# Fluorescent collagen hybridizing peptide for quantifying collagen denaturation in cortical bone

## TL;DR

This paper introduces a fluorescent peptide that can detect damaged collagen in bone, offering a new way to assess bone quality and fracture risk beyond traditional methods.

## Contribution

The study introduces R-CHP, a non-destructive method to quantify denatured collagen in mineralized bone.

## Key findings

- R-CHP fluorescence correlates strongly with collagen denaturation measured by the trypsin-hydroxyproline assay.
- Post-yield loading significantly increases collagen denaturation in bone.
- R-CHP localizes collagen damage at bone fracture surfaces.

## Abstract

Bone fracture risk is clinically assessed with bone mineral density (BMD); however, individuals with normal BMD also experience fractures, highlighting the need for complementary fracture risk assessment tools. While BMD remains the clinical gold standard, it fails to capture bone quality factors that contribute to fragility. Among these, collagen quality is essential for bone toughness, as it allows collagen to dissipate energy via stretching and uncoiling. When collagen is denatured, it loses its ability to deform, increasing fracture risk. This process is particularly relevant in aging, osteoporosis, and metabolic conditions such as diabetes, yet no clinical methods exist to quantify or localize denatured collagen in mineralized bone. This study introduces Collagen Hybridizing Peptide (CHP) as a tool to quantify denatured collagen in cortical bone. Here, we show that CHP fluorescence correlates strongly with collagen denaturation measured by established trypsin-hydroxyproline assay (r2 = 0.99) when applied to mineralized tissue subjected to heat treatment or mechanical loading. Confocal microscopy revealed a 55 % increase in collagen denaturation when tissue strain exceeded the yield point (p < 0.05). Our findings demonstrate that fluorescent CHP localizes high-strain regions to collagen denaturation on bone fracture surfaces, indicating that collagen damage occurs during post-yield failure. This non-destructive technique offers a powerful tool for assessing collagen quality, with potential applications in osteoporosis, diabetic bone fragility, and aging research. By advancing our ability to evaluate bone quality in cortical bone, R-CHP provides new method to study how denatures collagen affects bone resistance to fracture.

•R-CHP detects denatured collagen in bone without demineralization.•Confocal R-CHP fluorescence correlates with the well-established T-H assay.•High staining temperature improves R-CHP penetration in bone.•Post-yield loading significantly increases collagen denaturation in bone.•R-CHP localizes collagen damage at bone fracture surfaces.

R-CHP detects denatured collagen in bone without demineralization.

Confocal R-CHP fluorescence correlates with the well-established T-H assay.

High staining temperature improves R-CHP penetration in bone.

Post-yield loading significantly increases collagen denaturation in bone.

R-CHP localizes collagen damage at bone fracture surfaces.