Orientation of Collagen in Wound Healing*

Br. J. exp. Path. (1974) 55, 233.

ORIENTATION OF COLLAGEN IN WOUND HEALING*

M. LAUFER, C. ASHKENAZI, D. KATZ AND M. WOLMAN From the Departments of Plastic Surgery and Pathology, Chaim Sheba Medical Center, Tel Aviv University Medical School, Tel-Hashomer, Israel

Received for publication January 14, 1974

Summary.-Skin incisions were made in each of adult female white Yorkshire pigs. One set of 3 incisions was made on the back and one on the side of the animal. In each site one incision was longitudinal, another transverse to the length of the animal, and a third was longitudinal but at an angle of about 450 to the surface. Disposition of fibroblasts, new reticulin and collagen fibres was studied at 5, 15 and 30 days after the operations. No significant differences in fibre alignment were seen between the longitudinal and the transverse, and the back and the flank wounds. In the oblique wounds the cells and fibres ran in the direction of the incision and perpendicular to it rather than in relation to the direction of the pre-existing collagen. The data indicate that the orientation of cells and fibres might be due to chemotactic influence of the traumatized area rather than to mechanical factors or to pre-existing structural arrangement.

DERMAL COLLAGEN is known to form a 3-dimensional interlacing network (Kenedi et al., 1966). According to some authors (Gibson and Kenedi, 1967) collagen is arranged haphazardly when completely relaxed and only tension induces alignment of fibres. This alignment is in the direction of the force applied. The same authors (Gibson and Kenedi, 1969) noted, however, that there is directional quality to the extensibility of the skin and this cannot be easily reconciled with the notion of a totally haphazard arrangement. Thus it is not surprising that in another study the deformability of the skin was shown not to be a simple function of the pressure applied, as there are differences between various areas and between surgical biopsy material and skin obtained post mortem. The assumption that developmental factors (Langer's lines) (Forrester et al., 1969) or mechanical forces determine the collagen orientation does not seem to be based on acceptable experimental evidence (Gibson and Kenedi, 1967). In healing skin wounds collagen has been known to be laid down in a direction parallel to the surface. In a review, Schilling (1968) reported that the collagen fibres of scars align themselves in a direction parallel to that of tension or stress forces. The tensile strength and the breaking strength of scars have been shown to be proportional to the amount of collagen (Forrester et al., 1969; Levenson et al., 1965). Ordman and Gillman (1966) found that at an early stage of wound healing the reticulin fibres are deposited perpendicular to the skin surface rather than parallel to it. Only at a later stage is the collagen deposited parallel to the surface.

* This study was subvented by a research grant to Dr M. Wolman as an established investigator of the Chief Scientist's Bureau, Ministry of Health, Government of Israel. 22 234 M. LAUFER, C. ASHKENAZI, D. KATZ AND M. WOLMAN These findings suggest that the primary deposition of collagen is followed by a further slow process of remodelling (Levenson, 1969). It is clear that wound healing is associated with a more regular alignment of collagen than that present in the normal tissue. The present report describes experiments in which the nature ofthe determinant factors in the collagen orientation was studied.

MATERIALS AND METHODS Preliminary experiments were performed on rats but as the skin of the dorsum of the rat is extremely mobile and very hairy, adult female large Yorkshire pigs were used in the present studies. The animals were anaesthetized using thiopentone (Pentothal), ketamine (Ketalar) and halothane and each animal had 3 incisions on the dorsum and 3 on the flank (Fig. 1). At each site one incision was longitudinal and perpendicular to the surface, the second was transverse and perpendicular to the surface and the third was longitudinal and oblique, at an angle of 45° to the surface. The wounds were sutured with Supramid 5. Altogether 9 animals were operated on. Five days, 15 days and 30 days after operation 3 animals were sacrificed so that histological sections of 3 animals were available for each type of wound. Tissues were fixed in formalin and histological study was based on H. and E., reticulin and collagen staining procedures as well as examinations under crossed polars. TY7900 900 450 Al- 1 2 3

FIG. 1.-Skin wounds in pig; A, dorsum; B, flank. (1) is a transverse, (2) a longitudinal and (3) a longitudinal oblique wound.

RESULTS At an early stage of wound healing fibroblasts are mostly aligned perpendicular to the skin surface and parallel to the wound (Fig. 2); some are parallel to the surface. Reticulin and collagen fibres are also deposited, first mainly at rightangles and later mainly parallel to the surface in the perpendicular wounds. No striking difference was observed between the longitudinal and the transverse wounds and between the same on the back and on the flank. Thus in all the wounds made at rightangles to the surface connective tissue fibres were aligned either parallel or perpendicular to the skin surface.

EXPLANATION OF PLATES FIG. 2.- A healing transverse wound at 5 days. Alignment of cells within the wound at rightangles to the direction of collagen which runs here from side to side. H. and E. x 120. FIG. 3.-A healing oblique wound at 5 days. Alignment of cells and fibres follows the direction of the wound and lies at about 450 to the surrounding collagen. H. and E. x 120. FIG. 4.-A healing oblique wound after 15 days. Alignment of the new collagen at about 450 to the pre-existing fibres. H. and E., polarized light. x 120. BRMSH JouRN-.A. OF EXPERIMENTAL PATHOLoGY. Vol. 55, X-o. 3

Laufer, Ashkenazi, Katz and Wolman ORIENTATION OF COLLAGEN IN WOUND HEALING 235 In those wounds where the incision was made oblique the alignment of fibroblasts, reticulin and collagen fibres was mainly parallel and perpendicular to the wound rather than to the skin surface, as can be seen in Fig. 3 and 4.

DISCUSSION In the 3-dimensional network in the wound, or in the intact skin, resistance to stretching and tearing obviously depends on the alignment ofthe fibres. Examina- tion of any histological preparation of skin stained for collagen or studied by polarized light microscopy shows that the regular alignment of collagen is seen in many but not in all fibres. Still, the presence of an adequate number of collagen fibres running in one direction can render the skin less stretchable in one direction than in others. The well-known differences in the extensibility of skin in different directions indicate that, statistically, collagen is often aligned more in one than in other directions. The concept that collagen alignment is a function of alignment of another component such as fibroblasts, fibrin or capillaries, does not in reality answer any question. We are still faced with the problem as to which factors determine the alignment of that component. Obviously, the problem can be studied in the normal development ofundamaged skin of various areas. Wound healing, with its rapid scar formation, affords a good model for the study of factors influencing this " polarization ". The healing of cutaneous wounds is also of great importance in various branches of surgery. Several possible causes were considered: (a) Pre-existing alignment of collagen as the determining factor in the deposition of new collagen: this was practically ruled out as the early deposition of reticulin perpendicular to the surface bears no relation to the pre-existing alignment. Also, the lack of difference between longitudinal and transverse wounds speaks against this possibility. (b) Gravitational forces may play a role in the alignment of collagen. The absence of any difference between the wounds on the back and those on the flank rules out this suggestion. (c) That tension plays a major role in determining orientation of collagen could not be lightly discarded. It is obvious, however, that the tissue tension and the tension of a suture acting on a perpendicular to the surface and on an oblique wound are similar. Thus, the observation that collagen is aligned parallel and at rightangles to the wound and not to the surface indicates that the major factor determining collagen orientation is not tension. The only plausible explanation of the phenomenon seems to be that in the process of wounding chemical agents (" wound hormones ") are released into the tissue and that gradients of their concentrations play a major role in the fibrin. fibroblast and collagen alignment. Our observation might be of importance in surgery. It is believed that the resistance to tear of collagen fibres formed in the course of an oblique incision might be more than that obtained after an incision perpendicular to the surface. This possibility must be tested experimentally. Another point of practical importance is that this study indicates the need for a thorough search for chemotactic substances which induce collagen deposition and orientation. 2'36 M. LAUFER, C. ASHKENAZI, D. KATZ AND M. WOLMAN

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