Rapid developments in Regenerative Medicine and Tissue Engineering has witnessed an increasing drive toward clinical translation of breakthrough technologies. However, the progression of promising preclinical data to achieve successful clinical market authorisation remains a bottleneck. One hurdle for progress to the clinic is the transition from small animal research to advanced preclinical studies in large animals to test safety and efficacy of products. Notwithstanding this, to draw meaningful and reliable conclusions from animal experiments it is critical that the species and disease model of choice is relevant to answer the research question as well as the clinical problem. Selecting the most appropriate animal model requires in-depth knowledge of specific species and breeds to ascertain the adequacy of the model and outcome measures that closely mirror the clinical situation. Traditional reductionist approaches in animal experiments, which often do not sufficiently reflect the studied disease, are still the norm and can result in a disconnect in outcomes observed between animal studies and clinical trials. To address these concerns a reconsideration in approach will be required. This should include a stepwise approach using in vitro and ex vivo experiments as well as in silico modeling to minimize the need for in vivo studies for screening and early development studies, followed by large animal models which more closely resemble human disease. Naturally occurring, or spontaneous diseases in large animals remain a largely untapped resource, and given the similarities in pathophysiology to humans they not only allow for studying new treatment strategies but also disease etiology and prevention. Naturally occurring disease models, particularly for longer lived large animal species, allow for studying disorders at an age when the disease is most prevalent. As these diseases are usually also a concern in the chosen veterinary species they would be beneficiaries of newly developed therapies. Improved awareness of the progress in animal models is mutually beneficial for animals, researchers, human and veterinary patients. In this overview we describe advantages and disadvantages of various animal models including domesticated and companion animals used in regenerative medicine and tissue engineering to provide an informed choice of disease-relevant animal models.

Musculoskeletal injuries and chronic degenerative diseases commonly affect both athletic and sedentary horses and can entail the end of their athletic careers. The ensuing repair processes frequently do not yield fully functional regeneration of the injured tissues but biomechanically inferior scar or replacement tissue, causing high reinjury rates, degenerative disease progression and chronic morbidity. Regenerative medicine is an emerging, rapidly evolving branch of translational medicine that aims to replace or regenerate cells, tissues, or organs to restore or establish normal function. It includes tissue engineering but also cell-based and cell-free stimulation of endogenous self-repair mechanisms. Some regenerative medicine therapies have made their way into equine clinical practice mainly to treat tendon injures, tendinopathies, cartilage injuries and degenerative joint disorders with promising results. However, the qualitative and quantitative spatiotemporal requirements for specific bioactive factors to trigger tissue regeneration in the injury response are still unknown, and consequently, therapeutic approaches and treatment results are diverse. To exploit the full potential of this burgeoning field of medicine, further research will be required and is ongoing. This review summarises the current knowledge of commonly used regenerative medicine treatments in equine patients and critically discusses their use.

The treatment of tendon lesions with multipotent mesenchymal stromal cells (MSCs) is widely used in equine medicine. Cell sources of MSCs include bone marrow, as well as solid tissues such as adipose tissue. MSCs can be isolated from these solid tissues either by enzymatic digestion or by explant technique. However, the different preparation techniques may potentially influence the properties of the isolated MSCs. Therefore, the aim of this study was to investigate and compare the effects of these two different methods used to isolate MSCs from solid tissues.Equine adipose tissue, tendon and umbilical cord matrix served as solid tissue sources of MSCs with different stiffness and density. Subsequent to tissue harvest, MSCs were isolated either by enzymatic digestion with collagenase or by explant technique. Cell yield, growth, differentiation potential and tendon marker expression were analysed. At first passage, the MSC yield was significantly higher in enzymatically digested tissue samples than in explanted tissue samples, despite a shorter period of time in primary culture. Further analysis of cell proliferation, migration and differentiation revealed no significant differences between MSCs isolated by enzymatic digestion and MSCs isolated by explant technique. Interestingly, analysis of gene expression of tendon markers revealed a significantly higher expression level of scleraxis in MSCs isolated by enzymatic digestion. Both isolation techniques are feasible methods for successful isolation of MSCs from solid tissues, with no major effects on cellular proliferation, migration or differentiation characteristics. However, higher MSC yields were achieved in a shorter period of time by collagenase digestion, which is advantageous for the therapeutic use of MSCs. Moreover, based on the higher level of expression of scleraxis in MSCs isolated by enzymatic digestion, these cells might be a better choice when attempting tendon regeneration.

Aging is hypothesized to be associated with changes in tendon matrix composition which may lead to alteration of tendon material properties and hence propensity to injury. Altered gene expression may offer insights into disease pathophysiology and thus open new perspectives toward designing pathophysiology‐driven therapeutics. Therefore, the current study aimed at identifying naturally occurring differences in tendon micro‐morphology and gene expression of newborn, young and old horses. Age‐related differences in the distribution pattern of tendon fibril thickness and in the expression of the tendon relevant genescollagen type 1 (Col1), Col3, Col5, tenascin‐C, decorin, tenomodulin, versican, scleraxisandcartilage oligomeric matrix proteinwere investigated. A qualitative and quantitative gene expression and collagen fibril diameter analysis was performed for the most frequently injured equine tendon, the superficial digital flexor tendon, in comparison with the deep digital flexor tendon. Most analyzed genes (Col1, Col3, Col5, tenascin‐C, tenomodulin, scleraxis) were expressed at a higher level in foals (age ≤ 6 months) than in horses of 2.75 years (age at which flexor tendons become mature in structure) and older,decorinexpression increased with age. Decorin was previously reported to inhibit the lateral fusion of collagen fibrils, causing a thinner fibril diameter with increased decorin concentration. The results of this study suggested that reduction of tendon fibril diameters commonly seen in equine tendons with increasing age might be a natural age‐related phenomenon leading to greater fibril surface areas with increased fibrillar interaction and reduced sliding at the fascicular/fibrillar interface and hence a stiffer interfascicular/interfibrillar matrix. This may be a potential reason for the higher propensity to tendinopathies with increasing age.

Sheep are one of the most frequently used large animal models in stem cell research. However, minimal invasive or noninvasive sources of mesenchymal stem cells (MSCs) in sheep are scarce. In the light of the principles of the 3Rs (reduce, refine, replace), it would therefore be desirable to identify a minimally invasive or noninvasive ovine MSC source. In humans, the chorionic villi of the placenta, which can be noninvasively harvested as part of the afterbirth, have been identified as a rich source of MSCs. Therefore, in the present study, ovine placenta cotyledons, which have similar function and structure to human chorionic villi, were tested for their potential use as a noninvasive source of ovine MSCs. Through mincing of the placental cotyledons, collagenase digestion, and Ficoll density gradient centrifugation, combined with plastic adherence selection, MSCs were successfully isolated. Their morphological, immunophenotypical, and cellular growth characteristics, as well as their proliferation, differentiation, and migration potential, were evaluated and compared to the currently best-researched MSC source, bone marrow-derived stem cells. Ovine cotyledons were shown to be a reliable, abundant source for the noninvasive, pain- and risk-free harvest of MSCs. The collection procedure does not interfere with partum or the initial bonding phase between ewes and lambs and is therefore exempt from ethical debate. Ovine placenta cotyledon-derived MSCs exhibit multipotential characteristics and can be cryopreserved for later use.

Tendinopathies are painful, disabling conditions that afflict 25% of the adult human population. Filling an unmet need for realistic large-animal models, we here present an ovine model of tendon injury for the comparative study of adult scarring repair and fetal regeneration. Complete regeneration of the fetal tendon within 28 days is demonstrated, while adult tendon defects remained macroscopically and histologically evident five months post-injury. In addition to a comprehensive histological assessment, proteome analyses of secretomes were performed. Confirming histological data, a specific and pronounced inflammation accompanied by activation of neutrophils in adult tendon defects was observed, corroborated by the significant up-regulation of pro-inflammatory factors, neutrophil attracting chemokines, the release of potentially tissue-damaging antimicrobial and extracellular matrix-degrading enzymes, and a response to oxidative stress. In contrast, secreted proteins of injured fetal tendons included proteins initiating the resolution of inflammation or promoting functional extracellular matrix production. These results demonstrate the power and relevance of our novel ovine fetal tendon regeneration model, which thus promises to accelerate research in the field. First insights from the model already support our molecular understanding of successful fetal tendon healing processes and may guide improved therapeutic strategies.