Fatigue loading of tendon results in collagen kinking and denaturation but does not change local tissue mechanics

Abstract

Fatigue loading is a primary cause of tendon degeneration, which is characterized by the disruption of collagen fibers and the appearance of abnormal (e.g., cartilaginous, fatty, calcified) tissue deposits. The formation of such abnormal deposits, which further weakens the tissue, suggests that resident tendon cells acquire an aberrant phenotype in response to fatigue damage and the resulting altered mechanical microenvironment. While fatigue loading produces clear changes in collagen organization and molecular denaturation, no data exist regarding the effect of fatigue on the local tissue mechanical properties. Therefore, the objective of this study was to identify changes in the local tissue stiffness of tendons after fatigue loading. We hypothesized that fatigue damage would reduce local tissue stiffness, particularly in areas with significant structural damage (e.g., collagen denaturation). We tested this hypothesis by identifying regions of local fatigue damage (i.e., collagen fiber kinking and molecular denaturation) via histologic imaging and by measuring the local tissue modulus within these regions via atomic force microscopy (AFM). Counter to our initial hypothesis, we found no change in the local tissue modulus as a consequence of fatigue loading, despite widespread fiber kinking and collagen denaturation. These data suggest that immediate changes in topography and tissue structure - but not local tissue mechanics - initiate the early changes in tendon cell phenotype as a consequence of fatigue loading that ultimately culminate in tendon degeneration.

Keywords: Atomic force microscopy; Fatigue; Microscale mechanics; Second harmonic generation imaging; Tendon.

Figure 1

(A) Schematic of anatomical location of FCU. (B) Image of rat FCU tendon including adjacent pisiform (P) and muscle. Note: Anatomical locations of the FCU are identical between rats and humans. (C) Fatigue loading protocol.

Figure 2

Change in peak strain and secant modulus during fatigue of representative sample loaded to failure. Loading of subsequent samples was terminated prior to failure at a creep strain of 6%, which represents approximately 70% of the secondary creep phase.

Figure 3

Measurement of local collagen fiber orientation. (A) Representative subregion of SHG image (18×18 μm). (B) Binarized image of two-dimensional Fourier transform for the subregion. (C) Average collagen fiber orientation shown based on direction perpendicular to image of Fourier transform. (D) Representative image of fiber kinking (green arrows), which was defined as a >30 ° change in fiber orientation between adjacent subregions.

Figure 4

Minimal (A) collagen fiber kinking and (B) CHP staining was observed for fresh tendons, which sharply contrasts the significant (C) kinking and (D) denaturation seen in the fatigue-loaded samples. Scale bar = 1 mm.

Figure 5

(A) Significantly more collagen fiber kinking and CHP staining was observed in the fatigue-loaded samples. (B) Fiber kinking was evenly distributed between the CHP-positive and CHP-negative regions, suggesting no correlation between fiber kinking and collagen denaturation. * p<0.001 & ** p<0.0001 vs Fresh

Figure 6

No significant difference was found in the local tissue modulus either between fresh and fatigue-loaded samples or between CHP-positive and CHP-negative regions.