Equine Synovial Fluid Research Articles

Osteoarthritic sclerotic subchondral osteoblasts secreted elevated concentration of Fibulin-3 fragments in vitro

Osteoarthritic sclerotic subchondral osteoblasts secreted elevated concentration of Fibulin-3 fragments in vitro

Agreement of manual cell counts and automated counts of the scil Vet abc Plus+ hematology analyzer for analysis of equine synovial fluid

The purpose of this study was to determine whether the scil Vet abc Plus+ (SCIL Animal Care Company, Altorf, France), an impedance hematology analyzer, can accurately quantify and differentiate nucleated blood cells (NBCs) in equine synovial fluid. Synovial fluid samples (n=242) in different stages of experimentally induced inflammation were analyzed with and without hyaluronidase pretreatment and compared to manual hemocytometer counts and smear reviews. No significant effect of hyaluronidase pretreatment was observed. Total nucleated cell counts of the scil Vet abc Plus+ were significantly higher compared to the manual method (P=0.02), yet the difference was small and clinically irrelevant (ratio manual/automated count equal to 0.97 with 95% CI [0.95, 1.00]). Differential cell counts of the scil Vet abc Plus+ were not accurate. In conclusion, the scil Vet abc Plus+ hematology analyzer is highly accurate for quantification, but not accurate for differentiation of NBCs in equine synovial fluid.

Synovial fluid pretreatment with hyaluronidase facilitates isolation of CD44+ extracellular vesicles

Extracellular vesicles (EVs) in synovial fluid (SF) are gaining increased recognition as important factors in joint homeostasis, joint regeneration, and as biomarkers of joint disease. A limited number of studies have investigated EVs in SF samples of patients with joint disease, but knowledge on the role of EVs in healthy joints is lacking. In addition, no standardized protocol is available for isolation of EVs from SF. Based on the high viscosity of SF caused by high concentrations of hyaluronic acid (HA) – a prominent extracellular matrix component – it was hypothesized that EV recovery could be optimized by pretreatment with hyaluronidase (HYase). Therefore, the efficiency of EV isolation from healthy equine SF samples was tested by performing sequential ultracentrifugation steps (10,000g, 100,000g and 200,000g) in the presence or absence of HYase. Quantitative EV analysis using high-resolution flow cytometry showed an efficient recovery of EVs after 100,000g ultracentrifugation, with an increased yield of CD44+ EVs when SF samples were pretreated with HYase. Morphological analysis of SF-derived EVs with cryo-transmission-electron microscopy did not indicate damage by high-speed ultracentrifugation and revealed that most EVs are spherical with a diameter of 20–200 nm. Further protein characterization by Western blotting revealed that healthy SF-derived EVs contain CD9, Annexin-1, and CD90/Thy1.1. Taken together, these data suggest that EV isolation protocols for body fluids that contain relatively high amounts of HA, such as SF, could benefit from treatment of the fluid with HYase prior to ultracentrifugation. This method facilitates recovery and detection of CD44+ EVs within the HA-rich extracellular matrix. Furthermore, based on the findings presented here, it is recommended to sediment SF-derived EVs with at least 100,000g for optimal EV recovery.

Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis.

Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP) cells in equine synovial fluid (SF) and to determine the effect of fibroblast growth factor 2 (FGF-2) on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU) assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with or without FGF-2. Following monolayer expansion and serial immunophenotyping, cells were transferred to chondrogenic pellet cultures. Pellets were analyzed for chondrogenic mRNA expression and cartilage matrix secretion. There was a mean of 59.2 CFU/mL of SF. FGF-2 increased the number of population doublings during two monolayer passages and halved the population doubling times. FGF-2 did not alter the immunophenotype of SF-CPs during monolayer expansion, nor did FGF-2 compromise chondrogenesis. Hypertrophic phenotypic markers were not expressed in control or FGF-2 groups. FGF-2 did prevent the development of a “fibroblastic” cell layer around pellet periphery. FGF-2 significantly accelerates in vitro SF-CP expansion, the major hurdle to clinical application of this cell population, without detrimentally affecting subsequent chondrogenic capacity.

Characterization of mesenchymal stem cells derived from the equine synovial fluid and membrane.

BackgroundIsolation of mesenchymal stem cells (MSCs) in equines, has been reported for different tissues including bone marrow, adipose, umbilical cord, peripheral blood, and yolk sac. In regard to the MSCs derived from synovial fluid (SF) or membrane (SM), there is data available for humans, dogs, pigs, goats and horses. Especially in equines, these cells have being considered promising candidates for articular regeneration. Herein, we established and characterized MSCs obtained from equine SF and SM. Samples were obtained during arthroscopy and cultured using MEM (Minimum Essential Medium). MSCs were characterized by morphology and expression of specific markers for stem cells, pluripotency, inflammation, and cell cycle.ResultsThe medium MEM was more effective (97 % ± 2) to maintain both cultures. The cultures were composed by adherent cells with fibroblast-like shape, which had a growth pattern represented by a sigmoidal curve. After the expansion, the cells were analyzed by flow cytometry for stem cells, inflammatory, and cell cycle markers, and both lineages showed significant expression of CD45, Oct3/4, Nanog, CD105, CD90, CD34, CD117, CD133, TRA-1-81, VEGF, and LY6a. In contrast, there were differences in the cell cycle phases between the lineages, which was not observed in relation to the mitochondrial electrical potential.ConclusionGiven the large impact that joint pathology has on the athletic performance horses, our results suggested that the SF and SM are promising sources of stem cells with satisfactory characteristics of growth and gene expression that can be used in equine regenerative medicine.

Total nucleated cell and leukocyte differential counts in canine pleural and peritoneal fluid and equine synovial fluid samples: comparison of automated and manual methods.

Rapid and precise measurement of total and differential nucleated cell counts is a crucial diagnostic component of cavitary and synovial fluid analyses. The objectives of this study included (1) evaluation of reliability and precision of canine and equine fluid total nucleated cell count (TNCC) determined by the benchtop Abaxis VetScan HM5, in comparison with the automated reference instruments ADVIA 120 and the scil Vet abc, respectively, and (2) comparison of automated with manual canine differential nucleated cell counts. The TNCC and differential counts in canine pleural and peritoneal, and equine synovial fluids were determined on the Abaxis VetScan HM5 and compared with the ADVIA 120 and Vet abc analyzer, respectively. Statistical analyses included correlation, least squares fit linear regression, Passing-Bablok regression, and Bland-Altman difference plots. In addition, precision of the total cell count generated by the VetScan HM5 was determined. Agreement was excellent without significant constant or proportional bias for canine cavitary fluid TNCC. Automated and manual differential counts had R(2) <.5 for individual cell types (least squares fit linear regression). Equine synovial fluid TNCC agreed but with some bias due to the VetScan HM5 overestimating TNCC compared to the Vet abc. Intra-assay precision of the VetScan HM5 in 3 fluid samples was 2-31%. The Abaxis VetScan HM5 provided rapid, reliable TNCC for canine and equine fluid samples. The differential nucleated cell count should be verified microscopically as counts from the VetScan HM5 and also from the ADVIA 120 were often incorrect in canine fluid samples.

Determination of the unsaturated disaccharides of hyaluronic acid in equine synovial fluid by high-performance liquid chromatography and fluorescence detection.

BackgroundThe purpose of this study was to develop and validate an analytical method to determine the presence of hyaluronic acid derived disaccharides in equine synovial fluid.FindingsA high-performance liquid chromatography method for the determination of hyaluronic acid derived unsaturated disaccharides in equine synovial fluid was developed and validated. The method is based on the measurement of unsaturated disaccharides released by digestion of linear hyaluronic acid molecules. The method showed linearity (r2 = 0.996) over the full working concentration range 0.89-30 mg/l. Relative standard deviation of intra- and inter-day precision ranged from of 4.3-6.7% and 7.1-7.8% respectively. The detection limit was 0.3 mg/l corresponding to 20 mg/l in synovial fluid. Accuracy of the assay was 97-103%. This method was evaluated by determining the concentration of unsaturated disaccharides from hyaluronic acid in synovial fluid of horses with lameness in the metacarpo-/metatarsophalangeal joint localized with positive response to intra-articular anesthesia.ConclusionsThe described method is valid for determination of hyaluronic acid derived disaccharides in equine synovial fluid. This method was applied to a larger research project dealing with a new form of intra-articular therapy in horses with arthritic diseases.

Synovial Fluid Lubricant Properties Are Transiently Deficient After Arthroscopic Articular Cartilage Defect Repair With Platelet-Enriched Fibrin Alone and With Mesenchymal Stem Cells

Background:Following various types of naturally occurring traumatic injury to an articular joint, the lubricating ability of synovial fluid is impaired, with a correlated alteration in the concentration and/or structure of lubricant molecules, hyaluronan, and proteoglycan-4 (PRG4). However, the effect of arthroscopic cartilage repair surgery on synovial fluid lubricant function and composition is unknown.Hypothesis:Arthroscopic treatment of full-thickness chondral defects in horses with (1) platelet-enriched fibrin or (2) platelet-enriched fibrin + mesenchymal stem cells leads to equine synovial fluid with impaired lubricant function and hyaluronan and PRG4 composition.Study Design:Controlled laboratory study.Methods:Equine synovial fluid was aspirated from normal joints at a preinjury state (0 days) and at 10 days and 3 months following fibrin or fibrin + mesenchymal stem cell repair of full-thickness chondral defects. Equine synovial fluid samples were analyzed for friction-lowering boundary lubrication of normal articular cartilage (static and kinetic friction coefficients) and concentrations of hyaluronan and PRG4, as well as molecular weight distribution of hyaluronan. Experimental groups deficient in lubrication function were also tested for the ability of exogenous high–molecular weight hyaluronan to restore lubrication function.Results:Lubrication and biochemical data varied with time after surgery but generally not between repair groups. Relative to preinjury, kinetic friction was higher (+94%) at 10 days but returned to baseline levels at 3 months, while static friction was not altered. Correspondingly, hyaluronan concentration was transiently lower (−64%) and shifted toward lower molecular weight forms, while PRG4 concentration was increased (+210%) in 10-day samples relative to preinjury levels. Regression analysis revealed that kinetic friction decreased with increasing total and high–molecular weight hyaluronan. Addition of high–molecular weight hyaluronan to bring 10-day hyaluronan levels to 2.0 mg/mL restored kinetic friction to preinjury levels.Conclusion:Following arthroscopic surgery for cartilage defect repair, synovial fluid lubrication function is transiently impaired, in association with decreased hyaluronan concentration. This functional deficiency in synovial fluid lubrication can be counteracted in vitro by addition of high–molecular weight hyaluronan.Clinical Relevance:Synovial fluid lubrication is deficient shortly after arthroscopic cartilage repair surgery, and supplementation with high–molecular weight hyaluronan may be beneficial.

A Rapid Screen for Four Corticosteroids in Equine Synovial Fluid‡

Most antidoping method development in the equine industry has been for plasma and urine, though there has been recent interest in the analysis of synovial fluid for evidence of doping by intra-articular corticosteroid injection. Published methods for corticosteroid analysis in synovial fluid are primarily singleplex methods, do not screen for all corticosteroids of interest and are not adequately sensitive. The purpose of this study is to develop a rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS-MS) screening method for the detection of four of the most common intra-articularly administered corticosteroids--betamethasone, methylprednisolone, methylprednisolone acetate and triamcinolone acetonide. Sample preparation consisted of protein precipitation followed by a basified liquid-liquid extraction. LC-MS-MS experiments consisted of a six-min isocratic separation using a Phenomenex Polar-RP stationary phase and a mobile phase consisting of 35% acetonitrile, 5 mM ammonium acetate and 0.1% formic acid in nanopure water. The detection system used was a triple quadrupole mass analyzer with thermospray ionization, and compounds were identified using selective reaction monitoring. The method was validated to the ISO/IEC 17025 standard, and real synovial fluid samples were analyzed to demonstrate the application of the method in an antidoping context. The method was highly selective for the four corticosteroids with limits of detection of 1-3 ng/mL. The extraction efficiency was 50-101%, and the matrix effects were 14-31%. These results indicate that the method is a rapid and sensitive screen for the four corticosteroids in equine synovial fluid, fit for purpose for equine antidoping assays.

Effects of Omega-3 Long Chain Polyunsaturated Fatty Acid Supplementation on Equine Synovial Fluid Fatty Acid Composition and Prostaglandin E2

To determine if supplementation of omega-3 long chain polyunsaturated fatty acid (▪-3 LCPUFA) alters synovial fluid fatty acid composition and prostaglandin E2 (PGE2) concentration of mature, healthy horses. Twenty, nonpregnant light breed mares were assigned into one of three daily dietary treatments. Group 1 (MARINE) received 38 g total of the ▪-3 LCPUFA alpha-linolenic acid (ALA, 2 g), eicosapentaenoic acid (EPA, 7.6 g), and docosahexaenoic acid (DHA, 26.6 g) via a marine-derived supplement; group 2 (FLAX) received 38 g of ▪-3 ALA via a flaxseed supplement; and group 3 (CONT) did not receive additional ▪-3 LCPUFA. Blood was taken at baseline, 30, 60, and 90 days of supplementation and plasma separated. After 90 days of supplementation, 3 mL of synovial fluid was obtained through arthrocentesis. Plasma and synovial fluid were analyzed to identify fatty acid profiles and determine PGE2 concentration. MARINE synovial fluid fatty acids contained higher of EPA and DHA compared with the CONT group and higher DHA levels compared with FLAX group. Eicosapentaenoic acid was not detected in synovial fluid from the FLAX group. Prostaglandin E2 did not differ (P > .05) among horses; however, the MARINE group tended (P = .10) to have lower synovial PGE2 concentration compared with CONT horses. The presence of EPA and DHA only in MARINE synovial fluid and plasma suggests that direct supplementation of EPA and DHA is needed to modify fatty acid composition. A tendency for lower synovial PGE2 in healthy horses receiving oral EPA and/or DHA merits further investigation in the ▪-3 supplementation effect on prostaglandin production.

Do blood components affect the production of reactive oxygen species (ROS) by equine synovial cells in vitro?

Blood-derived products are commonly administered to horses and humans to treat many musculoskeletal diseases, due to their potential antioxidant and anti-inflammatory effects. Nevertheless, antioxidant effects have never been shown upon horse synovial fluid cells in vitro. If proved, this could give a new perspective to justify the clinical application of blood-derived products. The aim of the present study was to investigate the antioxidant effects of two blood-derived products - plasma (unconditioned blood product - UBP) and a commercial blood preparation (conditioned blood product - CBP)¹ - upon stimulated equine synovial fluid cells. Healthy tarsocrural joints (60) were tapped to obtain synovial fluid cells; these cells were pooled, processed, stimulated with lipopolysaccharide (LPS) or phorbol 12-myristate 13-acetate (PMA), and evaluated by flow cytometry for the production of reactive oxygen species (ROS). Upon addition of any blood-derived product here used - UBP and CBP - there was a significant decrease in the oxidative burst of synovial fluid cells (P&lt;0.05). There was no difference between UBP and CBP effects. In conclusion, treatment of stimulated equine synovial cells with either UBP or CBP efficiently restored their redox equilibrium.

Intra-articular ketamine administration in equine midcarpal joint: clinical, biochemical and cytological evaluations.

Summary Providing suitable analgesia following diagnostic and therapeutic arthroscopic surgeries, that are considered to cause some degree of postoperative pain, is a necessity in equine practice. The aim of the present study was to evaluate the equine synovial fluid biochemical and cytological changes as well as clinical assessments of the joint following intra-articular administration of ketamine. Six adult healthy donkeys were selected after clinical examination. Synovial fluid samples were taken from both middle carpal joints after routine preparation. Ketamine 2 mg/kg and 100 mg lidocaine 2% were administered to the right and left joints, respectively. Synovial fluid collection from the joints was performed at 12, 24, 48 and 192 h after medication. Cytological examination, total protein, glucose, specific gravity, alkaline phosphates (ALP), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), viscosity and quality of mucin clot were measured. Joint circumference, flexion test and lameness examination, stimulation of the joint skin area and radiographic examination were performed as clinical evaluations. Comparison of treatments was performed by nonparametric sign test and Wilcoxon rank sum test. Significance level was set to P≤0.05. In the ketamine group, increased joint circumference, positive flexion test and negative response to the ball-point pressure of the joint skin area were seen, unlike that of lidocaine. Mucin clot quality test and viscosity, the amount of total nucleated cell count (TNCC), mononuclear and neutrophil count, specific gravity, total protein content, ALP, AST and LDH of the ketamine treated joints revealed considerable differences between various sampling times compared to the 0 time and also between the ketamine and lidocaine groups (P>0.05). It was concluded that intra-articular ketamine administration in equine carpal joint resulted in acute inflammatory changes, and failed to demonstrate analgesia; therefore, it is not safe to the joint environment and is not recommended as a local analgesic following arthroscopic surgeries.

Effects of equine joint injury on boundary lubrication of articular cartilage by synovial fluid: Role of hyaluronan

To compare equine synovial fluid (SF) from injured and control joints for cartilage boundary lubrication function; concentrations of the putative boundary lubricant molecules hyaluronan (HA), proteoglycan 4 (PRG4), and surface-active phospholipids (SAPLs); relationships between lubrication function and composition; and lubrication restoration by addition of HA. Equine SF from normal joints, joints with acute injury, and joints with chronic injury were analyzed for boundary lubrication of normal articular cartilage (kinetic friction coefficient [μ(kinetic) ]). Equine SF samples were analyzed for HA, PRG4, and SAPL concentrations and HA molecular weight distribution. The effect of the addition of HA, of different concentrations and molecular weight, on the μ(kinetic) of equine SF samples from normal joints and joints with acute injury was determined. The μ(kinetic) of equine SF from joints with acute injury (0.036) was higher (+39%) than that of equine SF from normal joints (0.026). Compared to normal equine SF, SF from joints with acute injury had a lower HA concentration (-30%) of lower molecular weight forms, higher PRG4 concentration (+83%), and higher SAPL concentration (+144%). Equine SF from joints with chronic injury had μ(kinetic) , PRG4, and SAPL characteristics intermediate to those of equine SF from joints with acute injury and normal equine SF. Regression analysis revealed that the μ(kinetic) value decreased with increasing HA concentration in equine SF. The friction-reducing properties of HA alone improved with increasing concentration and molecular weight. The addition of high molecular weight HA (4,000 kd) to equine SF from joints with acute injury reduced the μ(kinetic) to a value near that of normal equine SF. In the acute postinjury stage, equine SF exhibits poor boundary lubrication properties, as indicated by a high μ(kinetic) . HA of diminished concentration and molecular weight may be the basis for this, and adding HA to deficient equine SF restored lubrication function.

Physiological Concentrations of Acute-Phase Proteins and Immunoglobulins in Equine Synovial Fluid

Synovial fluid (SF) is capable of reflecting infectious, immunological, or inflammatory joint conditions in horses by altering its composition and appearance. Although plasma and SF compositions are quantitatively different, this latter compartment reflects changes in plasma macromolecules. Therefore, changes in serum immunoglobulin protein concentrations tend also to alter intracapsular levels. Therefore, it is necessary to know the physiological concentrations of proteins present in SF. The aim of this study was to determine the levels of total protein, albumin, transferrin, haptoglobin, α1-acid glycoprotein, ceruloplasmin, and immunoglobulins A and G in SF of six healthy horses. The synovial proteinogram was obtained by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The SF proteins reached a maximum of 25% of serum concentrations, varying inversely with molecular weight of the protein, except for the ceruloplasmin.

Gambling on putative biomarkers of osteoarthritis and osteochondrosis by equine synovial fluid proteomics

Osteoarthritis (OA) and osteochondrosis (OC) are two of the main challenges in orthopedics, whose definitive diagnosis is usually based on radiographic/arthroscopic evidences. Their early diagnosis should allow preventive or timely therapeutic actions, which are generally precluded from the poor relationships occurring between symptomatologic and radiographic evidences. These limitations should be overcome by improving the knowledge on articular tissue metabolism and on molecular factors regulating its normal homeostasis, also identifying novel OA and OC biomarkers suitable for their earlier diagnoses, whenever clinical/pathological inflammatory scenarios between these joint diseases seem somewhat related. To identify proteins involved in their aetiology and progression, we undertook a differential proteomic analysis of equine synovial fluid (SF), which compared the protein pattern of OA or OC patients with that of healthy individuals. Deregulated proteins in OA and OC included components related to inflammatory state, coagulation pathways, oxidative stress and matrix damage, which were suggestive of pathological alterations in articular homeostasis, plasma-SF exchange, joint nutritional status and vessel permeability. Some proteins seemed commonly deregulated in both pathologies indicating that, regardless of the stimulus, common pathways are affected and/or the animal joint uses the same molecular mechanisms to restore its homeostasis. On the other hand, the increased number of deregulated proteins observed in OA with respect to OC, together with their nature, confirmed the high inflammatory character of this disease. Some deregulated proteins in OA found a verification by analyzing the SF of injured arthritic joints following autologous conditioned serum treatment, an emergent therapy that provides positive results for both human and equine OA. Being the horse involved in occupational/sporting activities and considered as an excellent animal model for human joint diseases, our data provide suggestive information for tentative biomedical extrapolations, allowing to overcome the limitations in joint size and workload that are typical of other small animal models.

The effect of blood contamination on equine synovial fluid analysis

Based on a systemic complete blood count and a synovial fluid sample, to develop a mathematical model enabling the approximation of corrected values for synovial fluid white blood cell (WBC) count, neutrophil percentage, and total protein concentration in samples of synovial fluid that were contaminated by blood. Peripheral venous blood and synovial fluid samples were obtained from ten horses. A pooled synovial fluid sample from each horse was separated into 2 mL aliquots, which were subsequently contaminated with a known percentage of autogenous blood (0 to 50% of the synovial fluid volume). A complete blood count, packed cell volume, total protein (TP) concentration, and differential cytological examination were performed in all the synovial fluid and venous blood samples. Regression analysis was used to generate a model to calculate non-contaminated synovial WBC count, TP concentration and synovial neutrophil percentage. Using a further five horses these models were applied in blinded fashion to contaminated synovial fluid samples. Calculated values were compared to non-contaminated measured values. Model results for synovial WBC count and TP concentration were not significantly different from measured values. Calculated neutrophil percentage of synovial fluid WBC was a mean of 6.6% higher than measured values and was significantly different. There was no effect of the severity of contamination (as a percentage of volume) on the ability of the models to predict any of the outcome variables. It is possible to calculate non-contaminated synovial fluid WBC and TP values, but not neutrophil percentage, from heavily contaminated samples. Further study would allow for improved prediction, validation and extrapolation to a wider horse population.

Comparison ofpH, Lactate, and Glucose Analysis of Equine Synovial Fluid using a Portable Clinical Analyzer with a Bench‐Top Blood Gas Analyzer

To compare agreement between a portable clinical analyzer and laboratory-based bench-top analyzer for analysis of pH, lactate, and glucose concentrations in synovial fluid. Prospective experimental study. Clinically normal horses (n=8); 6 horses euthanatized for reasons unrelated to the study; 11 horses that had synoviocentesis for reasons other than sepsis; 7 horses that had synoviocentesis for evaluation of sepsis; and 2 horses without recorded clinical data. Median age of horses was 8 years (range, 1 day to 24 years). Supernatant from each synovial fluid sample was analyzed for pH, lactate, and glucose concentrations using an ABL 705 laboratory-based bench-top analyzer and i-STAT portable clinical analyzer. Bland-Altman plots were constructed and concordance analysis performed to determine bias and agreement between the 2 analyzers. There was acceptable agreement between analyzers for lactate and glucose concentrations, with biases of 0.198 mmol/L and 9 mg/dL and concordance correlation coefficients of 0.97 and 0.96 for lactate and glucose, respectively. The agreement between analyzers for pH was not acceptable, with a bias of -0.057 and concordance correlation coefficient of 0.89. This study found that the portable clinical analyzer performed similarly to the bench-top blood gas analyzer for evaluation of lactate and glucose concentrations, but not pH, in synovial fluid.

Frictional Properties of the Meniscus Improve After Scaffold-augmented Repair of Partial Meniscectomy: A Pilot Study

To prevent further degeneration, it is desirable to fill a meniscal defect with a supportive scaffold that mimics the mechanics of native tissue. Degradable porous scaffolds have been used, but it is unclear whether the tissue that fills the site of implantation is mechanically adequate, particularly with respect to frictional performance. We therefore determined the frictional behavior of native and engineered meniscal replacement tissue from in vivo implantation over time. We evaluated boundary and mixed-mode friction coefficients of tissue generated in porous polyurethane scaffolds used to augment the repair of the meniscus of 13 skeletally mature sheep after partial meniscectomy. Implants were removed for evaluation at 3, 6, and 12 months. The friction coefficient, aggregate modulus, and hydraulic permeability were evaluated for tissue harvested from native meniscus adjacent to the implants, native meniscus from the intact contralateral knee, and repair tissue from the site of the scaffold implantation. The equilibrium friction coefficient (μ(eq)) was measured in the presence of a lubricant bath of either phosphate-buffered saline (PBS) or equine synovial fluid (ESF). Boundary μ(eq) in PBS of engineered meniscus improved with time and was similar to native tissue after 6 months. ESF enhanced lubrication for all samples at nearly all time points demonstrating the efficacy of ESF as a joint lubricant for repair tissue as well as native meniscus. Modulus increased and permeability decreased with implantation, likely as a result of tissue ingrowth. Promoting tissue ingrowth into porous scaffolds is a potential strategy for improving friction performance in meniscal repair.

Effects of Intraarticular Tramadol Administration on Biochemical and Cytological Properties of Equine Synovial Fluid: Comparison with Lidocaine

Problem statement: Diagnostic and therapeutic arthroscopic surgeries are procedures performed quite frequently in equine practice; and are considered to cause some degree of postoperative pain. The aim of the present study was to evaluate the equine synovial fluid biochemical and cytological changes following intra-articular administration of tramadol as a potential analgesic. Approach: Six adult healthy donkeys were selected after clinical examination. Synovial fluid samples were taken from both middle carpal joints after routine preparation. Tramadol 2 mg kg-1 and 100 mg lidocaine 2% were administered to the right and left joints respectively. Synovial fluid collection from the joints was performed at 12, 24, 48-192 h after medication. Cytological examination, total protein, glucose, specific gravity, Alkaline Phosphates (ALP), Aspartate Aminotransferase (AST), Lactate Dehydrogenase (LDH), viscosity and quality of mucin clot were measured. Comparison of treatments was performed by nonparametric sign test and Wilcoxon rank sum test. Significance level was set to p≤ 0.05. Results: Neither detectable lameness nor special side effect was observed throughout the study. Mucin clot quality test and viscosity, the amount of total nucleated cell count, glucose, ALP and LDH revealed no significant differences between various sampling times between the tramadol and lidocaine groups (P>0.05). Neutrophil count, total protein, specific gravity and AST activity were significantly different. Conclusion/Recommendations: Despite the slightly different results compared to the lidocaine, it seems that the injection of tramadol into the middle carpal joint has no adverse effects on the synovial fluid composition in this joint and it can be considered a good analgesic after arthroscopic surgery with the lowest side effects in horses.

A targeted lipidomics approach to the study of eicosanoid release in synovial joints

IntroductionArticular tissues are capable of producing a range of eicosanoid mediators, each of which has individual biological effects and may be affected by anti-inflammatory treatment. We set out to develop and evaluate a high performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) approach for the simultaneous analysis of multiple eicosanoid lipid mediators in equine synovial fluid (SF), and to illustrate its use for investigation of the in vivo effects of non-steroidal anti-inflammatory drug (NSAID) treatment.MethodsSynovial fluid samples were obtained from normal joints of 6 adult horses at baseline (0 hr) and at 8, 24 and 168 hours after experimental induction of transient acute synovitis, with horses treated once daily with oral NSAID (meloxicam, 0.6 mg/kg) or placebo. Following solid-phase extraction, SF lipid mediator quantitation was based on liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis, and results were compared between disease states using linear discriminant analysis (LDA) and analysis of variance (ANOVA) with multiple comparisons corrections.ResultsOf a total of 23 mediators targeted, 14 could be reliably identified and quantified in SF samples based on detection of characteristic fragment ions at retention times similar to those of commercial standards. LDA analysis of baseline, 8, 24 and 168 hour synovial fluid samples revealed a separation of these groups into discrete clusters, reflecting dynamic changes in eicosanoid release over the course of synovitis. Prostaglandin (PG) E2 was significantly lower in NSAID vs. placebo treated samples at all time points; PGE1, 11-hydroxyeicosatetraenoic acid (11-HETE) and 13,14-dihydro-15keto PGF2α were reduced at 8 and 24 hours by NSAID treatment; while 15-HETE, 6-keto PGF1α, PGF2α, 13,14-dihydro-15keto PGE2 and thromboxane B2 (TXB2) were reduced at the 8 hour time point only. An interesting pattern was seen for Leukotriene B4 (LTB4), NSAID treatment causing an initial increase at 8 hours, but a significant reduction by 168 hours.ConclusionsThe described method allows a comprehensive analysis of synovial fluid eicosanoid profiles. Eicosanoid release in inflamed joints as well as differences between NSAID treated and placebo treated individuals are not limited to PGE2 or to the early inflammatory phase.