Equine Joint Research Articles

Investigation of synovial fluid lubricants and inflammatory cytokines in the horse: a comparison of recombinant equine interleukin 1 beta-induced synovitis and joint lavage models

BackgroundLameness is a debilitating condition in equine athletes that leads to more performance limitation and loss of use than any other medical condition. There are a limited number of non-terminal experimental models that can be used to study early inflammatory and synovial fluid biophysical changes that occur in the equine joint. Here, we compare the well-established carpal IL-1β-induced synovitis model to a tarsal intra-articular lavage model, focusing on serial changes in synovial fluid inflammatory cytokines/chemokines and the synovial fluid lubricating molecules lubricin/proteoglycan 4 and hyaluronic acid. The objectives of this study were to evaluate clinical signs; synovial membrane and synovial fluid inflammation; and synovial fluid lubricants and biophysical properties in response to carpal IL-1β synovitis and tarsal intra-articular lavage.ResultsHyaluronic acid (HA) concentrations, especially high molecular weight HA, and synovial fluid viscosity decreased after both synovitis and lavage interventions. Synovial fluid lubricin concentrations increased 17–20-fold for both synovitis and lavage models, with similar changes in both affected and contralateral joints, suggesting that repeated arthrocentesis alone resulted in elevated synovial fluid lubricin concentrations. Synovitis resulted in a more severe inflammatory response based on clinical signs (temperature, heart rate, respiratory rate, lameness and joint effusion) and clinicopathological and biochemical parameters (white blood cell count, total protein, prostaglandin E2, sulfated glycosaminoglycans, tumor necrosis factor-α and CC chemokine ligands − 2, − 3, − 5 and − 11) as compared to lavage.ConclusionsSynovial fluid lubricin increased in response to IL-1β synovitis and joint lavage but also as a result of repeated arthrocentesis. Frequent repeated arthrocentesis is associated with inflammatory changes, including increased sulfated glycosaminoglycan concentrations and decreased hyaluronic acid concentrations. Synovitis results in more significant inflammatory changes than joint lavage. Our data suggests that synovial fluid lubricin, TNF-α, CCL2, CCL3, CCL5, CCL11 and sGAG may be useful biomarkers for synovitis and post-lavage joint inflammation. Caution should be exercised when performing repeated arthrocentesis clinically or in experimental studies due to the inflammatory response and loss of HA and synovial fluid viscosity.

4 The effects of clodronate disodium on normal and inflamed equine cartilage in vitro

Bisphosphonates (BP) are a group of drugs which modulate osteoclastic resorption and alter bone turnover. BP are only approved for use in navicular syndrome in adult horses, however, these drugs are being used off-label for their potential anti-inflammatory and pain-relieving effects. When the BP, clodronate disodium (CLO), is administered intramuscularly at the labeled dose, synovial fluid concentrations are detectable within 2 h of administration. There are currently no published studies evaluating the effects of CLO on equine joint tissues. This study evaluated the effects of CLO on normal and inflamed equine cartilage explants in vitro. Cartilage explants were collected from the femorotibial joints of 3 horses which underwent euthanasia for reasons other than stifle osteoarthritis. Only visually normal cartilage was selected for biopsy. The harvested cartilage from each femorotibial joint was subdivided into 24-well plates. Cartilage explants were subjected to 6 different treatments with 8 replicates each, after a 24 h equilibration period: Control (Con), CLO 5 ng/ml (C/lo), CLO 50 ng/ml (C/hi), rEqIL-1β 10 ng/ml (IL), C/lo plus IL (ILC/lo) and C/hi concentration plus IL (ILC/hi). Explant culture media was collected every 24 h and stored at −20°C until analysis of sulfated glycosaminoglycan (GAG) content and nitric oxide (NO) content using a Dimethylmethylene Blue (DMMB) assay and a Greiss reaction, respectively. At the conclusion of the experiment (96 h), cartilage explants were papain-digested and GAG were quantified using a DMMB assay. Data were analyzed using PROC MIXED procedure (SAS 9.4), where horses were considered a random effect, and treatment and day were fixed effects. For all analyses, values of P < 0.05 were considered significant. At d 1, d 2, and d 3, the IL treatment group had a significantly higher concentration of GAG within the media compared with the Con group ( P < 0.05), indicating the expected catabolic effect of IL-1β on the explants. Treatments with clodronate (C/lo, C/hi) alone did not result in an increase in GAG or NO within the media compared with the control indicating CLO did not have a cytotoxic effect. However, the addition of CLO to the IL-1β treated explants (ILC/lo, ILC/hi) did not result in a significant decrease in GAG or NO release into the media when compared with rEqIL-1β (IL) alone. No differences were observed between treatments when GAG content of cartilage explants was measured following papain-digestion. Our in vitro study suggests that CLO does not result in cartilage toxicity. However, no anti-inflammatory or chondroprotective effects were observed.

15 The effect of nutraceuticals on markers of equine joint health

Acute injury and degradation of joint function can limit the performance of horses. To evaluate the ability of a combination of nutraceuticals to impact joint health, an in vitro trial was designed utilizing cartilage (CART) and synovium (SYN) explants harvested aseptically from the carpal joint of 2 horses euthanized for unrelated reasons. CART and SYN explants (n = 3/horse/group) were created using a 6 mm dermal biopsy punch and cocultured in appropriate media with the CART explant in the well and the SYN explant in a trans well insert with a membrane allowing for the flow of media between the insert and the well. Tissues were randomly assigned to one of 5 culture groups: 1) Negative control; 2) Positive control (10ng/ml recombinant equine (re)IL-1β); 3) Low (10ng/ml reIL-1β, 0.2 ng/mL green tea (GT), 100 ng/mL hydrolyzed collagen (HC), 25 ng/mL cranberry powder (CP)); 4) Mid (10ng/ml reIL-1β, 2 ng/mL GT, 1 µg/mL HC, 250 ng/mL CP); or 5) High (10ng/ml reIL-1β, 20 ng/mL GT, 10 µg/mL HC, 2.5 µg/mL CP). Dosages were determined via a previous study. Tissues were cultured for 21 d, and media collected on d 3, 9, 15, and 21. On d 21 viable chondrocyte density (VCD) was determined. Tissue digests were tested for proteoglycan and collagen content. Culture media was analyzed for matrix metalloproteinase (MMP), prostaglandin e 2 (PGE2), nitric oxide (NO), proteoglycan content, interleukin-6 and 8 (IL-6, 8), and growth-related oncogene (GRO). Data were analyzed utilizing a one-way ANOVA in SAS 9.4. Data are presented in the table below. There were no treatment differences for any parameter in any of the culture groups with the exception of the positive and negative control groups. Differences by time existed with increases in inflammation over time in the positive control group and no changes in the negative control group. Significant time x treatment interactions existed only in the positive control group. No combination of nutraceuticals was able to mitigate the inflammatory challenge. At the high level, the combination appeared to have cytotoxic effects. Taken together, these data shed light on the challenges associated with developing an efficacious joint supplement for horses.

Synovial fluid mitochondrial DNA as a biomarker after naturally occurring intra-articular fracture

Synovial fluid mitochondrial DNA as a biomarker after naturally occurring intra-articular fracture

Microstructural changes at the osteochondral junction in naturally occurring osteoarthritis of the equine medial femorotibial joint

Purpose: Osteoarthritis (OA) is a progressive degenerative disease of the joint organ and affects all species. The medial femorotibial (MFT) joint is the knee compartment most commonly affected with OA in humans, dogs, and horses. We hypothesized that microCT (μCT) assessment of specimens with naturally occurring equine MFT-OA will provide new insight into the pathophysiology of OA in this joint. The aim of this study was to compare healthy and MFT-OA joint microarchitectural changes in both 2 and 3D μCT images. Methods: Equine medial femoral condyles (MFC; n = 24) and tibial plateaus (MTP; n = 24) were available in a tissue bank. Regions of interest (cranial and caudal) were scored for lesions. Articular surfaces were macroscopically scored for OA (cartilage fibrillation and erosion) and categorized into 2 groups (healthy and OA) for comparative purposes. MFCs and MTPs were scanned with μCT (Xtek HMXST 225, Nexus Metrology, Canada) and 2D and 3D images were assessed. Hyaline articular cartilage (HAC) was scored for fibrillation (0-3) and high density mineralized protrusions (HDMP) (0-3). The calcified cartilage/subchondral bone plate (ACC/SCP) was also assessed for radiolucencies and the subchondral trabecular bone (SCB) for sclerosis. Results: Seventeen healthy MFTs and 7 OA MFTs were studied. HAC fibrillation was more prevalent in the MFC OA group (100%) compared with the healthy MFC group (71%) and the scores (s) were also more severe: OA MFCs (s2- 29%, s3- 71%); healthy MFCs (s1- 29%, s2- 24%, s3- 18%). Although HAC fibrillation was almost equally observed in the MTP OA group (100%) and the healthy MTP group (94%), the scores were worse in the MTP OA group (s2- 29%, s3- 71%) compared with MTP healthy group (s1- 12%, s2- 43%, s3- 43%). HDMPs were observed frequently in both groups: in 57% OA MFCs and in 47% healthy MFCs. However, the HDMP scores were more severe in MFC OA (s1- 14%, s3- 43%) compared with healthy MFCs (s1- 12%, s2- 12%, s3- 23%). They were located almost exclusively in the cranial MFC (100% OA, 87% healthy) (Figure 1). Although HDMPs were more prevalent in the MTP OA group (43%) compared with the healthy MTP group (18%), the severity of the lesions was similar in both groups: OA MTPs (s1- 29%, s3- 14%); healthy MTPs (s1- 6%, s3- 12%). ACC/SCP radiolucency was never observed in either healthy or OA MFCs. However, ACC/SCP radiolucency was observed in the MTP: MTP OA (86%) and healthy (65%) and almost all were located in the caudal part of the plateau in both healthy (91%) and OA (100%) MTPs. SCB sclerosis had a similar prevalence in both healthy (82%) and OA (86%) MFCs but the distribution pattern was different. In the healthy MFCs, sclerosis was more frequent caudally (71%) whereas in OA MFCs, it was distributed equally in both regions (57%). At the tibial plateau, sclerosis was most frequently observed in MTP OA specimens (86%) and predominantly at the caudal site (86%). In healthy MTPs it was identified in 65% with a more balanced distribution between cranial (41%) and caudal (59%) sites. Conclusions: High density mineralized protrusions (HDMP) were observed protruding into macroscopically normal appearing cartilage and OA cartilage. However, they were more severe in OA joints. This confirms our previous identification of HDMP in equine post traumatic osteoarthritis (PTOA) of the carpal bones and extends them to the equine MFT where racing-induced PTOA is not a characteristic feature and suggests that HDMP is feature all forms of OA. ACC/SCP radiolucency was never observed in the MFCs but occurred in the caudal MTP of both groups, but more frequently in OA.

Cannabinoid receptors expression positively correlated with synovitis in horses with spontaneous osteoarthritis

Purpose: Osteoarthritis (OA) is a major cause of disabling pain in all species and palliative therapy with anti-inflammatory molecules is a common therapy but has many associated undesirable side-effects. There is increasing interest in the use of cannabinoids for OA therapy. The horse is a suitable large animal model for cartilage studies and information is lacking concerning the endocannabinoid system in the equine joint.Our objectives are (1) to investigate cannabinoid receptor (CB1 & CB2) expression pattern in the synovium of healthy and equine OA joints and (2) to study the relationship between cannabinoid receptor expression, synovitis and OA scores.

A case‐control study of the morphological measurements of equine distal interphalangeal joints affected by osseous cyst‐like lesions

SummaryBackgroundThe aetiology and effects of osseous cyst‐like lesions (OCLLs) affecting the distal interphalangeal (DIP) joint are poorly understood.ObjectivesTo describe the magnetic resonance (MR) imaging appearance of OCLLs of the DIP joint and morphological measurements of affected joints.Study designRetrospective case–control study.MethodsThe low‐field MR imaging appearance of OCLLs affecting the DIP joint diagnosed over a 5‐year period was reviewed. Each case was matched to two horses with foot lameness not affected by OCLLs. Morphological measurements of the articular surfaces were recorded.ResultsThere were 23 horses with OCLLs of the DIP joint (Group 1) and 45 controls (Group 2). The Group 1 horses had a total of 30 OCLLs. Twenty‐one OCLLs were present in the distal phalanx and 9 were present in the middle phalanx. Eight lesions were indentations of the articular surface and 22 had subchondral and trabecular bone involvement. OCLLs were commonly located in the centre of the articular surface of the distal phalanx. The dorsal‐palmar/plantar length of the articular surface of the distal and middle phalanges was greater in Group 1. The depth of the articular surface of the distal phalanx and the height of the articular surface of the middle phalanx were greater in the Group 1. 22/23 horses in Group 1 had evidence of osteoarthritis of the DIP joint compared to 22/45 in Group 2.Main limitationsSmall number of cases and small values of measurements.ConclusionsOCLLs of the DIP joint occur at consistent sites, most commonly the central third of the articular surface of the distal phalanx. Measurements of the joint surfaces suggest that DIP joints affected by OCLLs have different morphology than joints without OCLLs. These findings are consistent with the theory that OCLLs of the DIP joint could be associated with dysplasia of the joint.

DESCRIÇÃO ANATOMO-ULTRASSONOGRÁFICA DO CARPO EQUINO

O carpo equino é uma região anatômica que apresenta relevância clínica e cirúrgica aos veterinários, sendo o ultrassom uma modalidade de exame complementar útil para auxiliar no diagnóstico de afecções que envolvem as principais estruturas desta articulação. Devido à escassez de trabalhos que ilustrem os aspectos morfológicos da articulação cárpica hígida e também por consequência de sua alta complexidade, foi objetivo deste trabalho apresentar uma minuciosa descrição anátomo-ultrassonográfica comparada do carpo equino. Na primeira etapa deste estudo, foram utilizadas peças anatômicas provenientes de membros torácicos equinos de modo a priorizar estruturas de maior importância clínica dos aspectos dorsal, palmar, lateral e medial da articulação cárpica. Na segunda etapa, através do exame por ultrassonografia de dois cavalos, foram geradas imagens longitudinais e transversais das estruturas alvo para o estudo anatômico, as quais foram avaliadas quanto à espessura, ecogenicidade e ecotextura. A partir desses dois estudos, foi realizada a correlação das estruturas macroscópicas das peças com suas respectivas representações ultrassonográficas, levando ao desenvolvimento de imagens didáticas aliadas às descrições das estruturas musculoesqueléticas da articulação cárpica, de modo a auxiliar no estudo desta região.

Equine synovial fluid small non-coding RNA signatures in early osteoarthritis

BackgroundOsteoarthritis remains one of the greatest causes of morbidity and mortality in the equine population. The inability to detect pre-clinical changes in osteoarthritis has been a significant impediment to the development of effective therapies against this disease. Synovial fluid represents a potential source of disease-specific small non-coding RNAs (sncRNAs) that could aid in the understanding of the pathogenesis of osteoarthritis. We hypothesised that early stages of osteoarthritis would alter the expression of sncRNAs, facilitating the understanding of the underlying pathogenesis and potentially provide early biomarkers.MethodsSmall RNA sequencing was performed using synovial fluid from the metacarpophalangeal joints of both control and early osteoarthritic horses. A group of differentially expressed sncRNAs was selected for further validation through qRT-PCR using an independent cohort of synovial fluid samples from control and early osteoarthritic horses. Bioinformatic analysis was performed in order to identify putative targets of the differentially expressed microRNAs and to explore potential associations with specific biological processes.ResultsResults revealed 22 differentially expressed sncRNAs including 13 microRNAs; miR-10a, miR-223, let7a, miR-99a, miR-23b, miR-378, miR-143 (and six novel microRNAs), four small nuclear RNAs; U2, U5, U11, U12, three small nucleolar RNAs; U13, snoR38, snord96, and one small cajal body-specific RNA; scarna3. Five sncRNAs were validated; miR-223 was significantly reduced in early osteoarthritis and miR-23b, let-7a-2, snord96A and snord13 were significantly upregulated. Significant cellular actions deduced by the differentially expressed microRNAs included apoptosis (P < 0.0003), necrosis (P < 0.0009), autophagy (P < 0.0007) and inflammation (P < 0.00001). A conservatively filtered list of 57 messenger RNA targets was obtained; the top biological processes associated were regulation of cell population proliferation (P < 0.000001), cellular response to chemical stimulus (P < 0.000001) and cell surface receptor signalling pathway (P < 0.000001).ConclusionsSynovial fluid sncRNAs may be used as molecular biomarkers for early disease in equine osteoarthritic joints. The biological processes they regulate may play an important role in understanding early osteoarthritis pathogenesis. Characterising these dynamic molecular changes could provide novel insights on the process and mechanism of early osteoarthritis development and is critical for the development of new therapeutic approaches.

Concurrent Validity of Equine Joint Range of Motion Measurement: A Novel Digital Goniometer versus Universal Goniometer.

Simple SummaryWith the growth of the field of equine rehabilitation, there is an increased demand on rehabilitation clinicians to utilize simple and reliable objective outcome assessment tools. Goniometry, the measurement of joint angles, traditionally performed with a universal goniometer (UG), is a commonly utilized assessment tool in monitoring problems of the musculoskeletal system, as well as the progression of rehabilitation interventions. Goniometry has been demonstrated to be of value, being both reliable and valid, in humans and other animal species. There are different types of goniometers, each of which has some benefit, but varies in accessibility, cost, and user-friendliness. This study examined the use of a novel digital goniometer (DG) in the measurement of angles of two joints in a horse, and comparing the measurements to those taken with a UG. The study demonstrated significant differences in range of motion for the carpus and fetlock joint, a 2–3° difference comparing measurements on a sedated horse with corresponding radiographs, a “fair” to “excellent” inter-tester reliability and a “fair” to “almost perfect” intra-tester reliability. The novel DG was found to be easier to use than the UG. In conclusion, the novel DG may serve as a simple tool for measuring joint motion in equine rehabilitation patients.Goniometry is the measurement of joint angles with a conventional universal goniometer (UG) or a digital goniometer (DG). The UG is validated for use in dogs and cats. However, it demands both of the user’s hands when measuring. To avoid this, a novel type of DG has been developed, simplifying measurement by offering single-hand usage. The objective of this study is to examine the concurrent validity of the DG. The study consists of measurement with the DG and UG for flexion of the carpus and fetlock on ten horses, and with the DG in flexion and neutral positioning of the fetlock on a sedated horse and corresponding radiographs, intra- and inter-tester reliability and a survey on user-friendliness. The data were analyzed with ANOVA and intraclass correlation coefficient (ICC), with a significance of p < 0.05. The study showed significant differences in range of motion for the carpus and fetlock joint, a 2–3° difference comparing measurements on a sedated horse with corresponding radiographs, a “fair” to “excellent” inter-tester reliability and a “fair” to “almost perfect” intra-tester reliability, and the DG was easier to use than the UG. In conclusion, the novel DG may serve as a simple tool for measuring joint motion in equine rehabilitation patients.

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

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

Nerve growth factor in the equine joint

Nerve growth factor (NGF) is a neurotrophin with many functions. In humans, it is involved in inflammation, nerve growth, apoptosis and pain signalling. Increased concentrations of NGF in synovial fluid has been shown in humans and dogs with osteoarthritis. Despite osteoarthritis being a common problem in horses, no studies have previously been published on NGF in the equine joint. The aim of this study was to quantify NGF in equine synovial fluid from healthy joints, acutely inflamed septic joints and joints with structural changes associated with osteoarthritis. A secondary aim was to identify the localisation of NGF and its two receptors, TrkA and p75NTR, in healthy and osteoarthritic articular cartilage. NGF concentrations in synovial fluid from osteoarthritic joints (n = 27), septic joints (n = 9) and healthy joints (n = 16) were determined by ELISA. In addition, articular cartilage from osteoarthritic and healthy joints was examined for NGF, TrkA and p75NTR using immunohistochemistry staining.NGF was present in equine synovial fluid and articular cartilage. Compared to synovial fluid from healthy joints, NGF concentration was higher in synovial fluid from joints with structural osteoarthritic changes (P = 0.032) or acute septic inflammation (P = 0.006). In articular cartilage with severe osteoarthritic changes, there was more abundant positive immunohistochemistry staining for NGF and its receptors than in normal articular cartilage. Further studies should focus on identifying precursor forms of NGF, and on receptor expression and downstream signalling of TrkA and P75NTR in health and disease.

Macrophage Activation in the Synovium of Healthy and Osteoarthritic Equine Joints.

Synovitis is a major component of osteoarthritis and is driven primarily by macrophages. Synovial macrophages are crucial for joint homeostasis (M2-like phenotype), but induce inflammation (M1-like) when regulatory functions become overwhelmed. Macrophage phenotypes in synovium from osteoarthritic and healthy joints are poorly characterized; however, comparative knowledge of their phenotypes during health and disease is paramount for developing targeted treatments. This study compared patterns of macrophage activation in healthy and osteoarthritic equine synovium and correlated histology with cytokine/chemokine profiles in synovial fluid. Synovial histology and immunohistochemistry for M1-like (CD86), M2-like (CD206, IL-10), and pan macrophage (CD14) markers were performed on biopsies from 29 healthy and 26 osteoarthritic equine joints. Synovial fluid cytokines (MCP-1, IL-10, PGE2, IL-1β, IL-6, TNF-α, IL-1ra) and growth factors (GM-CSF, SDF-1α+β, IGF-1, and FGF-2) were quantified. Macrophage phenotypes were not as clearly defined in vivo as they are in vitro. All macrophage markers were expressed with minimal differences between OA and normal joints. Expression for all markers increased proportionate to synovial inflammation, especially CD86. Synovial fluid MCP-1 was higher in osteoarthritic joints while SDF-1 and IL-10 were lower, and PGE2 concentrations did not differ between groups. Increased CD14/CD86/CD206/IL-10 expression was associated with synovial hyperplasia, consistent with macrophage recruitment and activation in response to injury. Lower synovial fluid IL-10 could suggest that homeostatic mechanisms from synovial macrophages became overwhelmed preventing inflammation resolution, resulting in chronic inflammation and OA. Further investigations into mechanisms of arthritis resolution are warranted. Developing pro-resolving therapies may provide superior results in the treatment of OA.

Comparison of a 3-hole 4.5-mm Dynamic Compression Plate and a 7-hole 5.5-mm Y Locking Compression Plate for Arthrodesis of the Proximal Interphalangeal Joint in Horses—an Ex Vivo Biomechanical Study

The objective of this study was to compare the biomechanical properties in a single cycle axial loading test and the types of failures in two constructs (a 3-hole 4.5-mm dynamic compression plate (DCP) and 7-hole 5.5-mm Y locking compression plate (Y-LCP)) in equine proximal interphalangeal joint (PIJ) arthrodesis. One limb in each pair was randomly assigned to PIJ arthrodesis using a 3-hole 4.5-mm DCP combined with two transarticular 5.5-mm cortical screws, whereas the contralateral limb was submitted to PIJ arthrodesis using a 7-hole Y-shaped 5.0-mm LCP in conjunction with one transarticular 4.5-mm cortical screw inserted through the central plate hole. Cortical screws were inserted in lag fashion. Constructs were submitted to a single axial load cycle to failure. Construct stiffness, load, and deformation were analyzed. Dynamic compression plate and Y-LCP arthrodesis constructs did not differ significantly and were equally resistant to axial loading under the conditions studied (DCP and Y-LCP group stiffness, 5685.22 N/mm and 6591.10 N/mm, respectively). Arthrodesis of the PIJ using a DCP and two transarticular 5.5-mm cortical screws or a Y-LCP yielded biomechanically equivalent outcomes under the test conditions considered. However, Y-LCP provides less impact in the palmar/plantar bone. Application of Y-LCP with unicortical screws has equivalent biomechanical characteristics of DCP and may be a safe option for PIJ arthrodesis, where potential trauma secondary to applying bicortical screws in the palmar/plantar aspect of the pastern can be avoided.

Aquatic exercise and equine joint and bone metabolism.

Aquatic exercise and equine joint and bone metabolism.

Use of in vitro assays to identify antibiotics that are cytotoxic to normal equine chondrocytes and synovial cells.

Intra-articular (IA) antibiotic usage is prevalent in equine practice. However, recent emergence of antimicrobial resistance prompts re-evaluation of antibiotic selection, particularly when used prophylactically. Furthermore, many commonly used antibiotics exert direct cytotoxicity to equine cells, and appropriate IA doses have not been defined. To screen antibiotics in vitro as an initial assessment of cytotoxicity against normal equine joint cells in monolayer culture and explant tissues. In vitro experimental study. Chondrocytes and synovial cells were harvested from three horses and plated on 24-well plates (100000 cells/wells in triplicate) for 48hours prior to addition of antibiotics. Joint cells were exposed to antibiotics (n=15) at various doses (25-0.39mg/mL in complete DMEM media) for 24hours and viability was assessed by trypan blue dye exclusion. The half maximal inhibitory concentration (IC50) was determined for each antibiotic. Cartilage explants were obtained from 3 horses, minced and exposed to antibiotics (n=5) for 72hours. Live/dead staining was performed, and fluorescence was visualised using Olympus IX83 spinning disk confocal microscope. Percentage of live vs dead cells was quantified. Antibiotics from different antimicrobial classes expressed dose-dependent but variable cytotoxicity to equine joint cells in vitro. Aminoglycosides and doxycycline had the lowest IC50 (most toxic). Ampicillin sulbactam, imipenem, tobramycin, ceftiofur sodium and amoxicillin had IC50>25mg/mL for at least one cell line, representing potentially less cytotoxic alternatives. Further studies are necessary to extrapolate these in vitro data results to the in vivo joint environment. Targeted IA antibiotic therapy would involve selection of the safest antibiotics (highest IC50) with efficacy based on bacterial culture/sensitivity. Antimicrobial selection and evidence-based dosing may minimise damage to native articular cartilage and synovial cells and development of antimicrobial resistance when IA antibiotics are used in equine practice.

Assessment of Intra- and Inter-observer Measurement Variability in a Radiographic Metacarpophalangeal Joint Osteophytosis Scoring System for the Horse.

The study evaluated the intra- and inter-observer measurement variability of an osteophytosis metacarpophalangeal joint scoring system. Ten (n = 10) dorso/palmar, latero/medial, and oblique views of equine metacarpophalangeal joints affected by osteoarthritis were examined. Nine assessment points were graded (scale: 0–3) twice by five veterinary students (inexperienced group, I) and four equine veterinary surgeons (expert group, E). The grades for each of the nine factors were summed to obtain the osteophytosis score. The variability between the two measurements was −2.04 ± 3.5, 95% CI −3.04 to −1.03 for the I group. For the E group, they were 0 ± 1.43, 95% CI −0.45 to 0.45. In the evaluation of the same radiographs, the I group had a coefficient of variability (CV) of 37.29%. The correlation was r = 0.90%. The CV between groups was 28.85%. The mean difference between the two observations was -0.03 ± 0.29 in the E group and 0.22 ± 0.77 in the I group. The I group showed a greater CV when the score was low (r = −0.78) compared to the E group, where the CV was independent of severity of osteophytosis (r = −0.47). The osteophytosis scoring system is an easily applicable and feasible system to be used by observers with different levels of experience, but inexpert observers may need additional training or may need to be helped by reference images. These data are validated by the low inter- and intra-observer measurement variability results in the E group. Therefore, the scoring system proposed seems to be a repeatable instrument applicable to the radiographic score of the severity of metacarpophalangeal joint osteoarthritis.

A comparison between high density mineralised protrusions identified in the knees of cadavers and in patients of the osteoarthritis initiative

Purpose: Fatigue-induced microcracking is a common, naturally occurring phenomenon in articular calcified cartilage and subchondral bone (SCB). These microcracks are, in some cases, healed by infilling with and mineralisation of an aqueous matrix. High density mineralised protrusions (HDMP) of the same matrix from the tidemark mineralising front into hyaline articular cartilage (HAC) form as an aberrant by-product of this aresorptive repair mechanism. First discovered in equine joints, HDMPs have now been confirmed on the articular surfaces of femoral heads in humans with ochronotic and conventional osteoarthritis (OA).

Amikacin induces rapid dose-dependent apoptotic cell death in equine chondrocytes and synovial cells in vitro.

Equine veterinarians frequently inject aminoglycoside antibiotics intra-articularly, either to treat septic arthritis or for prophylaxis with other medications when injecting joints for osteoarthritis. Although aminoglycosides have been demonstrated to be toxic to equine mesenchymal stem cells (MSC), their effects on resident joint cells have not been previously investigated. Moreover, safe and effective intra-articular doses have not been defined. To determine effects of concentration, duration of exposure, pH and the presence of synovial fluid on the cytotoxic effects of amikacin on equine chondrocytes, synoviocytes and bone marrow- and adipose-derived MSC. In vitro experimental study. Four cell types were harvested from three donor horses and plated in triplicate wells for 48hours prior to the addition of amikacin. The effects of amikacin on cell viability were assessed for different exposure times, concentrations and with pH buffered or unbuffered in media, as well as in the presence of synovial fluid. Cell metabolism/viability was assessed by colorimetric MTT assay. Cell proliferation was assessed by live cell imaging. Cell viability was assessed using trypan blue and dimeric cyanine nucleic acid stain (yoyo-1). To determine the mechanism of cell death, apoptosis was evaluated using Annexin V and 7AAD staining with flow cytometric quantification. Induction of apoptotic cell death pathways was assessed using caspase-3 expression. Amikacin is cytotoxic to equine joint cells and MSC in a rapid, dose-dependent, pH-independent manner, which occurs primarily by apoptosis. Amikacin cytotoxicity was not mitigated by the addition of synovial fluid in vitro. Further studies are necessary to determine whether these in vitro results predict joint injury in live animal models. Amikacin at clinically applied doses induces rapid, pronounced cell death of equine joint cells. These findings suggest that amikacin doses currently used intra-articularly should be reconsidered pending in vivo joint titration studies.

Single and repeated intra-articular injections in the tarsocrural joint with allogeneic and autologous equine bone marrow-derived mesenchymal stem cells are safe, but did not reduce acute inflammation in an experimental interleukin-1β model of synovitis.

Allogeneic and autologous bone marrow-derived mesenchymal stem cells (BMDMSCs) have been administered in equine joints for their anti-inflammatory effects. However, allogeneic BMDMSC offer multiple clinical and practical advantages. Therefore, it is important to determine the relative effectiveness of allogeneic vs autologous BMDMSCs. The objective of the study was to compare the inflamed joint response to autologous vs allogeneic BMDMSCs injections, and to determine if either treatment generated an anti-inflammatory effect. Randomised controlled study. Bone marrow was harvested from eight horses. Autologous BMDMSCs and pooled allogeneic BMDMSCs were culture expanded, cryopreserved and thawed immediately prior to administration. Ten million autologous BMDMSCs were administered with 75ng rIL-1β into one tarsocrural joint and the contralateral tarsocrural joint received allogeneic BMDMSC plus 75ng rIL-1β. Repeat injections were performed with the same treatment administered into the same joint. Four additional horses received 75ng rIL-1β alone in a single tarsocrural joint. Clinical parameters (lameness, joint circumference and joint effusion) and synovial fluid parameters, including nucleated cell count (NCC), differential cell count, total protein (TP), prostaglandin E2 (PGE2 ) and C-reactive protein (CRP), were measured at baseline, 6, 12, 24, 72, 168 and 336hours post-injection. No difference was detected between autologous and allogeneic treatment groups with respect to subjective lameness, joint effusion, joint circumference, NCC, TP, differential cell count, CRP or PGE2 . Neither autologous nor allogeneic treatments resulted in an improvement in clinical or cytological parameters over that elicited by rIL-1β alone. A single dose of rIL-1β was evaluated and resulted in a severe synovitis which may have been too severe to observe a BMDMSC-mediated effect. This study revealed that allogeneic and autologous BMDMSCs resulted in an equivalent clinical and cytological response. Allogeneic and autologous BMDMSCs were equally ineffective in reducing the inflammatory response from acute rIL-1β-induced joint inflammation in horses.