Single injection of intra-articular autologous protein solution in horses with acute interleukin-1B-induced synovitis decreases joint pathology scores.

BACKGROUND: Autologous protein solution (APS) has been shown to decrease lameness in horses with osteoarthritis (OA). Synovitis is an early driver of OA, providing an opportunity to intervene in the progression of disease via intra-articular (IA) therapeutics. OBJECTIVES: The objective of this study was to investigate the effects of a single IA APS injection in horses with interleukin-1ß (IL-1ß)-induced synovitis. We hypothesised that APS would decrease joint swelling and lameness, improve synovial fluid parameters and improve joint pathology scores in horses compared with untreated controls. STUDY DESIGN: Randomised controlled in vivo experiment. METHODS: Synovitis was induced with IL-1ß (65 ng) in one randomly selected tarsocrural joint. Twenty-four hours later, joints were treated with APS (Pro-Stride®) (n = 12) or left as untreated controls (n = 6). Lameness examinations and joint circumference measurements were performed on Days 0 (prior to IL-1ß), 1 (prior to APS), 2, 4, 7 and 14. Synovial fluid, obtained on the same days, was analysed for protein concentration, nucleated cell count, and cytokine (IL-1ß, TNF-α, IFN-γ, IL-6, IL-10) and prostaglandin E2 (PGE2) concentrations. Gross pathology and synovial membrane histopathology scoring was performed on APS-treated (n = 5), untreated control (n = 4) and normal (n = 9) tarsocrural joints. RESULTS: APS did not decrease lameness or joint circumference compared with untreated controls. Synovial fluid parameters were not different between treatment groups. APS treatment did significantly decrease gross and histopathology scores. MAIN LIMITATIONS: Main limitations included the use of an induced model of the synovitis, inter-horse variability in the response to IL-1ß and likely variability in the constituents of APS from individual horses. CONCLUSIONS: APS treatment of tarsocrural joints with synovitis did not significantly improve lameness or alter synovial fluid parameters. APS did lead to significant improvement in gross joint appearance and synovial membrane histology suggesting that APS may have disease-modifying effects.

Prolonged administration of oral phenylbutazone and firocoxib in horses has no impact on selected cytokine and growth factor concentrations in platelet-rich plasma and autologous protein solution.

OBJECTIVE: To determine the effects of prolonged administration of the oral NSAIDs phenylbutazone and firocoxib on concentrations of cytokines and growth factors in platelet-rich plasma (PRP) and autologous protein solution (APS). ANIMALS: 6 adult University owned horses. METHODS: Horses were randomized to receive phenylbutazone (1 g, orally, q 12 h) or firocoxib (57 mg, orally, q 24 h) for 6 days. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before the administration of NSAIDs and at 7 days (1 day following cessation of NSAIDs). Horses underwent a two-week washout period, during which blood was obtained at 14 days and 21 days. The protocol was repeated with a crossover design. PRP and APS were analyzed for concentrations of platelets, leukocytes, and several cytokines (IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) and growth factors (PDGF, FGF-2, and TGF-ß1) using immunoassays. Plasma was evaluated for drug concentrations. RESULTS: No significant differences existed in concentrations of growth factors and cytokines before or after prolonged administration of NSAIDs. There were significant differences in concentrations of leukocytes and platelets in PRP compared to APS, with higher concentrations of leukocytes at the day 7 time point (T) in APS (phenylbutazone) and in concentrations of platelets in APS at T0 (firocoxib) and in APS at T7 (phenylbutazone). CLINICAL RELEVANCE: Veterinarians can recommend the administration of these oral NSAIDs prior to obtaining blood for PRP and APS provided a single-day washout period is instituted.

Effect of video angle on detection of induced front limb lameness in horses.

BACKGROUND: Lameness examinations are commonly performed in equine medicine. Advancements in digital technology have increased the use of video recordings for lameness assessment, however, standardization of ideal video angle is not available yielding videos of poor diagnostic quality. The objective of this study was to evaluate the effect of video angle on the subjective assessment of front limb lameness. A randomized, blinded, crossover study was performed. Six horses with and without mechanically induced forelimb solar pain were recorded using 9 video angles including horses trotting directly away and towards the video camera, horses trotting away and towards a video camera placed to the left and right side of midline, and horses trotting in a circle with the video camera placed on the inside and outside of the circle. Videos were randomized and assessed by three expert equine veterinarians using a 0-5 point scoring system. Objective lameness parameters were collected using a body-mounted inertial sensor system (Lameness Locator®, Equinosis LLC). Interobserver agreement for subjective lameness scores and ease of grading scores were determined. RESULTS: Induction of lameness was successful in all horses. There was excellent agreement between objective lameness parameters and subjective lameness scores (AUC of the ROC = 0.87). For horses in the "lame" trials, interobserver agreement was moderate for video angle 2 when degree of lameness was considered and perfect for video angle 2 and 9 when lameness was considered as a binary outcome. All other angles had no to fair agreement. For horses in the "sound" trials, interobserver agreement was perfect for video angle 5. All other video angles had slight to moderate agreement. CONCLUSIONS: When video assessment of forelimb lameness is required, a video of the horse trotting directly towards the video camera at a minimum is recommended. Other video angles may provide supportive information regarding lameness characteristics.

Plasma and synovial fluid extracellular vesicles display altered microRNA profiles in horses with naturally occurring post-traumatic osteoarthritis: an exploratory study.

OBJECTIVE: The objective of this study was to characterize extracellular vesicles (EVs) in plasma and synovial fluid obtained from horses with and without naturally occurring post-traumatic osteoarthritis (PTOA). ANIMALS: EVs were isolated from plasma and synovial fluid from horses with (n = 6) and without (n = 6) PTOA. METHODS: Plasma and synovial fluid EVs were characterized with respect to quantity, size, and surface markers. Small RNA sequencing was performed, and differentially expressed microRNAs (miRNAs) underwent bioinformatic analysis to identify putative targets and to explore potential associations with specific biological processes. RESULTS: Plasma and synovial fluid samples from horses with PTOA had a significantly higher proportion of exosomes and a lower proportion of microvesicles compared to horses without PTOA. Small RNA sequencing revealed several differentially expressed miRNAs, including miR-144, miR-219-3p, and miR-199a-3l in plasma and miR-199a-3p, miR-214, and miR-9094 in synovial fluid EVs. Bioinformatics analysis of the differentially expressed miRNAs highlighted their potential role in fibrosis, differentiation of chondrocytes, apoptosis, and inflammation pathways in PTOA. CLINICAL RELEVANCE: We have identified dynamic molecular changes in the small noncoding signatures of plasma and synovial fluid EVs in horses with naturally occurring PTOA. These findings could serve to identify promising biomarkers in the pathogenesis of PTOA, to facilitate the development of targeted therapies, and to aid in establishing appropriate translational models of PTOA.

Equine mesenchymal stem cell-derived extracellular vesicle productivity but not overall yield is improved via 3-D culture with chemically defined media.

OBJECTIVE: Mesenchymal stem cell (MSC) extracellular vesicles (EVs) have emerged as a biotherapeutic for osteoarthritis; however, manufacturing large quantities is not practical using traditional monolayer (2-D) culture. We aimed to examine the effects of 3-D and 2-D culture 2 types of media: Dulbecco modified Eagle medium and a commercially available medium (CM) on EV yield. ANIMALS: Banked bone marrow-derived MSCs (BM-MSCs) from 6 healthy, young horses were used. METHODS: 4 microcarriers (collagen-coated polystyrene, uncoated polystyrene, collagen-coated dextran, and uncoated dextran) were tested in static and bioreactor cultures, and the optimal microcarrier was chosen. The BM-MSCs were inoculated into a bioreactor with collagen-coated dextran microcarriers at 5,000 cells/cm2 or onto culture dishes at 4,000 cells/cm2 in either Dulbecco modified Eagle medium or CM media. Supernatants were obtained for metabolite and pH analysis. The BM-MSCs were expanded until confluent (2-D) or for 7 days (3-D) when the 48-hour EV collection period commenced using EV-depleted media. Extracellular vesicles were isolated and characterized via nanoparticle tracking analysis, Western blot, transmission electron microscopy, and protein quantification. The BM-MSCs were harvested, quantified, and immunophenotyped. RESULTS: The number of EVs isolated was not improved by 3-D culture or CM media, however, the CM 3-D condition improved the number of EVs produced per BM-MSC over the CM 2-D condition (mean ± SD: 306 ± 99 vs 37 ± 22, respectively). Glucose decreased and lactate and ammonium accumulated in 3-D culture. Surface markers of stemness exhibited reduced expression in 3-D culture. CLINICAL RELEVANCE: Optimization of our 3-D culture methods could improve BM-MSC expansion and thus EV yield.

Use of quantitative mass spectrometry-based proteomics and ELISA to compare the alpha 2 macroglobulin concentration in equine blood-based products processed by three different orthobiologic devices.

Introduction: Alpha 2 macroglobulin (A2M), a multi-functional protein in the plasma protease inhibitor class, regulates proinflammatory cytokines and the clearance of chondrodestructive enzymes in cases of joint injury and osteoarthritis (OA). The purpose of this study was to compare A2M concentrations in equine plasma samples processed by three commercial devices developed for stall-side regenerative joint therapy. Methods: Plasma samples were obtained from healthy adult horses (N = 13). Mass spectrometry analysis was used to determine the concentration of protein analytes in each sample. Selected reaction monitoring measured a specific A2M peptide as a surrogate of the whole A2M protein. A2M concentrations produced by each test device were compared for two sample types: a pre-concentrate or platelet-poor (PP) component and a final component for use in the horse. Results: There was no significant difference (p > 0.05) in the geometric mean (GM) concentration of A2M in the final concentration samples produced by the Alpha2EQ® device (N horses = 13) and the single-centrifugation PP samples produced by the Pro-Stride® APS (autologous protein solution) device (N = 13) and the Restigen® PRP (platelet-rich plasma) device (N = 11). When A2M content in final concentration samples produced by each device was compared, the Pro-Stride APS and Restigen PRP samples had significantly greater GM A2M content (p < 0.0001) compared to the Alpha2EQ samples, and the Pro-Stride APS final concentration samples had significantly greater GM A2M concentration (p < 0.0001) versus that for the Restigen PRP final samples. Discussion: This comparison demonstrated that the volume and A2M concentration of an Alpha2EQ final concentrate are no different than the volume and concentration of A2M in the PP from Pro-Stride or Restigen devices.

Single-dose nonsteroidal anti-inflammatory drugs in horses have no impact on concentrations of cytokines or growth factors in autologous protein solution and platelet-rich plasma.

OBJECTIVE: To determine the effects of a single dose of the NSAIDs phenylbutazone, firocoxib, flunixin meglumine, and ketoprofen on concentrations of growth factors and cytokines in autologous protein solution (APS) and platelet-rich plasma (PRP). ANIMALS: 6 adult university-owned horses. METHODS: For the first phase, 6 horses were randomized to receive ketoprofen (1,000 mg) or flunixin meglumine (500 mg) IV. Blood was obtained and processed for APS (Pro-Stride) and PRP (Restigen) before and 6 hours after administration of NSAIDs. Horses underwent a 2-week washout period, after which the protocol was repeated using a crossover design. For the second phase, following at least a 2-week washout period, the study protocol was repeated with phenylbutazone (1 g) or firocoxib (57 mg) administered orally. Plasma was collected 6 hours after administration for evaluation of drug concentrations, and APS and PRP were analyzed for concentrations of drug, platelets, leukocytes, and several growth factors and cytokines (PDGF, fibroblast growth factor, TGF-ß1, IL-1ß, IL-10, IL-6, IL-8, and tumor necrosis factor-α) before and 6 hours after administration of NSAIDs using immunoassays. RESULTS: There were no significant differences in concentrations of cytokines or growth factors before or after administration of any NSAID. There were significant differences in concentrations of leukocytes and platelets based on both product and time. NSAID concentrations in plasma were not significantly different from concentrations in APS and PRP. CLINICAL RELEVANCE: These results help guide clinicians on the appropriate use of these NSAIDs in conjunction with the processing of APS and PRP, which is unlikely to significantly alter the final product after single-dose administration.

Prospective, longitudinal assessment of subchondral bone morphology and pathology using standing, cone-beam computed tomography in fetlock joints of 2-year-old Thoroughbred racehorses in their first year of training.

BACKGROUND: Catastrophic injuries of the fetlock joints occur in Thoroughbred racehorses and are preceded by stress-induced bone injury. Early detection of subchondral bone injury is essential to prevent irreversible damage or bone failure. OBJECTIVES: To investigate the use of standing, robotic cone-beam computed tomography (CBCT) for assessing longitudinal changes in subchondral bone morphology and pathology of the fetlock joints associated with race training in young Thoroughbreds. STUDY DESIGN: Observational cohort study. METHODS: Forty-one 2-year-old Thoroughbred racehorses were recruited prior to the start of race training. Standing CBCT and radiographs of all 4 metacarpo-/metatarsophalangeal (MCP/MTP) joints were obtained at 0, 6 and 12 months. Hyperdensity, as an estimate of subchondral bone sclerosis, was measured in the distal third metacarpal (MC3)/metatarsal (MT3) bone and proximal phalanx (P1) at each time point on computed tomography. CBCTs were examined for subchondral bone pathology consisting of areas of hypodensity within regions of hyperdensity. RESULTS: Subchondral bone sclerosis increased significantly over time in the medial and lateral MC3/MT3 condyles and in the medial and lateral parasagittal grooves of MC3/MT3. The presence of subchondral bone pathology increased significantly over time in the medial and lateral palmar condyles of MC3/MT3, the lateral parasagittal groove, the medial dorsal condyle and the medial and lateral ridges of P1. MAIN LIMITATIONS: There was attrition of horses due to relocation, change in ownership, and retirement from racing. Husbandry, training regimens and racing schedules were not controlled for in the study. CONCLUSIONS: Standing CBCT is an efficient and effective screening tool for assessing subchondral bone morphology and identifying pathology of the fetlock joint in young Thoroughbred racehorses. CBCT may facilitate early detection of bone pathology allowing for timely intervention and prevention of more serious injuries.

Equine Autologous Conditioned Serum and Autologous Protein Solution.

Orthobiologics are used with increasing frequency in equine musculoskeletal disease to improve the quality of the repair tissue and prevent reinjury. Autologous blood-based products, or hemoderivatives, are made by processing the patient's blood using different systems to produce a final therapeutic product. Autologous conditioned serum (ACS) and autologous protein solution (APS) are commonly used to treat joint disorders and can also be used treat tendon and ligament injuries. Hemoderivatives contain increased concentrations of anti-inflammatory and immunomodulatory cytokines, and growth factors that help direct tissue healing and repair. The specifics of ACS and APS for treatment of musculoskeletal injuries are discussed.

Correlation of Arthroscopic Grading and Optical Coherence Tomography as Markers of Early Repair and Predictors of Later Healing Evident on MRI and Histomorphometric Assessment of Cartilage Defects Implanted with Chondrocytes Overexpressing IGF-I.

OBJECTIVE: Injury of articular cartilage is common, and due to the poor intrinsic capabilities of chondrocytes, it can precipitate joint degradation and osteoarthritis (OA). Implantation of autologous chondrocytes into cartilaginous defects has been used to bolster repair. Accurate assessment of the quality of repair tissue remains challenging. This study aimed to investigate the utility of noninvasive imaging modalities, including arthroscopic grading and optical coherence tomography (OCT) for assessment of early cartilage repair (8 weeks), and MRI to determine long-term healing (8 months). DESIGN: Large (15 mm diameter), full-thickness chondral defects were created on both lateral trochlear ridges of the femur in 24 horses. Defects were implanted with autologous chondrocytes transduced with rAAV5-IGF-I, autologous chondrocytes transduced with rAAV5-GFP, naïve autologous chondrocytes, or autologous fibrin. Healing was evaluated at 8 weeks post-implantation using arthroscopy and OCT, and at 8 months post-implantation using MRI, gross pathology, and histopathology. RESULTS: OCT and arthroscopic scoring of short-term repair tissue were significantly correlated. Arthroscopy was also correlated with later gross pathology and histopathology of repair tissue at 8 months post-implantation, while OCT was not correlated. MRI was not correlated with any other assessment variable. CONCLUSIONS: This study indicated that arthroscopic inspection and manual probing to develop an early repair score may be a better predictor of long-term cartilage repair quality following autologous chondrocyte implantation. Furthermore, qualitative MRI may not provide additional discriminatory information when assessing mature repair tissue, at least in this equine model of cartilage repair.

Identification of a previously unreported site of subchondral bone injury in the dorsodistolateral calcaneus in Thoroughbred racehorses.

BACKGROUND: Increasing accessibility and advancements in computed tomographic (CT) imaging improve understanding of the contributors to poor performance in the Thoroughbred racehorse. OBJECTIVES: To characterise an unreported site of tarsal subchondral bone injury (SBI) in Thoroughbred racehorses. STUDY DESIGN: Retrospective, cross-sectional analytical study. METHODS: Tarsal CT scans of 108 horses were reviewed for evidence of SBI in the dorsodistolateral calcaneus (DDLC). Signalment, including age, breed, sex, and discipline of horses, was recorded. The association of DDLC SBI with Thoroughbred racehorse compared to other breeds/disciplines was calculated. Nuclear scintigraphic scans of the hindlimbs of Thoroughbreds between 2007 and 2022 were also reviewed for increased radiopharmaceutical uptake (IRU) suggestive of DDLC SBI. RESULTS: Tarsal CT scans of 108 horses were analysed and lesion location and type were recorded. DDLC SBI was identified in 8/108 (7.3%) horses. All lesions were found in racing Thoroughbreds (Fischer's exact test p = 0.002) and in 20% (8/40) of racing Thoroughbreds that underwent tarsal CT. DDLC SBI was determined to be the primary cause of lameness in 3/8. Third tarsal bone fracture was considered the primary cause of lameness in 3/8 horses. A total of 1663 nuclear scintigraphic scans of 1603 Thoroughbred horses were reviewed. Increased IRU in the DDLC was present in 13 horses (0.9%). MAIN LIMITATIONS: Study limitations include the retrospective nature of the study, incomplete medical records, lack of sensitivity/specificity data for lesion identification on various imaging modalities or comparison to histopathology, and limited substantive follow-up information. CONCLUSIONS: The DDLC is a previously unreported site of SBI in the Thoroughbred racehorse. The lesion can be the primary source of lameness but may also be found in horses with other clinically relevant osseous injuries. This lesion may be underestimated in the Thoroughbred population. Further investigation is required to determine the true prevalence, clinical relevance, and prognosis in Thoroughbred racehorses with this injury.

CONTEXTO: A maior acessibilidade à tomografia computadorizada (TC) está aprimorando o conhecimento de fatores contribuintes à baixa performance de cavalos puro sangue inglês (PSI) de corrida. OBJETIVOS: Caracterizar um local não previamente descrito de lesão óssea subcondral (LOS) no tarso de cavalos PSI de corrida. DELINEAMENTO DO ESTUDO: Estudo analítico transversal retrospectivo. METODOLOGIA: TC do tarso de 108 cavalos foram revisadas para detectar evidência de LOS na região dorso-distal-lateral do calcâneo (DDLC). Informações de idade, raça, sexo e modalidade esportiva dos cavalos foram revisados. A presença de LOS na região DDLC em cavalos PSI de corrida foi comparada com a presença da mesma lesão em outras raças e modalidades esportivas. Cintilografia nuclear dos membros pélvicos de cavalos PSI de corrida entre 2007 e 2020 também foram revisadas para a presença de evidência de aumento de captação radiofarmacêutica (CRF) sugestiva de LOS na região DDLC. RESULTADOS: LOS na região DDLC for identificada em 8/108 (7.3%) dos equinos. Todas as lesões foram encontradas em PSI de corrida (teste exato de Fischer p = 0.002), e em 20% (8/40) dos PSI de corrida que tiveram o exame de TC feito. LOS na região DDLC foi determinada como a causa primária da claudicação em 3/8 dos casos. Fratura do terceiro osso do tarso foi considerada a causa primária de claudicação em 3/8 dos cavalos. 1663 exames de cintilografia de 1603 cavalos PSI de corrida foram revisados. Aumento da CRF na região DDLC estava presente em 13 equinos (0.9%). PRINCIPAIS LIMITAÇÕES: As limitações deste estudo incluem a natureza retrospectiva do mesmo, prontuários médicos incompletos, falta de dados de sensibilidade e especificidade da lesão nos métodos diagnósticos e comparação histológica, e informação de acompanhamento dos casos limitada. CONCLUSÕES: A região DDLC é um local de LOS não previamente descrito em cavalos PSI de corrida. A lesão pode ser a causa primária de claudicação, mas também pode ser encontrada em casos que apresentam outras lesões relevantes. Essa lesão pode ser subestimada em cavalos PSI de corrida. Mais investigações são necessárias para determinar a prevalência real, a relevância clínica e o prognóstico dessas lesões em cavalos PSI de corrida.

Administration and detection of gene therapy in horses: A systematic review.

Gene therapy uses genetic modification of cells to produce a therapeutic effect. Defective or missing genes can be repaired or replaced, or gene expression can be modified using a variety of technologies. Repair of defective genes can be achieved using specialized gene editing tools. Gene addition promotes gene expression by introducing synthetic copies of genes of interest (transgenes) into cells where they are transcribed and translated into therapeutic proteins. Protein production can also be modified using therapies that regulate gene expression. Gene therapy is currently prohibited in both human and equine athletes because of the potential to induce production of performance-enhancing proteins in the athlete's body, also referred to as "gene doping." Detection of gene doping is challenging and necessitates development of creative, novel analytical methods for doping control. Methods for detection of gene doping must be specific to and will vary depending on the type of gene therapy. The purpose of this paper is to present the results of a systematic review of gene editing, gene therapy, and detection of gene doping in horses. Based on the published literature, gene therapy has been administered to horses in a large number of experimental studies and a smaller number of clinical cases. Detection of gene therapy is possible using a combination of PCR and sequencing technologies. This summary can provide a basis for discussion of appropriate and inappropriate uses for gene therapy in horses by the veterinary community and guide expansion of methods to detect inappropriate uses by the regulatory community.

Comparing the immunomodulatory properties of equine BM-MSCs culture expanded in autologous platelet lysate, pooled platelet lysate, equine serum and fetal bovine serum supplemented culture media.

Joint injury often leads to cartilage damage and posttraumatic inflammation, which drives continued extracellular matrix degradation culminating in osteoarthritis. Mesenchymal stem cells (MSCs) have been proposed as a biotherapeutic to modulate inflammation within the joint. However, concerns have been raised regarding the immunogenicity of MSCs cultured in traditional fetal bovine serum (FBS) containing media, and the potential of xenogenic antigens to activate the immune system causing rejection and destruction of the MSCs. Xenogen-free alternatives to FBS have been proposed to decrease MSC immunogenicity, including platelet lysate (PL) and equine serum. The objective of this study was to compare the immunomodulatory properties of BM-MSCs culture-expanded in media supplemented with autologous PL (APL), pooled PL (PPL), equine serum (ES) or FBS. We hypothesized that BM-MSCs culture expanded in media with xenogen-free supplements would exhibit superior immunomodulatory properties to those cultured in FBS containing media. Bone marrow-derived MSCs (BM-MSCs) were isolated from six horses and culture expanded in each media type. Blood was collected from each horse to isolate platelet lysate. The immunomodulatory function of the BM-MSCs was assessed via a T cell proliferation assay and through multiplex immunoassay quantification of cytokines, including IL-1ß, IL-6, IL-8, IL-10, and TNFα, following preconditioning of BM-MSCs with IL-1ß. The concentration of platelet-derived growth factor BB (PDGF-BB), IL-10, and transforming growth factor-ß (TGF-ß) in each media was measured via immunoassay. BM-MSCs cultured in ES resulted in significant suppression of T cell proliferation (p = 0.02). Cell culture supernatant from preconditioned BM-MSCs cultured in ES had significantly higher levels of IL-6. PDGF-BB was significantly higher in APL media compared to FBS media (p = 0.016), while IL-10 was significantly higher in PPL media than ES and FBS (p = 0.04). TGF-ß was highest in APL media, with a significant difference in comparison to ES media (p = 0.03). In conclusion, expansion of equine BM-MSCs in ES may enhance their immunomodulatory abilities, while PL containing media may have some inherent therapeutic potential associated with higher concentrations of growth factors. Further studies are needed to elucidate which xenogen-free supplement optimizes BM-MSC performance.

Effects of regional limb perfusion technique on concentrations of antibiotic achieved at the target site: A meta-analysis.

Intravenous regional limb perfusions (RLP) are widely used in equine medicine to treat distal limb infections, including synovial sepsis. RLPs are generally deemed successful if the peak antibiotic concentration (Cmax) in the sampled synovial structure is at least 8-10 times the minimum inhibitory concentration (MIC) for the bacteria of interest. Despite extensive experimentation and widespread clinical use, the optimal technique for performing a successful perfusion remains unclear. The objective of this meta-analysis was to examine the effect of technique on synovial concentrations of antibiotic and to assess under which conditions Cmax:MIC ≥ 10. A literature search including the terms "horse", "equine", and "regional limb perfusion" between 1990 and 2021 was performed. Cmax (µg/ml) and measures of dispersion were extracted from studies and Cmax:MIC was calculated for sensitive and resistant bacteria. Variables included in the analysis included synovial structure sampled, antibiotic dose, tourniquet location, tourniquet duration, general anesthesia versus standing sedation, perfusate volume, tourniquet type, and the concurrent use of local analgesia. Mixed effects meta-regression was performed, and variables significantly associated with the outcome on univariable analysis were added to a multivariable meta-regression model in a step-wise manner. Sensitivity analyses were performed to assess the robustness of our findings. Thirty-six studies with 123 arms (permutations of dose, route, location and timing) were included. Cmax:MIC ranged from 1 to 348 for sensitive bacteria and 0.25 to 87 for resistant bacteria, with mean (SD) time to peak concentration (Tmax) of 29.0 (8.8) minutes. Meta-analyses generated summary values (θ) of 42.8 x MIC and 10.7 x MIC for susceptible and resistant bacteria, respectively, though because of high heterogeneity among studies (I2 = 98.8), these summary variables were not considered reliable. Meta-regression showed that the only variables for which there were statistically significant differences in outcome were the type of tourniquet and the concurrent use of local analgesia: perfusions performed with a wide rubber tourniquet and perfusions performed with the addition of local analgesia achieved significantly greater concentrations of antibiotic. The majority of arms achieved Cmax:MIC ≥ 10 for sensitive bacteria but not resistant bacteria. Our results suggest that wide rubber tourniquets and concurrent local analgesia should be strongly considered for use in RLP and that adequate therapeutic concentrations (Cmax:MIC ≥ 10) are often achieved across a variety of techniques for susceptible but not resistant pathogens.

Computed tomographic assessment of fracture characteristics and subchondral bone injury in Thoroughbred racehorses with lateral condylar fractures and their relationship to outcome.

OBJECTIVE: To investigate the effects of fracture characteristics and concurrent subchondral bone pathology identified with computed tomography (CT) on the racing performance of Thoroughbred racehorses with fractures of the MC3/MT3 lateral condyle. STUDY DESIGN: Retrospective cohort study. SAMPLE POPULATION: Thoroughbred racehorses (n = 50) with a fracture of the MC3/MT3 lateral condyle, which had preoperative CT and internal fixation performed. METHODS: Medical records were reviewed for age, sex, limb, and surgical treatment. Computed tomography scans were evaluated to determine fracture characteristics including length, whether the fracture was incomplete or complete, and displacement. The presence of subchondral bone injury (SBI), sesamoid bone fracture, articular comminution, and fragmentation in the joint was noted. Racing data was obtained from an online database. Univariable and multivariable analyses determined associations between independent variables and outcomes. RESULTS: Thirty-three (66%) horses raced after surgery. Horses with sesamoid bone fractures (P = .021), MC3/MT3 comminution (P = .016) and intra-articular fragmentation (P = .015) were less likely to race postoperatively. Concurrent SBI did not affect outcome. In the final multivariable model, sex (P = .015) and whether a fracture was incomplete or complete (P = .007) were the most significant predictors of racing postoperatively with females and horses with complete fractures being less likely to race. CONCLUSION: The prognosis for racing after a lateral condylar fracture is favorable but is decreased in horses with complete fractures and certain concurrent joint pathology. CLINICAL SIGNIFICANCE: Horses presenting with lateral condylar fractures commonly have concurrent joint pathology. Computed tomography can aid in preoperative evaluation and prognostication by enabling more complete fracture assessment.

A quantitative PCR screening method for adeno-associated viral vector 2-mediated gene doping.

Gene therapy is currently prohibited in human and equine athletes and novel analytical methods are needed for its detection. Most in vivo products use non-integrating, recombinant viral vectors derived from adeno-associated virus (AAV) to deliver transgenes into cells, where they are transcribed and translated into functional proteins. Although the majority of wild-type AAV (WTAAV) DNA is removed from recombinant AAV (rAAV) vectors, some sequences are conserved. The goal of this study was to develop a quantitative polymerase chain reaction (QPCR) screening test targeting conserved AAV sequences to enable theoretical detection of all rAAV gene therapy products, regardless of encoded transgenes while excluding the presence of WTAAV DNA in horses. Primer sets were developed and validated to target an AAV2 sequence highly conserved across rAAV viral vectors and a sequence only found in wild type AAV2 (WTAAV2). Six horses were administered an intra-articular injection of rAAV. Plasma and synovial fluid were collected on days 0, 1, 2, 4, 7, 14, 28, 56, and 84. Using QPCR, rAAV was detected in plasma for up to 2-4 days in all horses. rAAV DNA was detected for 28 days in synovial fluid from two horses for which synovial fluid samples were available. No WTAAV2 DNA was detected in any sample. This is the first study to develop a QPCR test capable of screening for rAAV vectors that may be used for gene doping in horses.

Comparative tribology II-Measurable biphasic tissue properties have predictable impacts on cartilage rehydration and lubricity.

Healthy articular cartilage supports load bearing and frictional properties unmatched among biological tissues and man-made bearing materials. Balancing fluid exudation and recovery under loaded and articulated conditions is essential to the tissue's biological and mechanical longevity. Our prior tribological investigations, which leveraged the convergent stationary contact area (cSCA) configuration, revealed that sliding alone can modulate cartilage interstitial fluid pressurization and the recovery and maintenance of lubrication under load through a mechanism termed 'tribological rehydration.' Our recent comparative assessment of tribological rehydration revealed remarkably consistent sliding speed-dependent fluid recovery and lubrication behaviors across femoral condyle cartilage from five mammalian species (equine/horse, bovine/cow, porcine/pig, ovine/sheep, and caprine/goat). In the present study, we identified and characterized key predictive relationships among tissue properties, sliding-induced tribological rehydration, and the modulation/recovery of lubrication within healthy articular cartilage. Using correlational analysis, we linked observed speed-dependent tribological rehydration behaviors to cartilage's geometry and biphasic properties (tensile and compressive moduli, and permeability). Together, these findings demonstrate that easily measurable biphasic tissue characteristics (e.g., bulk tissue material properties, compressive strain magnitude, and strain rates) can be used to predict cartilage's rehydration and lubricating abilities, and ultimately its function in vivo. STATEMENT OF SIGNIFICANCE: In healthy cartilage, articulation recovers fluid lost to static loading thereby sustaining tissue lubricity. Osteoarthritis causes changes to cartilage composition, stiffness, and permeability associated with faster fluid exudation and presumably poorer frictional outcomes. Yet, the relationship between mechanical properties and fluid recovery during articulation/sliding remains unclear. Through innovative, high-speed benchtop sliding and indentation experiments, we found that cartilage's tissue properties regulate its exudation/hydration under slow sliding speeds but have minimal effect at high sliding speeds. In fact, cartilage rehydration appears insensitive to permeability and stiffness under high fluid load support conditions. This new understanding of the balance of cartilage exudation and rehydration during activity, based upon comparative tribology studies, may improve prevention and rehabilitation strategies for joint injuries and osteoarthritis.

Evaluation of Autologous Protein Solution Injection for Treatment of Superficial Digital Flexor Tendonitis in an Equine Model.

Autologous protein solution (APS) has been used anecdotally for intralesional treatment of tendon and ligament injuries, however, its use in these injuries has never been studied in vivo. Our objective was to evaluate the effect of APS on tendon healing in an equine superficial digital flexor (SDF) tendonitis model. We hypothesized intralesional injection of APS would result in superior structural and biomechanical healing. SDF tendonitis was induced in both forelimbs of eight horses using collagenase injection. One forelimb was randomly assigned to receive an intralesional injection of APS, while the other was injected with saline. Ultrasonographic examinations were performed at weeks -1, 0, 2, 4, 8, and 12 following treatment. At 12 weeks, horses were euthanized and SDF samples harvested. Histologic evaluation, biomechanical testing, gene expression analysis, total glycosaminoglycan (GAG) and total DNA quantification were performed. Collagen type III (COL3A1) expression was significantly higher (p = 0.028) in saline treated tendon than in normal tendon. Otherwise, there were no significant differences in gene expression. There were no significant differences in histologic or ultrasonographic scores between groups. Mean total DNA content was significantly higher (p = 0.024) in saline treated tendons than normal tendons, whereas total DNA content was not significantly different between APS treated tendon and normal tendon. Elastic modulus was higher in APS treated than saline treated tendon, but the difference was not significant. Reduced expression of COL3A1 in APS treated tendon may indicate superior healing. Increased total DNA content in saline treated tendon may indicate ongoing healing processes, vs. APS treated tendons which may be in the later stages of healing. Limitations include a relatively short study period and inconsistency in size and severity of induced lesions. Intralesional injection of APS resulted in some improvements in healing characteristics.

Articular Cartilage Friction, Strain, and Viability Under Physiological to Pathological Benchtop Sliding Conditions.

In vivo, articular cartilage is exceptionally resistant to wear, damage, and dysfunction. However, replicating cartilage's phenomenal in vivo tribomechanics (i.e., high fluid load support, low frictions and strains) and mechanobiology on the benchtop has been difficult, because classical testing approaches tend to minimize hydrodynamic contributors to tissue function. Our convergent stationary contact area (cSCA) configuration retains the ability for hydrodynamically-mediated processes to contribute to interstitial hydration recovery and tribomechanical function via 'tribological rehydration'. Using the cSCA, we investigated how in situ chondrocyte survival is impacted by the presence of tribological rehydration during the reciprocal sliding of a glass counterface against a compressively loaded equine cSCA cartilage explant. When tribological rehydration was compromised during testing, by slow-speed sliding, 'pathophysiological' tribomechanical environments and high surface cell death were observed. When tribological rehydration was preserved, by high-speed sliding, 'semi-physiological' sliding environments and suppressed cell death were realized. Inclusion of synovial fluid during testing fostered 'truly physiological' sliding outcomes consistent with the in vivo environment but had limited influence on cell death compared to high-speed sliding in PBS. Subsequently, path analysis identified friction as a primary driver of cell death, with strain an indirect driver, supporting the contention that articulation mediated rehydration can benefit both the biomechanical properties and biological homeostasis of cartilage. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s12195-021-00671-2.