Understanding and modulating vessel formation in human rotator cuff tendon disease

Tears of the rotator cuff tendons are common and often debilitating causing pain and loss of upper limb function. Their prevalence continues to increase as populations age. Although the pathobiology of tendon disease is incompletely understood, it is increasingly evident that inflammation plays a critical role. Persistent immune cell infiltration, fibroblast activation and angiogenesis are cardinal features of rotator cuff tendon disease. Unfortunately, the surgical repair of rotator cuff tears fails in almost half of cases, often resulting in long-term pain and disability. To this end, the biomaterial augmentation of surgical repairs has gained in popularity. Current augments provide structural support to repairs but do not address the aberrant biology of torn tendons resulting in a compromised repair process. However, a new generation of biomaterials are currently under development. Produced by electrospinning these scaffolds and can alter cellular phenotypes through the provision of topographical cues. The successful tailoring of such biomaterials demands a detailed understanding of tendon disease pathobiology and cell-material interactions, yet the role of the endothelium in tendon disease remains hitherto unexplored. This thesis aimed to establish a clear need for a new generation of bioactive materials for rotator cuff repair. It then explored an entirely novel area of tendon pathobiology – the role of the endothelium, before attempting to modulate vessel growth through modifying the microscale architecture of electrospun scaffolds. Firstly, an a priori registered systematic review of all available clinical data for patch augmented repair found a similar complication rate between augmented (2.3%) and standard repair (2.1%). A meta-analysis confirmed an improvement in re-tear rate, and a subsequent subgroup analysis identified a moderate reduction in re-tear rate for synthetic (RR 0.4, 95% CI 0.25-0.64) and decellularised human allograft (RR 0.34, 95% CI 0.18-0.64) patches, but not for xenografts (RR 0.97, 95% CI 0.72-1.30) or human autografts (RR 0.69, 95% CI 0.401.18). This corresponded to a small improvement in pain and PROMS for synthetic patches, that was statistically significant but not clinically meaningful. Secondly, an in vitro co-culture model of tenocytes and endothelial cells, with or without the addition of peripheral blood leukocytes, was undertaken. During extended co-culture of healthy tenocytes, EC-tenocyte crosstalk increased mRNA expression of fibroblast activation markers including CD248, PDPN and VCAM-1, pro-inflammatory cytokines, extracellular matrix proteins and matrix proteases. This effect was not observed during EC co-cultures with diseased tenocytes. Cross-talk was bi-directional, tenocytes from healthy, but not diseased tendons, inhibited adhesion of lymphocytes and monocytes to the endothelium. Thirdly, the effect of electrospun scaffold micro-architecture, specifically fibre diameter and alignment, on the transcriptome of healthy and diseased tenocytes was characterised using bulk RNA sequencing. Aligned electrospun scaffolds with fibre diameters above 1000nm resulted in a downregulation of gene-sets related to fibroblast activation. Conversely, all randomly oriented scaffold and small diameter (~300nm) aligned scaffolds induced a relative activation of tenocytes. A more profound transcriptional response was observed for diseased tenocytes which were primed to respond to topographical cues. Despite the ability of scaffold architecture to modify fibroblast activation, no scaffold consistently altered the expression of genes involved in the direct regulation of angiogenesis by fibroblasts. This thesis has demonstrated that (1) current commercially available rotator cuff patches are not clinically efficacious, (2) endothelial-fibroblast crosstalk influences critical features of rotator cuff pathobiology including immune cell recruitment and fibroblast activation, and (3) the micro-architectural design of electrospun scaffolds modulates the activation status of tenocytes, but, within the parameters used in this work, does not appear to influence fibroblast derived angiogenic signaling.