Abstract
Summary Statement
Tendon micromechanics were investigated using 2 methods. When collagen deformation was measured directly, higher levels of inter-fibre sliding were observed than when tenocyte nuclei were tracked. This suggests that under high strain tenocytes become unattached from the collagen fibres.
Introduction
Fibre extension and inter-fibre sliding have both been reported during tendon extension, but fibre sliding is believed to be the predominant mechanism in normal healthy tendon function. Fatigue damage is known to result in structural changes and reduced mechanical properties, but its influence on micromechanics is unknown.
This work aimed:
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1.
To investigate the effect of fatigue loading on bovine digital extensor fascicle micromechanics, comparing fibre extension and fibre sliding, hypothesising that the relative importance of these may change due to fatigue damage.
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2.
To compare two techniques for characterising micromechanics: bleaching of a grid to directly measure collagen deformation, and using the cells as fiducial markers of fibre movement.
Methods
The tensional regions of healthy digital extensor tendons were removed within 24 hours of slaughter and frozen. Tendons were defrosted, hydrated and fascicles dissected and loaded into custom-designed chambers allowing the mechanical loading of fully hydrated tendon fascicles. Fascicles were loaded for 0, 300 or 900 cycles under creep conditions at a frequency of 1Hz and to a maximum applied stress of 25% of the mean UTS of the fascicles.
Fascicles were stained using either Acridine Orange to stain the cell nuclei or DTAF solution to stain the collagen. After DTAF staining, a grid consisting of 4 squares of side 50 μm was photo-bleached using the FRAP system on a Leica TCS SP2 confocal scanning microscope. To investigate micromechanics, fascicles were secured in a uniaxial rig and strained in 2% increments to 10% total strain at a rate of 1%s−1. Imaging was carried out at each increment and local strains calculated from grid deformation or nuclei movement.
Results
No significant changes in micromechanics were observed with increasing numbers of creep cycles, as measured with either technique. This was despite quite significant matrix damage being observed particularly after 900 cycles. When using the grid deformation measure of strains, a continual increase in fibre sliding was seen above 4% applied strain, correlating with the levelling off of intra-fibre strains. This same move towards dominant fibre sliding was not observed with techniques using the nuclei as fiducial markers. Using the nuclei as markers consistently reported significantly lower levels of fibre sliding than those measured from grid deformation at strains of 6% and above, under all creep conditions.
Discussion/Conclusion
The apparent absence of any effect of creep on the measured microstructural deformation may be a result of the localised nature of the measurement techniques. At sites where matrix structure broke down both the tracking of nuclei and the photo-bleaching of the grid proved problematic and it is these regions where the greatest degree of deformation would perhaps be expected, with remaining areas of the tissue stress-deprived. The smaller levels of fibre shear reported when measured through nuclei tracking suggests that the tenocytes may not be well adhered to the fibres and may be protected from some of the matrix deformation in response to loading.
