Development of a novel multiphysical approach for the characterization of mechanical properties of musculotendinous tissues

Abstract

At present, there is a lack of well-validated protocols that allow for the analysis of the mechanical properties of muscle and tendon tissues. Further, there are no reports regarding characterization of mouse skeletal muscle and tendon mechanical properties in vivo using elastography thereby limiting the ability to monitor changes in these tissues during disease progression or response to therapy. Therefore, we sought to develop novel protocols for the characterization of mechanical properties in musculotendinous tissues using atomic force microscopy (AFM) and ultrasound elastography. Given that TIEG1 knockout (KO) mice exhibit well characterized defects in the mechanical properties of skeletal muscle and tendon tissue, we have chosen to use this model system in the present study. Using TIEG1 knockout and wild-type mice, we have devised an AFM protocol that does not rely on the use of glue or chemical agents for muscle and tendon fiber immobilization during acquisition of transversal cartographies of elasticity and topography. Additionally, since AFM cannot be employed on live animals, we have also developed an ultrasound elastography protocol using a new linear transducer, SLH20-6 (resolution: 38 µm, footprint: 2.38 cm), to characterize the musculotendinous system in vivo. This protocol allows for the identification of changes in muscle and tendon elasticities. Such innovative technological approaches have no equivalent to date, promise to accelerate our understanding of musculotendinous mechanical properties and have numerous research and clinical applications.

Figure 1

Elasticity maps (15 µm × 15 µm) obtained from the AFM protocol for slow (A,B soleus: Sol) and fast (C,D EDL) twitch muscle fibers and tendon fibers (E,F) of wild-type (WT) and TIEG1 knockout (KO) mice. The muscle and tendon cartographies are represented by different colors which are representative of lower (blue/green) and higher (red/orange) elasticities.

Figure 2

Quantification of Young’s modulus obtained via AFM for wild-type (WT) and TIEG1 knockout (KO) soleus (SOL) and EDL muscle fibers (A) and tendon (B) fibers. ***p < 0.001 between indicated groups.

Figure 3

Transmission Electronic Microscopy (TEM) acquisition of soleus (Sol) and extensor digitorum longus (EDL) muscles isolated from wild-type (WT) and TIEG1 knockout (KO) mice. Longitudinal sections revealed a disorganized ultrastructure in both TIEG1 KO Sol and EDL muscles (B–D) compared to WT littermates (A–C).

Figure 4

Representative B-mode image of the mouse hindlimb (A) and the corresponding elastogram (B) showing the elasticity of the different tissues present within the hindlimb. The region of interest (ROI) analyzed for quantification of the Young’s modulus (E) is indicated (B).

Figure 5

Representative B-mode image of the mouse hindlimb (A) and the corresponding elastogram (B) showing the elasticity of the different tissues present within the hindlimb. The region of interest (ROI) for measurement of the Young’s modulus (E) of the Achilles tendon is indicated (B).

Figure 6

Principal component analysis of texture parameters from the B-mode acquisitions of muscle (A) and tendon (B) according to mouse genotype (1: TIEG1 knockout (KO) vs 2: wild-type (WT)). Significant differences for both tissues between the two genotypes are apparent. Quantification of muscle stiffness (C) and tendon stiffness (D) according to mouse genotype.

Figure 7

Hierarchical ascending classification (HAC) of the six muscles as a function of mouse genotype (wild-type (WT) vs TIEG1 knockout (KO)). Differences between Class I (CI) and Class II (CII) demonstrate a genotype effect on the different muscles.

Figure 8

Model indicating the orientation and placement of the ultrasound probe on the mouse hindlimb. This image was drawn by Frank M. Corl from the department of Biomedical and Scientific Visualization, Mayo Clinic. Used with permission of Mayo Foundation for Medical Education and Research, all rights reserved.