In situ multi-level analysis of viscoelastic deformation mechanisms in tendon collagen H.S. Gupta a , J. Seto b , S. Krauss b , P. Boesecke c , H.R.C. Screen a, * a School of Engineering and Materials Science, Queen Mary, University of London, Mile End Road, London E1 4NS, UK b Department of Biomaterials, Max Planck Institute of Colloids and Interfaces, D-14476 Potsdam-Golm, Germany c Beamline ID2, European Synchrotron Radiation Facility, F-38043 Grenoble, France article info Article history: Received 1 July 2009 Received in revised form 26 September 2009 Accepted 5 October 2009 Available online 12 October 2009 Keywords: Stress relaxation Small angle X-ray scattering Confocal microscopy Fibril strain Fibre strain Micromechanical testing abstract Tendon is a hydrated multi-level fibre composite, in which time-dependent behaviour is well established. Studies indicate significant stress relaxation, considered important for optimising tissue stiffness. How- ever, whilst this behaviour is well documented, the mechanisms associated with the response are largely unknown. This study investigates the sub-structural mechanisms occurring during stress relaxation at both the macro (fibre) and nano (fibril) levels of the tendon hierarchy. Stress relaxation followed a two-stage exponential behaviour, during which structural changes were visible at the fibre and fibril lev- els. Fibril relaxation and fibre sliding showed a double exponential response, while fibre sliding was clearly the largest contributor to relaxation. The amount of stress relaxation and sub-structural reorga- nisation increased with increasing load increments, but fibre sliding was consistently the largest contrib- utor to stress relaxation. A simple model of tendon viscoelasticity at the fibril and fibre levels has been developed, capturing this behaviour by serially coupling a Voigt element (collagen fibril), with two Max- well elements (non-collagenous matrix between fibrils and fibres). This multi-level analysis provides a first step towards understanding how sub-structural interactions contribute to viscoelastic behaviour. It indicates that nano- and micro-scale shearing are significant dissipative mechanisms, and the kinetics of relaxation follows a two-stage exponential decay, well fitted by serially coupled viscoelastic elements. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Tendon functions to transfer muscle forces to the skeleton as efficiently as possible, enabling minimal energy loss during load transfer while enabling enough extension to prevent injury be- tween the stiff bone and compliant muscle (Ker, 2007). Typical of many vertebrate connective tissues, it is highly hydrated at around 70% water (w/v), composed predominantly of collagen and proteo- glycans, and has an ordered hierarchical structure from the molec- ular to the organ level (Kastelic et al., 1978). While not all elements in the structural hierarchy are fully understood, the structural lev- els typically described (and used in the current study) are formed from angstrom-scale collagen molecules, tightly bound by collagen crosslinks to make nano-scale fibrils, which aggregate in turn to make micro-scale fibres, fascicles, and finally a tendon (Screen et al., 2004a). Each structural level is believed to act effectively as a fibre composite, with non-collagenous matrix components be- tween fibrils, fibres and fascicles acting to bind together the mate- rial, and assist in load transfer between the collagen units (Fratzl, 2003). Whilst the gross mechanical characteristics of tendon are well documented, the role of the structure in facilitating and controlling both static and time-dependent mechanical behaviour is less clear. The viscoelastic nature of tendon has been well described by a number of authors, and a high degree of stress relaxation has been reported (Kubo et al., 2002; Lynch et al., 2003; Woo et al., 1993). Viscoelastic properties are important for optimising tissue stiffness under different loading regimes and providing some damping to the loading response (Paxton and Baar, 2007). It is generally be- lieved that the non-collagenous matrix components of tendon are responsible for its viscoelastic nature, as a consequence of their high affinity with water (Elliott et al., 2003). Fluid flow and friction between matrix components have both been implicated (Yin and Elliott, 2004), and there has been considerable interest in model- ling the behaviour to try and improve understanding of the tissue response during stress relaxation (Fung, 1993; Sarver et al., 2003; Woo et al., 1993). In this context, Buehler (Buehler, 2006) has pio- neered a novel ab initio modelling approach to the mechanics of the collagen fibril, an especially promising route which reveals new nanostructural phenomena like localized deformation pulses. However, from a structural perspective, the relative contribution of different hierarchical components to mechanics remains unclear especially at the fibril and fibre level, with previous modelling 1047-8477/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.jsb.2009.10.002 * Corresponding author. Fax: +44 (0) 20 8983 3052. E-mail address: H.R.C.Screen@qmul.ac.uk (H.R.C. Screen). Journal of Structural Biology 169 (2010) 183–191 Contents lists available at ScienceDirect Journal of Structural Biology journal homepage: www.elsevier.com/locate/yjsbi