Ultrastructural viscoelasticity of fibrillar collagen identified by AFM Nano-Rheometry and direct indentation

Soft tissues exhibit predominantly time-dependent mechanical behavior critical for their biological function in organs like the lungs and aorta, as they can deform and stretch at varying rates depending on their function. Collagen type I serves as the primary structural component in these tissues. The viscoelastic characteristics of such tissues, stemming from diverse energy dissipation mechanisms across various length scales, remains poorly characterized at the nanoscale. Prior experimental investigations have predominantly centered on analyzing tissue responses largely attributed to interactions between cells and fibers. Despite many studies on tissue viscoelasticity from scaffolds to single collagen fibrils, the time-dependent mechanics of collagen fibrils at the sub-fibrillar level remain poorly understood. This pioneering study employs atomic force microscopy (AFM) nano-rheometry and indentation testing to examine the viscoelastic characteristics of individual collagen type I fibrils at the ultrastructural level within distinct topographical zones, specifically focusing on gap and overlap regions. Our investigation has unveiled that collagen fibrils display a viscoelastic response that replicates the mechanical behavior of the tissue at the macroscale. Further, our findings suggest a distinct viscoelastic behavior between the gap and overlap regions, likely stemming from variances in molecular organization and cross-linking modalities within these specific sites. The results of our investigation provide unequivocal proof of the temporal dependence of mechanical properties and provides unique data to be compared to atomistic models, laying a foundation for refining the precision of macroscale models that strive to capture tissue viscoelasticity across varying length scales. STATEMENT OF SIGNIFICANCE: Soft tissues such as the lungs and aorta depend on collagen to stretch and perform their functions, which involve continuous and dynamic deformation. Although these tissues are known to exhibit viscoelastic behavior, the mechanisms behind this at the fine scale of individual collagen fibril ultrastructure are not well understood. In this study, we used AFM nano-rheometry and direct indentation to be the first to directly measure the viscoelastic properties of collagen at the ultrastructural level. We discovered that single fibrils show time-dependent behavior similar to that of whole tissues, with distinct mechanical differences between regions likely due to variations in molecular organization and bonding. These insights advance our understanding of tissue mechanics and contribute to more accurate multi-scale modeling.