In healthy airways, mucus forms part of the innate immune response protecting the respiratory epithelium from damage by pathogens and environmental debris (Rose and Voynow, 2006). Conversely, in many respiratory diseases, mucus becomes part of the airway disease pathology. Mucus hypersecretion along with reduced clearance can cause blockage of the small airways, impairing gas exchange, promoting inflammation and becoming a culture medium for bacterial colonisation (Thornton et al., 2008). Recurrent airway obstruction (RAO) is a common yet poorly understood equine chronic respiratory disease where such altered mucus properties and clearance have been identified as major factors in the disease pathology (Davis and Rush, 2002; Gerber et al., 2000; Kaup et al., 1990; Robinson, 2001). The gel-forming mucins are largely responsible for the transport properties of mucus. The major equine airway gel-forming mucin in health is Muc5b and to a lesser extent Muc5ac; produced in specialised respiratory epithelial goblet cells and sub-mucosal glands (Rousseau et al., 2011b). Changes in mucin relative and net amounts and their macromolecular properties and interactions have been attributed to the altered physical properties of airway mucus in airways disease (Groneberg et al., 2002a; Jefcoat et al., 2001; Kirkham et al., 2002; Robinson et al., 2003; Sheehan et al., 1995).The project investigates the biochemical properties of mucins present in mucus from healthy horses and horses with RAO. This project identifies the anatomical presence of mucin-producing goblet cells and glands in fixed tissues from the respiratory tracts of healthy horses and subsequently examines mucin-production sites in respiratory tracts from horses with RAO. Finally the project investigates a methodology for the study of mucin production in airway cells harvested from live horses suffering from RAO.Our investigations confirmed that horses with RAO have more endotracheal mucus than healthy controls, and that Muc5b is the predominant mucin with Muc5ac also present in RAO horse mucus, both during symptomatic disease and when horses are asymptomatic. Mucins are produced in epithelial goblet cells and sub-mucosal glands dispersed throughout the length and circumference of the equine trachea and bronchi. Goblet cell hyperplasia occurs in symptomatic exposed RAO horse airways, although goblet cells are smaller than in asymptomatic RAO horse airways. Exposure to a dusty stable environment is associated with more goblet cells per length of bronchial compared to tracheal epithelium in all horses. RAO horses have larger sub-mucosal glands containing more mucin than control horses. Primary epithelial cell cultures grown at an air liquid interface are an alternative approach to study equine airway mucus, although the use of this culture system is in its early stages. We have developed novel ways to harvest equine airway epithelial cells (tracheal brushing) and shown it is possible to freeze cells collected via tracheal epithelial brushing in 20 % FBS and then culture to ALI at a later date.

Background: Long-term cyclic tensile testing with equine palmar/plantar tendons have not yet been performed due to problems in fixing equine tendons securely and loading them cyclically. It is well established that the biomechanical response of tendons varies during cyclic loading over time. The aim of this study was to develop a clamping device that enables repetitive cyclic tensile testing of equine superficial digital flexor tendon for at least 60 loading cycles and for 5 min. Results: A novel cryo-clamp was developed and built. Healthy and collagenase-treated pony SDFTs were mounted in the custom-made cryo-clamp for the proximal tendon end and a special clamping device for the short pastern bone (os coronale). Simultaneously with tensile testing, we used a biplanar high-speed fluoroscopy system (FluoKin) to track tendon movement. The FluoKin system was additionally validated in precision measurements. During the cyclic tensile tests of the SDFTs, the average maximal force measured was 325 N and 953 N for a length variation of 2 and 4 % respectively. The resulting stress averaged 16 MPa and 48 MPa respectively, while the modulus of elasticity was 828 MPa and 1212 MPa respectively. Length variation of the metacarpal region was, on average, 4.87 % higher after incubation with collagenase. The precision of the FluoKin tracking was 0.0377 mm, defined as the standard deviation of pairwise intermarker distances embedded in rigid bodies. The systems accuracy was 0.0287 mm, which is the difference between the machined and mean measured distance. Conclusion: In this study, a good performing clamping technique for equine tendons under repetitive cyclic loading conditions is described. The presented cryo-clamps were tested up to 50 min duration and up to the machine maximal capacity of 10 kN. With the possibility of repetitive loading a stabilization of the time-force-curve and changes of hysteresis and creep became obvious after a dozen cycles, which underlines the necessity of repetitive cyclical testing. Furthermore, biplanar high-speed fluoroscopy seems an appropriate and highly precise measurement tool for analysis of tendon behaviour under repetitive load in equine SDFTs.

Induced pluripotent stem cells (iPSC) have the capacity to self renew and differentiate into a myriad of cell types making them potential candidates for cell therapy and regenerative medicine. The goal of this thesis was to determine the characteristics of equine iPSC (eiPSC) that can be harnessed for potential use in veterinary regenerative medicine. Trauma to a horse’s limb often leads to the development of a chronic non-healing wound that lacks a keratinocyte cover, vital to healing. Thus, the overall hypothesis of this thesis was that eiPSC might offer a solution for providing wound coverage for such problematic wounds. Prior to considering eiPSC for clinical applications, their immunogenicity must be studied to ensure that the transplanted cells will be accepted and integrate into host tissues. The first objective of this thesis was to determine the immune response to eiPSC. To investigate the immunogenicity of eiPSC, the expression of major histocompatibility complex (MHC) molecules by the selected lines was determined, then the cells were used in an intradermal transplantation model developed for this study. While transplantation of allogeneic, undifferentiated eiPSC elicited a moderate cellular response in experimental horses, it did not cause acute rejection. This strategy enabled the selection of weakly immunogenic eiPSC lines for subsequent differentiation into lineages of therapeutic importance. Equine iPSC offer a potential solution to deficient epithelial coverage by providing a keratinocyte graft with the ability to differentiate into other accessory structures of the epidermis. The second objective of this thesis was to develop a protocol for the differentiation of eiPSC into a keratinocyte lineage. The protocol was shown to be highly efficient at inducing the anticipated phenotype within 30 days. Indeed, the eiPSC derived vi keratinocytes (eiPSC-KC) showed both morphologic and functional characteristics of primary equine keratinocytes (PEK). Moreover, the proliferative capacity of eiPSC-KC was superior while the migratory capacity, measured as the ability to epithelialize in vitro wounds, was comparable to that of PEK, suggesting exciting potential for grafting onto in vivo wound models. In conclusion, equine iPSC-derived keratinocytes exhibit features that are promising to the development of a stem cell-based skin construct with the potential to fully regenerate lost or damaged skin in horses. However, since eiPSC do not fully escape immune surveillance despite low MHC expression, strategies to improve engraftment of iPSC derivatives must be pursued. / Les cellules souches pluripotentes induites (iPSC) ont la capacité de s'auto renouveler et de se différencier en une myriade de types cellulaires, ce qui en fait des outils intéressants pour la thérapie cellulaire et la médecine régénérative. Le but de cette thèse était de déterminer les caractéristiques des iPSC équines (eiPSC) qui peuvent être exploitées pour l'usage potentiel en médecine régénérative vétérinaire. Chez le cheval, une plaie cutanée est souvent cicatrisée par seconde intention et est sujette à de nombreuses complications lorsque située sur le membre, notamment une épithélialisation lente. Ainsi, l'hypothèse globale de cette thèse était que les eiPSC pourraient offrir une solution novatrice de couverture pour de telles blessures. Avant d'envisager l’utilisation d'eiPSC à des fins cliniques, leur immunogénicité doit être étudiée afin de s'assurer que les cellules transplantées seront acceptées et intégrées dans les tissus du receveur. Le premier objectif de cette thèse était de définir la réponse immunitaire suscitée par les eiPSC. Afin d'étudier l'immunogénicité d'eiPSC, l'expression de molécules du complexe majeur d’histocompatibilité (MHC) des lignes choisies a été déterminée, puis les cellules ont été utilisées dans un modèle de transplantation intradermique développé pour cette étude. Bien que la transplantation allogénique d'eiPSC non différenciées ait induit une réponse cellulaire modérée chez les chevaux d'expérimentation, elle n'a pas provoqué de rejet. Cette stratégie a permis la sélection de lignées d'eiPSC faiblement immunogènes pour la différenciation ultérieure en des lignées d'importance thérapeutique. Les eiPSC représentent une solution intéressante et qui, par l’entremise du développement d’une lignée de kératinocytes, pourraient servir à la création d’une greffe ayant la capacité de former non seulement l’épithélium manquant mais aussi d'autres structures accessoires de l'épiderme. Le deuxième objectif de cette thèse était donc de iv développer un protocole pour la différentiation des eiPSC en lignée de kératinocytes. Un protocole visant cette différenciation fut ainsi développé et ce dernier a démontré une grande efficacité à produire le phénotype attendu dans une période de 30 jours. En effet, les kératinocytes dérivés d'eiPSC (eiPSC-KC) ont montré des caractéristiques morphologiques et fonctionnelles des kératinocytes primaires équins (PEK). En outre, la capacité de prolifération d'eiPSC-KC est supérieure tandis que la capacité migratoire, mesurée comme l'aptitude à cicatriser les plaies in vitro, est comparable à celle du PEK. En conclusion, les eiPSC-KC ont des caractéristiques intéressantes pour le développement d'un substitut cutané à base de cellules souches, ayant le potentiel de régénérer la peau perdue lors de trauma ou de maladie, chez le cheval. Cependant, parce que les eiPSC n'échappent pas totalement à la surveillance immunitaire, malgré une faible expression du MHC, des stratégies pour améliorer la prise de greffe eiPSC-KC doivent être élaborées.