MRI-Compatible Ex-Vivo Tensile Testing for Assessment of Passive Mechanical Properties of Whole Muscle

Accurate measurement of the passive mechanical properties of whole skeletal muscle is essential for physiological studies and biomechanical models. However, conventional ex-vivo tensile testing to measure these properties often relies on assumptions for stress and strain calculation. Specifically, strain is typically calculated from grip-to-grip displacement, and stress is found using muscle physiological cross-sectional area (PCSA) based on literature density values. This study aimed to develop a method directly quantifying muscle 3D geometry during stretching to measure its passive elastic modulus. Our MRI-compatible ex-vivo tensile testing framework integrated stepwise testing with high-field (9.4T) MRI. This enabled us to directly quantify whole-muscle 3D geometry, volume, and length during stretching. Ten rat tibialis anterior muscles underwent incremental lengthening (∼6% strain per step) with 4-min stress-relaxation holds while acquiring T2-weighted 3D MRI at each step. Engineering stress-strain data were calculated using MRI-based and conventional measurements. Grip-to-grip strain overestimated muscle strain by 13.7% compared to MRI-derived value, while considering MRI-based volumetric PCSA produced stresses 7.4% higher than the traditional gravimetric approach. These methodological differences increased the estimated passive elastic modulus by ∼40% versus conventional calculations. We also observed progressive muscle volume increases during testing (∼13.7% from slack length to ∼36% strain), with larger baseline volumes in specimens stored longer in Tyrode’s solution. Accounting for this volume change, true stress–strain results showed substantially higher modulus values than engineering estimates (1162 ± 519 vs. 745 ± 352 kPa at 30% strain). Overall, MRI-based geometry monitoring improved whole-muscle stress–strain characterization and revealed muscle volume increases during ex-vivo testing.