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

Area of Science:

• Biomedical Engineering
• Tissue Engineering
• Optical Imaging

Background:

• Assessing engineered tissue viability and function noninvasively is a critical challenge in tissue engineering.
• Label-free nonlinear optical molecular imaging (OMI) uses endogenous fluorescence and second harmonic generation (SHG) to evaluate metabolic activity and structural integrity.

Purpose of the Study:

• To demonstrate OMI techniques for assessing engineered skeletal muscle units (SMUs).
• To develop a novel method for evaluating SMUs prior to implantation.

Main Methods:

• Applied OMI, including nicotinamide adenine dinucleotide/flavin adenine dinucleotide fluorescence and SHG imaging, to control, steroid-supplemented, and metabolically stressed SMUs.
• Calculated redox ratio (RR) for metabolic activity and structure ratio for composition.
• Assessed function via tetanic force production and correlated OMI measures with functional outcomes.

Main Results:

• Redox ratios distinguished control from metabolically stressed SMUs.
• SHG signals differentiated control from steroid-supplemented SMUs.
• Steroid supplementation increased force generation, with a significant correlation between myosin density and force.

Conclusions:

• Label-free OMI shows potential for evaluating engineered skeletal muscle constructs noninvasively.
• OMI measures correlate with functional performance, potentially predicting post-implantation integration and regeneration.
• This methodology can aid in assessing tissue engineering technologies and confirming release criteria.