The influence of glycosaminoglycan proteoglycan side chains on tensile force transmission and the nanostructural properties of Achilles tendons

Abstract

This study investigates the nanostructural mechanisms that lie behind load transmission in tendons and the role of glycosaminoglycans (GAGs) in the transmission of force in the tendon extracellular matrix. The GAGs in white New Zealand rabbit Achilles tendons were enzymatically depleted, and the tendons subjected to cyclic loading at 6% strain for up to 2 hr. A nanoscale morphometric assessment of fibril deformation under strain was linked with the decline in the tendon macroscale mechanical properties. An atomic force microscope (AFM) was employed to characterize the D-periodicity within and between fibril bundles (WFB and BFB, respectively). By the end of the second hour of the applied strain, the WFB and BFB D-periodicities had significantly increased in the GAG-depleted group (29% increase compared with 15% for the control, p < .0001). No statistically significant differences were found between WFB and BFB D-periodicities in either the control or GAGdepleted groups, suggesting that mechanical load in Achilles tendons is uniformly distributed and fairly homogenous among the WFB and BFB networks. The results of this study have provided evidence of a cycle-dependent mechanism of damage accumulation. The accurate quantification of fibril elongation (measured as the WFB and BFB D-periodicity lengths) in response to macroscopic applied strain has assisted in assessing the complex structure-function relationship in Achilles tendon.differences in within-fibril and between-fibril-bundle D-periodicities, D-periodicity, force transmission in tendons, glycosaminoglycans, proteoglycan matrix, tendon structure-function relationships | INTRODUCTIONThe macroscopic stress-strain curve of tendons shows distinctive toe, heel and linear regions (Fratzl et al., 1998). Each of these regions has different instantaneously occurring mechanisms at different hierarchical levels in the tendon, such as quarter-staggered tropocollagen molecular elongation, changes in fibril axial D-periodicity, and the sliding of fibrils embedded in the hydrophilic proteoglycan matrix (Thorpe, Birch, Clegg, & Screen, 2013). At the nanoscale level, X-ray diffraction studies report that fibrils deform less than the whole tendon structure, suggesting a fibril sliding mechanism within the matrix

An AFM study of the nanostructural response of New Zealand white rabbit Achilles tendons to cyclic loading

“…(b) Elastic linear region (<5% strain)—sliding of fibrils and the removal of kinks in the molecular gap regions. (c) Inelastic linear region (>5% strain)—tropocollagen molecules slide past one another and GAGs detach from the core protein (Adapted from Al Makhzoomi, Kirk, Dye, & Allison, 2021)…”

“…All D‐periodicity lengths within a given tendon were pooled and referenced to the control D‐periodicity length to calculate the strain. Multiple regions along the axial direction of a fibril bundle (fiber) (20–50 μm distance) were investigated in a previous study (Al Makhzoomi, Kirk, Dye, & Allison, 2021) to show within‐fibril‐bundle and bundle‐to‐bundle variation.…”

“…After staining with fluorescent acridine orange (Eugene, Oregon, USA) solution (0.3 g/L for 45 min) followed by three repeated washings with phosphate‐buffered saline (PBS), the tendon ends were placed in freezing micro‐grips and mounted in a mechanical testing system (Instron 5848, MA, USA). The mechanical protocol describing the stretching of the tendons is reported previously (Supporting Information; Al Makhzoomi, Kirk, Dye, & Allison, 2021).…”

“…Random locations on the samples were imaged by AFM in search of fibril bundles. From each section, a minimum of five 3.5 μm × 3.5 μm regions of interest (ROI) were analyzed as described previously (Al Makhzoomi, Kirk, Dye, & Allison, 2021).…”

“…Multiple regions along the axial direction of a fibril bundle (fiber) (20-50 μm distance) were investigated in a previous study (Al Makhzoomi, Kirk, Dye, & Allison, 2021) to show within-fibril-bundle and bundleto-bundle variation.…”