/-0.17 vs 2.55+/-0.12, P<0.05) and the Mankin scores (3.03+/-0.28 vs 8.82+/-0.43, P<0.05). Intra-articular parecoxib injection also suppressed the synovial inflammation of ACLT joint compared to the ACLT/S group (3.92+/-0.41 vs 9.25+/-0.32, P<0.05). Moreover, glutamate and aspartate levels were also significantly reduced in the ACLT/P group compared to the ACLT/S group by parecoxib treatment (91.2+/-9.4% vs 189.5+/-17.0%, P<0.05 and 98.2+/-11.6% vs 175.3+/-12.4%, P<0.05, respectively). This study shows that intra-articular injection of COX-2 inhibitor parecoxib inhibits the ACLT-induced OA progression; it was accompanied by a reduction of glutamate and aspartate concentration in the ACLT joint dialysates. From our present results, we suggested that intra-articular parecoxib injection, in addition to the anti-inflammatory effect, inhibiting the EAAs' release, may also play a role in inhibiting the traumatic knee injury induced OA progression.