Quantification of age-related changes in the structure and mechanical function of skin with multiscale imaging

Abstract

The mechanical properties of skin change during aging but the relationships between structure and mechanical function remain poorly understood. Previous work has shown that young skin exhibits a substantial decrease in tissue volume, a large macro-scale Poisson's ratio, and an increase in micro-scale collagen fiber alignment during mechanical stretch. In this study, label-free multiphoton microscopy was used to quantify how the microstructure and fiber kinematics of aged mouse skin affect its mechanical function. In an unloaded state, aged skin was found to have less collagen alignment and more non-enzymatic collagen fiber crosslinks. Skin samples were then loaded in uniaxial tension and aged skin exhibited a lower mechanical stiffness compared to young skin. Aged tissue also demonstrated less volume reduction and a lower macro-scale Poisson's ratio at 10% uniaxial strain, but not at 20% strain. The magnitude of 3D fiber realignment in the direction of loading was not different between age groups, and the amount of realignment in young and aged skin was less than expected based on theoretical fiber kinematics affine to the local deformation. These findings provide key insights on how the collagen fiber microstructure changes with age, and how those changes affect the mechanical function of skin, findings which may help guide wound healing or anti-aging treatments.

Keywords: Biomechanics; Intrinsic Aging; Multi-scale; Multiphoton Microscopy; Second Harmonic Generation; Skin.

Fig. 1

Based on a reference unloaded configuration at both the 2D macro-scale (A; left) and 3D micro-scale (A; right), multiscale tissue kinematics were quantified at 10% (B) and 20% (C) Green strain in the direction of loading (ε_xx). The representative images shown were taken from young mouse skin. The white polygons correspond to tracked features and arrows represent the magnitude and direction of principal strains

Fig. 2

SHG and TPEF images of the undeformed microstructure of young (A; left) and aged (A; right) skin allow for visualization of different dermal structures, including dermal collagen (via SHG), hair follicles (via TPEF; red circles), and collagen crosslink autofluorescence (via TPEF). Using the SHG images (A; top), a trained CNN segmented collagen-positive pixels (A; middle), and collagen-positive masks were applied to SHG and TPEF (A; bottom) images. Within collagen-positive regions, the average collagen SHG intensity (B), collagen fiber directional variance (C), collagen-positive pixel density (D), and the average collagen crosslink TPEF intensity (E) were quantified. Although there was no significant difference in the intensity of the collagen SHG signal (B) or collagen-positive pixel density (D) with increased age, the fibers in aged tissue showed significantly higher non-enzymatic crosslink fluorescence (E) and were more randomly organized (C). All measurements were made in the undeformed configuration. N = 6 for both age groups, * represents p < 0.05

Fig. 3

The macro-scale mechanical response during uniaxial tensile stretch indicates aged tissue is less stiff and has less inward contraction at low strains. (A) Stress–strain curves for each sample were generated from the forces at static equilibrium measured during imaging. (B) Aged tissue had a lower Young’s modulus than young tissue that was significantly different at peak stiffness (Max E). (C) At 10% strain, aged tissue had significantly less macro-scale contraction in the direction orthogonal to the imposed stretch. N = 6 for both age groups, * represents p < 0.05

Fig. 4

Micro-scale volume ratio was measured for young (A; top) and aged (A; bottom) tissue from features that were tracked within the SHG image volumes (white polygons). Scalebar represents 100 μm. A substantial decrease in volume ratio with increasing stretch was observed for both age groups. (B) Volume reduction was generally greater for young samples and significantly so for 10% micro-scale Green strain (ε_xx) (C). N = 6 for both age groups, * represents p < 0.05

Fig. 5

At each 1 mm increment of displacement, the 3D directional variance of collagen fibers was computed from all collagen-positive pixels within an image volume. During stretching, the collagen fibers in both age groups realigned towards the direction of stretch (A), and slightly greater realignment occurred in aged samples at maximum Young’s modulus (B). The measured realignment was also found to be significantly overpredicted when compared to theoretical realignment assuming an affine relationship with tissue deformation (C). N = 6 for both age groups