J. COMP. PATH. 1975.VoL.85.
61
THE EPITHELIAL REACTION IN THE HEALING OF EXCISED CUTANEOUS WOUNDS IN THE DOG BY
E. W. WINSTANLEY Department of Clinical Veterinary Practices, School of Veterinary Medicine, University of Dublin, Trinity College, Ballsbridge, Dublin 4
INTRODUCTION
Much work has been devoted to the investigation of wound healing. During the last two decades a number of specialized studies have helped to reveal many facets of the behaviour of damaged epithelium. Recently, more sophisticated methods have been used. Nevertheless the light microscope and simple selective staining methods have a part to play in the investigation of the healing wound. The dog has not figured prominently in the field of wound healing and this is surprising in view of the part played by the dog in other fields of surgical investigation. The only extended accounts referring specifically to the dog are those of Carrel and co-workers (Carrel, 1910, 1921; Carrel and Hartmann, 1916; Carrel and du Nouy, 1921) and by Hartwell (1929, 1930). More recently Ordman and Gillman (1966) and Gillman (1968) have made histopathological studies on the healing excised wound in the pig. Despite these and other investigations (Dann, Glucksmann and Tansley, 1941; Lindquist, 1946; Winter, 1964) there are few detailed accounts devoted to the microscopical changes of the healing of the full thickness excised skin wound. This paper describes the epithelial response in uncovered full thickness excised skin wounds of the thoracic and metatarsal regions of the dog. MATERIALSANDMETHODS Animals and housing. Twelve male Collie dogs varying in age from 18 months to three years were kept each in a separate metal kennel on clean straw bedding. Anaesthetics. The animals were sedated by the i.v. injection of acetylpromazine (2 mg./ml.; Boots Pure Drugs Co. Ltd.) at a rate of O-25ml. per 4.5 kg. body weight. Anaesthesia was continued with halothane (Fluothane, Imperial Chemical Pharmaceuticals Ltd.) and oxygen by a semi-open technique. Wound preparation. Circular wounds were placed on the lateral thoracic wall and in the metatarsal region. A minimal amount of hair was removed from each site. The wounds were formed using a 13 mm. diameter stainless steel tube sharpened at one end. This was introduced at right angles to the skin surface and the lower tissue attachments were severed by means of scissors. The wounds penetrated well into the subcutaneous tissues. Woundprotection. A plastic bucket, without a bottom, was fastened over the animal’s head to prevent licking. There was no indication that any dog scratched its wounds and no precautions were taken. Biopsies. A total of 94 wounds was biopsied at the times indicated in Table 1. The biopsies consisted of the entire wound, the underlying tissue and approximately one cm. of surrounding skin.
62
E. W. WINSTANLEY
Histology. The biopsy specimens were gently flattened, pinned to cork boards to prevent warping and fixed in 10 per cent. form01 saline for 24 to 48 h. The specimens were embedded in paraffin wax and 8 pm. serial sections cut. Half of each block was sectioned perpendicular to the skin surface (sagittal) in the direction of the hair flow and the other half parallel to the skin surface (tangential). The numbered slides were stained with Harris’s haematoxylin and eosin, Gordon and Sweet’s method for reticulin, alcian blue for plasma cells, Verhoeff’s method for elastic tissue and van Gieson’s method for connective tissue. RESULTS
Normal Canine Epidermis in the Thoracic and Metatarsal
Regions
The canine epidermis is thin and does not show all the layers considered typical of mammalian epidermis. The epidermis of the thoracic skin consists of one or two cell layers and an outer stratum corneum. The epidermis of the metatarsal region is slightly thicker and an average of two to four cell layers are found beneath the stratum corneum. The surface is irregular and shows many small troughs and ridges. It is usually not possible to distinguish the outline of the individual cells. All the nuclei which are basophilic are mostly oval or round, but some are elongated. The nuclei of the outermost cells stain less intensely than those of the basal layer. The cytoplasm is mildly basophilic. Tonofibrils cannot be seen. Small numbers of melanocytes are present between the cells of the basal layer. Mitotic figures are not seen in normal epidermis. The stratum corneum is a clearly demarcated acidophilic layer, of about the same thickness as the cellular region. TABLE SUMMARY
Post-ofwatiue
Number
days
of biopsies from from metatarsus thorax
i
OF 94 BIOPSIES
OF EXCISED
I
Z-4
5-8
9-12
13-18
19-36
ooer 37
Total
4
10
66
5
:
i
7
45
Epithelial Reactions in the Thoracic and Metatarsal
WOUNDS
Regions
The epithelial response is similar in both thoracic and metatarsal wounds although variation in intensity occurs between wounds and within the individua1 wound. Day 1. Within a few hours of injury the epithelial cells close to the wound edge increase in size and begin to migrate either down the cut dermal edge or directly into the wound exudate (Figs 1 and 2). The epidermis around the circumference increases in thickness and is clearly divided into a basal layer, a stratum spinosum and a modified stratum corneum. All cells are hypertrophic, due mainly to an increase in the cytoplasm. Most of the thickened zone is formed by the basophilic stratum spinosum, many of the cells of which are distended by vacuoles which stain neither for glycogen nor for fat (Fig. 3). Close to the wound edge the outer layer of the epidermis differs from the normal stratum corneum (Fig. 3). The cells retain their nuclei and much of their
HEALING
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CUTANEOUS
WOUNDS
IN
DOG
63
cytoplasm. The layer appears as a deeply staining syncytium in which the nuclei are rod shaped, homogenous and markedly basophilic. The thickness of the layer varies: it may be several cells thick and swollen, but usually consists of a single or double layer of cells. There is an increase in the rate of mitotic division in the basal cells close to the wound. In the thoracic wounds epithelial cells push their way down the upper part of the dermal wall during the first 24 h. The distance travelled by the migrating cells varies and depends on local conditions. In some regions no cell movement occurs. The average distance of advance during the first 24 h. is about 75 urn. The migrating epithelium at this stage is in the form of a single tongue-like
Fig.
1. One day thoracic skin wound. The epithelial cells have moved down the cells are large and pale staining and The outer cells of the migrating shelf stratum corneum (a). HE. x 80.
epidermis is thickened and the cells hypertrophic. The cut dermal wall into the wound exudate. The advancing move just below the dense cellular layer of the exudate. retain their nuclei and are directly continuous with the
extension, two or three cells thick, but often the advancing cells are more diffuse and individual cells make their way down the dermal wall or into the wound exudate. A slightly different pattern occurs in the metatarsal wounds. In this region the exudate fills the wounds at an early stage and the migrating cells usually pass directly into the exudate rather than down the dermal wall (Figs 2 and 4). The epithelial cells tend to move forward as a single spur-like projection and only occasionally do individual cells explore alternative passages. The average distance covered during the first 24 h. is about 150 urn. The mode of epithelial migration is the same in all wounds. Cells from both the stratum spinosum and stratum corneum move into the wound, but the basal layer is not involved. The advancing cells are large and pale staining, and are
64
E. W.
WINSTANLEY
Fig. 2. One day metatarsal skin wound. A tongue of epithelial cells has passed directly into the wound exudate. The migrating cells are large and pale staining. The dark staining rod shaped nuclei of the cells of the upper syncytial layer are seen above the epithelial spur (a). HE. x 320.
Fig. 3. Two day thoracic skin wound. The epidermis is thickened and hydropic degeneration has taken place in many of the cells in the stratum spinosum (a). Migrating epithelial cells are moving into the wound exudate. HE. x 80.
HEALING
OF
Fig. 4. A 2 day metatarsal skin wound. passing directly into the wound epidermis. HE. x 80.
CUTANEOUS
IN
DOG
(a) Deep staining basal cells (b) pale staining exudate (c) stratum corneum separated from
Fig. 5. High power view of the broad advancing staining rod-shaped nuclei of the upper distance behind the tip. HE. x 320. E
WOUNDS
epithelial syncytial
65
migrating cells the rest of the
edge in a 2 day metatarsal wound. (a) Dark layer (b) mitosis in a lowermost cell a short
66
E. W.
WINSTANLEY
readily distinguished from the deeper staining cells of the basal layer (Figs 2 and 4). The migrating cells spill out over the end of the basal layer and advance by sliding across each other. In a few hours the advancing cells which come to rest on the wound tissues assume the appearance of basal layer cells and are capable of mitotic division after a further 48 h. Lying on top of the advancing spinosal cells and also moving into the wound is the upper syncytial layer of the stratum corneum (Figs 2 and 5). This layer frequently moves in advance of the other migrating cells (Fig. 6) and in some regions its cells are the only cells to move into the lesion during the early stages. The layer forms a direct continuation of the stratum corneum and extends into the wound as a single or double column of cells. The upper syncytial layer sometimes becomes completely separated from the cells of the stratum spinosum, an occurrence that appears to have little or no effect upon the subsequent migration (Fig. 4).
Fig. 6. The epithelial edge in an 8 day metatarsal skin wound. A well formed, though distorted, upper syncytial layer can be seen moving in front of the other epithelial cells which are advancing in the form of a thin wedge. HE. x 80.
Days 2 to 4. The epithelial cells around the wound edge attain their maximum size by the 2nd or 3rd day. In the stratum spinosum the outline of the individual cells and acidophilic tonofibrils can be seen. The cells in the upper region are less distinct, elongated and lie in a direction which is parallel to the skin surface. The hydropic degeneration in this layer is most pronounced between the 2nd and 4th days and extends for up to one mm. from the wound edge. After the 4th day the vacuoles rapidly disappear. The stratum spinosum is demarcated from the acidophilic stratum corneum in which the faint outlines of the cells and nuclei can be identified. This region possesses a refractive property not seen in other layers and the cells contain large accumulations of small round
HEALING
OF
CUTANEOUS
WOUNDS
IN
67
DOG
black keratohyaline granules. The outer layers of cornified flakes and strands are easily detached from the section. The migrating epithelial cells are derived from the cut epidermal edge and from the walls of hair follicles. They migrate further into the exudate or down the dermal.wall as single or multiple outgrowths (Figs 4 and 5). Some of the cells change their shape as they push between the surrounding cells. Days 5 to 8. The rate of mitotic division in the basal cells close to the wound reaches a maximum value at about the 6th day. By increased cell formation and cell hypertrophy the epidermis around the wound becomes markedly thickened (Figs 7 and 8). Well formed projections extend into the dermis from the lower surface of the thickened epithelium. A layer of homogenous acidophilic material lies in contact with the projections as they extend into the dermal tissue.
Fig.
7. The wound edge in a 6 day thoracic skin wound. edge (b) adventitious epithelial cells. HE. x 80.
(a) Thickening
ofthe
epithelium
at the wound
The greatest build up to cells occurs at the wound edge. By the 8th day an epithelial collar up to 60 cells thick encircles the wound. Cellular hyperplasia and hypertrophy extend for a distance of 5 to 10 mm. from the wound edge. The cells of the basal layer are strongly basophilic and are square or rectangular with oval or round nuclei. Over the crests of the ridges and in the troughs between them the cells and the nuclei are often elongated or pear shaped with their longitudinal axis at right angles to the basement membrane. In this form the cells do not undergo mitotic division. The basal layer is usually one cell thick, but when the epithelium is greatly thickened the lower three or four cell: layers are similar to those of the true basal cells. Although most cell divisions take place in the basal layer proper, mitotic figures are sometimes seen in the
68
E. W.
WINSTANLEY
modified cells which lie immediately above the basal layer itself. The stratum spinosum forms most of the thickened epidermis (Figs 4 and 8). It is weakly basophilic and the cells are larger than those of the basal layer. The nuclei are large and oval with a prominent nucleolus. By the 6th day a well formed out-growth of epithelial cells has advanced into the wound from the whole of the circumference, parallel to the surrounding epidermal surface and just below the upper dense cellular layer of the wound exudate (Fig. 8). Although at this stage the tip of the advancing cells is moving into the exudate, the tongue of newly formed epithelium behind this region rests on granulation tissue or occasionally, in the case of the thoracic wounds, on adipose tissue that has moved up into the wound cavity (Fig. 8). Many finger-like
Fig. 8. The edge of an 8 day thoracic skin wound. Thickened lower surface of the newly formed epithelium projects
(a) epidermis into the wound
and (b) hair follicles. exudate. HE. x 25.
The
processes and many individual cells extend from the lower surface of the new epithelium and pass into the wound tissue (Fig. 7). The tip of the advancing spur is wedge shaped and can be either sharp or blunt ended. All the migratory cells are large and elongated in the direction of movement. The lower cells of the epithelial spur stain as basal layer cells and melanoblasts occur between the cells. The outer cells form a distinct upper syncytical layer (Fig. 6), which is usually two or three cells thick, but occasionally is thicker and the cells are swollen. Days 9 to 12. Although a definite direction of migration has been established and most of the cells move over the wound in a plane which corresponds to that of the surrounding epidermis, a number of cells from the region just behind the advancing tip move downwards into the underlying tissues. Such cells usually
HEALING
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CUTANEOUS
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IN
69
DOG
survive for a short time only, but if the migrating epithelium encounters, for example, an area of infection or a region from which the exudate has become detached, the cells persist and form several finger-like projections which grow into the tissues of the wound. One of the projections undermines the offending area and its cells become established as the new tip of the migrating epithelial sheet (Fig. 9). Within a short time the connective tissue of the wound pushes up the epithelium and restores it to its original plane of migration. Apart from the region immediately behind the advancing edge the newly formed epithelium is clearly differentiated into basal layer, stratum spinosum and an upper stratum corneum (Fig. 10). Melanoblasts occur in the basal layer and move across the wound surface with the migrating cells.
Fig. 9. Epithelial surface ofan 18 day metatarsal skin wound. (a) Surface deep epithelium undermining surface layers. HE. x 20.
epithelial
growth
(b) thickened
Days 13 to 18. The thickness of the epithelium at the wound edge is reduced (Fig. 11) whereas the newly formed epidermis close to the wound edge is much thickened and prominent projections extend from the new epidermis into the tissues of the wound (Fig. 11). The projections are most numerous about the 18th day. Mitotic division continues to occur in the basal layer cells. Large quantities of keratohyaline granules occur in the lower layers of the stratum corneum (Fig. 10). The form of the outer layers of the stratum corneum depends upon whether or not the epidermis is covered with a scab. When the surface is so covered the cells retain their nuclei and form a syncytial layer, but when the surface is exposed the cells lose their nuclei and form a relatively thick layer of keratinized cells.
E. W. WINSTANLEY
Fig.
10. Epidermis in a 13 day thoracic between the cells of the strata HE. x 80.
Fig.
11. Edge of a 13 day thoracic the wound. HE. x 20.
skin wound. (a) Melanoblasts spinosum and (c) keratohyaline
skin wound.
The epidermis
in the basal layer (b) tonofibrils granules in the upper layers.
at the wound
edge is not as thick
as over
HEALING
OF
CUTANEOUS
WOUNDS
Fig.
12. A 24 day thoracic skin wound. The epidermis thickness. (a) Melanoblasts in the basal layer.
Fig.
13. Epidermis in a 36 day thoracic skin wound. Clearly Keratin strands overlie the surface. HE. x 80.
IN
surrounding Verhoeff’s
71
DOG
and over x 20.
differentiated
the wound
and reduced
is reduced
in thickness.
in
72
E. W. WINSTANLEY
Days 19 to 36. The wounds become covered with epithelium and once epithelization is complete there is a rapid fall in the rate of cell division. The epithelial projections gradually disappear leaving the regenerated epidermis as an almost uniform layer two or three times thicker than the normal epidermis of the region (Figs 12 and 13). Over 37 days. The epidermis in the region around the wound has an almost normal appearance, but even after 315 days it remains one or two cells thicker than normal. The surface of the regenerated epidermis becomes irregular and undulating, and deep depressions form in which keratin tends to accumulate. With time the epithelial thickness is reduced, but whereas after 315 days the stratum spinosum is lost in the thoracic wounds it is retained as a clearly discernible layer in the metatarsal wounds. In both regions the newly formed epidermis remains slightly thicker than the surrounding normal epidermis and possesses a thick stratum corneum. DISCUSSION
The healing of an excised wound involves both connective and epithelial tissue. Although it is convenient separately to describe the reactions of the two tissues, in reality the intensity and duration of the reactions are closely integrated. New connective tissue does not form in any appreciable quantity in the cavity of an excised wound until the 6th to the 8th day so that it is only at this time that epithelial cells can grow out to cover the surface of the wound. However, prior to the 6th day, intensive reactions take place in the epidermis surrounding the wound edge in preparation for the migration of epithelial cells. The reaction of the epidermis to injury is prompt. The most intense reactions take place close to the wound edge, but changes can be detected for a distance of at least 5 mm. into the surrounding skin. As a result of the cellular activity, the number of epidermal cell layers in the region around the wound increases until about the 8th day when, at the wound edge, over 50 layers are frequently present, and on some occasions over 90 cell layers are produced. The walls of the hair follicles increase in thickness and cells from these structures add significantly to the bulk of cells which are capable of migration. The accumulation of cells at the wound edge provides a reservoir of cells which are available for movement across the wound surface. The normal canine epidermis does not possess a stratum spinosum yet within a few hours of injury not only is a stratum spinosum produced, but the cells of the layer are morphologically similar to those seen in the typical mammalian epidermis and tonofibrils are present between the cells. It is the cells of the stratum spinosum that migrate to cover the surface of an excised wound and their large size plays an important part in epithelization. The migration of epithelial cells into the cavity of an excised wound begins at an early stage. The form assumed by the migrating spur of cells during the first 2 or 3 days is governed by the local conditions. If the exudate has not coagulated, the cells advance in the form of a thin wedge. On the other hand, when the exudate has coagulated and offers a firm resistance, the cells either tend to pile up at the edge or they move forward in the form of a thick wedge.
HEALING
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CUTANEOUS
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IN
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73
Most of the accounts in the literature state that the migrating epithelial cells advance across the wound surface in the form of a single celled layer (Peacock and van Winkle, 1970). This did not occur in the present studies, though it is possible that the non-aseptic environment could have influenced the method of advance. The cells which move into the wound and give rise to the new epidermis originate from the lower cells of the stratum spinosum. They are large pale staining cells and advance by rolling over the basal cells. When they come in contact with the underlying connective tissue, they assume the form of basal cells. These findings are in close agreement to those of Winter (1964) in young pigs. Although most of the migrating cells remain in contact with each other and advance as a distinct unit, a number of epithelial cells move independently and pass into the tissues of the wound. These cells arise both from the tip and from the lower surface of the advancing epithelial shelf and they may be instrumental in determining the plane and direction taken by the migrating epithelium across the wound. A layer of cells derived from the stratum corneum accompanies the migrating spinosal cells into the wound. In the conditions that exist at the wound edge, and later over the wound, the cells of the stratum corneum are able to retain their nuclei and form a syncytial layer. A similar collection of cells was described by Loeb (1898) and by Werner ( 1902) in the healing of excised wounds in the guinea pig. Loeb referred to the layer as the “upper protoplasmic layer” and both authors found that in small excised wounds of some 2 mm. in diameter the layer was able to spread quickly and covered the surface in a matter of days. Apart from these early descriptions no other reference to the layer has been found in the literature. This is surprising in view of the prominence that the layer can attain in the wounds of the dog. The present findings showed that the cells of the layer usually move a short distance in advance of the spinosal cells, but no indication was obtained that the layer has the ability to migrate independently as was suggested by Loeb and Werner. Although both these authors stated that the “upper protoplasmic layer” becomes incorporated in the scab and is later lost from the body, this course of events does not occur in the dog. Instead the layer persists and when the epithelium becomes differentiated the cells of the upper syncytial layer lose their nuclei and form part of the stratum corneum. Within 7 to 8 days a tongue of new epithelium passesdirectly into the wound and follows the lower border of the upper dense cellular layer of the exudate which by the 8th to the 12th day roughly corresponds to the plane of the surrounding epidermis. The passage of the epithelial cells through this region could be facilitated by the liberation of proteolytic enzymes due to the disintegration of cells in the upper cellular layer of the exudate. That a weakness exists at this level is indicated by the ease with which the layer becomes separated during the preparation of the slides. Apart from the region just behind the advancing edge, all the epithelium that lies over the wound is fully differentiated into epidermal strata. At first the thickest part of the newly formed epithelium is positioned close to the wound edge, but as the epithelium migrates across the wound surface so the region that
74
E. W. WINSTANLEY
shows the greatest number of layers passes from the wound edge and moves further and further into the wound. As this takes place the number of cell layers in the more peripheral regions becomes reduced. Prominent downgrowths develop on the lower surface of the newly formed epidermis after about the 12th day and reach their maximum size and extent between the 18th and the 21st day. The presence of these structures increases the number of basal cells and hence increases the supply of spinosal cells which are required for migration. The downgrowths also serve to increase the area of contact between the newly formed epidermis and the connective tissue of the wound at a time when the union between the two is likely to be weak. Peacock and van Winkle (1970) state that the projecting ridges do not persist, but by the 25th day become separated from the surface and form internally keratinizing epithelial pearls. This course of events did not occur in the dog: instead the size of the structures regressed until by the 36th day the lower border of the regenerated epidermis was relatively smooth. Once the new epidermis has been fully established the rate of cell division rapidly declines and there is a reduction in the size of the individual cells. The thickness of the epidermis is also reduced, but even after 315 days the epidermis over the wound is still thicker than the normal epidermis of the region. SUMMARY
The epithelial reaction which takes place in healing excised wounds in the thoracic and metatarsal regions of the dog is described. In both regions the behaviour of the epithelium is similar. Although the normal canine epidermis is thin a massive accumulation of cells occurs at the wound edge within 3 to 4 days. Cells from both the stratum spinosum and the stratum corneum migrate into the wound at an early stage. The bulk of the migrating epithelial cells move in the form of a shelf with a wedge-shaped edge, whereas some epithelial cells show independent movement and pass directly into the wound substance. A short distance behind the advancing edge the newly formed epithelium is differentiated into clearly defined strata. Whilst epithelial migration is taking place numerous projections or pseudo-rete pegs extend from the newly formed epidermis into the wound tissue F, but disappear when epithelization is complete. REFERENCES
Carrel, A. (19 10). The treatment
of wounds. Journal of the American Medical Association, 55,2148-2150. Carrel, A. (1921). Cicatrization of wounds: XII. Factors initiating regeneration. Journal of Experimental Medicine, 34, 425-434. Carrel, A., and Hartmann, A. (1916). The reaction between the size of the wound and the rate of its contraction. Journal of Ex@erimental Medicine, 24, 429-450. Carrel, A., and du Nouy, P. L. (1921). Cicatrization of wounds : XI. Latent period. Journal of Experilnental Medicine, 34, 339-348. Dann, L., Glucksmann, A., and Tansley, K. (1941). The healing of untreated experimental wounds. British Journal of Experimental Pathology, 22, l-9. Gillman, T. (1968). Healing of cutaneous wounds. The Glaxo Volume, 31, 5-3 1. Hartwell, S. W. (1929). A histologic study of healing with practical applications: I. Epithelial healing. Archives of Surgery, 19, 835-847.
HEALING
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WOUNDS
IN
DOG
75
Hartwell, S. W. (1930). A histologic study of healing with practical applications: II. Fibrous changes in wound healing. Archives of Surgery, 21, 76-96. Lindquist, G. (1946). The healing of skin defects: An experimental study in the white rat. Acta Chirurgica Scandinavicu, 94 (Suppl. 107), 1-163. Loeb, L. (1898). Uber regeneration des epithels. Archiv fur Entwicklungsmechanik der Organismen, 6, 297-364. Ordman, L. J., and Gillman, T. (1966). Studies in the healing of cutaneous wounds. Archives of Surgery, 93, 857-928. Peacock, E. E., and van Winkle, W. (1970). Epithelization and epithelialmesenchymal interactions. Chapter 2. pp. 17-48. Surgecv and Biology of Wound Reeuir, W. B. Saunders Company, Philadelphia. Werner, R. (1902). Experimentelle epithelstudien ueber wachstum regeneration, amitosen-und riesenzellen-bildung des epithels. Beitruge zur Klinischen Chirurgie, 34, l-84. Winter, G. D. (1964). Movement of Epithelial Cells over the Wound Surface. Advances in Biology qf Skin. Vol. 5, Chapter VII, pp. 113-127. W. Montagne and R. E. Billingham, Eds. Pergamon Press, Oxford. [Receivedfor publication,
&l&h
19th, 19741
61
THE EPITHELIAL REACTION IN THE HEALING OF EXCISED CUTANEOUS WOUNDS IN THE DOG BY
E. W. WINSTANLEY Department of Clinical Veterinary Practices, School of Veterinary Medicine, University of Dublin, Trinity College, Ballsbridge, Dublin 4
INTRODUCTION
Much work has been devoted to the investigation of wound healing. During the last two decades a number of specialized studies have helped to reveal many facets of the behaviour of damaged epithelium. Recently, more sophisticated methods have been used. Nevertheless the light microscope and simple selective staining methods have a part to play in the investigation of the healing wound. The dog has not figured prominently in the field of wound healing and this is surprising in view of the part played by the dog in other fields of surgical investigation. The only extended accounts referring specifically to the dog are those of Carrel and co-workers (Carrel, 1910, 1921; Carrel and Hartmann, 1916; Carrel and du Nouy, 1921) and by Hartwell (1929, 1930). More recently Ordman and Gillman (1966) and Gillman (1968) have made histopathological studies on the healing excised wound in the pig. Despite these and other investigations (Dann, Glucksmann and Tansley, 1941; Lindquist, 1946; Winter, 1964) there are few detailed accounts devoted to the microscopical changes of the healing of the full thickness excised skin wound. This paper describes the epithelial response in uncovered full thickness excised skin wounds of the thoracic and metatarsal regions of the dog. MATERIALSANDMETHODS Animals and housing. Twelve male Collie dogs varying in age from 18 months to three years were kept each in a separate metal kennel on clean straw bedding. Anaesthetics. The animals were sedated by the i.v. injection of acetylpromazine (2 mg./ml.; Boots Pure Drugs Co. Ltd.) at a rate of O-25ml. per 4.5 kg. body weight. Anaesthesia was continued with halothane (Fluothane, Imperial Chemical Pharmaceuticals Ltd.) and oxygen by a semi-open technique. Wound preparation. Circular wounds were placed on the lateral thoracic wall and in the metatarsal region. A minimal amount of hair was removed from each site. The wounds were formed using a 13 mm. diameter stainless steel tube sharpened at one end. This was introduced at right angles to the skin surface and the lower tissue attachments were severed by means of scissors. The wounds penetrated well into the subcutaneous tissues. Woundprotection. A plastic bucket, without a bottom, was fastened over the animal’s head to prevent licking. There was no indication that any dog scratched its wounds and no precautions were taken. Biopsies. A total of 94 wounds was biopsied at the times indicated in Table 1. The biopsies consisted of the entire wound, the underlying tissue and approximately one cm. of surrounding skin.
62
E. W. WINSTANLEY
Histology. The biopsy specimens were gently flattened, pinned to cork boards to prevent warping and fixed in 10 per cent. form01 saline for 24 to 48 h. The specimens were embedded in paraffin wax and 8 pm. serial sections cut. Half of each block was sectioned perpendicular to the skin surface (sagittal) in the direction of the hair flow and the other half parallel to the skin surface (tangential). The numbered slides were stained with Harris’s haematoxylin and eosin, Gordon and Sweet’s method for reticulin, alcian blue for plasma cells, Verhoeff’s method for elastic tissue and van Gieson’s method for connective tissue. RESULTS
Normal Canine Epidermis in the Thoracic and Metatarsal
Regions
The canine epidermis is thin and does not show all the layers considered typical of mammalian epidermis. The epidermis of the thoracic skin consists of one or two cell layers and an outer stratum corneum. The epidermis of the metatarsal region is slightly thicker and an average of two to four cell layers are found beneath the stratum corneum. The surface is irregular and shows many small troughs and ridges. It is usually not possible to distinguish the outline of the individual cells. All the nuclei which are basophilic are mostly oval or round, but some are elongated. The nuclei of the outermost cells stain less intensely than those of the basal layer. The cytoplasm is mildly basophilic. Tonofibrils cannot be seen. Small numbers of melanocytes are present between the cells of the basal layer. Mitotic figures are not seen in normal epidermis. The stratum corneum is a clearly demarcated acidophilic layer, of about the same thickness as the cellular region. TABLE SUMMARY
Post-ofwatiue
Number
days
of biopsies from from metatarsus thorax
i
OF 94 BIOPSIES
OF EXCISED
I
Z-4
5-8
9-12
13-18
19-36
ooer 37
Total
4
10
66
5
:
i
7
45
Epithelial Reactions in the Thoracic and Metatarsal
WOUNDS
Regions
The epithelial response is similar in both thoracic and metatarsal wounds although variation in intensity occurs between wounds and within the individua1 wound. Day 1. Within a few hours of injury the epithelial cells close to the wound edge increase in size and begin to migrate either down the cut dermal edge or directly into the wound exudate (Figs 1 and 2). The epidermis around the circumference increases in thickness and is clearly divided into a basal layer, a stratum spinosum and a modified stratum corneum. All cells are hypertrophic, due mainly to an increase in the cytoplasm. Most of the thickened zone is formed by the basophilic stratum spinosum, many of the cells of which are distended by vacuoles which stain neither for glycogen nor for fat (Fig. 3). Close to the wound edge the outer layer of the epidermis differs from the normal stratum corneum (Fig. 3). The cells retain their nuclei and much of their
HEALING
OF
CUTANEOUS
WOUNDS
IN
DOG
63
cytoplasm. The layer appears as a deeply staining syncytium in which the nuclei are rod shaped, homogenous and markedly basophilic. The thickness of the layer varies: it may be several cells thick and swollen, but usually consists of a single or double layer of cells. There is an increase in the rate of mitotic division in the basal cells close to the wound. In the thoracic wounds epithelial cells push their way down the upper part of the dermal wall during the first 24 h. The distance travelled by the migrating cells varies and depends on local conditions. In some regions no cell movement occurs. The average distance of advance during the first 24 h. is about 75 urn. The migrating epithelium at this stage is in the form of a single tongue-like
Fig.
1. One day thoracic skin wound. The epithelial cells have moved down the cells are large and pale staining and The outer cells of the migrating shelf stratum corneum (a). HE. x 80.
epidermis is thickened and the cells hypertrophic. The cut dermal wall into the wound exudate. The advancing move just below the dense cellular layer of the exudate. retain their nuclei and are directly continuous with the
extension, two or three cells thick, but often the advancing cells are more diffuse and individual cells make their way down the dermal wall or into the wound exudate. A slightly different pattern occurs in the metatarsal wounds. In this region the exudate fills the wounds at an early stage and the migrating cells usually pass directly into the exudate rather than down the dermal wall (Figs 2 and 4). The epithelial cells tend to move forward as a single spur-like projection and only occasionally do individual cells explore alternative passages. The average distance covered during the first 24 h. is about 150 urn. The mode of epithelial migration is the same in all wounds. Cells from both the stratum spinosum and stratum corneum move into the wound, but the basal layer is not involved. The advancing cells are large and pale staining, and are
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Fig. 2. One day metatarsal skin wound. A tongue of epithelial cells has passed directly into the wound exudate. The migrating cells are large and pale staining. The dark staining rod shaped nuclei of the cells of the upper syncytial layer are seen above the epithelial spur (a). HE. x 320.
Fig. 3. Two day thoracic skin wound. The epidermis is thickened and hydropic degeneration has taken place in many of the cells in the stratum spinosum (a). Migrating epithelial cells are moving into the wound exudate. HE. x 80.
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Fig. 4. A 2 day metatarsal skin wound. passing directly into the wound epidermis. HE. x 80.
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(a) Deep staining basal cells (b) pale staining exudate (c) stratum corneum separated from
Fig. 5. High power view of the broad advancing staining rod-shaped nuclei of the upper distance behind the tip. HE. x 320. E
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epithelial syncytial
65
migrating cells the rest of the
edge in a 2 day metatarsal wound. (a) Dark layer (b) mitosis in a lowermost cell a short
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readily distinguished from the deeper staining cells of the basal layer (Figs 2 and 4). The migrating cells spill out over the end of the basal layer and advance by sliding across each other. In a few hours the advancing cells which come to rest on the wound tissues assume the appearance of basal layer cells and are capable of mitotic division after a further 48 h. Lying on top of the advancing spinosal cells and also moving into the wound is the upper syncytial layer of the stratum corneum (Figs 2 and 5). This layer frequently moves in advance of the other migrating cells (Fig. 6) and in some regions its cells are the only cells to move into the lesion during the early stages. The layer forms a direct continuation of the stratum corneum and extends into the wound as a single or double column of cells. The upper syncytial layer sometimes becomes completely separated from the cells of the stratum spinosum, an occurrence that appears to have little or no effect upon the subsequent migration (Fig. 4).
Fig. 6. The epithelial edge in an 8 day metatarsal skin wound. A well formed, though distorted, upper syncytial layer can be seen moving in front of the other epithelial cells which are advancing in the form of a thin wedge. HE. x 80.
Days 2 to 4. The epithelial cells around the wound edge attain their maximum size by the 2nd or 3rd day. In the stratum spinosum the outline of the individual cells and acidophilic tonofibrils can be seen. The cells in the upper region are less distinct, elongated and lie in a direction which is parallel to the skin surface. The hydropic degeneration in this layer is most pronounced between the 2nd and 4th days and extends for up to one mm. from the wound edge. After the 4th day the vacuoles rapidly disappear. The stratum spinosum is demarcated from the acidophilic stratum corneum in which the faint outlines of the cells and nuclei can be identified. This region possesses a refractive property not seen in other layers and the cells contain large accumulations of small round
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black keratohyaline granules. The outer layers of cornified flakes and strands are easily detached from the section. The migrating epithelial cells are derived from the cut epidermal edge and from the walls of hair follicles. They migrate further into the exudate or down the dermal.wall as single or multiple outgrowths (Figs 4 and 5). Some of the cells change their shape as they push between the surrounding cells. Days 5 to 8. The rate of mitotic division in the basal cells close to the wound reaches a maximum value at about the 6th day. By increased cell formation and cell hypertrophy the epidermis around the wound becomes markedly thickened (Figs 7 and 8). Well formed projections extend into the dermis from the lower surface of the thickened epithelium. A layer of homogenous acidophilic material lies in contact with the projections as they extend into the dermal tissue.
Fig.
7. The wound edge in a 6 day thoracic skin wound. edge (b) adventitious epithelial cells. HE. x 80.
(a) Thickening
ofthe
epithelium
at the wound
The greatest build up to cells occurs at the wound edge. By the 8th day an epithelial collar up to 60 cells thick encircles the wound. Cellular hyperplasia and hypertrophy extend for a distance of 5 to 10 mm. from the wound edge. The cells of the basal layer are strongly basophilic and are square or rectangular with oval or round nuclei. Over the crests of the ridges and in the troughs between them the cells and the nuclei are often elongated or pear shaped with their longitudinal axis at right angles to the basement membrane. In this form the cells do not undergo mitotic division. The basal layer is usually one cell thick, but when the epithelium is greatly thickened the lower three or four cell: layers are similar to those of the true basal cells. Although most cell divisions take place in the basal layer proper, mitotic figures are sometimes seen in the
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modified cells which lie immediately above the basal layer itself. The stratum spinosum forms most of the thickened epidermis (Figs 4 and 8). It is weakly basophilic and the cells are larger than those of the basal layer. The nuclei are large and oval with a prominent nucleolus. By the 6th day a well formed out-growth of epithelial cells has advanced into the wound from the whole of the circumference, parallel to the surrounding epidermal surface and just below the upper dense cellular layer of the wound exudate (Fig. 8). Although at this stage the tip of the advancing cells is moving into the exudate, the tongue of newly formed epithelium behind this region rests on granulation tissue or occasionally, in the case of the thoracic wounds, on adipose tissue that has moved up into the wound cavity (Fig. 8). Many finger-like
Fig. 8. The edge of an 8 day thoracic skin wound. Thickened lower surface of the newly formed epithelium projects
(a) epidermis into the wound
and (b) hair follicles. exudate. HE. x 25.
The
processes and many individual cells extend from the lower surface of the new epithelium and pass into the wound tissue (Fig. 7). The tip of the advancing spur is wedge shaped and can be either sharp or blunt ended. All the migratory cells are large and elongated in the direction of movement. The lower cells of the epithelial spur stain as basal layer cells and melanoblasts occur between the cells. The outer cells form a distinct upper syncytical layer (Fig. 6), which is usually two or three cells thick, but occasionally is thicker and the cells are swollen. Days 9 to 12. Although a definite direction of migration has been established and most of the cells move over the wound in a plane which corresponds to that of the surrounding epidermis, a number of cells from the region just behind the advancing tip move downwards into the underlying tissues. Such cells usually
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survive for a short time only, but if the migrating epithelium encounters, for example, an area of infection or a region from which the exudate has become detached, the cells persist and form several finger-like projections which grow into the tissues of the wound. One of the projections undermines the offending area and its cells become established as the new tip of the migrating epithelial sheet (Fig. 9). Within a short time the connective tissue of the wound pushes up the epithelium and restores it to its original plane of migration. Apart from the region immediately behind the advancing edge the newly formed epithelium is clearly differentiated into basal layer, stratum spinosum and an upper stratum corneum (Fig. 10). Melanoblasts occur in the basal layer and move across the wound surface with the migrating cells.
Fig. 9. Epithelial surface ofan 18 day metatarsal skin wound. (a) Surface deep epithelium undermining surface layers. HE. x 20.
epithelial
growth
(b) thickened
Days 13 to 18. The thickness of the epithelium at the wound edge is reduced (Fig. 11) whereas the newly formed epidermis close to the wound edge is much thickened and prominent projections extend from the new epidermis into the tissues of the wound (Fig. 11). The projections are most numerous about the 18th day. Mitotic division continues to occur in the basal layer cells. Large quantities of keratohyaline granules occur in the lower layers of the stratum corneum (Fig. 10). The form of the outer layers of the stratum corneum depends upon whether or not the epidermis is covered with a scab. When the surface is so covered the cells retain their nuclei and form a syncytial layer, but when the surface is exposed the cells lose their nuclei and form a relatively thick layer of keratinized cells.
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Fig.
10. Epidermis in a 13 day thoracic between the cells of the strata HE. x 80.
Fig.
11. Edge of a 13 day thoracic the wound. HE. x 20.
skin wound. (a) Melanoblasts spinosum and (c) keratohyaline
skin wound.
The epidermis
in the basal layer (b) tonofibrils granules in the upper layers.
at the wound
edge is not as thick
as over
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12. A 24 day thoracic skin wound. The epidermis thickness. (a) Melanoblasts in the basal layer.
Fig.
13. Epidermis in a 36 day thoracic skin wound. Clearly Keratin strands overlie the surface. HE. x 80.
IN
surrounding Verhoeff’s
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and over x 20.
differentiated
the wound
and reduced
is reduced
in thickness.
in
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Days 19 to 36. The wounds become covered with epithelium and once epithelization is complete there is a rapid fall in the rate of cell division. The epithelial projections gradually disappear leaving the regenerated epidermis as an almost uniform layer two or three times thicker than the normal epidermis of the region (Figs 12 and 13). Over 37 days. The epidermis in the region around the wound has an almost normal appearance, but even after 315 days it remains one or two cells thicker than normal. The surface of the regenerated epidermis becomes irregular and undulating, and deep depressions form in which keratin tends to accumulate. With time the epithelial thickness is reduced, but whereas after 315 days the stratum spinosum is lost in the thoracic wounds it is retained as a clearly discernible layer in the metatarsal wounds. In both regions the newly formed epidermis remains slightly thicker than the surrounding normal epidermis and possesses a thick stratum corneum. DISCUSSION
The healing of an excised wound involves both connective and epithelial tissue. Although it is convenient separately to describe the reactions of the two tissues, in reality the intensity and duration of the reactions are closely integrated. New connective tissue does not form in any appreciable quantity in the cavity of an excised wound until the 6th to the 8th day so that it is only at this time that epithelial cells can grow out to cover the surface of the wound. However, prior to the 6th day, intensive reactions take place in the epidermis surrounding the wound edge in preparation for the migration of epithelial cells. The reaction of the epidermis to injury is prompt. The most intense reactions take place close to the wound edge, but changes can be detected for a distance of at least 5 mm. into the surrounding skin. As a result of the cellular activity, the number of epidermal cell layers in the region around the wound increases until about the 8th day when, at the wound edge, over 50 layers are frequently present, and on some occasions over 90 cell layers are produced. The walls of the hair follicles increase in thickness and cells from these structures add significantly to the bulk of cells which are capable of migration. The accumulation of cells at the wound edge provides a reservoir of cells which are available for movement across the wound surface. The normal canine epidermis does not possess a stratum spinosum yet within a few hours of injury not only is a stratum spinosum produced, but the cells of the layer are morphologically similar to those seen in the typical mammalian epidermis and tonofibrils are present between the cells. It is the cells of the stratum spinosum that migrate to cover the surface of an excised wound and their large size plays an important part in epithelization. The migration of epithelial cells into the cavity of an excised wound begins at an early stage. The form assumed by the migrating spur of cells during the first 2 or 3 days is governed by the local conditions. If the exudate has not coagulated, the cells advance in the form of a thin wedge. On the other hand, when the exudate has coagulated and offers a firm resistance, the cells either tend to pile up at the edge or they move forward in the form of a thick wedge.
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Most of the accounts in the literature state that the migrating epithelial cells advance across the wound surface in the form of a single celled layer (Peacock and van Winkle, 1970). This did not occur in the present studies, though it is possible that the non-aseptic environment could have influenced the method of advance. The cells which move into the wound and give rise to the new epidermis originate from the lower cells of the stratum spinosum. They are large pale staining cells and advance by rolling over the basal cells. When they come in contact with the underlying connective tissue, they assume the form of basal cells. These findings are in close agreement to those of Winter (1964) in young pigs. Although most of the migrating cells remain in contact with each other and advance as a distinct unit, a number of epithelial cells move independently and pass into the tissues of the wound. These cells arise both from the tip and from the lower surface of the advancing epithelial shelf and they may be instrumental in determining the plane and direction taken by the migrating epithelium across the wound. A layer of cells derived from the stratum corneum accompanies the migrating spinosal cells into the wound. In the conditions that exist at the wound edge, and later over the wound, the cells of the stratum corneum are able to retain their nuclei and form a syncytial layer. A similar collection of cells was described by Loeb (1898) and by Werner ( 1902) in the healing of excised wounds in the guinea pig. Loeb referred to the layer as the “upper protoplasmic layer” and both authors found that in small excised wounds of some 2 mm. in diameter the layer was able to spread quickly and covered the surface in a matter of days. Apart from these early descriptions no other reference to the layer has been found in the literature. This is surprising in view of the prominence that the layer can attain in the wounds of the dog. The present findings showed that the cells of the layer usually move a short distance in advance of the spinosal cells, but no indication was obtained that the layer has the ability to migrate independently as was suggested by Loeb and Werner. Although both these authors stated that the “upper protoplasmic layer” becomes incorporated in the scab and is later lost from the body, this course of events does not occur in the dog. Instead the layer persists and when the epithelium becomes differentiated the cells of the upper syncytial layer lose their nuclei and form part of the stratum corneum. Within 7 to 8 days a tongue of new epithelium passesdirectly into the wound and follows the lower border of the upper dense cellular layer of the exudate which by the 8th to the 12th day roughly corresponds to the plane of the surrounding epidermis. The passage of the epithelial cells through this region could be facilitated by the liberation of proteolytic enzymes due to the disintegration of cells in the upper cellular layer of the exudate. That a weakness exists at this level is indicated by the ease with which the layer becomes separated during the preparation of the slides. Apart from the region just behind the advancing edge, all the epithelium that lies over the wound is fully differentiated into epidermal strata. At first the thickest part of the newly formed epithelium is positioned close to the wound edge, but as the epithelium migrates across the wound surface so the region that
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shows the greatest number of layers passes from the wound edge and moves further and further into the wound. As this takes place the number of cell layers in the more peripheral regions becomes reduced. Prominent downgrowths develop on the lower surface of the newly formed epidermis after about the 12th day and reach their maximum size and extent between the 18th and the 21st day. The presence of these structures increases the number of basal cells and hence increases the supply of spinosal cells which are required for migration. The downgrowths also serve to increase the area of contact between the newly formed epidermis and the connective tissue of the wound at a time when the union between the two is likely to be weak. Peacock and van Winkle (1970) state that the projecting ridges do not persist, but by the 25th day become separated from the surface and form internally keratinizing epithelial pearls. This course of events did not occur in the dog: instead the size of the structures regressed until by the 36th day the lower border of the regenerated epidermis was relatively smooth. Once the new epidermis has been fully established the rate of cell division rapidly declines and there is a reduction in the size of the individual cells. The thickness of the epidermis is also reduced, but even after 315 days the epidermis over the wound is still thicker than the normal epidermis of the region. SUMMARY
The epithelial reaction which takes place in healing excised wounds in the thoracic and metatarsal regions of the dog is described. In both regions the behaviour of the epithelium is similar. Although the normal canine epidermis is thin a massive accumulation of cells occurs at the wound edge within 3 to 4 days. Cells from both the stratum spinosum and the stratum corneum migrate into the wound at an early stage. The bulk of the migrating epithelial cells move in the form of a shelf with a wedge-shaped edge, whereas some epithelial cells show independent movement and pass directly into the wound substance. A short distance behind the advancing edge the newly formed epithelium is differentiated into clearly defined strata. Whilst epithelial migration is taking place numerous projections or pseudo-rete pegs extend from the newly formed epidermis into the wound tissue F, but disappear when epithelization is complete. REFERENCES
Carrel, A. (19 10). The treatment
of wounds. Journal of the American Medical Association, 55,2148-2150. Carrel, A. (1921). Cicatrization of wounds: XII. Factors initiating regeneration. Journal of Experimental Medicine, 34, 425-434. Carrel, A., and Hartmann, A. (1916). The reaction between the size of the wound and the rate of its contraction. Journal of Ex@erimental Medicine, 24, 429-450. Carrel, A., and du Nouy, P. L. (1921). Cicatrization of wounds : XI. Latent period. Journal of Experilnental Medicine, 34, 339-348. Dann, L., Glucksmann, A., and Tansley, K. (1941). The healing of untreated experimental wounds. British Journal of Experimental Pathology, 22, l-9. Gillman, T. (1968). Healing of cutaneous wounds. The Glaxo Volume, 31, 5-3 1. Hartwell, S. W. (1929). A histologic study of healing with practical applications: I. Epithelial healing. Archives of Surgery, 19, 835-847.
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Hartwell, S. W. (1930). A histologic study of healing with practical applications: II. Fibrous changes in wound healing. Archives of Surgery, 21, 76-96. Lindquist, G. (1946). The healing of skin defects: An experimental study in the white rat. Acta Chirurgica Scandinavicu, 94 (Suppl. 107), 1-163. Loeb, L. (1898). Uber regeneration des epithels. Archiv fur Entwicklungsmechanik der Organismen, 6, 297-364. Ordman, L. J., and Gillman, T. (1966). Studies in the healing of cutaneous wounds. Archives of Surgery, 93, 857-928. Peacock, E. E., and van Winkle, W. (1970). Epithelization and epithelialmesenchymal interactions. Chapter 2. pp. 17-48. Surgecv and Biology of Wound Reeuir, W. B. Saunders Company, Philadelphia. Werner, R. (1902). Experimentelle epithelstudien ueber wachstum regeneration, amitosen-und riesenzellen-bildung des epithels. Beitruge zur Klinischen Chirurgie, 34, l-84. Winter, G. D. (1964). Movement of Epithelial Cells over the Wound Surface. Advances in Biology qf Skin. Vol. 5, Chapter VII, pp. 113-127. W. Montagne and R. E. Billingham, Eds. Pergamon Press, Oxford. [Receivedfor publication,
&l&h
19th, 19741
