ARTICLE |
Correspondence to: Cornelis J.F. Van Noorden, Dept. of Cell Biology and Histology, Academic Medical Center, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands. E-mail: c.j.vannoorden@amc.uva.nl
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Summary |
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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.
(J Histochem Cytochem 50:14691474, 2002)
Key Words: image analysis, quantitative microscopy, picrosirius red staining, collagen orientation, dermis, wound closure
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Introduction |
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CONNECTIVE TISSUE consists for the most part of collagen fibers. Collagen provides connective tissue with a unique combination of flexibility and tensile strength but is not very elastic (
Quantitative analyses of collagen have been described by
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Materials and Methods |
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Stretching of Piglet Skin
In (plastic) surgery, stretching of skin is frequently necessary to close large wounds. One of the methods is based on the use of the Sure Closure skin stretching device (Fig 1). The technique has been described by
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Sampling of Biopsies
At the beginning of the experiment and after 30 min of skin stretching, biopsies were taken at 0.5 cm from the wound margin. Biopsies had a size of 3 x 10 x 10 mm. After sampling of a biopsy specimen, it was placed on cork using water-soluble glue and the specimen was embedded in a solution of 7% gelatin (Merck; Darmstadt, Germany) in distilled water in a mold (
Sectioning and Staining of Sections
To keep the structure of the dermis intact during sectioning and to visualize collagen fibers separately, optimal section thickness was determined by varying section thickness between 3 and 30 µm. After determination of the optimal section thickness, staining methods were tested to specifically stain collagen fibers while other structures in the dermis were stained as little as possible. It is essential for image analysis to optimize contrast between collagen fibers and background (
Sections of the biopsy specimens were cut in a cryostat (Bright; Huntingdon, UK) at a low but constant speed to ensure constant section thickness at -25C. Sections were captured on glass slides and kept at -25C. A series of cryostat sections (3 and 5 µm thick) was also captured on adhesive tape (Scotch Tape 800; 3 M, Leiden, The Netherlands) before the sections were adhered to glass slides as described by
The following staining methods have been tested to select the method that stains collagen fibers with optimal contrast: Giemsa, Shoobridge, hematoxylin-eosin (H&E), Mallory, van Gieson, and picrosirius red. The picrosirius red-stained sections were analyzed with epipolarization microscopy according to
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Quantitative Analysis of the Length of Collagen Fibers in the Plane of Sections
Digitally captured images of sections were analyzed with the use of the image analysis software NIH Image (version 1.57; by Wayne Rassband and available via internet from http://rsb.info.nih.gov). The procedure of image analysis to quantitatively determine the length of collagen fibers in the plane of sections as a parameter for orientation of the fibers consisted of the following steps: (a) a gray level was determined automatically as threshold, to produce a binary image in which background is colored black and collagen fibers are colored white (Fig 2C). An iterative testing technique, based on a histogram that divides the data into two populations (
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Results |
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Preparation of cryostat sections of piglet skin biopsies was hampered by the fat layer underneath the dermis, which was sticky at 25C. The only way to maintain the integrity of epidermis and dermis in the sections during preparation was sectioning of the biopsies in the direction shown in Fig 3. In this way, sections included epidermis, dermis, and fat layer. We decided to cut sections at constant low speed with a thickness of 7 µm. These sections showed no artifacts and produced a good contrast between collagen fibers and background. Thinner sections caused destruction of tissue, whereas sections that were too thick did not allow discrimination of individual collagen fibers against the background after the various staining procedures. Sections that were captured on adhesive tape after sectioning were comparable in quality to sections that were captured directly on glass. A disadvantage of the use of tape was that the glue dissolved in acids that are present in a number of staining solutions, such as that of picrosirius red. Therefore, capturing of sections on tape was not performed. Sections cut from stretched skin did not retract or shrink because the biopsy specimens were excised during the period of rest when stretching forces were not exerted on the skin. The width and length of the sections corresponded with those of the face of the frozen tissue blocks.
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Testing of the various staining methods to obtain optimal contrast between collagen fibers and background had the following results. The Giemsa and H&E methods were not specific enough for collagen (Fig 4A). Collagen fibers could barely be discriminated. The Shoobridge, Mallory, and van Gieson staining methods were far better in that aspect. It was possible to discriminate collagen fibers in the sections. Unfortunately, these staining methods had the disadvantage that too low a contrast was obtained between collagen fibers and other structures in the sections.
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In addition to collagen fibers, glands and muscles were stained, so that no specific segmentation of collagen fibers could be performed. The picrosirius red staining method in combination with epipolarization microscopy appeared to be the most suitable method to visualize collagen fibers with high contrast. The unique aspect of the picrosirius red staining is that dye molecules bind in a specific way to collagen fibers, which enhances double refraction of the fibers. When sections stained with picrosirius red were analyzed by epipolarization microscopy, collagen fibers reflected clearly against a black background (Fig 4B).
The first step in the analysis of the digital images of picrosirius red-stained sections (Fig 2B) was to automatically determine a gray level as threshold to obtain binary images (Fig 2C). This step optimized contrast between collagen fibers and background and reduced misidentification due to incorrect positioning of collagen fibers in the plane of the sections. This misidentification can be recognized in Fig 2B as dark gray objects. After enumeration of the objects in the image (Fig 2D), best-fitting ovals surrounding each object were calculated. The major advantage of using the best-fitting oval, instead of, e.g., the circumference of the fibers, is the possibility of excluding the influence of irregularities in the contours of fibers and small branches from the results.
To test the procedure in an experimental setting, we used skin after 0 and 30 min of skin stretching. The collagen alignment indices are shown in Table 1 for five piglets. Control skin had a mean collagen alignment index of 76 ± 15. The mean collagen alignment index of stretched skin (30 min) was 132 ± 36, which was significantly different from the control skin (p<0.005). These quantitative data and their differences were in agreement with qualitative microscopic analysis of the sections (Fig 2A and Fig 2B).
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Discussion |
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The picrosirius red staining method, in combination with epipolarization microscopy as described by
The collagen alignment index appeared to be a good parameter to express changes in orientation of collagen fibers. The mean value in control skin was significantly lower than that in stretched skin, which was in agreement with data obtained with qualitative microscopic inspection. By using the quantitative method described here, it was possible to collect valid data on the orientation of collagen and changes in the orientation due to mechanical skin stretching (
Collagen fibers became rapidly orientated in the direction of the force exerted on the fibers. The molecular processes that are the basis of these rapid changes in orientation of ECM components are unknown but will be the subject of further studies. Understanding of these processes not only has histological importance but also is of major importance for the (plastic) surgeon. Knowledge about mechanical properties of skin helps the (plastic) surgeon to predict and optimize results of different treatments, e.g., to close large wounds.
Other quantitative methods to establish changes in the orientation of collagen have been described (
In conclusion, the present method is a relatively simple tool for the quantitative determination of orientation of collagen fibers in combination with structural information of connective tissue. It can be applied to study dynamics in the ECM induced by, e.g., mechanical stress. Moreover, the present quantitative method can be used for determination of the orientation of any other structural element in a tissue so long as a representative binary image can be created.
Received for publication November 26, 2001; accepted May 29, 2002.
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