Abstract

The skin is the largest organ in the body and is essential for protecting us from environmental stressors such as UV radiation, pollution, and pathogens. As we age, our skin undergoes complex changes that can affect its function, appearance, and health. These changes result from intrinsic (chronological) and extrinsic (environmental) factors that can cause damage to the skin’s cells and extracellular matrix. As higher-resolution microscopical techniques, such as Atomic Force Microscopy (AFM), are being deployed to support histology, it is possible to explore the biophysical properties of the dermal scaffold’s constituents, such as the collagen network. In this study, we demonstrate the use of our AFM-based quantitative nanohistology, performed directly on unfixed cryosections of 30 donors (female, Caucasian), to differentiate between dermal collagen from different age groups and anatomical sites. The initial 420 (10 × 10 μm2) Atomic Force Microscopy images were segmented into 42,000 (1 × 1 μm2) images before being classified according to four pre-defined empirical collagen structural biomarkers to quantify the structural heterogeneity of the dermal collagen. These markers include interfibrillar gap formation, undefined collagen structure, and registered or unregistered dense collagen fibrillar network with evident D-banding. The structural analysis was also complemented by extensive nanoindentation (∼1,000 curves) performed on individual fibrils from each section, yielding 30,000 indentation curves for this study. Principal Component Analysis was used to reduce the complexity of high-dimensional datasets. The % prevalence of the empirical collagen structural biomarkers between the papillary and reticular dermis for each section proves determinant in differentiating between the donors as a function of their age or the anatomical site (cheek or breast). A case of abnormal biological aging validated our markers and nanohistology approach. This case also highlighted the difference between chronological and biological aging regarding dermal collagen phenotyping. However, quantifying the impact of chronic and pathological conditions on the structure and function of collagen at the sub-micron level remains challenging and lengthy. By employing tools such as the Atomic Force Microscope as presented here, it is possible to start evaluating the complexity of the dermal matrix at the nanoscale and start identifying relevant collagen morphology which could be used toward histopathology standards.