Combining nano-physical and computational investigations to understand the nature of "aging" in dermal collagen

Abstract

The extracellular matrix of the dermis is a complex, dynamic system with the various dermal components undergoing individual physiologic changes as we age. Age-related changes in the physical properties of collagen were investigated in particular by measuring the effect of aging, most likely due to the accumulation of advanced glycation end product (AGE) cross-links, on the nanomechanical properties of the collagen fibril using atomic force microscope nano-indentation. An age-related decrease in the Young's modulus of the transverse fibril was observed (from 8.11 to 4.19 GPa in young to old volunteers, respectively, P<0.001). It is proposed that this is due to a change in the fibril density caused by age-related differences in water retention within the fibrils. The new collagen-water interaction mechanism was verified by electronic structure calculations, showing it to be energetically feasible.

Keywords: advanced glycation end products; aging; atomic force microscopy; collagen; nanomechanics; nanotechnology.

Figure 1

Age-related changes to components found within the dermal/epidermal ECM due to intrinsic and extrinsic aging processes. Abbreviations: AGE, advanced glycation end product; ECM, extracellular matrix; GAG, glycosaminoglycan; HLA, hyaluronic acid; LOX, lysyl oxidase; MMP, matrix metalloproteinase; RAGE, receptor for advanced glycation end products; ROS, reactive oxygen species; UV, ultraviolet.

Figure 2

Products formed from for enzymatic and nonenzymatic covalent cross-linking of collagen. Note: Data from Saito et al and Monnier. Abbreviations: LOX, lysyl oxidase; ROS, reactive oxygen species; GOLA, glyoxal lysine amide; GODIC, glyoxal imidazolimine cross-link; GOLD, glyoxal lysine dimer; DOLD, deoxyglucosone lysine dimer; MOLD, methylglyoxal lysine dimer; DOGDIC, deoxyglucosone imidazolium cross-link; MODIC, methylglyoxal imidazonlimine cross-link; HLNL, hydroxylysinonorleucine; DHLNL, dihydroxylysinonorleucine; LNL, lysinonorleucine; LKLN, lysino-5-ketonorleucine; HLKLN, hydroxylysino-5-kenonorleucine.

Figure 3

Histology sections stained in hematoxylin and eosin (A, C) and Pico Sirius Red (B, D) for a 28-year-old (A, B) and an 82-year-old volunteer (C, D), respectively.

Figure 4

SEM images of volunteers aged <32 years (A, B). AFM images of volunteers aged <32 years (C, D). SEM images of volunteers aged >60 years (E–G). AFM image of volunteers aged >60 years (H). Abbreviations: AFM, atomic force microscopy; SEM, scanning electron microscopy.

Figure 5

Nanoindentation of collagen fibrils. Notes: Indentation of a fibril in radial direction (A). Distribution of Young’s modulus for hydrated collagen fibrils for a young volunteer (B). Distribution of Young’s modulus for hydrated collagen fibrils for an old volunteer (C). Distribution of Young’s modulus for hydrated collagen fibrils for a young volunteer (D). Distribution of Young’s modulus for dehydrated collagen fibrils for an old volunteer (E). Relationship between Young’s modulus of dehydrated fibrils and age (F). Abbreviation: E, Young’s modulus.

Figure 6

I–VI show various optimized electronic structure calculations of glucosepane in coordination with a single water molecule. Intermolecular distance between the hydrogen bond acceptors and donors are labeled appropriately. Aliphatic side chain tails have been removed for clarity.

Figure 7

Illustrations for the three known modes of water interaction with collagen molecules: (A) covalently bonded to the molecule (a, green), water bridges between molecules (b, purple) and free-flowing water (c, blue). (B) The proposed mode of water interaction: water retention caused by glucosepane accumulation (d). (C) Proposed mechanism of water flow within the collagen fibril caused by nano-indentation.