Peter Milner

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 (O2) 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 O2 and consumes it, albeit at low rates. Changes in O2 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 O2 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 O2 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 O2 tension, in such a way as to produce significant effects on cell function. For this purpose, O2 probably acts via alteration in levels of reactive oxygen species. We discuss the possibility that O2 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 O2 and why its removal has such marked effects. Understanding the role of oxygen has implications for disease states in which O2 or ROS levels may be perturbed.

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

Summary 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 35S 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.

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.

Magnetic resonance imaging findings of equine solar penetration wounds.

The magnetic resonance (MR) imaging features, signalment, clinical history and outcome of 55 horses with a penetrating sole injury were evaluated. Our aim was to describe MR imaging findings within the hoof capsule, assess the utility of the technique and give recommendations for the optimal MR imaging protocol to evaluate such injuries. Data from five equine hospitals were analyzed retrospectively. The tract was more likely to be visualized in animals scanned within the first week postinjury. There was no significant predisposition based on breed, age, or gender. T2*W transverse sequences were the most useful for assessment of solar penetrations due to their orientation perpendicular to the deep digital flexor tendon, the reduced scanning time, and the T2* capability of enhancing magnetic susceptibility caused by hemorrhage.

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.

Stem cell-based therapies for bone repair.

This article provides an overview of the cellular and molecular events involved in bone repair and the current approaches to using stem cells as an adjunct to this process. The article emphasizes the key role of osteoprogenitor cells in the formation of bone and where the clinical applications of current research may lend themselves to large animal orthopaedics. The processes involved in osteogenic differentiation are presented and strategies for bone formation, including induction by osteogenic factors, bioscaffolds, and gene therapy, are reviewed.

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.

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

Summary 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-1β (IL-1β). 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-1β reduced HOAC cell viability and increased GAG media levels. Acidosis and IL-1β 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-1β but reduced in hypoxia and anoxia. Mitochondrial membrane potential was reduced in low oxygen, acidosis and IL-1β. Anoxia also resulted in intracellular acidosis. GSH/GSSG ratio was reduced in low oxygen conditions, acidosis and IL-1β. 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.

Serum snoRNAs as biomarkers for joint ageing and post traumatic osteoarthritis

The development of effective treatments for the age-related disease osteoarthritis and the ability to predict disease progression has been hampered by the lack of biomarkers able to demonstrate the course of the disease. Profiling the expression patterns of small nucleolar RNAs (snoRNAs) in joint ageing and OA may provide diagnostic biomarkers and therapeutic targets. This study determined expression patterns of snoRNAs in joint ageing and OA and examined them as potential biomarkers. Using SnoRNASeq and real-time quantitative PCR (qRT-PCR) we demonstrate snoRNA expression levels in murine ageing and OA joints and serum for the first time. SnoRNASeq identified differential expression (DE) of 6 snoRNAs in young versus old joints and 5 snoRNAs in old sham versus old experimental osteoarthritic joints. In serum we found differential presence of 27 snoRNAs in young versus old serum and 18 snoRNAs in old sham versus old experimental osteoarthritic serum. Confirmatory qRT-PCR analysis demonstrated good correlation with SnoRNASeq findings. Profiling the expression patterns of snoRNAs is the initial step in determining their functional significance in ageing and osteoarthritis, and provides potential diagnostic biomarkers and therapeutic targets. Our results establish snoRNAs as novel markers of musculoskeletal ageing and osteoarthritis.

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.