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.

Overuse injuries to dense collagenous tissues are common, but their etiology is poorly understood. The predominant hypothesis that micro-damage accumulation exceeds the rate of biological repair is missing a mechanistic explanation. Here, we used collagen hybridizing peptides to measure collagen molecular damage during tendon cyclic fatigue loading and computational simulations to identify potential explanations for our findings. Our results revealed that triple-helical collagen denaturation accumulates with increasing cycles of fatigue loading, and damage is correlated with creep strain independent of the cyclic strain rate. Finite-element simulations demonstrated that biphasic fluid flow is a possible fascicle-level mechanism to explain the rate dependence of the number of cycles and time to failure. Molecular dynamics simulations demonstrated that triple-helical unfolding is rate dependent, revealing rate-dependent mechanisms at multiple length scales in the tissue. The accumulation of collagen molecular denaturation during cyclic loading provides a long-sought “micro-damage” mechanism for the development of overuse injuries.

In vitro studies of cell-matrix interactions and the engineering of replacement materials for collagenous connective tissues require biocompatible scaffolds that replicate the high collagen density (15-25%/wt), aligned fibrillar organization, and anisotropic mechanical properties of native tissues. However, methods for creating scaffolds with these characteristics are currently lacking. We developed a new apparatus and method to create high density, aligned, and porous collagen scaffolds using a biaxial compression with porogens technique. These scaffolds have a highly directional structure and mechanical properties, with the tensile strength and modulus up to 100 times greater in the direction of alignment. We also demonstrated that the scaffolds are a suitable material for cell culture, promoting cell adhesion, viability, and an aligned cell morphology comparable to the cell morphology observed in native aligned tissues.

The function of ligaments and tendons is to support and transmit loads applied to the musculoskeletal system. These tissues are often able to perform their function for many decades; however, connective tissue disease and injury can compromise ligament and tendon integrity. A range of protein and non-protein constituents, combined in a complex structural hierarchy from the collagen molecule to the tissue and covering nanometer to centimeter length scales, govern tissue function, and impart characteristic non-linear material behavior. This review summarizes the structure of ligaments and tendons, the roles of their constituent components for load transfer across the hierarchy of structure, and the current understanding of how damage occurs in these tissues. Disease and injury can alter the constituent make-up and structural organization of ligaments and tendons, affecting tissue function, while also providing insight to the role and interactions of individual constituents. The studies and techniques presented here have helped to understand the relationship between tissue constituents and the physical mechanisms (e.g., stretching, sliding) that govern material behavior at and between length scales. In recent years, new techniques have been developed to probe ever smaller length scales and may help to elucidate mechanisms of load transfer and damage and the molecular constituents involved in the in the earliest stages of ligament and tendon damage. A detailed understanding of load transfer and damage from the molecular to the tissue level may elucidate targets for the treatment of connective tissue diseases and inform practice to prevent and rehabilitate ligament and tendon injuries. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

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