Aims

This aim of this study was to analyze the detection rate of rare pathogens in bone and joint infections (BJIs) using metagenomic next-generation sequencing (mNGS), and the impact of mNGS on clinical diagnosis and treatment.

Aims

The metabolic variations between the cartilage of osteoarthritis (OA) and Kashin-Beck disease (KBD) remain largely unknown. Our study aimed to address this by conducting a comparative analysis of the metabolic profiles present in the cartilage of KBD and OA.

Aims

To explore the efficacy of extracorporeal shockwave therapy (ESWT) in the treatment of osteochondral defect (OCD), and its effects on the levels of transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, -3, -4, -5, and -7 in terms of cartilage and bone regeneration.

Energy storing tendons such as the human Achilles and equine superficial digital flexor tendon (SDFT) are prone to age-related injury. Tendons have poor healing capacity and a lack of effective treatments can lead to ongoing pain, reduced function and re-injury. It is therefore important to identify the mechanisms underpinning age-related tendinous changes in order to develop more effective treatments. Our recent single cell sequencing data has shown that tendon cell populations have extensive heterogeneity and cells housed in the tendon interfascicular matrix (IFM) are preferentially affected by ageing. There is, however, a lack of established surface markers for cell populations in tendon, limiting the capacity to isolate distinct cell populations and study their contribution to age-related tendon degeneration. Here, we investigate the presence of the cell surface proteins MET proto-oncogene (MET), integrin subunit alpha 10 (ITGA10), fibroblast activation protein alpha (FAP) and platelet derived growth factor receptor alpha (PDGFRA) in the equine SDFT cell populations and their co-localisation with known markers. Using Western blot we validated the specificity of selected antibodies in equine tissue before performing immunohistochemistry to establish the location of the respective proteins in the SDFT. We subsequently used double labelling immunofluorescence with the established mural cell marker desmin (DES) to distinguish between tenocyte and mural cell populations. In situ, MET, ITGA10, and FAP presence was found in cells throughout the tendon whereas PDGFRA was present in cells within the IFM. Double labelling immunofluorescence with the mural cell marker DES showed lack of co-localisation between PDGFRA and DES suggesting PDGFRA is labelling an IFM cell population distinct from those associated with blood vessels. PDGFRA is a promising target for the specific cell sorting of IFM-localised tenocytes, enabling their isolation and subsequent characterisation. Acknowledgments: The authors acknowledge the Biotechnology and Biological Sciences Research Council (BB/W007282/1) for funding this work

Stem cells represent an exciting biological therapy for the management of many musculoskeletal tissues that suffer degenerative disease and/or where the reparative process results in non-functional tissue (‘failed healing’). The original hypothesis was that implanted cells would differentiate into the target tissue cell type and synthesise new matrix. However, this has been little evidence that this happens in live animals compared to the laboratory, and more recent theories have focussed on the immunomodulatory effects via the release of paracrine factors that can still improve the outcome, especially since inflammation is now considered one of the central processes that drive poor tendon healing. Because of the initial ‘soft’ regulatory environment for the use of stem cells in domestic mammals, bone and fat-derived stem cells quickly established themselves as a useful treatment for naturally occurring musculoskeletal diseases in the horse more than 20 years ago (Smith, Korda et al. 2003). Since the tendinopathy in the horse has many similarities to human tendinopathy, we propose that the following challenges and, the lessons learnt, in this journey are highly relevant to the development of stem cells therapies for human tendinopathy:. Source – while MSCs can be recovered from many tissues, the predominant sources for autologous MSCs have been bone and fat. Other sources, including blood, amnion, synovium, and dental pulp have also been commercialised for allogenic treatments. Preparation – ex vivo culture requires transport from a licensed laboratory while ‘minimally manipulated’ preparations can be prepared patient-side. Cells also need a vehicle for transport and implantation. Delivery – transport of cells from the laboratory to the clinic for autologous ex vivo culture techniques; implantation technique (usually by ultrasound-guided injection to minimise damage to the cells (or, more rarely, incorporated into a scaffold). They can also be delivered by regional perfusion via venous or arterial routes. Retention – relatively poor although small numbers of cells do survive for at least 5 months. Immediate loss to the lungs if the cells are administered via vascular routes. Synovially administered cells do not engraft into tendon. Adverse effects – very safe although needle tracts often visible (but do not seen to adversely affect the outcome). Allogenic cells require careful characterisation for MHC Class II antigens to avoid anaphylaxis or reduced efficacy. Appropriate injuries to treat – requires a contained lesion when administered via intra-lesional injection. Intrasynovial tendon lesions are more often associated with surface defects and are therefore less appropriate for treatment. Earlier treatment appears to be more effective than delayed, when implantation by injection is more challenging. Efficacy - beneficial effects shown at both tissue and whole animal (clinical outcome) level in naturally-occurring equine tendinopathy using bone marrow-derived autologous MSCs Recent (licenced) allogenic MSC treatment has shown equivalent efficacy while intra-synovial administration of MSCs is ineffective for open intra-synovial tendon lesions. Regulatory hurdles – these have been lighter for veterinary treatments which has facilitated their development. There has been greater regulation of commercial allogenic MSC preparations which have required EMA marketing authorisation

Intra-articular injection is a common way to deliver biologics to joints, but their effectiveness is limited by rapid clearance from the joint space. This barrier can be overcome by genetically modifying cells within the joint such that they produce anti-arthritic gene products endogenously, thereby achieving sustained, therapeutic, intra-articular concentrations of the transgene products without re-dosing. A variety of non-viral and viral vectors have been subjected to preclinical testing to evaluate their suitability for delivering genes to joints. The first transfer of a gene to a human joint used an ex vivo protocol involving retrovirally transduced, autologous, synovial fibroblasts. Recent advances in vector technology allow in vivo delivery using adeno-associated virus (AAV). We have developed an AAV vector encoding the interleukin-1 receptor antagonist (AAV.IL-1Ra) for injection into joints with osteoarthritis (OA). It showed efficacy and safety in equine and rat models of OA, leading to a recently-completed, investigator-initiated, Phase I, dose-escalation clinical trial in 9 subjects with mid-stage OA of the knee (. ClinicalTrials.gov. Identifier: NCT02790723). Three cohorts of three subjects with mild to moderate OA in the index knee were injected intra-articularly under ultrasound guidance with a low (10e11 viral genomes) medium (10e12 viral genomes) or high (10e13 viral genomes) dose of AAV.IL-1Ra and followed for one year. The data confirm safety, with evidence of sustained intra-articular expression of IL-1Ra and a clinical response in certain subjects. Funding for a subsequent Phase Ib trial involving 50 subjects (. ClinicalTrials.gov. Identifier: NCT05835895), expected to start later this year, has been acquired. Progress in this area has stimulated commercial activity and there are now at least seven different companies developing gene therapies for OA and a number of clinical trials are in progress. Acknowledgement: Clinical trial funded by US Department of Defense Clinical Trial Award W81XWH-16-1-0540

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

Osteoprogenitors on the inner layer of periosteum are the major cellular contributors to appositional bone growth and bone repair by callus formation. Previous work showed that periosteal-derived cells have little or no osteogenic activity under standard in vitro osteogenic culture conditions. This study was conducted to determine what growth factor(s) can activate periosteal osteogenic capacity. This study was conducted with IACUC approval. Periosteum from five equine donors was digested in collagenase for 3-4 hours at 37C. Isolated periosteal cells were maintained in DMEM/10% FBS medium and exposed to PDGF, Prostaglandin E2, BMP-2 and TGF-b3 at a range of concentrations for 72 hours. Changes in osteogenic gene expression (Runx2, OSX and ALP) were measured by qPCR. Periosteal cells were pre-treated with TGF-b3 or maintained in control medium were transferred into basal or osteogenic medium. Osteogenic status was assessed by Alizarin Red staining for mineralized matrix, ALP enzymatic activity and induction of osteogenic genes. PDGF, PgE2 and BMP-2 had little impact on expression of osteogenic markers by periosteal cells. In contrast, TGF-b3 stimulated significant increases in Osterix (over 100-fold) ALP expression (over 70-fold). Pre-treating periosteal cells with TGF-b3 for 72 hours stimulated rapid cell aggregation and aggregate mineralization once cells were transferred to osteogenic medium, while cells not exposed to TGF-b3 exhibited minimal evidence of osteogenic activity. This study indicate that TGF-b signaling is vital for periosteal osteogenic activity. Transient ‘priming’ of periosteal cells through TGF-b exposure was sufficient to activate subsequent osteogenesis without requiring ongoing growth factor stimulation. TGF beta ligands are secreted by many cell types, including periosteal progenitors and osteocytes, providing opportunities for both autocrine and paracrine pathways to regulate periosteal bone formation under homeostatic and reparative conditions

Joint tissues release extracellular vesicles (EVs) that potentially sustain joint homeostasis and contribute to osteoarthritis (OA) pathogenesis. EVs are putative novel therapeutics for OA, and transport biologically active molecules (including small non-coding RNAs (SNCRNAs)) between cells. This study identified altering SNCRNA cargo in EVs in OA which may act as early diagnostic markers and treatment targets.

OA was surgically induced in four skeletally mature Standardbred horses using an osteochondral fragment model in the left middle carpal joint. The right joint underwent sham surgery. Synovial fluid (SF) and plasma were obtained weekly throughout the 70-day study. EVs were isolated using size exclusion chromatography and characterised using nanoparticle tracking (Nanosight), and exosome fluorescence detection and tetraspanin phenotyping (Exoview). RNA was extracted from EVs derived from SF (sham and OA joints) and plasma collected at days 10, 35, 42, 49, 56, 63, and subjected to small RNA sequencing on a NovaSeq SP100 flow cell (Illumina).

Nanosight-derived EV characteristics of size and concentration were not significantly different following disease induction. The diameter of the temporal population of plasma and SF-derived exosomes changed significantly for CD9 and CD81 following OA induction with significant temporal, and disease-related changes in CD63 and CD81 protein expressin in plasma and SF.

In SF and plasma-derived EVs snoRNAs, snRNAs, tRNAs, lncRNA, y-RNA, piRNAs and scRNA were found. Following pairwise analysis of all-time points we identified 27 miRs DE in plasma and 45 DE miRs in SF. Seven were DE in plasma and SF; miR-451, miR-25, miR-215, miR-92a, miR-let-7c, miR-486-5p, miR-23a. In plasma and SF 35 and 21 snoRNAs were DE with four DE in plasma and SF; U3, snord15, snord46, snord58.

This work has identified alterations to OA EV sncRNAs in plasma and SF providing a greater understanding of the role of EVs in early OA.

Introduction. Advanced practice physiotherapists (APPs) manage the national low back and radicular pain pathway across the UK. A novel spinal APP-led same-day emergency care (SDEC) pathway in Nottingham, manages patients referred from community services and the emergency department (ED). Patients may attend ED in the belief their pain is due to sinister or ‘red flag’ pathology. Little data exists on prevalence of spinal ‘red flag’ pathologies within a secondary care setting. This paper aims to review the number of ‘red flag’ pathology identified by APP's on a same-day emergency care pathway. Methods. Retrospective data from 1 year of routinely collected information was extracted and analysed by two APPs. Counts were reported as a percentage of total patients seen on the SDEC unit over a one-year period and compared to nationally reported figures. A total of 2042 patients were assessed on the unit in 2021, of which, 293 (14%) had serious pathology identified. Patients were classified into type of serious pathology: myelopathy (126, 6.1%), fractures (72, 3.5%), cauda equine compression (40, 1.9%), infection (37, 1.8%), cancers (28, 1.3%), neurological conditions (14, 0.6%) and other (16, 0.8%) serious pathology. Conclusion. APP's working within an emergency pathway are highly likely to see and diagnose serious spinal pathology. The most common include cord or cauda equina compression, fractures, infection and cancers. Figures reported are slightly higher than previously documented. Knowledge and training to identify ‘red flags’ and robust pathways of escalation are essential in support of APP roles and services. Conflict of interest: No conflicts of interest. Sources of funding: No sources of funding

Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors.

Cite this article: Bone Joint Res 2022;11(8):561–574.

Aims

To explore the effect of different durations of antibiotics after stage II reimplantation on the prognosis of two-stage revision for chronic periprosthetic joint infection (PJI).

Osteochondrosis (OC) is a common joint disease that affects developing cartilage and subchondral bone in humans, and in multiple animal species including horses. It is an idiopathic localized joint disorder characterized by focal chondronecrosis and retention of growing cartilage that can lead to the formation of fissures, subchondral bone cysts or intra-articular fragments. OC is considered a complex multifactorial disease with chondrocyte biogenesis impairment mainly due to biochemical and genetic factors. Likewise, the molecular events involved in the OC are not fully understood. Moreover, the OC pathogenesis seems to be shared across species. In particular, equine OC and human juvenile OC share some symptoms, predilection sites and clinical presentation. In this study, by using the label-free mass spectrometry approach, proteome of chondrocytes isolated from equine OC fragments has been analysed in order to clarify some aspects of cell metabolism impairment occurring in OC. Equine chondrocytes isolated from 7 healthy articular cartilages (CTRL) and from 7 osteochondritic fragments (OC) (both obtained from metacarpo/metatarsophalangeal joints) were analysed. Proteins were extracted using urea and ammonium bicarbonate buffer, reduced, alkylated and digested with Trypsin/Lys-C Mix. Peptides were analysed using Q Exactive UHMR Hybrid Quadrupole-Orbitrap Mass Spectrometer (Thermo Scientific). All mass spectra of label-free samples analysed was set up to search against SwissProt human database (Homo sapiens) and SwissProt horse database (Equus caballus). One-way ANOVA was used for hypothesis testing. Proteins with a ≥1.5 fold change and with a FDR adjusted p value of ≤0.05 were defined as differentially expressed. Statistical analysis evidenced 41 proteins up-regulated in OC while 18 were down-regulated with respect to the CTRL. Functional analysis showed that up-regulated proteins in OC were related to extracellular matrix degradation, lysosome, apoptotic execution phase, unfolded protein response, hyaluronan and keratan sulfate degradation, oxidative stress response and negative regulation of BMP signalling pathway. The down-regulated proteins were associated with endochondral ossification, vitamin D in inflammatory disease, Wnt signalling pathway and ECM proteoglycans. Validation assays confirmed these findings. These findings may contribute to clarify the events determining the onset and progression of both equine and human OC. Imaging MS analysis of OC and healthy cartilage to analyse lipid and metabolomic changes occurring in OC cartilage is in progress

Calcium phosphates-based coatings have been widely studied to favour a firm bonding between orthopaedic implants and the host bone. To this aim, thin films (thickness below 1 μm) having high adhesion to the substrate and a nanostructured surface texture are desired, capable of boosting platelet, proteins and cells adhesion. In addition, a tunable composition is required to resemble as closely as possible the composition of mineralized tissues and/or to intentionally substitute ions having possible therapeutic functions. The authors demonstrated nanostructured films having high surface roughness and a composition perfectly resembling the deposition target one can be achieved by Ionized Jet Deposition (IJD). Highly adhesive nanostructured coatings were obtained by depositing bone-apatite like thin films by ablation of deproteinized bovine bone, capable of promoting host cells attachment, proliferation and differentiation. Here, biomimetic films are deposited by IJD, using biogenic and synthetic apatite targets. Since IJD deposition can be carried out without heating the substrate, application on heat sensitive polymeric substrate, i.e. 3D printed porous scaffolds, is investigated. Biogenic apatite coatings are obtained by deposition of deproteinized bone (bovine, ovine, equine, porcine) and compared to ones of stoichiometry hydroxyapatite (HAp). Coatings composition (FT-IR-ATR, FT-IR microscopy, XRD, EDS) and morphology (SEM, AFM) are tested for deposition onto metallic and 3D-printed polymeric substrates (polyurethane (PU)). Different post-treatment annealing procedures for metallic substrates are compared (350–425°C), to optimize crystallinity. Then, uniformity of substrate coverage and possible damage caused to the polymeric substrate are studied by SEM, DSC and FT-IR microscopy. Biogenic coatings are composed by carbonated HAp (XRD, FT-IR). Trace ions Na. +. and Mg. 2+. are transferred from deposition target to coating. All coatings are nanostructured, composed by nano-sized globular aggregates, of which morphology and dimensions depend on the target characteristics. As-deposited coatings are amorphous, but crystallinity can be tuned by post-treatment annealing. A bone-like crystallinity can be achieved for heating at ≥400°C, also depending on duration. When deposited on 3D-printed PU scaffolds, coatings, owing to sub-micrometric thickness, coat them entirely, without altering their fibre shape and porosity. Obtained biomimetic bone apatite coatings can be deposited onto a variety of metallic and polymeric biomedical devices, thus finding several perspective applications in biomedical field

Aims. The aim of this paper is to describe the impact of COVID-19 on spine surgery services in a district general hospital in England in order to understand the spinal service provisions that may be required during a pandemic. Methods. A prospective cohort study was undertaken between 17 March 2020 and 30 April 2020 and compared with retrospective data from same time period in 2019. We compared the number of patients requiring acute hospital admission or orthopaedic referrals and indications of referrals from our admission sheets and obtained operative data from our theatre software. Results. Between 17 March to 30 April 2020, there were 48 acute spine referrals as compared to 68 acute referrals during the same time period last year. In the 2019 period, 69% (47/68) of cases referred to the on-call team presented with back pain, radiculopathy or myelopathy compared to 43% (21/48) in the 2020 period. Almost 20% (14/68) of spine referrals consisted of spine trauma as compared to 35% (17/48) this year. There were no confirmed cases of cauda equine last year during this time. Overall, 150 spine cases were carried out during this time period last year, and 261 spine elective cases were cancelled since 17 March 2020. Recommendations. We recommend following steps can be helpful to deal with similar situations or new pandemics in future:. 24 hours on-call spine service during the pandemic. Clinical criteria in place to prioritize urgent spinal cases. Pre-screening spine patients before elective operating. Start of separate specialist trauma list for patients needing urgent surgeries. Conclusion. This paper highlights the impact of COVID-19 pandemic in a district general hospital of England. We demonstrate a decrease in hospital attendances of spine pathologies, despite an increase in emergency spine operations. Cite this article: Bone Joint Open 2020;1-6:281–286

Tenocytes from several mammal species have been shown to be prone to phenotypic drift at early sub-culture passages. In the present study we compared allogenic and xenogenic serum supplementation suitability as a supplement for the in vitro expansion of equine tenocytes (eTCs), in combination with the presence or absence of crowding conditions. eTCs were isolated from superficial digital flexor tendon and expanded in normal growth medium containing DMEM, 10% appropriate serum, 1% penicillin/streptomycin solution. Isolation was performed by migration method in growth medium containing the selected serum. Silver staining, densitometry, zymography, immunofluorescence, metabolic activity, proliferation, viability and morphology were performed after 3, 5 and 7 days in culture with a seeding density of 10,000 cells/cm2. Treatment conditions were equine serum (ES) or foetal bovine serum (FBS), with or without 75 μg/mL of crowding agent carrageenan (CR). Viability and metabolic activity of eTCs were affected by FBS. eTCs in ES reached higher cell density than in FBS in day 7, especially with CR. Morphology of eTCs was maintained under different sera. Silver staining on pepsin digested cell layers shows that collagen type I deposition rate is remarkably enhanced in the presence of CR in all conditions. Immunofluorescence showed increased expression for collagen I, III, V and VI in both sera in the presence of CR. Deposition of all collagen types but type VI was increased by ES supplementation. We conclude that ES in combination with CR can represent a reliable choice for the ex vivo expansion of eTCs

Cell-based scaffold-free tissue equivalents present a limited clinical translation as consequence of the delayed extracellular matrix (ECM) deposition due to the prolonged production time in vitro. Different combinations of media supplements such as ascorbic acid, growth factors, oxygen tension among others can modulate the cell fate or the ECM synthesis. New research lines are focusing on the use of macromolecular crowders (MMCs) as media supplement for cell sheet production due to their ability to increase ECM deposition by volume exclusion effect, pro-collagenases alosteric regulation, matrix self-assembly by confinement and diffusion limitation (most probably, modulating the interaction between the ECM, MMPs and TIMPs). Herein, different molecular weights and concentrations of a natural potential MMC (Crowder-A) have been tested in equine adipose-derived stem cell (eADSC) and human dermal fibroblast (hADF) cultures in comparison with other commonly used crowders such as carrageenan and the Ficoll™ cocktail 70 KDa and 400 KDa. The eADSCs were characterized according to the current criteria for horse MSCs. Tri-lineage and FACS analysis showed eADSC osteogenic and adipogenic potentials and the presence of the markers CD29, CD44, CD90. The screening of the aforementioned Crowder-A was performed in cultures of 15,000 cells / cm. 2. for the eADSCs and 25,000 cells / cm. 2. for the hADFs during 3, 5, and 7 days. Non-MMC conditions were used as negative controls. Collagen type I was analysed by SDS-PAGE. Other collagen types were studied by immunocytochemistry assays. Significant increase of some ECM components was observed in some concentrations and molecular weights of the Crowder-A

The aim of this work was the structural investigation of different type I collagen isoforms at atomic and nanoscale, as well as the evaluation of the impact of different fabrication treatments on the structural, mechanical and biological properties of collagen-based films. Raw type-I collagens from bovine hide (Typ-BH, CS, SYM) and equine tendon (TypE, TypCH and OPO) were analyzed. Materials were then used for fabricating air-dried films, obtained by: 1) dissolution in distilled water (HH); 2) dissolution in acidic medium (AA); 3) homogenization of acid solubilized fibers (HOM). Crosslinking treatments (DHT, DHT+EDC) were also adopted and studied. Analysis by Wide Angle (WAXS) and Small Angle (SAXS) X-ray Scattering was carried out at the XMI L@b (CNR-IC-Bari); Fourier Transform-IR and biological analysis was performed at UniSalento. WAXS and SAXS data on raw materials demonstrated the preferential orientation of collagen molecules and the preservation of hierarchical nanoscale architecture in equine tendon-derived collagens, in particular in chemically extracted, while randomly oriented molecules were found in bovine dermis collagens, together with a certain degree of salt contamination. Concerning equine collagen, we found that TypCH structure is influenced by crosslinking procedures at atomic scale, whereas both processing conditions and crosslinking treatments affect TypE collagen structure at atomic and nanoscale. WAXS, SAXS and FT-IR analyses showed that the HOM processing was the one which ensures a high content of structural super-organization of collagen into triple helices and a high crystalline domainof the final material. Crosslinking of the films by DHT/EDC combined treatment was shown to affect their mechanical stiffness, the latter depending on the collagen source and the specific processing conditions

Tendon injuries in both the human and horse represent a challenge due to persistent inflammation combined with inadequate reparative cells and a poorly organised extracellular matrix. The potential of mesenchymal stem cells (MSCs) in regenerating tendon injuries remains to be fully realised. The main mechanism of action by MSCs is considered to be primarily mediated via paracrine mechanisms. This may involve the production and release of extracellular vesicles (EVs) by stem cells with a sub-fraction of these EVs (<100 nm diameter) called exosomes that appear to be the main paracrine effectors. EVs can be readily prepared from MSCs and offer a clinically relevant therapy. However, EVs for tendon repair need to be fully characterised. The horse represents a highly relevant model of tendon and ligament injuries as it shares many features of mechanical loading, function and aetiopathology with the human. We have isolated and characterised EVs from equine MSCs for modulating tendon cell phenotype in an in vitro tendon injury model using IL-1ß. EVs can be isolated from IL-1ß stimulated MSCs although their levels are not significantly increased over controls suggesting that the nature of the stimulated EV cargo may be more important than absolute levels of released EVs

The paramount importance of synovial fluid in lubrication and protection of articular joints has long been recognized. Synovial fluid, a dialysate of plasma, forms an interface with both the synovium and cartilage and plays a crucial role in joint lubrication and bearing functions. In an osteoarthritic joint, damage to the articular cartilage causes modifications in the rheological properties of synovial fluid and, reducing the viscoelasticity and increasing the friction between articular surfaces. Viscosupplementation is a treatment for osteoarthritis that uses hyaluronic acid as a (visco)supplement to the diseased joint. The aim of this treatment is to restore the rheological properties of synovial fluid. Osteoarthritis is the most common disease affecting the joints in human population and among the most important causes of pain, disability and economic loss. Therefore, innovative methods are needed to more effectively treat osteoarthritis, directly addressing the disease process. Among various locomotor mechanisms that could serve to illustrate the integrated nature of functional morphology, perhaps none is more complex than the equine locomotor system. Confronting the need for evaluating the current methods to control joint disease, the horse provides an excellent animal model. As it suffers similar clinical manifestations to those seen in human, it may provide tentative biomedical extrapolations