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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 ?-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.

SUBMITTER: Zitnay JL

PROVIDER: S-EPMC5364439 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Zitnay Jared L JL Li Yang Y Qin Zhao Z San Boi Hoa BH Depalle Baptiste B Reese Shawn P SP Buehler Markus J MJ Yu S Michael SM Weiss Jeffrey A JA

Nature communications 20170322

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 collage ...[more]

PMID: 28327610

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Project description:It is well established that overstretch of arteries alters their mechanics and compromises their function. However, the underlying structural mechanisms behind these changes are poorly understood. Utilizing a recently developed collagen hybridizing peptide (CHP), we demonstrate that a single mechanical overstretch of an artery produces molecular-level unfolding of collagen. In addition, imaging and quantification of CHP binding revealed that overstretch produces damage (unfolding) among fibers aligned with the direction of loading, that damage increases with overstretch severity, and that the onset of this damage is closely associated with tissue yielding. These findings held true for both axial and circumferential loading directions. Our results are the first to identify stretch-induced molecular damage to collagen in blood vessels. Furthermore, our approach is advantageous over existing methods of collagen damage detection as it is non-destructive, readily visualized, and objectively quantified. This work opens the door to revealing additional structure-function relationships in arteries. We anticipate that this approach can be used to better understand arterial damage in clinically relevant settings such as angioplasty and vascular trauma. Furthermore, CHP can be a tool for the development of microstructurally-based constitutive models and experimentally validated computational models of arterial damage and damage propagation across physical scales.Statement of significanceArteries play a critical role by carrying oxygen and essential nutrients throughout the body. However, trauma to the head and neck, as well as surgical interventions, can overstretch arteries and alter their mechanics. In order to better understand the cause of these changes, we employ a novel collagen hybridizing peptide (CHP) to study collagen damage in overstretched arteries. Our approach is unique in that we go beyond the fiber- and fibril-level and characterize molecular-level disruption. In addition, we image and quantify fluorescently-labeled CHP to reveal a new structure-property relationship in arterial damage. We anticipate that our approach can be used to better understand arterial damage in clinically relevant settings such as angioplasty and vascular trauma.

Project description:Degenerative disc disease (DDD) is accompanied by structural changes in the intervertebral discs (IVD). Extra-cellular matrix degradation of the annulus fibrosus (AF) has been linked with degeneration of the IVD. Collagen is a vital component of the IVD. Collagen hybridizing peptide (CHP) is an engineered protein that binds to degraded collagen, which we used to quantify collagen damage in AF. This method was used to compare AF samples obtained from donors with no DDD to AF samples from patients undergoing surgery for symptomatic DDD. Fresh AF tissue was embedded in an optimal cutting temperature compound and cryosectioned at a thickness of 8 μm. Hematoxylin and Eosin staining was performed on sections for general histomorphological assessment. Serial sections were stained with Cy3-conjugated CHP and the mean fluorescence intensity and areal fraction of Cy3-positive staining were averaged for three regions of interest (ROI) on each CHP-stained section. Increases in mean fluorescence intensity (p = 0.0004) and percentage of positively stained area (p = 0.00008) with CHP were detected in DDD samples compared to the non-DDD samples. Significant correlations were observed between mean fluorescence intensity and percentage of positively stained area for both non-DDD (R = 0.98, p = 5E-8) and DDD (R = 0.79, p = 0.0012) samples. No significant differences were detected between sex and the lumbar disc level subgroups of the non-DDD and DDD groups. Only tissue pathology (non-DDD versus DDD) influenced the measured parameters. No three-way interactions between tissue pathology, sex, and lumbar disc level were observed. These findings suggest that AF collagen degradation is greater in DDD samples compared to non-DDD samples, as evidenced by the increased CHP staining. Strong positive correlations between the two measured parameters suggest that when collagen degradation occurs, it is detected by this technique and is widespread throughout the tissue. This study provides new insights into the structural alterations associated with collagen degradation in the AF that occur during DDD.

Dataset Information

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

Publications

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

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