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.

Angiogenesis is necessary for wound healing, tumorigenesis, implant inosculation, and homeostasis. In each situation, matrix structure and mechanics play a role in determining whether new vasculatures are able to establish transport to new or hypoxic tissues. Neovessel growth and directional guidance are sensitive to 3D matrix anisotropy and density, although the individual and integrated roles of these matrix features have not been fully recapitulated in vitro. We developed a tension-based method to align 3D collagen constructs seeded with microvessel fragments in matrices of three levels of collagen fibril anisotropy and two levels of collagen density. The extent and direction of neovessel growth from the parent microvessel fragments increased with matrix anisotropy and decreased with density. The proangiogenic effects of anisotropy were attenuated at higher matrix densities. We also examined the impact of matrix anisotropy in an experimental model of neovessel invasion across a tissue interface. Matrix density was found to dictate the success of interface crossing while interface curvature and fibril alignment were found to control directional guidance. Our findings indicate that complex configurations of matrix density and alignment can facilitate or complicate the establishment or maintenance of vascular networks in pathological and homeostatic angiogenesis. Further, we demonstrate a novel approach for tuning collagen anisotropy in thick constructs. This approach addresses gaps in tissue engineering and cell culture by supporting the inclusion of large multicellular structures in aligned constructs.

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The purpose of this study was to develop a microplate assay for quantifying denatured collagen by measuring the fluorescence of carboxyfluorescein bound collagen hybridizing peptides (F-CHP). We have shown that F-CHP binds selectively with denatured collagen, and that mechanical overload of tendon fascicles causes collagen denaturation. Proteinase K was used to homogenize tissue samples after F-CHP staining, allowing fluorescence measurement using a microplate reader. We compared our new assay to our previous image analysis method and the trypsin-hydroxyproline assay, which is the only other available method to directly quantify denatured collagen. Relative quantification of denatured collagen was performed in rat tail tendon fascicles subjected to incremental tensile overload, and normal and ostoeoarthritic guinea pig cartilage. In addition, the absolute amount of denatured collagen was determined in rat tail tendon by correlating F-CHP fluorescence with percent denatured collagen as determined by the trypsin-hydroxyproline assay. Rat tail tendon fascicles stretched to low strains (<7.5%) exhibited minimal denature collagen, but values rapidly increased at medium strains (7.5–10.5%) and plateaued at high strains (≥12%). Osteoarthritic cartilage had higher F-CHP fluorescence than healthy cartilage. Both of these outcomes are consistent with previous studies. With the calibration curve, the microplate assay was able to absolutely quantify denatured collagen in mechanically damaged rat tail tendon fascicles as reliably as the trypsin-hydroxyproline assay. Further, we achieved these results more efficiently than current methods in a rapid, high-throughput manner, with multiple types of collagenous tissue while maintaining accuracy.

The purpose of this study was to develop a microplate assay for quantifying denatured collagen by measuring the fluorescence of carboxyfluorescein bound collagen hybridizing peptides (F-CHP). We have shown that F-CHP binds selectively with denatured collagen, and that mechanical overload of tendon fascicles causes collagen denaturation. Proteinase K was used to homogenize tissue samples after F-CHP staining, allowing fluorescence measurement using a microplate reader. We compared our new assay to our previous image analysis method and the trypsin-hydroxyproline assay, which is the only other available method to directly quantify denatured collagen. Relative quantification of denatured collagen was performed in rat tail tendon fascicles subjected to incremental tensile overload, and normal and ostoeoarthritic guinea pig cartilage. In addition, the absolute amount of denatured collagen was determined in rat tail tendon by correlating F-CHP fluorescence with percent denatured collagen as determined by the trypsin-hydroxyproline assay. Rat tail tendon fascicles stretched to low strains (<7.5%) exhibited minimal denature collagen, but values rapidly increased at medium strains (7.5-10.5%) and plateaued at high strains (≥12%). Osteoarthritic cartilage had higher F-CHP fluorescence than healthy cartilage. Both of these outcomes are consistent with previous studies. With the calibration curve, the microplate assay was able to absolutely quantify denatured collagen in mechanically damaged rat tail tendon fascicles as reliably as the trypsin-hydroxyproline assay.Further, we achieved these results more efficiently than current methods in a rapid, highthroughput manner, with multiple types of collagenous tissue while maintaining accuracy.Keywords: collagen hybridizing peptide; denatured collagen; microplate assay; osteoarthritis; tendons and ligaments Lin et al.