Mesenchymal Stem Cell Conditioned Medium Modulates Inflammation in Tenocytes: Complete Conditioned Medium Has Superior Therapeutic Efficacy than Its Extracellular Vesicle Fraction.

Tendinopathy, a prevalent overuse injury, lacks effective treatment options, leading to a significant impact on quality of life and socioeconomic burden. Mesenchymal stem/stromal cells (MSCs) and their secretome, including conditioned medium (CM) and extracellular vesicles (EVs), have shown promise in tissue regeneration and immunomodulation. However, it remains unclear which components of the secretome contribute to their therapeutic effects. This study aimed to compare the efficacy of CM, EVs, and the soluble protein fraction (PF) in treating inflamed tenocytes. CM exhibited the highest protein and particle concentrations, followed by PF and EVs. Inflammation significantly altered gene expression in tenocytes, with CM showing the most distinct separation from the inflamed control group. Treatment with CM resulted in the most significant differential gene expression, with both upregulated and downregulated genes related to inflammation and tissue regeneration. EV treatment also demonstrated a therapeutic effect, albeit to a lesser extent. These findings suggest that CM holds superior therapeutic efficacy compared with its EV fraction alone, emphasizing the importance of the complete secretome in tendon injury treatment.

Characterisation of Extracellular Vesicles from Equine Mesenchymal Stem Cells.

Extracellular vesicles (EVs) are nanosized lipid bilayer-encapsulated particles secreted by virtually all cell types. EVs play an essential role in cellular crosstalk in health and disease. The cellular origin of EVs determines their composition and potential therapeutic effect. Mesenchymal stem/stromal cell (MSC)-derived EVs have shown a comparable therapeutic potential to their donor cells, making them a promising tool for regenerative medicine. The therapeutic application of EVs circumvents some safety concerns associated with the transplantation of viable, replicating cells and facilitates the quality-controlled production as a ready-to-go, off-the-shelf biological therapy. Recently, the International Society for Extracellular Vesicles (ISEV) suggested a set of minimal biochemical, biophysical and functional standards to define extracellular vesicles and their functions to improve standardisation in EV research. However, nonstandardised EV isolation methods and the limited availability of cross-reacting markers for most animal species restrict the application of these standards in the veterinary field and, therefore, the species comparability and standardisation of animal experiments. In this study, EVs were isolated from equine bone-marrow-derived MSCs using two different isolation methods, stepwise ultracentrifugation and size exclusion chromatography, and minimal experimental requirements for equine EVs were established and validated. Equine EVs were characterised using a nanotracking analysis, fluorescence-triggered flow cytometry, Western blot and transelectron microscopy. Based on the ISEV standards, minimal criteria for defining equine EVs are suggested as a baseline to allow the comparison of EV preparations obtained by different laboratories.

Fetal Immunomodulatory Environment Following Cartilage Injury-The Key to CARTILAGE Regeneration?

Fetal cartilage fully regenerates following injury, while in adult mammals cartilage injury leads to osteoarthritis (OA). Thus, in this study, we compared the in vivo injury response of fetal and adult ovine articular cartilage histologically and proteomically to identify key factors of fetal regeneration. In addition, we compared the secretome of fetal ovine mesenchymal stem cells (MSCs) in vitro with injured fetal cartilage to identify potential MSC-derived therapeutic factors. Cartilage injury caused massive cellular changes in the synovial membrane, with macrophages dominating the fetal, and neutrophils the adult, synovial cellular infiltrate. Correspondingly, proteomics revealed differential regulation of pro- and anti-inflammatory mediators and growth-factors between adult and fetal joints. Neutrophil-related proteins and acute phase proteins were the two major upregulated protein groups in adult compared to fetal cartilage following injury. In contrast, several immunomodulating proteins and growth factors were expressed significantly higher in the fetus than the adult. Comparison of the in vitro MSCs proteome with the in vivo fetal regenerative signature revealed shared upregulation of 17 proteins, suggesting their therapeutic potential. Biomimicry of the fetal paracrine signature to reprogram macrophages and modulate inflammation could be an important future research direction for developing novel therapeutics.

Molecular Mechanisms of Fetal Tendon Regeneration Versus Adult Fibrous Repair.

Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.

Age-related changes of tendon fibril micro-morphology and gene expression.

Aging is hypothesized to be associated with changes in tendon matrix composition which may lead to alteration of tendon material properties and hence propensity to injury. Altered gene expression may offer insights into disease pathophysiology and thus open new perspectives toward designing pathophysiology-driven therapeutics. Therefore, the current study aimed at identifying naturally occurring differences in tendon micro-morphology and gene expression of newborn, young and old horses. Age-related differences in the distribution pattern of tendon fibril thickness and in the expression of the tendon relevant genes collagen type 1 (Col1), Col3, Col5, tenascin-C, decorin, tenomodulin, versican, scleraxis and cartilage oligomeric matrix protein were investigated. A qualitative and quantitative gene expression and collagen fibril diameter analysis was performed for the most frequently injured equine tendon, the superficial digital flexor tendon, in comparison with the deep digital flexor tendon. Most analyzed genes (Col1, Col3, Col5, tenascin-C, tenomodulin, scleraxis) were expressed at a higher level in foals (age ≤ 6 months) than in horses of 2.75 years (age at which flexor tendons become mature in structure) and older, decorin expression increased with age. Decorin was previously reported to inhibit the lateral fusion of collagen fibrils, causing a thinner fibril diameter with increased decorin concentration. The results of this study suggested that reduction of tendon fibril diameters commonly seen in equine tendons with increasing age might be a natural age-related phenomenon leading to greater fibril surface areas with increased fibrillar interaction and reduced sliding at the fascicular/fibrillar interface and hence a stiffer interfascicular/interfibrillar matrix. This may be a potential reason for the higher propensity to tendinopathies with increasing age.

Cytokine-induced interleukin-1 receptor antagonist protein expression in genetically engineered equine mesenchymal stem cells for osteoarthritis treatment.

BACKGROUND: A combination of tissue engineering methods employing mesenchymal stem cells (MSCs) together with gene transfer takes advantage of innovative strategies and highlights a new approach for targeting osteoarthritis (OA) and other cartilage defects. Furthermore, the development of systems allowing tunable transgene expression as regulated by natural disease-induced substances is highly desirable. METHODS: Bone marrow-derived equine MSCs were transduced with a lentiviral vector expressing interleukin-1 receptor antagonist (IL-1Ra) gene under the control of an inducible nuclear factor-kappa B-responsive promoter and IL-1Ra production upon pro-inflammatory cytokine stimulation [tumor necrosis factor (TNF)α, interleukin (IL)-1ß] was analysed. To assess the biological activity of the IL-1Ra protein that was produced and the therapeutic effect of IL-1Ra-expressing MSCs (MSC/IL-1Ra), cytokine-based two- and three-dimensional in vitro models of osteoarthritis using equine chondrocytes were established and quantitative real-time polymerase chain reaction (PCR) analysis was used to measure the gene expression of aggrecan, collagen IIA1, interleukin-1ß, interleukin-6, interleukin-8, matrix metalloproteinase-1 and matrix metalloproteinase-13. RESULTS: A dose-dependent increase in IL-1Ra expression was found in MSC/IL-1Ra cells upon TNFα administration, whereas stimulation using IL-1ß did not lead to IL-1Ra production above the basal level observed in nonstimulated cells as a result of the existing feedback loop. Repeated cycles of induction allowed on/off modulation of transgene expression. In vitro analyses revealed that IL-1Ra protein present in the conditioned medium from MSC/IL-1Ra cells blocks OA onset in cytokine-treated equine chondrocytes and co-cultivation of MSC/IL-1Ra cells with osteoarthritic spheroids alleviates the severity of the osteoarthritic changes. CONCLUSIONS: Thus, pro-inflammatory cytokine induced IL-1Ra protein expression from genetically modified MSCs might represent a promising strategy for osteoarthritis treatment.

Inflammation-induced transgene expression in genetically engineered equine mesenchymal stem cells.

BACKGROUND: Osteoarthritis, a chronic and progressive degenerative joint disorder, ranks amongst the top five causes of disability. Given the high incidence, associated socioeconomic costs and the absence of effective disease-modifying therapies of osteoarthritis, cell-based treatments offer a promising new approach. Owing to their paracrine, differentiation and self-renewal abilities, mesenchymal stem cells (MSCs) have great potential for regenerative medicine, which might be further enhanced by targeted gene therapy. Hence, the development of systems allowing transgene expression, particularly when regulated by natural disease-dependent occuring substances, is of high interest. METHODS: Bone marrow-isolated equine MSCs were stably transduced with an HIV-1 based lentiviral vector expressing the luciferase gene under control of an inducible nuclear factor κB (NFκB)-responsive promoter. Marker gene expression was analysed by determining luciferase activity in transduced cells stimulated with different concentrations of interleukin (IL)-1ß or tumour necrosis factor (TNF)α. RESULTS: A dose-dependent increase in luciferase expression was observed in transduced MSCs upon cytokine stimulation. The induction effect was more potent in cells treated with TNFα compared to those treated with IL-1ß. Maximum transgene expression was obtained after 48 h of stimulation and the same time was necessary to return to baseline luciferase expression levels after withdrawal of the stimulus. Repeated cycles of induction allowed on-off modulation of transgene expression without becoming refractory to induction. The NFκB-responsive promoter retained its inducibility also in chondrogenically differentiated MSC/Luc cells. CONCLUSIONS: The results of the present study demonstrate that on demand transgene expression from the NFκB-responsive promoter using naturally occurring inflammatory cytokines can be induced in undifferentiated and chondrogenically differentiated equine MSCs. Copyright © 2016 John Wiley & Sons, Ltd.

Knot Security of 5 Metric (USP 2) Sutures: Influence of Knotting Technique, Suture Material, and Incubation Time for 14 and 28 Days in Phosphate Buffered Saline and Inflamed Equine Peritoneal Fluid.

OBJECTIVES: To evaluate knot security for 3 knot types created in 3 commonly used 5 metric suture materials incubated in physiological and pathological fluids. STUDY DESIGN: In vitro mechanical study. SAMPLE POPULATION: Knotted suture loops (n = 5/group). METHODS: Loops of 3 different suture materials (glycolide/lactide copolymer; polyglactin 910; polydioxanone) were created around a 20 mm rod using 3 knot types (square [SQ], surgeon's [SK], and triple knot [TK]) and were tested to failure in distraction (6 mm/min) after tying (day 0) and after being incubated for 14 and 28 days in phosphate buffered saline (PBS) or inflamed peritoneal fluid. Failure load (N) and mode were recorded and compared. RESULTS: For polydioxanone, significant differences in force to knot failure were found between SQ and SK/TK but not between SK and TK. The force required to break all constructs increased after incubation in phosphate buffered saline (PBS). With glycolide/lactide copolymer no differences in force to knot failure were observed. With polyglactin 910, a significant difference between SQ and TK was observed, which was not seen between the other knot types. Incubation in inflamed peritoneal fluid caused a larger and more rapid decrease in force required to cause knot failure than incubation in PBS. CONCLUSIONS: Mechanical properties of suture materials have significant effects on knot security. For polydioxanone, SQ is insufficient to create a secure knot. Additional wraps above a SK confer extra stability in some materials, but this increase may not be clinically relevant or justifiable. Glycolide/lactide copolymer had excellent knot security.

The impact of opioid administration on the incidence of postanaesthetic colic in horses.

Effective management of postoperative pain is essential to ensure patient welfare, reduce morbidity and optimize recovery. Opioids are effective in managing moderate to severe pain in horses but concerns over their adverse effects on gastrointestinal (GI) motility and associated increased colic risk limit their widespread use. Studies investigating the impact of systemic opioids on both GI motility and colic incidence in horses have yielded inconclusive outcomes. Therefore, this retrospective study aims to assess the influence of systemic administration of butorphanol, morphine, and methadone on post-anaesthetic colic (PAC) incidence. Horses undergoing general anaesthesia for non-gastrointestinal procedures that were hospitalized for at least 72 h post-anaesthesia were included in this study. Anaesthetised horses were stratified by procedure type into horses undergoing diagnostic imaging without surgical intervention, emergency or elective surgery. In addition, patients were grouped by opioid treatment regime into horses receiving no opioids, intraanaesthetic, short- (<24 h) or long-term (>24 h) postoperative opioids. Administered opioids encompassed butorphanol, morphine and methadone. The number of horses showing signs of colic in the 72 h after anaesthesia was assessed for each group. A total of 782 horses were included, comprising 659 undergoing surgical procedures and 123 undergoing diagnostic imaging. The overall PAC incidence was 15.1%. Notably, horses undergoing diagnostic imaging without surgery had a significantly lower PAC rate of 6.5% compared to those undergoing surgery (16.7%, p = 0.0146). Emergency surgeries had a significantly lower PAC rate of 5.8% compared to elective procedures (18%, p = 0.0113). Of the 782 horses, 740 received intraoperative opioids and 204 postoperative opioids, 102 of which long-term (≥24 h). Neither intraoperative (p = 0.4243) nor short-term postoperative opioids (p = 0.5744) increased PAC rates. Notably, only the long-term (≥24 h) administration of morphine significantly increased PAC incidence to 34% (p = 0.0038). In contrast, long-term butorphanol (5.3% PAC, p = 0.8482) and methadone (18.4% PAC, p = 0.6161) did not affect PAC rates. In summary, extended morphine administration was the only opioid treatment associated with a significantly increased risk of PAC.

Video Ethogram of Equine Social Behaviour.

Equine social behaviour studies face challenges stemming from the absence of a comprehensive ethogram with unequivocal standardised definitions and the resulting limits to data comparison across studies. To address these constraints, this ethogram offers researchers a standardised framework, defining thirty-seven distinct equine social behaviours supplemented by video examples for enhanced clarity. These definitions amalgamate insights from existing ethograms and are fine-tuned through meticulous video observations, encompassing contextual cues such as distinguishing between aggressive and playful circling based on ear position and facial expressions and communicative nuances to provide a detailed representation of equine social behaviours. Video recordings complement the standardised definitions by capturing the dynamic flow and sequence of social interactions. By providing a dynamic and detailed representation, videos allow researchers to observe the temporal aspects of behaviour, including the sequence, duration, and rhythm of interactions. These detailed data are crucial for interpreting social behaviours and unravelling the complexities of equine societies. Standardized and video-illustrated definitions of equine social behaviour facilitate clear and consistent communication between researchers, enabling cross-study comparisons regarding the impact of husbandry practices and health conditions on equine social behaviour, which, in turn, can facilitate the assessment and optimisation of management practices and equine welfare.

Proximity tracking using ultra-wideband technology for equine social behaviour research.

Sociopositive interactions with conspecifics are essential for equine welfare and quality of life. This study aimed to validate the use of wearable ultra-wideband (UWB) technology to quantify the spatial relationships and dynamics of social behaviour in horses by continuous (1/s) measurement of interindividual distances. After testing the UWB devices' spatiotemporal accuracy in a static environment, the UWB measurement validity, feasibility and utility under dynamic field conditions was assessed in a group of 8 horses. Comparison of the proximity measurements with video surveillance data established the measurement accuracy and validity (r = 0.83, p < 0.0001) of the UWB technology. The utility for social behaviour research was demonstrated by the excellent accordance of affiliative relationships (preferred partners) identified using UWB with video observations. The horses remained a median of 5.82 m (95% CI 5.13-6.41 m) apart from each other and spent 20% (median, 95% CI 14-26%) of their time in a distance ≤ 3 m to their preferred partner. The proximity measurements and corresponding speed calculation allowed the identification of affiliative versus agonistic approaches based on differences in the approach speed and the distance and duration of the resulting proximity. Affiliative approaches were statistically significantly slower (median: 1.57 km/h, 95% CI 1.26-1.92 km/h, p = 0.0394) and resulted in greater proximity (median: 36.75 cm, 95% CI 19.5-62 cm, p = 0.0003) to the approached horse than agonistic approaches (median: 3.04 km/h, 95% CI 2.16-3.74 km/h, median proximity: 243 cm, 95% CI 130-319 cm), which caused an immediate retreat of the approached horse at a significantly greater speed (median: 3.77 km/h, 95% CI 3.52-5.85 km/h, p < 0.0001) than the approach.

Between Leisure and Pressure-Veterinarians' Attitudes towards the Care of Competition Horses in Germany, Austria and Switzerland.

Equine veterinarians face a range of challenges when attending competition horses. Athletic goals may significantly impact veterinary decision making, and the veterinarian's work can be complicated by reputational considerations and rival opinions during an assessment of whether a horse is "fit to compete". Using an online questionnaire, we found that the majority of German, Austrian and Swiss equine veterinarians (N = 172) surveyed agreed that the owners of competition horses are more likely than owners of leisure horses to approach them with clear treatment ideas, and that the former have higher expectations of the medical services provided. The data also show that the veterinarian's reputation plays a more important role in the competition sphere. Using a case vignette, we established that, on the grounds of equine welfare, the majority of respondents indicated that they would decide against starting a dressage horse with low-grade lameness in a competition. Those respondents who indicated that they would approve a start of the dressage horse indicated that a horse with a low-grade lameness was fit enough "to compete". We conclude that clearer definitions of phrases, such as "fit to compete", may be helpful in guiding veterinarians as they discharge their professional responsibilities during competitions and reduce the reputational stress they experience in this working context.

Equine Social Behaviour: Love, War and Tolerance.

Sociality is an ethological need of horses that remained unchanged by domestication. Accordingly, it is essential to include horses' social behavioural requirements and the opportunity to establish stable affiliative bonds in equine management systems and welfare assessment. Thus, this systematic review aims to provide an up-to-date analysis of equine intraspecific social ethograms. A literature review yielded 27 papers that met the inclusion criteria by studying adult (≥2 years) equine social behaviour with conspecifics using a well-defined ethogram. Social interactions were observed in 851 horses: 320 (semi-)feral free-ranging, 62 enclosed (semi-)feral and 469 domesticated, living in groups averaging 9.1 (mean +/- 6.8 s.d., range: 2-33) horses. The ethograms detailed in these 27 studies included a total of 40 (mean: 12.8/paper, range: 2-23) social behaviours, of which 60% (24/40) were agonistic, 30% (12/40) affiliative, 7.5% (3/40) investigative and 2.5% (1/40) neutral. The 27 publications included 67.7% agonistic and only 26% affiliative, 5.1% investigative and 1.2% neutral social behaviours in their methodology, thus focusing predominantly on socio-negative interactions. The strong emphasis on agonistic behaviours in equine ethology starkly contrasts with the rare occurrence of agonistic behaviours in stable horse groups and the well-established importance of affiliative interactions for equine welfare. The nuanced and complex equine social behaviour requires refinement of the ethogram with a greater focus on affiliative, ambivalent and indifferent interactions and the role of social tolerance in equine social networks to advance equine welfare assessment.

Immortalized murine tenocyte cells: a novel and innovative tool for tendon research.

Primary tenocytes rapidly undergo senescence and a phenotypic drift upon in vitro monolayer culture, which limits tendon research. The Ink4a/Arf locus encodes the proteins p16Ink4a/Arf and p14ARF (p19ARF in mice) that regulate cell cycle progression and senescence. We here established an immortalized cell line using tenocytes isolated from Ink4a/Arf deficient mice (Ink4a/Arf-/-). These cells were investigated at three distinct time points, at low (2-5), intermediate (14-17) and high (35-44) passages. Wild-type cells at low passage (2-5) served as controls. Ink4a/Arf-/- tenocytes at all stages were comparable to wild-type cells regarding morphology, expression of tenogeneic genes (collagen type 1, 3 and 5, Scleraxis, Tenomodulin and Tenascin-C), and surface markers (CD29, CD44 and CD105) and form 3D tendon-like structures. Importantly, Ink4a/Arf-/- tenocytes maintained their phenotypic features and proliferation potential in culture for more than 40 passages and also following freeze-thaw cycles. In contrast, wild-type tenocytes underwent senescence starting in passage 6. These data define Ink4a/Arf-/- tenocytes as novel tool for in vitro tendon research and as valuable in vitro alternative to animal experiments.

Evaluation of the Potential of Umbilical Cord Mesenchymal Stromal Cell-Derived Small Extracellular Vesicles to Improve Rotator Cuff Healing: A Pilot Ovine Study.

BACKGROUND: Despite significant advancements in surgical techniques to repair rotator cuff (RC) injuries, failure rates remain high and novel approaches to adequately overcome the natural biological limits of tendon and enthesis regeneration of the RC are required. Small extracellular vesicles (sEVs) derived from the secretome of human multipotent mesenchymal stromal cells (MSCs) have been demonstrated to modulate inflammation and reduce fibrotic adhesions, and therefore their local application could improve outcomes after RC repair. PURPOSE: In this pilot study, we evaluated the efficacy of clinical-grade human umbilical cord (hUC) MSC-derived sEVs (hUC-MSC-sEVs) loaded onto a type 1 collagen scaffold in an ovine model of acute infraspinatus tendon injury to improve RC healing. STUDY DESIGN: Controlled laboratory study. METHODS: sEVs were enriched from hUC-MSC culture media and were characterized by surface marker profiling. The immunomodulatory capacity was evaluated in vitro by T-cell proliferation assays, and particle count was determined by nanoparticle tracking analysis. Twelve skeletally mature sheep were subjected to partial infraspinatus tenotomy and enthesis debridement. The defects of 6 animals were treated with 2 × 1010 hUC-MSC-sEVs loaded onto a type 1 collagen sponge, whereas 6 animals received only a collagen sponge, serving as controls. Six weeks postoperatively, the healing of the infraspinatus tendon and the enthesis was evaluated by magnetic resonance imaging (MRI) and hard tissue histology. RESULTS: CD3/CD28-stimulated T-cell proliferation was significantly inhibited by hUC-MSC-sEVs (P = .015) that displayed the typical surface marker profile, including the presence of the MSC marker proteins CD44 and melanoma-associated chondroitin sulfate proteoglycan. The local application of hUC-MSC-sEVs did not result in any marked systemic adverse events. Histologically, significantly improved Watkins scores (P = .031) indicated improved tendon and tendon-to-bone insertion repair after sEV treatment and lower postcontrast signal of the tendon and adjacent structures on MRI suggested less residual inflammation at the defect area. Furthermore, the formation of osteophytes at the injury site was significantly attenuated (P = .037). CONCLUSION: A local, single-dose application of hUC-MSC-sEVs promoted tendon and enthesis healing in an ovine model of acute RC injury. CLINICAL RELEVANCE: Surgical repair of RC tears generally results in a clinical benefit for the patient; however, considerable rerupture rates have been reported. sEVs have potential as a cell-free biotherapeutic to improve healing outcomes after RC injury.

Development, refinement, and validation of an equine musculoskeletal pain scale.

Musculoskeletal disease is a common cause of chronic pain that is often overlooked and inadequately treated, impacting the quality of life of humans and horses alike. Lameness due to musculoskeletal pain is prevalent in horses, but the perception of pain by owners is low compared with veterinary diagnosis. Therefore, this study aims to establish and validate a pain scale for chronic equine orthopaedic pain that is user-friendly for horse owners and veterinarians to facilitate the identification and monitoring of pain in horses. The newly developed musculoskeletal pain scale (MPS) was applied to 154 horses (mean age 20 ± 6.4 years SD) housed at an equine sanctuary, of which 128 (83%) suffered from chronic orthopaedic disease. To complete the MPS, the horses were observed and videotaped from a distance while at rest in their box or enclosure. In addition, they received a complete clinical and orthopaedic exam. The need for veterinary intervention to address pain (assessed and executed by the sanctuary independent from this study) was used as a longitudinal health outcome to determine the MPS's predictive validity. To determine the interrater agreement, the MPS was scored for a randomly selected subset of 30 horses by six additional blinded raters, three equine veterinary practitioners, and three experienced equestrians. An iterative process was used to refine the tool based on improvements in the MPS's correlation with lameness evaluated at the walk and trot, predictive validity for longitudinal health outcomes, and interrater agreement. The intraclass correlation improved from 0.77 of the original MPS to 0.88 of the refined version (95% confidence interval: 0.8-0.94). The refined MPS correlated significantly with lameness at the walk (r = 0.44, p = 0.001) and trot (r = 0.5, p < 0.0001). The refined MPS significantly differed between horses that needed veterinary intervention (mean MPS = 8.6) and those that did not (mean MPS = 5.0, p = 0.0007). In summary, the MPS showed good interrater repeatability between expert and lay scorers, significant correlation with lameness at the walk and trot, and good predictive validity for longitudinal health outcomes, confirming its ability to identify horses with orthopaedic health problems.

A Progress Report and Roadmap for Microphysiological Systems and Organ-On-A-Chip Technologies to Be More Predictive Models in Human (Knee) Osteoarthritis.

Osteoarthritis (OA), a chronic debilitating joint disease affecting hundreds of million people globally, is associated with significant pain and socioeconomic costs. Current treatment modalities are palliative and unable to stop the progressive degeneration of articular cartilage in OA. Scientific attention has shifted from the historical view of OA as a wear-and-tear cartilage disorder to its recognition as a whole-joint disease, highlighting the contribution of other knee joint tissues in OA pathogenesis. Despite much progress in the field of microfluidic systems/organs-on-a-chip in other research fields, current in vitro models in use do not yet accurately reflect the complexity of the OA pathophenotype. In this review, we provide: 1) a detailed overview of the most significant recent developments in the field of microsystems approaches for OA modeling, and 2) an OA-pathophysiology-based bioengineering roadmap for the requirements of the next generation of more predictive and authentic microscale systems fit for the purpose of not only disease modeling but also of drug screening to potentially allow OA animal model reduction and replacement in the near future.

Quality of Life within Horse Welfare Assessment Tools: Informing Decisions for Chronically Ill and Geriatric Horses.

Equine Quality of Life (QoL) is an important concern in decision making in veterinary medicine and is especially relevant for chronically ill or geriatric horses towards the end of their lives. To our knowledge, there is no currently available QoL assessment tool for chronically ill or geriatric horses that assesses equine QoL defined as the horse's evaluation of their life. However, tools exist to assess equine welfare in different contexts. Hence, the aims of this study were to analyse how equine welfare, QoL, well-being and happiness assessment tools label, define and operationalise the concepts and to discuss the tools' suitability to assess equine QoL in the context of end-of-life decisions for chronically ill or geriatric horses. Fourteen articles were found through a systematic literature search, describing ten equine welfare assessment tools and one approach to integrating equine QoL in veterinary practice that suggests QoL assessment parameters. We discuss that some welfare assessment tools have the potential to support the development of a QoL assessment tool informing decisions towards the end of horses' lives if they are adjusted to focus on the horses' experiences, to provide an integration into an overall QoL grade and are tailored to chronically ill or geriatric horses.

Tendinopathy: sex bias starts from the preclinical development of tendon treatments. A systematic review.

Tendinopathies are common overuse disorders that arise both in athletes and the general population. Available tendon treatments are used both for women and men without distinction. However, the existence of a sex-based difference in tendon biology is widely demonstrated. Since basic research represents the foundation for treatment development, an equal female-male representation should be pursued in preclinical studies. This systematic review quantified the current evidence by analyzing 150 studies on 8231 animals. Preclinical studies largely neglected the importance of sex, none analyzed sex-based differences, and only 4% of the studies reported disaggregated data suitable for the analysis of treatment results in males and females. There is an alarming female under-representation, in particular in the field of injective therapies. Despite the growing awareness on the importance of investigating treatments in both males and females, the investigated field proved resistant from properly designing studies including both sexes, and the lack of sex-representation remains critical.

Detection of synovial sepsis in horses using enzymes as biomarkers.

BACKGROUND: Synovial sepsis is a commonly occurring, potentially career-ending or even life-threatening orthopaedic emergency. Diagnosis of synovial sepsis is currently primarily based on synovial fluid analysis, which often leaves diagnostic ambiguity due to overlap of clinicopathological parameters between septic and aseptic inflammatory synovitis. OBJECTIVES: To evaluate the reliability of lysozyme (LYS), myeloperoxidase (MPO) and elastase (ELT) as biomarkers for synovial sepsis in horses using a photometric assay to measure increased enzyme activity. STUDY DESIGN: Prospective, single-blinded, analytical, clinical study. METHODS: Equine synovial samples were assigned to one of three groups: (1) healthy controls (n = 10), (2) aseptic (n = 27) and (3) septic synovitis (n = 30). The enzyme activity assays (LYS, MPO and ELT) were compared with standard synovial fluid parameters and broad-range bacterial 16S rDNA PCR. RESULTS: LYS and MPO activities were significantly different between septic synovial samples, and both aseptic and control samples (P < .001, LYS: confidence interval [CI]: 2.25-3.41, resp., 2.21-3.8, MPO: CI 0.752-1.6, resp., 0.639-1.81). LYS achieved a 100% sensitivity and 100% specificity in differentiating between septic and aseptic (cut-off value 751.4) or control (cut-off: 484.6) samples (P < .001). MPO reached 93.33% sensitivity, 100% specificity for distinguishing septic from control (cut-off value: 0.1254) synovial samples and 93.33% sensitivity, 81.48% specificity for discriminating between septic and aseptic (cut-off value: 0.1305) synovial samples (P < .001). ELT activity could not be measured in any synovial sample. Both the LYS and the MPO measurements showed a highly significant correlation with PCR (LYS r = .79, MPO r = .69), synovial leukocyte count (LYS r = .752, MPO r = .571), % neutrophils (LYS r = .751, MPO r = 0.663) and each other (r = .744, all P < .001). MAIN LIMITATIONS: Variation in horses' signalment, affected synovial structures and synovial fluid freezing times may have affected the discriminative power of this study. CONCLUSIONS: Increased MPO and LYS activities allow reliable, rapid diagnosis of synovial sepsis with high sensitivity and specificity.