First Empirical Evidence of Collagen Damage during Bone Fracture
Bone is a remarkable material, strong enough to withstand impressive forces without fracturing. Despite decades of research, however, our understanding of the molecular mechanisms underlying bone’s fracture resistance has remained incomplete.
It has long been hypothesized that collagen degradation occurs following bone fracture. Until recently, this hypothesis lacked empirical evidence due to a lack of molecular tools to measure collagen damage. Interestingly, a study published in 2022 by Corin Seelemann and Thomas Willett (University of Waterloo) has provided the first definitive proof of collagen denaturation in response to bone fracture. Their research was made possible using Collagen Hybridizing Peptides (CHPs) to visualize degraded collagen. Additionally, they investigated whether hydration is necessary for the fracture-mediated denaturation of collagen by testing dehydrated bone samples. They hypothesized that dehydration would change the nature of collagen denaturation during fracture, eliminating its contribution to fracture resistance.
To test whether collagen denaturation occurs as a result of bone fracture, and if collagen’s denaturation in this process serves as a molecular-level toughening mechanism, Seelemann and Willett employed F-CHP to allow direct visualization and quantification of collagen denaturation within the bone matrix. The researchers conducted fracture tests on samples of bovine cortical bone using the same chevron-notched four-point beam bending until the point of fracture. After fracturing the beams, they stained the fracture surfaces with F-CHP and imaged them via confocal microscopy. In addition to the standard hydrated samples, Seelemann and Willett prepared dehydrated bovine bone beams to test the effect of water on bone mechanical properties under the same bending protocol.
Fig. 3. a) Orientation of beam during 4-point bending b) Isometric view of fracture surface after beam fracture. Highlighted green area is fracture surface after beam is broken through 4-point bending
The results of their investigation are striking: They found clear evidence of increased CHP staining on fracture surfaces when compared to non-fractured areas, indicating significant collagen denaturation in response to fracture. The intensity of F-CHP staining correlated with the work-to-fracture (WFx) ratio, a measure of bone toughness, suggesting collagen denaturation plays a crucial role in energy dissipation during fracture. Importantly, they found similar staining patterns in human cortical bone samples, indicating that bone fracture leads to collagen denaturation across multiple species.
Fig. 8. Relationship between the % ROI stained with F–CHP of the hydrated specimens and the WFx of the specimens. P < 0.05 for hydrated beans determined based on a two-tailed student’s t-distribution.
Read More About the Study's Findings
Furthermore, the researchers observed that denaturation occurred primarily in regions of stable crack growth, indicating collagen denaturation helps prevent catastrophic failure by slowing down fracture propagations. Crucially, the dehydrated bone samples (below, bottom) showed significantly reduced F-CHP staining after fracture compared to the hydrated (below, top) samples.
Fig. 6. (left) Representative fracture surface of a specimen hydrated before fracture, stained with F–CHP. (Right) The same image thresholded based on the level determined from its polished control surface. Pixels that are above the threshold are white. Red circle with arrow indicates origin of fracture and direction of crack propagation. Red lines outline the triangular fracture surface. The blue line marks the edge of the rough textured ROI.
Fig. 7. (left) Representative fracture surface of a specimen dehydrated before fracture, stained with F–CHP. (Right) The same image thresholded based on the level determined from the polished control surface. Pixels that are above the threshold are white. Red circle with arrow indicates origin of fracture and direction of crack propagation. Red lines outline the triangular fracture surface.
This finding supports the hypothesis that water is essential for collagen to undergo sustained mechanical denaturation during fracture. In the absence of water, the collagen matrix appears to fail. The hypothesis is that during failure, hydrated collagen triple helices lose hydrogen bonding between alpha chains and these hydrogen bonds are transferred to nearby water molecules. In the absence of water in dehydrated conditions, the collagen alpha chains can no longer form hydrogen bonds with water leading to early failure.
The study by Seelemann and Willett (2022) represents a significant breakthrough in our understanding of bone fracture mechanics. By providing definitive evidence of collagen denaturation in response to fracture, this research confirms a long-standing hypothesis and opens up exciting new avenues for investigation. The use of F-CHPs as a tool to visualize and quantify collagen denaturation has proven to be invaluable in this study and is likely to play a critical role in future research aimed at elucidating the complex interplay between collagen, bone toughness, and fracture resistance.