Real-time monitoring and non-invasive correlation of tissue engineered skeletal muscle tissue contractile properties and structural development

<p dir="ltr">Three-dimensional (3D) engineered skeletal muscle tissues offer a promising in vitro model to study muscle development, disease, and drug response due to their ability to better replicate native muscle architecture and function compared to traditional two-dimensional systems. However, a major limitation in the field has been the lack of technologies that allow simultaneous, non-invasive, and real-time monitoring of both structural maturation and functional output within the same living tissue construct. Most current approaches are invasive or rely on endpoint analyses, preventing longitudinal assessment and limiting insights into the dynamic interplay between muscle architecture and contractility. This thesis aimed to bridge this gap by developing and validating an integrated, non-destructive platform capable of continuously assessing the functional performance and structural development of engineered skeletal muscle tissues over time.</p><p dir="ltr">To achieve this, a multi-faceted research program was undertaken, combining bioengineering, tissue culture, sensor development, and molecular biology. The study began by comparing in-house developed 3D printed tissue moulds with commercial muscle culture platforms, highlighting performance equivalence in force generation and tissue morphology while offering enhanced flexibility and scalability. A custom-designed printed circuit board (PCB) electrical stimulation system was then developed to deliver controlled excitation to 3D muscle tissues within multi-well plate formats. This was integrated with magnetic sensing systems and force transducers to allow non-invasive measurement of evoked twitch and tetanic contractions in real-time. The engineered muscle tissues, generated using the C2C12 murine myoblast cell line, were cultured for up to 18 days, during which their contractile force output, gene expression, and morphological characteristics were monitored.</p><p dir="ltr">Key findings of the thesis revealed that functional maturation, as indicated by increases in peak twitch and tetanic force, was significantly correlated with the expression of genes encoding excitation-contraction coupling proteins, particularly ryanodine receptor 1 (RYR1), voltage-gated calcium channel Cav1.1, and sodium channel Nav1.4. Interestingly, the content of structural proteins such as Myosin Heavy Chain (MyHC) did not directly correlate with force amplitude but showed strong relationships with contraction kinetics. Specifically, MyHC coverage predicted time to peak tension and half-relaxation time. Additional analysis demonstrated that nuclei density and myotube cross-sectional area were also associated with improved kinetic properties, suggesting that structural maturation impacts contraction timing more than absolute force output. Comparative evaluations confirmed that the custom-built hardware platform could match or exceed the capabilities of commercial systems in terms of sensitivity, consistency, and cost-effectiveness.</p><p dir="ltr">By developing a fully integrated, sensor-compatible culture environment for engineered muscle tissues, this thesis presents a significant advancement in tissue engineering methodology. The platform enables the concurrent monitoring of mechanical and morphological development in a scalable, high-throughput format. This innovation offers valuable applications not only in fundamental muscle biology but also in translational areas such as regenerative medicine, drug screening, and personalized disease modeling. In doing so, the work provides new insight into the temporal evolution of engineered muscle function and structure and establishes a robust framework for future studies aiming to emulate native muscle physiology in vitro.</p>