Advanced Glycation End-Products Reduce Collagen Molecular Sliding to Affect Collagen Fibril Damage Mechanisms but Not Stiffness

Abstract

Advanced glycation end-products (AGE) contribute to age-related connective tissue damage and functional deficit. The documented association between AGE formation on collagens and the correlated progressive stiffening of tissues has widely been presumed causative, despite the lack of mechanistic understanding. The present study investigates precisely how AGEs affect mechanical function of the collagen fibril – the supramolecular functional load-bearing unit within most tissues. We employed synchrotron small-angle X-ray scattering (SAXS) and carefully controlled mechanical testing after introducing AGEs in explants of rat-tail tendon using the metabolite methylglyoxal (MGO). Mass spectrometry and collagen fluorescence verified substantial formation of AGEs by the treatment. Associated mechanical changes of the tissue (increased stiffness and failure strength, decreased stress relaxation) were consistent with reports from the literature. SAXS analysis revealed clear changes in molecular deformation within MGO treated fibrils. Underlying the associated increase in tissue strength, we infer from the data that MGO modified collagen fibrils supported higher loads to failure by maintaining an intact quarter-staggered conformation to nearly twice the level of fibril strain in controls. This apparent increase in fibril failure resistance was characterized by reduced side-by-side sliding of collagen molecules within fibrils, reflecting lateral molecular interconnectivity by AGEs. Surprisingly, no change in maximum fibril modulus (2.5 GPa) accompanied the changes in fibril failure behavior, strongly contradicting the widespread assumption that tissue stiffening in ageing and diabetes is directly related to AGE increased fibril stiffness. We conclude that AGEs can alter physiologically relevant failure behavior of collagen fibrils, but that tissue level changes in stiffness likely occur at higher levels of tissue architecture.

Advanced glycation end-products: Mechanics of aged collagen from molecule to tissue

“…At the tissue level, ribose cross-linked specimens showed a clear loss of stress relaxation behavior, which is consistent with our previous studies [36,40] where AGEs were induced by methylglyoxal treatment, a substantially more reactive compound. This reduction in viscoelasticity can be attributed to changes at lower hierarchical levels, i.e.…”

“…Although the role of AGEs has been investigated mostly at the tissue level, a few studies reported AGE-related mechanical changes in collagen fibrils of tendon tissue. Our group [36] found a higher failure resistance in MGO treated collagen fibrils as well as a reduction in the lateral sliding of collagen molecules within fibrils, while fibril modulus remained unchanged. Svensson et al [21] observed a three-phase behavior pattern in the stress-strain curves of human collagen fibrils.…”

“…Advanced glycation end-products can dramatically affect collagen tissue viscoelasticity and molecular deformation [36]. On the basis of our early findings, the present study sought to clarify mechanisms behind these behaviors and verify that they occur in naturally aged, native tissue.…”

“…The ratio of intensities of the 2 nd and 3 rd order reflections was analyzed to assess changes in length of the overlap region and gap region during the ramp-to-failure experiments [36,39]. The control fibrils showed three distinct loading mechanism at the sub-fibrillar scale (Fig.…”

“…A fluorometric assay was carried out to assess the effects of the ribose treatment on the formation of AGEs in RTTFs and the different level of AGEs in tissues from human donors [36,53]. Ribose treated, ribose control fascicles and samples from human donors were vacuum dried (RTTF: 16 h, human: 30h) to determine the dry weight and then digested (RTTF: 16 h, human: 72h) at 65°C with 0.2 mg/ml papain (Sigma-Aldrich, P4762) in PBE buffer (100 mM phosphate, 10 mM EDTA, pH 6.5) [53].…”