High resolution melt electro-written scaffolds promote alignment of human skeletal muscle cells

Advanced tissue engineering (TE) strategies are vital to address challenging musculoskeletal conditions, such as volumetric muscle loss. These disorders impose a considerable economic burden and affect individuals' quality of life, highlighting the need for innovative treatments, such as TE, to address these challenges. Here, we examine how scaffold fibre orientation influences mechanical properties and cellular behaviour by utilising melt electrowriting (MEW) as a high-resolution 3D printing technique that combines aspects of electrospinning and melt based polymer deposition. In this work, we investigated the effects of fibre orientation in MEW scaffolds, and its effect on the scaffold mechanical properties as well as cell orientation and alignment. MEW scaffolds were mechanically characterised through uniaxial strain testing to determine critical parameters, including strain at failure, ultimate tensile strength, Young's modulus (<i>E</i>), fatigue rate, recovery time, and yield strain. These mechanical properties were analysed to define an optimal strain regime for transitioning from static to dynamic culture conditions under muscle-like cyclic loading, relevant to muscle's viscoelastic behaviour. In parallel, static cultures of primary human skeletal muscle myoblasts and normal human dermal fibroblasts (NHDFs) were grown on MEW scaffolds, with varying architectures, to study the effects of fibre aspect ratio on cell alignment. Cell alignment was visualised using DAPI/phalloidin staining and quantified with the ImageJ directionality plugin, enabling a systematic comparison of scaffold designs. This approach evaluates the potential of supportive scaffold architectures to promote aligned cell growth, offering insights into designing effective scaffolds for tissue regeneration.