Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates.

The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture.

Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes.

RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment.

RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes.

Abstract

Abstract

Purpose and background

Effective reassurance is an essential element of treatment for conditions that do not require further investigations, referrals and on-going monitoring. However, research defining what reassurance should consist of and how to deliver it is scarce. The aim of this review was to identify consultation-related processes that improved patients' outcomes, in order to build an evidence-based model of effective reassurance in primary care.

Early diagnosis of delayed- and non-union tibial fractures is difficult, but treatment options are available if timely data are available. Direct correlation between implant forces and healing status is difficult during stance phase loading due to soft tissue forces. This ongoing study seeks to find a minimal set of strain gauge sites needed to determine healing at any of several fracture sites, using isometric loading suitable for routine clinical usage. A series of instrumented tibial nails are being used to help determine whether an alternative technology can replace or augment existing routine methods for assessment of fracture healing.

In a prior study, a single strain gauge positioned close to the fracture site had produced mixed results. In the current study, a TRIGEN META NAIL, 10mm OD x 380mm long, was instrumented with 8 gauged sites spiraled down the nail at 34mm axial and 120deg angular separation (Gen1), and loaded in a Sawbone model in offset axial compression, 3 point bending and torque.

In order to gain early clinical results, and in a design informed by the Gen1 data, a set of instrumented nails have been made for an ovine wireless telemetry study (Gen3a), shortly to commence, in which the tibial nail has been over-gauged enabling multiple d.o.f. measurements to be made during gait, torque, axial compression and 3 point bending; the latter protocols offering more controlled patient postures. This study is to be followed by a similar human study (Gen3) involving five subjects (12 gauges per nail). Meanwhile, a parallel biomechanical study involving six nails with 20 gauges each is also planned.

In the Gen1 study, the strains diminished with distance from the fracture site and with out-of-plane sites during bending. During torque, however, the response was much more uniform for all strain sites. Significant increases in strains due to both loading regimes were seen in the fractured case vs. an intact bone.

Preliminary conclusions are that strains measured due to applied torque may offer a more sensitive and fracture site-independent means of assessing healing than induced bending. We now aim to confirm these observations in animal and human studies.

Tribology and wear of articular cartilage is associated with the mechanical properties, which are governed by the extracellular matrix (ECM). The ECM adapts to resist the loads and motions applied to the tissue. Most investigations take cartilage samples from quadrupeds, where the loading and motions are different to human. However, very few studies have investigated the differences between human and animal femoral head geometry and the mechanical properties of cartilage.

This study assessed the differences between human, porcine, ovine and bovine cartilage from the femoral head; in terms of anatomical geometry, thickness, equilibrium elastic modulus and permeability.

Diameter of porcine (3-6 months old), bovine (18-24 months old), ovine (4 years old) and human femoral heads were measured (n=6). Plugs taken out of the superior region of each femoral head and creep indentation was performed. The human femoral heads were obtained from surgery due to femoral neck fracture. Cartilage thickness was measured by monitoring the resistive force change as a needle traversed the cartilage and bone at a constant feed rate using a mechanical testing machine. The percentage deformation over time was determined by dividing deformation by thickness. A biphasic finite element model was used to obtain the intrinsic material properties of each plug. Data is presented as the mean ± 95% confidence limits. One-way ANOVA was used to test for significant differences (p < or = 0.05).

Significant differences in average femoral head diameter were observed between all animals, where bovine showed the largest femoral head. Human cartilage was found to be significantly thicker than cartilage from all quadrupedal hips. Human cartilage had a significantly larger equilibrium elastic modulus compared to porcine and bovine cartilage. Porcine articular cartilage was measured to be the most permeable which was significantly larger than all the other species. No significant difference in permeability was observed between human and the other two animals: bovine and ovine (Table 1).

The current study has shown that articular cartilage mechanical properties, thickness and geometry of the femoral heads differ significantly between different species. Therefore, it is necessary to consider these variations when choosing animal tissue to represent human.

Ischaemic preconditioning is a process by which exposure of a tissue to a short period of non-damaging ischaemic stress leads to resistance to the deleterious effects of a subsequent prolonged ischaemic stress. It has been extensively described in the heart, but few studies have examined the possibility that it can occur in skeletal muscle. We have used a rat model of ischaemia of one limb to examine this possibility. Exposure of the hind limb to a period of ischaemia of five minutes and reperfusion for five minutes significantly protected the tibialis anterior muscle against the structural damage induced by a subsequent period of limb ischaemia for four hours and reperfusion for one hour. This protection was evident on examination of the muscle by both light and electron microscopy. Longer or shorter times of prior ischaemia had no effect.