Quantitative, Label-Free Evaluation of Tissue-Engineered Skeletal Muscle Through Multiphoton Microscopy

The lack of tools for assessing engineered tissue viability and function in a noninvasive manner is a major regulatory and translational challenge facing tissue engineers. Label-free, nonlinear optical molecular imaging (OMI) has utilized endogenous nicotinamide adenine dinucleotide and flavin adenine dinucleotide fluorescence to indicate metabolic activity. Similarly, second harmonic generation (SHG) signals from myosin and collagen can measure overall muscle structural integrity and function. The purpose of this study was to demonstrate these OMI techniques for the first time in engineered skeletal muscle and to develop a novel method for evaluating our engineered skeletal muscle units (SMUs) before implantation. Three experimental groups were studied: Control, Steroid Supplemented, and Metabolically Stressed SMUs. After imaging and analysis in ImageJ, a redox ratio (RR) metric was calculated to indicate metabolic activity, and a structure ratio metric was calculated to reflect structural composition. In addition, function was evaluated as tetanic force production in response to electrical stimulation. In living tissues, the RRs successfully distinguished control and metabolically stressed SMUs in both monolayer and 3D form. OMI of myosin and collagen SHG similarly differentiated control SMUs from the steroid supplemented group. With respect to function, steroid supplementation significantly increased active force generation. When comparing functional and OMI measures, a significant correlation was present between overall myosin density and active force generation. This work demonstrates the potential for using label-free OMI to evaluate tissue-engineered skeletal muscle constructs. The positive correlation between structural OMI measures and force production suggests that OMI could potentially serve as an accurate predictor of functional behaviors, such as integration and tissue regeneration, after implantation. This noninvasive OMI methodology, demonstrated for the first time in engineered skeletal muscle, could prove invaluable for assessing our tissue engineering technology and confirming release criteria for validation.