Surgically-induced Osteoarthritis Research Articles

Differential gene expression analysis reveals pathways important in early post-traumatic osteoarthritis in an equine model

BackgroundPost-traumatic osteoarthritis (PTOA) is a common and significant problem in equine athletes. It is a disease of the entire joint, with the synovium thought to be a key player in disease onset and progression due to its role in inflammation. The development of effective tools for early diagnosis and treatment of PTOA remains an elusive goal. Altered gene expression represents the earliest discernable disease-related change, and can provide valuable information about disease pathogenesis and identify potential therapeutic targets. However, there is limited work examining global gene expression changes in early disease. In this study, we quantified gene expression changes in the synovium of osteoarthritis-affected joints using an equine metacarpophalangeal joint (MCPJ) chip model of early PTOA. Synovial samples were collected arthroscopically from the MCPJ of 11 adult horses before (preOA) and after (OA) surgical induction of osteoarthritis and from sham-operated joints. After sequencing synovial RNA, Salmon was used to quasi-map reads and quantify transcript abundances. Differential expression analysis with the limma-trend method used a fold-change cutoff of log2(1.1). Functional annotation was performed with PANTHER at FDR < 0.05. Pathway and network analyses were performed in Reactome and STRING, respectively.ResultsRNA was sequenced from 28 samples (6 preOA, 11 OA, 11 sham). “Sham” and “preOA” were not different and were grouped. Three hundred ninety-seven genes were upregulated and 365 downregulated in OA synovium compared to unaffected. Gene ontology (GO) terms related to extracellular matrix (ECM) organization, angiogenesis, and cell signaling were overrepresented. There were 17 enriched pathways, involved in ECM turnover, protein metabolism, and growth factor signaling. Network analysis revealed clusters of differentially expressed genes involved in ECM organization, endothelial regulation, and cellular metabolism.ConclusionsEnriched pathways and overrepresented GO terms reflected a state of high metabolic activity and tissue turnover in OA-affected tissue, suggesting that the synovium may retain the capacity to support healing and homeostasis in early disease. Limitations of this study include small sample size and capture of one point post-injury. Differentially expressed genes within key pathways may represent potential diagnostic markers or therapeutic targets for PTOA. Mechanistic validation of these findings is an important next step.

S-Allylmercaptocysteine Targets Nrf2 in Osteoarthritis Treatment Through NOX4/NF-κB Pathway.

PurposeThis study aimed to explore the potential role and mechanism of garlic-derived S-allylmercaptocysteine (SAMC), the major water-soluble fraction of garlic, in osteoarthritis (OA) both in vivo and in vitro.MethodsThe effect of SAMC in a surgical-induced OA model was examined by X-ray, staining, ELISA, and immunoblotting. Then the key role of Nrf2 by SAMC treatment in IL-1β stimulated chondrocytes in vitro was determined by gene-knockdown technique.ResultsSAMC could stabilize the extracellular matrix (ECM) by decreasing metalloproteinase (MMPs) expression to suppress type II collagen degradation in OA rats. The inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, were elevated in OA, which could be down-regulated by SAMC treatment. This effect was parallel with NF-κB signaling inhibition by SAMC. As oxidative stress has been shown to participate in the inflammatory pathways in OA conditions, the key regulator Nrf2 in redox-homeostasis was evaluated in SAMC-treated OA rats. Nrf2 and its down-stream gene NQO-1 were activated in the SAMC-treated group, accompanied by NAD(P)H oxidases 4 (NOX4) expression down-regulated. As a result, the toxic lipid peroxidation byproduct 4-hydroxynonenal (4HNE) was reduced in articular cartilage. In IL-1β-stimulated primary rat chondrocytes, which could mimic OA in vitro, SAMC could ameliorate collagen destruction, inhibit inflammation, and maintain redox-homeostasis. Interestingly, after Nrf2 gene knockdown by adenovirus, the protective effect of SAMC in IL-1β-stimulated chondrocytes disappeared.ConclusionOverall, our study demonstrated that SAMC targeted Nrf2 to protect OA both in vivo and in vitro, which would be a new pharmaceutical way for OA therapy.

Effect of autotaxin inhibition in a surgically-induced mouse model of osteoarthritis

Osteoarthritis (OA) is a degenerative joint disease with no approved disease modifying therapy. The enzyme autotaxin (ATX) converts lysophoshatidylcholine analogues to lysophosphatidic acid. Systemic inhibition of ATX reduces pain in animal models of OA; however, OA disease-modifying effects associated with ATX inhibition remain unknown. Here, we sought to determine whether local (knee joint) injection of an ATX inhibitor attenuates surgically-induced OA in mice. ATX expression was evaluated in human knee OA cartilage. Ten-week-old mice were subjected to surgically-induced OA. ATX inhibitor (PF-8380, 2.5ng/joint) was injected intra-articularly either at three and five weeks post-surgery or at two, four, six and eight weeks post-surgery and knee joints were evaluated by histopathology and immunohistochemistry to study the expression of catabolic and cell death markers. mRNA sequencing of human OA chondrocytes treated with/without ATX inhibitor was performed to identify differentially expressed transcripts, followed by pathway enrichment analysis. ATX expression was elevated in severely degenerated cartilage compared to less degenerated human OA cartilage. In surgically-induced OA mice, intra-articular injection of ATX inhibitor at three and five weeks post-surgery partially protected knee joints from cartilage degeneration. Interestingly, earlier and more frequent ATX inhibitor injections did not confer significant protection. Immunohistochemical analysis showed decreased expression of catabolic and apoptotic markers with two ATX inhibitor injections. mRNA sequencing followed by pathway analysis identified pathways related to cholesterol analogue metabolism and cell-cycle that could be modulated by ATX inhibition in human OA chondrocytes. Local delivery of ATX inhibitor partially attenuates surgically-induced OA in mice.

ALPHA defensin (human neutrophil peptide-1) slows surgically-induced osteoarthritis in association with promoting M1 to M2 macrophage polarization

Purpose: Emerging evidence suggests that macrophages play an important part in the low-grade inflammation implicated in the pathogenesis of osteoarthritis (OA). α-defensins are a group of low molecular weight molecules, released from apoptotic human neutrophils, which augment the antimicrobial capacity of macrophages while also having a significant ability to inhibit the biosynthesis of proinflammatory cytokines. In the current study, we evaluated the in vitro and in vivo effects of α-defensin (Human Neutrophil Peptide-1, HNP-1) on macrophage polarization and OA.

Shea Nut Oil Extracts Enhance the Intra-Articular Sodium Hyaluronate Effectiveness on Surgically Induced OA Progression in Rats.

Osteoarthritis (OA) progression is associated with joint pain and stiffness. Intra-articular hyaluronic acid (IAHA) injection in knee OA restores the viscoelasticity of the joint and prevents cartilage damage. Shea nut oil extract (SNO) was shown to provide chondroprotection on surgically-induced OA progression in rats. Here we aim to examine IAHA injection supplemented with SNO diet for a synergetic evaluation on the disease progression in OA rats. We employed an anterior cruciate ligament transection plus medial meniscectomy-induced knee OA rat model with up to 12 weeks of sign/behavior observation (knee width, weight-bearing) and histological assessments of joint damage. We found both IAHA and SNO alone significantly attenuated histological changes of cartilage degeneration and synovial reactions in these knee OA rats. Nonetheless, oral SNO alone mitigated OA pain and inflammation while IAHA alone had no significant impact on the weight-bearing test and knee joint swelling. Moreover, with IAHA-treated rats fed with oral SNO diet, additional anti-inflammatory and anti-nociceptive effects were found, which further enhanced and maintained IAHA protection. Given the differential phenotype of oral SNO vs. IAHA, a regimen of IAHA coupled with SNO supplement provides a long-term effect of IAHA treatment. Taken together, the SNO supplement can be safely used as an adjuvant diet for chronic symptomatic relief of OA coupled with IAHA management.

Berberine-mediated up-regulation of surfactant protein D facilitates cartilage repair by modulating immune responses via the inhibition of TLR4/NF-ĸB signaling

The innate immune system drives inflammatory joint damage in osteoarthritis (OA) and regulates cartilage repair. Berberine chloride (BBR) is an isoquinoline alkaloid that shows immunomodulatory activity in a variety of cell lines. However, the immunomodulatory mechanisms of BBR in chondrocytes during OA are largely unknown. Herein, we assessed the ability of BBR to mediate chondroprotection through its effects on innate immunity. We found that BBR up-regulated the expression of surfactant protein D (SP-D) in OA cartilage, a key regulator of inflammation and innate immunity both in the airways and extrapulmonary tissues, including joint cartilage. To further explore these findings, we used recombinant adeno-associated virus (rAAV)-mediated knockdown of SP-D. Silencing was assessed in rat model of surgically-induced OA in the presence or absence of BBR treatment, 10 weeks post-surgery. We observed a clear improvement in histological scores of BBR-treated animals compared to those treated with BBR and the rAAV-SP-D vector. In addition, animals co-treated with BBR + recombinant human SP-D (rhSP-D) exhibited significantly lower histological scores than those treated with BBR alone. BBR treatment led to significantly reduced immune cell infiltration mediated through TLR4, F4/80, CD68 and CD34, whilst SP-D silencing reversed this improvement. In contrast, rhSP-D treatment enhanced the protective phenotype. We further explored how BBR influences SP-D and other OA-associated genes in vitro. We observed an up-regulation of SP-D and a marked decline in TRAF6, TLR4, MD-2 and MyD88 expression, as well as NF-κB p65 and IκBα phosphorylation in chondrocytes treated with sodium nitroprusside. siRNAs specific for SP-D were able to partially reverse this phenotype, whilst both rhSP-D and the TLR4 inhibitor TAK-242 enhanced the effects. Together, these results are consistent with a model wherein SP-D has therapeutic potential for OA treatment. Concomitantly, BBR modulates immune responses and decreases cartilage degradation. These findings suggest that BBR achieves this function through releasing SP-D from MD2/SP-D complexes and through the inhibition of TLR4/NF-κB signaling.

Acute Synovitis after Trauma Precedes and is Associated with Osteoarthritis Onset and Progression.

Osteoarthritis (OA) is a whole-joint disease characterized by cartilage destruction, subchondral bone sclerosis, osteophyte formation, and synovitis. However, it remains unclear which part of the joint undergoes initial pathological changes that drives OA onset and progression. In the present study, we investigated the longitudinal alterations of the entire knee joint using a surgically-induced OA mouse model. Histology analysis showed that synovitis occurred as early as 1 week after destabilization of the medial meniscus (DMM), which preceded the events of cartilage degradation, subchondral sclerosis and osteophyte formation. Importantly, key pro-inflammatory cytokines such as IL-1β, IL-6, TNFα, and Ccl2, major matrix degrading enzymes including Adamts4, Mmp3 and Mmp13, as well as nerve growth factor (NGF), all increased significantly in both synovium and articular cartilage. It is notable that the inductions of these factors in synovium are far more extensive than those in articular cartilage. Results from behavioral tests demonstrated that sensitization of knee joint pain developed after 8 weeks, later than histological and molecular changes. In addition, the nanoindentation modulus of the medial tibiae decreased 4 weeks after DMM surgery, simultaneous with histological OA signs, which is also later than appearance of synovitis. Collectively, our data suggested that synovitis precedes and is associated with OA, and thus synovium may be an important target to intervene in OA treatment.

Inhibition of 4-aminobutyrate aminotransferase protects against injury-induced osteoarthritis in mice.

Recently we demonstrated that ablation of the DNA methyltransferase enzyme, Dnmt3b, resulted in catabolism and progression of osteoarthritis (OA) in murine articular cartilage through a mechanism involving increased mitochondrial respiration. In this study, we identify 4-aminobutyrate aminotransferase (Abat) as a downstream target of Dnmt3b. Abat is an enzyme that metabolizes γ-aminobutyric acid to succinate, a key intermediate in the tricarboxylic acid cycle. We show that Dnmt3b binds to the Abat promoter, increases methylation of a conserved CpG sequence just upstream of the transcriptional start site, and inhibits Abat expression. Dnmt3b deletion in articular chondrocytes results in reduced methylation of the CpG sequence in the Abat promoter, which subsequently increases expression of Abat. Increased Abat expression in chondrocytes leads to enhanced mitochondrial respiration and elevated expression of catabolic genes. Overexpression of Abat in murine knee joints via lentiviral injection results in accelerated cartilage degradation following surgical induction of OA. In contrast, lentiviral-based knockdown of Abat attenuates the expression of IL-1β-induced catabolic genes in primary murine articular chondrocytes in vitro and also protects against murine articular cartilage degradation in vivo. Strikingly, treatment with the FDA-approved small-molecule Abat inhibitor, vigabatrin, significantly prevents the development of injury-induced OA in mice. In summary, these studies establish Abat as an important new target for therapies to prevent OA.

Conditional deletion of E11/podoplanin in bone protects against load-induced osteoarthritis

BackgroundSubchondral bone (SCB) thickening is one of the earliest detectable changes in osteoarthritic joints and is considered a potential trigger for subsequent articular cartilage degeneration. In this manuscript, we examine whether disruption to the SCB osteocyte network contributes to the initiation and pathogenesis of osteoarthritis.MethodsWe examined expression patterns of the glycoprotein E11/podoplanin by immunohistochemical labelling in murine, human and canine osteoarthritis models. We also examined the effects of twice-weekly administration of Bortezomib, a proteasome inhibitor which stabilises osteocyte E11 levels, to C57/BL6 wild-type male mice (1 mg/kg/day) for 8 weeks after surgical destabilisation of the medial meniscus. By inducing osteoarthritis-like changes in the right knee joint of 12-week-old male E11 hypomorphic mice (and corresponding controls) using a post-traumatic joint loading model, we also investigated whether a bone-specific E11 deletion in mice increases joint vulnerability to osteoarthritis. Articular cartilage degradation and osteophyte formation were assessed by histology and in line with the OARSI grading system.ResultsOur studies reveal increased E11 expression in osteocytes of human and canine osteoarthritic SCB. We found that Bortezomib administration had no effect on surgically-induced osteoarthritis, potentially due to a lack of the expected stabilisation of E11 in the SCB. We also found, in concordance with our previous work, wild-type mice exhibited significant load-induced articular cartilage lesions on the lateral femoral condyle (p < 0.01) and osteophyte formation. In contrast, E11 hypomorphic mice did not develop osteophytes or any corresponding articular lesions.ConclusionsOverall, these data suggest that an intact osteocyte network in the SCB contributes to the development of mechanically-driven osteoarthritis. Further, the data presented here indicate that the molecular pathways that preserve the osteocyte network, such as those driven by E11, may be targeted to limit osteoarthritis pathogenesis.

RHEB gene therapy maintains the chondrogenic characteristics and protects cartilage tissue from degenerative damage during experimental murine osteoarthritis

Osteoarthritis (OA) is characterized by cartilage degeneration resulting from hypertrophic changes in chondrocytes caused by altered gene expression. The involvement of Ras homolog enriched in brain (RHEB) in OA regulation is unclear. Human knee articular cartilage samples - were analyzed for structural and biological changes by histology, immunohistochemistry, real time PCR and western blotting. OA-mouse model developed by surgical destabilization of the medial meniscus (DMM) were treated with adenovirus harboring Rheb gene to analyze onset and progression of OA. Histological scoring, immunohistochemistry, and TUNEL assay was performed to assess cartilage damage across the entire joint. Human and mouse OA cartilage is degenerated and has markedly reduced levels of RHEB. Human OA-degenerated chondrocytes (DC) exhibited a fibroblastic phenotype and 80 % of degenerative cartilage were senescent, with higher levels of reactive oxygen species (ROS). Gene expression analysis of DC revealed almost no COL2A1 expression and reduced SOX9 and RHEB expression. Transient transfection of RHEB rescued the DC phenotype and reduced senescence and ROS levels markedly. RHEB overexpression also increased COL2A1 and SOX9 expression. In an OA-mouse model, the Rheb protein level decreased as the severity of OA increased. Ectopic expression of Rheb using adenovirus in mouse-OA cartilage suppressed surgically-induced OA pathogenesis accompanied by modulation of Adamts5, Mmp 13, Col 10, and Col2a1 expression. Rheb induction significantly reduced apoptosis in OA-cartilage. RHEB plays an important role in maintaining the chondrogenic characteristics of chondrocytes, and has potential in preventing progression of OA in the destabilize the medial meniscus (DMM) mouse model of OA.

Disruption of glucocorticoid signalling in osteoblasts attenuates age-related surgically induced osteoarthritis

Aging is a major risk factor for osteoarthritis (OA). Skeletal expression and activity of the glucocorticoid-activating enzyme 11β-hydroxysteroid-dehydrogenase type 1 increases progressively with age in humans and rodents. Here we investigated the role of endogenous osteocytic and osteoblastic glucocorticoid (GC) signalling in the development of osteoarthritic bone and cartilage damage in mice. We utilized transgenic (tg) mice in which glucocorticoid signalling is disrupted in osteoblasts and osteocytes via overexpression of the glucocorticoid-inactivating enzyme, 11β-hydroxysteroid-dehydrogenase type 2. Osteoarthritis was induced in 10- and 22-week-old male transgenic mice (tg-OA, n=6/group) and their wildtype littermates (WT-OA, n=7-8/group) by surgical destabilization ofthemedial meniscus (DMM). Sham-operated mice served as controls (WT- & tg-Sham, n=3-5 and 6-8/group at 10- and 22-weeks of age, respectively). Sixteen weeks after DMM surgery, mice developed features of cartilage degradation, subchondral bone sclerosis and osteophyte formation. These changes did not differ between WT and tg mice when OA was induced at 10-weeks of age. However, when OA was induced at 22-weeks of age, cartilage erosion was significantly attenuated in tg-OA mice compared to WT-OA littermates. Similarly, subchondral bone volume (-5.2%, 95% confidence intervals (CI) -9.1 to-1.2%, P=0.014) and osteophyte size (-4.0mm2, 95% CI -7.5 to-0.5mm2, P=0.029) were significantly reduced in tg-OA compared to WT-OA mice. Glucocorticoid signalling in cells of the osteoblast lineage promotes the development of surgically-induced osteoarthritis in older, but not younger, male mice. These data implicate osteoblasts and osteocytes in the progression of DMM-OA, via a glucocorticoid-dependent and age-related pathway.

Dual functional nanoparticles containing SOX duo and ANGPT4 shRNA for osteoarthritis treatment.

In our previous studies, we found that adult stem cells transfected with sex-determining region Y-box (SOX)-9, -6 and -5 genes (SOX trio) enhanced chondrogenesis and suppressed the progression of osteoarthritis (OA). The inhibition of angiopoietin-like 4 (ANGPT4) is known to reduce levels of cartilage damaging enzymes, such as, matrix metalloproteinases (MMPs). In this study, we designed nanoparticles comprising dexamethasone-conjugated polyethylenimine (DEX PEI) complexed with minicircle plasmid (MC) harboring SOX duo (SOX-9, -6) and ANGPTL4 small hairpin RNA (shANG) [MC SOX9/6/shANG] in the expectation that transfection of these nanoparticles would enhance chondrogenesis of stem cells and suppress inflammation in OA. Adipose-derived stem cells (ADSCs) transfected with MC SOX9/6/shANG (MC SOX9/6/shANG-tADSCs) showed significantly higher expressions of COL2 gene and protein than MC SOX9/6-transfected ADSCs (MC SOX9/6-tADSCs) during in vitro chondrogenesis while both enhanced chondrogenesis in the absence of growth factor addition as compared with negative controls. Furthermore, the expressions of MMP13 and MMP3 genes were significantly more diminished in MC SOX9/6/shANG-tADSCs than in MC SOX9/6-tADSCs. In vivo experiments using surgically-induced OA rats showed MC SOX9/6/shANG-tADSC-treated rats had significantly lower levels of cyclooxygenase (COX-2) and MMP13 in synovial fluids than MC SOX9/6-tADSC-treated rats, but no significant difference was observed between them in histological appearances. Both groups showed significantly less joint destruction than control groups did. These results demonstrate that dual functional nanoparticles containing SOX duo and ANGPT4 shRNA enhance chondrogenesis of ADSCs and suppress inflammation in OA. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:234-242, 2020.

Attenuation of surgically-induced osteoarthritis (OA) by inhibition of autotaxin

Purpose: Osteoarthritis (OA) is a chronic, progressive joint disease leading to a poor quality of life. Previously, we have reported that selected Lysophoshatidylcholines (lysoPCs) are increased in animal models of OA (Datta et al., 2017, PMID: 28811491). We also identified that these particular metabolites may promote OA pathogenesis in an autotaxin (ATX) - dependent manner. ATX is an enzyme responsible for the conversion of lysoPC to the inflammatory mediator lysophosphatidic acid (LPA). Previous studies have also shown that ATX levels in plasma and synovial fluid correlate with the severity of knee osteoarthritis (Mabey et al., 2015, PMID: 25659292). Thus, we sought to identify if local pharmacological inhibition of ATX can attenuate surgically-induced OA in vivo and its mechanism of action in vitro. Methods: Nine week-old mice were subjected to surgically-induced OA by destabilizing medial meniscus (DMM surgery). ATX antagonist (PF -8380, Pfizer) was injected intra-articularly in the mouse knee joints at 2nd,4th 6th and 8th week of post surgery. Subsequent knee joints were collected at 10 weeks of post surgery and histopathological changes were evaluated. Primary human chondrocytes were treated with IL-1β/ATX antagonist, and the expression of catabolic markers were determined. Results: Local injection of ATX antagonist reduced the degree of cartilage degeneration in surgically induced OA models compared to saline injected controls. ATX antagonist also reduced the amount of synovitis as compared to controls. Immunohistochemical analysis of mouse knee joints showed decreased expression of catabolic markers (C1-2C and MMP 13) in ATX treated mice compared to control mice. In vitro, ATX antagonist attenuated IL-1β-induced increases in the expression of MMP13 Conclusions: Inhibition of ATX attenuates surgically-induced OA. We are currently investigating the mechanism of action of ATX in attenuating OA. Our data, to date, suggests pre-clinical efficacy of ATX to limit OA progression.

ARC (apoptosis repressor with caspase recruitment domain) deficient mice develops severe knee joint histopathology but demonstrates reduced mechanical allodynia after surgically-induced osteoarthritis

ARC (apoptosis repressor with caspase recruitment domain) deficient mice develops severe knee joint histopathology but demonstrates reduced mechanical allodynia after surgically-induced osteoarthritis

SOX-6, 9-Transfected Adipose Stem Cells to Treat Surgically-Induced Osteoarthritis in Goats.

We developed SOX-6, 9-transfected human adipose stem cells (SOX-6, 9ASCs) to treat osteoarthritis (OA). SOX-6, 9-transfection led to in vitro chondrogenesis of adipose stem cells (ASCs) comparable to that achieved by growth factor treatment. Intra-articular injection of SOX-6, 9-transfected ASCs reduced the progression of surgically-induced OA in goats. In vivo tracking of transfected cell suggested paracrine mode of action. We think that SOX-6, 9-transfected ASC provides a novel potential strategy for the treatment of OA.

Protective effects of quercetin against inflammation and oxidative stress in a rabbit model of knee osteoarthritis.

Hit, Lead & Candidate Discovery This study investigated the effects of a natural phenolic compound quercetin on surgical-induced osteoarthritis (OA) in rabbits. Forty-eight New Zealand White rabbits were used to establish OA model by Hulth modified method, and subsequently randomized into untreated OA group (treatment was drinking water), celecoxib treated group (celecoxib 100 mg kg-1 by gavage), and quercetin treated group (25 mg kg-1 by gavage). Sixteen nonoperated rabbits served as the normal controls (drinking water was given). The treatment (length: 4 weeks) started on the 5th week postoperation when the OA pathological changes were manifested. Expressions of superoxide dismutase (SOD), matrix metalloproteinase-13 (MMP-13) and tissue inhibitor of metalloproteinases-1 (TIMP-1) in serum, synovial fluid, and synovial tissue were measured at 8 and 12 weeks postoperation. Pathological analysis was performed with synovial tissue section and Osteoarthritis Research Society International histopathology grading and staging scores were determined. The quercetin treated group showed higher SOD and TIMP-1 expressions but lower MMP-13 expression than untreated OA group in the serum, synovial fluid and synovial tissues (p < .05). There was no significant difference in the SOD, MMP-13 and TIMP-1 expressions between the quercetin-treated and celecoxib-treated groups. The MMP-13/TIMP-1 ratio of the quercetin treated group was significantly lower than that of the untreated OA group (p < .05). Quercetin can up-regulate SOD and TIMP-1, down-regulate MMP-13, and improve the degeneration of OA through weakening the oxidative stress responses and inhibiting the degradation of cartilage extracellular matrix.

Effects of Hesperidin on H₂O₂-Treated Chondrocytes and Cartilage in a Rat Osteoarthritis Model.

BackgroundThe purpose of this research was to investigate the effects of hesperidin on hydrogen peroxide (H2O2)-induced chondrocytes injury and cartilage degeneration in a rat model of osteoarthritis (OA).Material/MethodsChondrocytes were isolated from rat knee joints and treated with hesperidin alone or combined with H2O2. Then, Cell Counting Kit-8 (CCK-8) assay was used to assess cell viability. Activity of reactive oxygen species (ROS) and levels of malondialdehyde (MDA) were estimated. Cell apoptosis was assessed by flow cytometry assay. In addition, gene expression levels were measured for caspase 3, tumor necrosis factor-alpha (TNF-α), interleukin-1beta (IL-1β), collagen type II (Col2a1), aggrecan, (sex-determining region Y)-box 9 (SOX9), matrix metalloproteinase (MMP)-13, and inducible nitric oxide synthase (iNOS) through quantitative real-time polymerase chain reaction (qPCR). To examine the effects on cartilage destruction in vivo, hesperidin or vehicle control were orally administrated in a surgically-induced osteoarthritis (OA) model.ResultsThe results indicated that hesperidin pretreatment of chondrocytes reduce H2O2-induced cytotoxicity and apoptosis. Hesperidin pretreatment decreased the formation of MDA and intracellular ROS, including chondrocyte apoptosis. Hesperidin also reversed the activity of H2O2 on inhibiting the Col2a1, aggrecan, and SOX9 gene expression and increasing the gene expression of caspase 3, IL-1β, TNFα, iNOS, and MMP13. In addition, hesperidin administration markedly attenuated cartilage destruction and reduced IL-1β and TNF-α levels in a surgically-induced OA model.ConclusionsOur study suggests that hesperidin can prevent H2O2-induced chondrocytes injury through its antioxidant effects in vitro and reduce cartilage damage in a rat model of OA.

Intra-articular administration of I\u03baB\u03b1 kinase inhibitor suppresses mouse knee osteoarthritis via downregulation of the NF-\u03baB/HIF-2\u03b1 axis

Activation of NF-κB signaling promotes osteoarthritis (OA) through the transcriptional induction of Hif-2α and catabolic enzymes. This study sought to examine whether inhibiting IκBα kinase (IKK) could suppress the development of surgically-induced OA of the knee in a mouse model. We employed BMS-345541 (4(2′-aminoethyl) amino-1, 8-dimethylimidazo (1,2-a) quinoxaline) as a selective inhibitor of the subunits of IKK. OA was created by resecting the medial collateral ligament and the medial meniscus in the knees of mice. The mice were then treated with an intra-articular injection of BMS-345541 (50 nM to 500 µM) or vehicle three times a week for 8 weeks. We found that the intra-articular administration of 500 nM and 5 µM BMS-345541 significantly suppressed OA development. In the BMS-345541-treated cartilage, there was a decrease in the phosphorylation of IκBα and the expression of Hif-2α, Mmp13, and Adamts5. In human articular chondrocytes, the IL-1β-enhanced expression of Hif-2α and catabolic factors were decreased by BMS-345541 treatment in dose-dependent manner. We conclude that the intra-articular administration of BMS-345541 at some concentrations may suppress the development of OA by downregulating signaling through the NF-κB–Hif-2α axis.

A Pathophysiological Validation of Collagenase II-Induced Biochemical Osteoarthritis Animal Model in Rabbit.

Current dilemma working with surgically-induced OA (osteoarthritis) model include inconsistent pathological state due to various influence from surrounding tissues. On the contrary, biochemical induction of OA using collagenase II has several advantageous points in a sense that it does not involve surgery to induce model and the extent of induced cartilage degeneration is almost uniform. However, concerns still exists because biochemical OA model induce abrupt destruction of cartilage tissues through enzymatic digestion in a short period of time, and this might accompany systemic inflammatory response, which is rather a trait of RA (rheumatoid arthritis) than being a trait of OA. To clear the concern about the systemic inflammatory response that might be caused by abrupt destruction of cartilage tissue, OA was induced to only one leg of an animal and the other leg was examined to confirm the presence of systemic degenerative effect. Although the cartilage tissues were rapidly degenerated during short period of time upon biochemical induction of OA, they did not accompanied with RA-like process based on the histology data showing degeneration of articular cartilage occurred only in the collagenase-injected knee joint. Scoring evaluation data indicated that the cartilage tissues in non-induced joint remained intact. Neutrophil count transiently increase between day 8 and day 16, and there were no significant change in other complete blood count profile showing a characteristics of OA disease. These study shows that biochemically induced cartilage degeneration truly represented uniform and reliable OA state.

Articular cartilage protection in Ctsk-/- mice is associated with cellular and molecular changes in subchondral bone and cartilage matrix.

Osteoarthritis (OA) is a degenerative disease and a major cause of chronic disability in aging individuals. Cathepsin K (CatK), encoded by the Ctsk gene, has been implicated in the pathogenesis of pycnodysostosis and osteoporosis. The use of a selective inhibitor of CatK was recently shown to delay OA progression in rabbits. However, the cellular mechanisms underlying these protective effects remain unexplored. We examined articular cartilage maintenance and joint bone remodeling using Ctsk null mice (Ctsk-/- ) which underwent destabilization of the medial meniscus (DMM). We found that Ctsk-/- mice displayed delayed remodeling of subchondral and calcified cartilage by osteoclasts and chodroclasts respectively in DMM-induced osteoarthritis. While WT mice displayed a more severe OA phenotype than Ctsk-/- mice at 16 weeks, higher subchondral bone volume and lower trabecular spacing were also observed in surgically-induced OA joints of Ctsk-/- mice. However, no differences were seen in non-surgical controls. During OA progression, TRAP+ osteoclast numbers were increased in both WT and Ctsk-/- mice. However, Ctsk-/- mice had fewer physis-derived chondroclasts than WT when OA was present. These data suggest that CatK may differentially regulate chondroclastogenesis in the growth plate. Targeted PCR arrays of RNA harvested from laser captured osteoclasts in the subchondral bone and chondroclasts in the growth plate demonstrated differential expression of Atp6v0d2, Tnfrsf11a, Ca2, Calcr, Ccr1, Gpr68, Itgb3, Nfatc1, and Syk genes between WT and Ctsk-/- mice at 8- and 16-weeks post-DMM. Our data provide insight into the cellular mechanisms by which cathepsin K deletion delays OA progression in mice.