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. 2018 Jul 18;8(1):10854.
doi: 10.1038/s41598-018-29060-y.

Dynamic Loading and Tendon Healing Affect Multiscale Tendon Properties and ECM Stress Transmission

Benjamin R Freedman  1   2   3   4 , Ashley B Rodriguez  1 , Ryan J Leiphart  1   2 , Joseph B Newton  1   2 , Ehsan Ban  5   6 , Joseph J Sarver  7 , Robert L Mauck  1   2 , Vivek B Shenoy  5   6 , Louis J Soslowsky  8   9
Affiliations

Dynamic Loading and Tendon Healing Affect Multiscale Tendon Properties and ECM Stress Transmission

Benjamin R Freedman et al. Sci Rep. .

Abstract

The extracellular matrix (ECM) is the primary biomechanical environment that interacts with tendon cells (tenocytes). Stresses applied via muscle contraction during skeletal movement transfer across structural hierarchies to the tenocyte nucleus in native uninjured tendons. Alterations to ECM structural and mechanical properties due to mechanical loading and tissue healing may affect this multiscale strain transfer and stress transmission through the ECM. This study explores the interface between dynamic loading and tendon healing across multiple length scales using living tendon explants. Results show that macroscale mechanical and structural properties are inferior following high magnitude dynamic loading (fatigue) in uninjured living tendon and that these effects propagate to the microscale. Although similar macroscale mechanical effects of dynamic loading are present in healing tendon compared to uninjured tendon, the microscale properties differed greatly during early healing. Regression analysis identified several variables (collagen and nuclear disorganization, cellularity, and F-actin) that directly predict nuclear deformation under loading. Finite element modeling predicted deficits in ECM stress transmission following fatigue loading and during healing. Together, this work identifies the multiscale response of tendon to dynamic loading and healing, and provides new insight into microenvironmental features that tenocytes may experience following injury and after cell delivery therapies.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Magnitude and duration of dynamic loading affects macroscale tendon properties. (a) Tendon, which connects muscle to bone, has a hierarchical structure that spans several length scales and comprises both a collagenous matrix and cellular components. Tendon cells maintain tensional homeostasis by balancing cell and ECM forces. (b) Force and displacement have a nonlinear relationship during tensile loading as disorganized fibers operative in the toe region become organized in the linear region. (c) Tendon groups were loaded at different load levels (zero, low, high) and cycle durations (0, 10, 1000) prior to multiscale property evaluation. (d) Tendons were preconditioned and then dynamically loaded for 0, 10, or 1000 cycles at either high (25–75% UTS) or low (2–10% UTS) loads. Following loading, tendons underwent a quasi-static ramp to 1% or 10% strain followed by a frequency sweep. For recovery experiments, tendons were allowed 1000 s of rest at 0% strain prior to a second quasi-static ramp. For non-recovery experiments, tendons were snap frozen at either 1% or 10% strain for microscale assessment. (e) The change in equilibrium stress was decreased following high magnitude long duration loading. (f) The dynamic modulus, |E*|, also decreased following long duration and high magnitude loading. (g) The strain at which collagen fiber re-alignment occurred was elevated due to long duration and high magnitude loading. Data shown as mean ± SD. N = 7–11/group. Lines indicate significant differences. Symbols indicate significant differences to quasi-static controls (unshaded).
Figure 2
Figure 2
Microscale tendon properties are altered with dynamic loading. (a) Multiphoton imaging was used to quantify collagen organization (white), amount of F-actin (purple), and nuclear aspect ratio (red) and disorganization (scale bar = 20 µm). (b) Collagen disorganization decreased with applied strain in all groups except those that had been subjected to high/1k loading, similar to (c) the nuclear disorganization. (d) The change in nuclear aspect ratio (ΔnAR) with applied strain also decreased in high magnitude loading groups. *Panels a–c: Data shown as mean ± SD. N = 7–11/group. Lines indicate significant differences. *Panel d: Data shown as mean ± SEM. N = 119–815 cells/group.
Figure 3
Figure 3
Multiscale tendon properties are altered with dynamic loading during tendon healing. (a) Healing tendons were evaluated for multiscale properties (macro and micro-scale) following dynamic loading. (b) Similar to uninjured tendon, the change in equilibrium stress was decreased in healing tendons due to high magnitude long duration loading. (c) Multiphoton imaging was used to assess collagen organization (white), amount of F-actin (purple), and nuclear aspect ratio (red) and disorganization following dynamic loading at different stages of healing (scale bar = 20 µm). (d) Collagen disorganization was strain responsive with dynamic loading in uninjured tendons, but was not responsive in healing tendons. (e) The change in nuclear aspect ratio decreased following high magnitude dynamic loading in uninjured and week-6 post-injury groups, but increased in the week-2 post-injury healing groups. *Panels b,d: Data shown as mean ± SD. N = 7–11/group. Lines indicate significant differences. Symbols indicate significant differences (#) or trends ($) compared to quasi-static loading samples (0 cycles). *Panel e: Data shown as mean ± SEM. N = 119–1012 cells/group.
Figure 4
Figure 4
Tendon healing and dynamic loading affect cell-generated stress transmission through the surrounding ECM. (a) Cell-generated stress transmission through the surrounding ECM decayed more rapidly in (b) healing tendon and in tendons subjected to high magnitude dynamic loading, as evidenced by (c) the decreased exponent of displacement decay η. (d) When scaling the ECM stress transmission decay rate by tissue cellularity, the effective ECM stress transmission decay rate was more similar between groups. U indicates displacment (μm) from the cell perimeter. “Quasi” indicates quasi-static loading, “low/1k” indicates low magnitude loading for 1000 cycles, and “high/1k” indicates high magnitude loading for 1000 cycles.
Figure 5
Figure 5
Dynamic loading and tendon healing affect multiscale tendon properties and cell-generated stress transmission through the ECM. Summary of findings highlighting differences in matrix stress transmission (SECM, color gradients), change in nuclear aspect ratio with applied strain (ΔnAR), healing, F-actin, matrix disorganization (CSD), nuclear disorganization (nCSD), loading magnitude, and loading duration. Each row indicates a healing group. The left column is the response during quasi-static loading, in contrast to the right column during high magnitude long duration loading.
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