Despite a massive body of knowledge which has been produced related to the mechanisms guiding muscle regeneration, great interest still moves the scientific community toward the study of different aspects of skeletal muscle homeostasis, plasticity, and regeneration. Indeed, the lack of effective therapies for several physiopathologic conditions suggests that a comprehensive knowledge of the different aspects of cellular behavior and molecular pathways, regulating each regenerative stage, has to be still devised. Hence, it is important to perform even more focused studies, taking the advantage of robust markers, reliable techniques, and reproducible protocols. Here, we provide an overview about the general aspects of muscle regeneration and discuss the different approaches to study the interrelated and time-dependent phases of muscle healing.

Tissue engineering is a multidisciplinary approach focused on the development of innovative bioartificial substitutes for damaged organs and tissues. For skeletal muscle, the measurement of contractile capability represents a crucial aspect for tissue replacement, drug screening and personalized medicine. To date, the measurement of engineered muscle tissues is rather invasive and not continuous. In this context, we proposed an innovative sensor for the continuous monitoring of engineered-muscle-tissue contractility through an embedded technique. The sensor is based on the calibrated deflection of one of the engineered tissue’s supporting pins, whose movements are measured using a noninvasive optical method. The sensor was calibrated to return force values through the use of a step linear motor and a micro-force transducer. Experimental results showed that the embedded sensor did not alter the correct maturation of the engineered muscle tissue. Finally, as proof of concept, we demonstrated the ability of the sensor to capture alterations in the force contractility of the engineered muscle tissues subjected to serum deprivation.

The measurement of the mechanical properties is a crucial point for new engineered muscle tissues. The final aim is to implant these tissues to substitute or restore the functionality of impaired muscles, so that functional properties as close as possible to the healthy native muscles are required. We developed an engineered skeletal muscle tissue, X-MET, whose strong point is to be created without any endogenous component. This construct is able to contract spontaneously as well as to respond to electrical stimulation. In this work, we developed an experimental system to measure for the first time, the power developed by the X-MET. The power was measured by applying the isovelocity contraction technique. This technique has never been applied on muscle engineered tissues so far, so the aim of this work was to find out the optimal stimulation parameters. Once determined the range of displacement and velocity of shortening for which the X-MET was able to develop power, we proceeded looking at the optimal parameters allowing the production of its maximum power. Preliminary tests showed that the X-MET generates the optimal power when stimulated to shorten 3% of its ideal length at a speed of 0.2 L0/s.

Adherent cells perceive mechanical feedback from the underlying matrix and convert it into biochemical signals through a process known as mechanotransduction. The response to changes in the microenvironment relies on the cell’s mechanical properties, including elasticity, which was recently identified as a biomarker for various diseases. Here, we propose the design, development, and characterization of a new system for the measurement of adherent cells’ strain drop, a parameter correlated with cells’ elasticity. To consider the interplay between adherent cells and the host extracellular matrix, cell stretching was combined with adhesion on substrates with different stiffnesses. The technique is based on the linear stretching of silicone chambers, high-speed image acquisition, and feedback for image centering. The system was characterized in terms of the strain homogeneity, impact of collagen coating, centering capability, and sensitivity. Subsequently, it was employed to measure the strain drop of two osteosarcoma cell lines, low-aggressive osteoblast-like SaOS-2 and high-aggressive 143B, cultured on two different substrates to recall the stiffness of the bone and lung extracellular matrices. Results demonstrated good substrate homogeneity, a negligible effect of the collagen coating, and an accurate image centering. Finally, the experimental results showed an average strain drop that was lower in the 143B cells in comparison with the SaOS-2 cells in all the tested conditions.

Peripheral nerve injuries pose a significant clinical challenge due to the limited self-repair capacity and the complexity of neural tissue architecture. Tissue engineering strategies applied to the peripheral nerve system aim to restore functional nerve constructs by combining scaffolds, cells, and biochemical cues to recreate the native microenvironment. This work aimed to propose the electrical conductivity as a functional readout of structural and biological remodeling in engineered peripheral nerve scaffolds, along with functional and molecular evaluations. To this end, bioimpedance measurements were combined with equivalent circuit modeling to track state-dependent changes across different levels of tissue organization. Murine sciatic nerves were decellularized and recellularized with neural populations to generate engineered constructs, and their electrical properties were assessed using broadband bioimpedance spectroscopy. Distinct impedance profiles were observed across control, decellularized, and recellularized samples, reflecting structural and functional changes associated with cell removal and repopulation. Furthermore, a multilayer series RC circuit model was implemented to accurately reproduce the measured spectra, enabling the extraction of layer-specific electrical parameters. Analysis of these parameters revealed that decellularization reduces compartmental resistances and increases inter-layer coupling, whereas recellularization restores outer-layer resistances and reduces coupling, consistent with functional tissue organization. Overall, the results demonstrate that bioimpedance provides a readout of the scaffold biological state and cellular integration, and that equivalent circuit modeling offers a quantitative framework to link structural remodeling to electrical function in engineered peripheral nerve tissues.

Sarcoglycanopathies are heterogeneous proximo-distal diseases presenting severe muscle alterations. These diseases are caused by mutations in genes coding for one of the four sarcoglycan transmembrane proteins, which form the sarcoglycan complex (SGC). Little is known about the different roles of the SGC beyond the dystrophin glycoprotein complex (DGC) structural role. Here, we show that SGC proteins are enriched at the post-synaptic membrane of neuromuscular junctions (NMJs). Using a mouse model lacking the beta-sarcoglycan subunit, we describe for the first time that the loss of the SGC in the NMJ area results in alterations of pre- and postsynaptic membrane, as well as a significant reduction of membrane potential. Moreover, using different denervated wild-type mouse models, we demonstrate that enrichment of sarcoglycans within the NMJ occurs only after innervation, suggesting a nerve-dependent sarcoglycan expression. Altogether, our findings suggest that pathological decline should no longer be understood only in terms of sarcolemma damage but also in terms of sarcoglycans' participation in the NMJ. Henceforth, our work paves the way for the identification of new mechanisms involving sarcoglycans and new approaches for the treatment of sarcoglycanopathies.