Three-dimensional analysis of load-dependent changes in the orientation of dermal collagen fibers in human skin: A pilot study.

The availability of quantitative structural data on the orientation of collagen fibers is of crucial importance for understanding the behavior of connective tissues. These fibers can be visualized using a variety of imaging techniques, including second harmonic generation (SHG) microscopy. However, characterization of the collagen network requires the accurate extraction of parameters from imaging data. To this end, several automated processes have been developed to identify the preferred orientation of collagen fibers. Common methods include fast Fourier transforms and curvelet transforms, but these tools are mostly used to infer a single preferred orientation. The purpose of this pilot study was to develop an easy procedure for comprehensively comparing multiple human skin samples with the goal of analyzing load-dependent changes via SHG microscopy. We created a 3D model based upon 2D image stacks that provide fiber orientation data perpendicular and parallel to the plane of the epidermis. The SHG images were analyzed by CurveAlign to obtain angle histogram plots containing information about the multiple fiber orientations in each single image. Subsequently, contour plots of the angle histogram intensities were created to provide a useful visual plotting method to clearly show the anomalies in the angle histograms in all samples. Our results provided additional details on how the collagen network carries a load. In fact, analysis of SHG images indicated that increased stretch was accompanied by an increase in the alignment of fibers in the loading direction. Moreover, these images demonstrated that more than one type of preferred orientation is present. In particular, the 3D network of fibers appears to have two preferred orientations in the planes both perpendicular and parallel to the plane of the epidermis.