Injected human umbilical cord-derived mesenchymal stromal cells do not appear to elicit an inflammatory response in a murine model of osteoarthritis.

OBJECTIVE: This study investigated the effect of hUC-MSCs on osteoarthritis (OA) progression in a xenogeneic model. DESIGN: Male, 10 week-old C57BL/6 mice underwent sham surgery (n = 15) or partial medial meniscectomy (PMM; n = 76). 5x105 hUC-MSCs (from 3 donors: D1, D2 and D3) were phenotyped via RT-qPCR and immunoprofiling their response to inflammatory stimuli.They were injected into the mouse joints 3 and 6 weeks post-surgery, harvesting joints at 8 and 12 weeks post-surgery, respectively. A no cell 'control' group was also used (n = 29). All knee joints were assessed via micro-computed tomography (µCT) and histology and 10 plasma markers were analysed at 12 weeks. RESULTS: PMM resulted in cartilage loss and osteophyte formation resembling human OA at both time-points. Injection of one donor's hUC-MSCs into the joint significantly reduced the loss of joint space at 12 weeks post-operatively compared with the PMM control.This 'effective' population of MSCs up-regulated the genes, IDO and TSG6, when stimulated with inflammatory cytokines, more than those from the other two donors.No evidence of an inflammatory response to the injected cells in any animals, either histologically or with plasma biomarkers, arose. CONCLUSION: Beneficial change in a PMM joint was seen with only one hUC-MSC population, perhaps indicating that cell therapy is not appropriate for severely osteoarthritic joints. However, none of the implanted cells appeared to elicit an inflammatory response at the time-points studied. The variability of UC donors suggests some populations may be more therapeutic than others and donor characterisation is essential in developing allogeneic cell therapies.

Differences in plasma and peritoneal fluid proteomes identifies potential biomarkers associated with survival following strangulating small intestinal disease.

BACKGROUND: Strangulating small intestinal disease (SSID) carries a poor prognosis for survival in comparison to other types of colic, particularly if resection is required. Identification of markers which aid early diagnosis may prevent the need for resection, assist with more accurate prognostication and/or support the decision on whether surgical intervention is likely to be successful, would be of significant welfare benefit. OBJECTIVES: To apply an unbiased methodology to investigate the plasma and peritoneal fluid proteomes in horses diagnosed with SSID requiring resection, to identify novel biomarkers which may be of diagnostic or prognostic value. STUDY DESIGN: Prospective clinical study. METHODS: Plasma and peritoneal fluid from horses presented with acute abdominal signs consistent with SSID was collected at initial clinical examination. Samples from eight horses diagnosed with SSID at surgery in which resection of affected bowel was performed and four control horses subjected to euthanasia for orthopaedic conditions were submitted for liquid chromatography tandem mass spectrometry. Protein expression profiles were determined using label-free quantification. Data were analysed using analysis of variance to identify differentially expressed proteins between control and all SSID horses and SSID horses which survived to hospital discharge and those which did not. Significance was assumed at P≤0.05. RESULTS: A greater number of proteins were identified in peritoneal fluid than plasma of both SSID cases and controls, with 123 peritoneal fluid and 13 plasma proteins significantly differentially expressed (DE) between cases and controls (P<0.05, ≥2 fold change). Twelve peritoneal fluid proteins (P<0.036) and four plasma proteins (P<0.05) were significantly DE between SSID horses which survived and those which did not. MAIN LIMITATIONS: A low number of samples were analysed, there was variation in duration and severity of SSID and only short-term outcome was considered. CONCLUSIONS: Changes in peritoneal fluid proteome may provide a sensitive indicator of small intestinal strangulation and provide biomarkers relevant to prognosis.

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.

Clinical Research Abstracts of the British Equine Veterinary Association Congress 2015.

REASONS FOR PERFORMING STUDY: Colic remains a life-threatening condition in the horse. Ischaemia and reperfusion following correction of small intestinal strangulation may produce oxidative stress. The ability to withstand oxidative stress depends on antioxidant levels and may be linked to horse survival. OBJECTIVES: To measure peripheral antioxidant levels in horses undergoing exploratory laparotomy with small intestinal strangulation. STUDY DESIGN: Case-control study. METHODS: Blood and plasma were collected from horses undergoing exploratory laparotomy for small intestinal strangulation and stored at -80°C. Controls involved non-colic horses. Total plasma glutathione was measured spectrophotometrically at 412 nm using the 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB, Ellman's reagent) reaction. Samples containing scavenger (to remove reduced glutathione, GSH) were used to measure oxidised glutathione (GSSG). Glutathione reductase (GR) activity (u/l) was measured as the rate of GSH production at 412 nm. Glutathione peroxidise (GPx) activity (u/l) was measured as the change in optical density (340 nm) following the consumption of NADPH after GSSG production. All assays were purchased from BioAssay Systems (Hayward, California). Clinical data including arterial blood gas analysis were collected on admission. RESULTS: Glutathione reductase activity in horses with strangulating small intestinal lesions was significantly reduced compared to control horses (12.2 ± 1.1 u/l vs. 15.9 ± 0.8 u/l, P = 0.03, n = 6) whereas GPx activity did not significantly differ between colic and control horses (155.7 ± 48.7 u/l vs. 167.3 ± 30.1 u/l, P = 0.84, n = 6). Total glutathione, reduced or oxidised glutathione did not differ significantly between control and colic horses. A positive correlation existed between GR activity and Ca(2+) (r = 0.93) and K(+) (r = 0.75) whereas a strong negative correlation was present between GR activity and HCO3 (-) (r = -0.92) and PaCO2 (r = -0.96). CONCLUSIONS: Reduced plasma glutathione reductase activity with small intestinal strangulation indicates oxidative stress and may be related to systemic electrolyte/bicarbonate abnormalities. Ethical animal research: Study approval No. VREC219a. Explicit owner informed consent for inclusion of animals in this study was not stated. SOURCE OF FUNDING: Supported by the School of Veterinary Sciences, University of Liverpool. Competing interests: None declared.

Factors associated with survival to hospital discharge following endoscopic treatment for synovial sepsis in 214 horses.

REASONS FOR PERFORMING STUDY: To determine risk factors involved in survival to hospital discharge of cases of synovial sepsis. OBJECTIVES: Investigate pre-, intra- and post operative factors involved in short-term survival of horses undergoing endoscopic treatment for synovial sepsis. STUDY DESIGN: Retrospective case series. METHODS: Clinical data were obtained for horses (>6 months old) undergoing endoscopic surgery as part of management for synovial sepsis over a 7-year period in a single hospital population. Descriptive data were generated for pre-, intra- and post operative variables. Multivariable logistic regression analysis was used to develop 3 models related to presurgical, surgical and post surgical stages of management with outcome defined as survival to hospital discharge. RESULTS: Two hundred and fourteen horses were included. In Model 1 (preoperative variables), increased preoperative synovial fluid total protein (TP) was associated with nonsurvival (OR 0.88, 95% CI 0.83-0.94, P<0.001) whereas the presence of a wound on admission was associated with survival (OR 4.75, 95% CI 1.21-18.65, P = 0.02). Model 2 (intraoperative variables) revealed that factors associated with decreased survival were anaesthetic induction outside of normal working hours (OR 0.36, 95% CI 0.15-0.88 P = 0.02) and presence of moderate/severe synovial inflammation at surgery (OR 0.28, 95% CI 0.12-0.67, P = 0.004). Model 3 (post operative variables) showed that increased post operative synovial fluid TP (OR 0.94, 95% CI 0.90-0.98, P = 0.013) and undertaking more than one endoscopic surgery for treatment (OR 0.19, 95% CI 0.05-0.70, P = 0.005) were associated with nonsurvival. Cut-off values for predicting survival were 55-60 g/l for preoperative and 50-55 g/l for post operative TP measurements. CONCLUSIONS: This study has identified factors associated with altered likelihood of survival to hospital discharge following endoscopic surgery for synovial sepsis. Prognosis for survival to hospital discharge can be based on evidence from this study at the key stages of management of horses with synovial sepsis.

Outcome of horses with synovial structure involvement following solar foot penetrations in four UK veterinary hospitals: 95 cases.

REASONS FOR PERFORMING STUDY: The factors associated with outcome following solar foot penetration involving synovial structures treated using endoscopic lavage have not been described in the UK population. OBJECTIVES: To provide descriptive data on horses with synovial contamination or sepsis following solar penetration in 4 UK equine referral hospitals and to identify specific factors associated with the outcome. STUDY DESIGN: Retrospective case series. METHODS: Data were collected from 4 veterinary hospitals. Follow-up data were obtained via a telephone questionnaire. Two multivariable logistic regression models were generated. Model 1 included all horses with synovial contamination following foot penetration undergoing surgical treatment, with the outcome variable being euthanasia during hospitalisation. Model 2 included all horses surviving anaesthesia, with the outcome variable being failure to return to pre-injury athletic function. RESULTS: Ninety-five horses were included. Overall, 56% of horses survived to discharge and 36% of horses returned to pre-injury athletic function. Model 1 included penetration of the central frog sulcus (odds ratio [OR] 10, 95% confidence interval [CI] 1.9-51.8), concurrent distal phalanx involvement (OR 32, 95% CI 2.6-101.9), increasing days to presentation (OR 1.2, 95% CI 1.0-1.3) and hospital. Model 2 included increasing days to presentation (OR 1.1, 95% CI 1.1-1.6), breed (OR 32, 95% CI 2.2-135.4), more than one surgery (OR 5.6, 95% CI 1.0-32.7) and hospital. CONCLUSIONS AND POTENTIAL RELEVANCE: Synovial involvement following solar foot penetration has a guarded prognosis for survival to discharge and a poor prognosis for return to pre-injury athletic function. Penetration of the central sulcus of the frog and distal phalanx involvement are associated with euthanasia during hospitalisation. Delayed referral and hospitalisation are associated with both euthanasia and failure to return to pre-injury athletic function. Breed and more than one surgery are associated with failure to return to pre-injury athletic function. These data may assist veterinary surgeons and owners to make evidence-based decisions when managing cases with synovial involvement following solar foot penetration.

Oxygen and pH-sensitivity of human osteoarthritic chondrocytes in 3-D alginate bead culture system.

OBJECTIVE: To identify the effect of alterations in physical parameters such as oxygen and pH on processes associated with cellular redox balance in osteoarthritic chondrocytes. METHOD: Human osteoarthritic chondrocytes (HOAC) were isolated from total knee arthroplasty samples and cultured in 3-D alginate beads in four different oxygen tensions (<1%, 2%, 5% and 21% O2), at pH 7.2 and 6.2 and in the presence or absence of 10 ng/ml, interleukin-1ß (IL-1ß). Cell viability, media glycosaminoglycan (GAG) levels, media nitrate/nitrate levels, active matrix metalloproteinase (MMP)-13 and intracellular adenosine triphosphate (ATPi) were measured over a 96-h time course. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential, intracellular pH and reduced/oxidised glutathione (GSH/GSSG) were additionally measured after 48-h incubation under these experimental conditions. RESULTS: Hypoxia (2% O2) and anoxia (<1% O2), acidosis (pH 6.2) and 10 ng/ml IL-1ß reduced HOAC cell viability and increased GAG media levels. Acidosis and IL-1ß increased nitrite/nitrate release, but increases were moderate at 2% O2 and significantly reduced at <1% O2. ATPi was significantly reduced following hypoxia and anoxia and acidosis. At 48 h cellular ROS levels were increased by acidosis and IL-1ß but reduced in hypoxia and anoxia. Mitochondrial membrane potential was reduced in low oxygen, acidosis and IL-1ß. Anoxia also resulted in intracellular acidosis. GSH/GSSG ratio was reduced in low oxygen conditions, acidosis and IL-1ß. CONCLUSIONS: This study shows that oxygen and pH affect elements of the redox system in HOAC including cellular anti-oxidants, mitochondrial membrane potential and ROS levels.

Deep digital flexor tendon injury within the hoof capsule; does lesion type or location predict prognosis?

The type and location of deep digital flexor tendon (DDFT) lesions may be important in predicting outcome. The objectives of this study were to determine the frequency of different types of DDFT lesions within the hoof capsule and to determine whether lesion type predicts return to athletic activity. Lesions of the DDFT were divided into: core lesions, dorsal border lesions and parasagittal splits. Lesion location was documented, and follow-up information was obtained by telephone survey at least 18 months after diagnosis. Of 168 horses with primary DDFT injury, 54 horses had dorsal border lesions, 59 had parasagittal splits and 55 had core lesions. Twenty-five per cent of all horses returned to previous levels of athletic activity within 18 months of MRI evaluation. Horses with complete splits or core lesions of the DDFT were significantly less likely to return to some level of athletic activity than horses with dorsal border lesions P<0.001. Dorsal border lesions of the DDFT appear to have a better prognosis than core lesions or parasagittal splits. This study provides additional information that may help clinicians predict the prognosis for different types of DDFT injury.

Distribution of purinergic P2X receptors in the equine digit, cervical spinal cord and dorsal root ganglia.

Purinergic pathways are considered important in pain transmission, and P2X receptors are a key part of this system which has received little attention in the horse. The aim of this study was to identify and characterise the distribution of P2X receptor subtypes in the equine digit and associated vasculature and nervous tissue, including peripheral nerves, dorsal root ganglia and cervical spinal cord, using PCR, Western blot analysis and immunohistochemistry. mRNA signal for most of the tested P2X receptor subunits (P2X1-5, 7) was detected in all sampled equine tissues, whereas P2X6 receptor subunit was predominantly expressed in the dorsal root ganglia and spinal cord. Western blot analysis validated the specificity of P2X1-3, 7 antibodies, and these were used in immunohistochemistry studies. P2X1-3, 7 receptor subunits were found in smooth muscle cells in the palmar digital artery and vein with the exception of the P2X3 subunit that was present only in the vein. However, endothelial cells in the palmar digital artery and vein were positive only for P2X2 and P2X3 receptor subunits. Neurons and nerve fibres in the peripheral and central nervous system were positive for P2X1-3 receptor subunits, whereas glial cells were positive for P2X7 and P2X1 and 2 receptor subunits. This previously unreported distribution of P2X subtypes may suggest important tissue specific roles in physiological and pathological processes.

Short-term temporal alterations in magnetic resonance signal occur in primary lesions identified in the deep digital flexor tendon of the equine digit.

REASONS FOR PERFORMING STUDY: Primary lesions of the deep digital flexor tendon (DDFT) within the digit are an important cause of lameness diagnosed using magnetic resonance imaging (MRI) but appearance of these lesions over time has not been documented. OBJECTIVES: To determine whether the magnetic resonance (MR) appearance of different primary DDFT lesions alter over a 6 month period and whether lesion type is a determinant of these changes. METHODS: Cases included had lameness attributable to a primary lesion involving the DDFT in the digit diagnosed on MRI. Lesions were typed into parasagittal, dorsal border and core lesions. Approximate volumes and intensities were quantified for each lesion type using T2* scan sequences. Follow-up examinations and measurements were repeated at 3 and 6 month periods following conservative management. RESULTS: Twenty-three horses fitted the inclusion criteria. Lesion distribution included: parasagittal (n = 7), dorsal border (n = 11) and core lesions (n = 5). No association was found between age of horse, degree of lameness and lesion type. Only dorsal border lesions showed statistically significant reduction both in volume (initial scan: 0.18 ± 0.14 cm(3) ) at 3 months (0.11 ± 0.10 cm(3) , P<0.05) and 6 months (0.05 ± 0.05 cm(3) , P<0.01) and ratiometric intensity (initial scan: 4.06 ± 1.54) at 6 months (2.00 ± 0.43; P<0.01). Parasagittal and core lesions showed no difference in lesion volume or ratiometric intensity. Lameness improved in all lesion types following conservative management. CONCLUSIONS: Dorsal border lesions of the DDFT show reduction in both volume and intensity whereas parasagittal and core lesions do not. POTENTIAL RELEVANCE: Lesion typing may be important in predicting lesion behaviour and short-term outcome using MR imaging.

Regulation of SOX9 in normal and osteoarthritic equine articular chondrocytes by hyperosmotic loading.

OBJECTIVES: SOX9 is a transcription factor that is essential for cartilage extracellular matrix (ECM) formation. Osteoarthritis (OA) is characterised by a loss of cartilage ECM. In chondrocytes SOX9 gene expression is regulated by osmotic loading. Here we characterise SOX9 mRNA regulation through static and cyclical application of hyperosmotic conditions in normal and OA monolayer equine chondrocytes. Furthermore, we investigate whether extracellular signal-regulated protein kinase (ERK)1/2 mitogen-activated protein kinases (MAPK) pathways have a role in this regulation of SOX9. METHODS: Equine chondrocytes harvested from normal or OA joints were subjected to different osmotic loading patterns as either primary (P0) or passaged (P2) cells. The involvement of MEK-ERK signalling was demonstrated by using pharmacological inhibitors. In addition SOX9 gene stability was determined. Levels of transcripts encoding SOX9, Col2A1 and aggrecan were measured using qRT-PCR. De novo glycosaminoglycan synthesis of explants was determined with (35)S sulphate during static hyperosmolar loading. RESULTS: MEK-ERK signalling increases glycosaminoglycans (GAG) synthesis in explants. Static hyperosmotic conditions significantly reduced SOX9 mRNA in normal P2 and OA P0 but not normal P0 chondrocytes. SOX9 mRNA was stabilised by hyperosmotic conditions. Cyclical loading of normal P2 and OA P0 but not normal P0 cells led to an increase in SOX9 gene expression and this was prevented by MEK1/2 inhibition. CONCLUSIONS: The response to osmotic loading of SOX9 mRNA is dependent on the nature of the osmotic stimulation and the chondrocyte phenotype. This variation may be important in disease progression.

Oxygen and reactive oxygen species in articular cartilage: modulators of ionic homeostasis.

Articular cartilage is an avascular tissue dependent on diffusion mainly from synovial fluid to service its metabolic requirements. Levels of oxygen (O(2)) in the tissue are low, with estimates of between 1 and 6%. Metabolism is largely, if not entirely, glycolytic, with little capacity for oxidative phosphorylation. Notwithstanding, the tissue requires O(2) and consumes it, albeit at low rates. Changes in O(2) tension also have profound effects on chondrocytes affecting phenotype, gene expression, and morphology, as well as response to, and production of, cytokines. Although chondrocytes can survive prolonged anoxia, low O(2) levels have significant metabolic effects, inhibiting glycolysis (the negative Pasteur effect), and also notably matrix production. Why this tissue should respond so markedly to reduction in O(2) tension remains a paradox. Ion homeostasis in articular chondrocytes is also markedly affected by the extracellular matrix in which the cells reside. Recent work has shown that ion homeostasis also responds to changes in O(2) tension, in such a way as to produce significant effects on cell function. For this purpose, O(2) probably acts via alteration in levels of reactive oxygen species. We discuss the possibility that O(2) consumption by this tissue is required to maintain levels of ROS, which are then used physiologically as an intracellular signalling device. This postulate may go some way towards explaining why the tissue is dependent on O(2) and why its removal has such marked effects. Understanding the role of oxygen has implications for disease states in which O(2) or ROS levels may be perturbed.

The role of mitochondrial reactive oxygen species in pH regulation in articular chondrocytes.

OBJECTIVE: To examine the effect of O(2) and the role, and source, of reactive oxygen species (ROS) on pH regulation in articular chondrocytes. METHODS: Cartilage from equine metacarpo/tarsophalangeal joints was digested (collagenase) to isolate chondrocytes and loaded with 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein, a pH-sensitive fluorophore. O(2) tension was maintained using Eschweiler tonometers and a Wosthoff gas mixer. Cells were exposed to agents which alter ROS levels, mitochondrial inhibitors and/or inhibitors of protein phosphorylation. ROS levels were determined by dichlorofluorescein and mitochondrial membrane potential measured using JC-1. RESULTS: pH homeostasis was dependent on ROS. Na(+)/H(+) exchanger (NHE) activity was inhibited at low O(2) tension (acid efflux reducing from 2.30+/-0.05 to 1.27+/-0.11mMmin(-1) at 1%). NHE activity correlated with ROS levels (r(2)=0.65). ROS levels were increased by antimycin A (with levels at 1% O(2) tension increasing from 59+/-9% of the value at 20% to 87+/-7%), but reduced by rotenone, myxothiazol and diphenyleneiodonium. Hypoxia induced depolarisation of the mitochondrial membrane potential (with JC-1 red-green fluorescence ratio at 1% O(2) tension decreasing to 40+/-10% of the value at 20%). The response to changes in O(2) and to antimycin A was inhibited by staurosporine, wortmanin and calyculin A. CONCLUSION: The fall in ROS levels in hypoxia reduces the ability of articular chondrocytes to regulate pH, inhibiting NHE activity via changes in protein phosphorylation. The site of ROS generation is likely to be mitochondrial electron transport chain complex III. These effects are important to understanding normal chondrocyte function and response to altered O(2) tension.

The effect of O2 tension on pH homeostasis in equine articular chondrocytes.

OBJECTIVE: To determine the effects of varying O(2) on pH homeostasis, based on the hypothesis that the function of articular chondrocytes is best understood at realistic O(2) tensions. METHODS: Cartilage from equine metacarpophalangeal/tarsophalangeal joints was digested with collagenase to isolate chondrocytes, and then loaded with the pH-sensitive fluorophore 2',7'-bis-2-(carboxyethyl)-5(6)-carboxylfluorescein. The radioisotope(22)Na(+) was used to determine the kinetics of Na(+)/H(+) exchange (NHE) and the activity of the Na(+)/K(+) pump, and ATP levels were assessed with luciferin assays. Levels of reactive oxygen species (ROS) were determined using 2',7'-dichlorofluorescein diacetate. RESULTS: The pH homeostasis was unaffected when comparing tissue maintained at 20% O(2) (the level in water-saturated air at 37 degrees C) with that at 5% O(2) (which approximates the normal level in healthy cartilage); however, an O(2) tension of <5% caused a fall in intracellular pH (pH(i)) and slowed pH(i) recovery following acidification, an effect mediated via inhibition of NHE activity (likely through acid extrusion by NHE isoform 1). The Na(+)/K(+) pump activity and intracellular ATP concentration were unaffected by hypoxia, but the levels of ROS were reduced. Hypoxic inhibition of NHE activity and the reduction in ROS levels were reversed by treatment with H(2)O(2), Co(2+), or antimycin A. Treatment with calyculin A also prevented hypoxic inhibition of NHE activity. CONCLUSION: The ability of articular chondrocytes to carry out pH homeostasis is compromised when O(2) tensions fall below those normally experienced, via inhibition of NHE. The putative signal is a reduction in levels of ROS derived from mitochondria, acting via altered protein phosphorylation. This effect is relevant to both physiologic and pathologic states of lowered O(2), such as in chronic inflammation.