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Objective: This study aimed to examine the temporal activation of NF-κB and its relationship to the development of pain-related sensitivity and behavioral changes in a non-invasive murine knee loading model of PTOA., Method: Following knee injury NF-κB activity was assessed longitudinally via in vivo imaging in FVB. Cg-Tg (HIV-EGFP,luc)8Tsb/J mice. Measures of pain-related sensitivity and behavior were also assessed longitudinally for 16 weeks. Additionally, we antagonized NF-κB signaling via intra-articular delivery of an IκB kinase two antagonist to understand how local NF-κB inhibition might alter disease progression., Results: Following joint injury NF-κB signaling within the knee joint was transiently increased and peaked on day 3 with an estimated 1.35 p/s/cm 2 /sr (95% CI 0.913.1.792 p/s/cm 2 /sr) fold increase in signaling when compared to control joints. Furthermore, injury resulted in the long-term development of hindpaw allodynia. Hyperalgesia withdrawal thresholds were reduced at injured knee joints, with the largest reduction occurring 2 days following injury (estimate of between group difference 129.1 g with 95% CI 60.9,197.4 g), static weight bearing on injured limbs was also reduced. Local delivery of an NF-κB inhibitor following joint injury reduced chondrocyte death and influenced the development of pain-related sensitivity but did not reduce long-term cartilage degeneration., Conclusion: These findings underscore the development of behavioral changes in this non-invasive loading model of PTOA and their relationships to NF-κB activation and pathology. They also highlight the potential chondroprotective effects of NF-κB inhibition shortly following joint injury despite limitations in preventing the long-term development of joint degeneration in this model of PTOA., (Copyright © 2020 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Knee osteoarthritis (OA) is a common degenerative disease. Intra-articular administration of flurbiprofen is frequently employed in clinic to treat OA, while repeated injections are required because of the limited effective duration. To improve therapeutic outcome and prolong the treatment interval, a poly( ε -caprolactone- co -lactide)- b -poly(ethylene glycol)- b -poly( ε -caprolactone- co -lactide) (PCLA-PEG-PCLA) triblock copolymer based flurbiprofen thermosensitive gel for the sustained intra-articular drug delivery was designed in this study. The anti-OA effects of this flurbiprofen thermogel were investigated on collagenase II-induced rat knee OA model by multiple approaches and compared with that of conventional sodium hyaluronate and flurbiprofen injecta. In vitro drug release studies indicated that flurbiprofen was sustained released from the thermosensitive gel for more than three weeks. This sustained drug release system exerted comparable short-term analgesic effects and distinctly improved long-term analgesic efficacy in terms of the increased percentage of the total ipsilateral paw print intensity and the reduced Knee-Bend scores of OA rats. The inflammatory response was attenuated in the samples of flurbiprofen gel treated group by showing decreased IL-1, IL-6, and IL-11 levels in the joint fluid and down-regulated IL-1, IL-6, IL-11, COX-2, TNF-α, and NF-κB/p65 expression in the articular cartilages. The results suggest the suitability of thermosensitive copolymer PCLA-PEG-PCLA for sustained intra-articular effects of flurbiprofen and provide in vivo experimental evidence for potential clinical application of this flurbiprofen delivery system to better management of OA cases.

Objectives: Pharmacological options for treating osteoarthritis (OA) are limited and alternative treatments are required. Given the clinical data indicating that granulocyte macrophage-colony stimulating factor (GM-CSF) may be a therapeutic target in human OA, we evaluated different treatment regimens with a neutralizing anti-GM-CSF monoclonal antibody (mAb) in an experimental OA model to determine their effectiveness on amelioration of pain and disease., Methods: The collagenase-induced osteoarthritis (CiOA) model was induced in C57BL/6 mice, followed by different treatment regimens of anti-GM-CSF mAb or isotype control. Anti-CCL17 mAb treatment was also administered continually during the late stage of CiOA. Pain-related behavior (change in weight distribution of hind limbs), and disease (cartilage damage and osteophyte size) were assessed., Results: Blocking GM-CSF only during early synovitis in CiOA prevented pain and disease development. Once OA pain was established, regardless of the treatment regimen, anti-GM-CSF mAb treatment rapidly and efficiently ameliorated it; however, unless the treatment was continued, pain returned and disease progressed. Continual late stage blockade of GM-CSF was able to ameliorate pain (between-group difference: -6.567; 95% confidence interval (CI): -10.12, -3.011) and suppress cartilage damage (P = 0.0317, 95% CI: -1.75, -0.0556). Continual late stage blockade of CCL17 showed similar effects on pain and disease development., Conclusions: Early and short-term GM-CSF neutralization is effective at preventing CiOA pain and disease development but, once pain is evident, continual GM-CSF blockade is required to prevent pain from returning and to suppress disease progression in mice. These data reinforce the potential benefits of anti-GM-CSF (and anti-CCL17) mAb therapy in OA and should inform further clinical trials., (Copyright © 2020 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objectives: To examine the effect of the circadian gene Clock on posttranscriptional function and pro-inflammatory mechanisms in osteoarthritis (OA)., Methods: The cartilage from Clock mutant mice was assessed using histology, (OA) score, and real-time polymerase chain reaction (PCR) quantification of key pro-inflammatory genes. Nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB) translocation, posttranslational state and expression levels during day and night conditions were assessed using immunoblot and IP. The regulation of transcription by Clock in cartilage tissue was assessed by using chromatin immunoprecipitation (ChIP) and luciferase assays. Total acetylation level and pattern over 24 h were quantified using immunoblot and real-time PCR. Finally, the effects of exogenous Clock nanoparticle treatment were quantified by histology and immunoblot., Results: The Clock mutation significantly promoted the degradation of cartilage and the expression of the key pro-inflammatory mediators, IL-1β, IL-6 and MCP-1. The Clock mutation significantly promoted NFκB nuclear translocation. The circadian protein CLOCK positively regulates NFκB at the transcriptional level by binding the E-box domain. The Clock mutation significantly inhibited the total lysine acetylation level in cartilage and inhibited NFκB acetylation at the Lys310 residue but promoted phosphorylation at the Ser276 residue. The forced expression of Clock in vivo inhibited NFκB activation by increasing acetylation and decreasing phosphorylation levels and by decreasing cartilage damage and inflammation., Conclusions: This study demonstrates the mutation of Clock promotes inflammatory activity by mediating the posttranscriptional regulation of NFκB in OA pathogenesis., (Copyright © 2019 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: The objective of this pilot study was to determine time point(s) at which maximum concentration of fluticasone propionate (Cmax) occurs in synovial fluid and plasma in Beagle dog knees after intra-articular injection of EP-104IAR., Design: EP-104IAR is composed of fluticasone propionate drug crystals coated with heat-treated polyvinyl alcohol (PVA) to result in extended release properties. Thirty-two Beagle dogs had an injection of EP-104IAR into the knee joint at 2 different dose levels (0.6 mg and 12 mg). Outcome measures included plasma, synovial fluid, and articular cartilage fluticasone propionate concentrations as well as histological analysis of cartilage and synovium at a variety of time points up to 58 days postdosing., Results: Intra-articular administration of 0.6 and 12 mg EP-104IAR was well tolerated. Early minor abnormalities found on microscopy resolved by the end of the study. There were no quantifiable concentrations of fluticasone propionate in plasma of animals administered 0.6 mg at any of the sampling time points. Highest concentrations in plasma following 12 mg administration occurred 1 day postdose and declined with a half-life of approximately 45 days. Highest concentrations of fluticasone propionate in synovial fluid and cartilage generally occurred 5 days postdose in both dose groups and declined with a half-life of approximately 11 to 14 days., Conclusions: EP-104IAR is capable of providing a safe and prolonged local exposure to a corticosteroid in the synovial joint while minimizing systemic exposure, with peak exposures occurring within a matter of days after dosing before declining in all tissues in a predictable manner.

The use of intra-articular corticosteroids for traumatic arthritis and osteoarthritis (OA) is common in the horse. The beneficial and deleterious effects of the principal corticosteroids used betamethasone esters (Celestone [Soluspan], methylprednisolone acetate [Depo Medrol], and triamcinolone acetonide [TA] [Vetalog or Kenalog]) have been defined for the horse. While TA has both disease-modifying as well as symptom-modifying effects, methyl prednisolone acetate has deleterious effects on the articular cartilage. Studies in traumatically injured joints show the same rationale (suppression of deleterious mediators associated with inflammation) and positive results from the use of TA in both equine and human patients. Studies in the experimental equine OA model allow for more in-depth knowledge of disease-modifying effects. Recent insights allow us to understand posttraumatic OA as an early consequence of joint injury that may require a more aggressive and proactive treatment approach than commonly applied to date., Competing Interests: None., (Thieme Medical Publishers 333 Seventh Avenue, New York, NY 10001, USA.)

Abnormalities in joint shape are increasingly considered a critical risk factor for developing osteoarthritis in life. It has been shown that mechanical forces during prenatal development, particularly those due to fetal movements, play a fundamental role in joint morphogenesis. However, how mechanical stimuli are sensed or transduced in developing joint tissues is unclear. Stretch-activated and voltage-gated calcium ion channels have been shown to be involved in the mechanoregulation of chondrocytes in vitro In this study, we analyse, for the first time, how blocking these ion channels influences the effects of mechanical loading on chick joint morphogenesis. Using in vitro culture of embryonic chick hindlimb explants in a mechanostimulation bioreactor, we block stretch-activated and voltage-gated ion channels using, respectively, gadolinium chloride and nifedipine. We find that the administration of high doses of either drug largely removed the effects of mechanical stimulation on growth and shape development in vitro , while neither drug had any effect in static cultures. This study demonstrates that, during joint morphogenesis, mechanical cues are transduced-at least in part-through mechanosensitive calcium ion channels, advancing our understanding of cartilage development and mechanotransduction.This article is part of the Theo Murphy meeting issue 'Mechanics of development'., (© 2018 The Authors.)

Objective: To identify if synovial fluid prostaglandin E 2 increases in response to a single intra-articular dose of bupivacaine in the normal canine stifle., Results: There were no significant differences in synovial fluid prostaglandin E 2 (PGE 2 ) concentrations between treatment groups or over time within bupivacaine or saline groups. Samples requiring ≥ 3 arthrocentesis attempts had significantly higher PGE 2 concentrations compared to samples requiring 1 or 2 attempts. Following correction for number of arthrocentesis attempts, PGE 2 concentrations were significantly higher than baseline at 24 and 48 h in the bupivacaine group; however there were no significant differences between the bupivacaine and saline groups. In normal dogs, a single bupivacaine injection did not cause significant synovial inflammation, as measured by PGE 2 concentrations, compared to saline controls. Future research should minimize aspiration attempts and include evaluation of the synovial response to bupivacaine in clinical cases with joint disease.

Study Design: Experimental study., Objective: The aim of this study was to evaluate the effect of nandrolone decanoate (ND) on the time taken for bone consolidation in dogs undergoing tibial tuberosity advancement surgery (TTA)., Materials and Methods: Seventeen dogs that underwent TTA surgery were randomly divided into two groups: group C (TTA; 9 stifles), and group TTA+ND (TTA and systemic administration of ND; 8 stifles). Three observers (two radiologists and an orthopaedic surgeon), assessed bone consolidation by visual inspection of serial radiographs at intervals of 21 days following surgery., Results: There were no differences in median weight and age between groups, nor between the medians of the variables right and left stifle. Only weight and age values were normally distributed. The other variables, right and left stifle and time to consolidation, showed non-normal distribution. Meniscal injury was present in all animals in group C and all animals in group TTA+ND. There was a significant difference between time to consolidation in groups C and TTA+ND (p <0.05). One animal in the group TTA+ND showed increased libido. Kappa agreement among observers on radiographs was 0.87., Conclusion: Administration of ND reduces time to bone consolidation in dogs undergoing TTA.

The objective of the study was to establish the dose-response relationship for robenacoxib, a selective cyclooxygenase (COX)-2 inhibitor, in a urate crystal model of acute synovitis. In a randomized partial Latin square design trial, 12 beagle dogs were administered orally single doses of robenacoxib (0.5, 1, 2, 4 and 8 mg/kg), placebo and the positive control meloxicam (0.1 mg/kg), 3 h after injection of sodium urate crystals into a stifle joint. Dogs were assessed for weight bearing on a force plate and by subjective clinical orthopaedic observations. Robenacoxib produced dose-dependent improvement in weight bearing, and decreased pain on palpation and joint swelling, over the dose range 0.5-2 mg/kg with no further increase in effect over the range 2-8 mg/kg. For weight bearing on the force plate, the ED 50 of robenacoxib was 0.6-0.8 mg/kg. The onset of action and time to peak effect of robenacoxib were faster (respectively, 2-2.5 h and 3-5 h) than for meloxicam (respectively, 3 h and 6 h). Robenacoxib significantly inhibited COX-2 at all doses, with dose-related activity. Robenacoxib did not inhibit COX-1 over the dose range 0.5-4 mg/kg, but produced transient inhibition at 8 mg/kg. In conclusion, oral administration of robenacoxib over the dose range 0.5-8 mg/kg demonstrated significant analgesic and anti-inflammatory efficacy in dogs., (© 2016 The Authors. Journal of Veterinary Pharmacology and Therapeutics Published by John Wiley & Sons Ltd.)

Objective: To investigate the role of advanced glycation end products (AGEs) in cartilage degeneration in vivo and determine the influence of the peroxisome proliferator-activated receptor-γ (PPARγ) agonist pioglitazone on AGEs-induced osteoarthritis (OA) in a rabbit model., Design: Thirty-two rabbits were separated into four groups (n = 8 each) and received 500 μL of 123, 350, or 1000 mmol/L D-ribose or Phosphate buffered saline (PBS) solution administered to the right stifle joint via intra-articular injection twice a week. All the rabbits ran 500 m on treadmills every day. Another 16 rabbits were administered 1000 mmol/L D-ribose and divided into 2 groups (n = 8) that received either placebo or pioglitazone administered orally at 20 mg/kg/day. Eight weeks later, cartilage damage was evaluated macroscopically, histologically, and biochemically., Results: Artificially increasing the AGEs level and exercise load resulted in cartilage damage and dose-dependent downregulation of PPARγ expression. The efficacy of pioglitazone treatment was tested in a rabbit OA model, and a clear chondroprotective effect was revealed by macro- and microscopic assessments., Conclusion: Elevating AGEs in rabbits can accelerate the articular cartilage degradation that occurs with physical exercise, and pioglitazone can reduce the severity of the AGEs-induced OA in a rabbit model., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: The primary goal of this study was to test the disease-modifying effect of blocking a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 with a neutralizing monoclonal antibody (mAb) starting 4 weeks after destabilization of the medial meniscus (DMM) in the mouse. We also investigated whether ADAMTS-5 blockade reversed mechanical allodynia and decreased monocyte chemoattractant protein (MCP)-1 production by dorsal root ganglia (DRG) cells., Methods: Ten-week old male C57BL/6 mice underwent DMM surgery and were either left untreated or treated with anti-ADAMTS-5 mAb or IgG2c isotype control mAb starting 4 weeks after surgery. Knees were collected for histopathology 4 or 12 weeks later. Mechanical allodynia was monitored biweekly in the ipsilateral hind paw through 16 weeks. DRG were collected and cultured 8 weeks after DMM for analysis of MCP-1 production., Results: By 4 weeks after DMM, mild cartilage degeneration was evident in the medial compartment, small osteophytes were present, and subchondral bone sclerosis was established. By 16 weeks after surgery, significant cartilage deterioration was apparent on the medial tibial plateaux and medial femoral condyles, osteophyte size had increased, and subchondral bone sclerosis was maintained. Treatment with ADAMTS-5 mAb from week 4 to 16 after surgery slowed cartilage degeneration and osteophyte growth but did not affect subchondral bone sclerosis. Moreover, ADAMTS-5 blockade resulted in temporary reversal of mechanical allodynia, which correlated with decreased MCP-1 production by cultured DRG cells., Conclusions: This study suggests therapeutic efficacy of an ADAMTS-5 mAb in the DMM model, when therapy starts early in disease., (Copyright © 2015 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The objective of this study was to investigate the effects of selective inducible nitric oxide synthase and neuronal nitric oxide synthase inhibitors on cartilage regeneration. The study involved 27 Wistar rats that were divided into five groups. On Day 1, both knees of 3 rats were resected and placed in a formalin solution as a control group. The remaining 24 rats were separated into 4 groups, and their right knees were surgically damaged. Depending on the groups, the rats were injected with intra-articular normal saline solution, neuronal nitric oxide synthase inhibitor 7-nitroindazole (50 mg/kg), inducible nitric oxide synthase inhibitor amino-guanidine (30 mg/kg), or nitric oxide precursor L-arginine (200 mg/kg). After 21 days, the right and left knees of the rats were resected and placed in formalin solution. The samples were histopathologically examined by a blinded evaluator and scored on 8 parameters. Although selective neuronal nitric oxide synthase inhibition exhibited significant (P = 0.044) positive effects on cartilage regeneration following cartilage damage, it was determined that inducible nitric oxide synthase inhibition had no statistically significant effect on cartilage regeneration. It was observed that the nitric oxide synthase activation triggered advanced arthrosis symptoms, such as osteophyte formation. The fact that selective neuronal nitric oxide synthase inhibitors were observed to have mitigating effects on the severity of the damage may, in the future, influence the development of new agents to be used in the treatment of cartilage disorders.

Objective: The mechanisms linking obesity and osteoarthritis (OA) are not fully understood and have been generally attributed to increased weight, rather than metabolic or inflammatory factors. Here, we examined the influence of fatty acids, adipokines, and body weight on OA following joint injury in an obese mouse model., Methods: Mice were fed high-fat diets rich in various fatty acids (FA) including saturated FAs (SFAs), ω-6 polyunsaturated FAs (PUFAs), and ω-3 PUFAs. OA was induced by destabilising the medial meniscus. Wound healing was evaluated using an ear punch. OA, synovitis and wound healing were determined histologically, while bone changes were measured using microCT. Activity levels and serum cytokines were measured at various time-points. Multivariate models were performed to elucidate the associations of dietary, metabolic and mechanical factors with OA and wound healing., Results: Using weight-matched mice and multivariate models, we found that OA was significantly associated with dietary fatty acid content and serum adipokine levels, but not with body weight. Furthermore, spontaneous activity of the mice was independent of OA development. Small amounts of ω-3 PUFAs (8% by kcal) in a high-fat diet were sufficient to mitigate injury-induced OA, decreasing leptin and resistin levels. ω-3 PUFAs significantly enhanced wound repair, SFAs or ω-6 PUFAs independently increased OA severity, heterotopic ossification and scar tissue formation., Conclusions: Our results indicate that with obesity, dietary FA content regulates wound healing and OA severity following joint injury, independent of body weight, supporting the need for further studies of dietary FA supplements as a potential therapeutic approach for OA., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Hyaluronan (HA) is present in the extracellular matrix of all body tissues, including synovial fluid in joints, in which it behaves as a filter that buffers transmission of mechanical forces to nociceptor nerve endings thereby reducing pain. Using recombinant systems, mouse-cultured dorsal root ganglia (DRG) neurons and in vivo experiments, we found that HA also modulates polymodal transient receptor potential vanilloid subtype 1 (TRPV1) channels. HA diminishes heat, pH and capsaicin (CAP) responses, thus reducing the opening probability of the channel by stabilizing its closed state. Accordingly, in DRG neurons, HA decreases TRPV1-mediated impulse firing and channel sensitization by bradykinin. Moreover, subcutaneous HA injection in mice reduces heat and capsaicin nocifensive responses, whereas the intra-articular injection of HA in rats decreases capsaicin joint nociceptor fibres discharge. Collectively, these results indicate that extracellular HA reduces the excitability of the ubiquitous TRPV1 channel, thereby lowering impulse activity in the peripheral nociceptor endings underlying pain.

The influence of administration of the antioxidant complexes consisting of nonenzymatic antioxidants (alpha-tocopherol acetate preparation) and enzymatic antioxidants (ceruloplasmin) has been studied in rabbits with experimental arthritis. The introduction of alpha-tocopherol acetate (at a daily dose of 4 mg) improved metabolic processes in the organism (decreased in the rate of erythrocyte precipitation, total leukocytes and their stub and segmental forms; increased in erythrocyte count; reduced the glycosaminoglycan content as determined from uronic acid and hexose level; decreased ceruloplasmin activity and malonic dialdehyde level ion blood serum, all at p < 0.05), thus favoring reduction in the total activity of the inflammatory process as judged from hematological and biochemical data. Intra-articular introduction of ceruloplasmin (1.5 mg/kg, once per week) positively influenced the state of joint structures in damaged knee joints of the animals: decreased the activity of ceruloplasmin (from 5.28 ± 0.06 to 3.94 ± 0.01 AU), and malonic dialdehyde level (0.18 ± 0.02 to 0.08 ± 0.01 μM) in the articular fluid (all at p < 0.05). These effects are probably related to the elimination of inefficiency of the antioxidant system in the synovial medium, thus preventing inflammatory destruction of articular tissues, hindering the development of pannus, and assisting the activation of reparative regeneration of connective tissue structures.

Augmentation of implants with polymethylmethacrylate (PMMA) bone cement in osteoporotic fractures is a promising approach to increase implant purchase. Side effects of PMMA for the metaphyseal bone, particularly for the adjacent subchondral bone plate and joint cartilage, have not yet been studied. The following experimental study investigates whether subchondral PMMA injection compromises the homeostasis of the subchondral bone and/or the joint cartilage.Ten mature sheep were used to simulate subchondral PMMA injection. Follow-ups of 2 (4 animals) and 4 (6 animals) months were chosen to investigate possible cartilage damage and subchondral plate alterations in the knee. Evaluation was completed by means of high-resolution peripheral quantitative computed tomography (HRpQCT) imaging, histopathological osteoarthritis scoring, and determination of glycosaminoglycan content in the joint cartilage. Results were compared with the untreated contralateral knee and statistically analyzed using nonparametric tests.Evaluation of the histological osteoarthritis score revealed no obvious cartilage damage for the treated knee; median histological score after 2 months 0 (range 4), after 4 months 1 (range 5). There was no significant difference when compared with the untreated control site after 2 and 4 months (P = 0.23 and 0.76, respectively). HRpQCT imaging showed no damage to the metaphyseal trabeculae. Glycosaminoglycan measurements of the treated joint cartilage after 4 months revealed no significant difference compared with the untreated cartilage (P = 0.24).The findings of this study support initial clinical observation that PMMA implant augmentation of metaphyseal fractures appears to be a safe procedure for fixation without harming the subchondral bone plate and adjacent joint cartilage.

Background: The present preliminary study describes concentration time courses of the NSAID carprofen in the plasma and synovial fluid in a microfrature sheep model after transcutaneous treatments with a novel application device (Vetdrop®). To treat circumscribed inflammatory processes a transcutaneous application device could potentially be beneficial. After transcutaneous application normally lower systemic concentrations are measured which may reduce the incidence of side effects, whereas efficacy is still maintained. In this study carprofen was used based on its capacity to provide analgesia after orthopaedic procedures in sheep and it is considered that it may have a positive influence on the healing of cartilage in low concentrations., Results: In all transcutaneously treated animals, carprofen plasma concentrations exceeded those of synovial fluid, although plasma levels remained significantly reduced (300-fold) as compared to carprofen administered intravenously. Furthermore, in contrast to the intravenously treated animals, a modest accumulation of carprofen in plasma and synovial fluid was observed in the transcutaneously treated animals over the 6-week treatment period., Conclusions: The transcutaneously administered carprofen using the Vetdrop® device penetrated the skin and both, plasma- and synovial concentrations could be measured repeatedly over time. This novel device may be considered a valuable transcutaneous drug delivery system.

Objective: To assess effects of zoledronic acid on biomarkers, radiographic scores, and gross articular cartilage changes in dogs with induced osteoarthritis., Animals: 21 purpose-bred hound-type dogs., Procedures: The left stifle joint of each dog was examined arthroscopically to determine initial articular cartilage status, which was followed by cranial cruciate ligament (CrCL) transection to induce osteoarthritis. Dogs were assigned to 3 groups (control group, low dose [10 μg of zoledronic acid/kg], or high dose [25 μg of zoledronic acid/kg). Treatments were administered SC every 3 months for 1 year beginning the day after CrCL transection. Serum and synovial fluid samples and radiographs were obtained 0, 1, 3, 6, 9, and 12 months after transection. At 12 months, each joint was scored for cartilage defects. Serum and synovial fluid biomarkers of bone and cartilage turnover (bone-specific alkaline phosphatase, type I and II collagen, carboxy-propeptide of type II collagen, and chondroitin sulfate 846) were analyzed with ELISAs., Results: The high-dose group had fewer total articular defects and lower severity scores in CrCL-transected stifle joints than did the control group. In addition, the high-dose group had significantly less change in collagenase cleavage of type I or II collagen in the synovial fluid at 1 and 3 months after CrCL transection than did the control group and also had greater changes in bone-specific alkaline phosphatase in synovial fluid at 3 months after CrCL transection than did the control group., Conclusions and Clinical Relevance: Zoledronic acid had a chondroprotective effect in dogs with a transected CrCL.

The purpose of this study was to determine the effects of melatonin intervention on gastrocnemius remodeling in rats with collagenase-induced knee instability. Type VII collagenase was injected into the right knee to induce joint laxity with cartilage destruction. Melatonin (MT; 10 mg/kg) injection was performed twice daily subcutaneously, and treadmill exercise (Ex; 11 m/min) was conducted for 1 hr/day at a frequency of 5 days/wk for 4 wks. The gastrocnemius mass, which was reduced with collagenase injection only (Veh), was increased with collagenase injection with melatonin treatment with and without exercise in the early phase, and the mass in both limbs was significantly different in the Veh compared with the MT group. However, there was an increase in the relative muscle weight to body weight ratio in the Veh group at the advanced stage. Insulin-like growth factor receptor (IGF-IR) was downregulated in the Veh group, whereas IGF-IR was upregulated in the MT and MT + Ex groups. Joint laxity induced enhancement of autophagic proteolysis (LC3 II) in the muscle, which was recovered to values similar to those in the normal control group (Con) compared with those in the MT and MT+Ex groups. Although intra-articular collagenase increased the total C/EBP homology protein (CHOP) levels at 1 wk and decreased them at 4 wks in the Veh group, CHOP in the nucleus was upregulated continuously. Prolonged melatonin treatment with and without exercise intervention suppressed nuclear localization of ATF4 and CHOP with less activation of caspase-3, at the advanced phase. Moreover, the interventions promoted the expression of myosin heavy chain (MHC) isoforms under the control of myogenin. Concomitant with a beneficial effect of melatonin with and without exercise, step length of the saline-injected limb and the collagenase-injected supporting side was maintained at values similar to those in control rats. Taken together, the findings demonstrate that melatonin with and without exercise accelerate remodeling of the gastrocnemius through inhibition of nuclear CHOP in rats with collagenase-induced knee instability., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Objective: To evaluate the effects of sequential anesthesia of the individual compartments of the equine stifle joint on lameness induced by intra-articular deposition of interleukin (IL)-1β., Animals: 6 horses., Procedures: For each horse, baseline hind limb lameness was first evaluated. A randomly selected compartment of 1 stifle joint was then injected with IL-1β to induce synovitis and lameness; subsequently, the same compartment was anesthetized with 2% mepivacaine hydrochloride, and lameness was reevaluated. Two weeks later, baseline lameness was evaluated, and lameness was similarly induced; thereafter, the 2 synovial compartments of the stifle joint not injected with IL-1β were anesthetized sequentially in random order (ie, first and second blocks); lameness was evaluated after each block. Finally, the IL-1β-treated compartment was anesthetized (third block); lameness was again evaluated. This second experiment was repeated for the contralateral stifle joint 2 weeks later. Throughout the study, lameness was quantified objectively by assessing vertical pelvic movement asymmetry with a wireless, inertial sensor-based system., Results: Intra-articular deposition of IL-1β induced lameness in all injected limbs. In the first experiment, anesthesia of the compartment injected with IL-1β resulted in a significant decrease in lameness, with vertical pelvic movement asymmetry approaching baseline. In the second experiment, lameness improved significantly after the second and third blocks and was almost completely abolished after all 3 synovial compartments were anesthetized., Conclusions and Clinical Relevance: In horses, lameness caused by a lesion in 1 compartment of a stifle joint can be improved more by instillation of local anesthetic solution into that compartment than by anesthesia of the other compartments.

Objective: Although articular cartilage has evolved to facilitate joint mobilization, severe loading can induce chondrocyte apoptosis, which is related to the progression of osteoarthritis (OA). To avoid apoptosis, chondrocytes synthesize heat-shock proteins (HSPs). This study was undertaken to examine the roles of Hsp70 and Hsp90 in biomechanically induced OA, and the possibility of using Hsp90 inhibition as an intervention strategy for OA management., Methods: OA was biomechanically induced in rats by means of strenuous running. Disease progression was compared between running rats treated with Hsp90 inhibitor and untreated running controls. Hsp70 and Hsp90 protein levels in articular cartilage were determined by Western blotting. OA progression was monitored using contrast-enhanced micro-computed tomography to measure cartilage degradation and subchondral bone changes and single-photon-emission computed tomography to examine synovial macrophage activation and histologic features., Results: Chronic cartilage loading led to early OA development, characterized by degeneration of cartilage extracellular matrix. In vivo Hsp90 inhibition resulted in increased Hsp70 synthesis, which suggests that Hsp90 activity limits Hsp70 production. Hsp90 inhibitor treatment increased cartilage sulfated glycosaminoglycan levels to concentrations even beyond baseline and protected against cartilage degradation, stimulated subchondral bone thickness, and suppressed macrophage activation., Conclusion: Our findings indicate that Hsp90 plays a pivotal role in biomechanically induced chondrocyte stress responses. Intervention strategies that inhibit Hsp90 can potentially protect or improve cartilage health and might prevent OA development., (Copyright © 2013 by the American College of Rheumatology.)

Background/rationale: Introducing new or testing existing drugs in an attempt to modify the progress of osteoarthritis (OA) is of paramount importance., Questions/purposes: This study aims to determine the effect exerted by Calcitonin on the progress of early-stage osteoarthritic lesions., Methods: We used 18, skeletally mature, white, female, New Zealand rabbits. OA was operatively induced in the right knee of each animal by the complete dissection of the anterior cruciate ligament, complete medial meniscectomy and partial dissection of the medial collateral ligament. Postoperatively, animals were divided into two groups. Starting on the ninth postoperative day and daily thereafter, group A animals (n = 9) received 10 IU oculus dexter (o.d.) of synthetic Calcitonin IntraMuscularly (I.M.); group B animals (n = 9) received equal volume of saline o.d. Three animals from each group were sacrificed at 1, 2 and 3 months following treatment's initiation. The extent and the grade of OA were assessed macroscopically, histologically and by radiographs, Computed Tomography (CT) and Magnetic Resonance Imaging (MRI)-scans. The Osteoarthritis Research Society International (OARSI) score, incorporating histological and macroscopic information, was calculated for each knee., Results: Osteoarthritic changes in group A animals were less severe and progressed less rapidly when compared with those of group B animals (sham). This difference was statistically significant in the first and second month (P = 0.05), but not in the third month (P = 0.513)., Conclusions: I.M. administration of Calcitonin seems to delay the progress of early-stage osteoarthritic lesions induced by mechanical instability in a rabbit experimental model., (Copyright © 2013 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

Objective: To clarify the roles of hyaluronan (HA) in joint inflammation and the process of joint destruction, using 4-methylumbelliferone (4-MU), an inhibitor of HA synthesis, in a mouse model of collagen-induced arthritis (CIA) and in a monolayer culture of fibroblast-like synoviocytes (FLS) derived from patients with rheumatoid arthritis., Methods: DAB/1J mice were immunized with type II collagen. The effects of 4-MU were evaluated by the physiologic arthritis score, paw swelling, the histologic arthritis score, and expression of matrix metalloproteinase 3 (MMP-3) and MMP-13 in chondrocytes and synovial tissue. In vitro, the effect of 4-MU on messenger RNA and protein expression of MMP-1 and MMP-3 was determined. The effects of 4-MU on HA deposition and on serum/medium concentrations of HA were analyzed using biotinylated HA binding protein staining and an HA binding assay, respectively., Results: Treatment with 4-MU in mice with CIA dramatically decreased the severity of arthritis (based on the arthritis score), paw thickness, and histopathologic changes. MMP-3 and MMP-13 expression in chondrocytes and synovial cells was significantly inhibited by 4-MU in vivo. Treatment with 4-MU also inhibited MMP-1 and MMP-3 expression in tumor necrosis factor α-stimulated FLS, in a dose-dependent manner. The 4-MU-induced decreases in the serum HA concentration in mice with CIA and in "medium" and "pericellular" HA concentrations in cultured FLS support the contention that the inhibitory mechanism of 4-MU is mediated by HA suppression., Conclusion: Reduced disease activity induced by 4-MU in mice with CIA revealed HA to be a crucial regulator in the course of arthritis. Therefore, 4-MU is a potential therapeutic agent in arthritis, and its inhibitory mechanism is possibly mediated by suppression of HA synthesis., (Copyright © 2013 by the American College of Rheumatology.)

Objective: To investigate the role of the antiinflammatory neuropeptide cortistatin in chronic pain evoked by joint inflammation., Methods: Thermal and mechanical hyperalgesia was evoked in mouse knee joints by intraplantar injection of tumor necrosis factor α and intraarticular infusion of Freund's complete adjuvant, and the analgesic effects of cortistatin, administered centrally, peripherally, and systemically, were assessed. In addition, the effects of cortistatin on the production of nociceptive peptides and the activation of pain signaling were assayed in dorsal root ganglion cultures and in inflammatory pain models. The role of endogenous cortistatin in pain sensitization and perpetuation of chronic inflammatory states was evaluated in cortistatin-deficient mice. Finally, the effect of noxious/inflammatory stimuli in the production of cortistatin by the peripheral nociceptive system was assayed in vitro and in vivo., Results: Expression of cortistatin was observed in peptidergic nociceptors of the peripheral nociceptive system, and endogenous cortistatin was found to participate in the tuning of pain sensitization, especially in pathologic inflammatory conditions. Results showed that cortistatin acted both peripherally and centrally to reduce the tactile allodynia and heat hyperalgesia evoked by arthritis and peripheral tissue inflammation in mice, via mechanisms that were independent of its antiinflammatory action. These mechanisms involved direct action on nociceptive neurons and regulation of central sensitization. The analgesic effects of cortistatin in murine arthritic pain were linked to binding of the neuropeptide to somatostatin and ghrelin receptors, activation of the G protein subunit Gαi , impairment of ERK signaling, and decreased production of calcitonin gene-related peptide in primary nociceptors., Conclusion: These findings indicate that cortistatin is an antiinflammatory factor with potent analgesic effects that may offer a new approach to pain therapy in pathologic inflammatory states, including osteoarthritis and rheumatoid arthritis., (Copyright © 2013 by the American College of Rheumatology.)

Objective: Lyme arthritis (LA) is characterized by infiltration of inflammatory cells, mainly neutrophils (polymorphonuclear cells [PMNs]) and T cells, into the joints. This study was undertaken to evaluate the role of the neutrophil-activating protein A (NapA) of Borrelia burgdorferi in eliciting inflammation and in driving the adaptive immune response., Methods: Levels of NapA, interferon-γ (IFNγ), interleukin-17 (IL-17), and T cell-attracting chemokines were assessed by enzyme-linked immunosorbent assay in synovial fluid from patients with LA. The profile of T cells recruited into the synovia of patients with LA was defined by fluorescence-activated cell sorting analysis. NapA was intraarticularly injected into rat knees, and the cells recruited in synovia were characterized. The role of NapA in recruiting immune cells was confirmed by chemotaxis assays using a Transwell system., Results: NapA, IFNγ, IL-17, CCL2, CCL20, and CXCL10 accumulated in synovial fluid from patients with LA. Accordingly, T cells obtained from these patients produced IFNγ or IL-17, but notably, some produced both cytokines. NapA promoted neutrophil and T lymphocyte recruitment both in vitro and in vivo. Interestingly, the infiltration of T cells not only resulted from the chemotactic activity of NapA but also relied on the chemokines produced by PMNs exposed to NapA., Conclusion: We provide evidence that NapA functions as one of the main bacterial products involved in the pathogenesis of LA. Accordingly, we show that, at very early stages of LA, NapA accumulates and, in turn, orchestrates the recruitment of inflammatory cells into the joint cavity. Thereafter, with the contribution of recruited cells, NapA promotes the infiltration of T cells producing IL-17 and/or IFNγ., (Copyright © 2013 by the American College of Rheumatology.)

The study aimed to investigate the effect of the oral administration for 15 days of either Echinacea (E) or genuphil (a composite of chondroitin sulphate, glucosamine and methyl sulfonyl methane [GCM]) nutraceutical supplements on female rat model of acute or chronic arthritis induced by bacterial outer membrane protein (OMP) from faecal flora of healthy and rheumatic humans. Anti-cyclic citrullinated peptide (anti-CCP2), C-reactive protein (CRP) and rheumatoid factor (RF) values increased (p < 0.05) in both arthritic groups as compared to normal values. The rheumatic markers anti-CCP2, CRP and RF values decreased significantly in E- and GCM-treated groups compared to arthritic none-treated acute or chronic groups. The results of RF values of GCM-treated groups in acute and chronic models decreased exhibiting no statistical difference compared with the normal value. Histological examinations of the hind paw sections revealed moderate inflammation, oedema and mild proliferation of synovial cells in acute arthritic rats and more damage to cartilage and bone with severe inflammation in chronic ones. Echinacea acute treated group showed edema with proliferated synovial membrane and partial damage in cartilage and bone. While in the E-chronic treated group, rough edge with destructed cartilage and bone existed. However, the acute GCM group revealed mild cartilage damage. But the chronic GCM group showed mild synovial cells proliferation and revealed no inflammation with mild cartilage damage edge. Results demonstrated the OMP arthropathic property and through promising light on arthritis treatment using E- or GCM, with the advantage of GMC results over that of E-. The composite GCM is needed for further studies over the dose and duration to assess its preventive effects against the bacterial OMP arthrogenicity.

Objectives: To investigate whether treatment with a bisphosphonate would influence the subchondral bone plate stiffness and the development of cartilage damage in Dunkin Hartley guinea pigs, which develop osteoarthritis (OA) spontaneously., Method: Fifty-six 3-month-old male Dunkin Hartley guinea pigs were randomized into a baseline group and six groups receiving either the bisphosphonate risedronate (30 µg/kg) or vehicle five times a week for 6, 12, or 24 weeks. The medial condyle of the right stifle joint was investigated by histology, using the Osteoarthritis Research Society International (OARSI) score, along with static and dynamic histomorphometry. The subchondral bone plate of the left tibia was tested mechanically with indentation testing. Degradation products of C-terminal telopeptides of type II collagen (CTX-II) were measured in serum., Results: The OARSI score did not differ between risedronate-treated and control animals at any time point. The fraction of bone surfaces covered with osteoclasts (Oc.S/BS) was significantly suppressed in risedronate-treated animals at all time points, as were the fractions of mineralizing surfaces (MS/BS) and osteoid-covered surfaces (OS/BS), and also serum CTX-II. This was accompanied by a significant increase in the epiphyseal content of calcified tissue and in the thickness of the subchondral bone plate. However, this did not result in a stiffer subchondral bone at any time point., Discussion: The risedronate treatment inhibited osteoclastic resorption of calcified cartilage in the primary spongiosa under the epiphyseal growth plate, explaining the risedronate-mediated decrease in CTX-II. Moreover, the serum CTX-II level was not related to the OA-induced articular cartilage degradation seen in this model., Conclusions: Risedronate did not influence the OARSI score and subchondral plate stiffness, but decreased serum CTX-II in Dunkin Hartley guinea pigs.

Objective: The objectives were to investigate the in vivo effects of treatment with rebamipide on pain severity and cartilage degeneration in an experimental model of rat osteoarthritis (OA) and to explore its mode of action., Materials and Methods: OA was induced in rats by intra-articular injection of monosodium iodoacetate (MIA). Oral administration of rebamipide was initiated on the day of MIA injection, 3 or 7 days after. Limb nociception was assessed by measuring the paw withdrawal latency and threshold. We analyzed the samples macroscopically and histomorphologically, and used immunohistochemistry to investigate the expression of matrix metalloproteinase-13 (MMP-13), interleukin-1β (IL-1β), hypoxia-inducible factor-2α (HIF-2α), inducible nitric oxide synthase (iNOS), and nitrotyrosine in knee joints. Real-time quantitative reverse transcription-polymerase chain reaction was used to quantify the mRNA for catabolic and anticatabolic factors in human OA chondrocytes., Results: Rebamipide showed an antinociceptive property and attenuated cartilage degeneration. Rebamipide reduced the expression of MMP-13, IL-1β, HIF-2α, iNOS, and nitrotyrosine in OA cartilage in a dose-dependent manner. Nitrotyrosine expression in the subchondral bone region was decreased in the rebamipide-treated joints. mRNA expression of MMP-1, -3, and -13, and ADAMTS5 was attenuated in IL-1β-stimulated human OA chondrocytes. By contrast, rebamipide induced the mRNA expression of tissue inhibitor of metalloproteinase-1 and -3., Conclusion: The results show the inhibitory effects of rebamipide on pain production and cartilage degeneration in experimentally induced OA. The suppression of oxidative damage and the restoration of extracellular matrix homeostasis of articular chondrocyte suggest that rebamipide is a potential therapeutic strategy for OA., (Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.)

Intra-articular delivery of therapeutics to modulate osteoarthritis (OA) is challenging. Delivery of interleukin-1 receptor antagonist (IL-1Ra), the natural protein inhibitor of IL-1, to modulate IL-1-based inflammation through gene therapy or bolus protein injections has emerged as a promising therapy for OA. However, these approaches suffer from rapid clearance and reduced potency over time. Nano/microparticles represent a promising strategy for overcoming the shortcomings of intra-articular drug delivery. However, these delivery vehicles are limited for delivery of protein therapeutics due to their hydrophobic character, low drug loading efficiency, and harsh chemical conditions during particle processing. We designed a new block copolymer that assembles into submicron-scale particles and provides for covalently tethering proteins to the particle surface for controlled intra-articular protein delivery. This block copolymer self-assembles into 300 nm-diameter particles with a protein tethering moiety for surface covalent conjugation of IL-1Ra protein. This copolymer particle system efficiently bound IL-1Ra and maintained protein bioactivity in vitro. Furthermore, particle-tethered IL-1Ra bound specifically to target synoviocyte cells via surface IL-1 receptors. Importantly, IL-1Ra nanoparticles inhibited IL-1-mediated signaling to equivalent levels as soluble IL-1Ra. Finally, the ability of nanoparticles to retain IL-1Ra in the rat stifle joint was evaluated by in vivo imaging over 14 days. IL-1Ra-tethered nanoparticles significantly increased the retention time of IL-1Ra in the rat stifle joint over 14 days with enhanced IL-1Ra half-life (3.01 days) compared to that of soluble IL-1Ra (0.96 days) and without inducing degenerative changes in cartilage structure or composition., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Objective: To investigate the chondroprotective effect of cyclooxygenase 2 (COX-2) inhibition in experimental osteoarthritis (OA)., Methods: The expression of prostaglandin E2 synthetic enzymes was examined by immunostaining of tibial cartilage from mice with surgically induced knee joint instability and from OA patients undergoing total knee arthroplasty. The effect of orally administered celecoxib (10 mg/kg/day and 30 mg/kg/day) or vehicle alone in mice was examined 12 weeks after the induction of OA. To investigate the involvement of COX-1 and COX-2 in OA development, we also created the model in COX-1-homozygous-knockout (Ptgs1-/-) mice and COX-2-homozygous-knockout (Ptgs2-/-) mice. OA severity was assessed using a grading system developed by our group and by the Osteoarthritis Research Society International scoring system., Results: In mouse and human OA cartilage, the expression of the inducible enzymes COX-2 and microsomal prostaglandin E synthase 1 (mPGES-1) was enhanced, while that of the constitutive enzymes COX-1, cytosolic PGES, and mPGES-2 was suppressed. Daily celecoxib treatment did not prevent cartilage degradation or osteophyte formation during OA development in the mouse model. Furthermore, neither Ptgs1-/- mice nor Ptgs2-/- mice exhibited any significant difference in OA development as compared to wild-type littermates., Conclusion: The two COX enzymes differ in terms of regulation of their expression during OA development. Nevertheless, experiments using inhibitor and genetic deficiency demonstrated a lack of chondroprotective effect of COX-2 inhibition in the mouse surgical OA model., (Copyright © 2012 by the American College of Rheumatology.)

Objectives: (1) assess the molecular weight dependence of hyaluronan's (HA) cartilage boundary lubricating ability, alone and in combination with proteoglycan 4 (PRG4), at physiological concentrations; (2) determine if HA and PRG4 interact in solution via electrophoretic mobility shift assay (EMSA)., Methods: The cartilage boundary lubricating ability of a broad range of MW HA (20 kDa, 132 kDa, 780 kDa, 1.5 MDa, and 5 MDa) at 3.33 mg/ml, both alone and in combination with PRG4 at 450 μg/ml, was assessed using a previously described cartilage-on-cartilage friction test. Static, μ(static, Neq), and kinetic, <μ(kinetic, Neq)>, were calculated. An EMSA was conducted with PRG4 and monodisperse 150 kDa and 1,000 kDa HA., Results: Friction coefficients were reduced by HA, in a MW-dependent manner. Values of <μ(kinetic, Neq)> in 20 kDa HA, 0.098 (0.089, 0.108), were significantly greater compared to both 780 kDa, 0.080 (0.072, 0.088), and 5 MDa, 0.079 (0.070, 0.089). Linear regression showed a significant correlation between both μ(static, Neq) and <μ(kinetic, Neq)>, and log HA MW. Friction coefficients were also reduced by PRG4, and with subsequent addition of HA; however the synergistic effect was not dependent on HA MW. Values of <μ(kinetic, Neq)> in PRG4, 0.080 (0.047, 0.113), were significantly greater than values of PRG4+various MW HA (similar in value, averaging 0.040 (0.033, 0.047)). EMSA indicated that migration of 150 kDa and 1,000 kDa HA was retarded when combined with PRG4 at high PRG4:HA ratios., Conclusions: These results suggest alterations in HA MW could significantly affect synovial fluid's cartilage boundary lubricating ability, yet this diminishment in function could be circumvented by physiological levels of PRG4 forming a complex, potentially in solution, with HA., (Copyright © 2011 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.)

The purpose of this study was to investigate several inflammatory mediators and cartilage degradation molecules as possible biomarkers of joint lesion in the anterior cruciate ligament transection (ACLT) model of osteoarthritis in rats. We also assessed whether the treatment with the anti-inflammatory agent tin protoporphyrin IX (SnPP) reduces the progression of disease. Our results indicate that serum levels of interleukin (IL)-6 and PGE(2) are significantly increased in ACLT rats 10 weeks after surgery, whereas the increases in IL-1β and tumor necrosis-α were not significant. In addition, our data suggest that IL-17 is the main pro-inflammatory cytokine in the ACLT joint. We have shown that serum C-telopeptide of type II collagen (CTX-II) is a biomarker better than cartilage oligomeric matrix protein, whereas hyaluronic acid showed no correlation with disease progression. Administration of SnPP (i.p. 12 mg/kg BW/day for 10 weeks) reduces the severity of structural changes and the levels of CTX-II, IL-6, and PGE(2) in serum and IL-17 and PGE(2) in ACLT knees. These effects on PGE(2) are dependent on the down-regulation of cyclooxygenase-2. Our findings indicate that systemic and local inflammatory mediators participate in the rat ACLT model and anti-inflammatory strategies may help to reduce the progression of joint lesion., (Copyright © 2011 Orthopaedic Research Society.)

Objective: Galectin 3, an endogenous β-galactoside-binding lectin, plays an important role in the modulation of immune responses. The finding that galectin 3 is present in the inflamed synovium in patients with rheumatoid arthritis suggests that the protein is associated with the pathogenesis of this disease. We undertook this study to investigate the influence of galectin 3 deficiency in a murine model of arthritis., Methods: Wild-type (WT) and galectin 3-deficient (galectin 3(-/-) ) mice were subjected to antigen-induced arthritis (AIA) through immunization with methylated bovine serum albumin. The concentration of serum cytokines (interleukin-6 [IL-6] and tumor necrosis factor α [TNFα]) and antigen-specific antibodies was evaluated using a cytometric bead array platform and enzyme-linked immunosorbent assay (ELISA). Cellular IL-17 responses were examined by flow cytometry, ELISA, and enzyme-linked immunospot assay., Results: The joint inflammation and bone erosion of AIA were markedly suppressed in galectin 3(-/-) mice as compared with WT mice. The reduced arthritis in galectin 3(-/-) mice was accompanied by decreased levels of antigen-specific IgG and proinflammatory cytokines. The frequency of IL-17-producing cells in the spleen was reduced in galectin 3(-/-) mice as compared with WT mice. Exogenously added recombinant galectin 3 could partially restore the reduced arthritis and cytokines in galectin 3(-/-) mice., Conclusion: Our findings show that galectin 3 plays a pathogenic role in the development and progression of AIA and that the disease severity is accompanied by alterations of antigen-specific IgG levels, systemic levels of TNFα and IL-6, and frequency of IL-17-producing T cells. To our knowledge, this is the first report of in vivo evidence that galectin 3 plays a crucial role in the development of arthritis., (Copyright © 2011 by the American College of Rheumatology.)

To evaluate the efficacy and safety of intraarticular propolis compared with systemic antibiotic treatment in an experimental septic arthritis model. Thirty-two rabbits were infected intraarticularly by Staphylococcus aureus. The rabbits were randomly divided into four groups, including a control group and three experimental groups. Drainage was the only procedure performed in group I (control group). The animals were treated with daily intramuscular cefazolin sodium (75 mg/kg) for 7 days in group II. In group III, intraarticular ethanolic extract of propolis (0.5 mg/ml) was injected to the infected knees under sterile conditions on days 7, 14, and 21 after drainage. In group IV, the rabbits received both intramuscular cefazolin sodium as in group II and intraarticular ethanolic extract of propolis as in group III. After 8 weeks, the animals were killed and joint histopathological and scanning electron microscopic parameters were assessed. The best clinical score was obtained in group IV. There were statistically significant differences among all the groups (P<0.05). The highest total score of the histological examination was found in group I and the best total score was obtained in group IV. There were statistically significant differences among the groups when we evaluated the scores of the parameters as loss of chondrocytes, loss of matrix, and pannus in-growth (P<0.05). But there was no significant difference among the groups for the scores of cloning of the chondrocytes (P>0.05). The highest scanning electron microscopy score was found in group I and the best score was obtained in group IV. Our results confirm the safety and efficacy of intraarticular propolis and synergistic effect of propolis when used with cefazolin in an experimental septic arthritis model.

We examined the therapeutic effect of high molecular weight hyaluronic acid (HA) on the progression of joint pain and cartilage degeneration in a rabbit osteoarthritis (OA) model. The OA model was induced by partial meniscectomy. In the time course study, cartilage degeneration was assessed at 3, 7 and 14 days after operation. In the therapeutic study, HA or loxoprofen (LOX) was administered for 14 days beginning four days after operation (after the onset of knee pain and cartilage degeneration). Knee pain was assessed by weight distribution on the hind paw, and cartilage damage and MMP production in the joints were evaluated 18 days after surgery. In the time course study, severe cartilage damage was found three days after operation. In the treatment study, weight-bearing on the injured paw in the control group decreased with time from four days after the operation. However, HA or LOX treatment beginning four days after the operation normalized the reduced hind paw weight distribution, and PGE(2) production was inhibited by HA treatment and LOX treatment. HA significantly inhibited cartilage degeneration, whereas LOX did not. HA also suppressed the production of MMP in joints. Treatment of HA after the onset of cartilage destruction and pain showed a cartilage protective effect as well as an analgesic effect.

Objective: To determine whether treatment with an antiresorptive drug in combination with an antiinflammatory drug reduces periarticular bone and soft tissue adaptations associated with the progression of posttraumatic secondary osteoarthritis (OA)., Methods: We used in vivo microfocal computed tomography (micro-CT) to map bony adaptations and in vivo micro-magnetic resonance imaging (micro-MRI) to examine joint inflammation in a rat model of surgically induced OA secondary to knee triad injury. We examined the arthroprotective effects of the bisphosphonates alendronate and risedronate and the nonsteroidal antiinflammatory drug (NSAID) meloxicam., Results: Micro-CT revealed reduced levels of periarticular trabecular bone loss in animals with knee triad injury treated with the bisphosphonate drugs alendronate or risedronate, or the NSAID meloxicam, compared with untreated animals. Alendronate treatment reduced bony osteophyte development. While risedronate as a monotherapy did not positively impact osteophytogenesis, combination therapy with risedronate and meloxicam reduced osteophyte severity somewhat. Micro-MRI revealed an increased, diffuse water signal in the epiphyses of untreated rats with knee triad injury 8 weeks after surgery, suggestive of a bone marrow lesion-like stimulus. In contrast, meloxicam-treated rats showed a significant reduction in fluid signal compared with both bisphosphonate-treated groups 8 weeks after surgery. Histologic analysis qualitatively confirmed the chondroprotective effect of both bisphosphonate treatments, showing fewer degradative changes compared with untreated rats with knee triad injury., Conclusion: Our findings indicate that select combinations of bisphosphonate and NSAID drug therapy in the early stages of secondary OA preserve trabecular bone mass and reduce the impact of osteophytic bony adaptations and bone marrow lesion-like stimulus. Bisphosphonate and NSAID therapy may be an effective disease-modifying drug regimen if administered early after the initial injury.

Objective: To determine the relationship between serum cortisol concentration and pain severity as measured by force platform gait analysis in dogs with experimentally induced synovitis of the stifle joint., Animals: 10 healthy hound-type dogs., Procedures: Dogs underwent 2 study phases. In the first phase, serum cortisol concentration, systolic arterial blood pressure, heart rate, and gait data were obtained at 0 (first sample), 2.5, 5, 7.5, and 10 hours. In the second phase, the same data were gathered immediately before (0 hours) and 2.5, 5, 7.5, and 10 hours after induction of acute urate synovitis in the left stifle joint. Data were statistically evaluated to compare changes in variable values over time and to determine the accuracy of serum cortisol measurements for diagnosis of acute orthopedic pain., Results: Following induction of synovitis, ground reaction forces were significantly decreased relative to preinduction values at 2.5, 5.0, 7.5, and 10.0 hours and serum cortisol concentration was significantly increased at 2.5 hours. A cortisol concentration of >or= 1.6 microg/dL indicated pain with a 91% sensitivity and 35% specificity., Conclusions and Clinical Relevance: In this model, cortisol concentration may be useful for diagnosing pain in dogs. Although, with a cutoff of >or= 1.6 microg/dL, pain would be detected in most dogs with pain, some pain-free dogs would also be identified as having pain. Conversely, dogs with a serum cortisol of < 1.6 microg/dL would be unlikely to have pain. Validation of this diagnostic test in a large, heterogeneous group of clinical patients is necessary.

To compare the susceptibility to toxicity of semicarbazide hydrochloride (SEM-HCl) between young and adult rats, 3- and 20-week-old female SD rats were given a diet containing SEM-HCl at 0, 500, or 1,000 ppm and 0 or 1,000 ppm, respectively, for 4 weeks. Half of the animals were then maintained on basal diet for a further 2 weeks as recovery groups. Only in young rats was deformation of the knee joints as well as thorax and tail observed at 500 and 1,000 ppm. Histopathologically, severe osteochondral lesions, such as disarrangement and thickening of the epiphyseal cartilage and deformation of articular cartilage, were observed, but the severity of these lesions became reduced during the recovery period. In adult rats, osteochondral lesions were relatively mild. Fissures in the cartilage matrix of the tibia were characteristic of adult rats, and in these, reduction of severity was not obvious in the recovery group. In the thoracic aorta, the appearance of elastic laminae was altered only in young rats in both the 4-week treatment and recovery groups. These results suggest that growing animals are more susceptible to toxicity of SEM-HCl than adults are. Effects and the induced lesions link to the developing stage of the target organs.

The objective of this study was to determine the primary articular tissue target of doxycycline and minocycline. Synoviocytes-cartilage cocultures (n = 4) were treated with MMP-13 (25 ng/mL medium) or IL-1 (1.0 ng/mL medium) for 24 h. Doxycycline (4.3, 0.43, 0.043 microM) or minocycline (10, 1.0 or 0.1 microM) were then added and cultures were continued for 96 h. Cartilage and media were analyzed for GAG content. Quantitative PCR was used to measure cartilage MMP-3, MMP-13, aggrecan, COL2A1, ADAMTS-4, and ADAMTS-5 expression, and synoviocyte MMP-3, MMP-13, ADAMTS-4, and ADMATS-5 expression. Total and active MMP-3, MMP-13, and ADAMTS 4/5 enzymes were measured in culture medium. All concentrations of doxycycline and minocycline diminished GAG accumulation in the media. All concentrations of minocycline, but only the highest concentration of doxycycline decreased MMP-3 and MMP-13 expression in synoviocytes but not cartilage, and basal ADAMTS-5 mRNA levels in both synoviocytes and cartilage. Only minocycline decreased active MMP-13 protein in synoviocytes. In summary, the protective effects of tetracycline compounds are more pronounced in synoviocytes than cartilage, and following minocycline compared to doxycycline. Studies to determine the molecular mechanism of action of the tetracyclines in synoviocytes might lead to the design of targeted therapeutics for the treatment of OA or RA.

Recent studies have indicated that there may be a correlation between acute chondrocyte damage and joint degeneration reminiscent of early-stage osteoarthritis (OA). P188 surfactant has been shown to acutely restore the integrity of damaged chondrocytes; however, its long-term efficacy is unknown. The hypothesis of this study was that a single injection of P188 into a traumatized joint would acutely repair damaged cell membranes and maintain their viability in the long term. Twelve rabbits were divided into two groups, with and without P188, and sacrificed 4 days after tibiofemoral (TF) impact. Another six rabbits were sacrificed after 6 weeks and divided into two groups, with and without P188 treatment immediately posttrauma. Treatment with P188 increased the viable cell density 4 days posttrauma. A higher density of viable cells was also documented 6 weeks posttrauma in the treated versus untreated limb. The results of the current study confirm the acute efficacy of P188 treatment, and may suggest long-term efficacy of treatment, but additional studies are still needed to investigate the chronic implications of the acute repair of cells in the traumatized joint.

A ninety-day toxicity study of semicarbazide hydrochloride (SEM-HCl) was conducted in male and female Wistar Hannover GALAS rats fed diet containing the compound at concentration of 0, 250, 500 and 1000 ppm. Suppression of body weight gain and food consumption was found in both sexes at 1000 ppm throughout the study. Enlargement and deformation of knee joints were obvious at 500 and 1000 ppm from week 3, together with deformation of the thorax and tail. Histopathologically, disarrangement of chondrocytes and fissures in the cartilage matrix were apparent at all doses tested in epiphyseal and articular cartilage. The severity of these lesions increased dose-dependently, accompanied by increased connective tissues and bone deformation at high doses. Additionally, compact bones at 1000 ppm became thin, suggesting loss of bone mass. In the thoracic aorta, the edges of elastic laminae became rough and the interlaminar spaces were altered from a fibrillar to a rod or globular appearance. No abnormalities were detected in any other organs. Taken together, toxicological effects of subchronic exposure to SEM-HCI were mainly observed in bone, cartilage and the aorta, with the no-observed-adverse-effect-level estimated from the present histopathological examination of less than 250 ppm in both sexes.

Female athletes have a significantly higher rate of anterior cruciate ligament (ACL) injury than their male counterparts. Sex steroid hormones are considered to have an influence as risk factors for female ACL injuries. We hypothesized that estrogen and progesterone have specific and synergistic influences on the composition of extracellular matrix in ACL. By ovariectomy (OVX) followed by subcutaneous estradiol (E2) and/or progesterone (P4) replacement, 40 female rats were divided into 5 groups: E2, P4, combined E2 and P4 (EP), OVX control, and sham group. After 30 days, using undecalcified sections of knee joints in conjunction with immunofluorescence staining of estrogen receptor alpha and beta (ERalpha and ERbeta), collagen types 1 and 3, and cartilage oligomeric matrix protein (COMP), the immunoreactivities of these proteins in two distinct parts of ACL, proximal and middle portions, were compared semiquantitatively among experimental groups. By E2 replacement, the expressions of ERalpha in ACL fibroblasts were elevated compared to the OVX group. At the proximal portion, the immunoreactivities of type 1 collagen by E2 replacement, type 3 collagen by P4 replacement, and COMP by E2 or P4 replacement were significantly reduced. At the middle portion, the immunoreactivity of type 3 collagen was significantly elevated by E2 replacement. However, no differences were observed between the sham and OVX groups. These findings suggest that ACL is ER-dependent and that ovarian hormones alter ligament tissue composition, especially at the proximal portion. Female hormonal influences are partly involved in the biological properties of ACL.

Reasons for Performing Study: There are no published results of subchondral cystic lesions (SCLs) in the medial femoral condyle (MFC) treated with arthroscopic injection of corticosteroids into the lining of the cyst., Objectives: 1) To determine the success rate for treatment of SCLs in the MFC with arthroscopic injection of the fibrous tissue of the cyst with corticosteroids. 2) To identify any factors that may predict outcome., Hypotheses: Injection of the fibrous tissue of SCLs of the MFC with corticosteroids utilising arthroscopic guidance yields a similar or higher chance for intended performance than does arthroscopic debridement as previously reported; this technique will be effective for treating SCLs in older horses., Methods: Horses with clinical and radiographic evidence of a SCL in the MFC were injected with corticosteroids under arthroscopic guidance, and case records and radiographs were reviewed retrospectively. A telephone survey of referring veterinarians, owners and trainers was conducted., Results: Thirty-five of 52 (67%) cases were classified as successful involving 73 SCLs of which 56 (77%) were classified as successful. There was no significant association between age group (age3 years) and outcome, or cyst configuration and outcome. Significantly more unilateral SCLs (28/31 [90%] SCLs) were classified as successful than bilateral (28/42: 67%). There were significant differences in outcome based on the surgeon operating the case and an association between pre-existing radiographic findings of osteophytes and negative outcome., Conclusions: Injection of SCLs utilising arthroscopic guidance is an effective alternative method of surgical treatment of SCL., Potential Relevance: This technique offers a similar chance of success as has been reported with debridement and may allow for a shorter period of convalescence. If unsuccessful, the option remains to debride the cyst in a second surgery.

Combined effects of hyaluronate and indomethacin in the treatment of rabbits with antigen-induced arthritis (AIA) were evaluated by assessing joint swelling, C-reactive protein (CRP) and prostaglandin E(2) (PGE(2)) levels with periodic intra-articular (ia) injections of hyaluronate alone (HA group) and with either a low or high concentration of indomethacin (LI-HA or HI-HA group). End-point analyses included matrix metalloproteinases-3 (MMP-3) activity and macroscopic and histological joint examinations. Results demonstrated that treatment in LI-HA and HI-HA groups resulted in statistically significant suppression of CRP, PGE(2, )and MMP-3 in comparison with those of HA group. Inhibition of serum CRP was only observed in LI-HA group. The order of serum MMP-3 inhibition was LI-HA>HI-HA>HA. Based on macroscopic and histological analyses of pannus formation, hyperplasia, inflammation, joint leakage and erosion, and loss of proteoglycan, the only statistically significant improvement was shown in LI-HA group compared to HA group and HI-HA group compared to control group.

Objective: To investigate whether oral calcitonin treatment influences the increases in type II collagen (CII) degradation and related surface erosions of articular cartilage in ovariectomized rats., Methods: Fifty rats were randomly allocated into 1 of the 5 following intervention groups: sham-operated, ovariectomy, ovariectomy plus subcutaneously implanted 17beta-estradiol pellet, ovariectomy plus 2 mg/kg salmon calcitonin plus 50 mg/kg 5CNAC (carrier), or ovariectomy plus 50 mg/kg 5CNAC. Each treatment was administered for 9 weeks after the ovariectomy. Blood samples for biochemical marker analysis were collected from fasting animals at baseline, on day 3, and after 1, 2, 4, 6, and 9 weeks. CII degradation was quantified by specific immunoassay, and the changes in severity scores of articular cartilage erosions were visualized and scored in histologic sections of the knees., Results: Ovariectomy resulted in a marked increase in serum levels of C-telopeptide of type II collagen (CTX-II) (P < 0.001), which could be effectively reversed by 17beta-estradiol supplementation. Oral administration of calcitonin elicited similar decreases in serum levels of CTX-II (P < 0.001). Histologic scoring of cartilage erosion showed significantly less cartilage erosion in calcitonin-treated ovariectomized rats versus control ovariectomized rats that were untreated or treated with 5CNAC alone. (P < 0.01)., Conclusion: The in vivo effects of calcitonin in rats suggest that calcitonin is able to counteract CII degradation and the accompanying structural disintegration of articular cartilage promoted by estrogen deficiency. Clinical assessment of the chondroprotective potential of calcitonin in postmenopausal women seems warranted.

Objective: To investigate the hypothesis that Freund's complete adjuvant (CFA) plays an essential role in the induction of collagen-induced arthritis in mice, by testing whether CFA by itself is able to induce arthritis in interferon-gamma receptor-knockout (IFNgammaR-KO) mice., Methods: IFNgammaR-KO and wild-type mice were sensitized with a single intradermal injection of CFA containing heat-killed Mycobacterium butyricum. Flow cytometric analysis and in vitro osteoclastogenesis assays were performed on blood, spleen, and bone marrow cells. Tumor necrosis factor (TNF) levels were measured in the serum, and levels of RANKL, osteoprotegerin (OPG), and TNFalpha in the synovium were determined by quantitative reverse transcriptase-polymerase chain reaction. Effects of treatment with the TNFalpha antagonist etanercept were assessed., Results: Symptoms of arthritis appeared in IFNgammaR-KO mice but not in wild-type mice, and reached an incidence of 55%. The onset coincided with an expansion of CD11b+ splenocytes that spontaneously produced TNFalpha and with increased osteoclastogenesis in spleen and blood cells. Expansion of CD11b+ splenocytes and osteoclast precursor cells was more pronounced in arthritic than in nonarthritic mice. There was a >100-fold increase in the RANKL:OPG ratio in the synovia of CFA-sensitized mice compared with those of naive animals. Treatment with etanercept prevented the development of arthritis and mitigated the increased expansion of myeloid cells as well as the increase in osteoclast precursor numbers in the spleen and blood., Conclusion: These results indicate that sensitization of mice with CFA creates a condition in which dysregulation of a single cytokine leads to arthritis by triggering TNFalpha-driven osteoclastogenesis.

Objective: To determine the role of the terminal complement complex (TCC) in the development of experimental antigen-induced arthritis (AIA) and the therapeutic effects of human anti-C5 single-chain Fv (scFv)., Methods: Two different anti-C5 scFv, one that inhibits both release of C5a and assembly of the TCC (TS-A 12/22) and another that selectively blocks formation of the TCC (TS-A 8), were injected at the onset of AIA. The effects of these scFv on disease severity were evaluated for up to 21 days and compared with the effects of injection of an unrelated scFv. AIA was also established in C6-deficient and C6-sufficient PVG rats to obtain further information on the role of the TCC in this model., Results: TS-A 12/22 and TS-A 8 proved to be equally effective in reducing joint swelling, cell counts and tumor necrosis factor alpha levels in synovial lavage fluids, and the degree of histomorphologic changes compared with the effects of the unrelated scFv. TS-A 12/22 and TS-A 8 prevented the deposition of C9 but not that of C3, confirming the ability of the 2 scFv to neutralize C5. Administration of the 2 anti-C5 scFv after AIA onset also reduced disease severity. In C6-deficient rats with AIA, disease activity was reduced markedly compared with that in C6-sufficient rats., Conclusion: These 2 human anti-C5 scFv could represent potential therapeutic reagents to be used in patients with rheumatoid arthritis. In addition, the finding that TS-A 8 was as effective as TS-A 12/22 in reducing disease severity suggests that the TCC is mainly responsible for the joint inflammation and damage observed in AIA.

Objective: To determine the effects of chronic deficiency of growth hormone (GH) and insulin-like growth factor 1 (IGF-1) on osteoarthritis (OA) severity., Methods: Thirty-five rats were divided into 4 treatment groups at 4 weeks of age: 1 control group (normal GH/IGF-1 levels [heterozygous]) and 3 groups of dwarves with a genetic mutation that results in GH deficiency. The first dwarf group received GH for 64 weeks (GH replete) and the second received GH until 14 weeks of age, followed by saline for 50 weeks (adult-onset GH/IGF-1 deficiency [AO-GHD]). The third dwarf group received saline injections only (lifetime GH deficient [GHD]). Sections of the medial knee joint compartment were graded and measured histologically; data were summarized using factor analysis, and treatment effects were assessed using analysis of variance and adjusting for body weight., Results: Terminal IGF-1 levels and body weights were significantly affected by treatment (P = 0.002 and P < 0.001, respectively). Factor analysis yielded a total of 5 factors, the first 3 of which were not significantly affected by treatment. Factor 4 (weighted by medial tibial plateau articular cartilage width and area) was significantly affected by treatment (P < 0.012), with larger values in the AO-GHD group than in the GHD group (P < 0.05). Factor 5 (weighted primarily by articular cartilage structure and loss of toluidine blue staining scores) also was significantly affected by treatment (P < 0.001), and was significantly lower (less severe lesions) in the GH replete group than in all other treatment groups (P < 0.05). Despite the presence of cartilage lesions, osteophytes and subchondral sclerosis were not observed in GH/IGF-1-deficient animals., Conclusion: These results indicate that chronic GH/IGF-1 deficiency causes an increased severity of articular cartilage lesions of OA without the bony lesions normally seen in this disease.