Experimental Model Of Osteoarthritis Research Articles

MicroRNA-17-5p contributes to osteoarthritis progression by binding p62/SQSTM1.

Autophagy has been reported to be widely involved in the pathogenesis of osteoarthritis (OA). Increasing evidence suggested the important role of microRNAs (miRs) in the progression of OA. However, the functional role of miR-17-5p in OA development has remained to be fully elucidated. First, a mouse model of OA was established and the relative level of miR-17-5p was determined using PCR. Safranin O-fast green staining was applied to determine cartilage degeneration. TargetScan software and a dual luciferase reporter assay were applied to determine potential target genes of miR-17-5P. Autophagy measurement was performed using green fluorescent protein-microtubule-associated protein 1 light chain 3 (LC3) dot analysis. The results demonstrated that the relative expression of miR-17-5p was significantly decreased in OA model mice. In addition, the level of miR-17-5p was decreased in SW1353 human chondrosarcoma cells treated with interleukin-1β. Furthermore, autophagy was found to be suppressed in the knee joints of experimental OA model mice. The dual luciferase reporter assay confirmed that p62/sequestosome 1 was a target gene of miR-17-5p. Of note, miR-17-5p inhibitor-induced reduction of LC3 dots was markedly reversed by knockdown of p62 in SW1353 cells. In conclusion, decreased miR-17-5p expression in chondrocytes induced autophagy mainly through suppressing the expression of p62, thereby contributing to OA progression.

Chondroitin sulfate and glucosamine sulfate associated to photobiomodulation prevents degenerative morphological changes in an experimental model of osteoarthritis in rats.

The aim of this study was to compare the effects of combined treatment with chondroitin sulfate and glucosamine sulfate (CS/Gl) and photobiomodulation (PBM) on the degenerative process related to osteoarthritis (OA) in the articular cartilage in rats. Forty male Wistar rats were randomly divided into four groups: OA control group (CG); OA animals submitted to PBM treatment (PBM); OA animals submitted to CS/Gl treatment (CS/Gl); OA submitted to CS/GS associated with PBM treatments (GS/Gl + PBM). The CS/Gl started 48h after the surgery, and they were performed for 29 consecutive days. Moreover, PBM was performed after the CS/Gl administration on the left joint. Morphological characteristics and immunoexpression of interleukin 10 (IL-10) and 1 beta (IL-1β) and collagen type II (Col II) of the articular cartilage were evaluated. The results showed that all treated groups (CS/Gl and PBM) presented attenuation signs of degenerative process (measured by histopathological analysis) and lower density chondrocytes [PBM (p = 0.0017); CS/Gl (p = 0.0153) and CS/Gl + PBM (p = 0.002)]. Additionally, CS/Gl [associated (p = 0.0089) or not with PBM (p = 0.0059)] showed significative lower values for OARSI grade evaluation. Furthermore, CS/GS + PBM decreased IL-1β protein expression (p = 0.0359) and increased IL-10 (p = 0.028) and Col II imunoexpression (p = 0.0204) compared to CG. This study showed that CS/Gl associated with PBM was effective in modulating inflammatory process and preventing the articular tissue degradation in the knees OA rats.

Diagnostic value of combined serum marker changes and quantitative MRI evaluation of cartilage volume of tibial plateau in a surgically-induced osteoarthritis dog model.

ObjectiveTo evaluate the combined diagnostic value of two serum osteoarthritis (OA) markers and quantitative magnetic resonance imaging (MRI) evaluation of the cartilage volume of the tibial plateau in a canine model of experimental OA.MethodsA total of 18 male Beagle dogs were used in this longitudinal study. OA was surgically induced via anterior cruciate ligament transection (ACLT) of the right knee in 10 dogs. The remaining eight dogs formed the sham operation control group and underwent the same procedure without ACLT. At various times after surgery, enzyme-linked immunosorbent assay was used to measure serum C-telopeptide of type II collagen (CTX-II) and type X collagen (ColX) levels. Quantitative evaluation of the tibial plateau volume was undertaken using MRI and ImageJ software.ResultsThe serum CTX-II levels were significantly higher in the OA group at weeks 8, 12 and 16 after surgery, but not at week 4, compared with the control group. The serum ColX levels in the OA group were significantly higher than in the control group at weeks 8 and 12. The tibial plateau cartilage volumes in the OA group were significantly lower than in the control group at weeks 8 and 16.ConclusionSerum CTX-II and ColX levels combined with quantitative MRI evaluation of the tibial plateau cartilage volume in a canine model of OA demonstrated the potential to detect and monitor OA progression.

Mechanisms of Osteoarthritic Pain. Studies in Humans and Experimental Models.

Pain due to osteoarthritis (OA) is one of the most frequent causes of chronic pain. However, the mechanisms of OA pain are poorly understood. This review addresses the mechanisms which are thought to be involved in OA pain, derived from studies on pain mechanisms in humans and in experimental models of OA. Three areas will be considered, namely local processes in the joint associated with OA pain, neuronal mechanisms involved in OA pain, and general factors which influence OA pain. Except the cartilage all structures of the joints are innervated by nociceptors. Although the hallmark of OA is the degradation of the cartilage, OA joints show multiple structural alterations of cartilage, bone and synovial tissue. In particular synovitis and bone marrow lesions have been proposed to determine OA pain whereas the contribution of the other pathologies to pain generation has been studied less. Concerning the peripheral neuronal mechanisms of OA pain, peripheral nociceptive sensitization was shown, and neuropathic mechanisms may be involved at some stages. Structural changes of joint innervation such as local loss and/or sprouting of nerve fibers were shown. In addition, central sensitization, reduction of descending inhibition, descending excitation and cortical atrophies were observed in OA. The combination of different neuronal mechanisms may define the particular pain phenotype in an OA patient. Among mediators involved in OA pain, nerve growth factor (NGF) is in the focus because antibodies against NGF significantly reduce OA pain. Several studies show that neutralization of interleukin-1β and TNF may reduce OA pain. Many patients with OA exhibit comorbidities such as obesity, low grade systemic inflammation and diabetes mellitus. These comorbidities can significantly influence the course of OA, and pain research just began to study the significance of such factors in pain generation. In addition, psychologic and socioeconomic factors may aggravate OA pain, and in some cases genetic factors influencing OA pain were found. Considering the local factors in the joint, the neuronal processes and the comorbidities, a better definition of OA pain phenotypes may become possible. Studies are under way in order to improve OA and OA pain monitoring.

Isoimperatorin ameliorates osteoarthritis by downregulating the mammalian target of rapamycin C1 signaling pathway.

Osteoarthritis (OA) is the most common disease of the joints, and is characterized by the breakdown of cartilage and degradation of the extracellular matrix. OA causes a high level of patient suffering and incurs large societal costs; however, the current strategies for treating OA are restricted due to limited understanding of the underlying molecular and cellular mechanisms. In the present study, the beneficial effects of isoimperatorin (Iso) were investigated using an experimental mouse model of OA, and its mechanism of action on primary chondrocytes was elucidated. Destabilization of the medial meniscus was performed on 8‑week‑old male mice to induce OA in the knees. Iso (500mg/g/day) was intragastrically administered for 4weeks. Degeneration of articular cartilage was assessed by histology using the Osteoarthritis Research Society International scoring system. The expression of matrix metalloproteinase (MMP)13, Runt‑related transcription factor (Runx)2, type X collagen (Col X) and vascular endothelial growth factor (VEGF) in the knee joints was examined by immunohistochemistry. Invitro, murine primary chondrocytes were treated with various concentrations of Iso, followed by 10ng/ml interleukin‑1. The mRNA expression levels of MMP13, Runx2, Col X and VEGF were determined by reverse transcription‑quantitative polymerase chain reaction. The levels of autophagy and mammalian target of rapamycin (mTOR) signaling were determined by western blotting. Iso significantly ameliorated the severity of articular cartilage degradation in mice with experimental OA. The expression levels of MMP13, Runx2, Col X and VEGF were reduced in Iso‑treated mice. In murine primary chondrocytes, Iso also reduced MMP13, Runx2, Col X and VEGF expression, and activated autophagy by downregulating the mTOR complex1 (mTORC1) signaling pathway. Therefore, the results of the present study demonstrated that Iso ameliorates OA‑induced pathological alterations by delaying chondrocyte deterioration, activating autophagy and inhibiting mTORC1, which suggests that Iso may have therapeutic potential for attenuating articular cartilage degradation and treating OA.

Osteoarthritis pain: What are we learning from animal models?

All experimental models of osteoarthritis (OA)-like joint damage are accompanied by behaviors indicative of pain. In experimental knee OA, evoked pain responses to exogenously applied stimuli suggest that animals become sensitized to mechanical stimuli. Neurobiological techniques including electrophysiology and invivo calcium imaging confirm that joint damage is associated with mechanical stimuli through peripheral sensitization. Several mediators present in the OA joint can cause peripheral sensitization, most notably the neurotrophin nerve growth factor (NGF). Furthermore, experimental OA is associated with neuroinflammation in the peripheral nervous system and central nervous system (CNS), including macrophage infiltration of the dorsal root ganglia and microglial activation in the spinal cord. Increasingly, researchers are employing models that are slowly progressive, and this approach has revealed that distinct pain mechanisms operate in a time-dependent manner, which may have important translational significance. While the study of pain in experimental OA is rapidly evolving, with the application of increasingly sophisticated techniques to assess pain and unravel the neurobiology of its genesis, important gaps and limitations in our current approaches exist, which our research community needs to address.

Osteoartróza jako součást metabolického syndromu?

Osteoarthritis (OA) is the most frequent joint disease, whereas etiopathogenesis of OA is not entirely clarified. It is a heterogeneous disorder and genetic as well as biomechanical, endocrine and inflammatory effects may be involved in its origin. The author examines the problems concerning relationships between the metabolic syndrome and OA, and states that the prevalence of metabolic syndrome in patients with OA is higher than in those without OA (59 % vs 23 %). It remains problematic that one of the main components of metabolic syndrome is obesity which in itself is a risk factor for arthrosis development in the weight-bearing joints, not for OA in the hands. After adjustment for BMI the relationships between the metabolic syndrome and OA are less expressed. Over the last decade evidence has been gained about adipose tissue being the source of numerous cytokines and adipokines which may cause inflammation of low-activity synovial tissue, sometimes also called "meta-inflammation." The most data was gathered on leptin, resistin, adiponectin and visfatin. Mostly there were serum levels of these adiponectins assessed and the results were sometimes inconsistent. Two studies have been published this year presenting a histological and immunohistochemical evaluation of the fat stored in 2 tissues right in the joints of patients with metabolic syndrome and OA, and the fat on an experimental OA model, concluding that the secretion activity of the potentially pro-inflammatory adipokines through adipocytes may differ in the synovial membrane, infrapatellar fat body and in abdominal fat. It is evident that the components of metabolic syndrome and OA can share a common pathological process which is an "adipose tissue associated inflammation." The changed secretion profile of pro-inflammatory adipokines is present in obese individuals, an older population and postmenopausal women, the populations at high risk for both metabolic syndrome and OA. Significant mechanical loads may stimulate OA of the knee joints in obese patients, however not of the hands, and further differences between arthrosis of the knee joints and the hands. The adipose tissue induced inflammation is the common pathological mechanism for both metabolic syndrome and OA and it may account for some of the variations.Key words: adiponectins - metabolic syndrome - osteoarthritis.

Chitosan in viscosupplementation: in vivo effect on rabbit subchondral bone

BackgroundTo investigate the effect of intra-articular injection of Chitosan (Cs) added to hyaluronic acid (HA) on subchondral bone during osteoarthritis (OA), microarchitectural parameters and mineral density were measured in a rabbit model of early OA. A novel hybrid hydrogel adding reacetylated Cs of fungal origin to HA was compared to high molecular weight HA commercial formulation.MethodEighteen rabbits underwent unilateral anterior cruciate ligament transection (ACLT) and were divided into three groups (Saline-group, HA-group and Hybrid-group) depending on the intra-articular injection compound. Eight contralateral knees were used as non-operated controls (Contralateral-group). Micro-computed tomography was performed six weeks post-ACLT to study subchondral bone microarchitectural parameters and mineral density at an early stage of OA development.ResultsCartilage thickness mean value was reduced only in Saline-group compared to Contralateral-group. When the Hybrid-group was compared to Saline-group, subchondral bone microarchitectural parameters (trabecular thickness and trabecular bone volume fraction) were significantly changed; subchondral bone plate and trabecular bone mineral densities (bone mineral density and tissue mineral density) were reduced. When the Hybrid-group was compared to HA-group, subchondral bone microarchitectural parameters (subchondral plate thickness and trabecular thickness) and trabecular bone mineral densities (bone mineral density and tissue mineral density) were significantly decreased.ConclusionConclusion: Compared to HA alone, the novel hybrid hydrogel, constituted of Cs added to HA, enhanced microarchitectural parameters and mineral density changes, leading to subchondral bone loss in a rabbit model of early experimental OA.

Evaluation of the chondroprotective action of N-acetylglucosamine in a rat experimental osteoarthritis model.

It has been demonstrated that oral administration of N-acetylglucosamine (GlcNAc) alleviates the symptoms of osteoarthritis (OA). The aim of the present study was to elucidate the molecular mechanisms for the chondroprotective action of GlcNAc in OA. Biomarkers for type II collagen degradation and synthesis were evaluated, as were histopathological changes, using a rat anterior cruciate ligament transection (ACLT)-induced OA model. Changes in the expression of genes in the cartilage were assessed via DNA microarray and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The results indicated that ACLT induced histopathological changes of articular cartilage, whereas oral administration of GlcNAc (1,000 mg/kg/day for 28 days) significantly suppressed these changes. Additionally, GlcNAc significantly decreased levels of a type II collagen degradation marker in sera compared with that in the ACLT group, although there were no significant changes in the levels of a type II collagen synthesis marker. Furthermore, DNA microarray and reverse transcription-quantitative polymerase chain reaction results demonstrated that GlcNAc treatment downregulated the expression of periostin, which is likely involved in the degradation of cartilage, whereas GlcNAc upregulated the expression of lipocalin 2, which is involved in the regulation of chondrocyte proliferation and differentiation. In conclusion, the results of the present study suggest that GlcNAc is able to suppress the histopathological changes induced by OA and exhibits a chondroprotective action by inhibiting type II collagen degradation in the articular cartilage, possibly via modulation of the expression of inflammatory and chondroprotective molecules, including periostin and lipocalin 2.

WISP1/CCN4 aggravates cartilage degeneration in experimental osteoarthritis.

Increased Wisp1 expression was previously reported in experimental and human osteoarthritis (OA). Moreover, adenoviral overexpression of Wisp1 in naïve mouse knee joints resulted in early OA-like cartilage lesions. Here, we determined how the matricellular protein WISP1 is involved in the pathology that occurs in the complex osteoarthritic environment with aging and experimental OA in wild type (WT) and Wisp1-/- mice. WT and Wisp1-/- mice were aged or experimental OA was induced with intraarticular collagenase injection, destabilization of the medial meniscus (DMM) or anterior cruciate ligament transection (ACLT). Joint pathology was assessed using histology and microCT. Protease expression was evaluated with qRT-PCR and activity was determined by immunohistochemical staining of the aggrecan neoepitope NITEGE. Protease expression in human end-stage OA synovial tissue was determined with qRT-PCR after stimulation with WISP1. With aging, spontaneous cartilage degeneration in Wisp1-/- was not decreased compared to their WT controls. However, we observed significantly decreased cartilage degeneration in Wisp1-/- mice after induction of three independent experimental OA models. While the degree of osteophyte formation was comparable between WT and Wisp1-/- mice, increased cortical thickness and reduced trabecular spacing was observed in Wisp1-/- mice. In addition, we observed decreased MMP3/9 and ADAMTS4/5 expression in Wisp1-/- mice, which was accompanied by decreased levels of NITEGE. In line with this, stimulation of human OA synovium with WISP1 increased the expression of various proteases. WISP1 plays an aggravating role in the development of post-traumatic experimental OA.

Viscossuplementação intra‐articular de ácidos hialurônicos em modelo experimental de osteoartrite

ObjectiveTo analyze, from the immunohistochemical perspective, the effects of hyaluronic acid of different molecular weights in an experimental model of osteoarthritis in rabbits. MethodsForty‐four male California rabbits were randomly assigned to three different groups (PR, S, and P) and submitted to the resection of the anterior cruciate ligament of the right knee. Three weeks after the surgical procedure, three intra‐articular weekly injections were carried out with low‐molecular‐weight native hyaluronic acid (Hyalgan®) to PR group, high molecular weight branched chain hyaluronic acid (Synvisc®) to group S, and saline solution 0.9% to group P. All animals were sacrificed 12 weeks after the surgical procedure, and the tibial plateaus of the infiltrated knees were then dissected. Histological sections of cartilage from the tibial plateau support areas were stained with immunohistochemical markers in order to investigate the amount of metalloproteases (MMPs 3 and 13) and their inhibitors (TIMPs 1 and 3). The staining intensity was quantified on a Zeiss Imager.Z2 Metasystems microscope and analyzed by Metafer4 Msearch software. ResultsThe chondroprotective effect of the hyaluronic acids used in the study was demonstrated when compared to the control group. However, the comparison between them presented no significant statistical difference regarding chondroprotection. ConclusionThe injection of saline solution demonstrated signs of OA development, while adding native hyaluronic acid of low molecular weight (Hyalgan®) and hyaluronic acid of high molecular weight (Synvisc®) protected the articular cartilage in this model of OA.

Local intra-articular injection of resveratrol delays cartilage degeneration in C57BL/6 mice by inducing autophagy via AMPK/mTOR pathway

Autophagy is an essential cellular homeostasis mechanism that was found to be compromised in aging and osteoarthritis (OA) cartilage. Previous studies showed that resveratrol can effectively regulate autophagy in other cells. The purpose of this study was to determine whether the chondroprotective effect of resveratrol was related to chondrocyte autophagy and to elucidate underlying mechanisms. OA model was induced by destabilization of the medial meniscus (DMM) in 10-week-old male mice. OA mice were treated with resveratrol with/without 3-MA for 8 weeks beginning 4 weeks after surgery. The local intra-articular injection of resveratrol delayed articular cartilage degradation in DMM-induced OA by OARSI scoring systems and Safranin O-fast green. Resveratrol treatment increased Unc-51-like kinase1, Beclin1, microtubule-associated protein light chain 3, hypoxia inducible factor-1α, phosphorylated AMPK, collagen-2A1, Aggrecan expressions, but decreased hypoxia inducible factor-2α, phosphorylated mTOR, matrix metalloproteinases13 and a disintegrin and metalloproteinase with thrombospondin motifs 5 expressions. The effects of resveratrol were obviously blunted by 3-MA except HIF and AMPK. These findings indicate that resveratrol intra-articular injection delayed articular cartilage degeneration and promoted chondrocyte autophagy in an experimental model of surgical DMM-induced OA, in part via balancing HIF-1α and HIF-2α expressions and thereby regulating AMPK/mTOR signaling pathway.

Culture-expanded allogenic adipose tissue-derived stem cells attenuate cartilage degeneration in an experimental rat osteoarthritis model.

Mesenchymal stem cell (MSC)-based cell therapy is a promising avenue for osteoarthritis (OA) treatment. In the present study, we evaluated the efficacy of intra-articular injections of culture-expanded allogenic adipose tissue-derived stem cells (ADSCs) for the treatment of anterior cruciate ligament transection (ACLT) induced rat OA model. The paracrine effects of major histocompatibility complex (MHC)-unmatched ADSCs on chondrocytes were investigated in vitro. Rats were divided into an OA group that underwent ACLT surgery and a sham-operated group that did not undergo ACLT surgery. Four weeks after surgery mild OA was induced in the OA group. Subsequently, the OA rats were randomly divided into ADSC and control groups. A single dose of 1 × 106 ADSCs suspended in 60 μL phosphate-buffered saline (PBS) was intra-articularly injected into the rats of the ADSC group. The control group received only 60 μL PBS. OA progression was evaluated macroscopically and histologically at 8 and 12 weeks after surgery. ADSC treatment did not cause any adverse local or systemic reactions. The degeneration of articular cartilage was significantly weaker in the ADSC group compared to that in the control group at both 8 and 12 weeks. Chondrocytes were co-cultured with MHC-unmatched ADSCs in trans-wells to assess the paracrine effects of ADSCs on chondrocytes. Co-culture with ADSCs counteracted the IL-1β-induced mRNA upregulation of the extracellular matrix-degrading enzymes MMP-3 and MMP-13 and the pro-inflammatory cytokines TNF-α and IL-6 in chondrocytes. Importantly, ADSCs increased the expression of the anti-inflammatory cytokine IL-10 in chondrocytes. The results of this study indicated that the intra-articular injection of culture-expanded allogenic ADSCs attenuated cartilage degeneration in an experimental rat OA model without inducing any adverse reactions. MHC-unmatched ADSCs protected chondrocytes from inflammatory factor-induced damage. The paracrine effects of ADSCs on OA chondrocytes are at least part of the mechanism by which ADSCs exert their therapeutic activity.

Increased expression of CCN4/WISP1 in osteoarthritic articular cartilage is epigenetically regulated and disrupts cartilage homeostasis

Background Previously, we described increased expression of Wnt-1-induced signaling protein 1 (Wisp1) in murine synovium and cartilage after induction of two experimental osteoarthritis (OA) models. Wisp1 is a downstream target of the canonical Wnt signaling pathway that has been shown to play a pivotal role in the etiopathology of OA. In line with this, we have observed increased breakdown of the articular cartilage after overexpression of Wisp1 in naive mouse knee joints, whereas Wisp1-/- mice revealed decreased cartilage degeneration in three independent experimental OA models compared to wild type controls. Together, these data indicate a direct correlation between Wisp1 and OA in mice. Objectives In the current study we set out to characterize the relation between expression of WISP1 and human OA. Methods Articular cartilage from preserved and degenerated OA areas was collected from 39 Caucasian end-stage OA patients. Cartilage from non-OA-diagnosed individuals was collected after femoral neck fractures. Cartilage degeneration was classified according to the Mankin scoring system. DNA was isolated to determine correlation between WISP1 expression and methylation profiles using Generalized Linear Mixed Model (GLMM). RNA expression levels were determined with microarray analysis and RNA sequencing. Immunohistochemical staining was used to determine WISP1 protein expression. Recombinant WISP1 was added to human chondrocyte microparticles, and cartilage extracellular matrix deposition was determined by measuring cartilage microparticle size and Safranin O/Fast Green staining. Results We observed increased WISP1 expression in cartilage of OA patients compared to non-OA-diagnosed controls. Moreover, within OA patients, both WISP1 mRNA and protein expression were significantly increased in OA-affected cartilage compared to preserved regions of the same joint, and WISP1 expression significantly correlated with Mankin score. Interestingly, we found that positional CpG dinucleotides were hypomethylated in cartilage of OA-affected areas as compared to unaffected areas from the same joint, which correlated with increased RNA expression as determined with both microarray analysis and RNA sequencing analysis. Of note, methylation levels of a CpG affecting WISP1 transcription were found to highly significantly correlate to a single nucleotide polymorphism (SNP) at the WISP1 locus. Next, to investigate effects of increased WISP1 levels on chondrocyte microparticles, we added human recombinant WISP1. This resulted in a significantly decreased deposition of cartilage extracellular matrix as reflected by decreased microparticle circumference, and a strongly decreased proteoglycan content, suggesting that increased WISP1 levels are detrimental to cartilage. Conclusions The expression of WISP1, both RNA and protein, is increased in OA-affected as compared to preserved articular cartilage. This increased expression is inversely correlated with methylation levels of a positional CpG for WISP1, which was found to be under the influence of a SNP at the WISP1 locus. Together, our results suggest that tight regulation of WISP1 expression via methylation is essential to maintain cartilage homeostasis. Disclosure of Interest None declared

Automated assessment of bone changes in cross-sectional micro-CT studies of murine experimental osteoarthritis.

ObjectiveThe degradation of articular cartilage, which characterises osteoarthritis (OA), is usually paired with excessive bone remodelling, including subchondral bone sclerosis, cysts, and osteophyte formation. Experimental models of OA are widely used to investigate pathogenesis, yet few validated methodologies for assessing periarticular bone morphology exist and quantitative measurements are limited by manual segmentation of micro-CT scans. The aim of this work was to chart the temporal changes in periarticular bone in murine OA by novel, automated micro-CT methods.MethodsOA was induced by destabilisation of the medial meniscus (DMM) in 10-week old male mice and disease assessed cross-sectionally from 1- to 20-weeks post-surgery. A novel approach was developed to automatically segment subchondral bone compartments into plate and trabecular bone in micro-CT scans of tibial epiphyses. Osteophyte volume, as assessed by shape differences using 3D image registration, and by measuring total epiphyseal volume was performed.ResultsSignificant linear and volumetric structural modifications in subchondral bone compartments and osteophytes were measured from 4-weeks post-surgery and showed progressive changes at all time points; by 20 weeks, medial subchondral bone plate thickness increased by 160±19.5 μm and the medial osteophyte grew by 0.124±0.028 μm3. Excellent agreement was found when automated measurements were compared with manual assessments.ConclusionOur automated methods for assessing bone changes in murine periarticular bone are rapid, quantitative, and highly accurate, and promise to be a useful tool in future preclinical studies of OA progression and treatment. The current approaches were developed specifically for cross-sectional micro-CT studies but could be applied to longitudinal studies.

Evaluating the Protective Effects and Mechanisms of Diallyl Disulfide on Interlukin-1β-Induced Oxidative Stress and Mitochondrial Apoptotic Signaling Pathways in Cultured Chondrocytes.

The protective effects and mechanisms of DADS on IL-1β-mediated oxidative stress and mitochondrial apoptosis were investigated in C28I2 human chondrocytes. The effect of various concentrations of DADS (1, 5 10, 25, 50, and 100 μM) on C28I2 cell viability was evaluated in different times (2, 4, 8, 16, and 24 h) to obtain the non-cytotoxic concentrations of drug by MTT-assay. The protective effect of non-toxic concentrations of DADS on experimentally induced oxidative stress and apoptosis by IL-1β in C28I2 was evaluated. The effects of DADS on IL-1β-induced intracellular ROS production and lipid peroxidation were detected and the proteins expression of Nrf2, Bax, Bcl-2, caspase-3, total and phosphorylated JNK, and P38 MAPKs were analyzed by Western blotting. The mRNA expression of detoxifying phase II/antioxidant enzymes including heme oxygenase-1, NAD(P)H quinine oxidoreductase, glutathione S-transferase-P1, catalase, superoxide dismutase-1, glutathione peroxidase-1, -3, -4 were evaluated by reverse transcription-polymerase chain reaction. DADS in 1, 5, 10, and 25 μM concentrations had no cytotoxic effect after 24 h. Pretreatment with DADS remarkably increased Nrf2 nuclear translocation as well as the genes expression of detoxifying phase II/antioxidant enzymes and reduced IL-1β-induced elevation of ROS, lipid peroxidation, Bax/Bcl-2 ratio, caspase-3 activation, and JNK and P38 phosphorylation. DADS could considerably reduce IL-1β-induced oxidative stress and consequent mitochondrial apoptosis, as the major mechanisms of chondrocyte cell death in an experimental model of osteoarthritis. It may be considered as natural product in protecting OA-induced cartilage damage in clinical setting. J. Cell. Biochem. 118: 1879-1888, 2017. © 2017 Wiley Periodicals, Inc.

Síntese e caracterização de nanopartículas de ouro conjugadas com curcumina e seus efeitos na osteoartrite experimental induzida

RESUMO: A Osteoartrite (OA) é uma denominação clínica para uma combinação de condições patológicas que envolvem a degeneração progressiva da cartilagem articular e remodelação de osso subcondral. A curcumina, um potente agente anti-inflamatório, têm sido extensivamente estudada, no entanto não oferece boa biodisponibilidade sistêmica. Nanopartículas de ouro (AuNPs) apresentam aplicações potenciais na administração de substâncias terapêuticas aumentando a eficiência do transporte de fármacos. O objetivo deste estudo foi realizar a síntese e caracterização de um sistema conjugando as AuNPs à curcumina e avaliar seu potencial terapêutico em um modelo experimental de OA em camundongos por desestabilização do menisco medial (DMM). As AuNPs foram conjugadas com curcumina e os sistemas foram caracterizados por espectroscopia no UV-VIS, espalhamento de luz dinâmico (DLS) e determinação do potencial zeta. Formou-se 4 grupos de oito animais cada, denominados A, B, C, D que receberam injeção intra-articular de AuNPs, curcumina, AuNP-curcumina e solução fisiológica, respectivamente. Após 7 semanas, a cartilagem da articulação-femoro- tibio-patelar (AFTP) foi avaliada em uma variação de escore de 0 a 24. A conjugação de AuNP-curcumina mostrou boa estabilidade e aplicação terapêutica, mas não diferiu significativamente (P>0,05) dos grupos A e B, no entanto, mostrou menor valor de escore e significância (P<0,001) em relação ao grupo controle. Os resultados deste trabalho mostram a importância do desenvolvimento de novos nanofármacos. Neste caso a conjugação de AuNPs com a curcumina permitiu a obtenção de um nanofármaco com sugestivo potencial para aplicação no tratamento da OA.

Nerve growth factor blockade for the management of osteoarthritis pain: what can we learn from clinical trials and preclinical models?

Anti-nerve growth factor (NGF) antibodies hold tremendous potential for the management of osteoarthritis pain, but clinical trials have revealed serious adverse effects that are incompletely understood. This review discusses clinical trial results along with preclinical studies that have assessed NGF blockade in experimental osteoarthritis, in order to provide insight for future studies. Systematic reviews have revealed that anti-NGF therapy, including tanezumab, is efficacious in improving pain and function, but serious adverse events, including rapidly progressive osteoarthritis and osteonecrosis, resulted in a moratorium on trials that was only recently lifted. Within the past year, preclinical testing has revealed effects of NGF blockade on both pain behaviors and joint structure in experimental models of osteoarthritis. Similar to clinical trial results, these studies in laboratory animals demonstrated analgesic efficacy of NGF blockade. Interestingly, several animal studies have suggested detrimental effects on joint integrity as a result of treatment, particularly when treatment is started early in the disease, when joint damage is mild to moderate. NGF blockade continues to represent a promising new approach for the treatment of osteoarthritis pain, but the actual benefits and risks remain to be fully elucidated. Preclinical models may suggest patient populations that could be best served while limiting side-effects, but future work should further investigate the mechanisms of benefits and unwanted side-effects.

Glial activation in the collagenase model of nociception associated with osteoarthritis

BackgroundExperimental osteoarthritis entails neuropathic-like changes in dorsal root ganglia (DRG) neurons. Since glial activation has emerged as a key player in nociception, being reported in numerous models of neuropathic pain, we aimed at evaluating if glial cell activation may also occur in the DRG and spinal cord of rats with osteoarthritis induced by intra-articular injection of collagenase.MethodsOsteoarthritis was induced by two injections, separated by three days, of 500 U of type II collagenase into the knee joint of rats. Movement-induced nociception was evaluated by the Knee-Bend and CatWalk tests during the following six weeks. Glial fibrillary acidic protein (GFAP) expression in satellite glial cells of the DRG was assessed by immunofluorescence and Western Blot analysis; the pattern of GFAP and activating transcription factor-3 (ATF-3) expression was also compared through double immunofluorescence analysis. GFAP expression in astrocytes and IBA-1 expression in microglia of the L3–L5 spinal cord segments was assessed by immunohistochemistry and Western Blot analysis. The effect of the intrathecal administration of fluorocitrate, an inhibitor of glial activation, on movement-induced nociception was evaluated six weeks after the first collagenase injection.ResultsGFAP expression in satellite glial cells of collagenase-injected animals was significantly increased six weeks after osteoarthritis induction. Double immunofluorescence showed GFAP upregulation in satellite glial cells surrounding ATF-3-positive neurons. In the spinal cord of collagenase-injected animals, an ipsilateral upregulation of GFAP and IBA-1 was also observed. The inhibition of glial activation with fluorocitrate decreased movement- and loading-induced nociception.ConclusionCollagenase-induced knee osteoarthritis leads to the development of nociception associated with movement of the affected joint and to the activation of glial cells in both the DRG and the spinal cord. Inhibition of glial cell activation by fluorocitrate decreases these osteoarthritis-associated nociceptive behaviours. These results suggest that glial cell activation may play a role in the development of chronic pain in this experimental model of osteoarthritis.

Effect of strontium ranelate on pain behavior in an experimental model of osteoarthritis.

Strontium ranelate (SrRan) is a drug usually prescribed to treat osteoporosis, with proven effects of decreasing the risk of fractures and an indication of reducing the progression of osteoarthritis (OA). This study aimed to investigate the effects of SrRan as either a prophylactic or a treatment drug, using an OA rat model to assess pain behavior. A monoiodoacetate (MIA)-induced knee joint OA model in Wistar rats was used. Thirty Wistar rats (both sexes, 60 days old) were distributed in five groups of 6 rats each: the control group, that received no intervention; a prophylactic group, that received oral administration of 25 mg·kg-1·day-1 of SrRan for 28 days before induction of OA; a group treated with 25 mg·kg-1·day-1 of SrRan for 28 days after OA induction; a group treated with 50 mg·kg-1·day-1 during 28 days after OA induction; and a group that received oral saline for 28 days after induction. The assessment of pain behavior was performed considering articular incapacitation (weight-bearing test), mechanical hyperalgesia (Randall Selitto test) and motor activity (rotarod test), on days 0, 7, 14, 21, and 28. This experiment did not yield a significant difference when comparing the group that received SrRan prophylactically with the groups treated with 25 or 50 mg·kg-1·day-1 and the group that received oral saline. Thus, SrRan did not provide analgesia in either treated rats or as a prophylactic drug with the tested doses. Higher doses should be tested further to achieve possible significant results.