Burns, 1, 13-22
Laser wound healing compared with other surgical modalities Kumio Hishimoto, The First Surgical Department,
University of Tokyo, Tokyo, Japan
R. James Rockwell, jun. Directing Physicist of Laser Laboratory and Associate Professor of Laser Science, Department of Dermatology and Otolaryngology and Maxillofacial Surgery, University of Cincinnati
Robert A. Epstein Directing Engineer of Laser Laboratory, University of Cincinnati
James P. Fidler Consultant for the Shriners Burns Institute, Director of Laser Surgery at the Laser Laboratory, and Assistant Professor of Surgery,. University of Cincinnati, Department of Surgery Summary
In this study the measured rate of healing of identical excisional wounds produced by the carbon dioxide laser and three other surgical modalities, viz.: conventional scalpel blade, electrosurgical (Bovie) unit, and plasma torch are compared. Differences in the gross and microscopic findings are discussed and partially explained. These studies would indicate that portable and easily controllable lasers have the potentiality of becoming a new type of practical surgical-knife and haemostatic instrument. THE laser is currently being investigated in numer-
ous research departments as a new type of surgical instrument. The development of the high-power continuous wave (CW) laser has prompted its trial application as an instrument for thermocoagulation and excision of tissues. Specifically, the CW carbon-dioxide laser which emits a beam of invisible radiation in the far infra-red spectrum has been used experimentally to incise, dissect and coagulate tissues. Although it is stressed that laser surgery at present remains in an investigative stage of development, the laser has been examined for various surgical procedures on highly vascularized tissues such as blood vessel tumours (Goldman and Rockwell, 1971X liver,
(Mullins et al., 1968; Roberts and Brayshaw 1969) gastric mucosa (Goodale et al., 1971) skin Hall et al., 1971), and the brain (Stellar et al., 1970). The purpose of these studies has been to determine whether laser surgical procedure may be conducted with less haemorrhage and tissue damage than is encountered with the conventional scalpel or thermoscalpel; namely electrosurgery. cryosurgery, or plasma surgery. Since each of the various surgical instruments causes an effect upon the rate of wound healing, the present study was undertaken to compare the wound healing rates obtained with the carbondioxide laser, the standard cold-knife scalpel, the high-frequency electrosurgical (Bovie) scalpel and an experimental plasma-torch scalpel. The open wound system as proposed by Grill0 and Potsaid (1958) and other investigators and (Abercrombie et al., 1954; Billingham Russell, 1956) was used for these investigations. Fuil-thickness 2 cm-square dermal defects were allowed to close by secondary intention via the processes of epithelialization and contraction. A comparative study was designed to follow the healing rate as determined by intermittent measurement of the reduction in wound area until closure.
INSTRUMENTATION The laser system (Polanyi and Bredemeier, 1969) used in these experiments was a carbon-dioxide gas laser specifically developed for use in surgical research (Fig. 1). The laser system produces a continuous beam in the far infra-red spectrum at a wavelength of 10.6 micrometres. The device provides its useful output at the distal end of a
Fig. 1.-Carbon dioxide surgical laser system. Beam is conducted via multiple reflections, through the flexible articulating arm to the handpiece. Beam is focused by a lens located in the handpiece. surgical manipulating arm. The laser beam is focused by an Irtran-II lens with a focal length of 51 mm attached in the handpiece. The laser power available at the handpiece may be varied from zero to a maximum of 35 watts. In this range, the spot-size diameter of the focused laser beam varies from 0.4 to 0.6 mm. The maximum power density is 18.8 kw per cm2 at the focal plane of the lens. A focused laser beam of 10 watts providing an approximate power density of 6.9 kw per cm2 was used for excising the skin in these investigations. Preliminary studies had indicated that this produces an optimum incision of the skin in terms of accuracy and speed while producing a minimum of thermal damage. Haemostasis was accomplished, when necessary, by coagulation with a partially focused 10 watt beam at a position approximately 1 cm beyond the focal plane of the lens. The handpiece of the surgical arm was wrapped with a sterile drape for aseptic procedures.
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The high-frequency electrosurgical scalpel used for this comparative study was the conventional Bovie Unit. The electrosurgical skin excisions were performed using a cutting control setting of ‘30’ and a haemostatic control setting of ‘2’ (or ‘moderate ‘). This cutting current produced light dehydration of severed tissue edges to arrest capillary oozing. The plasma scalpel used for the comparative study was an experimental thermosurgical device (Brayshaw; Hishimoto et al., 1969) which employs a high-radiofrequency current (80 megahertz) to generate from neutral argon gas a tiny thermal plasma with a minimum temperature of 7000 K”. A 1 x 8 mm hyperthermal .jet of ionized gas, the plasma, is emitted from the handpiece tip. Interaction of this plasma will vaporize tissue, seal the vessels by heat, and produce a sharp non-haemorrhagic incision. For the comparative study, operational parameters were determined by trial incision which resulted in reliable haemostasis, with minimal marginal charring. Skin incisions were made using an argon gas pressure of 10 lb per in2 through a 0.007 in bore in the surgical handpiece. This combination produced a 6-8 mm long, needlelike plasma jet. Haemorrhage was controlled by holding the plasma jet l-2 mm above the bleeding point. The conventional scalpel or cold knife used for a control in these investigations was a regular new no. 10 steel blade. In an attempt to avoid the influence of suture material on the healing process, further bleeding was controlled by gauze compression. MATERIAL AND METHODS Wister strains of male albino rats with a body weight range of 300400 g were used in these studies. The animals were anaesthetized with an intraperitoneal injection of 45 mg per kg body weight of sodium pentobarbital. The skin on the back of the animal was clipped, shaved and then washed with a hexachlorophene preparation. Full-thickness wounds measuring 2 x 2 cm were made on both sides of the back. The excisional plane lie deep to the panniculus carnosus muscle, but anterior to the deep fascia. The excision of tissue was performed with the carbon-dioxide laser on one side and on the other side with: 1. A cold knife in group I. 2. An electrosurgical unit in group II (Fig. 2). 3. A plasma scalpel in group III. Eight animals comprised each group. The wounds were not dressed. Sanitary living conditions were maintained in an attempt to prevent
wound infections. No supportive treatment, either antibiotics or topical antiseptics, was administered. Crusts were left in place to fall off spontaneously. The wounds were inspected every day. Outlines of the wounds were directly traced on to a transparent celluloid film with a marking pencil at the time of cutaneous excision and at regular time intervals varying from 1 to 28 days. The area of the wound was determined by placing the celluloid tracing over a linear graph paper and
were stained with haematoxylin Masson’s trichrome.
and eosin, and by
RESULTS Healing in a cold-scalpel wound (group 1) The healing pattern was quite similar to that described by Abercrombie et al. (1954), and other investigators (Billingham and Russell, 1956; Grill0 and Potsaid, 1958, 1961). The fresh wound became dry and was covered by a thin brown film
skin excision study using the carbon dioxide laser (left side) and the electrosurgical unit (right side) immediately after surgery.
counting the total number of smallest square subdivisions which were included in the area enclosed by the wound outline. The area of the open wound was expressed as a percentage of the initial wound area by a simple ratio. The mean area and standard deviation were then computed from the data obtained from the 8 animals in each group for various days after cutaneous excision. The significant difference in the areas between the unhealed paired wounds in each group was determined by Student’s t test. A comparison of the areas in the paired groups was calculated by dividing the respective ratios; both expressed as a percentage of the initial wound area. The mean ratio and standard deviation were computed for. each group at varying time intervals. In this way the area of wound healing was determined and compared as a function of time. In additional groups of animals with similarly paired wounds, incisional biopsies were obtained from the wound edge, including the wound floor, immediately after operation and on the second, seventh, fourteenth and thirtieth postoperative days. Specimens were fixed in 10 per cent formalin solution and embedded in paraffin. Sections
Fig. 3.-Wound contraction curves for group I. The curve of solid black data points represents thecontraction of thelaser-scalpel wound. Thecurveof triangular date points represents thecontractionof thecold-knife wounds. Each data point represents the mean value of the data obtained from eight wounds. The standard
deviation of the mean is represented by the vertical bar through the data point. There is a statistically significant difference between the two contraction curves on the fifth to seventeenth day.
in several hours. This formed a tough crust covering the whole area of the open wound in 48 hours and usually remained in place until the seventh to tenth postoperative day when it separated from the underlying granulation tissue and eventually fell off. Wound-closure rates became uniform and rapid after the third to fifth day(&. 3).
A mean value of the cold-knife wound-area measured as a percentage of the original woundarea was obtained for each day and the standard deviation of the mean was determined. In group I, the wound was found diminished to : 1. 31.4 h6.9 per cent of its original size by the seventh day, 2. 20.8 &5.7 per cent by the tenth day, 3. 8.6 h1.7 per cent by the fourteenth day, and, 4. 5.0 kl.5 per cent by the twenty-first day. The wound then showed a gradual decrease in contraction rate, and the remaining granulation area was eventually covered by the advancing epidermal tissue from the wound margin to first form a reddish and then pale stellate scar by the end of one month. Healing in an electrosurgical wound (group II) Immediately after excision, the edge and base of the electrosurgical wound appeared pale, desiccated, with a thin brownish coagulated surface. A scab formed within 24 hours and usually remained in situ for lo-12 days with a progressive gain in thickness and firmness. Wound contraction proceeded rapidly between the seventh to tenth day (Fig. 5). After the scab sloughed, the granulation area continued to diminish by contraction which proceeded at an increased rate until the fourteenth to eighteenth day, followed slowly by epidermization. The electrosurgical wound was reduced to : 1. 71.6 511.4 per cent of its original area on the seventh, 2. 50.5f9.9 per cent on the tenth, 3. 18.3 k31.2 per cent on the fourteenth, and, 4. 3.8 + 1.5 per cent on the twenty-first day. Healing in a plasma-scalpel wound (group Ill) Immediately after surgery, the inspected plasmascalpel wound revealed a uniform black wound edge measuring less than 1 mm in thickness. The wound was covered with desiccated charred thermocoagulated tissue which formed a thick hard scab during the next few days. Erythema and oedema usually developed in the skin adjacent to the wound edge within 3-5 days after surgery. This lesion eventually became necrotic and was gradually demarcated to form a picture-frame eschar around the open wound. The eschar delayed the shrinkage of the wound until it sloughed sometime between the tenth to fifteenth day, after which contraction was observed to progress rapidly and permit complete epidermal coverage by the end of the fourth week.
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A markedly retarded contraction rate was observed in the plasma-scalpel wound compated to all other scalpel wounds during the first 21 days (Fig. 5). The plasma wound contracted only to : 1. 80.0 k7.3 per cent of the original area by the seventh day, 2. 74.3 k9.0 per cent by the tenth day, 3. 43.3 f5.4 per cent by the fourteenth day, and, 4. 16.7 14.1 per cent of the initial wound area remained unhealed by the twenty-first day. Healing in a laser-scalpel wound (groups I, II and Ill) The laser wound appeared dry, brownish and of normal thickness immediately following the surgical procedure. Within a few hours the wound usually became oedematous and anexudate formed to a greater degree than in the electrosurgical wound but to a lesser degree than in the cold-knife wound. Thermocoagulation of th : wound margin left a brownish surface more prominent than the electrosurgical wound (Fig. 2). These minor morphological differences, however, became difficult to detect after scab formation which usually occurred within 24 hours. Only by close observation could an irregular incision in the laser wound be discerned. This difference was attributed to the technical problems associated in moving the bulky articulated arm in a uniform straight line over the intended incisional path. Subsequent gross morphology and healing rate was roughly identical to that of the electrosurgical wound (Fig. 5). The area of the laser wound contracted to: 1. 67.3 h7.6 per cent, 70.1~8.5 per cent and 62.5+8.7 per cent of its original area in each group by the seventh day;
Fig. 4.-The laser-scalpel wound (left) versus the coldknife wound (right) on the fourteenth postoperative day.
Hishimoto et al. : Laser Wound Healing
2. 54.7 -t8.l per cent, 52.45 11.5 per cent and 47.7 -k-7.3 per cent by the tenth day; 3. 22.2*3.1 per cent, 18.1 13.1 per cent, and 15.8 t4.5 per cent by the fourteenth day; and, 4. 7.0 t2.2 per cent, 3.8 +6.7 per cent and 7.8 1 2.0 per cent by the twenty-first day.
The laser versus the cold knife (group I) (Figs. 3, 4) There was a delay in contraction of area in the laser wound as compared with the cold-knife wound. When the data of the two modalities were compared at various times after surgery there was statistically a highly significant difference in the mean contraction rate beginning between the fifth to seventh day (P. 0901). When the areas of each wound were compared in the individual animal at various times after
Fig. S.-Wound contraction curves for group II. The curve of solid black data pointsrepresents thecontraction of the laser-scalpel wounds. Thecurveof triangular data points represents the contraction of the electrosurgical wounds. Each data point represents the mean value of the data obtained from 8 animals. There was found no significant difference between the two contraction curves.
Fig. 6.-The laser-scalpel wound (left) versus the electrosurgical wound (right) on the fourteenth postoperative day.
The gross appearance between the scars from the wound made with these different types of surgical scalpels showed no difference 2-10 months after operation with an exception that the healing scar of the plasma-scalpel wound has a tendency to be of slightly larger area than the other scalpel wounds. Table I.-Time
to initial wound
The laser versus the electroscalpel (group II) (Figs. 5, 6) When the laser-scalpel wound is compared with the electrosurgical wound, the contraction curves
Mean value of normalized area ratio? Group II Group I (laser/Bovie) (laser/cold knife) 1.6&0.24 2.3hO.47 2.BhO.88 2.3hO.84 1.450.57
5 7 10 14 21 * Normalized
the laser wound was found to be, on the average, I .6 to 2.8 times larger than the coldknife-wound with the maximum area difference on the tenth day (Table I-group I). Thus, the laser wound contracts at a slower rate than the cold-knife-wound with maximum retardment occurring on the tenth post-incisional day.
t Variance expressed as standard deviation
1.0&0.2 1.0+0.2 1.0*0.2 1 .010,06 1.4*0.3
Group Ill (plasma/laser) 1 ,l +O,ll 1.3kO.16 1 .6+0.15 3.6Jr2.15 2.2 +0.62
No statistically show a marked similarity. significant differences were found between the contractions of the two wounds using the mean value of wound areas expressed as a percentage of the respective initial wound areas (P>O.l). The mean ratios of the laser and electrosurgical wound-areas obtained from the individual animals maintained a value of 1.0 with a standard deviation ranging from 0.06 to 0.27 until closure of the wound (Table I-group II). Thus, the contraction process appears to proceed at a similar rate throughout the entire healing period until the two wounds are epithelized. The laser versus the plasma scalpel (group III) (Figs. 7, 8) Contraction of the laser wound proceeded at a much faster rate than the plasma wound. There was statistically ‘highly significant ’ (PP>OGM) differences in mean percentages of contractions between the
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laser and plasma wounds on the seventh through twenty-eighth days. The plasma wound area was found to be, on the average, 1.3, 1.6, 3.6 and 2.2 times larger than the laser wound on the seventh, tenth, fourteenth and twenty-first days respectively (Table Z-group III). Thus, the laserwound coverage proceeds at a significantly faster rate than the plasma-torch wound coverage. HISTOLOGICAL OBSERVATION Following incision with the carbon dioxide laserscalpel the cutaneous margin reveals nonspecific changes markedly similar to that observed in electrosurgical wounds (Figs. 9, 10). The cut margin in both thermoscalpel wounds shows a thin homogeneous band of charred tissue with an underlying narrow zone of thermocoagulation necrosis which extends into the epidermal, dermal and hypodermal tissues and includes the skin appendages. Immediately adjacent is a cutaneous zone which presents the same histological characteristics as a second- or third-degree skin bum. This is composed of basophilic necrotic and necrobiotic cells which preserve the original skin architecture. Precise definition of these zones, however, is difficult owing to the inhomogeneities in the transitional regions. There are multiple fine vacuoles within and between the cells in the zones surrounding the incision edge. The exposed cut surface of the capillaries or small vessels in the deep dermal and hypodermal tissues is sealed with a charred thrombotic mass and the intravascular erythroThe histological cytes are conglomerated. characteristics present in the fresh laser-scalpel wound compare both qualitatively and in the
Fig. ‘I.--Wound contraction curves for group II. The curve of solid black data points represents the contraction of the laser-scalpel wounds. The curve of triangular data points represents the contraction of the plasma-scalpel wounds. Each data point represents the mean value of the data obtained from 8 animals. The standard deviation of the mean is represented by the vertical bar through the data point. A significant difference is found statistically between the two curves from the seventh to twenty-eighth days.
Fig. I.-The laser-scalpel wound (left) versus the plasma-scalpel wound (right) on the fourteenth postoperative day.
relative degrees of thermal tissue damage to the incisional wound produced by the electrosurgical scalpel. Both appear indistinguishable upon routine microscopy. The wound produced by the plasma scalpel microscopically displays an irregular, torn appearance (Fig. 1 I ). The tissue destruction by charring and coagulation in the cutaneous margin is greater in the case of the plasma scalpel than for the electrosurgical or laser methods. There are vacuolized lesions in tissues adjacent to the cutaneous margin which must be differentiated from the intracellular or intercellular vacuoles generated along the path of the incision produced by either the laser or the electrosurgical modalities
The healing process is histologically similar in the wounds produced by all three thermoscalpels. In the charred and burned tissue zones of the cutaneous margin, necrotization of the irreversibly damaged tissues and demarcation of the necrotized tissue zone is apparent within a few days. The demarcation line between reversible and irreversible thermal injury becomes recognizable both grossly and microscopically by the third post-excisional day. At this time a thin filmlike scab is formed from exudative products and migrated cells of the wound. The mass of charred and necrotic tissues constitute a principal part of the thick firm eschar in all thermoscalpel wounds. The healing processes observed microscopically in the laser wound are found synchronously in
Fig. 9.-Cutaneous margin incised with the carbon dioxide-laser-scalpel. Note the charring and coagulation necrosis. Haematoxylin-eosin stain. ( ‘: 115.)
Fig. lO.-Cutaneous margin incised with the electrosurgical knife. Morphology is similar to the carbon diocide laser wound. Haematoxylin-eosin stain ( i 115.)
Fig. 1 I.-Cutaneous margin incised with the plasma scalpel. Note the broad zone of carbonization and coagulation necrosis. Characteristic vacuolization is seen in the dermal and hypodermal tissues. Haematoxylin-eosin stain. ( 2 115.)
12.-Laser-scalpel wound on the twenty-first postoperative day. Histology appears indistinguishable with the electrosurgical type of wound healing. Haematoxylin-eosin stain. (7 115.)
20 the electrosurgical wound from both quantitative and qualitative criteria, and differences in wound healing is difficult to differentiate microscopically (Fig. 12). Healing of the plasma-scalpel wound eventually yields the same histological findings as that of the laser-scalpel wound, but proceeds at a much slower rate. Histologically there is no unique effect in the process of epidermization of the laser-scalpel wound when compared with that of the coldknife, electrosurgical, or plasma-surgical wounds. Morphological findings suggestive of ionizing radiation effects were not detected in the healing processes produced by the laser, plasma torch, or the electrosurgical unit. CONSIDERATIONS In these experiments, the closure rate of an open granulating wound was used for comparative quantitative studies on wound healing. Suitable precautions were taken throughout to minimize the influence of secondary factors which could alter closure rates. The secondary locational, topographical, dimensional, infectious and therapeutic factors involved in the wounds were avoided in part by making two identical, symmetrical wounds simultaneously on the back of the same animal. No parenteral or topical treatments were administered. Although severe infection was rare, the small number of animals which did form an abscess or develop sepsis were excluded from the series. Mild infections, however, did not affect the general rate or pattern of the wound contraction. Discrepancies in data between the laser wound and the other three groups is attributed to seasonal or colonial factors. Hence, comparisons of the woundclosure rate is only significant when made within each group. Wound healing is also affected by the technical skill of the surgeon. Variations due to sugical skill were minimized since each wound was made by the same surgeon who was experienced in using all four modalities. The full-thickness cold-knife incision was performed in a single stroke to expose the deep fascial layer. In the other surgical modalities, the optimum incisional techniques were determined by preliminary investigations. The methods were developed empirically to produce a full-thickness, 2.5-3.0 mm deep incision in the most rapid but least traumatic manner. Dissection of the skin flap from the deep fascial layer was removed by dissection by using the particular surgical instrument under consideration. These studies indicate that the precision of performing an incision with the laser scalpel
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presently available is inferior to the electrosurgical unit. This is mainly attributed to limitations in flexibility and manipulation of the cumbersome articulating arm which permits manoeuvrability of the laser beam. There is difficulty maintaining the beam at its focal point and precisely moving the beam along a proposed incision line. The flexibility and compact design of the plasma-scalpel handpiece also accounted for the increased facility in incisional performance of this scalpel compared to the carbon dioxide laser scalpel. However, incisions performed with the plasma scalpel were more difficult than those produced with the electrosurgical scalpel. This was in part due to an insufficient plasma torch output which did not produce rapid and sharp separation of tissue. In addition, it was also difficult to move the plasma scalpel precisely along the intended incisional path. The plasma scalpel accomplished haemostatic control over capillary oozing along the wound edge only by carbonization of tissue to a greater extent than both the other thermoscalpels. There is also moderate difficulty in directing the plasma jet on a bleeding vessel. These investigations have shown that the contraction of the laser-scalpel wound proceeds at the same rate as the electrosurgical wound; is slower than the conventional cold-knife wound; but is faster than the rate of coverage of the plasma-scalpel wounds. The contraction process in wounds healing by secondary intention has been previously explained (Watts et al., 1958; Grill0 and Potsaid, 1961) by the proliferation and migration of fibroblast cells which originate in the narrow marginal zone of the open wound edge. Tissue reaction to a highfrequency electric current, a carbon dioxide laser beam, or to a hyperthermal plasma is substantially thermal. Gross and histological examination of the wound edges have shown non-specific carbonization, coagulation, necrosis, and thermal tissue reactions similar to those produced by the usual Possible non-thermal biological heat burn. effects which are often attributed to the electromagnetic fields of the laser beam or plasma were not detected. The effects of a high-frequency electric current producing vasoconstriction, and of the hyperbaric-plasma jet causing vacuolization in the wound margin do play some minor modifying roles in the process of wound healing and should be taken into account. These studies have indicated that the major influence on closure retardation in the thermoscalpel wounds can be attributed to the thermal
Hishimoto et al.
injury as a function of the duration and degree of the thermal insult as well as the peripheral conduction of the heat away from the site of exposure. The thermally injured tissue forms a line of demarcation between the reversible and irreversible dermal injury which separates the zone of tissue capable of recovery and the necrotized tissue which eventually sloughs in the form of an eschar. The wound closure is affected by the formation of a firm eschar at the cut edge which inhibits the contraction and epithelization processes. Heat transfer in tissues occurs primarily by conduction and, owing to the relative low thermal conductivity of the skin, the temperature gradient in the edges of an incision produced by the carbon dioxide laser will be very large. We have previously (Mullins et al., 1968) shown that the temperature rise measured 5 mm from an incision in rat skin with a focused 20 watt carbon dioxide laser beam moving at a velocity of 20 mm per set was not greater than 2-3’C and lasted only about 2.5 seconds. This produces a temperature gradient in the 1 mm region immediately adjacent to the incision in the order of 75” C per mm. As a result, the thermal injury is limited to a very narrow (200-500 micrometres) zone of the wound margin since the temperature rise in the tissues adjacent to this zone will remain below the temperature-exposure-threshold criteria as determined by Moritz and Henriques (1947) and Moritz (1967) to devitalize tissue. Heat produced by the plasma scalpel is transferred to the peripheral tissues by convection and conduction. Experimental data of Roberts and Brayshaw (1969) have demonstrated a temperature increase which remains within 2-3°C at 2 mm peripheral to the incision. The broad necrotic band which occurs in plasma surgery may be explained by the prolonged exposure duration from lack of cutting power and from formation of vacuoles in the cutaneous margin. In contrast, the high-frequency electrosurgical unit produces a heating effect which is not localized but broadly extends throughout the neighbouring tissues. This can cause vasoconstriction and local ischaemia, and possibly injure the wall and induce a thrombotic occlusion of the vessels peripheral to the wound edge. Owing to a high energy distribution, tissue damage can reach to a considerable distance, as indicated by Shaw (1966). It is of interest that our data suggests that the electrosurgical wound, despite these shortcomings, shows a superiority in respect to healing ability in comparison to the plasma scalpel. Our investigations indicate that the present
carbon dioxide laser system is comparable to the conventional electrosurgical (Bovie) unit with respect to localized tissue necrosis and wound contraction rate. The impaired healing of the carbon dioxide laser wound, in comparison to the cold-knife scalpel wound, can be attributed primarily to the dermal tissue injury caused by the delivery of excessive thermal energy. Acknowledgements We wish to express our sincere appreciation to Dr Leon Goldman and Dr V. E. Siler for their valuable advice and encouragement during this research project and to Mr Ronald Dreffer, Marilyn Franzen and John Johnson for their invaluable technical assistance.
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Elland and Bethesda