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Viability of skin subjected to deep partial skin thickness thermal damage: experimental studies
Bums (1991) 17,(I), 17-24 Printed in Great Britain
17
Viability of skin subjected to deep partial skin thickness thermal damage: experimental studies J. Smahel Division of Hand, Plastic and Reconstructive Switzerland
Secoda y
Surgery, Department
tisw lossinbum wounds (due to necrosis in thezoneof stasis) is
infer-p&d as a sequel of progressive vascular occulsion and dehydration of fhermally damaged tissue. In thisstudyon rafs
Introduction Contact with heat may cause three types of pathological changes in the skin - superficial, deep partial and full skin thickness thermal damage. Superficial damage involves relatively minor changes that are spontaneously reversible. Deep partial skin thickness damage results in pathological processes that, as a rule, end in loss of the affected tissue about 1 week after the trauma. Full skin thickness damage characteristically means immediate tissue necrosis. Heat sources that give rise to full skin thickness damage also produce deep partial and superficial damage concentric to the superficial coagulation necrosis, so that the wound shows three zones reflecting different degrees of damage. Jackson (1953) introduced the terms ‘zone of coagulation’, ‘zone of stasis’ and ‘zone of hyperaemia’. Efforts to reduce tissue loss from bums to a minimum have, for obvious reasons, concentrated on deep partial skin thickness damage (the zone of stasis) which is thought to be potentially reversible (Jackson, 1969). Loss of this zone is considered to be due to progressive vascular occlusion resulting in ischaemia and tissue dehydration. Numerous attempts to prevent or reverse the progressive vascular occlusion with vasoactive agents or various other drugs have generally proved unsuccessful (for survey see Zimmerman and Krizek, 1984; Arturson, 1985; Smahel, 1985a). The experiments with thromboxane inhibition have given promising results (Robson et al., 1979,198O; Heggers et al., 1985). The opposite has, however, also been reported (Ehrlich, 0 1991 Butterworth-Heinemann Ltd 0305/4179/91/010017-08
of Surgery, Zurich University
Medical School, Zurich,
1984). Zawacki (1974a,b) and Saranto et al. (1983) are of the opinion that dehydration plays a key role in secondary tissue loss. One charaderistic feature of the burn wound is the late onset of healing. It may be assumed that burn wounds initially lack the stimulus that triggers the healing process. The phenomenon probably has its cause in the gradual character of thermal damage which hampers the diffusion of chemical messengers (Winter, 1975). Delayed healing may be another factor in determining the fate of tissues in the zone of stasis (Smahel, 1985b). The present study was designed to establish the effect of early revascularization on the fate of skin that has been subject to deep partial skin thickness thermal damage.
Materials and methods Female SW 50 strain rats (n= 160), weighing 280-310 g, were used. They were kept in single cages and given food and water ad libitum. All experimental procedures were done under ether anaesthesia.
The bums Bums were produced using the model developed earlier (Smahel, 1976). The backs of the animals were shaved with electric clippers and cleaned with benzine. A pneurnodenna was produced by injecting 40-50 ml of air under the dorsal skin including the panniculus camosus; this resulted in a single ovoid air cell. The pneumoderma was transilluminated with a powerful light source to locate the major skin vessels before two symmetrical areas 20mm apart were selected that did not include major vessels. Aniials in whom the areas selected for burn wounds were found to be in the growing stage of the hair cycle (anagen) were replaced. Bums were produced by applying a heated 50-g brass weight of l9mrn diameter. The combination of a temperature of 60°C and 15-s exposure resulted in deep partial skin thickness damage to the whole skin area. After a IO-min interval to allow for spontaneous cooling of the wound the pneumoderma was collapsed by withdrawing the air.
Bums (1991) Vol. 17/No. 1
18
Figure 1. Excised bum wound (right) and control wound (left).
Figure 2. Bum wound with replanted skin (right), control wound on left.
Surgical procedures One of the bums served as an untreated control on each animal, the other was excised close to its outer margin. Tissues removed included both the skin and the thin muscular layer (panniculus carnosus). Excision started with a scalpel and continued with blunt scissors; care was taken not to traumatize the loose suprafascial connective tissue which was left in situ (Figure 7). The panniculus carnosus and adipose tissue were then removed from the excised skin disc. Replantation of the burned skin into the wounds from which it had been excised was done on 80 animals each in two variations. Variation A involved replanting the excised skin disc as a free ti.~Uthickness skin graft. For variation B the excised skin was first split into two layers of approximately equal thickness and then replanted as a two-layer graft. A simple device assisted the splitting operation (Smahel, 1986). The skin is held firm on a perforated stage by applying negative pressure and split by taking the blade through tangentially. All grafts were held in place with eight symmetrical sutures using 6-O silk (Fimre 2). Variation B also included experiments where only the lower or upper skin layer was replanted. All bums were given the same dressing, consisting of a layer of petroleum jelly gauze followed by gauze padding and adhesive tape. They were checked daily and renewed as necessary. To prevent damage to the wounds, the animals were put in protective rubber vests (Smahel, 1984). As the replanted wounds were unable to epithelialize spontaneously the closure of the wounds was examined by covering them with autologous split skin grafts starting within 3 weeks after surgery. For this purpose, split skin of variable thickness was removed with a small dermatome from either the caudal part of the dorsum or the abdominal wall. The grafts were held in place by applying a dressing only.
the vessels, 4 x 4 cm squares of dorsal skin with the bum at the centre were excised, fixed in Camoy’s solution and cleared in glycerin. Following assessment of the whole preparation under a stereomicroscope, the glycerin was removed in some cases by washing in water and 40-pm frozen sections were produced. For histological examination the material was fixed in formalin and embedded in paraffin. Sections (6-10 pm) were produced and stained with haematoxylin and eosin (HE) and by the van Gieson technique (vG) using the Hansen modification.
Investigations Investigations continued for up to 6 weeks after producing the burns and comprised clinical investigations, visualization of vessels in healing wounds and histological studies. NO fixed time schedule was required, as healing of the treated wound was always compared with healing of the control wound in the same animal. Vessels were visualized in one-third of the animals by perfusing the vascular system with black Indian ink and gelatin that had been heated to 55°C (Smahel, 1976). On average 3Oml of the ink plus gelatin was injected into the ascending aorta per animal. Following gelation of the ink in
Results Observations relating to control wounds Immediately after exposure the burned skin showed notable paleness, followed after some minutes by a reddish pink colouration. Oedema reached a peak after about 4 h and then slowly regressed, to disappear completely during the next few days. Parallel to the reduction in oedema the bum dried out and towards the end of the first week all wounds were found to be covered with brownish scabs. Perfusion of the vascular system on day 2 after producing the burn showed complete vascular occulsion with sludged blood. Signs of healing were not noted until the end of the first week at the earliest, when capillary proliferation and granulation tissues developed in the lower margin of the wound as the first stage in demarcation of necrosis (Figure 3). Skin loss was full thickness in all wounds. The necrosis had characteristically a dish-shaped form. At the periphery of the bum, only the skin was involved, at the centre also the subcutaneous fat layer and part of the panniculus carnosus. Spontaneous sloughing of necrotic tissues started during week 3 after producing the bum (Figure 4). Wound contraction had started in the meanwhile, with the wounds getting considerably smaller. When the necrotic tissue had sloughed off, the wounds were filled with granulation tissue, with epithelialization already starting in the periphery. The final outcome was a small atrophic scar. Observations relating to experimental variation A The paleness of the burned skin after excision persisted during the first few days following replantation. The surface then began to dry out, similar to the control wounds, and a scab formed. Characteristically replanted wounds showed little oedema. Capillary and cell proliferation analogous to that noted in control wounds was evident in the wound base by day 3 after producing the bum. Vascular proliferation showed rapid progression, with capillaries invading the
19
Smahel: Skin viability after thermal damage
Figure 3. Proliferation of capillaries (filled with Indian ink) and cells at lower margin of subcutis (arrows). Note also occluded vessels at lower margin of corium. Control wound, day 8 after producing the bum. (vG, x 65.)
Figure 4. Spontaneous sloughing of full-thickness necrotic skin; granulation tissue in wound base. Control wound, day 20 after producing the bum. (vG, x 110.)
Figure 5. Newly formed vascular networks in base of excised bum wound, day 8 postoperation. Unstained total skin preparation seen from below; vessels filled with Indian ink. ( x 5.)
Bums (1991) Vol. 17/No. 1
20
Figure6.
Revascularization of replanted corium. Variation A, day 10 postoperation. section, vessels visualized with Indian ink. (HE, x 110.)
Frozen
Figures.
Wound with replanted skin (variation A) following separation of necrotic tissue. On the left, beginning of spontaneous epithelialization from wound margin, day 18 postoperation.
Figure 7. Demarcation of necrotic corium layer (arrow). Variation
A, day 12 postoperation. (vG,
x
65).
replanted skin. Assessment at the beginning of the second week after producing the burn showed dense capillary networks in the wound base (Figure 5) and advanced revascularization of the replanted skin. Considerably more vessels had filled on perfusion in replanted skin than usually
seen in the skin; they generally ran at right angles to the skin surface (Figwe6). During the second week, demarcation of the necrotic area started in the graft, which had by now united with the wound base. The lower half of the replanted skin was, as a rule, revascularized and survived, the upper half became necrotic and dried to a scab (Fiere 7). At this stage, the difference in wound contraction also became evident, with control wounds growing progressively smaller whilst the regrafted wound areas remained almost the original size. Spontaneous sloughing of the necrotic surface corium layer was very slow, often extending into week 4 after producing the bum. The surviving corium layer that became exposed showed local differences in blood supply; epithelium had already formed in the margins of the wound (Figure 8). The healed corium showed a moderate degree of mononuclear infiltration and no appreciable change in its collagen structures. Hair follicles and sebaceous glands on the other hand had degenerated almost completely as the graft healed (Fipre 9). Small groups of surviving epithelial cells at the periphery of the graft were the exception. As healing progressed, the surviving coriun
Smahel: Skin viability after thermal damage
Figure 9. Degeneration of hair follicles and sebaceous postoperation. (HE, x 220.)
glands
in replanted
corium,
day
18
Figure 10. Granulation tissue masking the healed corium layer, day 20 postoperation (variation A). (vG, x 220.)
Figure 11. Healing of lower, necrosis of upper corium layer, day 16 postoperation (variation B). (vG, x 65).
tissue (F&u-e IO).
in much the same way as in variation A, except that surface drying of the replanted skin was faster. Healing also gave the same results as in variation A, so that the lower layer of replanted skin was revascularized and survived, whilst the upper layer became necrotic (F&we II). As there was no organic relationship between the two layers in this case, the long process of sloughing off the necrotic tissue was avoided. It proved easy to establish the best moment for
layer began to be covered with granulation Spontaneous epjtheli~~tion from ‘the
wound margins progressed only slowly and was still in its initial stage at the end of the 6-week observation period.
Observations
relating
to experimental
variation
B
The split skin layers adhered when superimposed and acted as a single graft when replanted. Wound healing proceeded
22
Burns (1991) Vol. 17/No. 1
Figure 12. Superficial skin graft lifted up to assess healing. Variation B.
Figure 13. Layer (marked by arrows) connecting healed corim (below) and split skin (above), day 6 postoperation.
(vG, x ZZO.)
removing the surface graft, which served as a biological dressing; all it needed was to lift it up slightly and assess the revascularization of the underlying corium layer (Fimre 12). Removal of the upper graft, usually during the third week after producing the bum, exposed the healed corium layer beneath: this had a smooth surface with evidence of a fairly regular blood supply. As healing progressed further, masking of the healed corium layer with granulation tissue
Figure 14. Maturation of connecting layer between coriurn (below) and split skin (above), 2 weeks postoperation. (vG, x IIO.)
began. With this variation, too, the epitheliai adnexa degenerated completely. Spontaneous epithelialization from the margins of the wound was slightly better than with variation A, but the wounds still had not closed by the end of the 6-week observation period. The attempts at replanting either the lower or the upper split skin layer on its own essentially ended in failure. Revascularization did in fact start in wounds grafted with
23
Smahel: Skin viability after thermal damage
Figure 15. Healing complete in control wound (left) and replanted wound (right), 6 weeks postoperation. Replantation with split skin was in two stages. Note absence of contraction replanted wound.
in
the lower layer, but rapid drying of the graft soon put an end to the process. When only the upper layer was replanted revascularization did not develop, with small islets of corium surviving only in exceptional cases. Observations relating to wound closure with autologous split skin Wound closure with autologous split skin was effected in weeks 3 and 4 after producing the bum, when healing of the replanted corium was complete. The grafts healed perfectly if transplanted to a corium free from necrotic changes where masking with granulation tissue had started. These requirements were fairly easily met in group B, whereas in group A transplantation frequently had to be delayed or done in two stages as necrotic tissue still adhered. The connection between healed corium and transplanted split skin initially consisted of cell-rich connective tissue traversed by numerous vessels (Figctre 13). This later matured into collagen tissue, with the frequency of vessels markedly reduced (Figctre 74). The main characteristic of all treated wounds, marked reduction of wound contraction, persisted also when the split skin had healed (Figure 15).
Discussion Standardization of experimental bums is a well-known problem, particularly when relatively low temperatures are used. Use of the pneumoderma experimental model has eliminated some of the variables that in addition to the temperature of the heat source and the application time determine the degree of thermal damage. The pneumoderma technique ensures that the bum is limited to the skin, i.e. the pneumoderma wall, the deeper structures such as fascia and skeletal musculature are protected from damage. The model also makes it possible to position burns in the same relative position to major blood vessels. It had been noted during development of the model that the onset of healing was earlier in bums containing relatively large vessels. Finally the technique also ensures that skin tension is the same when both bums are made. Other variables, such as dermal blood supply, skin hydration, skin temperature at the moment the bum is produced and variations in skin thickness did however remain, and it was probably due to these that the depth of the bum varied slightly from animal to animal. It was possible to compensate for this to some
extent by always comparing the progress of excised and control wounds in the same animal. Under the given experimental conditions the combination of 15-s exposure time and a temperature of 60°C resulted in two-zone bums, with the zone of stasis involving the whole skin. Burns of this type, called zone-of-stasis burns by Saranto et al. (1983, are widely used in experimental studies. From the methodological point of view they offer the advantage that spontaneous healing leads to full thickness skin loss and the efficacy of measures to save the zone of stasis is easily assessed. Experimental methods to explore the effect of delayed onset of healing in wounds with loss of the zone of stasis were based on the realization that excision of a burn wound down to bleeding vessels, i.e. at least the zone of hyperaemia, promptly initiates healing. The phenomenon is well known with tangential excision and radical excision of small bums in clinical practice. The above experiments provide further substantiation. In the control wounds, vascular proliferation did not start before the end of the first week after producing the burn; in excised bum wounds the process came into full effect on day 3. The experiments substantiate the theory that delayed onset of healing plays a role in determining the fate of tissues with deep partial skin thickness damage. They also demonstrate clearly that the deeper layer of skin with deep partial skin thickness bums can be saved through early excision and replantation. In the control wounds, spontaneous healing regularly resulted in full thickness skin loss; in wounds treated with replanted skin more than half the corium was revascularized and survived. The outstanding feature was that wound contraction was almost completely prevented. The epithelial adnexa degenerated in the healing process. These findings bring to mind the work of Brauer and Spira (1966), who grafted burned skin onto freshly produced wounds in pigs. They compared their results to the healing of thin dermal grafts. The revascularization of replanted skin was always by second intention, i.e. due to invasion with new vessels (Smahel, 1977). This mode of revascularization characteristically involves a large number of vessels penetrating the graft at right angles to the skin surface and the fairly regular progress of revascularization from below to above. The fact the revascularization was consistently by second intention suggests that the proliferating capillaries in the wound bed did not anastomose with the vessels of the graft. The question arises as to how far the result of the grafting techniques used reflects the degree of thermal damage, i.e. the viability of the different tissue elements in the zone of stasis. On the one hand the experiments substantiated Zawacki’s view (1974a) that thermal damage in the zone of stasis had a gradient. They also showed, however, that this gradient differs depending on the tissue component involved. The endothelium appears to be most sensitive to heat trauma, for here damage extended to the lower margin of the zone of stasis, which explains why revasculanzation was always by second intention. The epithelial cells only degenerated in the course of the healing process and were therefore less sensitive to heat trauma, and the collagen structures of the corium evidently tolerated heat better than the other tissues. This implies that tangential excision, with its depth determined by vascular damage, goes too deep where damage to the corium is concerned. The evident conclusion that the outcome of replantation reflects differences in the degree of thermal damage to the zone of stasis has to be treated with some caution. Similar
24 results are known with the healing of free full thickness skin grafts where revascularization is by second intention; the grafts show deep desquamation and epithelial adnexa are generally lost (Smahel, 1977). The outcome of replantation might thus also reflect the mode of revascularization. This assumption is however negated by the experimental variation where only the superficial layer of burned skin was replanted and revascularization of the graft did not occur. Zawacki (1974a) saw similar results with upper layer grafts in bums. The almost complete absence of oedema in burns treated by excising and replanting the skin, potentially of significance in clinical practice, is explained by the fact that blood supply to the leaking vessels was stopped early. Total loss of the epithelial part of replanted skin could be to some extent specific to rat skin. In all the animals used in the experiments the skin was in the resting stage of the hair cycle, in telogen. In this stage the follicles are short, extending about two-thirds into the corium. They were therefore largely in the part of the corium that always became necrotic. At the same time rat skin does not include sweat glands, the secretory parts of which extend down to the sub&is and are therefore most likely to survive a skin bum. The absence of spontaneous epithelialization in replanted corium was overcome by the simplest experimental solution, split thickness skin grafting. From the biological point of view the use of split thickness skin was really unnecessary as there was sufficient corium in the wounds. It may be assumed that another solution would be to use cultured epithelium. The time when the wounds would be ready to be supplied with epithelium - after 2 or 3 weeks - would coincide well with the time required to culture the epithelium. The main purpose in including variation B was to assess separate grafting of either the lower or the upper layer of the burned skin. Among other things the variation showed that the lower layer will heal only if dehydration is prevented. In our experiment this was effectively achieved by the superficial layer of burned skin. Another reason for including variation B was that it comes closest to the situation liable to arise when the method would be used in clinical practice. Clinical use of the method might be considered for ‘deep dermal bums’ where tangential excision has already proved effective. The experiments have shown that this excision removes part of the corium that still has biological value and that replantation of this part may upgrade deep dermal bums to superficial dermal bums. It certainly seems feasible that the excised skin layer could also heal into another wound; the experiments done by Brauer and Spira (1966) seem to point in that direction.
Bums(l991) Vol. 17lNo.l
References Arturson M. G. (1985) The pathophysiology
of severe thermal injury.]. Bum Cure Rehabd 6, 129. Brauer R. 0. and Spira M. (1966) Full-thickness burns as source for donor graft in the pig. Plast. Reconstr. Surg. 37,~~ Ehrlich H. P. (1984) Anti-inflammatory drugs in the vascular response to burn injury. J. Trauma 24, 311. Heggers J. P., Robson M. C. and Zachary L. S. (1985) Thromboxane inhibitors for the prevention of progressive dermal ischemia due to the thermal injury. J. Bum Cure Rehbil. 6,466. Jackson D.MacG. (1953) The diagnosis of the depth of burning. Br. ]. Surg. 40, 588. Jackson D.MacG. (1969) Second thoughts on the burn wound. 1. Trauma 9, 839. Robson M. C., DelBeccaro E. J. and Heggers J. P. (1979) The effect of prostaglandins on the dermal microcirculation after burning, and the inhibition of the effect by specific pharmacological agents. Plast. Reconstr. Surg 63, 781. Robson M. C., DelBeccaro E. J., Heggers J. P. et al. (1980) Increasing dermal perfusion after burning by decreasing thromboxane production. J Trauma 20, 722. Saranto J. R., Rubayi S. and Zawacki B. E. (1983) Blisters, cooling, antithromboxanes, and healing in experimental zone-of-stasis bums. J Trauma 23, 927. Smahel J. (1976) Bum wounds. A new experimental model. Chir. %.stiui 3,231. Smahel J. (1977) The healing of skin grafts. Clin. Pk. Surg. 4,409. Smahel J. (1984) A protective rat vest. Letter to the Editor. Pkzst. ficonstr. Surg. 73, 859. Smahel J. (1985a) Pathophysiologie der Verbrennungswunde. Handchimrgie 17,340. Smahel J. (1985b) Replantation of thermally damaged tissue. In: May S. R. and Dogo G. (eds), Cure of the Bum Wound. Basle: Karger, p. 157. Smahel J. (1986) Device for splitting skin in small laboratory animals. Eur. 1. P&. Surg. 9,36. Winter G. D. (1975) Histological aspects of burn wound healing. Burns 1.191. Zawacki B. E. (1974a) The natural history of reversible burn injury. Surg. Gynecol. Obstet. 139,867. Zawacki B. E. (1974b) Reversal of capillary stasis and prevention of necrosis in burns. Ann Surg. 180,98. Zimmerman T. J. and Krizek T. J. (1984) Thermally induced dermal injury: a review of pathophysiologic events and therapeutic intervention. J. Bum Cure Rehabil. 5,913.
Paper accepted 20 August
1990.
Acknowledgement Thanks are expressed to MS B. Jentsch for her technical assistance in performing the surgical procedures and the histological examinations.
Correspondence shod be aa%emd to: Dr J. Smahel, Division of Reconstructive Surgery, University Hospital, CH-8091 Zurich, Switzerland.
17
Viability of skin subjected to deep partial skin thickness thermal damage: experimental studies J. Smahel Division of Hand, Plastic and Reconstructive Switzerland
Secoda y
Surgery, Department
tisw lossinbum wounds (due to necrosis in thezoneof stasis) is
infer-p&d as a sequel of progressive vascular occulsion and dehydration of fhermally damaged tissue. In thisstudyon rafs
Introduction Contact with heat may cause three types of pathological changes in the skin - superficial, deep partial and full skin thickness thermal damage. Superficial damage involves relatively minor changes that are spontaneously reversible. Deep partial skin thickness damage results in pathological processes that, as a rule, end in loss of the affected tissue about 1 week after the trauma. Full skin thickness damage characteristically means immediate tissue necrosis. Heat sources that give rise to full skin thickness damage also produce deep partial and superficial damage concentric to the superficial coagulation necrosis, so that the wound shows three zones reflecting different degrees of damage. Jackson (1953) introduced the terms ‘zone of coagulation’, ‘zone of stasis’ and ‘zone of hyperaemia’. Efforts to reduce tissue loss from bums to a minimum have, for obvious reasons, concentrated on deep partial skin thickness damage (the zone of stasis) which is thought to be potentially reversible (Jackson, 1969). Loss of this zone is considered to be due to progressive vascular occlusion resulting in ischaemia and tissue dehydration. Numerous attempts to prevent or reverse the progressive vascular occlusion with vasoactive agents or various other drugs have generally proved unsuccessful (for survey see Zimmerman and Krizek, 1984; Arturson, 1985; Smahel, 1985a). The experiments with thromboxane inhibition have given promising results (Robson et al., 1979,198O; Heggers et al., 1985). The opposite has, however, also been reported (Ehrlich, 0 1991 Butterworth-Heinemann Ltd 0305/4179/91/010017-08
of Surgery, Zurich University
Medical School, Zurich,
1984). Zawacki (1974a,b) and Saranto et al. (1983) are of the opinion that dehydration plays a key role in secondary tissue loss. One charaderistic feature of the burn wound is the late onset of healing. It may be assumed that burn wounds initially lack the stimulus that triggers the healing process. The phenomenon probably has its cause in the gradual character of thermal damage which hampers the diffusion of chemical messengers (Winter, 1975). Delayed healing may be another factor in determining the fate of tissues in the zone of stasis (Smahel, 1985b). The present study was designed to establish the effect of early revascularization on the fate of skin that has been subject to deep partial skin thickness thermal damage.
Materials and methods Female SW 50 strain rats (n= 160), weighing 280-310 g, were used. They were kept in single cages and given food and water ad libitum. All experimental procedures were done under ether anaesthesia.
The bums Bums were produced using the model developed earlier (Smahel, 1976). The backs of the animals were shaved with electric clippers and cleaned with benzine. A pneurnodenna was produced by injecting 40-50 ml of air under the dorsal skin including the panniculus camosus; this resulted in a single ovoid air cell. The pneumoderma was transilluminated with a powerful light source to locate the major skin vessels before two symmetrical areas 20mm apart were selected that did not include major vessels. Aniials in whom the areas selected for burn wounds were found to be in the growing stage of the hair cycle (anagen) were replaced. Bums were produced by applying a heated 50-g brass weight of l9mrn diameter. The combination of a temperature of 60°C and 15-s exposure resulted in deep partial skin thickness damage to the whole skin area. After a IO-min interval to allow for spontaneous cooling of the wound the pneumoderma was collapsed by withdrawing the air.
Bums (1991) Vol. 17/No. 1
18
Figure 1. Excised bum wound (right) and control wound (left).
Figure 2. Bum wound with replanted skin (right), control wound on left.
Surgical procedures One of the bums served as an untreated control on each animal, the other was excised close to its outer margin. Tissues removed included both the skin and the thin muscular layer (panniculus carnosus). Excision started with a scalpel and continued with blunt scissors; care was taken not to traumatize the loose suprafascial connective tissue which was left in situ (Figure 7). The panniculus carnosus and adipose tissue were then removed from the excised skin disc. Replantation of the burned skin into the wounds from which it had been excised was done on 80 animals each in two variations. Variation A involved replanting the excised skin disc as a free ti.~Uthickness skin graft. For variation B the excised skin was first split into two layers of approximately equal thickness and then replanted as a two-layer graft. A simple device assisted the splitting operation (Smahel, 1986). The skin is held firm on a perforated stage by applying negative pressure and split by taking the blade through tangentially. All grafts were held in place with eight symmetrical sutures using 6-O silk (Fimre 2). Variation B also included experiments where only the lower or upper skin layer was replanted. All bums were given the same dressing, consisting of a layer of petroleum jelly gauze followed by gauze padding and adhesive tape. They were checked daily and renewed as necessary. To prevent damage to the wounds, the animals were put in protective rubber vests (Smahel, 1984). As the replanted wounds were unable to epithelialize spontaneously the closure of the wounds was examined by covering them with autologous split skin grafts starting within 3 weeks after surgery. For this purpose, split skin of variable thickness was removed with a small dermatome from either the caudal part of the dorsum or the abdominal wall. The grafts were held in place by applying a dressing only.
the vessels, 4 x 4 cm squares of dorsal skin with the bum at the centre were excised, fixed in Camoy’s solution and cleared in glycerin. Following assessment of the whole preparation under a stereomicroscope, the glycerin was removed in some cases by washing in water and 40-pm frozen sections were produced. For histological examination the material was fixed in formalin and embedded in paraffin. Sections (6-10 pm) were produced and stained with haematoxylin and eosin (HE) and by the van Gieson technique (vG) using the Hansen modification.
Investigations Investigations continued for up to 6 weeks after producing the burns and comprised clinical investigations, visualization of vessels in healing wounds and histological studies. NO fixed time schedule was required, as healing of the treated wound was always compared with healing of the control wound in the same animal. Vessels were visualized in one-third of the animals by perfusing the vascular system with black Indian ink and gelatin that had been heated to 55°C (Smahel, 1976). On average 3Oml of the ink plus gelatin was injected into the ascending aorta per animal. Following gelation of the ink in
Results Observations relating to control wounds Immediately after exposure the burned skin showed notable paleness, followed after some minutes by a reddish pink colouration. Oedema reached a peak after about 4 h and then slowly regressed, to disappear completely during the next few days. Parallel to the reduction in oedema the bum dried out and towards the end of the first week all wounds were found to be covered with brownish scabs. Perfusion of the vascular system on day 2 after producing the burn showed complete vascular occulsion with sludged blood. Signs of healing were not noted until the end of the first week at the earliest, when capillary proliferation and granulation tissues developed in the lower margin of the wound as the first stage in demarcation of necrosis (Figure 3). Skin loss was full thickness in all wounds. The necrosis had characteristically a dish-shaped form. At the periphery of the bum, only the skin was involved, at the centre also the subcutaneous fat layer and part of the panniculus carnosus. Spontaneous sloughing of necrotic tissues started during week 3 after producing the bum (Figure 4). Wound contraction had started in the meanwhile, with the wounds getting considerably smaller. When the necrotic tissue had sloughed off, the wounds were filled with granulation tissue, with epithelialization already starting in the periphery. The final outcome was a small atrophic scar. Observations relating to experimental variation A The paleness of the burned skin after excision persisted during the first few days following replantation. The surface then began to dry out, similar to the control wounds, and a scab formed. Characteristically replanted wounds showed little oedema. Capillary and cell proliferation analogous to that noted in control wounds was evident in the wound base by day 3 after producing the bum. Vascular proliferation showed rapid progression, with capillaries invading the
19
Smahel: Skin viability after thermal damage
Figure 3. Proliferation of capillaries (filled with Indian ink) and cells at lower margin of subcutis (arrows). Note also occluded vessels at lower margin of corium. Control wound, day 8 after producing the bum. (vG, x 65.)
Figure 4. Spontaneous sloughing of full-thickness necrotic skin; granulation tissue in wound base. Control wound, day 20 after producing the bum. (vG, x 110.)
Figure 5. Newly formed vascular networks in base of excised bum wound, day 8 postoperation. Unstained total skin preparation seen from below; vessels filled with Indian ink. ( x 5.)
Bums (1991) Vol. 17/No. 1
20
Figure6.
Revascularization of replanted corium. Variation A, day 10 postoperation. section, vessels visualized with Indian ink. (HE, x 110.)
Frozen
Figures.
Wound with replanted skin (variation A) following separation of necrotic tissue. On the left, beginning of spontaneous epithelialization from wound margin, day 18 postoperation.
Figure 7. Demarcation of necrotic corium layer (arrow). Variation
A, day 12 postoperation. (vG,
x
65).
replanted skin. Assessment at the beginning of the second week after producing the burn showed dense capillary networks in the wound base (Figure 5) and advanced revascularization of the replanted skin. Considerably more vessels had filled on perfusion in replanted skin than usually
seen in the skin; they generally ran at right angles to the skin surface (Figwe6). During the second week, demarcation of the necrotic area started in the graft, which had by now united with the wound base. The lower half of the replanted skin was, as a rule, revascularized and survived, the upper half became necrotic and dried to a scab (Fiere 7). At this stage, the difference in wound contraction also became evident, with control wounds growing progressively smaller whilst the regrafted wound areas remained almost the original size. Spontaneous sloughing of the necrotic surface corium layer was very slow, often extending into week 4 after producing the bum. The surviving corium layer that became exposed showed local differences in blood supply; epithelium had already formed in the margins of the wound (Figure 8). The healed corium showed a moderate degree of mononuclear infiltration and no appreciable change in its collagen structures. Hair follicles and sebaceous glands on the other hand had degenerated almost completely as the graft healed (Fipre 9). Small groups of surviving epithelial cells at the periphery of the graft were the exception. As healing progressed, the surviving coriun
Smahel: Skin viability after thermal damage
Figure 9. Degeneration of hair follicles and sebaceous postoperation. (HE, x 220.)
glands
in replanted
corium,
day
18
Figure 10. Granulation tissue masking the healed corium layer, day 20 postoperation (variation A). (vG, x 220.)
Figure 11. Healing of lower, necrosis of upper corium layer, day 16 postoperation (variation B). (vG, x 65).
tissue (F&u-e IO).
in much the same way as in variation A, except that surface drying of the replanted skin was faster. Healing also gave the same results as in variation A, so that the lower layer of replanted skin was revascularized and survived, whilst the upper layer became necrotic (F&we II). As there was no organic relationship between the two layers in this case, the long process of sloughing off the necrotic tissue was avoided. It proved easy to establish the best moment for
layer began to be covered with granulation Spontaneous epjtheli~~tion from ‘the
wound margins progressed only slowly and was still in its initial stage at the end of the 6-week observation period.
Observations
relating
to experimental
variation
B
The split skin layers adhered when superimposed and acted as a single graft when replanted. Wound healing proceeded
22
Burns (1991) Vol. 17/No. 1
Figure 12. Superficial skin graft lifted up to assess healing. Variation B.
Figure 13. Layer (marked by arrows) connecting healed corim (below) and split skin (above), day 6 postoperation.
(vG, x ZZO.)
removing the surface graft, which served as a biological dressing; all it needed was to lift it up slightly and assess the revascularization of the underlying corium layer (Fimre 12). Removal of the upper graft, usually during the third week after producing the bum, exposed the healed corium layer beneath: this had a smooth surface with evidence of a fairly regular blood supply. As healing progressed further, masking of the healed corium layer with granulation tissue
Figure 14. Maturation of connecting layer between coriurn (below) and split skin (above), 2 weeks postoperation. (vG, x IIO.)
began. With this variation, too, the epitheliai adnexa degenerated completely. Spontaneous epithelialization from the margins of the wound was slightly better than with variation A, but the wounds still had not closed by the end of the 6-week observation period. The attempts at replanting either the lower or the upper split skin layer on its own essentially ended in failure. Revascularization did in fact start in wounds grafted with
23
Smahel: Skin viability after thermal damage
Figure 15. Healing complete in control wound (left) and replanted wound (right), 6 weeks postoperation. Replantation with split skin was in two stages. Note absence of contraction replanted wound.
in
the lower layer, but rapid drying of the graft soon put an end to the process. When only the upper layer was replanted revascularization did not develop, with small islets of corium surviving only in exceptional cases. Observations relating to wound closure with autologous split skin Wound closure with autologous split skin was effected in weeks 3 and 4 after producing the bum, when healing of the replanted corium was complete. The grafts healed perfectly if transplanted to a corium free from necrotic changes where masking with granulation tissue had started. These requirements were fairly easily met in group B, whereas in group A transplantation frequently had to be delayed or done in two stages as necrotic tissue still adhered. The connection between healed corium and transplanted split skin initially consisted of cell-rich connective tissue traversed by numerous vessels (Figctre 13). This later matured into collagen tissue, with the frequency of vessels markedly reduced (Figctre 74). The main characteristic of all treated wounds, marked reduction of wound contraction, persisted also when the split skin had healed (Figure 15).
Discussion Standardization of experimental bums is a well-known problem, particularly when relatively low temperatures are used. Use of the pneumoderma experimental model has eliminated some of the variables that in addition to the temperature of the heat source and the application time determine the degree of thermal damage. The pneumoderma technique ensures that the bum is limited to the skin, i.e. the pneumoderma wall, the deeper structures such as fascia and skeletal musculature are protected from damage. The model also makes it possible to position burns in the same relative position to major blood vessels. It had been noted during development of the model that the onset of healing was earlier in bums containing relatively large vessels. Finally the technique also ensures that skin tension is the same when both bums are made. Other variables, such as dermal blood supply, skin hydration, skin temperature at the moment the bum is produced and variations in skin thickness did however remain, and it was probably due to these that the depth of the bum varied slightly from animal to animal. It was possible to compensate for this to some
extent by always comparing the progress of excised and control wounds in the same animal. Under the given experimental conditions the combination of 15-s exposure time and a temperature of 60°C resulted in two-zone bums, with the zone of stasis involving the whole skin. Burns of this type, called zone-of-stasis burns by Saranto et al. (1983, are widely used in experimental studies. From the methodological point of view they offer the advantage that spontaneous healing leads to full thickness skin loss and the efficacy of measures to save the zone of stasis is easily assessed. Experimental methods to explore the effect of delayed onset of healing in wounds with loss of the zone of stasis were based on the realization that excision of a burn wound down to bleeding vessels, i.e. at least the zone of hyperaemia, promptly initiates healing. The phenomenon is well known with tangential excision and radical excision of small bums in clinical practice. The above experiments provide further substantiation. In the control wounds, vascular proliferation did not start before the end of the first week after producing the burn; in excised bum wounds the process came into full effect on day 3. The experiments substantiate the theory that delayed onset of healing plays a role in determining the fate of tissues with deep partial skin thickness damage. They also demonstrate clearly that the deeper layer of skin with deep partial skin thickness bums can be saved through early excision and replantation. In the control wounds, spontaneous healing regularly resulted in full thickness skin loss; in wounds treated with replanted skin more than half the corium was revascularized and survived. The outstanding feature was that wound contraction was almost completely prevented. The epithelial adnexa degenerated in the healing process. These findings bring to mind the work of Brauer and Spira (1966), who grafted burned skin onto freshly produced wounds in pigs. They compared their results to the healing of thin dermal grafts. The revascularization of replanted skin was always by second intention, i.e. due to invasion with new vessels (Smahel, 1977). This mode of revascularization characteristically involves a large number of vessels penetrating the graft at right angles to the skin surface and the fairly regular progress of revascularization from below to above. The fact the revascularization was consistently by second intention suggests that the proliferating capillaries in the wound bed did not anastomose with the vessels of the graft. The question arises as to how far the result of the grafting techniques used reflects the degree of thermal damage, i.e. the viability of the different tissue elements in the zone of stasis. On the one hand the experiments substantiated Zawacki’s view (1974a) that thermal damage in the zone of stasis had a gradient. They also showed, however, that this gradient differs depending on the tissue component involved. The endothelium appears to be most sensitive to heat trauma, for here damage extended to the lower margin of the zone of stasis, which explains why revasculanzation was always by second intention. The epithelial cells only degenerated in the course of the healing process and were therefore less sensitive to heat trauma, and the collagen structures of the corium evidently tolerated heat better than the other tissues. This implies that tangential excision, with its depth determined by vascular damage, goes too deep where damage to the corium is concerned. The evident conclusion that the outcome of replantation reflects differences in the degree of thermal damage to the zone of stasis has to be treated with some caution. Similar
24 results are known with the healing of free full thickness skin grafts where revascularization is by second intention; the grafts show deep desquamation and epithelial adnexa are generally lost (Smahel, 1977). The outcome of replantation might thus also reflect the mode of revascularization. This assumption is however negated by the experimental variation where only the superficial layer of burned skin was replanted and revascularization of the graft did not occur. Zawacki (1974a) saw similar results with upper layer grafts in bums. The almost complete absence of oedema in burns treated by excising and replanting the skin, potentially of significance in clinical practice, is explained by the fact that blood supply to the leaking vessels was stopped early. Total loss of the epithelial part of replanted skin could be to some extent specific to rat skin. In all the animals used in the experiments the skin was in the resting stage of the hair cycle, in telogen. In this stage the follicles are short, extending about two-thirds into the corium. They were therefore largely in the part of the corium that always became necrotic. At the same time rat skin does not include sweat glands, the secretory parts of which extend down to the sub&is and are therefore most likely to survive a skin bum. The absence of spontaneous epithelialization in replanted corium was overcome by the simplest experimental solution, split thickness skin grafting. From the biological point of view the use of split thickness skin was really unnecessary as there was sufficient corium in the wounds. It may be assumed that another solution would be to use cultured epithelium. The time when the wounds would be ready to be supplied with epithelium - after 2 or 3 weeks - would coincide well with the time required to culture the epithelium. The main purpose in including variation B was to assess separate grafting of either the lower or the upper layer of the burned skin. Among other things the variation showed that the lower layer will heal only if dehydration is prevented. In our experiment this was effectively achieved by the superficial layer of burned skin. Another reason for including variation B was that it comes closest to the situation liable to arise when the method would be used in clinical practice. Clinical use of the method might be considered for ‘deep dermal bums’ where tangential excision has already proved effective. The experiments have shown that this excision removes part of the corium that still has biological value and that replantation of this part may upgrade deep dermal bums to superficial dermal bums. It certainly seems feasible that the excised skin layer could also heal into another wound; the experiments done by Brauer and Spira (1966) seem to point in that direction.
Bums(l991) Vol. 17lNo.l
References Arturson M. G. (1985) The pathophysiology
of severe thermal injury.]. Bum Cure Rehabd 6, 129. Brauer R. 0. and Spira M. (1966) Full-thickness burns as source for donor graft in the pig. Plast. Reconstr. Surg. 37,~~ Ehrlich H. P. (1984) Anti-inflammatory drugs in the vascular response to burn injury. J. Trauma 24, 311. Heggers J. P., Robson M. C. and Zachary L. S. (1985) Thromboxane inhibitors for the prevention of progressive dermal ischemia due to the thermal injury. J. Bum Cure Rehbil. 6,466. Jackson D.MacG. (1953) The diagnosis of the depth of burning. Br. ]. Surg. 40, 588. Jackson D.MacG. (1969) Second thoughts on the burn wound. 1. Trauma 9, 839. Robson M. C., DelBeccaro E. J. and Heggers J. P. (1979) The effect of prostaglandins on the dermal microcirculation after burning, and the inhibition of the effect by specific pharmacological agents. Plast. Reconstr. Surg 63, 781. Robson M. C., DelBeccaro E. J., Heggers J. P. et al. (1980) Increasing dermal perfusion after burning by decreasing thromboxane production. J Trauma 20, 722. Saranto J. R., Rubayi S. and Zawacki B. E. (1983) Blisters, cooling, antithromboxanes, and healing in experimental zone-of-stasis bums. J Trauma 23, 927. Smahel J. (1976) Bum wounds. A new experimental model. Chir. %.stiui 3,231. Smahel J. (1977) The healing of skin grafts. Clin. Pk. Surg. 4,409. Smahel J. (1984) A protective rat vest. Letter to the Editor. Pkzst. ficonstr. Surg. 73, 859. Smahel J. (1985a) Pathophysiologie der Verbrennungswunde. Handchimrgie 17,340. Smahel J. (1985b) Replantation of thermally damaged tissue. In: May S. R. and Dogo G. (eds), Cure of the Bum Wound. Basle: Karger, p. 157. Smahel J. (1986) Device for splitting skin in small laboratory animals. Eur. 1. P&. Surg. 9,36. Winter G. D. (1975) Histological aspects of burn wound healing. Burns 1.191. Zawacki B. E. (1974a) The natural history of reversible burn injury. Surg. Gynecol. Obstet. 139,867. Zawacki B. E. (1974b) Reversal of capillary stasis and prevention of necrosis in burns. Ann Surg. 180,98. Zimmerman T. J. and Krizek T. J. (1984) Thermally induced dermal injury: a review of pathophysiologic events and therapeutic intervention. J. Bum Cure Rehabil. 5,913.
Paper accepted 20 August
1990.
Acknowledgement Thanks are expressed to MS B. Jentsch for her technical assistance in performing the surgical procedures and the histological examinations.
Correspondence shod be aa%emd to: Dr J. Smahel, Division of Reconstructive Surgery, University Hospital, CH-8091 Zurich, Switzerland.