Rabbit Anterior Cruciate Ligament Transection Research Articles

Therapeutic effect of induced pluripotent stem cell -derived extracellular vesicles in an in vitro and in vivo osteoarthritis model.

Osteoarthritis (OA) is a multifactorial joint disease associated with the deterioration of chondrocytes and inflammation. Treatment of OA is only aimed at reducing pain and improving joint function. Recently, extracellular vesicles (EVs) secreted from stem cells have emerged as a cell regenerative tool in several degenerative diseases, including OA. We hypothesised that induced pluripotent stem cell (iPSC)-derived EVs would be beneficial for regenerating chondrocytes and OA therapy. Therefore, we aimed to investigate iPSC-EVs' effects on chondrocyte behaviour in an interleukin 1 beta (IL-1β)-induced in vitro OA model and anterior cruciate ligament transection (ACLT)-induced in vivo OA model of rabbit articular cartilage. The iPSC-EVs were isolated by sequential ultracentrifugation from a 48-h-incubated conditional medium of iPSC. The isolated iPSC-EVs were characterised by transmission electron microscopy, western blot analyses, and dynamic light scatter. The effects of iPSC-EVs on the viability of human primary chondrocytes and cell senescence were analysed. Premature senescence of cells was induced by long-term incubation with low doses of hydrogen peroxide. To investigate the therapeutic effect of iPSC-EVs on OA chondrocytes in vitro, IL-1β was used to induce chondrocyte damage. Inflammatory macrophages were activated from THP-1 monocytes to observe the impact of iPSC-EV on macrophage polarisation. The phenotypes of the macrophages exposed to iPSC-EVs were evaluated by ELISA and western blot analyses. The primary chondrocytes were co-cultured with different phenotypes of macrophages to observe the expression of collagen II and catabolic enzymes in chondrocytes. iPSC-EVs were injected intraarticularly into the rabbit with an ACLT-induced OA model. The progression of lesions was assessed through macroscopic and histopathological studies. We showed that iPSC-EVs significantly stimulated the proliferation of primary human chondrocytes and suppressed cell senescence by regulating the expression of p21 and collagen II. iPSC-EVs reduced matrix degradation enzymes and IL-6 expression and attenuated IL-1β-mediated cell death of chondrocytes. Furthermore, iPSC-EVs modulated macrophage polarisation, resulting in the rescue of damaged chondrocytes in an inflammatory microenvironment. In the rabbit ACLT model, the OA-like lesions, including inflammation, subchondral bone protrusion, and articular cartilage destruction, were ameliorated by iPSC-EV. A histopathological study consistently revealed that iPSC-EVs attenuated ACLT-mediated alteration of MMP13 and ADAMTS5 and collagen II expression. iPSC-EVs protected chondrocytes by enhancing cell proliferation, suppressing premature senescence, and maintaining homeostasis of collagen II synthesis and matrix degradation enzymes such as matrix metalloproteinases (MMPs) and ADAMTS5. iPSC-EVs also reduced cell death in IL-1β-mediated chondrocyte cell damage. In the rabbit ACLT-induced OA model, iPSC-EV injection reduced cartilage destruction, as indicated by the upregulation of collagen II and down-regulation of MMP13 and ADAMTS5. Overall, our results suggest that iPSC-EVs possess therapeutic potential and may be used as an OA treatment option. This study highlights the potential of iPSC-EVs as a therapeutic option for chondrocyte regeneration and OA treatment.

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.

Injectable recombinant block polymer gel for sustained delivery of therapeutic protein in post traumatic osteoarthritis

Protein-based biomaterials offer several advantages over synthetic materials, owing to their unique stimuli-responsive properties, biocompatibility and modular nature. Here, we demonstrate that E5C, a recombinant protein block polymer, consisting of five repeats of elastin like polypeptide (E) and a coiled-coil domain of cartilage oligomeric matrix protein (C), is capable of forming a porous networked gel at physiological temperature, making it an excellent candidate for injectable biomaterials. Combination of E5C with Atsttrin, a chondroprotective engineered derivative of anti-inflammatory growth factor progranulin, provides a unique biochemical and biomechanical environment to protect against post-traumatic osteoarthritis (PTOA) onset and progression. E5C gel was demonstrated to provide prolonged release of Atsttrin and inhibit chondrocyte catabolism while facilitating anabolic signaling in vitro. We also provide in vivo evidence that prophylactic and therapeutic application of Atsttrin-loaded E5C gels protected against PTOA onset and progression in a rabbit anterior cruciate ligament transection model. Collectively, we have developed a unique protein-based gel capable of minimally invasive, sustained delivery of prospective therapeutics, particularly the progranulin-derivative Atsttrin, for therapeutic application in OA.

Intra-Articular Adeno-Associated Virus-Mediated Proteoglycan 4 Gene Therapy for Preventing Posttraumatic Osteoarthritis.

Lubricin, a glycoprotein encoded by the proteoglycan 4 (PRG4) gene, is an essential boundary lubricant that reduces friction between articular cartilage surfaces. The loss of lubricin subsequent to joint injury plays a role in the pathogenesis of posttraumatic osteoarthritis. In this study, we describe the development and evaluation of an adeno-associated virus (AAV)-based PRG4 gene therapy intended to restore lubricin in injured joints. The green fluorescent protein (GFP) gene was inserted the PRG4 gene to facilitate tracing the distribution of the transgene product (AAV-PRG4-GFP) in vivo. Transduction efficiency of AAV-PRG4-GFP was evaluated in joint cells, and the conditioned medium containing secreted PRG4-GFP was used for shear loading/friction and viability tests. In vivo transduction of joint tissues following intra-articular injection of AAV-PRG4-GFP was confirmed in the mouse stifle joint in a surgical model of destabilization of the medial meniscus (DMM), and chondroprotective activity was tested in a rabbit anterior cruciate ligament transection (ACLT) model. In vitro studies showed that PRG4-GFP has lubricin-like cartilage-binding and antifriction properties. Significant cytoprotective effects were seen when cartilage was soaked in PRG4-GFP before cyclic shear loading (n = 3). Polymerase chain reaction and confocal microscopy confirmed the presence of PRG4-GFP DNA and protein, respectively, in a mouse DMM (n = 3 per group). In the rabbit ACLT model, AAV-PRG4-GFP gene therapy enhanced lubricin expression (p = 0.001 vs. AAV-GFP: n = 7-14) and protected the cartilage from degeneration (p = 0.014 vs. AAV-GFP: n = 9-10) when treatments were administered immediately postoperation, but efficacy was lost when treatment was delayed for 2 weeks. AAV-PRG4-GFP gene therapy protected cartilage from degeneration in a rabbit ACLT model; however, data from the ACLT model suggest that early intervention is essential for efficacy.

Quantification of Cartilage Surface Degeneration by Curvature Analysis Using 3D Scanning in a Rabbit Model.

Accurate analysis to quantify cartilage morphology is critical for evaluating degenerative conditions in osteoarthritis (OA). Three-dimensional (3D) optical scanning provides 3D data for the entire cartilage surface; however, there is no consensus on how to quantify it. Our purpose was to validate a 3D method for evaluating spatiotemporal alterations in degenerative cartilages in a rabbit OA model by analyzing their curvatures at various stages of progression. Twelve rabbits underwent anterior cruciate ligament transection (ACLT) unilaterally and were divided into 4 groups: 4 weeks control, 4 weeks OA, 8 weeks control, and 8 weeks OA. 3D scanning, India ink staining, and histological assessments were performed in all groups. In 3D curvature visualization, the surfaces of the condyles were divided into 8 areas. The standard deviations (SD) of mean curvatures from all vertices of condylar surfaces and subareas were calculated. Regarding the site of OA change, curvature analysis was consistent with India ink scoring. The SD of mean curvature correlated strongly with the India ink Osteoarthritis Research Society International (OARSI) score. In curvature histograms, the curvature distribution in OA was more scattered than in control. Of the 8 areas, significant OA progression in the posterolateral part of the lateral condyle (L-PL) was observed at 4 weeks. The histology result was consistent with the 3D evaluation in terms of representative section. This study demonstrated that 3D scanning with curvature analysis can quantify the severity of cartilage degeneration objectively. Furthermore, the L-PL was found to be the initial area where OA degeneration occurred in the rabbit ACLT model.

Low-Molecular-Weight Collagen Peptide Ameliorates Osteoarthritis Progression through Promoting Extracellular Matrix Synthesis by Chondrocytes in a Rabbit Anterior Cruciate Ligament Transection Model.

This study examined whether the oral administration of low-molecular-weight collagen peptide (LMCP) containing 3% Gly-Pro-Hyp with >15% tripeptide (Gly-X-Y) content could ameliorate osteoarthritis (OA) progression using a rabbit anterior cruciate ligament transection (ACLT) model of induced OA and chondrocytes isolated from a patient with OA. Oral LMCP administration (100 or 200 mg/kg/day) for 12 weeks ameliorated cartilage damage and reduced the loss of proteoglycan compared to the findings in the ACLT control group, resulting in dose-dependent (p < 0.05) improvements of the OARSI score in hematoxylin & eosin (H&E) and Safranin O staining. In microcomputed tomography analysis, LMCP also significantly (p < 0.05) suppressed the deterioration of the microstructure in tibial subchondral bone during OA progression. The elevation of IL-1βand IL-6 concentrations in synovial fluid following OA induction was dose-dependently (p < 0.05) reduced by LMCP treatment. Furthermore, immunohistochemistry illustrated that LMCP significantly (p < 0.05) upregulated type II collagen and downregulated matrix metalloproteinase-13 in cartilage tissue. Consistent with the in vivo results, LMCP significantly (p < 0.05) increased the mRNA expression of COL2A1 and ACAN in chondrocytes isolated from a patient with OA regardless of the conditions for IL-1βinduction. These findings suggest that LMCP has potential as a therapeutic treatment for OA that stimulates cartilage regeneration.

Sustained delivery of pgrn-derivative atsttrin via e5c hydrogel protects cartilage and bone quality in a rabbit model of post-traumatic osteoarthritis

Purpose: We have developed a protein-engineered thermo-responsive hydrogel, comprised of elastin (E) and cartilage oligomeric matrix protein coiled-coil (C), capable of providing sustained delivery of our chondroprotective engineered progranulin (PGRN)-derivative, Atsttrin (Tang et al., Science, 2011). Here, we characterize the impact of hydrogel delivered Atsttrin on chondrocyte viability and metabolism in vitro and describe the protective effects of injectable E5C hydrogel and Atsttrin-loaded E5C hydrogel in a rabbit anterior cruciate ligament transection (ACLT) induced model of post-traumatic osteoarthritis.

Periodic injections of adipose-derived stem cell sheets attenuate osteoarthritis progression in an experimental rabbit model

BackgroundSubcutaneous adipose tissue represents an abundant source of multipotent adult stem cells named as Adipose-derived stem cells (ADSCs). With a cell sheet approach, ADSCs survive longer, and can be delivered in large quantities. We investigated whether intra-articular ADSC sheets attenuated osteoarthritis (OA) progression in a rabbit anterior cruciate ligament transection (ACLT) model.MethodsFabricating medium containing ascorbate-2-phosphate was used to enhance collagen protein secretion by the ADSCs to make ADSC sheets. At 4 weeks after ACLT, autologous ADSC sheets were injected intra-articularly into the right knee (ADSC sheets group), and autologous cell death sheets treated by liquid nitrogen were injected into the left knee (control group). Subsequent injections were administered once weekly. Femoral condyles were compared macroscopically and histologically.ResultsMacroscopically, OA progression was significantly milder in the ADSC sheets than in the control groups. Histologically, control knees showed obvious erosions in the medial and lateral condyles, while cartilage was retained predominantly in the ADSC sheets group. Immunohistochemically, MMP-1, MMP-13, ADAMTS-4 were less expressive in the ADSC sheets than in the control groups.ConclusionsPeriodic ADSC sheets injections inhibited articular cartilage degeneration without inducing any adverse effects. A large quantity of autologous ADSCs delivered by cell sheets homed to the synovium and protected chondrocytes.

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.

Deep learning-based segmentation from histology allows for automated quantification of calcified cartilage morphology in a rabbit model of post-traumatic osteoarthritis

Purpose: Calcified cartilage (CC) undergoes constant remodeling due to two competing events: 1) mineralization of the deep non-calcified cartilage that advances the tidemark, and 2) new bone formation through endochondral ossification that advances the cement line. However, CC modifications in the presence of osteoarthritis (OA) progression are not well understood. In this study, we investigate CC morphology during OA progression and aging in the rabbit anterior cruciate ligament transection (ACLT) model.

Increasing Concentration of Sinovial: Effect on Cartilage Protection in a Rabbit ACLT Model.

This study aimed to test the hypothesis that administration of increasing doses of Sinovial (hyaluronic acid [HA]), would exhibit a dose-dependent effect on the prevention of cartilage degradation, without local and systemic toxicity. Twenty-seven adult rabbits were subjected to anterior cruciate ligament transection (ACLT). Two Sinovial products containing HA concentrations of 1.6% and 2.4% were used as active treatment, and 0.9% saline was used as control and injected intra-articularly 7 days post ACLT. Radiographs were taken prior to surgery, at injection and sacrifice times. After euthanasia, 8 weeks postsurgery, knee joints were observed macroscopically using India ink staining with OARSI (Osteoarthritis Research Society International) scoring and histologically using modified Mankin scoring. The synovial membranes were analyzed using Cake classification. No intraoperative complications were observed. One week postinjection, 4 animals in the HA 2.4% group developed subcutaneous nodules that disappeared spontaneously. No inflammation of the synovial membrane was ever observed. The control group exhibited the maximum uptake of India ink 2.22 ± 0.14. HA groups exhibited a dose-dependent (P = 0.02) reduction in India ink uptake: 1.75 ± 0.17 for HA 1.6% and 1.58 ± 0.14 for HA 2.4%. The most marked dose-dependent effect of this study was a reduction of modified Mankin score for HA groups, with the 2.4% treatment achieving a statistically significant improvement as compared with the control group (7.19 ± 0.85 for saline, 4.65 ± 0.66 for HA 1.6%, and 3.53 ± 0.59 for HA 2.4%; P = 0.005). A dose-dependent protective effect on cartilage was observed after injection of both HA solutions.

Early degeneration of the meniscus revealed by microbiomechanical alteration in a rabbit anterior cruciate ligament transection model

BackgroundThe microbiomechanical properties of the meniscus influence the cell response to the surrounding biomechanical environment ​and are beneficial to understand meniscus repairing and healing. To date, however, this information remains ambiguous. This study aims to characterise the microbiomechanical properties of the meniscus after degeneration in a rabbit anterior cruciate ligament transection (ACLT) model and to analyse the corresponding histology at the macroscale and chemical composition. MethodsTwenty New Zealand white rabbits were used. Menisci were collected from the knee joints 4 and 8 weeks after the ACLT and from those of the corresponding control groups. The central portions of both medial and lateral menisci were investigated using atomic force microscopy, histological study, and an energy-dispersive spectrometer. The evaluation was conducted regionally within the inner, middle, and outer sites from the top layer (facing the femoral surface) to the bottom layer (facing the tibial surface) in both the lateral and medial menisci to obtain the site-dependent properties. ResultsAt 4 weeks after surgery, the dynamic elastic modulus at the microlevel increased significantly at both the top and bottom layers compared with the intact meniscus (P ​= ​0.021). At 8 weeks after surgery, the stiffening occurred in all regions (P ​= ​0.030). The medial meniscus showed greater change than the lateral meniscus. All these microbiomechanical alterations occurred before the histological findings at the macroscale. ConclusionThe microbiomechanical properties in the meniscus changed significantly after ACLT and were site dependent. Their alterations occurred before the histological changes of degeneration were observed. The Translational Potential of this ArticleThe results of our study indicated that degeneration promoted meniscus stiffening. Thus, they provide a better understanding of the disease process affecting the meniscus. Our results might be beneficial to understand how mechanical forces distribute throughout the healthy and pathologic joint. They indicate the possibility of early diagnosis using a minimally invasive arthroscopic tool, as well as they might guide treatment to the healthy and pathologic meniscus and joint.

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.

Inhibitory effect of lenvatinib, a multiple receptor tyrosine kinase inhibitor, on osteoarthritis

Purpose: With the initial damage to articular cartilage due to aging or trauma as its origin, osteoarthritis (OA) progresses towards a disease state causing cartilage degeneration, synovitis, and osteophyte formation. The angiogenic factor vascular endothelial growth factor (VEGF) is involved in synovitis and angiogenesis in cartilage tissue, and in recent studies, anti-VEGF therapies have been reported to suppress synovitis and OA. In this study, we investigated the effects of Lenvatinib, an angiogenesis inhibitor targeting VEGFR/FGFR, on synovitis and osteophyte formation in a rabbit model. Methods: Anterior cruciate ligament transection (ACLT) was performed on one knee of each rabbit to induce traumatic OA and to allow the evaluation of pain behavior in terms of weight-bearing asymmetry. Lenvatinib was administered orally from 5 weeks to 8 weeks after ACLT. We classified the rabbits into 4 groups according to the following Lenvatinib dosages: group A=no administration (n=12); group B=0.3 mg/kg/day (n=15); group C=1.0 mg/kg/day (n=14); and group D=3.0 mg/kg/day (n=13). We measured the weight distribution ratio of the damaged versus undamaged limbs using the Linton incapacitance tester (Linton Instrumentation) for pain evaluation at 4, 6, 8, 10, and 12 weeks after ACLT. Rabbits were sacrificed at 12 weeks after ACLT. The femoral condyles were examined macroscopically. We checked and evaluated osteophyte formation using an osteophyte formation score developed by Tibesku. Histological sections of the synovium were stained for hematoxylin and eosin and evaluated using a synovitis score developed by Kreen. In addition, adverse events including body weight gain rate before and after administration of Lenvatinib were evaluated. Results: The weight distribution ratio at 12 weeks postoperatively was 38.9 ± 5.2% in group A, 47.3 ± 3.4% in group B, 47.4±3.0% in group C, and 42.8±5.2% in group D, and significant differences were observed between A and B (P <0.01) and between A and C (P <0.01). Osteophyte formation score was 2.1±0.5 in group A, 1.2±1.1 in group B, 1.2±0.7 in group C, and 1.0±0.6 in group D, and significant differences were observed between A and B (P <0.05), A and C (P <0.05), and A and D (P<0.01). The synovitis score was 4.3±1.4 in group A, 2.6±0.9 in group B, 2.7±1.0 in group C, and 2.7±1.1 in group D, and significant differences were observed between A and B (P <0.05), A and C (P <0.05), and A and D (P<0.05). There was no significant difference in body weight gain rate between the non-administered group and the administered groups, and no adverse events were detected. Conclusions: In this study, our results suggest that oral administration of Lenvatinib may inhibit synovitis and reduce pain in a rabbit ACLT model. We previously reported that both intravenous and intra-articular administrations of bevacizumab, an anti-VEGF antibody, inhibit OA in a similar model. Lenvatinib is a safe orally administrable VEGF inhibitor which is easier to administer than other VEGF inhibitors and has the potential to be applied clinically. It may also be a more effective angiogenesis inhibitor because of its higher selectivity towards VEGFR and FGFR than other angiogenesis inhibitors, as existing VEGF inhibitors are known to enhance FGF-2 production by the inhibition of VEGF and consequently enhance VEGFR signaling.View Large Image Figure ViewerDownload Hi-res image Download (PPT)

Activation of a chondrocyte volume-sensitive Cl− conductance prior to macroscopic cartilage lesion formation in the rabbit knee anterior cruciate ligament transection osteoarthritis model

SummaryObjectiveThe anterior cruciate ligament transection (ACLT) rabbit osteoarthritis (OA) model confers permanent knee instability and induces joint degeneration. The degeneration process is complex, but includes chondrocyte apoptosis and OA-like loss of cartilage integrity. Previously, we reported that activation of a volume-sensitive Cl− current (ICl,vol) can mediate cell shrinkage and apoptosis in rabbit articular chondrocytes. Our objective was therefore to investigate whether ICl,vol was activated in the early stages of the rabbit ACLT OA model.DesignAdult Rabbits underwent unilateral ACLT and contralateral arthrotomy (sham) surgery. Rabbits were euthanized at 2 or 4 weeks. Samples were analyzed histologically and with assays of cell volume, apoptosis and electrophysiological characterization of ICl,vol.ResultsAt 2 and 4 weeks post ACLT cartilage appeared histologically normal, nevertheless cell swelling and caspase 3/7 activity were both significantly increased compared to sham controls. In cell-volume experiments, exposure of chondrocytes to hypotonic solution led to a greater increase in cell size in ACLT compared to controls. Caspase-3/7 activity, an indicator of apoptosis, was elevated in both ACLT 2wk and 4wk. Whole-cell currents were recorded with patch clamp of chondrocytes in iso-osmotic and hypo-osmotic external solutions under conditions where Na+, K+ and Ca2+ currents were minimized. ACLT treatment resulted in a large increase in hypotonic-activated chloride conductance.ConclusionChanges in chondrocyte ion channels take place prior to the onset of apparent cartilage loss in the ACLT rabbit model of OA. Further studies are needed to investigate if pharmacological inhibition of ICl,vol decreases progression of OA in animal models.

The paracrine effect of adipose-derived stem cells inhibits osteoarthritis progression.

BackgroundThis study aimed to determine whether intra-articularly injected adipose-derived stem cells (ADSCs) inhibited articular cartilage degeneration during osteoarthritis (OA) development in a rabbit anterior cruciate ligament transection (ACLT) model. The paracrine effects of ADSCs on chondrocytes were investigated using a co-culture system.MethodsACLT was performed on both knee joints of 12 rabbits. ADSCs were isolated from the subcutaneous adipose tissue. ADSCs with hyaluronic acid were intra-articularly injected into the left knee, and hyaluronic acid was injected into the right knee. The knees were compared macroscopically, histologically, and immunohistochemically at 8 and 12 weeks. In addition, cell viability was determined using co-culture system of ADSCs and chondrocytes.ResultsMacroscopically, osteoarthritis progression was milder in the ADSC-treated knees than in the control knees 8 weeks after ACLT. Histologically, control knees showed obvious erosions in both the medial and lateral condyles at 8 weeks, while cartilage was predominantly retained in the ADSC-treated knees. At 12 weeks, the ADSC-treated knees showed a slight suppression of cartilage degeneration, unlike the control knees.Immunohistochemically, MMP-13 expression was less in the ADSC-treated cartilage than in the control knees. The cell viability of chondrocytes co-cultured with ADSCs was higher than that of chondrocytes cultured alone. TNF-alpha-induced apoptotic stimulation was similar between the two groups.ConclusionsIntra-articularly injected ADSCs inhibited cartilage degeneration progression by homing to the synovium and secreting a liquid factor having chondro-protective effects such as chondrocyte proliferation and cartilage matrix protection.

A review on current animal models of osteoarthritis research; paving the way for the development of novel disease-modifying anti-osteoarthritic agents

Purpose: Osteoarthritis (OA) is one of the most prevalent chronic conditions worldwide. Pathogenesis of idiopathic OA has not yet been fully understood. The time course of disease onset is slow and disease progression is extremely variable. Therefore, the development of OA is difficult to study in humans, and animal models seem to be invaluable research tools that enable the researchers not only to further their understanding of disease pathogenesis but also to pave the way for the development of novel therapeutic agents. The present study aims to review available OA animal models and induction mechanisms. Advantages and disadvantages of the species commonly used and OA induction methods will also be discussed in detail. Methods: The researchers reviewed available OA animal models and induction mechanisms including intra-articular injections of enzymes, and chemicals as well as surgical inductions of OA. Literature searches were conducted on electronic databases, and the following MeSH terms were used: Osteoarthritis animal models, Osteoarthritis Induction Mechanisms, Papain, Collagenase, Hyaluronidase, Monosodium Iodoacetate, Anterior Cruciate Ligament Transection (ACLT), and Meniscectomy. Results: Enzymatically/ chemically induced OA models have found to exhibit various effects on joint physiology. Papain and Monosodium Iodoacetate have found to specifically alter chondrocyte metabolism and induce significant chondrocyte apoptosis and joint inflammation; whereas, collagenase-induced models showed histological damages to ligaments and tendons. However, some studies were skeptical regarding the use of Monosodium Iodoacetate induction method for pathophysiological studies of human OA. Cartilage degeneration in collagenase-induced models is thought to be associated with the direct ingestion of collagen in cartilage and the inflammatory reaction in the joint tissue. However, studies suggested that genetic differences in the composition of proteoglycans and arrangement of collagen fibers may influence the sensitivity of the cartilage. Surgically-induced animal models (ACLT, and Meniscectomy) were found to be one of the most widely used OA induction methods. In comparison with other OA induction methods, surgically-induced OA showed a higher disease onset and disease severity. Most studies reported sequential events of cartilage degeneration, subchondral sclerosis, and osteophyte formation in both rat and rabbit ACLT and Meniscectomy models. The underlying initiating mechanism in surgically-induced OA models was found to be the altered mechanical loading which is the same initiating cause in human secondary OA. Conclusions: All animal models and induction mechanisms result in morphological changes that resemble human pathology in some stages of the disease. Differences are particularly in time of onset of the disease and the speed of disease progression. Moreover, molecular mechanisms of joint structural damage are distinct in different animal models and induction mechanisms. Each animal model and induction mechanism used in OA research has its own advantages and disadvantages. Thus, choosing the best model which mimics human etiology and addresses the aim of the study seems to be a crucial step in conducting research in this area. Hopefully, the present study provides a framework for choosing the best animal model and paves the way for the development of novel disease-modifying agents.

The biophysical mechanisms of altered hyaluronan concentration in synovial fluid after anterior cruciate ligament transection

The residence time of hyaluronan (HA) in knee joint synovial fluid (SF) was investigated using a rabbit anterior cruciate ligament transection (ACLT) model. The aims of this study were to assess, at 7 and 28 days after surgery, the 1) HA concentration and molecular mass (M(r) ) distribution in the SF, 2) endogenous replenishment of HA after saline washout, 3) HA residence times in the SF, and 4) synovium and subsynovium cellularity of the knee joints of rabbits subjected to ACLT, compared to sham-operated and nonoperated control joints. Adult NZW rabbits underwent ACLT or sham surgery on one hind limb, while each contralateral limb was the nonoperated control. On day 7 or 28 after surgery, the joints were aspirated for SF, lavaged with saline, and injected with saline or polydisperse HA, and samples were obtained for analysis at set time points up to 8 hours after injection. Joint fluid samples were analyzed for the concentration and M(r) distribution of HA to calculate the HA residence time constant. Analysis of HA concentrations and M(r) distributions showed 1) loss of high-M(r) HA in the SF on day 7 and a shift toward a lower-M(r) distribution on day 28, 2) endogenous replenishment of high-M(r) HA after washout, and 3) M(r) -dependent loss of HA from the knee joints after ACLT, particularly on day 7 postsurgery. The HA residence time decreased with decreasing HA M(r) (residence time ∼27 hours with an M(r) load of 7,000-2,500 kd, to ∼7 hours with an M(r) load of 250-50 kd). HA residence time also decreased (by ∼70%) in the knee joints on day 7 after ACLT. The subsynovium of the joints subjected to ACLT displayed increased cellularity and neovascularization on days 7 and 28 postsurgery. The residence time of HA in the SF is transiently decreased after ACLT, suggesting that a biophysical transport mechanism is responsible for the altered composition of the SF after joint injury or during inflammation.

Evaluation of the Chondroprotective Effects of Glucosamine and Fish Collagen Peptide on a Rabbit ACLT Model Using Serum Biomarkers

The aim of this study was to investigate the correlations of severity of osteoarthritis (OA) and serum biomarkers including keratan sulfate (KS), hyaluronic acid (HA) and chondroitin sulfate (CS) 846 epitope. We also investigated the effect of glucosamine and fish collagen peptide (FCP) on OA. OA was induced in 12 rabbits (12 weeks of age) by anterior cruciate ligament transection (ACLT). After the surgery, the rabbits were orally administered FCP (F group), glucosamine (G group) or FCP and glucosamine (FG group) for 4 weeks. The control group was provided water ad libitum (C group). Blood was collected before surgery (pre-ACLT) and before euthanasia (post-ACLT) for serum marker measurement. Biomarker levels were measured by using commercial kits. We evaluated OA severity both macroscopically and histologically. Macroscopic evaluation showed mildly eroded condylar surfaces in the C group. Histological findings were significantly different from the FG and other groups. There were no significant differences between each group at post-ACLT in terms of serum KS, HA and CS 846. Histological assessment and serum biomarker measurements performed at post-ACLT showed a significant correlation between HA concentration and OA severity. Variations in the CS 846 concentration at pre-ACLT and post-ACLT were significantly correlated with OA severity. Administration of glucosamine and FCP had chondroprotective effects in the ACLT model. Serum biomarker concentrations were significantly correlated with cartilage injury. Serum biomarker measurement would be useful for monitoring articular cartilage damage in the clinical setting.

Correlation of Plasma Amino Acid Concentrations and Chondroprotective Effects of Glucosamine and Fish Collagen Peptide on the Development of Osteoarthritis

We investigated the correlation of plasma amino acid concentrations and the effects of glucosamine and fish collagen peptide (FCP) on osteoarthritis (OA). OA was induced according to the rabbit anterior cruciate ligament transection (ACLT) model. After surgery, the rabbits were orally administered FCP (F group), glucosamine (G group) or both (FG group) for 4 weeks. The control group (C group) was not administered anything. Blood was collected before surgery (pre-ACLT) and before euthanasia (post-ACLT). Changes in the alanine, threonine and methionine concentrations were significant between pre- and post-ACLT. The correlation between the histological assessment and arginine concentration post-ACLT was significant. These findings indicate that measurement of plasma amino acids is useful for evaluation of the efficacy of intervention for OA.