Extrafibrillar diffusion and intrafibrillar swelling at the nanoscale are associated with stress relaxation in the soft collagenous matrix tissue of tendons

The mechanical behaviour of hierarchically structured soft biological tissues like tendon and cartilage shows time-dependent properties. The origin of this phenomenon is undoubtedly related to the nano- and microscale levels of structural hierarchy, but the exact mechanism is not known. Understanding this phenomenon could help us understand normal physiological tendon mechanics and how these alter with tendon degeneration, inflammation or disease. Here we measure the micro- and nanoscale structural changes in tendons during stress relaxation, using a multi-scale strain imaging method (combining confocal scanning microscopy and synchrotron small angle X-ray scattering) together with in situ mechanical testing. We tracked both the transverse (fibre swelling) as well as axial (fibre elongation) strain in both the microscale fibres (similar to 50 mu m diameter) and the nanoscale fibrils (similar to 100 nm diameter). We find that macroscopic stress relaxation is accompanied by a transverse expansion of nano-fibrils together with an (opposite) reduction of diameter in the micron-scale fibres. The expansion of the fibrils at the nanoscale is more than that required for volume conservation, suggesting a stress-induced diffusion of free fluid molecules from the extrafibrillar to intrafibrillar space during stress relaxation. We propose a simple diffusion thinning mechanism whereby the proteoglycans gel layer coating the fibrils releases loosely bound water molecules upon stress-induction. The simultaneous diffusion thinning and associated water diffusion from extra- to intra-fibrillar compartments is proposed to be the driving mechanism for time-dependent behaviour in hierarchical connective tissues like tendon.