Characterisation of key proteoglycans in the cranial cruciate ligaments (CCLs) from two dog breeds with different predispositions to CCL disease and rupture.

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds.

Insulin-like growth factor binding protein (IGFBP6) is a cross-species tendon marker.

The main challenge in tendon injury management is suboptimal tissue healing that fails to re-establish original tendon function. Tissue bioengineering is a promising approach for tendon therapy, with potential to improve its functional outcomes. However, evaluation criteria for tissue-engineered tendon are unclear due to the lack of specific markers of differentiated tendon. The study aim was to identify a panel of genes that characterised tendons in comparison to cartilage or muscles and validate those genes, both in human and key species used as models for tendon diseases. Gene expression profiling of rat tendon and cartilage in whole-tissue samples and primary tenocytes and chondrocytes was undertaken using two independent microarray platforms. Genes that demonstrated high expression correlation across two assays were validated by qRT-PCR in rat tendon relative to cartilage and muscle. Five genes demonstrating the highest tendon-related expression in the validation experiment (ASPN, ECM1, IGFBP6, TNMD, THBS4) were further evaluated by qRT-PCR in ovine, equine and human tissue. The group of tendon markers, identified by unbiased transcriptomic analysis of rat musculoskeletal tissues, demonstrated species-dependent profiles of expression. Insulin-like growth factor binding protein 6 (IGFBP6) was identified as the only universal tendon marker. Further investigation in equine tendon showed that IGFBP6 expression was not affected by ageing or tendon function but decreased in anatomical regions subjected to elevated compressive force. IGFBP6 is a robust cross-species marker of tendon phenotype and may find application in evaluation of tendon physiology and guided differentiation of permissive cells towards functional tenocytes.

Emergence of carriage of CTX-M-15 in faecal Escherichia coli in horses at an equine hospital in the UK; increasing prevalence over a decade (2008-2017).

BACKGROUND: This study investigated changes over time in the epidemiology of extended-spectrum ß-lactamase (ESBL) producing Escherichia coli within a single equine referral hospital in the UK. Faecal samples were collected from hospitalised horses in 2008 and 2017, processed using selective media and standard susceptibility laboratory methods. A novel real-time PCR with high resolution melt analysis was used to distinguish blaCTX-M-1 and blaCTX-M-15 within CTX-M-1 group. RESULTS: In 2008, 457 faecal samples from 103 horses were collected, with ESBL-producing E. coli identified in 131 samples (28.7, 95% CI 24.6-33.1). In 2017, 314 faecal samples were collected from 74 horses with ESBL-producing E. coli identified in 157 samples (50.0, 95% CI 44.5-55.5). There were 135 and 187 non-duplicate ESBL-producing isolates from 2008 and 2017, respectively. In 2008, 12.6% of isolates belonged to CTX-M-1 group, all carrying blaCTX-M-1, whilst in 2017, 94.1% of isolates were CTX-M-1 group positive and of these 39.2 and 60.8% of isolates carried blaCTX-M-1 and blaCTX-M-15, respectively. In addition, the prevalence of doxycycline, gentamicin and 3rd generation cephalosporin resistance increased significantly from 2008 to 2017 while a decreased prevalence of phenotypic resistance to potentiated sulphonamides was observed. CONCLUSIONS: The real-time PCR proved a reliable and high throughput method to distinguish between blaCTX-M-1 and blaCTX-M-15. Furthermore, its use in this study demonstrated the emergence of faecal carriage of CTX-M-15 in hospitalised horses, with an increase in prevalence of ESBL-producing E. coli as well as increased antimicrobial resistance to frequently used antimicrobials.

Variations during ageing in the three-dimensional anatomical arrangement of fascicles within the equine superficial digital flexor tendon.

BiTendons are constructed from collagenous fascicles separated by endotenon/interfascicular matrix (IFM). Tendons may be specialised for precision movement or to store energy during locomotion and for the latter the elasticity of the endotenon/IFM is particularly important. The equine superficial digital flexor tendon (SDFT) is a dedicated energy-storing tendon with a similar function to the human Achilles tendon. Classical anatomical descriptions portray fascicles as longitudinally arranged distinct anatomical structures. In the present study, using three-dimensional reconstruction from whole tissue slices and histological sections, the fascicles of the equine SDFT were found to adopt a complex interweaved arrangement. Fascicles were found to fully and partially converge and diverge within the tendon and fascicle bundles were observed. Fascicle morphology was not homogenous with narrowing, broadening and twisted fascicles observed in addition to relatively straight fascicles. The number of fascicle bundles observed in cross-section increased from the proximal to the distal end of the tendon, whilst the number of fascicles decreased with age in the proximal region. Fascicular patterns were not similar between the left and right limbs, across different regions or at different ages. A decrease in thickness of the endotenon/IFM between fascicles with age was found in the distal tendon region. The results provide a rationale for considering fascicular organisation when diagnosing and treating tendon injuries, for bioengineering tendon and when modelling tendon function.

A systems biology approach to defining regulatory mechanisms for cartilage and tendon cell phenotypes.

Phenotypic plasticity of adult somatic cells has provided emerging avenues for the development of regenerative therapeutics. In musculoskeletal biology the mechanistic regulatory networks of genes governing the phenotypic plasticity of cartilage and tendon cells has not been considered systematically. Additionally, a lack of strategies to effectively reproduce in vitro functional models of cartilage and tendon is retarding progress in this field. De- and redifferentiation represent phenotypic transitions that may contribute to loss of function in ageing musculoskeletal tissues. Applying a systems biology network analysis approach to global gene expression profiles derived from common in vitro culture systems (monolayer and three-dimensional cultures) this study demonstrates common regulatory mechanisms governing de- and redifferentiation transitions in cartilage and tendon cells. Furthermore, evidence of convergence of gene expression profiles during monolayer expansion of cartilage and tendon cells, and the expression of key developmental markers, challenges the physiological relevance of this culture system. The study also suggests that oxidative stress and PI3K signalling pathways are key modulators of in vitro phenotypes for cells of musculoskeletal origin.

Age-related changes in mesenchymal stem cells identified using a multi-omics approach.

Mesenchymal stem cells (MSC) are capable of multipotent differentiation into connective tissues and as such are an attractive source for autologous cell-based treatments for many clinical diseases and injuries. Ageing is associated with various altered cellular phenotypes coupled with a variety of transcriptional, epigenetic and translational changes. Furthermore, the regeneration potential of MSCs is reduced with increasing age and is correlated with changes in cellular functions. This study used a systems biology approach to investigate the transcriptomic (RNASeq), epigenetic (miRNASeq and DNA methylation) and protein alterations in ageing MSCs in order to understand the age-related functional and biological variations, which may affect their applications to regenerative medicine. We identified no change in expression of the cellular senescence markers. Alterations were evident at both the transcriptional and post-transcriptional level in a number of transcription factors. There was enrichment in genes involved in developmental disorders at mRNA and differential methylated loci (DML) level. Alterations in energy metabolism were apparent at the DML and protein level. The microRNA miR-199b-5p, whose expression was reduced in old MSCs, had predicted gene targets involved in energy metabolism and cell survival. Additionally, enrichment of DML and proteins in cell survival was evident. Enrichment in metabolic processes was revealed at the protein level and in genes identified as undergoing alternate splicing. Overall, an altered phenotype in MSC ageing at a number of levels implicated roles for inflamm-ageing and mitochondrial ageing. Identified changes represent novel insights into the ageing process, with implications for stem cell therapies in older patients.

RNA binding proteins regulate anabolic and catabolic gene expression in chondrocytes.

OBJECTIVE: Regulation of anabolic and catabolic factors is considered essential in maintaining the homoeostasis of healthy articular cartilage. In this study we investigated the influence of RNA binding proteins (RNABPs) in this process. DESIGN: Using small interfering RNA (siRNA), RNABP expression was knocked down in SW1353 chondrosarcoma cells and human articular chondrocytes. Gene expression and messenger RNA (mRNA) decay of anabolic (SOX9, Aggrecan) and catabolic (matrix metalloproteinase (MMP)13) factors were analysed using reverse transcription quantitative polymerase chain reaction (RT-qPCR). RNA-electromobility shift assays (EMSAs) were used to investigate RNABP interactions with the SOX9 mRNA 3' untranslated region (UTR). Immunohistochemical localisation of MMP13 and the RNABP human antigen R (HuR) was performed in E13.5 and E16.5 mouse embryo sections. RESULTS: SOX9 mRNA, mRNA half-life and protein expression were increased with siRNA targeting the RNABP tristetraprolin (TTP) in both HACs and SW1353s. TTP knockdown also stimulated aggrecan mRNA expression but did not affect its stability. RNA-EMSAs demonstrated that adenine uracil (AU)-rich elements in the SOX9 mRNA 3'UTR interacted with chondrocyte proteins with three specific elements interacting with TTP. HuR knockdown significantly increased MMP13 expression and also regulated the expression of a number of known transcriptional repressors of MMP13. HuR was ubiquitously expressed within mouse embryos yet displayed regional down-regulation within developing skeletal structures. CONCLUSION: This study demonstrates for the first time how RNABPs are able to affect the balance of anabolic and catabolic gene expression in human chondrocytes. The post-transcriptional mechanisms controlled by RNABPs present novel avenues of regulation and potential points of intervention for controlling the expression of SOX9 and MMP13 in chondrocytes.

A test of the universal applicability of a commonly used principle of hoof balance.

This study used a UK trimming protocol to determine whether hoof balance is achieved (as defined by equivalence of geometric proportions) in cadaver limbs (n = 49) and two cohorts of horses (shod, n = 6, and unshod, n = 20; three trimming cycles). To determine equivalence, dorsal hoof wall length (DHWL), distance from the heel buttress to the centre of pressure (HBUT-COP) and distance from dorsal toe to centre of rotation (DT-COR) were calculated as a proportion of bearing border length (BBL) using digital photography. Geometric proportions were tested using Fieller's test of equivalence with limits of difference of 2.8%. In 22 cadaver limbs the location of external COR and COP was also mapped radiographically to the extensor process of the third phalanx and the centre of rotation of the distal interphalangeal joint. Equivalence of geometric proportions was not present following trimming in cadaver limbs or in the two cohorts. Although the dorsal hoof wall to heel wall ratio improved in cadaver and unshod horses after trimming, dorsal hoof wall and lateral heel parallelism was absent in all groups and COP was not consistently in line with the extensor process. Increased COP-COR distance occurred in shod horses and may relate to solar arch flattening. Palmar heel migration, however, occurred more in unshod horses. The study shows that equivalence of geometric proportions as a measure of static hoof balance was not commonly present and widely published measures and ratios of hoof balance rarely occurred in this sample population of horses.

Ageing does not result in a decline in cell synthetic activity in an injury prone tendon.

Advancing age is a well-known risk factor for tendon disease. Energy-storing tendons [e.g., human Achilles, equine superficial digital flexor tendon (SDFT)] are particularly vulnerable and it is thought that injury occurs following an accumulation of micro-damage in the extracellular matrix (ECM). Several authors suggest that age-related micro-damage accumulates due to a failure of the aging cell population to maintain the ECM or an imbalance between anabolic and catabolic pathways. We hypothesized that ageing results in a decreased ability of tendon cells to synthesize matrix components and matrix-degrading enzymes, resulting in a reduced turnover of the ECM and a decreased ability to repair micro-damage. The SDFT was collected from horses aged 3-30 years with no signs of tendon injury. Cell synthetic and degradative ability was assessed at the mRNA and protein levels. Telomere length was measured as an additional marker of cell ageing. There was no decrease in cellularity or relative telomere length with increasing age, and no decline in mRNA or protein levels for matrix proteins or degradative enzymes. The results suggest that the mechanism for age-related tendon deterioration is not due to reduced cellularity or a loss of synthetic functionality and that alternative mechanisms should be considered.

Antimicrobial resistance in bacteria from horses: Epidemiology of antimicrobial resistance.

Antimicrobial resistance poses a significant threat to the continued successful use of antimicrobial agents for the treatment of bacterial infections. While the epidemiology of antimicrobial resistance in bacteria from man has been studied extensively, less work has been undertaken in companion animals, particularly horses. Methicillin-resistant Staphylococcus aureus has been identified as a cause of infections, with a low prevalence of nasal carriage by horses in the community but higher for hospitalised horses. Molecular characterisation has shown methicillin-resistant Staphylococcus aureus strains either to be predominantly of types associated with horses or of sequence type ST398. Antimicrobial-resistant Escherichia coli (including multidrug-resistant and extended spectrum ß-lactamase-producing isolates) have caused infections and been documented in faecal carriage by horses, with many significant resistance mechanisms identified. More sporadic reports and molecular characterisation exist for resistance in other bacteria such as enterococci, Salmonella, Acinetobacter and Pseudomonas species. Limited work has been undertaken evaluating risk factors and much of the epidemiology of antimicrobial resistance in bacteria from horses remains to be determined.

Comprehensive protein profiling of synovial fluid in osteoarthritis following protein equalization.

OBJECTIVE: The aim of the study was to characterise the protein complement of synovial fluid (SF) in health and osteoarthritis (OA) using liquid chromatography mass spectrometry (LC-MS/MS) following peptide-based depletion of high abundance proteins. DESIGN: SF was used from nine normal and nine OA Thoroughbred horses. Samples were analysed with LC-MS/MS using a NanoAcquity™ LC coupled to an LTQ Orbitrap Velos. In order to enrich the lower-abundance protein fractions protein equalisation was first undertaken using ProteoMiner™. Progenesis-QI™ LC-MS software was used for label-free quantification. In addition immunohistochemistry, western blotting and mRNA expression analysis was undertaken on selected joint tissues. RESULTS: The number of protein identifications was increased by 33% in the ProteoMiner™ treated SF compared to undepleted SF. A total of 764 proteins (462 with≥2 significant peptides) were identified in SF. A subset of 10 proteins were identified which were differentially expressed in OA SF. S100-A10, a calcium binding protein was upregulated in OA and validated with western blotting and immunohistochemistry. Several new OA specific peptide fragments (neopeptides) were identified. CONCLUSION: The protein equalisation method compressed the dynamic range of the synovial proteins identifying the most comprehensive SF proteome to date. A number of proteins were identified for the first time in SF which may be involved in the pathogenesis of OA. We identified a distinct set of proteins and neopeptides that may act as potential biomarkers to distinguish between normal and OA joints.

Tendon overload results in alterations in cell shape and increased markers of inflammation and matrix degradation.

Tendon injury is thought to involve both damage accumulation within the matrix and an accompanying cell response. While several studies have characterized cell and matrix response in chronically injured tendons, few have assessed the initial response of tendon to overload-induced damage. In this study, we assessed cell response to cyclic loading. Fascicle bundles from the equine superficial digital flexor tendon were exposed to cyclic loading in vitro, designed to mimic a bout of high-intensity exercise. Changes in cell morphology and protein-level alterations in markers of matrix inflammation and degradation were investigated. Loading resulted in matrix damage, which was accompanied by cells becoming rounder. The inflammatory markers cyclooxygenase-2 and interleukin-6 were increased in loaded samples, as were matrix metalloproteinase-13 and the collagen degradation marker C1,2C. These results indicate upregulation of inflammatory and degradative pathways in response to overload-induced in vitro, which may be initiated by alterations in cell strain environment because of localized matrix damage. This provides important information regarding the initiation of tendinopathy, suggesting that inflammation may play an important role in the initial cell response to tendon damage. Full understanding of the early tenocyte response to matrix damage is critical in order to develop effective treatments for tendinopathy.

A topographic anatomical study of the equine epiploic foramen and comparison with laparoscopic visualisation.

REASONS FOR PERFORMING STUDY: There are no previous studies correlating the anatomy of the equine epiploic foramen and its defining structures with laparoscopic images. OBJECTIVES: The purpose of this study was to describe the precise anatomy of the epiploic foramen and omental vestibule, to quantify the dimensions of the epiploic foramen and to relate these findings to laparoscopic images. STUDY DESIGN: Descriptive study of cadaver material and laparoscopic images. METHODS: Thirty-two horses subjected to euthanasia for reasons unrelated to colic were studied. Two cadavers were used to make vascular casts of the coeliac artery and portal vein. In 30 cadavers the epiploic foramen and omental vestibule were cast with a polyurethane prepolymer immediately after euthanasia. The cast served as a landmark during dissection and the circumference of the epiploic foramen was measured from these casts. Histology was performed on structures defining the epiploic foramen in 2 horses. Laparoscopic images from 6 standing right flank procedures were reviewed. RESULTS: The defining structures of the epiploic foramen and omental vestibule are the hepatoduodenal ligament, hepatogastric ligament and the gastropancreatic and hepatopancreatic folds. The hepatoduodenal ligament has a secondary fold which forms the ventral border of the epiploic foramen, consisting of a central connective tissue core of mainly elastin fibres. The hepatic artery for part of its course is incorporated in the hepatoduodenal ligament. All these structures are clearly visible laparoscopically. The mean circumference of the epiploic foramen is 11.6 ± 2.6 cm and its circumference is positively correlated with body weight but is unrelated to age or gender. CONCLUSIONS: Several clinically relevant structures delineate the epiploic foramen. Its defining structure consists, in part, of elastin fibres. Anatomical and laparoscopic knowledge may assist surgeons in developing interventions to treat diseases involving the epiploic foramen.

Elastin content is high in the canine cruciate ligament and is associated with degeneration.

Cruciate ligaments (CLs) are primary stabilisers of the knee joint and canine cranial cruciate ligament disease (CCLD) and rupture is a common injury. Elastin fibres, composed of an elastin core and fibrillin containing microfibrils, are traditionally considered minor components of the ligament extracellular matrix (ECM). However, their content and distribution in CLs is unknown. The purposes of this study were to determine the elastin content of canine CLs and to ascertain its relationship to other biochemical components and histological architecture. Macroscopically normal CLs were harvested from Greyhounds (n=11), a breed with a low risk of CCLD. Elastin, collagen and sulfated glycosaminoglycan content were measured and histological scoring systems were developed to quantify ECM changes using a modified Vasseur score (mVS) and oxytalan fibre (bundles of microfibrils) staining. Elastin contents were 9.86 ± 3.97% dry weight in the cranial CL and 10.79 ± 4.37% in the caudal CL, respectively, and did not alter with advancing histological degeneration. All CLs demonstrated mild degenerative changes, with an average mVS score of 11.9 ± 3.3 (maximum 24). Increasing degeneration of the ligament ECM showed a positive correlation (r=0.690, P<0.001) with increased oxytalan fibre staining within the ECM. Elastin is an abundant protein in CLs forming a greater proportion of the ligament ECM than previously reported. The appearance of oxytalan fibres in degenerative CL ECM may reflect an adaptive or reparative response to normal or increased loads. This finding is important for future therapeutic or ligament replacement strategies associated with cranial CL injury.

Evaluation of cartilage, synovium and adipose tissue as cellular sources for osteochondral repair.

Osteochondral lesions are a major cause of pain and disability in several species including dogs, horses and human beings. The objective of this study was to assess three potential sources of canine cells for their osteochondral regenerative potential. Cartilage, synovium and adipose tissue cells were grown in pellet culture in chondrogenic or osteogenic media. Cartilage-derived pellets displayed the best chondrogenic differentiation as indicated by significantly higher COL2A1 and SOX9 mRNA expression, greater glycosaminoglycan content, and higher retention of Safranin-O stain compared to the synovium and adipose-derived cells. Following application of the osteogenic media, all three cell sources exhibited small areas of positive alizarin red staining. Poor intracellular alkaline phosphatase activity was found in all three cell types when stimulated although osteocalcin and RUNX2 expression were significantly increased. Cells isolated and cultured from canine articular cartilage retained their specific chondrocytic phenotype. Furthermore, canine adipocytes and synovial cells did not undergo chondrogenic differentiation and did not exhibit evidence of multipotency. Although osteogenic differentiation was initiated at a genomic level, phenotypic osteoblastic differentiation was not observed. The findings of this study suggest that cells isolated from canine adipose tissue and synovium are sub-optimal substitutes for chondrocytes when engineering articular cartilage in vitro.