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.

Figure 1. Collagen mechanical damage and CHP binding.

(a) Simple schematic of triple-helical collagen organization in intact tendon microfibrils based on the Hodge–Petruska model. (b) Intact RTT fascicles were stretched in uniaxial tension to initiate mechanical damage within the tissue structure, followed by incubation with single-strand CF-CHP to allow triple helix-mediated hybridization to unfolded collagen strands, and washing to remove unbound CHP.

Figure 2. Fluorescence intensity and stress in tail tendon fascicles at incremental strains.

(a) Average stress–strain curves for each of the strain groups tested (n=3 each curve). Fascicle mechanical behaviour encompassed the range of linear behaviour to tissue failure. For clarity, the standard error bars are only shown for the last 1% strain of each curve. (b) Representative whole-sample fluorescence scans of 5, 10.5 and 15% strain samples, and the corresponding brightfield image for the 15% strain sample, clearly showing an increase in CF-CHP staining with increased strain. In these images, fascicles have been folded on microscope slides showing the stretched section on the left half of the image and the clamped ends on the right half of the image. Arrows indicate the approximate location of clamping. Scale bars, 2 mm. (c) Mean pixel intensity (n=5, 0% strain; n=3, 1–15% strain, mean±s.e.m.) quantified from fluorescence images of loaded fascicles (left axis) and the average percentage of collagen digested by trypsin (right axis) from fascicles in each group (0, 5, 7.5, 9, 10.5, 15% strain, n=5, mean±s.e.m.). The orange dotted lines in a and c indicate the approximate transition strain from the linear region of the stress–strain curve to the onset of damage as identified by the deviation of the stress–strain curve from linearity, which correlates with onset of CF-CHP intensity.

Figure 3. Multiphoton fluorescence localization of CF-CHP in mechanically damaged fascicles.

Multiphoton images of collagen SHG (white) and CF two-photon excited fluorescence (TPEF, green). (a) Multiphoton z-stack combining both SHG and CF TPEF through the imaging depth for a single fascicle stretched to 7.5% strain. The presence of CF fluorescence through the 80 μm depth confirmed that CHP binding was not limited to the tissue surface. SHG and CF TPEF signals are separated in panels b and c. (b) Banded CHP staining pattern in a 15% strain sample revealed inhomogeneity in molecular level collagen damage. This pattern was prevalent in samples across the entire range of strains tested. (c) Images of the end of a sample that was cut using sharp scissors. Note the minimal CHP staining. Scale bars 100 μm for all panels.

Figure 4. Transmission electron micrographs of NP-CHP binding to damaged collagen fibrils.

Raw images are in the left column, while corresponding annotated images are in the right column. Yellow dots represent the identified NP-CHPs. (a) Damaged tissue from a 12% strain fascicle exhibited a high density of NP-CHP binding and a large amount of visible fibril disruption (that is, loss of d-banding pattern). The few areas with remaining d-banding pattern are outlined in white lines. (b) Undamaged tendon fascicle incubated with NP-CHP. (c) Damaged tissue from a 12% strain fascicle incubated with scrambled sequence NP-^SCHP, showing low levels of non-specific binding. (d) Overall NP-CHP densities averaged from 5 randomly selected images from each group (mean±s.e.m.). Inset—Number of NP-CHP per μm² inside and outside the areas with preserved d-banding (outlined areas in panel a) in the images of 12% strain fascicles (mean±s.e.m.). *indicates statistical significance from the undamaged group with P<0.05. Scale bars, 200 nm.

Figure 5. CF-CHP binding to fatigue loaded fascicles.

(a) Mean fluorescence intensity versus fatigue loading condition for samples loaded between 1 and 5% strain at 0.1 and 1.0 Hz (n=3, mean±s.e.m.). Statistically significant intensity increase from the controls was observed in 0.1 Hz samples after 100 cycles and in 1.0 Hz samples after 1,000 cycles. (b) Representative wide-field fluorescence images reveal different CHP staining patterns between the 0.1 and 1.0 Hz samples. The staining images help explain the apparent overall high intensity of 0.1 Hz samples, as CHP was bound to a larger area in 0.1 Hz samples compared to 1.0 Hz samples tested for the same number of cycles. Arrows indicate approximate location of clamps. Scale bars, 2 mm.

Figure 6. Simulations of tensile strain applied to collagen triple helix.

We investigated two possible mechanisms for damage to the collagen triple helix under tension that would allow CHP binding: peptide backbone break of one or more α-chains, referred to as the tension-dominant case, and pull-out of a single α-chain, referred to as the shear-dominant case. These cases were investigated using steered MD simulations for a homotrimeric collagen model peptide made of 10 Gly–X–Y units derived from the crosslink region (930 Lys) of rat and mouse type I, α1 collagen chain. (a) Schematic of the two possible loading mechanisms that were investigated in the numerical simulations: the tension-dominant case (i) and the shear-dominant case (ii). (b) The force–strain curves of the collagen molecule under tension-dominant and shear-dominant loading. The thicker curves (dark blue and dark red) are results of a moving average with a 400 ps window width. (c) Simulation snapshots taken before and after each relaxation simulation for the shear-dominant case. Starting conformations were obtained after a specific level of strain (ɛ) in the shear-dominant test as indicated in panel b. Water molecules and ions are not shown for clarity. (d) SASA, a measure of triple helix unfolding, as a function of applied strain for the shear-dominant case. Structural changes to the triple helix started to take place at 13.6% strain, and by 15.9% strain nearly maximum SASA was reached. Mean±s.d., computed during the last 20 ns of relaxation for each simulation.

Figure 7. Simulations of tensile strain applied to confined full-length collagen.

(a) MD simulation snapshots of the mechanical response of the full-length collagen confined in a microfibril under deformations of different strains. Close-ups of the C-terminus of the center molecule are inserted for different strain states with other molecules displayed by gray points as the coordinates of their backbone atoms. Water molecules and ions are not show for clarity. Scale bar, 100 nm. (b) The force–strain (f-ɛ) curve of the center collagen molecule under uniaxial deformation with a strain rate of 10⁸ s⁻¹. The plot and error bar are results of average value and s.d. with a 40 ps window width. Snapshots in a are indicated by arrows in the plot with corresponding colors. (c) The peak force (f_peak) of the f–ɛ curve measured under different strain rates (dɛ/dt) in simulations (mean±s.d.). Data points are fitted according to an Extended Bell Model.