Mechanical overload decreases the thermal stability of collagen in an in vitro tensile overload tendon model

Abstract

Musculoskeletal soft tissue injuries are very common, yet poorly understood. We investigated molecular-level changes in collagen caused by tensile overload of bovine tail tendons in vitro. Previous investigators concluded that tensile tendon rupture resulted in collagen denaturation, but our study suggests otherwise. Based on contemporary collagen biophysics, we hypothesized that tensile overload would lead to reduced thermal stability without change in the nativity of the molecular conformation. The thermal behavior of collagen from tail tendons ruptured in vitro at two strain rates (0.01 s(-1) and 10 s(-1)) was measured by differential scanning calorimetry (DSC). The 1,000-fold difference in strain rate was used since molecular mechanisms that determine mechanical behavior are thought to be strain rate-dependent. DSC revealed that the collagen in tensile overloaded tendons was less thermally stable by 3 degrees to 5 degrees C relative to undamaged controls and was not denatured since there was no change in enthalpy of denaturation. The decrease in thermal stability occurred throughout the overloaded regions, independent of rupture site, and was greater in specimens ruptured at the lower strain rate. The deformation mechanism apparently involves disruption of the lattice structure of the collagen fibrils and greatly increases the molecular freedom of the collagen molecules, leading to reduced thermal molecular stability and the previously reported increased proteolysis. This has important implications for understanding soft tissue injuries, disease etiology and treatment, and for developing tissue engineered products with improved durability.

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

“…While some evidence existed for damage at the molecular level accompanying damage at the fibril level, this was only suggested for highly damaged tissues and was presented as necessarily preceded by fibrillar disruption1521. However, our combined evidence of CHP binding under subfailure loading conditions (Figs 2, 3 and 5), CHP binding to intact fibril structures (Fig.…”

“…For example, collagen fibrils in ruptured20 or repeatedly overloaded tendons are more susceptible to proteolytic digestion by trypsin than collagen from unloaded tendons15161721. Since trypsin can efficiently digest non-triple-helical structure, this was considered a sign that mechanical overload may have caused unfolding of the triple-helical collagen molecules.…”

“…However, trypsin has been also reported to digest triple-helical collagen molecules by targeting their thermally unstable domain, if the collagen molecules are not assembled into the fibril structure2223. Therefore it is unclear whether the increased susceptibility to trypsin digestion in overloaded tendons was the result of damage to collagen molecules or to collagen fibrils15161721, although additional experiments involving differential scanning calorimetry and low temperature digestion seem to suggest molecular damage in some instances17.…”

“…To date, studies that examined mechanical damage to collagen fibrils in tendon were performed on severely damaged samples (ruptured, or with repeated sub-rupture overloading)151617202124, and it is unclear whether collagen denaturation plays a role in initiation of damage or it is merely a secondary outcome of the failure at the fibril, fiber and/or tissue levels. There is no direct and conclusive experimental evidence to indicate the extent or location of molecular level failure of collagen during moderate subfailure loading, or the mechanism of failure at the molecular level.…”

Differences in collagen cross-linking between the four valves of the bovine heart: a possible role in adaptation to mechanical fatigue

“…If the rate of the cellular synthesis and degradation of collagen is constant among all of these tissues then, at any given time, tissues experiencing higher loads will contain more damaged collagen than will tissues experiencing lower loads. Damage at the molecular level may increase molecular spacing, resulting in molecular destabilization via the "polymer-ina-box" theory (39,56,57). Our results suggest that leaflet tissues under higher in vivo loads are less thermally stable at the molecular level.…”