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

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

Gene therapy with insulin-like growth factor-1 (IGF-1) increases matrix production and enhances chondrocyte proliferation and survival in vitro. The purpose of this study was to determine whether arthroscopically-grafted chondrocytes genetically modified by an adenovirus vector encoding equine IGF-1 (AdIGF-1) would have a beneficial effect on cartilage healing in an equine femoropatellar joint model. A total of 16 horses underwent arthroscopic repair of a single 15 mm cartilage defect in each femoropatellar joint. One joint received 2 × 10. 7. AdIGF-1 modified chondrocytes and the contralateral joint received 2 × 10. 7. naive (unmodified) chondrocytes. Repairs were analysed at four weeks, nine weeks and eight months after surgery. Morphological and histological appearance, IGF-1 and collagen type II gene expression (polymerase chain reaction, in situ hybridisation and immunohistochemistry), collagen type II content (cyanogen bromide and sodium dodecyl sulphate-polyacrylamide gel electrophoresis), proteoglycan content (dimethylmethylene blue assay), and gene expression for collagen type I, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, aggrecanase-1, tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) and TIMP-3 were evaluated. Genetic modification of chondrocytes significantly increased IGF-1 mRNA and ligand production in repair tissue for up to nine weeks following transplantation. The gross and histological appearance of IGF-1 modified repair tissue was improved over control defects. Gross filling of defects was significantly improved at four weeks, and a more hyaline-like tissue covered the lesions at eight months. Histological outcome at four and nine weeks post-transplantation revealed greater tissue filling of defects transplanted with genetically modified chondrocytes, whereas repair tissue in control defects was thin and irregular and more fibrous. Collagen type II expression in IGF-1 gene-transduced defects was increased 100-fold at four weeks and correlated with increased collagen type II immunoreaction up to eight months. Genetic modification of chondrocytes with AdIGF-1 prior to transplantation improved early (four to nine weeks), and to a lesser degree long-term, cartilage healing in the equine model. The equine model of cartilage healing closely resembles human clinical cartilage repair. The results of this study suggest that cartilage healing can be enhanced through genetic modification of chondrocytes prior to transplantation

The rheological properties of synovial fluid (SF) are largely attributed to the presence of high molecular weight hyaluronic acid (HA). In normal SF, HA has been shown to be an anti-inflammatory molecule able to increase the viscosity and promote endogenous production of HA. The aim of the present report was to investigate the possible effect of HA concentration in rheological properties (elastic modulus, G´ and viscous modulus, G´´) of osteoarthritic equine SF. For this purpose, SF from intercarpal, metacarpophalangeal and distal interphalangeal joint was aspirated by aseptic arthrocentesis from 60 Warmblood horses. For determining HA concentrations in equine SF samples, a commercially available ELISA kit was used. Additionally, full rheological sample characterization was carried out with an AR-G2 rheometer (TA Instruments Ltd., UK) in order to measure the elastic G´ and viscous G´´ moduli, at horse's body (37.5 ºC) temperature. The ANOVA findings revealed statistically significant main effects of the factors Joint Type (p = 0.001), and main effects of covariates Age (p = 0.019) and HA (p < 0.001) on the mean values of logG” and logG' measurements. Interpreting the coefficients of the covariate HA, a positive correlation of HA was detected on the response logG” and logG' measurements. Collectively, these data illustrate the role of HA in equine pathological SF

Summary Statement. Transportation media and injection protocol have implications for the viability of MSCs used for intra-lesional treatment of tendon injuries. Every effort should be made to implant cells within 24h of laboratory re-suspension, using a needle bore larger than 21G. Introduction. Intra-lesional implantation of autologous mesenchymal stem cells (MSCs) has resulted in significant improvements in tendon healing in experimental animal models. Intra-tendinous injection of MSCs into naturally-occurring equine tendon injuries has been shown to be both safe and efficacious. 1. and these protocols can assist in the translation to the human. Efficient transfer of cells from the laboratory into the tissue requires well validated techniques for transportation and implantation. The aim of this study was to determine the influence of transport media and injection procedure on cellular damage. Methods. Bone marrow derived MSCs (n=3 horses) were prepared and expanded as described. 1. Cells were suspended in 0.5mL of experimental media at 2.5×10. 6. cells/mL and stored at 4–8°C for 24, 48 and 72h. Experimental media were: bone marrow aspirate (BMA); cell culture media (DMEM); equine serum; equine plasma; isotonic saline; hyaluronic acid (HA); platelet-rich plasma (PRP) and frozen (in 90% serum, 10% DMSO). In addition, cells suspended in DMEM were injected through a 19G, 21G or 23G needle and cell viability, proliferation and apoptosis were analysed using trypan blue, alamarBlue® and Annexin-V assays respectively. Results. There was no significant difference in overall viability at 24h storage in any media, however cell death was most rapid when cells were suspended in BMA, PRP and serum. Viability was greatest at all time points when cells were frozen. Cell proliferation was similar following storage for 24 and 72h in all media, except for 24h in serum, wherein proliferation was enhanced. There was no significant decrease in viability immediately following injection but 21G and 23G needles resulted in a marked increase in apoptotic cells compared to 19G and non-injected controls after 24h when re-seeded for culture. All needle gauges resulted in a marked decrease in cell proliferation immediately post-injection with recovery by 2h post-injection. Conclusions. Although there is, as yet, no guidance on the storage of MSCs, it has been suggested that in vitro storage of hematopoietic stem or progenitor cells should not exceed 2h. 2. This suggestion is impractical both for current equine therapeutic use and when considering future, commercial applications of MSC therapy in humans, because of the necessity to transport the cells from a remote licensed facility to the clinic. Our data suggest an upper limit of 24h for transportation, whereas for transportation of greater duration than 24h, cells should ideally be frozen, to maximise viability. An increased number of dead cells potentially has two adverse consequences; first, a reduced efficacy and second, the presence of dead cell debris may induce inflammation. While the first can be compensated for by higher cell numbers, this compounds the problems of the second. This study reinforces the importance of limiting the delay between preparation of cells for shipment from the laboratory and implantation in the clinic and suggests that an injection procedure while not causing immediate cell death can cause significant delayed cell death if small bore needles are used

Summary Statement. Extended expansion of cells derived from equine articular cartilage reveal maintenance of chondrogenic potency and no evidence of senescence up to 100 population doublings. The data suggests the reclassification of these cells from progenitor cells to stem cells. Introduction. One sign of ‘in vitro aging’ is the diminishing capacity for cell division. In contrast to embryonic stem cells that show no loss of proliferative potency, the maximal population doublings (PD) for mesenchymal stem cells (MSCs) in vitro is reported to be between 30 and 40 replications 1,2,3. We have isolated a population of chondroprogenitor cells from articular cartilage of several species, including equine4. These cells have demonstrated functional equivalence in their differentiation capacity when compared with MSCs but have the advantage of retaining the highly desirable stable (permanent) chondrocyte phenotype. In this study, we examined the age-related capacity of these cells for extended division and retention of potency. Methods. Chondroprogenitors were isolated from equine articular cartilage by adhesion onto fibronectin5. Cells were isolated from both skeletally immature (1 year-old) and mature animals (8 year-old). Clonal and polyclonal cell lines (at least 5 of each for each age) were cultured in the presence of 10% FCS, 1ng/ml TGFb-1 & 2.5 ng/ml FGF-2. Cells were seeded at low density and passaged weekly. Results. Chondroprogenitors from both animals reached over 40 (mean) PD in 50 days with growth remaining linear. Little difference in growth rates was observed between clonal and polyclonal cell lines. For the mature animal, 96% of cells were BrDU positive at 22 PD whilst none of cells were (senescence associated) β-gal positive. At 44 PD, 88% of cells were BrDU positive and just 15% of cells were β-gal positive. Three clonal and three polyclonal cell lines from the mature animal were cultured beyond the 50-day time point. At 120 days, cells reached up to 90 PD with the same pattern of linear growth observed. When tested at 70 PD, 79% of these cells were still BrDU positive (range 55–97%) and just 11% of cells were β-gal positive (range 2–22%). Furthermore, little difference in cell morphology was observed throughout this extended expansion. At 70 PD, we found that both clonal and polyclonal cell lines in monolayer culture were still expressing the chondrogenic transcription factor; Sox-9. Expression of genes for aggrecan and collagen type II was also detected in cells that were chondrogenically induced for 72 hours. Discussion & Conclusions. We have demonstrated for the first time the extended expansion of cells derived from articular cartilage that retain chondrogenic potency. These equine cells have since been cultured to over 100 PD without evidence of senescence. One hundred PD is equivalent to 1 × 1030 cells originating from a single cell. We have previously reported that the human equivalents of these cells surpass MSCs in doubling capacity but senesce at approximately 60 PD6. The properties of these equine chondroprogenitor cells make them ideal candidates for allogeneic cell therapy for articular cartilage repair. In addition, the data suggest the reclassification of these cells from progenitor cells to stem cells

Introduction. The equine SDFT tendon is a complex hierarchal structure that transmits force from muscle to bone and stores energy through its stretching and recoiling action. It is a common site of pathology in athletic horses. Our aim was to describe the ultrastructural anatomy of the SDFT as part of a larger programme to understand the structure-functional relationship of this tendon. Materials and Methods. Fifteen SDFT from different aged horses, sectioned transversely (2–3 mm thickness) and then photographed using Canon EOS 5D Mark III (100 mm focal length). Images processed through ImageJ and IMOD software for 3D reconstruction. Samples were also taken from the proximal, middle and distal part of the SDFT from a foetal, one and nine years old horse, processed for H&E staining and sectioned longitudinally in series into 20 sections (5µm), additionally the mid metacarpal region of one year old was fully sectioned into 250 sections. The entire cut surface on the slide was imaged and transformed to one collated image using Inkscape. Using IMOD collated photos transformed to mrc file (Z-stack) and in order to reconstruct 3D forms. Results. A tertiary fascicle was defined as a bundle of collagen fibres surrounded by a well-defined interfascicular matrix IFM (width 34.56 µm +/− 16.43 (St.Dev)). Secondary fascicles were defined as subdivisions of the tertiary fascicles (IFM width 11.1 µm +/− 4.01 (St.Dev)) (n=2). Using this classification we found that the numbers of the secondary and tertiary fascicles were not continuous through the tendon in a proximal to distal regions of the tendon. The histological 3D anatomy manifests similar fascicular structure in all ages, but their fascicular contours were less irregular in aged and in the mid-metacarpal region. The 3D anatomy of the mid-metacarpal tendon demonstrated heterogeneous fascicles, which had helical arrangement in their longitudinal axis. Discussion. Secondary and tertiary fascicles are heterogeneous in numbers, shapes and interconnections with each other in different regions. Fascicles appear to branch from proximal to distal through the tendon and are not always continuous through the tendon length. Some fascicles intercommunicating with each other and have helical configuration. Understanding the 3D anatomy will facilitate understanding of tendon structure-function relationships and injury predisposition

Osteoarthritis (OA) is an important cause of pain, disability and economic loss in humans, and is similarly important in the horse. Recent knowledge on post-traumatic OA has suggested opportunities for early intervention, but it is difficult to identify the appropriate time of these interventions. The horse provides two useful mechanisms to answer these questions: 1) extensive experience with clinical OA in horses; and 2) use of a consistently predictable model of OA that can help study early pathobiological events, define targets for therapeutic intervention and then test these putative therapies. This paper summarises the syndromes of clinical OA in horses including pathogenesis, diagnosis and treatment, and details controlled studies of various treatment options using an equine model of clinical OA

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.

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

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

We compared the quality of debridement of chondral lesions performed by four arthroscopic (SH, shaver; CU, curette; SHCU, shaver and curette; BP, bipolar electrodes) and one open technique (OPEN, scalpel and curette) which are used prior to autologous chondrocyte implantation (ACI). The ex vivo simulation of all five techniques was carried out on six juvenile equine stifle joints. The OPEN, SH and SHCU techniques were tested on knees harvested from six adult human cadavers. The most vertical walls with the least adjacent damage to cartilage were obtained with the OPEN technique. The CU and SHCU methods gave inferior, but still acceptable results whereas the SH technique alone resulted in a crater-like defect and the BP method undermined the cartilage wall. The subchondral bone was severely violated in all the equine samples which might have been peculiar to this model. The predominant depth of the debridement in the adult human samples was at the level of the calcified cartilage. Some minor penetrations of the subchondral end-plate were induced regardless of the instrumentation used. Our study suggests that not all routine arthroscopic instruments are suitable for the preparation of a defect for ACI. We have shown that the preferred debridement technique is either open or arthroscopically-assisted manual curettage. The use of juvenile equine stifles was not appropriate for the study of the cartilage-subchondral bone interface

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

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

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

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

Introduction. Tendons are critical to mobility, and are susceptible to degeneration through injury and ageing. Type I collagen is the most abundant protein in vertebrates; it is the main structural protein of the extracellular matrix in numerous musculoskeletal tissues, including tendons. Type I collagen predominantly is a heterotrimer, which consists of two alpha-1 chains and one alpha-2 chain (α1). 2. (α2) encoded by the COL1A1 and COL1A2 genes, respectively. However, type I collagen can form homotrimers (α1). 3. which are protease-resistant, and are associated with age-related musculoskeletal diseases, fibrotic and connective tissue pathologies. Transforming growth factor beta (TGFβ) enhances collagen (I) gene expression, is involved in tendon mechanobiology and repair processes, while its effect on homotrimer formation is unknown. Our aim is to investigate the relative expressions of collagen (I) α1 and α2 polypeptide chains in tenocytes (tendon fibroblasts) stimulated with TGFβ. Materials and Methods. Included RT-qPCR to measure the relative expression of COL1A1 and COL1A2 genes. [. 14. C]-proline metabolic labelling was used to measure the expression of the collagen (I) α1 and α2 polypeptide chains. These techniques were performed in equine superficial digital flexor tendon (SDFT) tenocytes (n=3) and murine tail tendon tenocytes (n=3) with different concentrations of TGFβ (0.01 ng/ml-100 ng/ml). Results. There was an increase in both COL1A1 and COL1A2 gene expression when stimulated with TGFβ in both cell types. In equine tenocytes the gene expression ratio of COL1A1:COL1A2 increased from 1.73 ± 0.75 to 7.87 ± 2.9 (p=0.003) when stimulated with 100 ng/ml of TGFβ3. TGFβ upregulated collagen (I) protein in both cell types. In equine tenocytes (n=3) when stimulated with 100 ng/ml of TGFβ3, the α1:α2 protein chain ratio increased from 1.93 ± 0.54 to 3.02 ± 0.32 (p=0.059) in comparison with serum-starved cells, which alongside the changes in gene expression, may be indicative of collagen (I) homotrimer production. Discussion. There were biosynthetic alterations in collagen production, and putative collagen (I) homotrimer when equine tenocytes were stimulated with 100 ng/ml TGFβ3. Future work will focus isolating different collagens by repeated differential salt precipitation. The level of TGFβ receptors and Smad signaling molecules will be also analysed using RT-qPCR and western blotting

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

Introduction. Tendon is prone to degeneration through ageing and injury and current therapies are largely ineffective. The recent identification of a cell population within tendon with stem cell-like characteristics holds potential for regeneration of tendon. The local stem cell environment (niche) is important for stem cell maintenance and function. This study aims to characterize extracellular matrix (ECM) components of the stem cell niche in equine tendon, which is prone to age-related degeneration and rupture. Materials and Methods. Putative tendon stem cells (TSCs) were isolated from equine superficial digital flexor tendon by low-density plating and differential adhesion to fibronectin. Cells were analysed by flow cytometry using antibodies to mesenchymal stem cell markers, as well as qRT-PCR for stem cell and tenogenic markers. The multipotency of cells was assessed using tri-lineage differentiation assays. ECM components of the tenocyte and TSC niche were analysed using radio-isotope labelling, immunohistochemistry and histology. Results. Putative TSCs were able to form colonies, and both tenocytes and TSCs expressed CD90, CD105 and CD73 as determined by flow cytometry. However, TSCs did not exhibit increased expression of stem cell marker genes when compared with tenocytes. TSCs and tenocytes both displayed osteogenic and chondrogenic differentiation, however not adipogenic differentiation. Tenocytes and TSCs labelled with 14C-labelled amino acids both displayed similar labelling profiles. Histological analysis of tendon tissue highlighted the varied structure and composition of tendon, with tenascin C expression confined to the interfascicular matrix. Discussion. TSCs do not highly express stem cell markers when compared with tenocytes, indicating that these cells may not be true stem cells. In addition the similar labelling profiles of the two cell types indicates that a stem cell population has not been differentially isolated, however the tri-lineage differentiation assays suggest the cells may possess some stem cell-like properties. It is possible that the equine tendon cell population consists of a heterogeneous mixture of cells at different stages of differentiation

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