A Quantitative Method to Determine the Orientation of Collagen Fibers in the Dermis

Abstract

We have developed a quantitative microscopic method to determine changes in the orientation of collagen fibers in the dermis resulting from mechanical stress. The method is based on the use of picrosirius red-stained cryostat sections of piglet skin in which collagen fibers reflect light strongly when epipolarization microscopy is used. Digital images of sections were converted into binary images that were analyzed quantitatively on the basis of the length of the collagen fibers in the plane of the section as a measure for the orientation of the fibers. The length of the fibers was expressed in pixels and the mean length of the 10 longest fibers in the image was taken as the parameter for the orientation of the fibers. To test the procedure in an experimental setting, we used skin after 0 and 30 min of skin stretching. The orientation of the fibers in sections of control skin differed significantly from the orientation of fibers in sections of skin that was stretched mechanically for 30 min [76 +/- 15 (n=5) vs 132 +/- 36 (n=5)]. The method described here is a relatively simple way to determine (changes in) the orientation of individual collagen fibers in connective tissue and can also be applied for analysis of the orientation of any other structural element in tissues so long as a representative binary image can be created.

Deep Tissue Injury in Development of Pressure Ulcers: A Decrease of Inflammasome Activation and Changes in Human Skin Morphology in Response to Aging and Mechanical Load

“…To further analyze changes occurring in dermis of young and aged individuals in response to loading we utilized a quantitative microscopic method to assess changes in the orientation of collagen fibers using a protocol adapted from Noorlander et al [33]. To quantitatively determine the orientation of collagen fibers in the dermis we converted digital images of sections into binary images and analyzed them on the basis of the measured length of the collagen fibers in the plane of the section as a measure for the fibers orientation [33]. The orientation of collagen fibers in sections of aged skin loaded for 2 and 4 hrs differed significantly when their length was measured compared to control, unloaded, aged skin ( Figure 2, C, D ).…”

“…The slides were mounted using Fluorescein-FragEL™ Mounting Media. Digitally captured picrosirius red stained images of sections were analyzed with ImageJ (version 1.47; http://rsbweb.nih.gov/ij/) using a protocol adapted from Noorlander et al [33]. Briefly, images were processed using a binary filter, resulting fiber outlines were then fitted with ellipses, then mean length of the major axis was then determined as a measurement for orientation of the bundles of aligned collagen fibers in the plane of the section [33].The mean value of this length parameter was based on three images per section, with a total of 15 specimens per age group, in three serial sections of a specimen was used as a collagen alignment index; the collagen alignment indices of control and loaded skin were compared and differences were analyzed.…”

Clinical and histopathological evaluation of collagen fiber orientation in patients with oral submucous fibrosis

“…The collagen fibril assembly and collagen fiber bundle formation take place, outside the cell, in inlets formed between fibroblast cell processes in a precise configuration. [16] The alignment of cells and collagen fibers is a result of the forces generated by the cell-mediated gel contraction. [12] And in scars, the unidirectional alignment of collagen fibers occurs in as a sequence of mechanical stress with the fibers getting oriented in the direction of force exerted on the fibers.…”

Qualitative and quantitative evaluation of total and types I and III collagens in patients with ventral hernias

“…Many scientific studies have shown a direct relationship between hernias and a smaller amount of type I and type III collagens in this anatomical region [3,15,24].…”