Investigation of dermal collagen nanostructures in Ehlers-Danlos Syndrome (EDS) patients

Abstract

Ehlers-Danlos syndromes (EDS) represent a group of rare genetic disorders affecting connective tissues. Globally, approximately 1.5 million individuals suffer from EDS, with 10,000 reported cases in Canada alone. Understanding the histological properties of collagen in EDS has been challenging, but advanced techniques like atomic force microscopy (AFM) have opened up new possibilities for label-free skin imaging. This approach, which explores Type I collagen fibrils at the nanoscale, could potentially enhance EDS diagnosis and our knowledge of collagen type I-related connective tissue disorders. In the current study, we have employed AFM to examine ex-vivo skin biopsies from four individuals: one with classical EDS (cEDS), one with hypermobile EDS (hEDS), one with hEDS and Scleroderma (hEDS-Scleroderma), and one healthy control. Picrosirius red (PS) staining was used to highlight collagen differences in the samples. For each case, 14 images and 1400 force curves were obtained, with seven images and 700 force curves representing healthy collagen (PS-induced red staining) and the rest showcasing disrupted collagen (yellow staining). The results showed that PS staining was uniform throughout the control section, while cEDS and hEDS displayed localized areas of yellow staining. In the case of hEDS-Scleroderma, the yellow staining was widespread throughout the section. AFM images revealed irregular collagen fibrils in the disrupted, yellow-stained areas, contrasting with aligned and well-registered collagen fibrils in healthy, red-stained regions. Additionally, the study assessed the ability of non-AFM specialists to differentiate between healthy and disrupted collagen in AFM images, yielding substantial agreement among raters according to Fleiss's and Cohen's kappa scores (0.96 and 0.79±0.1, respectively). Biomechanical analysis revealed that normal healthy collagen exhibited a predominant population at 2.5 GPa. In contrast, EDS-affected collagen displayed subpopulations with lower compressive elastic modulus, indicating weaker collagen fibrils in EDS patients. Although these findings pertain to a limited number of cases, they offer valuable insights into the nanoscale collagen structure and biomechanics in individuals with EDS. Over time, these insights could be developed into specific biomarkers for the condition, improving diagnosis and treatment for EDS and related connective tissue disorders.

Fig 1. The histological sections of control, cEDS, hEDS, and hEDS-Scleroderma patients stained with H&E and PS.

(A). H&E-stained histological sections showing thick, organized collagen bundles for control skin and disordered, thin ones in the disrupted regions. The areas marked as *, **, and *** show the heterogeneous collagen density throughout the section for the sample obtained from hEDS, Scleroderma patient. (B). Histological sections from all four groups were stained with PS and imaged under polarized. Control samples showed red stains throughout the section, whereas cEDS and hEDS exhibited localized islets of yellow stained area. In the case of hEDS-Scleroderma, the yellow stain is widespread throughout the section. (C). Pie-chart distribution of the yellow/red stained areas ratio for each patient. Scale bars are 500μm.

Fig 2. Correlated AFM and PS-stained images, identifying intact and compromised regions within the skin samples.

(A) showcases selected AFM images from areas of PS-stained samples referred to as red on the right and yellow on the left. All fibrils in the red-stain area of all four cases were structurally homogeneous with long, aligned, and unidirectional collagen fibrils. In contrast, yellow-stained, disrupted areas exhibited random orientation and structural disorganization in the collagen matrix. (B)presents Complementary AFM images from the disrupted area (yellow stained) from cEDS, hEDS, and hEDS-Scleroderma samples, highlighting the random orientation of the collagen fibrils along with wrinkled, swollen, and amorphous collagen matrix (highlighted with stars).

Fig 3. Collagen fibrils’ Young’s moduli distributions of skin section’s healthy and disrupted area.

(A). Shows an unimodal distribution of Young modulus for control skin in healthy and disrupted regions. This peak shifts gradually to the smaller value, indicating weaker collagen when transiting from control samples to cEDS (indicated by a star). This shift is more pronounced when moving to hEDS and hEDS scleroderma samples. The red dotted line presents the healthy collagen distribution in the control group. (B). Summary table of the collagen Young’s moduli medians and their prevenances. At 0.05 level, all Younge modulus distributions between healthy and disrupted collagen are significant. Additionally, disrupted and healthy distribution within the same group is significant except for the control skin.