Non-Invasive, Quantitative, and Label-Free Characterization of Tissue Engineered Skeletal Muscle

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As researchers seek to translate exciting technologies for the regeneration of damaged tissue, the lack of non- invasive and sterile methods for quantitatively evaluating the quality of engineered tissues prior to use in patients presents a significant challenge. Relatively few engineered tissue products have received FDA approval, so many translational technologies have no antecedent as a guide for navigating the path from bench to bedside. The proposed research intends to validate Optical Molecular Imaging (OMI) as a tool for characterizing the viability of engineered skeletal muscle during and following fabrication, and predict successful implantation that could also have broad applicability to other tissue engineering products. Current evaluation of engineered skeletal muscle utilizes invasive histological staining or non-sterile functional testing. Neither method avoids irrevocable damage of precious samples while simultaneously providing the quantitative data required for rigorous product quality testing. To address this issue, we propose to use nonlinear OMI to evaluate the metabolic activity and structural integrity of engineered skeletal muscle tissues during our tissue fabrication process. Specifically, the fluorescence intensity of endogenous flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide (NADH) will be detected and quantified through label-free OMI as a measure of metabolic activity and cell viability. Additionally, the second harmonic generation signatures generated by myosin in muscle sarcomeres and collagen fibers in the extracellular matrix will be quantified as a measure of structural organization correlated with functional outcomes. By combining these two label free imaging techniques, we will evaluate the metabolic and structural status of engineered skeletal muscle tissues during fabrication. These characterized engineered tissues will then be implanted into a regenerative environment to examine the ability of the proposed imaging method to predict successful repair of damage. It is expected that the gold standards for evaluating skeletal muscle in vitro can be replicated non-invasively and sterilely by this method. If successful for evaluating skeletal muscle, this technique could be applied to other engineered tissues, allowing for reliable evaluation of a wide variety of tissue engineering technologies prior to use as a repair modality.