Willems, Sarah (2024) Engineered artificial niches for skeletal muscle regeneration in vitro. [Tesi di dottorato]

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Abstract

Skeletal muscle tissue engineering aims to provide a model of the muscle for regenerative medicine applications. In this work, the aim was to replicate the intricate microenvironment of the muscle tissue, using an engineered artificial in vitro niche. The dissertation delves into the characterisation of nanogrooved substrates for skeletal muscle tissue niches, as a foundation for cultivating the cells. The study progresses from an initial material characterisation to the formation of a three-dimensional (3D) skeletal muscle tissue construct in vitro, by using the microenvironmental cues within an artificial niche. In the first phase, nanogrooved substrates are thoroughly investigated to characterise their suitability for skeletal muscle tissue cultures. This investigation involves topographical analysis of the nanogrooves using atomic force microscopy (AFM), followed by an analysis of the adhesive and elastic properties of the surface. The biocompatibility of these substrates was assessed using C2C12 murine myoblast cells. Subsequently, the microenvironmental cues of the artificial niche are harnessed to guide the cells into a self-assembled 3D muscle construct. The resulting constructs are extensively characterised using immunofluorescence and focused ion beam scanning electron microscopy (FIB SEM). A third part of the dissertation explores the mechanical identity of cells cultivated on the engineered niche. This evaluation encompasses the assessment of actinin tension within the cells and the determination of the Young’s Modulus of the cell using AFM measurements. Considering this mechanical evaluation at the construct formation stage, revealed that the muscle constructs exhibit a Young’s Modulus comparable to that of native skeletal muscle, highlighting the success of the self-organisation approach in recapitulating the structural and mechanical characteristics for skeletal muscle tissue. This dissertation contributes valuable insights into the complex cell-material crosstalk, by using nanotopographical cues, mechanical properties and biochemical signals in skeletal muscle tissue engineering. The findings in this study provide a foundation for the development of advanced strategies in regenerative medicine and hold promise for applications in tissue repair and regeneration studies.

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