0003-9969/83/040287-05$03.00/O Copyright Q 1983 Pergamon Press Ltd
Archs oral Biol. Vol. 28, No. 4, pp. 287-291, 1983 Printed in Great Britain. All rights reserved
A COMPARATIVE HISTOLOGICAL STUDY OF WOUND HEALING FOLLOWING CO2 LASER AND lCONVENTIONAL SURGICAL EXCISION OF CANINE BUCCAL MUCOSA ISHEILA E. FISHER, J. W. Department
FRAME, R. M. BROWNE and R. M. D. TRANTER Oral Surgery and Oral Medicine and Department of Oral Pathology, The Dental School, Birmingham, B4 6NN and Dudley Road Hospital, Birmingham, England
Summary-The histological changes following removal of 2 cm discs of buccal mucosa by CO2 laser and conventional surgery were studied over a period of 42 days. The laser wound differed from the conventional wound: there was minimal damage to the adjacent tissue; initially a reaction was less; coagulum of denatured protein formed on the surface; the inflammatory fewer myofibroblasts were present and there was little wound contraction; less collagen was formed and epithelial regeneration was delayed and more irregular.
which incorporated a 2cm diameter hole was placed over the buccal mucosa at each test site. The surgical excision was performed using a scalpel and fine dissecting scissors and extended through the lamina propria just beneath the basement membrane. Haemostasis was achieved using pressure packs and fine diathermy forceps. On the buccal mucosa of the opposite cheek, two wounds were created using a continuous laser beam at both the 10 and 20 W power settings. The beam was applied until the 2 cm diameter patch of mucosa at the centre of the template had been vaporized. The surgical and laser wounds were left exposed in the mouth, this differing slightly from the human clinical situation where biopsy wounds are normally sutured. The animals were fed on liquidized food for 1 week and then on a standard laboratory diet of meat and soft biscuits. One dog was killed by an overdose of sodium pentobarbitone at each of the following time intervals after surgery: immediately, 2, 4, 7, 10, 14, 28 and 42 days. A full thickness cheek specimen containing the mucosal wound was dissected out from the 3 experimental sites in each animal and fixed in 10 per cent neutral buffered form01 saline for at least 48 hr. After paraffin wax-embedding, sections were prepared across the middle of each wound and stained with haematoxylin and eosin and Van Gieson technique.
Surgical excision using the scalpel is an established technique for removing oral mucosa affected by leukoplakia or similar ,conditions (Kramer, El-Labban and Lee, 1978; Pindborg, 1980). However. the contraction and scarring which occurs during wound healing may subsequently restrict oral function. The CO2 laser now offers an alternative means of eradicating oral mucosal :Zesions. The CO2 laser beam has a wavelength of 10.6 km and is absorbed by water, resulting in vaporiza,tion of the intra- and extra-cellular fluid and disintegration of the cells (Hall et al., 1971a; Hall, Hill and Beach, 1971b). Clinical studies in man have shown that there is little post-operative swelling; the treated area heals with less contraction and scarring than a surgically excised wound (Strong et a[., 1973; Tuffin and Carruth, 1980). Although there are several reports on the effects of the CO2 laser on tissue, these histological investigations have been concerned with incisions and focused spot impacts, mainly on the skin (Hall, 1971; Hall et al., 1971a,b; Mester et ul., 1971; h4ihashi et al., 1976). The healing mechanism following vaporization of larger areas of oral mucosa has not been investigated. Our aim was to eltamine in an animal model the histological changes during healing after removal of patches of oral mucosa using the CO2 laser, and to compare these with similar surgically excised wounds.
MATER1 &LS AND METHODS Eight adult beagle dogs weighing between 8 and 13 kg were used. Anlesthesia was induced by intravenous injection of sodium pentobarbitone and was maintained with nitrous oxide, oxygen and halothane administered via a cuffed oral endotracheal tube. Three lesions were created in the mouth of each animal : a conventional surgical wound in one cheek, and two laser wounds in the other cheek. In order to standardize the size of the wounds, a metal template A.0
Visual inspection revealed satisfactory healing without infection, although the 2 types of wound behaved differently. The surgically-excised areas contracted considerably and had rolled margins which became flatter with time, but scarring was still visible at 48 days. The laser wounds initially had a buff-coloured base with adherent carbonized tissue fragments. Subsequently, there was little contraction and the edge of the wound was level with the adjacent tissue from an early stage. After 28 days, there was little evidence of the original injury.
Sheila E. Fisher et (11
Histological examination confirmed that the tissue changes extended beyond the mucosa into the superficial muscle fibres and were typical of healing by secondary intention. In the immediate specimen, scattered polymorphonuclear leucocytes were present both on the wound surface and perivascularly. The surface fibrinous coagulum was thickest at 2 days (Fig. 1). Entrapped within it were numerous polymorphonuclear leucocytes which also formed a diffuse infiltrate throughout the base of the wound, although in the deeper layers the infiltrate was predominantly perivascular. Degeneration of the endothelial cells lining the most superficial blood vessels was prominent. Even at this stage, early myofibroblast differentiation had commenced, most of the spindle-shaped cells being aligned parallel to the wound surface. Myofibroblasts were identifiable by their ovoid, vesicular nuclei and abundant, faintly granular cytoplasm. The myofibroblast proliferation became increasingly more prominent beneath the wound surface and reached its maximum after 10 days. The cells were orientated parallel to the surface and were separated into columns by newly-formed blood vessels disposed at right angles to the surface (Fig. 2). The wounds showed evidence of progressive contraction from 7 days onwards. There was a diffuse inflammatory cell infiltrate which consisted mainly of polymorphonuclear leucocytes with only scattered lymphocytes and plasma cells. This progressively diminished during the first 14 days, when re-epithelialization was almost complete. Epithelial cell migration over the surface and mitosis were first apparent at 4 days. The new epithelial layer was thinner than the adjacent epithelium and contained only shallow rete ridges (Fig. 3). This appearance was still evident at 42 days. Only a small number of myofibroblasts were present beneath the epithelialized wound and their reduction in number was accompanied by a gradual increase in the amount of newly-formed collagen. Laser wounds
Little difference was observed between the patterns of healing following tissue removal by the laser at the 10 and 20 W settings, and a single description will be presented. In general, the laser wounds were of uniform depth but were shallower than the surgical defects. Initially, the surface was composed of a layer of homogeneous basophilic coagulum beneath which was a narrow zone where the collagen showed altered staining with eosin. In some superficial areas, dessicated brown coagulum was present. No polymorphonuclear leucocytes were observed in any of the immediate specimens. A fibrinous coagulum accumulated on the surface during the next few days and was thickest after 7 days (Fig. 4). Polymorphonuclear leucocytes were entrapped within this and were the predominant cells in the inflammatory infiltrate in the underlying tissue. The infiltrate reached its maximum after 4 days and was only mild. The endothelial cells lining the superficial blood vessels exhibited degenerative changes after 2 days but, at 4 days, the endothelial cell proliferation was prominent.
Epithelial cell migration was apparent at 4 days and continued until completed at 28 days. The regenerated epithelium was thinner than in the adjacent undamaged tissue and showed only shallow rete ridges even after 42 days. The process of epithelial cell migration over the damaged surface seemed to be less well controlled than in the surgical defects and in most specimens areas of fibrinous coagulum were covered by the epithelium at the edges of the wound. In healing by secondary intention in conventional wounds, the epithelial cells normally proliferate along the boundary between the surface coagulum and the underlying granulation tissue. Few myofibroblasts were present beneath the damaged surface, in contrast to the large numbers present in the surgical sites. However, small numbers appeared in the newly-constituted lamina propria following epithelialization at the periphery of the wounds (Fig. 5). As the epithelialization progressed, a greater area of healing tissue contained myofibroblasts although these were in general restricted to the subepithelial tissue and were few. After the completion of epithelialization at 28 days, a thin band of myofibroblasts was present beneath the epithelium across the full width of the wound (Fig. 6). These cells were fewer at 42 days, when there was some reformation of collagen.
The differences between the two types of wounds were significant. The zone of tissue damage adjacent to the laser defects was narrow, and is believed to be related to the mechanism of tissue destruction (Hall et al., 197la,b). A laser wound is not a burn, but rather there is almost instantaneous vaporization of the intra-cellular fluid and thus disintegration of cell structure. Such cell destruction might not release the chemical mediators of inflammation. At the same time, vaporization of extracellular fluid takes place together with denaturation of the structural proteins. The thin layer of denatured collagen on the surface of the lasered area probably acts as an impermeable dressing in the immediate post-operative period and reduces the degree of tissue irritation from the oral contents. The marked difference in the extent and intensity of the inflammatory cell infiltrate between the two types of wound and the delay in the formation of a surface fibrinous coagulum at the lasered site support this hypothesis. Generally, the tissue architecture around the lasered area showed little alteration throughout the healing period. The lack of contraction may well account for the delay in complete re-epithelialization, because the surface area to be covered is greater. As epithelial migration started at approximately the same time in the two types of defects, the greater surface area of the laser wound requires a longer period of time for epithelialization to reach completion. However, such a hypothesis must be based upon the assumption that there is no difference in the mitotic rate of the epithelium in the two situations. As the degree of inflammatory infiltrate and thus presumably of tissue irritation is greater in the surgical area, this assumption may not be correct.
Healing of laser and surgical wounds The minimal degree of contraction following laser irradiation is probably a consequence of the small numbers of myofibroblasts present. This cell is now accepted as the effector of wound contraction (Gabbiani, Ryan and Majno, 1971; Montandon, D Andiran and Gabbiani, 1977). Following surgical excision, myofibroblasts were present in large numbers parallel to the surface so that contraction in their longitudinal axis resulted in reduction in the size of the defect. In contrast, only a few myofibroblasts were seen following laser irradiation and these were largely restricted to the newly-constituted lamina propria after each part of the wound had been re-epithelialized. In addition, the distribution of these cells in the tissue lacked orientation, and thus their contraction would have little effect on the overall dimensions of the defect. The reason for the small number of myofibroblasts in the laser wound is not clear although it is probably related to the minor degree of tissue irritation. wish to thank Coherent Ltd for the loan of the 450 series carbon dioxide surgical laser.
Gabbiani G., Ryan ence of modified
G. B. and Majno G. (1971) The presfibroblasts in granulation tissue and
their possible role in wound contraction. Experientia 27, 549-550. Hall R. R. (1971) The healing of tissues incised by a carbon dioxide laser. Br. J. Surg. 58, 222-225. Hall R. R., Beach A. D., Baker E. and Morison P. C. A. (1971a) Incision of tissue by carbon dioxide laser. Nature, Lond. 232, 13 l-l 32. Hall R. R., Hill D. W. and Beach A. D. (1971b) A carbon dioxide surgical laser. Ann. R. Coil. Surg. Engl. 48, 181~188. Kramer I. R. H., El-Labban N. and Lee K. W. (1978) The clinical features and risk of malignant transformation in sublingual keratosis. Br. dent. J. 144, 171-180. Mester E., Spiry T., Szende B. and Tota J. G. (1971) Effect of laser rays on wound healing. Am. J. Surg. 122, 532-535. Mihashi S., Jako G. J., Incze J., Strong M. S. and Vaughan C. W. (1976) Laser surgery in otolaryngology-interaction of CO2 laser and soft tissue. Ann. N.Y. Acud. Sci. 267, 263-294. Montandon D., D’Andiran G. and Gabbiani G. (1977) The mechanism of wound contraction and epithelialisation: clinical and experimental studies. C/in. Plast. Surg. 4, 325-346. Pindborg J. J. (1980) Oral Cancer and Precancer. Chap. 23, p. 148. Wright, London. Strong M. S., Jako G. J., Polanyi T. and Wallace R. A. (1973) Laser surgery in the aerodigestive tract. Am. J. Suru. 126. 529-533. Tuffin”J. R. and Carruth J. A. S. (1980) The carbon dioxide surgical laser. Br. denr. J. 149, 255-258.
Sheila E. Fisher
Fig. 1. Surgical wound, 2 days. The wound surface is covered by a thick, fibrinous coagulum densely infiltrated by polymorphonuclear leucocytes. Darkly-staining degenerate endothehal cells line the superficial blood vessels. In the deeper layers, the inflammatory cell infiltrate is predominantly perivascular. Haematoxylin and eosin. x 28 Fig. 2. Surgical wound, 7 days. This area, just deep to the surface coagulum, contains numerous myofibroblasts aligned parallel with the surface of the wound which is towards the top of the field. They are separated into columns by newly-formed capillaries running at right angles to the surface. Haematoxylin and eosin. x 64 Fig. 3. Surgical wound, 28 days. The surface is completely covered by a layer of stratified squamous epithehum which is thinner than normal epithelium and has fewer, shallower rete ridges. Scattered myofibroblasts are present in the lamina propria. Haematoxylin and eosin. x 64 Fig. 4. Laser wound, 7 days. The surface is now covered by a thin fibrinous coagulum. scattered polymorphonuclear leucocytes are present throughout the tissue. Haematoxylin x 64
a few eosin.
Fig. 5. Laser wound, 14 days. Part of the healing margin of the wound covered by a newly-formed layer of stratified squamous epithehum. Randomly-orientated myofibroblasts are present in the lamina propria. Haematoxylin and eosin. x 64 Fig. 6. Laser wound, 28 days. The surface is completely covered by a layer of stratified squamous epithelium which is thinner than the adjacent epithehum and has fewer, shallower rete ridges. Scattered myofibroblasts are present in the superficial layers of the lamina propria. Haematoxylin and eosin. x 64
of laser and surgical