Department of Plastic Surgery, Churchill Hospital, Oxford, and NuffieM Department of Surgery, Oxford University

Transcription

1 REVASCULARISATION AND PHAGOCYTOSIS IN FREE FAT AUTOGRAFTS: AN EXPERIMENTAL STUDY By BRUNO ROSSATTI Department of Plastic Surgery, Churchill Hospital, Oxford, and NuffieM Department of Surgery, Oxford University IN the field of contour restoration, free fat autografts have been valuable in building out localised facial depressions and also in the correction of more deforming conditions such as facial lipodystrophy. Surgeons are well aware that A case of bilateral facial lipodystrophy in a woman aged 26 years. A, Original condition. B, Twenty days after bilateral free fat autografts. C, Appearance fifteen months after operation. (Mr Eric Peet's case.) it is necessary to over-correct the deformity to compensate for the shrinkage of fat graft which occurs in all cases. The amount of shrinkage which will occur in a given case is quite unpredictable (see illus. A, B, and c). Sometimes the fat graft completely disappears. There is ample clinical evidence that there is less shrinkage if the adipose tissue is transplanted with dermis. The fat and dermis graft is becoming more popular for this reason. There are still gaps in our knowledge regarding the processes at work which produce this disappearance of free fat autografts. The purpose of this paper is to describe the results of observations made on the behaviour of free fat autografts in rabbits. Some writers assume that, in free fat autografts, adipose cells do not survive, but are replaced by histiocytes which, by ingesting the fat liberated in the graft, become the new fat ceils. They therefore conclude that regeneration of fat tissue occurs in the graft (Rehn, I9I 9 ; Hilse, I928 ; Burkhardt, I94I). Others maintain that the fat ceus survive at least in part (Gurney, I938; Lyndon Peer, I95O, 35

2 3 6 BRITISH JOURNAL OF PLASTIC SURGERY Until now, little attention has been paid to the process of revascularisation of these grafts, which certainly plays an important part in the survival of transplants. The mode and the rate at which a new blood supply is established has not been fully investigated. In addition, the relationship between revascularisation and degenerative changes in fat graft s has not been considered. It is with these aspects of free fat autotransplantation that the present contribution is concerned. I95I, 1956 ). MATERIALS AND METHODS In this investigation a series of twenty-five aduk rabbits was used. Autografting of fat tissue was carried out in sterile conditions. The adipose tissue was removed from the groin and transplanted into the ear or into the dorsum of the animal. All grafts used were of similar size and shape, being cut in the form of a cylinder of adipose tissue 40 ram. in length and IO mm. in diameter. The graft was inserted into a pocket made between skin and perichondrium by blunt dissection in the ear, or subcutaneously on the dorsum. The ear was chosen as the main site of grafting because it does not contain any fat tissue, is well vascularised, and reproduces conditions similar to those of the usual graft in humans, in whom a common site is between bone and skin, or between fascia and skin. The animals were sacrificed at varying intervals up to four months after grafting, and the vascular network of grafted ears was injected either with Indian ink or with neoprene latex immediately after death. From the material injected with Indian ink, whole-mount preparations were made ; small portions of graft were also fixed and embedded in paraffin. Sections were cut at xo to I5O microns. The neoprene latex preparations were obtained by corrosion following the usual technique which leaves a cast of the vascular network, Sections, 6 to IO microns thick, were cut from material fixed in Carnoy or Susa and stained by the Mallory-Azan method or by hmmatoxylin and eosin. In three animals trypan blue was injected subcutaneously at daily intervals for ten to twelve days prior to sacrifice in order to study the phagocytic activity in grafted areas at different periods of time. Grafts were removed and fixed in formol saline and Carnoy. Frozen sections of this material were also stained with Sudan III for fat tissue. RESULTS The first signs of revascularisation in free fat autografts implanted in the rabbit's ear occurred between the second and fourth days. At this stage only a few connections between the vessels of the host area and the graft were established. These early anastomoses were developed from newly formed capillaries in the host tissues surrounding the graft and connected to pre-existing branches of its vascular network. The blood supply at this stage was obviously insufficient and rudimentary. The fact that the peripheral portion of the graft presented a vascular network partially full of blood is not sufficient evidence of the existence of a circulation of blood through that area. Indeed, a sufficient venous drainage was not yet available and consequently the blood filling the peripheral capillary bed was more or less stagnant. Up to the fifth day, intravascular injections of latex or Indian ink did not show a dense peripheral network (Fig. i). The formation

3 REVASCULARISATION AND PHAGOCYTOSIS IN FREE FAT AUTOGRAFTS 37 of more numerous and larger anastomoses with the existing vessels of the graft proceeded steadily from the periphery towards the central part from the fifth to the fifteenth day after implantation. Between the twelfth and fifteenth days the outer portion of the graft possessed a well-developed vascular network similar to that of normal fat tissue. At this stage the central portion of the graft, still completely deprived of a blood supply, had undergone necrosis, appearing as a roundish amorphous area, full of debris (Fig. 2). Sections of the peripheral part of the graft demonstrated that a number of adipose cells were well preserved ; and intercellular capillaries and veins appeared filled with Indian ink (Fig. 3). The formation of cystic spaces containing fat liberated from cells had already occurred. Between the twentieth and thirtieth days the number of vessels in the remnant of the graft had further increased, and between the thirtieth and fiftieth days the remaining part of the graft appeared to have acquired a rich blood supply. At this stage the process of revascularisation seems to be complete. Preparations obtained by corrosion after latex injection show a very dense capillary network at the site of the graft, but the arrangement of these vessels does not resemble that of normal fat tissue (Fig. 4); but their number and disposition may reflect the requirements of the grafted area at this stage. Indeed, from the beginning of the third week after grafting an intense process of phagocytosis was present in the peripheral area of the graft, where already a good revascularisation of the graft had occurred during the first two weeks after transplantation. At the site of the graft a large number of macrophages and giant cells ingesting debris and fat tissue were present (Figs. 5 and 6). Staining with Sudan III shows clearly that the giant cells and macrophages contain fat globules in their cytoplasm. Intravital injections with trypan blue demonstrate likewise the large number of macrophages in the grafted area and that the giant cells possessed phagocytic activity (see Fig. 5). This degenerative process continued during the following weeks and led to the almost complete absorption of the grafted fat tissue. Towards the end of the third month that process of phagocytosis had nearly ceased and the surviving fat tissue had assumed a normal arrangement and appearance (Fig. 7)- DISCUSSION From the present experimental study it is evident that the first signs of revascularisation of free fat autografts appear between the second and fourth day after transplantation, which seems to be rather a long period of time for the adipose cells to survive at body temperature. This consideration alone is sufficient to indicate that the number of cells which are likely to be irreparably damaged must necessarily be high. Indeed, the central portion of the graft receives a blood supply when irreversible degenerative changes have already occurred. This leads to total loss of the central portion of the graft, after ten to fourteen days ; while in the periphery of the graft blood-vessels continue to grow. At first this outer zone appears to have escaped this autolytic process because of the earlier revascularisation, but it suffers a subsequent degeneration accompanied by phagocytosis. The reason for this degeneration remains obscure but could possibly be due to slight but irreparable damage to the graft before adequate revascularisation occurs. At this stage the vascular requirements of the graft are twofold : (I) the supply of nutrition to the surviving fat cells, and (2) the supply of the large number of cells involved in the process of phagocytosis designed to remove cellular debris

4 38 BRITISH JOURNAL OF PLASTIC SURGERY ~IGS. I to 4 [See opposite page for legends

5 REVASCULARISATION AND PHAGOCYTOSIS IN FREE FAT AUTOGRAFTS 39 and fat. Two weeks after grafting it is clear that those undamaged cells which received an early blood supply will survive. After this time the revascularisation is concerned with the extensive process of phagocytosis which continues in the rabbit for almost twenty weeks. In man, where proportionally larger grafts are usually carried out, the degenerative changes will presumably continue for a much longer period of time. Rehn (I912), Neuhof (I923), Hilse (I928), Burkhardt (I94I), and others, noticed regeneration of fat tissue in autografts in mammals as a direct result of the phagocytic activity of histiocytes which became the new fat cells. Such regeneration of fat tissue, commencing seven to eight weeks after transplantation, could not be confirmed in this investigation. In appropriate preparations, macrophages containing both trypan blue and stained fat droplets were often observed. Transition forms which could prove the transformation of these elements into adipose cells were not observable. It would seem more acceptable to assume that fat, freed from cells and most likely chemically altered following transplantation, is metabolised or at any rate eliminated as debris. Neither in these animals nor in man is there any increase in bulk that could be taken as evidence of regeneration of adipose tissue after a certain period of time from grafting. It is more common to observe a progressive decrease in volume, indicating the slow but continuous process of phagocytosis. The purely clinical observations made in man of the decreased tendency to shrinkage of dermo-fat autografts is probably explained by the presence in the dermis of numerous capillary loops opening on its surface, which encourages an early revascularisation through direct anastomoses with small vessels of the host tissues. SUMMARY The process of revascularisation in free fat autografts was studied in a series of twenty-five adult rabbits with different injection techniques and histological methods. The slow rate of revascularisation is the cause of the loss of the central portion of the graft. Many adipose cells of the peripheral part of the transplant Fig. i.--early stage of revascularisation of free fat autograft in rabbit's ear, five days after implantation. Only a few peripheral vessels are filled with Indian ink, and this shows that some anastomoses are already established but that a circulation has not yet been formed, x 2o. f, fat graft ; c, cartilage ; s, skin. Fig. 2.--Indian ink injection of a fat graft fifteen days after implantation. Peripheral zone of graft is well vascularised ; central portion has at this stage undergone degeneration. About two-thirds of the graft possesses a good blood supply, x IO. s, skin ; c, cartilage ; h, cyst ; na, necrotic area. Fig. 3.~Microscopic appearance of peripherai portion of the graft fifteen days after implantation. Some fat ceils seem well preserved. Histiocytes are already infiltrating the graft. Note that intercellular capillaries and veins are filled with Indian ink. x 3oo. Fig. 4.--Neoprene-latex preparation fifteen days after grafting. A thick felt of newly formed capillaries at the site of the graft (nv). 35-

6 40 BRITISH JOURNAL OF PLASTIC SURGERY FIGS. 5 to 7 Fig. 5.--Intravital staining with trypan blue of a fat graft fifty days after implantation. Numerous macrophages and giant cells containing both globules of fat and particles oftrypan blue in their cytoplasm are scattered throughout the graft, x 2oo. m, macrophages; g, giant cells intravitally stained. Fig. 6.--Section of the peripheral portion of the graft at forty days. Huge giant cells showing phagocytic activity. (Mallory Azan.) x 36o. g, giant cell ; h~ fat cyst. Fig. 7.--Normal fat cells (fc) three months after transplantation. Phagocytic activity is still present. Sudan III-H~ematoxylin staining, x 18o. i, histiocytes.

7 REVASCULARISATION AND PHAGOCYTOSIS IN FREE FAT AUTOGRAFTS 41 are also affected. The process of phagocytosis of this peripheral zone which follows and extends over a period of several months is responsible for the further loss of the fat graft. Different aspects of fat transplantation are briefly discussed. I wish to express my gratitude to Dr E. H. Leach of the Department of Physiology of this University for facilities provided in his laboratory, and to Mr E. W. Peer, F.R.C.S., for helpful criticism and advice during the preparation of this paper. REFERENCES BVRI<HAROT, L. (1941). Dtsch. Z. Chit., 254, 372. GtlRNE, C. E. (1938). Surgery, 3, 679. HILSE, A. (1928). Beitr. path. Anat., 79, 592. NEUHOF, H. (1923). " The Transplantation of Tissues." London : D. Appleton & Co. PEER, L. A. (195o). Plast. reconstr. Surg., 5, (1956). Transpl. Bull., 3, 147. PEER, L. A., and WALKER, J. C. (1951). Plast. reconstr. Surg., 7, 74- REHI% L. (1912). Arch. klin. Chit., x, (1919). In E. Lexer, " Die Freien Transplantationen." Neue Deutsche Chirurgie, vol. 26A. Stuttgart : Enke.