Molecular level detection and localization of mechanical damage in collagen enabled by collagen hybridizing peptides

Abstract

Mechanical injury to connective tissue causes changes in collagen structure and material behaviour, but the role and mechanisms of molecular damage have not been established. In the case of mechanical subfailure damage, no apparent macroscale damage can be detected, yet this damage initiates and potentiates in pathological processes. Here, we utilize collagen hybridizing peptide (CHP), which binds unfolded collagen by triple helix formation, to detect molecular level subfailure damage to collagen in mechanically stretched rat tail tendon fascicle. Our results directly reveal that collagen triple helix unfolding occurs during tensile loading of collagenous tissues and thus is an important damage mechanism. Steered molecular dynamics simulations suggest that a likely mechanism for triple helix unfolding is intermolecular shearing of collagen α-chains. Our results elucidate a probable molecular failure mechanism associated with subfailure injuries, and demonstrate the potential of CHP targeting for diagnosis, treatment and monitoring of tissue disease and injury.

Microplate assay for denatured collagen using collagen hybridizing peptides

“…20 This curve reliably determined absolute amounts of denatured collagen from CHP fluorescence, which alone can only measure relative amounts. The T-H method requires over 64 hours of assay time, 4 and is prone to error with its complicated multistep protocol. The CHP microplate assay contrasts with this by requiring less than 24 hours of bench time following creation of the calibration curve, requiring only a simple staining process followed by tissue digestion.…”

“…This is likely due to the fact that 9% strain was in the region of strain where the samples departed linear stress-strain behavior which is associated with permanent tissue damage. 4 Therefore, sample-to-sample variation alone could result in a relatively large changes in the amount of denatured collagen compared to low strains and the plateau region at high strains. In addition, the amount of denatured collagen was similar to that found by Zitnay et al 4 for the same levels of damage.…”

“…Captured images were thresholded using Otsu’s method 14 to isolate the stained fascicle region, and the mean pixel intensity value was calculated, as per our previous method. 4 Image processing was performed using MATLAB (2016b, MathWorks, Natick, MA).…”

“…For example, it is known that denatured collagen plays a major role in osteoarthritis, 2 and more recently, has been shown to occur during mechanical damage to tendons. 3,4 Therefore, the ability to rapidly and accurately quantify denatured collagen would be a powerful asset for the study of many diseases and injuries.…”

“…The fascicles were imaged using widefield fluorescence microscopy to measure and quantify fluorescence intensity and by extension collagen damage. 4 While this method was effective at detecting and localizing denatured collagen in a tissue sample, it is unable to quantify the exact amount of denatured collagen, which is important for elucidating trends and mechanisms during disease onset and progression.…”