Mechanical strain as possible physical crosslinker of natural-derived hydrogels obtained from decellularized extracellular matrix

Skeletal muscle is the most abundant tissue in the body; its extensive damage can occur in cases of traumatic injury, volumetric muscle loss, tumor ablation, infection or inherited disorders such as muscular dystrophy or congenital defects. Current treatment options include the application of non-bioabsorbable synthetic materials, but these do not follow the physiological muscle growth nor the active tissue functionality. An alternative solution can be the use of tissue engineering, a discipline that aims to regenerate tissues and organs, combining biocompatible materials, patient cells, and chemical and mechanical stimuli. Thus, tissue engineering of skeletal muscle has been proposed as a potential regenerative method for the restoration of injured muscles. In the recent years, several types of scaffolds and materials have been tested to develop in vitro engineered skeletal muscle, and hydrogels, especially those composed of natural components, demonstrated to be valuable biomaterials for skeletal muscle tissue engineering purposes. Hydrogels are generally soft materials, that need additional crosslinking to increase their biomechanical characteristics. The aim of this thesis is to analyse the effects of mechanical stimulation, a less common approach then the often harmful physical and chemical methods, on the crosslinking of hydrogels obtained from skeletal muscle decellularized extracellular matrix (dECM). The main goal is to determine the possible increase of dECM derived hydrogels stiffness, evaluating how imposed deformation may help the material self-assembly, and how it modifies hydrogel conformation. A honeycomb-structured scaffold made of 3% w/v dECM hydrogel was used as possible biomaterial for skeletal muscle implants. Through a bioreactor based on a system of pumps and hydrostatic pressure, the samples were subjected to an increasing percentage of strain during hydrogel polymerization phase. Hydrogels stimulated with 4.5% and 7% of strain were analysed and compared with samples polymerized without application of mechanical stimuli. Through a computational model, the biomaterial behaviour was analysed, and a marker tracing assay was used to compare computational prediction and experimental data. Measurements of stiffness of stimulated hydrogels were performed using atomic force microscopy, whereas microscopic visualization of the arrangement of hydrogel collagen fibers at the imposed deformation were used to analysed scaffold architecture and alignment. Finally, human cells were included in the hydrogels before polymerization and mechanical stimulation to determine feasibility and safety of the methodology.