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

Abstract

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.

A real-time monitoring platform of myogenesis regulators using double fluorescent labeling

“…These tools include assessment of physical properties and function such as contractility through displacement analysis [ 36 ], skeletal muscle thickness [ 45 , 46 ] and myoblast differentiation [ 46 ] through electrical impedance. Additionally, multiple imaging modalities have been applied to assess muscle biology, including bioluminescence imaging for luciferase tagged cell transplantation [ 47 ], second harmonic generation (SHG) imaging to quantify muscle striation patterns [ 48 , 49 ], MRI to assess oxidative phosphorylation in muscle [ 50 ], and multi-photon imaging for the assessment of myoblast oxidation level [ 51 ].…”

Nonlinear Optical Microscopy: From Fundamentals to Applications in Live Bioimaging

“…In the last decade, it has been demonstrated that non-invasive nonlinear microscopy represents a promising strategy for label-free live imaging of 3D engineered tissues. In fact, several studies had been performed to characterize morphology ( Hofemeier et al, 2016 ; Nguyen et al, 2017 ; Syverud et al, 2017 ; Costa Moura et al, 2018 ; Kaushik et al, 2019 ; Moura et al, 2019 ), functionality ( Hofemeier et al, 2016 ; Li et al, 2017 ; Syverud et al, 2017 ; Cong et al, 2019 ; Okkelman et al, 2020 ), composition and distribution of chemicals ( Hofemeier et al, 2016 ; Li et al, 2017 ; Syverud et al, 2017 ; Costa Moura et al, 2018 ; Moura et al, 2019 ; Sood et al, 2019 ), invasion, infiltration and mechano-regulation of cellular constructs ( Hofemeier et al, 2016 ; Nguyen et al, 2017 ; Syverud et al, 2017 ; Costa Moura et al, 2018 ; Kaushik et al, 2019 ; Moura et al, 2019 ; Sood et al, 2019 ), which have been collected in Table 4 . A representative study made by Hofemeier et al (2016) carried out a multi-spectral CARS and SHG imaging on an engineered bone tissue from stem cell differentiation toward osteogenic phenotype within 3 weeks of culture.…”

“…A representative study made by Hofemeier et al (2016) carried out a multi-spectral CARS and SHG imaging on an engineered bone tissue from stem cell differentiation toward osteogenic phenotype within 3 weeks of culture. Similarly, Syverud et al (2017) generated a fully-differentiated musculoskeletal tissue and exploited NLO microscopy with TPEF for imaging the autofluorescence of NAD(P)H, and SHG to identify collagen and myosin fibers. This work established label-free NLO microscopy as a valid alternative to collagen and myosin immunostaining, even if the tissue construct was fixed and sliced before the acquisitions, and only monolayered cells were imaged in their entirety and vitality.…”

A tissue engineering approach for repairing craniofacial volumetric muscle loss in a sheep following a 2, 4, and 6-month recovery

“…Muscle progenitor cells including satellite cells were isolated as described previously [23,24,[29][30][31][32][33][34]. Briefly, muscle biopsies between 3g and 3.5g were sanitized in 70% ethanol and finely minced with a razor blade.…”

“…A subset of the SMUs, termed "sentinel SMUs", was reserved for in vitro characterization including biomechanical testing and histology. Approximately 24-48 hours after 3D formation, contractile properties of the single SMUs (not modularly fused) were measured as described previously [23,[29][30][31][32][33]35,36]. Briefly, contractions were elicited through field stimulation with a platinum electrode and measured by an optical force transducer (World Precision Instruments, cat.…”