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

Anterior cruciate ligament (acl) reconstruction is one of the most commonly performed procedures in orthopedics for acl injury. While literature suggest short-term good-to-excellent functional results, a significant number of long-term studies report unexplained early oa development, regardless type of reconstruction. The present study reports the feasibility analysis and development of a clinical protocol, integrating different methodologies, able to determine which acl reconstruction technique could have the best chance to prevent oa. It gives also clinicians an effective tool to minimize the incidence of early oa. A prospective clinical trial was defined to evaluate clinical outcome, biochemical changes in cartilage, biomechanical parameters and possible development of oa. The most common reconstruction techniques were selected for this study, including hamstring single-bundle, single-bundle with extraarticular tenodesis and anatomical double-bundle. Power analysis was performed in terms of changes at cartilage level measurable by mri with t2 mapping. A sample size of 42 patients with isolated traumatic acl injury were therefore identified, considering a possible 10% to follow-up. Subjects presenting skeletal immaturity, degenerative tear of acl, other potential risk factors of oa and previous knee surgery were excluded. Included patients were randomized and underwent one of the 3 specified reconstruction techniques. The patients were evaluated pre-operatively, intra-operatively and post-operatively at 4 and 18 months of follow-up. Clinical evaluation were performed at each time using subjective scores (koos) and generic health status (sf-12). The activity level were documented (marx) as well as objective function (ikdc). Preliminary results allow to verify kinematic patterns during active tasks, including level walking, stair descending and squatting using dynamic roentgen sterephotogrammetric analysis (rsa) methodology before and after the injured ligament reconstruction. Intra-operative kinematics was also available by using a dedicated navigation system, thus to verify knee laxity at the time of surgery. Additionally, non-invasive assessment was possible both before the reconstruction and during the whole follow-up period by using inertial sensors. Integrating 3d models with kinematic data, estimation of contact areas of stress patterns on cartilage was also possible. The presented integrate protocol allowed to acquired different types of information concerning clinical assessment, biochemical changes in cartilage and biomechanical parameters to identify which acl reconstruction could present the most chondroprotective behavior. Preliminary data showed all the potential of the proposed workflow. The study is on-going and final results will be shortly provided

Summary Statement. Conventional imaging techniques lack the ability to objectively assess early stages of intervertebral disc degeneration, characterised by glycosaminoglycan loss. This study shows that MRI T2∗ mapping correlates positively with GAG content and that it provides continuous measurements for disc degeneration. Introduction. Early degenerative changes arise in the nucleus pulposus (NP) and are characterised by a loss of glycosaminoglycans (GAG). Early disc degeneration (DD) could possibly be treated with upcoming regenerative therapies (e.g. with stem cells and/or growth factors). In order to evaluate degeneration and treatments, a sensitive diagnostic tool is needed. While conventional magnetic resonance imaging (MRI) and x-ray techniques can detect late stages of DD, these techniques lack the ability to detect early degenerative changes. Recently, T2∗ mapping has been proposed as a new technique to evaluate early IVD degeneration, yet the correlation with GAG content and histological features has not been previously investigated. The objective of this study was to determine the value of T2∗ mapping in diagnosing DD by correlating this technique with the biochemical composition of IVDs. Materials & Methods. Six caprine lumbar spines obtained from an in vivo study and two healthy goat spines from the local abattoir, encompassing a total of 48 IVDs, were examined using sagittal standard T2-weighted and T2∗ mapping MRI protocols at 1.5 Tesla. Regions of interest (ROIs) were drawn on the T2∗ maps, covering the IVD. Based on T2 weighted MRI, discs were morphologically classified using the Pfirrmann score. Histological and macroscopic features were evaluated based on grading scales adapted for goat DD. Finally, GAG content was determined using colorimetric analysis (DMMB assay). Correlations between variables were analysed using Pearson correlation (r) coefficients (parametric data) or Spearman's rho (ρ) coefficients (non-parametric data). Results. The mean GAG content in the NP was 450 μg/mg dry weight (range 20–730 μg/mg dry weight) and the mean histological grade was 2.2 (range 0–6), corresponding with relatively mild disc degeneration. A linear positive correlation was observed between T2∗ and NP GAG content (r = 0.65, p < 0.001). T2∗ in the NP decreased linearly with increasing degeneration as assessed with macroscopic (ρ = 0.33, p < 0.05) and histological (ρ = −0.45, p < 0.05) grading, as well as with the Pfirrmann scoring system (ρ = −0.67, p < 0.001). Discussion. T2∗ mapping is a relatively new MRI technique which allows for measurements on a continuous scale, is acquired in less time than T2 mapping and minimises observer bias compared to grading systems. Although limited by a small sample size (n=48), this study showed a relatively good, linear correlation between T2∗ and GAG content in the NP, suggesting that T2∗ mapping may be an efficient and reliable tool for the objective assessment of proteoglycan content in early DD. Furthermore, with minor software modifications, it can be implemented on a standard 1.5 Tesla clinical MRI scanner. Future research should aim at optimizing the efficiency and user-friendliness of the T2∗ mapping protocol as well as yielding an even stronger correlation between T2∗ mapping and glycosaminoglycan content in human IVD tissue