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    Biomechanics

    Biaxial mechanical testing to characterize tissue strains during in vivo loading in mdx and healthy diaphragm muscle

    (D-128) Biaxial mechanical testing to characterize tissue strains during in vivo loading in mdx and healthy diaphragm muscle

    Friday, October 13, 2023
    3:30 PM – 4:30 PM PDT
    Location: Exhibit Hall - Row D - Poster # 128

      Co-Author(s)

    • RS

      Ridhi Sahani

      Graduate Student
      Universtiy of Virginia, United States

      • Presenting Author(s)

    • KH

      Kaitlyn R. Hixson (she/her/hers)

      Undergraduate Student
      University of Virginia
      Charlottesville, Virginia, United States

      • Primary Investigator(s)

    • SB

      Silvia Blemker

      Professor of Biomedical Engineering
      University of Virginia, United States

    Introduction::

    Duchenne muscular dystrophy (DMD) is a degenerative neuromuscular condition resulting from a lack of dystrophin protein at the muscle cell membrane. Increased susceptibility to contraction induced damage leads to an accumulation of extracellular matrix (ECM) components such as collagen during the development of fibrosis in the muscle, leading to impaired function. Due to its role as a primary inspiratory muscle and its near-constant use, the diaphragm muscle is especially impacted in DMD, and the mdx mouse diaphragm muscle replicates the fibrosis and impairment seen in patients. The diaphragm is an anisotropic muscle that sustains biaxial loads, making it vital to measure passive properties in both the along- and cross- fiber directions to understand in vivo pathological changes. Previous studies show that diaphragm muscle is stiffer in the cross- relative to along-muscle fiber direction,1,2 with greater tissue stiffness and collagen alignment in mdx compared with healthy (WT) diaphragm muscle.3 Additionally, sonomicrometry revealed along-fiber shortening and cross-fiber lengthening in the diaphragm muscle of mdx and WT mice during tidal breathing, with smaller in vivo strains in mdx compared with WT mice.4 To fully understand the in vivo tissue properties of the fibrotic diaphragm, testing methods must first be established to replicate natural loading conditions. The goals of this study were to (1) develop methods to replicate in vivo strains during ex vivo biaxial testing in diaphragm muscle tissue and (2) examine relationships between applied and tissue strains in both mdx and WT mice.



    Materials and Methods:: All protocols were approved by University of Virginia’s IACUC. 6-month-old C57BL/6J male mice (WT, n=6) and 6-month-old dystrophin knockout mice (mdx, n=5), were euthanized and the diaphragm was excised and placed in a 30mM solution of BDM to prevent cross-bridge formation. Two 5 by 5 mm squares were cut from the costal diaphragm and tissue thickness was measured with a laser displacement sensor (Keyence, Itasca, IL) for one sample. The other sample was loaded onto a Cellscale Biotester for biaxial testing with 5 mm rakes placed in the tissue on each side, forming a square. Graphite powder was sprinkled on the tissue sample to allow for strain tracking. Samples were preloaded to 10 mN in the along- and cross-fiber directions before initiating the stretch protocol. Strains were applied cyclically at 1% strain per second and an equal recovery period with five stretches per test followed by a five-minute hold. The in vivo ratios used were -1:0.5, -1:1, and -1:1.5, in the along- relative to cross-fiber direction, from sonomicrometry measurements.4 The prescribed strains were -5%:2.5%, -5%:5%, and -5%:7.5%. Images from each test’s final stretch cycle were strain tracked using specialized code from the Barocas lab5 utilizing direct image correlation to obtain a deformation gradient for each image of each test. A custom MATLAB (Mathworks, Natick, MA) script calculated Green-Lagrange strain values from the deformation gradient for each image. Tissue strains obtained from deformation gradients were plotted against strain applied by the rakes in the along- and cross-fiber directions throughout each test.

    Results, Conclusions, and Discussions::

    Altered mechanical behavior was observed in the disease group relative to the control group. In the along-fiber direction the WT and the mdx samples had nearly identical relationships between applied and tissue strain throughout the duration of each strain ratio test (Figure 1C). In the cross-fiber direction the mdx samples had a markedly lower tissue strain compared to WT samples for the duration of each strain ratio test (Figure 1C). The average strains of both groups fell below the unity line throughout each test, indicating there was lower tissue strain than applied strain. Our finding of lower ex vivo cross-muscle fiber tissue strains in mdx relative to WT mice is consistent with the results of sonomicrometry experiments, where lower in vivo cross-fiber strains were measured in mdx diaphragm muscle compared to WT.4 Further, greater cross-muscle fiber collagen fiber alignment seen in mdx compared with WT diaphragm muscle may be implicated in the lower cross-muscle fiber tissue strains.3 Larger differences between the disease groups in the cross- relative to along-fiber strains show the necessity of biaxial testing, not just along-fiber uniaxial testing, to characterize altered passive properties. Using physiologically representative strains for mechanical testing aids in understanding the contribution of altered passive properties to the overall decrease in muscle function seen in DMD. Additionally, the consistent discrepancy between the applied and tissue strain may be an artifact of the tissue’s attachment to the Biotester. The rakes piercing the tissue could be moving within the holes they created during the stretch cycle, or the holes themselves disrupt the native muscle architecture which creates areas of large strain heterogeneity, both of which would transmit less strain to the center of the tissue.6,7 The discrepancy between the applied and tissue strain indicates that tracking strain by the movement of edge attachment points could introduce large amounts of error due to potential artifacts introduced by the rakes. Overall, this experiment establishes a method to replicate in vivo loading during ex vivo biaxial experiments and demonstrates differences in cross-fiber passive properties of dystrophic relative to healthy diaphragm muscle, further connecting benchtop experiments with pathological function.



    Acknowledgements (Optional): :

    References (Optional): :

    1.         Strumpf, R. K., Humphrey, J. D. & Yin, F. C. Biaxial mechanical properties of passive and tetanized canine diaphragm. American Journal of Physiology-Heart and Circulatory Physiology 265, H469–H475 (1993).


    2.         Boreik, A., Kelly, N., Rodarte, J. & Wilson, T. Biaxial constitutive relations for the passive canine diaphragm | Journal of Applied Physiology. https://journals.physiology.org/doi/full/10.1152/jappl.2000.89.6.2187 (2000).


    3.         Sahani, R., Wallace, C. H., Jones, B. K. & Blemker, S. S. Diaphragm muscle fibrosis involves changes in collagen organization with mechanical implications in Duchenne muscular dystrophy. Journal of Applied Physiology 132, 653–672 (2022).


    4.         Wallace, C. H. Novel experimental-modeling coupled framework to elucidate the impact of collagen on diaphragm muscle mechanics in Duchenne muscular dystrophy. (University of Virginia, 11/21).


    5.         Robust Image Correlation Based Strain Calculator for Tissue Systems (20130022, Dr. Victor Barocas) available from Technology Commercialization. https://license.umn.edu/product/robust-image-correlation-based-strain-calculator-for-tissue-systems.


    6.         Eilaghi, A., Flanagan, J. G., Brodland, G. W. & Ethier, C. R. Strain Uniformity in Biaxial Specimens is Highly Sensitive to Attachment Details. Journal of Biomechanical Engineering 131, (2009).


    7.         Malcolm, D. T. K., Nielsen, P. M. F., Hunter, P. J. & Charette, P. G. Strain measurement in biaxially loaded inhomogeneous, anisotropic elastic membranes.