Development of an in vitro model of skeletal muscle wound healing

Skeletal muscle has a high regenerative capacity, injuries trigger a regenerative program that restores tissue function to a level indistinguishable to the pre-injury state. However, in some cases where significant trauma occurs, such as injuries seen in military populations, the regenerative process is overwhelmed and cannot restore full function. The potential clinical interventions which have been presented to date, can be used to promote regeneration and prevent the formation of non-regenerative defects, have arisen from in depth understanding of the underpinning regenerative physiology. Robust and reproducible techniques and systems for modelling complex tissue responses are therefore essential to promote the discovery of effective clinical interventions. Tissue engineering has been highlighted as such a method, allowing the generation of three-dimensional in vivo like tissues. These engineered models have the potential to supplement animal models allowing rapid screening of potential clinical interventions, as these models are more easily manipulated genetically and pharmacologically. Reducing the associated cost and complexity, whilst increasing access to models for laboratories without animal facilities. Therefore, this thesis presents a series of experiments designed to support the generation of a human tissue engineered skeletal muscle capable of functional regeneration post injury, to produce a system within which regenerative physiology can be examined and potential therapies assessed. Initially we demonstrate that BaCl2 can be used to create an effective myotoxic injury in monolayer cultures and is suitable for use with cell lines (C2C12) and primary human donor myocytes. These monolayer cultures display only a partially regenerative response to injury, which could not be improved upon by changing the culture conditions. Therefore, it was hypothesised that to mimic the regenerative response to BaCl2 seen in vivo a 3D environment was required to support regeneration. We therefore aimed to translate this monolayer model into an engineered tissue. In order to generate reproducible and repeatable measures of skeletal muscle utilising primary human cells there is a requirement for systems that can generate engineered tissues in a way that is economical with cell number and reproducible between laboratories and repeats. To achieve this we scaled an existing C2C12 type I collagen based system from 500µL to 50µL volume, reducing the cellular requirement by an order of magnitude and allowing human myogenic precursor cells to be used to generate a functional human engineered skeletal muscle. In addition, this work created a method to produce moulds through additive manufacture, reducing the reliance of engineered models on bespoke parts, potentially reducing repeat variability. Finally, at the reduced scale the addition of the basement membrane supplement Matrigel® was examined. Matrigel® was shown to increase morphological and functional maturity of the tissues and later shown to be essential for regeneration post injury. With a reproducible base model of engineered skeletal muscle, which could be generated from primary human cells or cell lines we examined the response of these tissue to injury. C2C12 engineered tissues showed complete functional and morphological regeneration following injury, only when engineered tissues contained Matrigel®. This demonstrated the absolute requirement for a basement membrane organised into a 3D environment. Additional complexity was attempted through the addition of human immune cells in the form of peripheral blood mononuclear cells. Although these immune cells were not maintained within the tissues, soluble factors were shown to drive an effect post injury. Finally, we looked to translate this work into a human engineered model of skeletal muscle. Engineered muscles created from explant biopsy cells showed poor reproducibility between donors preventing basic explant biopsy isolations being used. We addressed these difficulties using magnetic association cell sorting, for the marker CD56, and media supplementation with fibroblast growth factor 2 (FGF-2) and B-27 supplement. Cell sorting allowed extended expansion of myogenic cells and supplementation was shown to improve myogenesis and increase function of engineered tissues. These engineered human skeletal muscles regenerated function and morphology following BaCl2 injury producing an ex vivo model of human skeletal muscle regeneration. In addition, these engineered tissues contained a population of Pax7+ cells that expanded following injury and demonstrated MyoD population dynamics similar to those seen in vivo. This thesis outlines the progress from a simple monolayer injury model to an engineered human skeletal muscle capable of functional regeneration following injury, built upon an open source 3D printed mould system.