Effect of aggrecan degradation on the nanomechanics of hyaluronan in extra-fibrillar matrix of annulus fibrosus: A molecular dynamics investigation

Highlights

• Tensile and Compressive tests conducted on a representative atomistic model of hyaluronan.

• To account for Aggrecan degradation, hydration levels are varied from 0 – 75% water by weight.

• Hydration, owing to presence of aggrecan, is a major contributing factor to the nanoscale mechanics of Hyaluronan.

• Formation of intramolecular H-bonds reduces the load transfer across the hyaluronan-water interface under tensile loading.

• Formation of hyaluronan-water H-bonds enhance the load transfer across the hyaluronan-water interface under compressive loading.

Abstract

Intervertebral Disc (IVD) Degeneration is one of the primary causes of low back pain among the adult population – the most significant cause being the degradation of aggrecan present in the extra-fibrillar matrix (EFM). Aggrecan degradation is closely associated with loss of water content leading to an alteration in the mechanical behaviour of the IVD. The loss in water content has a significant impact on the chemo-mechanical interplay of IVD biochemical constituents at the fundamental level. This work presents a mechanistic understanding of the effect of hydration, closely associated with aggrecan degradation, on the nanoscale mechanical behaviour of the hyaluronan present in the EFM of the Annulus Fibrosus. For this purpose, explicit three-dimensional molecular dynamics analyses of tensile and compressive tests are performed on a representative atomistic model of the hyaluronan present in the EFM. To account for the degradation of aggrecan, hydration levels are varied from 0 to 75% by weight of water. Analyses show that an increase in the hydration levels decreases the elastic modulus of hyaluronan in tension from ~4.6 GPa to ~2.1 GPa. On the other hand, the increase in hydration level increases the elastic moduli in axial compression from ~1.6 GPa in un-hydrated condition to ~6 GPa in 50% hydrated condition. But as the hydration levels increase to 75%, the elastic modulus reduces to ~3.5 GPa signifying a shift in load-bearing characteristic, from the solid hyaluronan component to the fluid component. Furthermore, analyses show a reduction in the intermolecular energy between hyaluronan and water, under axial tensile loading, indicating a nanoscale intermolecular debonding between hyaluronan and water molecules. This is attributed to the ability of hyaluronan to form stabilizing intra-molecular hydrogen bonds between adjacent residues. Compressive loading, on the other hand, causes intensive coiling of hyaluronan molecule, which traps more water through hydrogen bonding and aids in bearing compressive loads. Overall, study shows that hydration level has a strong influence on the atomistic level interactions between hyaluronan molecules and hyaluronan and water molecules in the EFM which influences the nanoscale mechanics of the Annulus Fibrosus.

Introduction

The intervertebral discs (IVDs) are soft cartilaginous tissues that lie between the vertebral bones in the spine, helping in transmitting load and motion through the spine. They consist of a fibrous outer jacket called the Annulus Fibrosus (AF), and inner gel part called Nucleus Pulposus (NP). The Annulus Fibrosus is mainly responsible for transferring bending and twisting motion through the spine by undergoing a combination of tensile, compressive and shear deformation. The Annulus is able to undergo such complex deformation mechanism because it has a very specialized, hierarchical and fibre-reinforced lamellated composite structure with fibres arranged in an angle-ply orientation (Raj, 2008a). The fibrillar part is mainly composed of collagen and the non-fibrillar part, commonly referred to as the extra-fibrillar matrix (EFM), is primarily composed of large aggregating proteoglycans, mostly Aggrecan, forming large conjugates with Hyaluronan (Urban and Roberts, 1995). The proteoglycans form a network of interconnected negatively charged macromolecules creating an imbalance of ion concentration between the tissue and the surrounding fluid. This creates an osmotic gradient primarily responsible for tissue hydration. Thus, the mechanical response of the AF can be deemed to be a combination of the mechanical response of the fibrillar part, the extra-fibrillar part and the osmotic pressure (Ehlers et al., 2009; Sun and Leong, 2004). A simplified schematic of the hierarchical structure of EFM is shown in Fig. 1.

Ageing, lifestyle-related disorders and abnormal loading conditions causes biochemical changes like degradation of aggrecan which causes a loss of fixed charges which causes a reduction in the water content in the IVD (Adams et al., 2000; Ahmed et al., 2012; Weiler et al., 2011). This significantly alters the mechanical response of the intervertebral disc. The alteration in the mechanical behaviour in the AF affects the mechanical response of the fibril as well as the extra-fibrillar components (Derby and Akhtar, 2015; Roughley et al., 2006; Singh et al., 2009). Existing studies have focused on the mechanical behaviour of healthy and degenerated in annulus fibrosus as a single entity (Fujita et al., 1997; O'Connell et al., 2012, 2009) and on the mechanical behaviour of the extra-fibrillar matrix with their dependence on age and location and nature of loads applied (Cortes et al., 2013; Cortes and Elliott, 2012). But, the chemo-mechanical interplay between the molecular components in the extra-fibrillar matrix and their role on the mechanics of a healthy and degenerated annulus fibrosus has not been properly established. Although existing explicit molecular dynamics (MD) based studies have contributed to the understanding of the mechanics of collagen fibrils at the molecular scale (Buehler, 2008, 2006; Gautieri et al., 2012, 2011, 2008), MD based analyses on the nanoscale mechanics of the EFM constituents have not been performed. Such analyses could bring forth important evidence regarding the role of mechanical interactions between the biomolecular constituents on the mechanics of the EFM in a healthy and degenerated annulus fibrosus.

Therefore, this work examines the effect of hydration, which is closely associated with aggrecan degradation, on the nanoscale mechanical behaviour of the hyaluronan component of the extra fibrillar matrix of annulus fibrosus under tension and compression using explicit three-dimensional molecular dynamics simulations. Furthermore, present study also analyzes the intramolecular and intermolecular interaction energies in conjunction with the mechanical behaviour to gain a comprehensive understanding of structural interaction behaviour between hyaluronan and water molecules during deformation. Such analyses could bring forth important understanding regarding the nanomechanical behavior of hyaluronan which has implications in gaining significant insights into the mechanistic effects of degenerative disorders in the IVD and will aid in developing therapies for its treatment.