Department of Zoology, University of Birmingham

Transcription

1 STUDIES ON THE VIABILITY OF MAMMALIAN SKIN AUTOGRAFTS AFTER STORAGE AT DIFFERENT TEMPERATURES By F. J. PEPPER, Ph.D. Department of Zoology, University of Birmingham IN guinea-pigs and other laboratory animals full-thickness skin autografts (i.e., those which include the full thickness of the corium) rarely "take " well. In the early stages after transplantation they are often disfigured by patches of dry gangrene or ulceration. This shortcoming is understandable : the graft will be deprived of a blood circulation until new capillaries have penetrated the corium by an upward sprouting from those already present on the graft bed, and the length of time required for the establishment of an adequate circulation will be roughly proportional to the thickness of the graft. Before vascularisation is completed the epidermis of the graft may therefore undergo ischa:mic autolysis. With thin skin grafts (" split-thickness " grafts) the blood-vessels do in fact establish a circulation quickly enough to prevent these pathological changes. In thick grafts, however, they do not always do so, and little patches of ulceration and dry gangrene may develop accordingly. It is known (and confirmed by experiments described in this paper) that within the temperature range o to 37 C. the length of time which skin can survive without food and oxygen is a function of its temperature ; the lower the temperature within this range, the longer the survival time. It was suggested by Medawar (1945) that a form of dressing which kept the graft surface cool would be advantageous to the healing of thick grafts. Grafts are usually held in place by pressure pads of cotton-wool, gauze, or other materials which retain heat and keep the graft at body temperature, i.e., at that temperature which is least favourable for its survival. The experiments described in this paper were designed to obtain quantitative information on the survival of skin in an ischmmic condition at various temperatures. An investigation was made of the effect of storage in vitro at temperatures between o and 37 C. in order to find the maximum storage life at different temperatures. The storage conditions roughly simulated those under which thick grafts exist in the early stages following transplantation, before a blood circulation is established. In the past a considerable amount of experimental work has been carried out on the storage of skin at low temperatures for different lengths of time prior to transplantation, but some of the literature on this work tends to be contradictory and some of the reports are unconvincing. Much of the early experimental work was confused by the fact that, after storage, some of the skin was grafted autoplastically and some homoplastically. (For reports of previous work see : Brewer, 1882 ; Wentscher, 19o3 ; Webster, 1944 ; Strumia and Hodge, 1945 ; Baxter and Entin, 1948 ; Billingham and Medawar, 1952.) No reports could be found on storage at temperatures higher than 8 C., and it was therefore decided in this present work to investigate the effect of storage at o, 13, 22, 3 o, and 37 C. The rabbit was used as the experimental animal since it is possible to prepare a larger raw area on the chest wall of the rabbit than on that of the guinea-pig, and 250

2 MAMMALIAN SKIN AUTOGRAFTS it is only when skin grafts are transplanted to a sufficiently large raw area that they can exhibit epithelial outgrowth, which is a useful indication of survival in the early stages following transplantation. METHOD Rabbits of both sexes were used and all exceeded 5 lb. in weight; they differed in coat colour. In one series of experiments thin grafts were cut from the dorsal surface of the ear, and after storage they were transplanted autoplastically to a raw area on the lateral chest wall. In some animals several grafts were cut from the ear at the same time and were stored at different temperatures for equal lengths of time, before transplantation to the same raw area in a single operation. In other animals skin grafts were cut from the ears at different times and were all stored at the same temperature before transplantation to a raw area in a single operation. In the second series of experiments, rectangular pieces of skin, including the full thickness of the corium, were cut from the lateral abdominal wall and were stored at various temperatures. After the storage period, small thin slices of Thiersch graft thickness were cut from these pieces of skin and were transplanted autoplastically to the chest wall. (By a "Thiersch" graft is meant one which includes the epidermis and the upper part of the corium, but excludes the lower part of the corium and the hair follicle bases.) The transplants were examined from time to time for indications of survival. OPERATIVE TECHNIQUE The rabbit was anmsthetised with Nembutal, supplemented by open ether amesthesia. In the first set of experiments the dorsal surface of the ear was shaved carefully and was cleansed with soap and water and with I½ per cent. Cetavlon. The skin surface was then treated with surgical spirit and allowed to dry, and was finally smeared thinly with sterile vaseline. A sterile, sharp-edged cork borer, of diameter 9 mm., was pressed on to the skin surface to define the limit of the graft which was excised by means of a No. 12 scalpel blade. The skin removed included the full thickness of corium, but ear skin contains much less corium than body skin and is only about ½ mm. thick in the rabbit. On removal from the ear, each piece of skin was transferred to a separate storage bottle, in which it was maintained at a constant temperature for the required length of time. The donor site on the ear was sprayed with sterile sulphadiazine powder from an insufflator and left to heal. The skin was stored in stoppered weighing bottles of diameter 0.8 in. and height 1.5 in. Into the bottom of each weighing bottle a circular piece of filter paper was fitted, and the bottles were then sterilised. Immediately before use, the filter paper in the bottle was moistened with three drops of freshly boiled distilled water, and the piece of ear skin was laid dermal-side-down on the moist filter paper. The glass stopper, having sterile vaseline smeared round the frosted glass surface, was replaced immediately and was sealed by applying melted wax round the rim. The bottles were stored in the appropriate incubators or refrigerators in order to maintain them at temperatures of 37, 3 o, 22, i3, and o C. respectively. 25i

3 252 BRITISH JOURNAL OF PLASTIC SURGERY At the end of the storage periods the pieces of skin were autografted to a raw area on the lateral wall of the chest overlying the ribs. The raw area, of size approximately 5 cm, square, was prepared by the technique described by Billingham and Medawar (I95I). The skin was stripped off at the level just superficial to the vascular plane overlying the panniculus carnosus muscle. The ear skin autografts were so arranged that they were of approximately equal distance from each other and from the edges of the raw area. The operation site was sprayed with sterile sulphadiazine powder and was dressed in the way outlined by Billingham and Medawar (I95I). The grafts were inspected on the ninth day post-operatively and on every subsequent third or fourth day. In the second set of experiments, the rabbit was anmsthetised and the hair was clipped and shaved from the lateral wall of the abdomen. The skin surface was cleansed and prepared in the way described for the ear. A thick, rectangular piece of skin, of dimensions approximately 3 cm. by I cm. and including the full thickness of corium, was then cut from this region. The donor area was closed with interrupted sutures, painted with a I per cent. solution of iodine in 7o per cent. alcohol, and sprayed with sterile sulphadiazine powder when dry. The piece of excised skin was cut into two or three pieces, which were stored in one or more sterile glass boiling tubes fitted with rubber stoppers. The tubes (which had been prepared previously and sterilised) contained a number of glass beads at the bottom and about 5 ml. of Ringer's solution. The pieces of skin (which had rolled over on their inner surfaces, corium to corium) were dropped into the tubes and beached on the glass beads, the top layer of which was moistened by, but not :immersed in, the Ringer's solution. The rubber stoppers were replaced immediately and the tubes were stored at the appropriate temperatures for the required length of time. At the end of the storage period the pieces of skin were removed and pinned corium-side-down, in a stretched condition, upon a sterile cork. Thiersch grafts were cut as thinly as possible from the surface of the skin and were transplanted autoplastically to a raw area on the chest in the manner described for ear skin grafts. In most of the animals used in this second series of experiments, ear skin and abdominal skin grafts were stored by their respective methods at the same temperature for the same period of time, and two or three grafts of each type were transplanted together to the same raw area for comparison. The wounds were dressed as in the first set of experiments and were inspected at the ninth and every subsequent third or fourth day until the region was completely healed. Healing was brought about to a large extent by contracture, which caused close approximation of the edges of the wounded area and the boundaries of the surviving grafts. When healing was complete, dressings were no longer applied, but the grafts were inspected at frequent intervals in order to observe hair growth on the grafts and to compare its pigmentation with that of the hair growing on the donor site, i.e., to observe whether or not the melanocytes in the hair bulbs had been.affected by storage. OBSERVATIONS In some animals it was difficult to be sure, at the first naked-eye inspection on the ninth day, whether or not the grafts were surviving ; diagnosis was therefore Often deferred until the fifteenth day. The first indication of successful storage of

4 MAMMALIAN SKIN AUTOGRAFTS 2,53 the grafts was given by epithelial outgrowth from the surviving transplants. From those which had suffered little ill-effect from storage, epithelial outgrowth could be detected at the ninth day ; from those which had suffered more, epithelial outgrowth appeared to be delayed for a day or two and then to proceed more slowly. Ultimate survival or non-survival of a graft could be judged macroscopically, and in most animals it was found unnecessary to remove biopsy specimens. Diagnosis of survival based on mere epithelial outgrowth was in all cases except one confirmed by a later growth of hair on the grafts. The time at which hair growth was first noted on the surviving grafts varied between twenty and twentysix days post-operatively, and within these limits the time of appearance of hair seemed to bear little relation to the duration of storage or to the temperature prevailing during the storage period. The observations are summarised by Table I and show that within the temperature range o to 37 C. the viability of stored skin varies inversely with the temperature and with the duration of storage, the lower temperatures being more favourable for any one storage period, and the shorter storage periods the more favourable for any one temperature. H TABLE I Survival of Thin Grafts cut from Thick Pieces of Abdominal Skin stored at Different Temperatures x indicates survival. o indicates no survival Figures along the top row indicate number of days of storage. I IOlII I'~ 7 c x I o o o 3o c x o x o o Xo OO I ]3o 35 Only at a temperature of 37 C. did the method of storage of thin pieces of ear skin prove to be superior to that used for the storage of thick pieces of abdominal skin from which Thiersch grafts were later cut for transplantation. At all other temperatures used in this investigation the method of storage of abdominal skin proved to be as good as, or better than, that employed for ear skin, and therefore

5 254 BRITISH JOURNAL OF PLASTIC SURGERY only the results of abdominal skin storage are tabulated for temperatures below 37 C. (Table II). One possible explanation of the shorter survival of thick skin at 37 C. is the harmful effects of the autolydc breakdown products of the thick corial layer. The storage lifetime at o C. (about five weeks) was unexpectedly short for both methods. 1 TABLE II Survival of Transplanted Skin stored as Thin Grafts of Ear Skin I IOIII21314ISI6 18 i± 2o 2 2± 2A L C' i o.o It was noted in this investigation, as in that of Billingham et al. (I95I), that after transplantation surviving autografts expand during the period of contraction.of the raw area which forms the graft bed and that (to quote from their paper) " during expansion the mouths of the hair follicles become separated to an extent proportional to the degree of linear magnification, and the hair pelt becomes.correspondingly more sparse." In the present investigation the majority of grafts of both types eventually grew a pelt of hair of the same depth of pigmentation as that growing on the donor site. The few which grew patches of unpigmented hair did so at very scattered temperatures and storage periods. CONCLUSION The results of these experiments indicate that skin will survive storage in vitro for up to twenty-one days at I3 C., up to eleven days at 22 C., and up to seven days at 3o C. The conditions under which the skin was stored are physiologically comparable to those prevailing in the early stages following transplantation when the graft is still largely avascular. The figures obtained in the experiments are, however, systematic under-estimates of the expected periods of survival of transplants maintained in vivo at equivalent temperatures, because in this investigation all the pieces of stored skin had to undergo a further period of ischmmia while being used as grafts to test their survival. Bearing this in mind, it is suggested that a form of dressing which would keep the surface of a thick autograft at a temperature of about 3o C., while maintaining adequate pressure.on the graft, would promote its survival and successful "take." This surface temperature would probably not chill the deeper planes of the graft enough to impede vascularisation. 1 It should be emphasised that the temperature described as " o C." was exactly o C., and this temperature may be decidedly less favourable for survival than an ordinary refrigerator temperature of 3 to 5 C.

6 MAMMALIAN SKIN AUTOGRAFTS 255 An attempt was made to test this idea on guinea-pigs in the following manner. In eleven anmsthetised guinea-pigs two full-thickness disc-shaped grafts were removed from the thigh and were transplanted autoplastically to the beds from which similar grafts had been cut on the lateral chest wall. (For details of the grafting technique, see Billingham and Medawar, I95I.) The transplants were sprayed with sterile sulphadiazine powder. One was used as a "warm" graft (i.e., it was covered by the usual type of dressing which retains heat), and the other was used as a " cold" graft (i.e., one whose surface was intended to be kept relatively cool). A I½ in. wide strip of Lastonet (an elastic open-mesh bandage) was wrapped round the chest in the stretched condition and held in place with a row of suture clips where the two ends of the strip met. A watery rubber latex solution was applied over the Lastonet to the region surrounding each graft in order to give the dressings a certain degree of rigidity and to prevent wrinkling. Lastonet provided the only covering for the "cold" graft, but over the " warm" graft a thick pad of surgical gauze was stuck into place with latex. The grafts were inspected macroscopically after seven days ; specimens for histological examination were taken at the seventh or twelfth day post-operatively. The results indicated that the supposedly " cool surface" grafts were not at an advantage in comparison with the warm grafts--in fact, in a few cases they seemed to be at a disadvantage. The temperature difference between the graft surfaces was determined by means of thermocouples lying on the grafts while they were covered by their respective dressings. The temperature difference between the warm and cold grafts on each animal was found to be as low as 1.5 to 1.7 C.--an insignificant temperature differential. Any differences between the two grafts could therefore probably be attributed to differences in the degree of pressure applied to them in the early stages after transplantation, since with the dressings of the type employed it was not easy to regulate the pressure so that it was exactly equal on the two grafts. As already explained, it seemed legitimate to deduce from the storage experiments that it should be advantageous for the survival of a thick graft to maintain its surface temperature at about 30 C. while maintaining adequate pressure on the graft. One possible means of fulfilling both the temperature and the pressure requirements for efficient" take" of the grafts would be to have the dressing in the form of a rubber bag through which water of a constant known temperature could circulate, and by means of which the required pressure could be applied to the grafts. It is impracticable to test the efficiency of such a device on laboratory animals. The author believes that dressings making use of a similar principle have been tested clinically in at least one hospital (personal communication) but with little success. This can probably be attributed to the thickness of the grafts used (which had a fairly thick layer of fat at the base), and to the low temperature employed, i.e., about o C. The writer hopes that these experiments will be repeated with grafts in which the layer of fat is reduced, and with a temperature of 22 to 3 C., which should not prejudice vascularisation. SUMMARY Investigation has been made of the effect of storing rabbit skin in vitro at different temperatures for different periods of time prior to autoplastic transplantation. Two methods of storage were used and the results compared: in

7 256 BRITISH JOURNAL OF PLASTIC SURGERY (a) thin autografts of ear skin were stored and grafted without further treatment ; in (b) thick pieces of abdominal skin were stored and thin grafts were cut from them for transplantation. Storage temperatures of o, I3, 22, 3 o, and 37 C. were employed. After transplantation to a prepared raw area on the lateral chest wall, the grafts were kept under observation for evidence of epkhelial survival, hair growth, and pigmentation of the hair. The grafts survived for not longer than five weeks at a storage temperature of o C. ; twenty-one days at 13 C. ; eleven days at 22 C. ; seven days at 3 C. ; and three to five days (according to the method used) at 37 C. From a consideration of the results it is suggested that the "take " of thick autografts of skin might be facilkated by maintaining the surface of the graft at a temperature in the neighbourhood of 3o C. An attempt was made to test this hypothesis on guinea-pigs, but it was abandoned because of the technical difficulties involved in keeping the graft surface at a sufficiently low temperature ; these difficulties should not, however, affect a clinical trial. The writer wishes to express sincere thanks to Professor P. B. Medawar, who supervised the work recorded in this paper and gave assistance at all stages. REFERENCES ]baxter, H., and ENTIN, M. A. (1948). Plast. reconstr. Surg., 3, 303. ]3ILLINGHAM, R. E., and MEDAWAR, F. B. (1951). ft. exp. Biol., 28, (1952). Ibid., 29, 454. ]3ILLINGHAM, R. E., KROHN, P. L., and MEDAWAR, P. ]3. (1951). Brit. reed..7, i, ]3REWER, E. P. (I882). Med. Rec., 2z, 483. MEDAWA% P. ]3. (1945)- Brit. rned. Bull., 3, 79. STRUMIA, M. M., and HODGE, C. C. (1945). Ann. Surg., 21, 860. WEBSTER, J. P. (1944). Ann. Surg., z2o, 431. WENTSCHER, W. (19o3). Dtsch. Z. Chit., 7 o, 21.