Abstract Tendon injuries and tendinopathy are a growing problem in the aging but physically active population as well as athletes. Tendons have highly ordered matrix and undergo complex changes during the remodelling phase of tendon healing. Moreover, anaerobic metabolism and poor vascular network contribute to slow adaptation of tissue to the remodelled matrix which consequently results in slow and compromised healing. Such a destitute and slow healing process necessitates development of new and effective therapies and to combine therapies to obtain possibly synergistic effects. Addressing this clinical requirement, the work presented in this thesis investigates the role of two emerging treatment options, platelet rich plasma (pRP) and mechanical loading, on tendon healing. The effects of PRP, a rich autologous source of growth factors, on tendon cells was studied by modelling important stages of tendon healing in vitro. Key parameters such as cellular migration, chemotaxis, viability and senescence were investigated by means of different culturing and staining techniques together with microscopic analyses. PRP significantly increased migration and chemotaxis in human pnmary tenocyte culture. Moreover, PRP protected human tenocytes against challenging environments created by known tendon damaging drugs, dexamethasone and, ciprofloxacin, as well as the injury relevant condition of hypoxia. 11 Concurrently, an in vitro rat tail tendon injury model and static loading device was developed to assess the effect of static mechanical loading and PRP on the biochemical and biomechanical properties of tendon at the tissue level. This in vitro system was also used to investigate the synergistic effects of PRP and mechanical loading on tendon healing. Both PRP and mechanical loading helped to improve the biomechanical and biochemical properties of damaged tendon in vitro. In conclusion, the positive effects of PRP on key cellular parameters such as cell survival, migration and chemotaxis and also mechanical and biochemical properties of tendon tissue make it an important option for faster and less invasive tendon treatment. Additionally, an in vitro tendon injury model together with the mechanical loading device provide a new tool to investigate the mechanical boundary conditions suitable for treating different types of tendon disorders. The findings from the current study points towards the. significant contribution of PRP and mechanical loading to the healing process in tendons and could serve as a promising starting point for developing integrated therapeutic modalities to improve the quality and speed of recovery from tendon injury. 111.

The motivation for this PhD thesis was to understand the molecular basis of how cells regulate the formation of an organised and mechanically strong extracellular matrix (ECM). In tendon this process begins during embryogenesis with the appearance of bundles of narrow-diameter (~30 nm) collagen fibrils that are parallel to the tendon long axis. At the onset of collagen fibrillogenesis, the cells elongate, the fibrils are constrained within plasma membrane channels with their ends contained in tension-sensitive actin-stabilised plasma membrane protrusions. The mechanism by which actin is reorganised during cell elongation and the formation of tension-sensitive plasma membrane protrusions is poorly understood. The small GTPase RhoA is the major regulator of actin reorganisation into stress fibres, which have been implicated in mechanotransduction, ECM assembly and remodelling. The hypothesis tested by this PhD thesis was that the organisation and tensioning of extracellular collagen fibrils is generated on a blueprint of tensioned actin filaments within the cell. Rho activity is regulated specifically by Rho GTPase activating proteins (RhoGAPs). By comparing the global gene expression of tendon tissues at different developmental stages, Arhgap28, a novel RhoGAP, which is expressed during tendon development but not during postnatal maturation, was identified.Arhgap28 belongs to a large family of RhoGAPs containing the closely related members, Arhgap6 and Arhgap18, which have previously been shown to regulate RhoA and stress fibre formation. Arhgap28 expression was upregulated in embryonic fibroblasts cultured in a 3D, tensioned embryonic tendon-like construct compared to monolayer culture. Arhgap28 expression was further enhanced during the development of mechanical strength and stiffness of the tendon constructs, but downregulated when the tension in tendon constructs was released. Overexpression of a C-terminal V5-tagged Arhgap28 protein caused a reduction in RhoA activation and disruption of stress fibre assembly. Modulation of Rho signalling using lysophosphatidic acid and Y27632 showed that collagen remodelling by cells in collagen gels and tendon constructs is regulated by RhoA signalling. A tissue-wide qPCR analysis identified Arhgap28 in several tissues including tendon, bone, and skin. An Arhgap28 reporter mouse (Arhgap28gt) and an Arhgap28 knockout mouse (Arhgap28del) were also studied to investigate the role of Arhgap28 in tissue organisation in vivo. Arhgap28gt mice showed Arhgap28 expression in bones at E18.5. Homozygous Arhgap28del mice were viable, appeared normal but expressed a truncated Arhgap28 transcript, which if translated, would produce a protein lacking the RhoGAP domain. Therefore, it was hypothesised that knockout mice were normal due to compensation from another RhoGAP. Overexpression of Arhgap6 in Arhgap28-null bone tissues was confirmed. Upregulation in RhoA expression was also detected, further suggesting that Arhgap28 regulates RhoA. Interestingly, a microarray comparison of bone tissues from wild type and Arhgap28-null mice showed that genes linked to bone dysplasia are downregulated in Arhgap28-null bone. Together, these results suggest that formation of a strong and organised collagen ECM is mediated by RhoA-generated cellular tension and that Arhgap28 and Arhgap6 might be co-regulators of this process.

Degenerative disorders of the rotator cuff tendons account for nearly 75% of all shoulder pain, causing considerable pain and morbidity. Given the strong correlation between age and tendinopathy, and unprecedented population aging, these disorders will become increasingly prevalent. Improved understanding of tendon degeneration will guide the development of future diagnostic and treatments, and is therefore urgently needed. However, the aetiology and pathology of rotator cuff tendinopathy remain unclear. The complicated mechanical environment of the rotator cuff is hypothesised to influence the susceptibility of the tendons to degeneration and tearing. Studies have reported biological adaptations in torn cuff tendons indicative of increased compressive loading within the tendon. The material adaptations of healthy and degenerative cuff tendons are largely unreported but will provide further insight into the role of the mechanical environment in rotator cuff aetiology and pathology. This thesis examined the material adaptations of healthy and diseased tendons to explore the role of mechanical loading in rotator cuff pathology. The material adaptations of healthy animal tendons, and healthy and delaminated human cadaveric rotator cuff tendons, in response to different loading environments were characterised. The effects of age, tears, steroid injection and subacromial decompression surgery on the structural adaptations of human cuff tendons were also studied, as was the effect of tendon cell proliferation on the mechanical properties and degradation behaviour of collagen scaffolds. Loading environment significantly affected the structural adaptations of healthy tendons. Regions exposed to compressive and shear strains exhibited thinner fibres, shorter crimp lengths and thinner, less aligned fibrils compared with regions exposed to tensile strains alone. In healthy rotator cuff tendons, the inhomogeneous loading environment produced topographically inhomogeneous structural adaptations. The tendons of a delaminated rotator cuff exhibited less topographical variation in properties and thinner, less aligned fibrils compared with healthy cuff tendons. Torn cuff tendons exhibited thinner fibrils and shorter crimp lengths compared with control samples. These adaptations were identifiable early in the disease progression, and neither steroid injection nor subacromial decompression surgery significantly influenced these adaptations at seven weeks post‐treatment. Significant correlations between decreasing dimensions and increasing tear size were found when age was included as a confounding factor, reflecting the importance of age and tear size in determining the material properties of tendons. Tendon cell proliferation influenced the mechanical properties and degradation behaviour of the collagen scaffolds, emphasising the integral role of cells in the functional adaptation of biological materials. These results demonstrate the effect of mechanical environment on the material adaptations of tendons. They also indicate the importance of the complicated mechanical environment experienced by the rotator cuff tendons in predisposing the tendons to degeneration and tearing. The observed material adaptations of degenerative and torn tendons suggest that rotator cuff pathology is associated with increased levels of compressive and/or shear strains within the tendon. These changes begin early in the disease progression and neither steroid injection nor sub‐acromial decompression surgery are capable of reversing the changes in the timeframe investigated. These findings highlight the urgent clinical need for pre‐rupture diagnostic techniques for the detection of early pathological changes in the rotator cuff. They also emphasize the requirement for new intervention strategies that restore the healthy mechanical environment and reverse early pathological adaptations in order to prevent catastrophic failure of the tendons.