STRUCTURAL CHANGES IN STRETCHED COLLAGEN FIBRILS AS A FUNCTION OF RELAXATION TIMES

Abstract

The collagen fibrils are the main building block of connective tissues in mammals where they fulfill both structural and mechanical roles. The structure of a fibril is based on collagen molecules that self-assemble into micro-fibrils and sub-fibrils stabilized by hydrogen bonds and covalent crosslinks. The non-integer staggering of collagen molecules results in a characteristic D-band pattern along the fibril with a periodicity of 67nm. Fibrils were extracted from bovine extensor tendons, around 40 microns long segments were isolated (n=14) and glued, imaged with AFM before manipulation and then stretched between 5% and 20% strain, held at that strain for 150 seconds (n=8), 1 second (n=3), or 1500 seconds (n=3) then released. The manipulated fibrils were then imaged using AFM to characterize morphological changes. To assess whether stress relaxation led to denaturation (uncoiling) of collagen molecules, fluorescent collagen hybridizing peptide (CHP, a probe specific to denatured collagen) was applied to the samples and binding was measured using a Zeiss LSM 710 upright confocal microscope. We observed that stretching followed by stress relaxation did not break the fibrils and did not change significantly the value of the D-band. However, our fluorescence microscopy results indicate that some collagen molecules unfold within the fibril due to the stretching and relaxation protocol. AFM mapping of the fibrils cross section indicates a correlation between molecular denaturation as measured through fluorescence and loss of cross-sectional area. In addition, the mechanical energy density for denaturing a full single fibril was estimated. To our knowledge, this is the first demonstration of molecular denaturation within a single collagen fibril during a non-disruptive tensile test.