Muscle and tendon injuries are prevalent and range from minor sprains and strains to traumatic, debilitating injuries. However, the interactions between these tissues during normal development, injury, and recovery remain unclear. Three-dimensional tissue models that incorporate both tissues and a physiologically relevant junction between muscle and tendon may aide in understanding how the two tissues interact.

To address this lack of knowledge, we used tissue specific extracellular matrix (ECM) derived from muscle and tendon to determine how cells of each tissue interact with the microenvironment of the opposite tissue resulting in junction specific features. ECM materials were derived from the Achilles tendon and gastrocnemius muscle, decellularized, and processed to form tissue specific hydrogels. C2C12 myoblasts and tendon fibroblasts were encapsulated in tissue-specific ECM hydrogels to determine cell-matrix interactions and the effects on a muscle-tendon junction marker, paxillin and type XXII collagen. C2C12s seeded in tendon ECM had the highest expression of paxillin, and both tissue specific ECM hydrogels had higher expression compared to type I collagen. Tendon fibroblasts had higher paxillin expression in tissue specific ECM hydrogels comparted to type I collagen. Type XXII collagen expression was not upregulated by tissue specific ECM hydrogels compared to type I collagen. Using tissue specific ECM allowed for the deconstruction of the cell-matrix interactions to study the expression of MTJ specific proteins.

Next, we used the tendon ECM with C2C12 myoblasts in a novel hydrogel tissue bioreactor. After 2 or 4 weeks of static culture or stimulated culture with 10% cyclic strain applied for 3 hours a day, effects from mechanical stimulation of the muscle-tendon junction constructs was quantified. Mechanical stimulation of the constructs promoted MTJ protein expression, which was also increased in tECM hydrogel constructs.

Finally, we aimed to quantify the structural features at the myotendinous junction in different muscle groups with tissue clearing methods. The FLASH clearing method effectively cleared the tissue so that paxillin antibody staining could be visualized throughout the native myotendinous junction without physical sectioning. Using confocal imaging, the interface in the different muscle groups was assessed in two different planes to quantify the width of the paxillin junction. In both cases, gastrocnemius muscle-tendon junction width was greater than in the other muscle groups. In threedimensions, more features of the junction such as interdigitations between fibers were visualized. Furthermore, the data collected allowed for 3D visualization and modelling of the specific junctions, which can be used in future studies to inform others about specific features at the interface and to improve computational models of the muscle-tendon interface.

Overall, this research used tissue specific ECM hydrogels to isolate individual matrix-cell interactions that mimic the myotendinous junction. After isolating specific interactions between cells and tissue micro-environments, a novel bioreactor was designed and utilized to study the effect of mechanical stimulation on the tissue constructs. Lastly, methodologies for visualizing the myotendinous junction in three dimensions without physical disruption of the native tissue, were developed and used to quantify features in different muscle groups. This research aids in the development of multi-tissue models of the muscle-tendon interface by establishing the importance of a junction specific region and quantifying the complexity of the native junction.