Journal of Histochemistry and Cytochemistry, Vol. 50, 1469-1474, November 2002, Copyright © 2002, The Histochemical Society, Inc.


ARTICLE

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

Maril L. Noorlandera, Paris Melisb, Ard Jonkera, and Cornelis J.F. Van Noordena
a Academic Medical Center, University of Amsterdam, Departments of Cell Biology and Histology, Amsterdam, The Netherlands
b Plastic, Reconstructive and Handsurgery, Amsterdam, The Netherlands

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


  Summary
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 50:1469–1474, 2002)

Key Words: image analysis, quantitative microscopy, picrosirius red staining, collagen orientation, dermis, wound closure


  Introduction
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 (Junqueira et al. 1993 ). In (plastic) surgery, research into the properties of skin is of major importance to optimize wound closure. In addition to wound healing and vascularization, tensile strength plays a major role. When mechanical tension is applied, skin appears to be able to stretch beyond its natural yield and this unique property provides opportunities for closure of large wounds (Gibson et al. 1965 ; Stark 1977 ; Hirshowitz et al. 1993 ). Because 85% of the dermis of the skin consists of collagen, this extracellular matrix (ECM) component is an important topic in studies of changes in the skin caused by mechanical stretching. Thus far, results of studies of the mechanical properties of collagen have been described mostly as qualitative histological observations (Gibson et al. 1965 ; Pasyk et al. 1982 ; Van Rappard et al. 1988 ; Gibson 1990 ; Timmenga and Das 1992 ).

Quantitative analyses of collagen have been described by Ferdman and Yannas 1993 , Moragas et al. 1998 , and De Vries et al. 2000 . These methods provide general information only for, e.g., orientation of collagen fibers, but not for that of individual collagen fibers. The method in the present study was developed to enable quantitative determination of changes in the orientation of individual collagen fibers in the dermis. The skin used to develop this method was obtained in a study of the effects of skin stretching to close large wounds in piglets. In this study we investigated the changes that take place in the various layers of skin when skin is stretched beyond its natural yield over a time span of 30 min by means of mechanical forces (Melis et al. 2001a, Melis et al. 2002 ). The present quantitative method is based on specific staining of collagen fibers with picrosirius red, which allows the visualization of individual collagen fibers on the basis of double refraction of light against a black background when epipolarization microscopy is used (Junqueira et al. 1979 ). Because of the large contrast, it is possible to create representative binary images to establish the length of collagen fibers quantitatively in the plane of sections using digital microscopy. In this way, changes in orientation resulting from skin stretching can be determined.


  Materials and Methods
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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 Hirshowitz et al. 1993 and has been applied by us on piglet skin as described in detail by Melis et al. 2001 , Melis et al. 2002 . Briefly, a square piece of skin of 9 x 9 cm was excised on the sides of the piglets under general anesthesia. Two straight needles of the skin-stretching device were placed through the dermis opposite each other and at 0.5 cm distance from the wound margin. The skin-stretching device was then placed in position with its U-shaped arms anchored behind the intradermal needles. In this way, the hooks of the arms abut against the intradermal needles which, in turn, distribute the stretching force along the length of the wound margin. By turning the tension screw of the device, approximation of the skin and subcutaneous tissue was performed with a maximal applied force of 2.5 kg. The wounds were closed using the principle of cycle loading. This method involved 4 min of stretching of the skin, followed by 1 min of relaxation by unlocking the system to restore blood circulation in the stretched skin tissue. This cycle was repeated six times over a total period of 30 min.



View larger version (112K):
[in this window]
[in a new window]
 
Figure 1. Experimental setup for skin stretching in young piglets. The Sure Closure Skin Stretching device is placed in the wound margins. The ends of the two steel needles (P), which distribute the stretching force along the length of the wound margin, are shown. By turning the tension screw (T), approximation of the skin is performed.

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 (Van Noorden and Vogels 1986 ). In this way, orientation of the biopsy and the cutting edge for sectioning were determined. The gelatin-embedded biopsy in the mold was fixed by freezing as quickly as possible in liquid nitrogen at -196C. Biopsies were stored at -80C.

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 (Chieco et al. 2001 ).

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 Van Noorden and Vogels 1986 to keep the structure of the tissue in the sections intact.

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 Junqueira et al. 1979 . Sections were placed between two crossed polarization filters (polarizer slider AH2-KPO and analyzer slider AH2-AN45; Olympus, Tokyo, Japan), and images of serial sections were captured with a video camera (4910; Cohu, Tucson, AZ) at a low magnification (x5) (Fig 2A and Fig 2B). Images of 768 x 512 pixels were made of areas with a size of 1.2 mm x 0.8 mm of the reticular dermis. One pixel covered a surface of 1.6 µm x 1.6 µm in the section.



View larger version (53K):
[in this window]
[in a new window]
 
Figure 2. Epipolarization microscopic image of control skin (A) and stretched skin (B) after staining with picrosirius red. Collagen of control skin appears to have a randomly distributed orientation, with relatively small white areas representing collagen (A). Orientation of the collagen fibers in stretched skin is more parallel in the field of the section (B). Bar = 100 µm. Binary image showing white collagen fibers against a black background (C). All white objects in the image are numbered (D). For each white object, the best-fitting oval is calculated and the shortest and longest diameters (minor and major, respectively) are determined (E).

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 (Ridler and Calvard 1979 ), was used to produce the binary image; (b) an analysis was performed to number all white objects in the image (Fig 2D); (c) the best fitting oval was calculated for each numbered white object (Fig 2E); (d) the shortest and longest diameters of the oval were determined in numbers of pixels and were named minor and major axis, respectively; (e) these axes were calculated for all white objects and results were copied into a spreadsheet; (f) data in the spreadsheet (Excel 3.0; Microsoft, Seattle WA) were sorted on the basis of the length of the major axis, with the longest major axis at the top and the shortest at the bottom; (g) the mean length of the 10 longest major axes was determined. This mean length was considered to be the parameter for orientation of collagen fibers in the plane of the section. The mean value of the parameter, based on three serial sections of a specimen, was called the collagen alignment index; (h) the collagen alignment indices of control and stretched skin were compared and differences were analyzed, using a paired t-test with p=0.05 as level of significance.


  Results
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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.



View larger version (12K):
[in this window]
[in a new window]
 
Figure 3. Drawing of a piglet skin tissue block and a section cut from the block. The vectors indicate the direction of the stretching force and the direction of cutting the section.

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.



View larger version (124K):
[in this window]
[in a new window]
 
Figure 4. Sections of piglet skin after staining with H&E (A) and picrosirius red in combination with epipolarization microscopy (B). Both figures are at the same magnification. Bar = 100 µm.

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).


 
View this table:
[in this window]
[in a new window]
 
Table 1. Collagen alignment indices (± SD) after 0 and 30 min stretching of the skin of five piglets


  Discussion
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

The picrosirius red staining method, in combination with epipolarization microscopy as described by Junqueira et al. 1979 , appeared to be the most suitable method to visualize individual collagen fibers and to quantitatively determine their length in the plane of tissue sections. Only collagen fibers were visualized and other structures in the skin, such as glands and muscles, were not visible. The large contrast of the stained fibers against a black background not only enabled qualitative analysis of orientation of the fibers but also enabled quantitative digital image analysis. In this study, most of the collagen fibers reflected in red. This is an indication that these collagen fibers are relatively thick because thin collagen fibers appeared as yellow or green (Junqueira et al. 1982 ). Some collagen fiber endings appeared yellow or green, indicating that these endings are thinner (Fig 4B). A possible explanation could be that, as a result of mechanical stress, collagen fibers rupture.

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 (Melis et al. 2002 ). Whether these changes in orientation of collagen fibers in the plane of sections were a consequence of rapid changes in the spatial distribution of collagen fibers or elongation of collagen fibers by, e.g., aligning of individual fibrils, is not yet completely clear. However, the study of Melis et al. 2002 and the data from the present study suggest that rapid changes in the orientation of collagen fibers take place as a consequence of mechanical stress.

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 (Ferdman and Yannas 1993 ; Moragas et al. 1998 ; De Vries et al. 2000 ). The methods described by Ferdman and Yannas 1993 and De Vries et al. 2000 are based on the analysis of light scatter. These two methods enable analysis of the mean orientation of collagen in a tissue area, but they do not allow the determination of orientation of individual collagen fibers, which is possible with the method described here. Moragas et al. 1998 described a quantitative method based on digital skeletalization of contours of collagen fibers. With this method, changes in orientation and structure of individual collagen fibers can, in principle, be determined. However, skeletalization of individual collagen fibers appeared not to be representative because the procedure yielded a large number of dendrites or small branches owing to irregularities in the contours of collagen fibers in the stained sections and adherence to other fibers or bifurcation of fibers. When the longest major axes are calculated, this phenomenon does not play a role because of the use of an oval by which involvement of roughness of contours is eliminated. The major axes appeared to be calculated simply and validly on the basis of the binary image.

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.
  Literature Cited
Top
Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

Chieco P, Jonker A, Van Noorden CJF (2001) Image Cytometry. Oxford, BIOS

De Vries HJC, Enomoto DNH, Van Marle J, Van Zuijlen PPM, Mekkes JR, Bos JD (2000) Dermal organization in scleroderma: the fast Fourier transform and the laser scatter method objectify fibrosis in nonlesional as well as lesional skin. Lab Invest 80:1281-1289[Medline]

Ferdman AG, Yannas , IV (1993) Scattering of light from histologic sections: a new method for the analysis of connective tissue. J Invest Dermatol 100:710-716[Abstract]

Gibson T (1990) The physical properties of skin. In Converse JM, ed. Reconstructive Plastic Surgery. Vol I. 2nd ed Philadelphia, WB Saunders, 207-220

Gibson T, Kenedi RM, Craik JE (1965) The mobile micro-architecture of dermal collagen: a bio-engineering study. Br J Surg 52:764-770[Medline]

Hirshowitz B, Lindenbaum E, Har-Shai Y (1993) A skin-stretching device for harnessing of the viscoelastic properties of the skin. Plast Reconstruct Surg 92:260-270[Medline]

Junqueira LCU, Bignolas G, Brentani RR (1979) Picrosirius staining plus polarization microscopy, a specific method for collagen detection in tissue sections. Histochem J 11:447-455[Medline]

Junqueira LC, Carneiro J, Kelly RO (1993) Functionele Histologie. 6th ed Utrecht, Bunge

Junqueira LCU, Montes GS, Sanchez EM (1982) The influence of tissue section thickness on the study of collagen by the picrosirius-polarization method. Histochemistry 74:153-156[Medline]

Melis P, Noorlander ML, Bos KE (2001) Tension decrease during skin stretching in undermined and not undermined skin: an experimental study in piglets. Plast Reconstruct Surg 107:1201-1205[Medline]

Melis P, Noorlander ML, Van der Horst CMAM, Van Noorden CJF (2002) Rapid alignment of collagen fibers in the dermis of undermined and not undermined skin stretched with a skin stretching device. Plast Reconstruct Surg 109:674-680[Medline]

Moragas A, Garcia–Bonafé M, Sans M, Torán N, Huguet P, Martín–Plata C (1998) Image analysis of dermal collagen changes during skin aging. Anal Quant Cytol Histol 20:493-499[Medline]

Pasyk KA, Austad ED, McClatchey KD, Cherry GW (1982) Electron microscopic evaluation of guinea pig skin and soft tissues expanded with a self-inflating silicon implant. Plast Reconstruct Surg 70:37-45[Medline]

Ridler TW, Calvard (1979) Picture thresholding using an iterative selection method. IEEE Transact Syst Man Cybern 8:630-632

Stark HL (1977) Directional variations in the extensibility of human skin. Br J Plast Surg 30:105-114[Medline]

Timmenga EJF, Das PK (1992) Histomorphological observations on dermal repair in expanded rabbit skin: a preliminary report. Br J Plast Surg 45:503-507[Medline]

Van Noorden CJF, Vogels IMC (1986) Enzyme histochemical reactions in unfixed and undecalcified cryostat sections of mouse knee joints with special reference to arthrithic lesions. Histochemistry 86:127-133[Medline]

Van Rappard JH, Sonneveld GJ, Borghouts JM (1988) Histological changes in soft tissues due to tissue expansion. Facial Plast Surg 5:280-286[Medline]





This Article
Abstract
Full Text (PDF)
Alert me when this article is cited
Alert me if a correction is posted
Services
Similar articles in this journal
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Noorlander, M. L.
Articles by Van Noorden, C. J.F.
Articles citing this Article
PubMed
PubMed Citation
Articles by Noorlander, M. L.
Articles by Van Noorden, C. J.F.


Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]