Synovial Concentrations Research Articles

Phenylbutazone concentrations in synovial fluid following administration via intravenous regional limb perfusion in the forelimbs of six adult horses.

Pain management is critical to equine welfare with non-steroidal anti-inflammatory drugs (NSAID) commonly used in horses. However, systemic NSAID use is limited by harmful gastrointestinal and renal side effects. Intravenous regional limb perfusion (IVRLP) is a technique used in horses that produces high, local antibiotic concentrations while limiting systemic circulation. NSAID-IVRLP would be a novel method of local pain management while limiting systemic NSAID side effects. To date, NSAID-IVRLP administration has not been reported in horses. This study aimed to identify the pharmacokinetics and local complications associated with using the NSAID phenylbutazone (PBZ) for IVRLP in six standing adult horses. PBZ-IVRLP, at a dose of 2.2 mg/kg PBZ, was performed in a randomly assigned forelimb cephalic vein in 6 standing, healthy adult horses. A placebo-IVRLP was performed in the contralateral forelimb for comparison. Systemic serum and radiocarpal joint synovial fluid PBZ concentrations were identified at various timepoints (before IVRLP T-0 h, just after tourniquet removal T-0.5, 1.5, 3, 5, 12, 16, and 24 h post IVRLP) for non-compartmental pharmacokinetic analysis and concentration over time curves. Local complications associated with PBZ-IVRP were evaluated for up to 7 days following PBZ-IVRLP using physical and ultrasonographic assessment. On day 7 horses were humanely euthanized with histology performed on both forelimbs at PBZ-IVRLP and placebo-IVRLP administration sites. Non-compartmental pharmacokinetics for PBZ, and its major metabolite oxyphenbutazone (OBZ), were determined for serum and synovial fluid. Synovial PBZ concentrations (mean ± SD; 1.9 ± 2.1 μg/mL) were significantly lower (p = 0.03; CI,0.46-7.36) than serum PBZ concentrations (5.8 ± 5.1 μg/mL) at any time point. Physical and ultrasonographic measurements were not significantly different between PBZ- and placebo-IVRLP forelimbs. The most common histologic findings included focal deep dermal/subcutaneous hemorrhage and edema. Two horses showed perivasculitis and one horse showed a resolving thrombus in the cephalic vein of the PBZ-IVRLP limb. This horse also had severe perivasculitis and fibrinosuppurative dermatitis/panniculitis in the placebo-IVRLP limb. PBZ-IVRLP pharmacokinetics at a 2.2 mg/kg dose showed no benefit compared to systemic PBZ administration in standing adult horses. Local complications associated with PBZ-IVRLP were similar to placebo-IVRLP on physical and ultrasonographic evaluation.

Longitudinal evaluation of fentanyl concentrations in equine plasma and synovial fluid following application of transdermal fentanyl patches over one carpal joint.

To determine if transdermally delivered fentanyl can achieve greater concentrations of fentanyl in synovial fluid when applied over a synovial structure. Randomized, experimental study. Six healthy adult horses. Each horse had two 100 μg/h fentanyl matrix patches applied on the dorsal aspect of one, randomly assigned, carpometacarpal joint (CMCJ) for 48 h. Whole blood and bilateral synovial samples from the intercarpal joint were obtained at 0, 2, 6, 12, 24, 36 and 48 h. Fentanyl concentrations were measured with liquid chromatography-mass spectrometry. All subjects achieved detectable concentrations of fentanyl in both plasma and synovial fluid. Time to peak synovial and plasma concentration was 12 h. At 6 h, the synovial concentration in the untreated carpus (0.104 ng/mL ± 0.106) was lower than plasma fentanyl concentrations 0.31 ± 0.27 (p = .036). At 12 h, both treated (0.55 ng/mL ± 0.3) and untreated (0.53 ng/mL ± 0.28) synovial fluid fentanyl concentrations were lower than plasma (0.87 ng/mL ± 0.48) concentrations (p < .001 and p = .001, respectively). Synovial concentrations of fentanyl did not differbetween treated and untreated joints (p > 0.608 for all time points). Application of fentanyl matrix patches directly over the CMCJ did not result in increased fentanyl concentrations in the synovial fluid of the treated intercarpal joint in normal horses. There is likely no analgesic advantage to placing fentanyl patches directly over the affected joint, as it did not result in increased synovial concentrations at the tested site.

Effect of dipotassium-ethylenediaminetetraacetic acid or lithium-heparin treatments and storage times on selected clinicopathologic analytes in equine synovial fluid.

Sample processing methods and storage time affect the outcome of biochemical analysis. We aimed to assess the effects of dipotassium-ethylenediaminetetraacetic acid (K2-EDTA) and lithium-heparin treatments and storage times on selected analytes in equine synovial fluid (SF). Approximately 2 mL of SF from each horse (n = 7) were collected via femoropatellar joint arthrocentesis into K2-EDTA-treated bottles (K2-EDTA group), lithium-heparin-treated bottles (heparin group), and plain bottles (control group). The pH was determined using an electronic bench pH meter. The total nucleated cell count (TNCC) of samples was determined by hemocytometer method, while total protein (TP) concentrations, and lactate dehydrogenase (LDH) and alkaline phosphatase (ALP) activities of the samples were determined spectrophotometrically at 2, 8, 24, 48, and 168 hours postcollection while being maintained at approximately 4°C. TP concentrations in the anticoagulant-treated groups remained stable for 48 hours. TNCCs were stable for 8 hours. However, after 2 hours, ALP, LDH, and pH varied significantly (P < 0.05). At 2 hours, mean ALP and LDH activities were significantly elevated in the lithium-heparin treatment samples, while the activity of these analytes was similar in the K2-EDTA and control groups. At 8 hours, the TNCC and pH were significantly elevated in K2-EDTA treated groups, while values were similar in lithium-heparin and control groups. No significant variation was seen in TP values at 2 hours, irrespective of treatment. The analytes-except for TP-became unstable within a few hours postcollection. Lithium-heparin and K2-EDTA treatments significantly altered ALP, LDH, TNCCs, and pH but not the TP concentrations of equine SF. Studies establishing reference intervals for these analytes based on the anticoagulant used are warranted to limit misinterpretations in clinical or research settings.

Knee Osteotomy Decreases Joint Inflammation Based on Synovial Histology and Synovial Fluid Analysis

To examine the biological changes in the joints of patients with knee osteoarthritis (OA) before and after around-knee osteotomy (AKO), focusing on synovial fluid (SF) and synovial pathological changes. Patients who underwent AKO for medial compartment knee OA between 2019 and 2021 were examined. SF and synovium were obtained at the time of AKO and plate removal after bone union (mean, 16.8 months [range: 11-38 months] postoperatively). SF volume and interleukin (IL)-6 concentrations in SF were assayed using enzyme-linked immunosorbent assay. Synovitis was assessed histologically using a semiquantitative scoring system. Macrophage infiltration was assessed by immunohistochemistry using a semiquantitative score for F4/80 expression. The M1/M2 ratio was calculated using percentage of cells positive for CD80 and CD163. The expression of proinflammatory cytokines was assessed by the percentage of IL-1β- and IL-6-positive cells. The number of vascular endothelial growth factor-positive luminal structures was counted to assess angiogenesis. The change in each parameter was compared before and after AKO using the Wilcoxon matched-pairs signed-rank test. Twenty-four knees of 21 patients were included. SF volume and IL-6 concentration significantly decreased postoperatively (12.6 ± 2.1 mL vs 4.2 ± 0.6 mL; P < .0001 and 50.5 ± 8.6 pg/mL vs 20.7 ± 3.8 pg/mL; P= .0001, respectively). A significant reduction in synovitis score (P= .0001), macrophage infiltration (P < .0003), M1/M2 ratio (P < .0007), angiogenesis (P < .0001), and the percentage of IL-1β- and IL-6-positive cells in the intima (P < .008 and P < .002, respectively) was found after AKO. SF volume and IL-6 concentrations in the SF decreased and inflammatory synovium pathology improved after AKO. In addition to biomechanical changes, the biological environment of the joint can be improved after AKO. Level IV, retrospective therapeutic case series.

Better post-operative outcomes at 1-year follow-up are associated with lower levels of pre-operative synovitis and higher levels of IL-6 and VEGFA in unicompartmental knee arthroplasty patients.

Osteoarthritis (OA) is associated with inflammation, and residual inflammation may influence outcomes following knee arthroplasty. This may be more relevant for patients undergoing unicompartmental knee arthroplasty (UKA) due to larger remaining areas of native tissue. This study aimed to: (1) characterise inflammatory profiles for medial UKA patients and (2) investigate whether inflammation markers are associated with post-operative outcomes. This prospective, observational study has national ethics approval. Bloods, synovial fluid, tibial plateaus and synovium were collected from medial UKA patients in between 1 January 2021 and 31 December 2021. Cytokine and chemokine concentrations in serum and synovial fluid (SF) were measured with multiplexed assays. Disease severity of cartilage and synovium was assessed using validated histological scores. Post-operative outcomes were measured with Oxford Knee Score (OKS), Forgotten Joint Score (FJS-12) and pain scores. The study included 35 patients. SF VEGFA was negatively correlated with pre-operative pain at rest (r -0.5, p = 0.007), and FJS-12 at six-week (r 0.44, p = 0.02), six-month (r 0.61, p < 0.01) and one-year follow-up (r 0.63, p = 0.03). Serum and SF IL-6 were positively correlated with OKS at early follow-up (serum 6weeks, r 0.39, p = 0.03; 6months, r 0.48, p < 0.01; SF 6weeks, r 0.35, p = 0.04). At six weeks, increased synovitis was negatively correlated with improvements in pain at rest (r -0.41, p = 0.03) and with mobilisation (r -0.37, p = 0.047). Lower levels of synovitis and higher levels of IL-6 and VEGFA were associated with better post-operative outcomes after UKA, which could be helpful for identifying UKA patients in clinical practice. Level IV case series.

Poster 121: Association Between MRI Findings in ACL Injury and Subacute Synovial Fluid Inflammatory Biomarker Levels at the Time Of Surgery

Objectives: To Investigate the association between MRI findings following anterior cruciate ligament (ACL) injury and synovial fluid biomarker profile at the time of surgery Methods: Patients undergoing surgery for ACL injury from October 2011 to December 2021 were included in a database of subjects who had synovial fluid samples collected at the time of surgery. From this cohort, 82 subjects with acute ACL injury were retrospectively selected who met the following inclusion criteria: no history of prior knee trauma to suggest chronic ACL injury, history of acute non- contact knee injury, MRI examination of the knee performed within 3 weeks of knee injury, subsequent knee surgery performed between 4 and 12 weeks of knee injury, and no additional ligament tear requiring surgical treatment. All subjects underwent a preoperative MRI examination of the knee using multi-planar, multi-contrast fast spin-echo sequences. Synovial fluid was aspirated from the operative knee prior to surgical incision. Concentrations of inflammatory biomarkers including regulation on activation normal T cell expressed and secreted (RANTES), interleukin 6 (IL-6), vascular endothelial growth factor (VEGF), tissue inhibitor matrix metalloproteinase 1 (TIMP1), interleukin 1 receptor agonist (IL1-RA) and matrix metalloproteinase 3 (MMP-3) were measured. The MRI examinations of all subjects were reviewed by a fellowship trained musculoskeletal radiologist to assess the presence and severity of various MRI features of acute and chronic knee injury using the validated Anterior Cruciate Ligament Osteoarthritis Score (ACLOAS) system. Nonparametric Wilcoxon tests and Fisher’s exact test were used to compare demographic factors, MRI findings, clinical symptoms, and synovial fluid inflammatory biomarkers between clusters. K-means cluster analysis was used to identify 2 distinct clusters of subjects with acute ACL injury based on biomarkers concentrations in synovial fluid. Results: The mean age of the cohort was 31.6 ± 8 years, average body mass index (BMI) was 25.5 ± 4.4 kg/m2. There 42.7% (n=35) of the cohort was female and 85.4% (n=70) were non-smokers. Two distinct clusters were identified: cluster 1 included 45 subjects and was marked by older subjects (33.2 ± 9.1 years, p=.038) with lower levels of all inflammatory synovial fluid biomarkers. Cluster 2 included 37 subjects and was marked by younger patients (29.7 ± 6.1 years, p=.038) with a more pro-inflammatory synovial fluid biomarker profile, higher ACLOAS Hoffa’s synovitis score (p=.001), and a higher incidence of subchondral bone fractures (54%, p=.002). There were no differences between clusters in time from injury to surgery (p=), BMI (p=.141), or ACLOAS meniscus (p=.086), cartilage (p=.730), or effusion scores (p=.202) Conclusions: Given the variable outcomes seen following ACL reconstruction (ACLR) surgery, a better understanding of the pathophysiology following ACL injury is needed. This study found distinct cluster of patients who exhibited a higher inflammatory reaction at the time of surgery as well as more severe findings on preoperative MRI. These findings could be used to identified clinical parameters associated with more inflammation at the time of surgery and their effect on clinical outcomes of ACLR.

Quantification of chondroitin sulfate, hyaluronic acid and N-glycans in synovial fluid – A technical performance study

ObjectiveTo validate a quantitative high performance liquid chromatography (HPLC) assay for chondroitin sulfate (CS) and hyaluronic acid (HA) in synovial fluid, and to analyze glycan-patterns in patient samples. DesignSynovial fluid from osteoarthritis (OA, n ​= ​25) and knee-injury (n ​= ​13) patients, a synovial fluid pool (SF-control) and purified aggrecan, were chondroitinase digested and together with CS- and HA-standards fluorophore labelled prior to quantitative HPLC analysis. N-glycan profiles of synovial fluid and aggrecan were assessed by mass spectrometry. ResultsUnsaturated uronic acid and sulfated-N-acetylgalactosamine (ΔUA-GalNAc4S and ΔUA-GalNAc6S) contributed to 95% of the total CS-signal in the SF-control sample. For HA and the CS variants in SF-control the intra- and inter-experiment coefficient of variation was between 3–12% and 11–19%, respectively; tenfold dilution gave recoveries between 74 and 122%, and biofluid stability test (room temperature storage and freeze-thaw cycles) showed recoveries between 81 and 140%. Synovial fluid concentrations of the CS variants ΔUA-GalNAc6S and ΔUA2S-GalNAc6S were three times higher in the recent injury group compared to the OA group, while HA was four times lower. Sixty-one different N-glycans were detected in the synovial fluid samples, but there were no differences in levels of N-glycan classes between patient groups. The CS-profile (levels of ΔUA-GalNAc4S and ΔUA-GalNAc6S) in synovial fluid resembled that of purified aggrecan from corresponding samples; the contribution to the N-glycan profile in synovial fluid from aggrecan was low. ConclusionsThe HPLC-assay is suitable for analyzing CS variants and HA in synovial fluid samples, and the GAG-pattern differs between OA and recently knee injured subjects.

Effects of a 10% dimethyl sulfoxide solution on radiocarpal joint amikacin pharmacokinetics during intravenous regional limb perfusion in standing sedated horses.

To determine the effect of a 10% dimethyl sulfoxide (DMSO) solution on the peak concentration (CMAX ) of amikacin in the radiocarpal joint (RCJ) during intravenous regional limb perfusion (IVRLP) compared with 0.9% NaCl. Randomized crossover study. Seven healthy adult horses. The horses underwent IVRLP with 2 g of amikacin sulfate diluted to 60 mL using a 10% DMSO or 0.9% NaCl solution. Synovial fluid was collected from the RCJ at 5, 10, 15, 20, 25, and 30 minutes after IVRLP. The wide rubber tourniquet placed on the antebrachium was removed after the 30 min sample. Amikacin concentrations were quantified by a fluorescence polarization immunoassay. The mean CMAX and time to peak concentration (TMAX ) of amikacin within the RCJ were determined. A one-sided paired t-test was used to determine the differences between treatments. The significance level was p < .05. The mean ± SD CMAX in the DMSO group was 1361.8 ± 593 μg/mL and in the 0.9% NaCl group it was 860 ± 481.6 μg/mL (p = .058). Mean TMAX using the 10% DMSO solution was 23 and 18 min using the 0.9% NaCl perfusate (p = .161). No adverse effects were associated with use of the 10% DMSO solution. Although there were higher mean peak synovial concentrations using the 10% DMSO solution no difference in synovial amikacin CMAX between perfusate type was detected (p = .058). Use of a 10% DMSO solution in conjunction with amikacin during IVRLP is a feasible technique and does not negatively affect the synovial amikacin levels achieved. Further research is warranted to determine other effects of using DMSO during IVRLP.

A Gel-Based Proteomic Analysis Reveals Synovial α-Enolase and Fibrinogen β-Chain Dysregulation in Knee Osteoarthritis: A Controlled Trial.

The identification of synovial fluid (SF) biomarkers that could anticipate the diagnosis of osteoarthritis (OA) is gaining increasing importance in orthopaedic clinical practice. This controlled trial aims to assess the differences between the SF proteome of patients affected by severe OA undergoing Total Knee Replacement (TKR) compared to control subjects (i.e., subjects younger than 35, undergoing knee arthroscopy for acute meniscus injury). The synovial samples were collected from patients with Kellgren Lawrence grade 3 and 4 knee osteoarthritis undergoing THR (study group) and young patients with meniscal tears and no OA signs undergoing arthroscopic surgery (control group). The samples were processed and analyzed following the protocol defined in our previous study. All of the patients underwent clinical evaluation using the International Knee Documentation Committee (IKDC) subjective knee evaluation (main outcome), Knee Society Clinical Rating System (KSS), Knee injury and Osteoarthritis Outcome Score (KOOS), and Visual Analogue Scale (VAS) for pain. The drugs' assumptions and comorbidities were recorded. All patients underwent preoperative serial blood tests, including complete blood count and C-Reactive Protein (CRP). The synovial samples' analysis showed a significantly different fibrinogen beta chain (FBG) and alpha-enolase 1 (ENO1) concentration in OA compared to the control samples. A significant correlation between clinical scores, FBG, and ENO1 concentration was observed in osteoarthritic patients. Synovial fluid FBG and ENO1 concentrations are significantly different in patients affected by knee OA compared with non-OA subjects.

Concentration-dependent effects of leptin on osteoarthritis-associated changes in phenotype of human chondrocytes

ABSTRACT Metabolic syndrome is a risk factor for osteoarthritis. Elevated leptin levels have been implicated as a potential cause of this association. Previous studies have shown that supra-physiological leptin concentrations can induce osteoarthritis-like changes in chondrocyte phenotype. Here, we tested the effects of leptin in the concentration range found in synovial fluid on chondrocyte phenotype. Chondrocytes isolated from macroscopically normal regions of cartilage within osteoarthritic joints from patients undergoing knee arthroplasty, all with body mass index >30 kg/m2 were treated with 2-40 ng/ml leptin for 24 h. Chondrocyte phenotype marker expression was measured by RT-qPCR and western blot. The role of HES1 in mediating the effects of leptin was determined by gene knockdown using RNAi and over-expression using adenoviral-mediated gene delivery. Treatment of chondrocytes with 20 or 40 ng/ml leptin resulted in decreased SOX9 levels and decreased levels of the SOX9-target genes COL2A1 and ACAN. Levels of HES1 were lower and ADAMTS5 higher in chondrocytes treated with 20 or 40 ng/ml leptin. HES1 knockdown resulted in increased ADAMTS5 expression whereas over-expression of HES1 prevented the leptin-induced increase in ADAMTS5. An increase in MMP13 expression was only evident in chondrocytes treated with 40 ng/ml leptin and was not mediated by HES1 activity. High concentrations of leptin can cause changes in chondrocyte phenotype consistent with those seen in osteoarthritis. Synovial fluid leptin concentrations of this level are typically observed in patients with metabolic syndrome and/or women, suggesting elevated leptin levels may form part of the multifactorial network that leads to osteoarthritis development in these patients.

Vancomycin Concentrations in Synovial Fluid Do Not Reach Chondrotoxic Thresholds After Anterior Cruciate Ligament Reconstruction With Vancomycin-Soaked Autologous Soft Tissue Grafts: An In Vivo Prospective Observational Study in Humans

Background: Studies have revealed that vancomycin soaking of the anterior cruciate ligament (ACL) graft can drastically reduce the incidence of postoperative infections after ACL reconstruction. However, it remains unknown whether the chondrotoxic threshold of vancomycin in synovial fluid is exceeded during this process. Several studies investigated the chondrotoxic properties of vancomycin in in vitro experiments and described a concentration of 1000 µg/mL as the critical threshold. Purpose/Hypothesis: The purpose of the study was to measure the vancomycin concentration in synovial fluid after ACL reconstruction with vancomycin-soaked autografts. It was hypothesized that intra-articular vancomycin concentrations in the synovial fluid would not reach the chondrotoxic threshold of 1000 µg/mL after vancomycin soaking of autologous semitendinosus tendon and soft tissue quadriceps tendon grafts for ACL reconstruction. Study Design: Cohort study; Level of evidence, 3. Methods: The study enrolled 10 patients undergoing ACL reconstruction using 4-strand semitendinosus tendon autografts and 10 patients undergoing ACL reconstruction using soft tissue quadriceps tendon autografts. Before implantation, each harvested graft was intraoperatively wrapped in gauze swabs that had been soaked in a 5-mg/mL vancomycin solution. After wound closure, an aspirate of 5 mL of synovial fluid was taken from each patient. The vancomycin concentration of the aspirate was analyzed using high-performance liquid chromatography–tandem mass spectrometry. Spearman rho correlation coefficients were used to identify relationships between the parameters, and the t test was used to test for differences between graft types. A P value of <.05 was considered statistically significant. Results: The study included 20 patients (14 women and 6 men; age, 29.35 ± 11.3 years). The mean vancomycin concentration measured in the synovial fluid was 23.23 ± 21.68 µg/mL, with a minimum concentration of 2.32 µg/mL and a maximum concentration of 71.56 µg/mL. No significant difference was found between the 2 graft types (P = .911). Significant positive correlation (r = 0.644; P < .05) was observed only between the vancomycin concentration and the mean duration from initiation of vancomycin soaking of semitendinosus tendon grafts to implantation (13.4 ± 6 minutes). No correlations were observed between the vancomycin concentration and the duration from implantation to fluid aspiration or between the vancomycin concentration and the graft diameter (median, 8.5 mm; range, 6.0-10.0 mm) for both graft types. Conclusion: Chondrotoxic vancomycin concentrations ≥1000 µg/mL were not reached in any aspiration of synovial fluid after ACL reconstruction using soft tissue autografts that were intraoperatively soaked in a 5-mg/mL vancomycin solution. Against the backdrop of multiple studies that showed significantly reduced infection rates after ACL reconstruction when vancomycin soaking was used, this study suggests that the chondrotoxic properties of this method are negligible because of its submarginal intra-articular concentrations.

Association of Dipeptidylpeptidase 4 (CD26) With Chondrocyte Senescence and Radiographic Progression in Knee Osteoarthritis.

To evaluate the association of dipeptidylpeptidase 4 (DPP-4; also known as CD26) with cellular senescence of human cartilage and progression of knee osteoarthritis (OA). Articular cartilage sections and chondrocytes were acquired from 35 individuals undergoing total knee replacement for OA to evaluate the following: 1) the association between OA severity and established senescence markers (senescence-associated β-galactosidase activity and p16), which was quantified using immunohistochemistry and flow cytometry (n=19 samples); 2) the coexpression of DPP-4 with established senescence markers, which was assessed using flow cytometry; and 3) expression levels of anabolic and catabolic genes, senescence-related genes, and senescence-associated secretory phenotypes in DPP-4+ and DPP-4- cells, which were isolated using fluorescence-activated cell sorting or magnetic-activated cell sorting (n=16 samples). The concentration of soluble DPP-4 was measured in samples of synovial fluid and samples of plasma from the Prediction of Osteoarthritis Progression cohort and then evaluated for association with the severity of radiographic knee OA at baseline (n=65 samples) and the progression of structural radiographic OA (n=57 samples) over a 3-year period. DPP-4 expression was associated with higher senescence-associated β-galactosidase activity, p16 expression, senescence-related gene and catabolic gene (ADAMTS5, MMP13, IL6, and IL8) expression, higher senescence-associated secretory phenotype secretion, and lower anabolic gene (COL2A1 and ACAN) expression in primary chondrocytes. Synovial fluid DPP-4 concentration was associated with radiographic OA progression (odds ratio 105.32; P=0.015), proteases (synovial fluid matrix metalloproteinase 1 and matrix metalloproteinase 3), aggrecan degradation (synovial fluid sulfated glycosaminoglycan), indicators of activated macrophages (synovial fluid CD14 and CD163), and inflammation (synovial fluid interleukin-6). Our study identifies DPP-4 as a key surface marker in senescent chondrocytes and a predictor of radiographic OA progression.

Serum nerve growth factor in horses with osteoarthritis-associated lameness.

Nerve growth factor (NGF) is a neurotrophin that is increased in osteoarthritic joints of horses. In humans, NGF has been associated with pain, and both synovial and serum NGF concentrations are increased in osteoarthritic patients. Studies in humans also have shown that serum NGF concentration can increase with stress. Serum NGF concentration should be evaluated in horses with osteoarthritis-associated lameness. Quantify and compare serum NGF concentration in horses with osteoarthritis-associated lameness and sound horses. Additionally, the impact of short-term stress on serum NGF concentration was investigated. Lame horses with radiographic evidence of osteoarthritis (n = 20), lame horses without radiographic changes in the affected joint (n = 20) and sound horses (n = 20). In addition, horses with acute fractures (n = 9) were sampled. To determine the effect of stress, serum from horses subjected to a stressful event (transportation, n = 5; stress confirmed by increased serum cortisol concentration) was analyzed. Cross-sectional clinical study (lame, sound, and fracture cohorts) and experimental longitudinal study (stress cohort). Serum NGF concentration was determined using a quantitative sandwich ELISA. Serum NGF concentration was increased in lame horses with radiographic evidence of osteoarthritis (P < .0001; median, 238 pg/mL; interquartile range [IQR], 63-945 pg/mL) and in lame horses without radiographic evidence of osteoarthritis in the painful joint (P < .05; median, 31 pg/mL; IQR, 31-95 pg/mL) compared with sound horses (median, 31 pg/mL; IQR, 31-46 pg/mL). Serum NGF concentration did not increase with short-term stress and was low in horses with fracture-associated pain. Serum NGF concentration was high in the cohort with advanced osteoarthritis and should be investigated as a marker for osteoarthritis-associated pain.

Increased Effusion Synovitis for Those With a Dysregulated Inflammatory Response After an Anterior Cruciate Ligament Injury.

Introduction The progression to posttraumatic osteoarthritis (PTOA) after an anterior cruciate ligament (ACL) injury is likely multifactorial, involving biological, mechanical, and psychosocial factors. Following acute joint trauma, there appears to be a subset of patients that demonstrate a dysregulated inflammatory response. This pro-inflammatory phenotype, or "Inflamma-type," is characterized by an amplified pro-inflammatory response combined with a lack of attendant anti-inflammatory responseand has been observed following both an ACL injury and an intra-articular fracture. The aims of this study were to: 1) compare magnetic resonance imaging (MRI)-measured effusion synovitis between those with vs. without a dysregulated inflammatory response, and 2) assess the correlations between effusion synovitis and synovial fluid concentrations of proinflammatory cytokines, degradative enzymes, and synovial fluid biomarkers of cartilage degradation. Methods A cluster analysis was previously performed with synovial fluid concentrations of biomarkers of inflammation and cartilage degradation from 35 patients with acute ACL injuries. Patients were then categorized into two groups: a pro-inflammatory phenotype ("Inflamma-type") and those with a more normal inflammatory response to injury (NORM). Effusion synovitis measured from each patient's preoperative clinical MRI scan was compared between the Inflamma-type and NORM groups using an independent, two-tailed t-test. In addition, Spearman's rho non-parametric correlations were calculated to evaluate the relationship between effusion synovitis and each of the synovial fluid concentrations of pro-inflammatory cytokines, degradative enzymes, and biomarkers of cartilage degradation and bony remodeling. Results Effusion synovitis was significantly greater for the Inflamma-type (10.9±3.8 mm) than the NORM group (7.4±4.4 mm, p=0.04, Cohen's d=0.82). Effusion synovitis significantly correlated with matrix metalloproteinase-3 (rho=0.63, p<0.001), matrix metalloproteinase-1 (rho=0.50, p=0.003), and sulfated glycosaminoglycan (rho=0.42, p=0.01). No other significant correlations were present. Conclusion Effusion synovitis was significantly greater for those that demonstrated a dysregulated inflammatory response after acute ACL injury than those with a more normal response to injury. Effusion synovitis was also found to significantly correlate with synovial fluid concentrations of degradative enzymes and a biomarker of early cartilage degradation. Future work is needed to determine if non-invasive methods, such as MRI or ultrasound, may accurately identify patients within this pro-inflammatory phenotypeand whether this subset is more prone to more rapid PTOA changes after injury.

Synovial fluid mitochondrial DNA concentration reflects the degree of cartilage damage after naturally occurring articular injury

To evaluate mitochondrial DNA (mtDNA) release from injured chondrocytes and investigate the utility of synovial fluid mtDNA concentration in early detection of posttraumatic osteoarthritis. We measured mtDNA release using four models of osteoarthritis: in vitro interleukin-1β stimulation of cultured equine chondrocytes, ex vivo mechanical impact of bovine cartilage explants, in vivo mechanical impact of equine articular cartilage, and naturally occurring equine intraarticular fracture. In our in vivo model, one group was treated with an intraarticular injection of the mitoprotective peptide SS-31 following cartilage injury. mtDNA content was quantified using qPCR. For naturally occurring cases of joint injury, clinical data (radiographs, arthroscopic video footage) were scored for criteria associated with degenerative joint disease. Chondrocytes released mtDNA in the acute time frame following inflammatory and mechanical cellular stress in vitro. mtDNA was increased in equine synovial fluid following experimental and naturally occurring injury to the joint surface. In naturally occurring posttraumatic osteoarthritis, we found a strong positive correlation between the degree of cartilage damage and mtDNA concentration (r=0.80, P=0.0001). Finally, impact-induced mtDNA release was mitigated by mitoprotective treatment. Changes in synovial fluid mtDNA occur following joint injury and correlate with the severity of cartilage damage. Mitoprotection mitigates increases in synovial fluid mtDNA suggesting that mtDNA release may reflect mitochondrial dysfunction. Further investigation of mtDNA as a potentially sensitive marker of early articular injury and response to mitoprotective therapy is warranted.

Effects of a Single Intra-Articular Injection of 2% Lidocaine or 0.5% Bupivacaine on Synovial Fluid Acute Phase Protein Concentrations in Healthy Horses

The purpose of this study was to compare the extent of inflammation response in the middle carpal joints of healthy horses following intra-articular injection of 2% lidocaine, 0.5% bupivacaine, or 0.9% saline solution. The right middle carpal joint of 20 horses was injected with 5 mL of 0.5% bupivacaine (GB, n = 10) or 5 mL of 2% lidocaine (GL, n = 10). The left middle carpal joint of horses was used as a control (5 mL 0.9% saline). Serum and synovial fluid (SF) were aseptically collected before and at predetermined times after each injection. Serum and synovial fluid protein, albumin, transferrin, haptoglobin, ceruloplasmin, α1-antitripsin, and α1-acid glycoprotein concentrations were measured by sodium dodecyl sulfate polyacrylamide gel electrophoresis and compared among treatments. The results were submitted to analysis of variance using the SAS statistical program, and means were compared by the Student–Newman–Keuls test (P < .05). Both lidocaine and bupivacaine induced serum and SF changes indicative of inflammation, but the magnitude of those changes was more pronounced for lidocaine. Administration of 0.9% saline also induced an inflammatory reaction, but the magnitude of these changes was less pronounced than those caused by GB and GL. The results suggested that bupivacaine is safer than lidocaine for intra-articular injection in horses. Saline solution should not be used as an adjunct to intra-articular injections in horses.

Plasma and Synovial Fluid Cell-Free DNA Concentrations Following Induction of Osteoarthritis in Horses.

Biomarkers for osteoarthritis (OA) in horses have been extensively investigated, but translation into clinical use has been limited due to cost, limited sensitivity, and practicality. Identifying novel biomarkers that overcome these limitations could facilitate early diagnosis and therapy. This study aimed to compare the concentrations of synovial fluid (SF) and plasma cell-free DNA (cfDNA) over time in control horses with those with induced carpal OA. Following an established model, unilateral carpal OA was induced in 9 of 17 healthy Thoroughbred fillies, while the remainder were sham-operated controls. Synovial fluid and plasma samples were obtained before induction of OA (Day 0) and weekly thereafter until Day 63, and cfDNA concentrations were determined using fluorometry. The SF cfDNA concentrations were significantly higher for OA joints than for sham-operated joints on Days 28 (median 1430 μg/L and 631 μg/L, respectively, p = 0.017) and 63 (median 1537 μg/L and 606 μg/L, respectively, p = 0.021). There were no significant differences in plasma cfDNA between the OA and the sham groups after induction of carpal OA. Plasma cfDNA measurement is not sufficiently sensitive for diagnostic purposes in this induced model of OA. Synovial fluid cfDNA measurement may be used as a biomarker to monitor early disease progression in horses with OA.

Pharmacokinetics of intra-articular buprenorphine in horses with lipopolysaccharide-induced synovitis.

The objective of this study was to describe the pharmacokinetics of intra-articular (IA) administered buprenorphine in horses with lipopolysaccharide (LPS)-induced synovitis. Radiocarpal synovitis was induced in six healthy adult horses with the IA injection of LPS (0.5ng/joint) on two occasions in a randomized cross-over design. Treatments (IA buprenorphine (IAB) at 5μg/kg plus intravenous saline; and intravenous buprenorphine (IVB) at 5μg/kg plus IA saline) were administered 4h following LPS injection. Concentrations of buprenorphine were assessed in plasma and synovial fluid (SF) at 0.5, 2, 6, 12, and 24 h after administration. Pharmacokinetic parameters after IVB and IAB in plasma and synovial fluid were calculated using a nonlinear mixed effects model. IAB was detectable in SF of all horses at 24 h [median concentration of 6.2 (3.46-22.6) ng/mL]. IAB resulted in a median plasma concentration of 0.59 (0.42-1.68) ng/mL at 0.5h and was detectable in all subjects for up to 6 h and in two horses for up to 12 h. IVB resulted in SF concentrations detected up to 6 h in all horses [median concentration of 0.12 (0.07-0.82) ng/mL]. Results suggest that IA buprenorphine remains present in the inflamed joint for at least 24 h and systemic absorption occurs.

Complement system dysregulation in synovial fluid from patients with persistent inflammation following anterior cruciate ligament reconstruction surgery

IntroductionPatients with anterior cruciate ligament injury are at high risk of posttraumatic osteoarthritis and their response to reconstructive surgery and rehabilitation vary. Proteins identified in the orchestration of the acute inflammatory response may be predictive of patient outcomes. ObjectiveAn unbiased, bottom-up proteomics approach was used to discover novel targets for therapeutics in relation to dysregulation in the orchestration of inflammatory pathways implicated in persistent joint inflammation subsequent to joint trauma. MethodsSynovial fluid was aspirated from patients at 1 week and 4 weeks after anterior cruciate ligament reconstruction (ACLR) and interleukin 6 (IL-6) concentrations were quantified by enzyme-linked immunosorbent assay. Patients were segregated into IL-6low and IL-6high groups based on IL-6 concentrations in synovial fluid at 4-weeks postoperation and proteins in synovial fluid were analyzed using qualitative, bottom-up proteomics. Abundance ratios were calculated for IL-6high and IL-6low groups as 4 weeks postoperation:1 week postoperation. ResultsA total of 291 proteins were detected in synovial fluid, 34 of which were significantly (P < .05) differentially regulated between groups. Proteins associated with the classical and alternative complement cascade pathways were increased in the IL-6high compared to IL-6low group. Insulin-like growth factor-binding protein 6 (IGFBP-6) was increased by nearly 60-fold in the IL-6low group. ConclusionsPatients segregated by IL-6 concentration in synovial fluid at 4 weeks post-ACLR demonstrated differential regulation of multiple pathways, providing opportunities to investigate novel targets, such as IGFBP-6, and to take advantage of therapeutics already approved for clinical use in other diseases that target inflammatory pathways, including the complement system.

Hyaluronic Acid in Synovial Fluid Prevents Neutrophil Activation in Spondyloarthritis

Spondyloarthritis (SpA) patients suffer from joint inflammation resulting in tissue damage, characterized by the presence of numerous neutrophils in the synovium and synovial fluid (SF). As it is yet unclear to what extent neutrophils contribute to the pathogenesis of SpA, we set out to study SF neutrophils in more detail. We analyzed the functionality of SF neutrophils of 20 SpA patients and 7 disease controls, determining ROS production and degranulation in response to various stimuli. In addition, the effect of SF on neutrophil function was determined. Surprisingly, our data show that SF neutrophils in SpA patients have an inactive phenotype, despite the presence of many neutrophil-activating stimuli such as GM-CSF and TNF in SF. This was not due to exhaustion as SF neutrophils readily responded to stimulation. Therefore, this finding suggests that one or more inhibitors of neutrophil activation may be present in SF. Indeed, when blood neutrophils from healthy donors were activated in the presence of increasing concentrations of SF from SpA patients, degranulation and ROS production were dose-dependently inhibited. This effect was independent of diagnosis, gender, age, and medication in the patients from which the SF was isolated. Treatment of SF with the enzyme hyaluronidase strongly reduced the inhibitory effect of SF on neutrophil activation, indicating that hyaluronic acid that is present in SF may be an important factor in preventing SF neutrophil activation. This finding provides novel insights into the role of soluble factors in SF regulating neutrophil function and may lead to the development of novel therapeutics targeting neutrophil activation via hyaluronic acid or associated pathways.