OBSERVATIONS ON THE GROWTH OF REFRIGERATED. By ADRIAN E. FLATT, M.A., M.B.(Cantab.)

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1 OBSERVATIONS ON THE GROWTH OF REFRIGERATED SKIN GRAFTS By ADRIAN E. FLATT, M.A., M.B.(Cantab.) DURING the last thirty years several comments have appeared in the literature concerning the effects of cooling on the rate of tissue growth. In general it can be said that most observers consider that cooling subsequently stimulates tissues to greater growth activity. In I912 Carrel, when discussing the tissue culture of pieces of skin, said: " When they cease to proliferate I place them in cold storage for several days in a state of latent life, then when I replace these tissues in a medium suitable for their culture I have observed a new and active growth of cellular proliferation:" A year later de Martigny stated: " We have noticed that tissues preserved for several days at least in cold storage give us better results when they are used." Lake, in his paper on frostbite, published in i917, said : "Tissues may be stored at a temperature of I2 C. for periods up to at least seven days without losing any of their vitality, either functional or vegetative, and, indeed, in some cases appear to have increased in vitality over the controls which were implanted immediately." More recently Strumia and Hodge (x945), working with human skin grafts, noticed that the refrigerated grafts appeared to do better than their fresh counterparts. In Case x they record : "... the takes appeared to be proliferating more than the grafts that were transplanted without freezing." In Case 3 they state : " Those (grafts) which had been frozen appeared to be somewhat healthier than the control grafts at similar stages and were proliferating more than usual." Matthews (I945), working at about the same time, thought that refrigeration accelerated fibroblastic proliferation, thus speeding up the rate of take. On the other hand, Davis, in the discussion which followed the reading of a paper by Webster (I944), stated that in his opinion there was a latency of epithelial growth of a week or longer in previously refrigerated tissue, although the blood supply became established and "take" occurred. Baxter, Entin and More, in a paper published in I948, stated : "We have noticed the lag that follows grafting of chilled or frozen skin, but do not consider it to be intrinsically a delay of healing." No explanation appears to have been put forward to account for this increased vitality of cooled tissues, and the following paragraphs are an attempt to provide such an explanation. Skin kept in a refrigerator, being divorced from all nutrition, must either die: of " starvation " or must suspend its metabolic activity to such a degree that it requires no nutrition if it is to remain alive. Cramer (I93 o) has shown, however, that although skin stored at o C. has virtually suspended metabolism, there is still a minimal degree of activity. Since metabolism is not wholly suspended at o C., a certain degree of catabolism must be occurring, therefore inevitably leading to the death of individual cells. In addition to this slow but steady destruction of cells by metabolites, the death of other cells from old age must be occurring. In any piece of skin which is being stored, the length of life of similar cells is about the same, but the cells are of different ages and a certain proportion will die during each day of storage. 28

2 GROWTH OF REFRIGERATED SKIN GRAFTS 2 9 Thus it can be seen that at o C. there is a steady reduction in the number of viable cells in the stored skin, and ultimately a point will be reached at which the number of viable cells is so reduced that they will be insufficient to regenerate the whole skin even when placed on a suitable nutritive bed. Until this irreversible point is reached, however, there will be a steady increase in the products of cell destruction retained within the stored skin, and the diffusion of these products throughout the graft will be accelerated immediately the skin is restored to normal body temperature. Therefore a graft which has not reached the irreversible point will, within a short time of being placed on its recipient area, have all its viable cells bathed in the products of the destruction of their dead fellows. Tissue culture experiments have shown that the products of cell destruction have a very stimulating effect on cell growth. Tissue hunger due to inadequacy of oxygen and other nutritive substances necessary to life is known to be a powerful stimulant to mitosis, and skin which has been stored may well be potentially in a very active stage of cell division. It is therefore possible that the increased vitality of the skin which has been cooled is due to the combined stimulating effects of tissue hunger and retained metabolites. There have been very few comments in the literature concerning the effect of refrigeration upon the method of consolidation and subsequent growth of refrigerated skin grafts as compared with fresh skin grafts. In I9O3 Wentscher published two hand-drawn illustrations showing the histology of skin which he had preserved for seven and fourteen days. Kubyani published two photo-micrographs in I93o showing sections of biopsies taken from grafts stored for eight and twelve days. During I948 Baxter et al. published a large series of photo-micrographs showing the effects of refrigeration and cooling upon the method and rate of consolidation of skin grafts. They tended to concentrate upon the changes to be observed in the dermis and concluded that both freezing and cooling produced a considerable degree of replacement of the deeper dermal elements by fibrous tissue. My own observations have been directed more towards studying the effect of cooling upon the survival and consolidation of the actual epithelial cells of the graft. If the preceding assumption is correct there is a steady accumulation of altered proteins in the graft throughout its storage period, and it is probable that this accumulation of waste products would have some effect upon the growth of the surviving epithelial cells after the implantation of the graft. It is more probable that any effects to be observed will be in the later stages of consolidation rather than in the first ten days of grafting. During this time the process of consolidation follows certain well-defined steps, and little variation in this can be expected if the graft is to survive. Fig. I shows a biopsy taken four days after a graft stored for sixty-eight days at o C. had been placed on an infected granulating area. There does not appear to be any significant difference from a biopsy taken of a fresh graft four days after grafting. The superficial layers of the epithelial cells have already degenerated. The intermediate cells are poorly staining and show much distortion. The deeper epithelial cells can be more clearly recognised as a regular layer of cells. The dermis is infiltrated with many leucocytes and occasional red blood cells. Polymorphonuclear leucocytes, plasma cells, and lymphocytes and several eosinophil cells can be seen in the stained section, although they are not so easily distinguished in the photograph.

3 30 BRITISH JOURNAL OF PLASTIC SURGERY The presence of these eosinophils and giant cells is interesting, as Baxter et al. (1948) have recorded that eosinophils and giant cells are present in the biopsies of skin grafts which have been preserved by freezing. They noted a similar though less intense response in grafts which had been preserved by prolonged chilling. They believe that the cold temperatures modify the proteins of the skin so that they become " foreign," thereby provoking a typical reaction when the graft is implanted. It would seem more logical to assume that the death of individual cells would produce the altered proteins since the total protein content of the skin would be altered if the causal factor was the temperature. If this alteration took place throughout the skin it is difficult to see how any epithelial cells would survive and subsequently grow when implanted. FIG. I Skin stored at o C. for sixty-eight days. Biopsy taken four days after grafting. ( 280.) Several biopsies were obtained from grafts which had been stored for varying lengths of time. One of these biopsies was taken when the graft had been in place for four weeks, and the remainder were taken eight weeks after grafting. The times of storage varied from fifty to four days. Examination of the sections shows an interesting feature which can be correlated, I believe, with the effects of the length of time of storage. These specimens were collected over several months, but in order to eliminate as many errors as possible the sections were all cut and stained with ha~matoxylin and eosin at one time. These photomicrographs can show only a small portion of each slide, but they have been chosen to illustrate the characteristic points of each biopsy. In general the thickness of both the keratin and epithelial layers increases with the storage time. The section of skin stored for four days is indistinguishable from a section of normal skin, while the biopsy of skin stored for fifty days shows a gross keratin layer and a thick faintly staining epithelial layer. The intermediate sections show a proportional thickening of these layers as the storage time increases. The fifty-days skin shows nuclei in the keratin layer and karyorrhexis of its upper epithelial cells. This karyorrhexis is also present in the twenty-eight-days skin. The biopsy of skin stored for fifteen days does not show either keratosis or karyorrhexis, but the majority of its superficial cells are dead

4 GROWTH OF REFRIGERATED SKIN GRAFTS 3I FIG. 2 FIG. 3 Skin stored at o C. for fifty days. Biopsy taken eight weeks after successful grafting on infected granulations. ( 112.) H i g h p o w e r ( x 420) o f Fig. 2 to show keratinisation, karyorrhexis, a n d chromatolysis. FIG. 4 FIG. 5 Skin stored at o C. for t w e n t y - e i g h t days. Biopsy taken eight weeks after successful graftm g on infected granulations. ( x II2.) A large portion of the keratin layer h a s broken away. Skin stored at o C. for fifteen days. Biopsy taken eight weeks after successful grafting on infected granulations. T h e r e is vacuolation o f the superficial epithelial cells a n d a mild degree o f chromatolysis of i n t e r m e d i a t e cell layer. ( x I i 2 )

5 32 BRITISH JOURNAL OF PLASTIC SURGERY and vacuolated. This vacuolation can be seen in some of the cells of the nine-days skin, but the nuclei of the majority of these cells are well stained. The chromatolysis of the intermediate cell layer is most marked in the oldest skin and becomes progressively less as the length of storage time decreases. There is no chromatolysis of the cells of the skin stored for four days. It is not suggested that the changes noted in these sections are specific to the appropriate times of storage, but it is considered that the overall picture is one of increasingly precarious existence of the epithelial cells as the storage time increases. The keratosis present is similar to that seen in sections of thin scar epithelium and is generally considered to be evidence of a short epithelial cell life-cycle. The FIG. 6 Skin stored at o C, for nine days, Biopsy taken four weeks after successful grafting on infected granulations, Apart from vacuolation of some ceils and chromatolysis of a few others, the general picture is one of normal skin. ( II2.) FIG. 7 Skin stored at o C. for four days. Biopsy taken eight weeks after successful grafting on infected granulations. The epithelium of this section is indistinguishable from that of normal skin. ( 112.) karyorrhexis of the older sections and the increase of chromatolysis with storage age also indicate weak powers of survival of the cells. These abnormalities would seem to indicate that the basal cells were multiplying rapidly but that their progeny had only a short duration of life. The explanation of these histological changes is probably that the longer the skin is stored the more precarious is the life of the surviving cells owing to the accumulation of breakdown products. When these cells are placed in a normal environment they revive and proliferate rapidly, and it is to be expected that the longer the storage the more prolonged will be the effect of the accumulated waste products and the refrigeration. Another series of sections, which are not illustrated, show that skin stored for as long as 22o days does not develop this excessive epithelial proliferation and keratin formation during the storage period. Thus it can be concluded that these changes occur in the graft after implantation. No views can be expressed as to the probable duration of these changes, since no biopsies were obtained longer than two months after implantation.

6 GROWTH OF REFRIGERATED SKIN GRAFTS 33 These observations were prompted by a clinical investigation into the use of refrigerated grafts to provide skin cover for grossly infected granulations. The results of this investigation, which have been discussed elsewhere (Flatt, I948), show that stored grafts can survive and take in spite of a degree of infection which is usually assumed to be too great for normal successful fresh skin grafting. Previous publications would seem to suggest that three weeks is the longest practicable storage time for skin grafts, but the illustrated biopsies show that epithelial coverage for infected granulations can be supplied by skin stored for considerably longer periods of time. Arey (193 o) has pointed out that leucocytes are known to elaborate definite growth-producing substances and that there is abundant evidence that dead cellular matter, particularly dead granulations, yields a powerful growth-promoting substance. These growth stimulants will be present in quantity to react on any graft placed on infected granulations. If the hypothesis put forward in the opening paragraphs is correct, it will be seen that the stored graft which is placed on infected granulations is provided with both intracellular and extracellular growth stimuli and its surviving cells are in a potentially active state owing to a prolonged period of tissue hunger. My thanks are due to Professor Kilner and the surgeons of the Plastic Surgery Unit, Stoke Mandeville, for the facilities afforded me while working on the Unit, and to Professor Harris and Mr Fozzard of the Department of Anatomy, Cambridge University, for the photographs. REFERENCES AREY, L. B. (I93O). Physiol. Rev., to, 547. BAXTER, H., ENTIN, M., and MORE, R. H. (1948). Plast. Co" Recons. Surg., 3, II. CARREL, A. (1912). J. Amer. reed. Ass., 59, 523. CRAMER~ W. (193o). Imperial Canadian Research Fund. Ninth Report, 21. DE MARTIGNY, F. (1913). Cong. Franc. Chir., 26, 252. FLATT, A. E. (1948). Lancet, 2, 249. KUBYANI, E. (193o). Arch. kiln. Chir., I6Z, 502. LAKE, N. C. (1917). Lancet, 2, 557. MATTHEWS, D. N. (1945)- Lancet, x, 775. STRUMIA, M. M., and HODGE, C. C. (1945). Ann. Surg., 12i, 860. WEBSTER, J. P. (1944). Ann. Surg., x2o, 431. WENTSCHER~ J. (19o3). Dtsch. Z. Chit., 7o, 2I.