Freezing does not alter multiscale tendon mechanics and damage mechanisms in tension

Abstract

It is common in biomechanics to use previously frozen tissues, where it is assumed that the freeze-thaw process does not cause consequential mechanical or structural changes. We have recently quantified multiscale tendon mechanics and damage mechanisms using previously frozen tissue, where damage was defined as an irreversible change in the microstructure that alters the macroscopic mechanical parameters. Because freezing has been shown to alter tendon microstructures, the objective of this study was to determine if freezing alters tendon multiscale mechanics and damage mechanisms. Multiscale testing using a protocol that was designed to evaluate tendon damage (tensile stress-relaxation followed by unloaded recovery) was performed on fresh and previously frozen rat tail tendon fascicles. At both the fascicle and fibril levels, there was no difference between the fresh and frozen groups for any of the parameters, suggesting that there is no effect of freezing on tendon mechanics. After unloading, the microscale fibril strain fully recovered, and interfibrillar sliding only partially recovered, suggesting that the tendon damage is localized to the interfibrillar structures and that mechanisms of damage are the same in both fresh and previously frozen tendons.

Keywords: damage; freeze-thaw; mechanics; multiscale testing; tendon.

Figure 1

Mechanical loading profile schematic. (A) Definition of Δ = diagnostic – baseline was used to quantify damage when loaded to 6% strain after preconditioning (PC). Fascicle was allowed to rest for 40 min at the reference length before loading to failure. The blue dots represent when confocal images were taken to quantify fibril-level deformations. EL (end of loading), SR (start of rest), and ER (end of rest) marked on the figure were used for calculating fibril-level recovery. (B) Representative stress–strain curve with fascicle-level parameters. All the loading and unloading rates were 1%/second. (C) Representative image of tendon used to measure microscale mechanics with four photobleached lines: (1) the image corresponds to the reference state before loading; (2) at EL (end of loading), the fibril strain (ε) and interfibrillar sliding (γ) increased compared with the reference image; (3) immediately after unloading, at SR (start of rest), the interfibrillar sliding partially recovered; and, (4) at ER (end of rest), the interfibrillar sliding was not fully recovered.

Figure 2

Fascicle-level tensile behavior with baseline and diagnostic. For any of the parameters, there was no difference between the fresh and frozen groups (P > 0.07). Transition and inflection point strain both increased, consistent with our previous findings. These results suggest that freezing does not alter macroscale tendon mechanics and damage mechanisms. The error bars represent SEM.

Figure 3

Fascicle-level tensile behavior in Δ. At the fascicle level, tendons experienced a permanent change in mechanical parameters Δ ≠ 0 for both groups. For any of the parameters, there was no difference between the fresh and frozen groups (P > 0.07). The error bars represent SEM.

Figure 4

Transverse strain recovery. (A) Transverse strain fully recovered over time for both groups. The dashed line represents when samples were unloaded. (B) The transverse τ was not significantly different between the groups (P > 0.80). Similarly, the constant C averaged −0.11 ± 0.12 and −0.13 ± 0.04 for the fresh and frozen groups, respectively, with no difference between the two groups (P > 0.47). The error bars represent SEM.

Figure 5

Fibril-level deformation of tendon fascicles. (A) Fibril strain fully recovered for both groups, suggesting that the collagen fibrils are undamaged. On the other hand, (B) interfibrillar sliding only partially recovered, exhibiting damage at the fibril level, suggesting that tendon damage is localized to the interfibrillar structures and that the mechanisms of tendon damage are the same in both fresh and frozen tendons. The dashed line represents when samples were unloaded. (C) The interfibrillar sliding c was not significantly different in the two groups (P > 0.98). Similarly, the constant D averaged 9.60 ± 4.21 and 7.40 ± 2.94 for fresh and frozen, respectively, with no difference between two groups (P > 0.19). The error bars represent SEM.

Figure 6

Decomposed interfibrillar sliding recovery. Interfibrillar sliding recovery was decomposed to elastic, viscoelastic, and nonrecoverable portions. For all three components, there was no significant difference between the two groups (P > 0.78). The error bars represent SEM.

Figure 7

SEM images of collagen fibrils for the fresh and frozen groups. The qualitative observation of SEM images at two different magnifications—6000× and 35,000×—shows that the fibril morphology and organization are very similar between the two groups. The red box at the low magnification represents the area imaged for the high magnification.