The tissue organisation of dermal collagen is gaining importance as a contributing factor both in development and ageing, as well as in skin maturation processes. In this work we aim to study different representative parameters of this structural organisation in 45 human skin samples of assorted ages, by means of image analysis. The variation of these parameters on the basis of age was assessed using several regression models (linear, quadratic and cubic). The area occupied by collagen was significantly reduced as a function of age in the papillary dermis (R 2 = 0.437, P < 0.0001), as well as the thickness of the collagen bundles (R 2 = 0.461, P < 0.0001), following statistical models of cubic and quadratic regression, respectively. The width of the papillary dermis increased in a significant manner over a linear regression model (R 2 = 0.26, P < 0.0001). In the reticular dermis, the cubic regression indicated a significant decline (R 2 = 0.392, P = 0.002) of the area filled with collagen according to the age. Both collagen thickness and bundle orientation parameters fit a quadratic regression over the age in a significant way (R 2 = 0.433 and R 2 = 0.334, respectively, both P < 0.0001). The width of the reticular dermis followed also a significant quadratic distribution according to age (R 2 = 0.193, P = 0.011). These parameters could partially explain the lifelong functional changes taking place in the skin and propose a baseline providing a useful entry point for future investigation.

Measurement of collagen bundle orientation in histopathological samples is a widely used and useful technique in many research and clinical scenarios. Fourier analysis is the preferred method for performing this measurement, but the most appropriate staining and microscopy technique remains unclear. Some authors advocate the use of Haematoxylin-Eosin (H&E) and confocal microscopy, but there are no studies comparing this technique with other classical collagen stainings. In our study, 46 human skin samples were collected, processed for histological analysis and stained with Masson's trichrome, Picrosirius red and H&E. Five microphotographs of the reticular dermis were taken with a 200× magnification with light microscopy, polarized microscopy and confocal microscopy, respectively. Two independent observers measured collagen bundle orientation with semiautomated Fourier analysis with the Image-Pro Plus 7.0 software and three independent observers performed a semiquantitative evaluation of the same parameter. The average orientation for each case was calculated with the values of the five pictures. We analyzed the interrater reliability, the consistency between Fourier analysis and average semiquantitative evaluation and the consistency between measurements in Masson's trichrome, Picrosirius red and H&E-confocal. Statistical analysis for reliability and agreement was performed with the SPSS 22.0 software and consisted of intraclass correlation coefficient (ICC), Bland-Altman plots and limits of agreement and coefficient of variation. Interrater reliability was almost perfect (ICC > 0.8) with all three histological and microscopy techniques and always superior in Fourier analysis than in average semiquantitative evaluation. Measurements were consistent between Fourier analysis by one observer and average semiquantitative evaluation by three observers, with an almost perfect agreement with Masson's trichrome and Picrosirius red techniques (ICC > 0.8) and a strong agreement with H&E-confocal (0.7 < ICC < 0.8). Comparison of measurements between the three techniques for the same observer showed an almost perfect agreement (ICC > 0.8), better with Fourier analysis than with semiquantitative evaluation (single and average). These results in nonpathological skin samples were also confirmed in a preliminary analysis in eight scleroderma skin samples. Our results show that Masson's trichrome and Picrosirius red are consistent with H&E-confocal for measuring collagen bundle orientation in histological samples and could thus be used indistinctly for this purpose. Fourier analysis is superior to average semiquantitative evaluation and should keep being used as the preferred method.