Novel method reveals heterogeneous micro-scale response of sclera collagen bundles to homogeneous macro-scale stretch

Abstract

Purpose : Collagen has a natural waviness called crimp that largely determines the nonlinear mechanical behavior of many ocular tissues. However, almost nothing is known about how this waviness changes with stretch. We have developed a novel method for observing and measuring collagen crimp changes during stretch. Our goal was to quantify the effects of macro-scale stretch on the micro-scale stretch and waviness of collagen fiber bundles of sclera.

Methods : 3 sheep eyes were acquired from a local abattoir and processed within 12 hours. The eyes were cryosectioned axially (30µm). The equatorial sclera was mounted to a uniaxial stretcher (Fig1A). Six samples were imaged with polarized light microscopy at various levels of macro-scale stretch and analyzed for collagen fiber orientation (Fig1B-D). Using manual markings, the local micro-scale stretch and waviness were tracked through different levels of stretch for several bundles of each section. Waviness was defined as the normalized SD of the fiber orientations along the bundle. Linear mixed effect models were used to test the association between bundle stretch and loss of waviness (uncrimping).

Results : We tracked the stretch and waviness of 21 bundles over an average of 9 stretch levels. Waviness decreased significantly with increasing micro-scale stretch (p<0.0001, Fig2A). Bundles had variable initial waviness, though all bundles uncrimped with sufficient stretch. Even within the same sample under homogeneous macro-scale stretch, bundles varied in responses, uncrimping at different levels of stretch and at different rates (Fig2B).

Conclusions : Our results indicate that the sclera response to macro-scale stretch is highly inhomogeneous at the micro-scale, in both the stretch level at which fiber bundles uncrimp and their uncrimping rate. Understanding the micro-scale response to mechanical loading is important for determining the role of collagen architecture on the macro-scale tissue biomechanics, and for understanding the biomechanical environment of scleral cells and their contributions to tissue growth and remodeling.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.

 

Fig 1. Tissue was held by clamps (A) and imaged at different levels of stretch. Panels B-D shows the micro-scale fiber orientation of a region (arrow in A) at increasing levels of stretch.

 

Fig 2. Stretch vs. waviness for all samples pooled (A) and for 3 example collagen bundles from 1 sample of eye 1 (B).