Tendons are collagen-based fibrous tissues that connect muscles to bones. Tendon injury is common, but currently available treatments for damaged tendons are limited, and often fail to restore pre-injury function. The cellular and molecular mechanisms underlying tendon formation are not completely understood, and a detailed understanding of the processes involved would facilitate new therapeutic strategies. Work presented in this thesis had two aims: to investigate the role and mechanisms of cell-generated force during tendon development, and to examine the effect of external force applied to tendon tissue. The rationale to investigate micro-mechanical aspects of tendon development came from observations that tendons are intrinsically tensioned, presumably to transmit force efficiently. Tension is also critical for the formation and regulation of the primary cell-matrix interaction structures seen in embryonic tendon, known as fibripositors. In initial experiments cell-generated force was disrupted with the non-muscle myosin II (NMII) inhibitor blebbistatin. NMII inhibition prevented formation of tendon tissue in a 3D cell culture system (tendon-construct). Cell-contraction was also shown to shape the ECM and generate a crimped collagen structure characteristic of tendon. These observations highlight the importance of cell-generated force in tendon development. Further investigation using novel microscopy techniques suggested that fibripositors contained newly formed collagen fibrils, and that fibrils in the ECM were internalised and fragmented in a NMII/MT1-MMP dependent pathway. It is proposed that these fragmented fibrils are re-secreted into the matrix, where they seed fibril growth. This pathway is disrupted in MT1-MMP deficient mice, which have half normal size tendons with fewer collagen fibrils. Investigations using a mechanical rig showed that external application of force to tendon-constructs stimulated matrix maturation that more closely mimicked embryonic development. Taken together these results show that both cell-generated force and external force have important roles during tendon development.Experiments in this thesis aimed to elucidate the role of force at the tissue level (generation of mechanical properties, crimp structure), the microscopic level (cell-matrix interactions) and the molecular level (role of NMII and MT1-MMP in ECM collagen fibril handling). Given the complexity of the developmental system being investigated the multi-level experimental approach used here is necessary to generate a holistic understanding of the important developmental processes involved. Therapeutic tissue regeneration may be facilitated by application of external force or modulation of cell-generated force. Furthermore, the identification of a fibril amplification pathway has implications for medical conditions characterised by excessive collagen fibril formation (fibrosis, some cancers) or by slow or inadequate fibril formation (tendon healing).
|Date of Award||1 Aug 2012|
- The University of Manchester
|Supervisor||Karl Kadler (Supervisor)|