Histological characteristics of the healing process of frozen skin allograft used in the treatment of burns

Vol. 22. No. 3, pp. Science Ltd for ISBI. All Printed 0305-4179196

Bum

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0 1996 Else&r

206-.211,1996 rights reserved in Great Britain $15.00 + 0.00

ELSEVIER

Histological characteristics of the healing frozen skin allograft used in the treatment T. Ornil, 0. Kawanamil,

process of of burns

K. Matsudalp2, A. Tsujiiz, M. Kawai2, H. Henmi2 and V. J. Ferrans3

IPathology and Clinical Research Laboratory, Nippon Medical School, Second Hospital, Kawasaki-shi, Japan, 2Department of Emergency and Critical Care Medicine, Nippon Medical School, Tokyo, Japan and 3Pathology Section, National Heart, Lung and Blood Institute, National Institutes of Health, Bethesda, MD, USA

Combined transplantation of skin autograft and allograff was used for the treatmenf of severe burns. The allografts were obtained from cadavers and were pretreated with 15 per cent glycerol for 2 h af 4°C then frozen at - 80°C until used. Patches of autograffs were placed over the burns and were covered by a stretched mesh of allograffs. Biopsy samples of transplanted skin were obtained from 5 days to 4 weeks after grafting. Sections were examined by histological and immunohistochemical strainings. Af 4 days, the epidermal-dermal junction of allografted skin was separated due to migfafion of epifheliul cells derived from aufograff epidermis or from skin appendages of recipient dermis. At 2 weeks, dermal fibroblasfs and capillaries proliferated in autografts.At 3 weeks, the dermal components of the allograft were covered by epithelial cells from recipient tissue and were invaded byfibroblasts andcapillaries. At 4 weeks, allograffed skin was replaced by granulation tissue, which mediated the adhesion of the graffs to the underlying tissue. Skin allograffs with a freeze-thawing pretreafment provide an appropriate matrix for the epithelial relining and for the growfh of granulation tissue in burned skin.

Bums,Vol. 22, No. 3, 206-211, 1996

Introduction The treatment of deep dermal bums often requires skin autografts, which usually are successful”‘. When adequate amounts of healthy skin are not available for autografting, the patients are often faced with excessivelossof fluids and with opportunistic infections leading to fatal sepsis.To prevent these complications, allograft3”, artificial skin’ and even xenograft skin‘r8 have been used. However, the results of such grafts can be unsatisfactory. We have used transplantation9,10of skin autografts and allografts for the treatment of severely burned patients. The skin for these allografts was obtained from cadavers, treated with glycerol, frozen quickly and stored at - 80°C10-14. The frozen allograft skin was thawed and stretched in a mesh7,‘0-‘3that was placed over the areaof the bum upon which small autograft skin patches had been placed. This method of grafting showed a number of interesting characteristics:they functioned sufficiently well to protect against infection and to control fluid loss, and they provoked only a very limited immune reaction15. In

addition, the allograft skin guided and facilitated the synthesis of new collagen fibres16, the formation of granulation tissue” that played a critical role for the adhesion of the grafts to the recipient skin”. This communication presents a morphological and immunohistochemica1study of thesegrafts, with emphasison a comparison of the adhesion processin skin autografts and allografts.

Materials

and methods

Patients Six patients (five male and one female) who had deep dermal bums or full skin thicknessbums were admitted to the Nippon Medical School Emergency Center. Four of the six patients had flame bums (all men, mean age 44 years; age range 23-58 years) and the other two (a boy and a girl, each 2 years old) had scaldbums. Eachof the patients had a satisfactory clinical course after undergoing the grafting procedure described below, and all the grafts were considered to be functionally adequate. Methods After debridement of the bum, autograft skin (approximately 0.4 x 0.4 in (10mm) wide and 12/1000 in (0.30mm) thick) taken from their own body surface was transplanted in smallpatches. Then the surface of the bum was covered with a mesh of allograft skin and a compression bandage was applied over the graft (Figure 7). According to the standard criteria of the Skin BankI4 of Nippon Medical School, piecesof allograft skin of 15/1000 to 18/1000 in (0.375-0.450 mm) thick and approximately 4 x 12 in (100 x 300 mm) wide had been obtained from the thighs or back of cadavers within 2 h of death. They were immersedin 15 per cent glycerol at 4°C for 2 h then stored at - 80°C as described previously10,‘2,‘4, and thawed in saline(37°C) before use. Pieces of normal control and thawed skin were fixed with buffered 10 per cent formalin and examined by light microscopy after staining as described below. Biopsies were taken of autografts and allografts at 5 days and 2, 3 and 4 weeksafter transplantation. These sampleswere also fixed with formalin and embeddedin paraffin wax. Sections 4 pm were stained with haematoxylin and eosin (H&E), periodic acid-methenamine silver (PAM), and periodic

Omi

et al.: Morphology

of skin

allografts

acid-Schiff (PAS) methods. Digestion with diastases was performed on sections from samples that showed a positive PAS reaction. The avidin-biotin complex (ABC) immunoperoxidase method was used in conjunction with the following antibodies: (I) low molecular weight keratin cocktail antibody (LMK; 1: 25 dilution; Triton Bioscience, Alameda, CA, USA)‘9*zo; (2) total keratin antibody (TK; I : 400 dilution; DAKO, Tokyo, Japan)19; (3) proliferating cell nuclear antigen (PCNA; 1: 20 dilution; DAKO) Z1;and (4) Factor-VIII-related antigen (F-VIII; 1: 450 dilution; DAK0)22. The reaction product was visualized with diaminobenzidine tetrahydrochloride (DAB) and hydrogen peroxide. This study was approved by the Ethics Committee of Nippon Medical School.

Results The histological findings in the biopsy specimenstaken from the autografts and the allografts did not differ significantly among the patients at any given time period. For this reason, the results obtained in individual patients are not presented separately. Histology of normal and frozen control skin In comparisonwith normal control skin, tissuespretreated by freezing and thawing appearedgenerally homogeneous after staining with H&E. These tissues were attenuated, especially in the epidermal layer. The nuclei of epidennal and interstitial cells were relatively condensed, and some nuclei appeared even pyknotic. The cytoplasm of epiderma1cells gave a positive reaction for TK. In contrast, the reaction for LMK was negative in the epidermis of both normal control and frozen skin. The epithelial cells of the sweat glands and ducts and the central portions of the hair follicles were positive for LMK in both normal control and frozen skin. In frozen skins, the dermal fibroblasts were relatively thin, spindle-shapedand densely eosinophilic. The dermal collagen fibres appeared very compact. The dermal capillaries were collapsed and contained no red blood cells. The endothelial cells appeared thin. In normal control skin, a positive reaction for PCNA was observed in a very few nuclei of epidermal basal and parabasalcells. This reaction was only rarely observed in the epithelial cells of skin appendages,dermal fibroblasts and capillary endothelial cells. However, a positive PCNA reaction was not observed in any of the cells in glycerol-treated frozen skin. The reaction for F-VIII was strongly positive in the endothelial cellsof normal control skin, but was negative in those of glycerol-treated frozen skin. Histology of skin autografts At 5 days after transplantation, the cytoplasm of the grafted epidermal cellsbecamediffusely pale (Figure 2) and contained coarse glycogen granules (PAS-positive and diastase-digestible). Most epidermal cells, particularly those in the basaland parabasallayers, showed a strongly positive reaction for PCNA. The reaction for TK was positive in the entire epidermis throughout the time periods included in this study. The suprabasalepidermal cells frequently gave a positive reaction for LMK. This reaction was as strong as that shown by the sweat glands and ducts. The PAM stain revealed the basement membrane at the epidermal-dermal junction to be of normal thickness. The edge of the grafted dermal collagenous tissueappearedto be attached to the underlying bed of the

207

recipient tissue. However, narrow spaceswere, occasionally found along the boundary. Mononuclear inflammatory cells were only rarely found in the dermis of the autograft. The vascular lumina in the grafted skin were greatly dilated and contained numerous red blood cells. The cytoplasm of the endothelial cells in these vessels usually was swollen and often gave a negative reaction for F-VIII, especially in small vessels. Their nuclei were enlarged and gave a positive reaction for PCNA. Anastomosesbetween the blood vesselsof the graft and those of the recipient tissue were not histologically apparent. Fibroblasts were enlarged and were frequently reactive for PCNA, particularly in the upper dermis of the grafted skin. This reaction was also seen in the epithelial cells of skin appendagesof grafted and recipient tissues. At 2 weeks after transplantation, the basal cells, which were enlarged, eosinophilic and cuboidal-shaped, underwent marked proliferation, forming single or miltiple layers that extended over the fibrin strands covering the surfacesof the bums. The nuclei of these cellswere highly reactive for PCNA, especially at the edges of the grafts. The upper dermisof the skin autografts showed perivascular oedema. Capillaries were dilated and often sprouted into adjacent areas of connective tissue. The endothelial cells in these vesselsshowed strongly positive reactions for PCNA and F-VIII. The fine bundlesof collagen in these regions were reactive with the PAM and PAS stains, in contrast to their diffuse and extensive reaction in normal controls. The border between the recipient tissue was clearly shown due to the lack of the reaction to PAM stain (Figure 3). Fibroblasts were enlarged and proliferated around small blood vessels. A mean 78 per cent of all fibroblasts gave a positive reaction for PCNA (Figure4). At 3 weeks after transplantation, the epidermis of the graft becamehighly acanthotic, although the comification of the surfaceremained incomplete. Approximately half of the cells in the basalportion of the epidermisgave a weakly positive reaction for PCNA. The subepidennal basement membrane appeared irregular and indistinct. Approximately 50 per cent of the fibroblasts and endothelial cells were positive for PCNA. The fibroblasts tended to be arranged parallel to the surface of the skin, while many of the capillarieswere arranged perpendicularly. Giant cellsof the foreign body type and macrophagescontaining fragmented cellular debris accumulated along the skin appendagesat the borders of the grafts. At 4 weeks after transplantation, the epidermis appeared mature and intact, and the epidermal-dermal junction showed a distinct, PAM-positive basementmembrane. The dermis occasionally contained localized foci of fine bundles of collagen fibres. Most capillaries appeared engorged. The endothelial cells and the fibroblasts were negative for PCNA. Histology of skin allografts At 5 days after transplantation, the nuclei of the epidermal cells and the dermal mesenchymalcells appearedindistinct and pyknotic in pretreated skin allografts. Their reaction for TK was similar to that of normal control skin. Variable numbers of lymphocytes and neutrophils were scattered throughout these grafts. Fibrin strandswere present at the borders between grafted and recipient tissues.The capillaries in the dermis of the allografts were collapsed.Bundles of collagen fibres appeared homogeneous and degenerate (Figure 5). All the cellsin the grafted skin were negative for PCNA. Cells in the recipient skin showed a strongly

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Burns: Vol. 22, No. 3, 1996

Figure 1. Macroscopic view at 1 week after transplantation of autograft patches (+) which were covered with allograft mesh. Biopsies were taken from areas of both autograft and allograft.

. . \-

t I

. .

b -.‘-

*

Figurea. Autograft at 2 weeks. Proliferating cell nuclear antigen (immunoperoxidase reaction) is demonstrated in basal layers of epidermis (A), dermal fibroblasts (A) and capillary CC) endothelial cells. Epithelial cells derived from recipient skin appendages have begun to extend along the surface of the recipient dermis. ( x 680.)

u

L I

-

l *.

-

Figure 2. Autograft at 5 days. The cytoplasm of many of the suprabasal epidermal cells is pale and enlarged. (H&E stain, x 680.) Figure 5. Allograft at 5 days. The allografted skin (G) appears generally amorphous and homogeneous, and the dermal capillaries are collapsed. (H&E stain, x 340.)

positive reaction for PCNA and proliferated from the areas of the skin appendages (which had survived the bum injury). These epithelial cells were cuboidal in shape and

Figurea. Autograft at 2 weeks. The autografted dermis contains fine bundles of collagen fibres, especially just beneath the epidermis. Capillaries are numerous and dilated. The epidermis appears acanthotic. The grafted skin is separated by fibrin(+) exudates from the underlying recipient tissue. The autograft is sharply demarcated from the underlying recipient tissue by newly formed hands of connective tissue (1). (PAM stain, x 340.)

formed a layer that extended over the recipient connective tissue bed exposed to air (Figures). Moreover, sheetsof these cuboidal epithelial cells migrated into the allograft skin, often separating its epidermal layer from the underlying tissues. Part of the dermis of the allograft then became incorporated into recipient tissues that were covered by the new epidermal cells that originated either from adjacent autografted skin or from skin appendagesof recipient skin (Figure6). These epidermal cells were positive for both TK and LMK. This pattern of keratin distribution was maintained throughout the periods examined in this study. At this period of time, the basal surfaces of these cuboidal epithelial cells lacked PAMpositive basementmembranes.Endothelial cells in collapsed capillaries in the dermis of the allografts and in subjacent recipient tissue were negative for F-VIII. Fibrin thrombi were occasionally found in the dilated lumina of

Omi et al.: Morphology

of skin allografts

Figure 6. Allograft at 5 days. Epidermal cells derived from recipient skin (R) have formed layers of cuboidal-shaped cells (A) that separate the grafted epidermis (G) from the underlying dermis. (H&E rstain, x 680.)

Figure 7. Allograft at 2 weeks. Sequestrated epidermis of allograft overEies the new epidermis of recipient origin. The dermis in the centre and the left side of the photograph contained relatively numerouscapillaries and other mesenchymal cells. In

contrast,the right quartershowsthe remainingcomponentsof allografteddermis.Squamous metaplasia(M) isevident in some areas.(H&E stain, x 340.) small vesselsin recipient tissues.Fibroblasts and endothelial cells in recipient tissuesoften were positive for PCNA. At 2 weeks after transplantation, the allografted epidermis was always completely sequestrated (with or without dermal components of the graft). A new outgrowth of epidermal sheathing developed from remnants of the skin appendagesof host tissue. The remaining skin appendageswere connected to eachother by thin bundles of collagen fibres, and their epithelial cells occasionally showed squamousmetaplasia (Figure7). About one-third of the regenerating epidermalcells were PCNA positive. A PAM-positive basementmembrane was present, but was discontinuous. Very few remnants of cells were observed in the dermis of the grafts. Only a few fibroblasts and capillaries had extended into the dermis of the allograft from adjacent areasof recipient tissue (Figure7). Both the fibroblasts and the endothelial cells increased in number with the time elapsed after grafting. Most of these capillaries were dilated, and two-thirds of their endothelial cells gave a positive reaction for F-VIII. At 3 weeks after transplantation, the new epidermis of

209

Figure 8. Allograft at 3 weeks. Dermal capillaries and fibroblasts are numerous in the oedematous connective tissue matrix. Capillary endothelial cells are enlarged and often show budding and sprouting in various directions. (H&E stain, x 1360.)

Figure 9. Allograft at 4 weeks. The epidermis is characterized by hyperkeratosis and acanthosis. Capillary endothelial cells are positive for F-VIII, and the vessels are perpendicular to the skin surface. The fibroblasts are proliferating parallel to the skin surface. (Immunoperoxidase stain, x 680.)

recipient origin became acanthotic and covered the remaining aliografted dermis, which appeared basophiiic and contained increasing numbers of fibroblasts and capillaries. Numerous capillaries were located just below the new epidermis. Capillaries showed branching and sprouting towards the surrounding tissues(Figure8). Giant cells of the foreign body type occasionally were phagocytosing fragments of epithelial cells that had undergone squamousmetaplasia. At 4 weeks after transplantation, the capillaries tended to be arranged perpendicularly to the epidermal surface. Most endothelial cells of these vesselswere positive for F-VIII. A large number of fibroblasts arranged parallel to the surface were located among these vessels(Figure 9). PAM-positive basement membranes became obvious along the entire epidermal sheets.However, some epiderma1 basal cells in these areas appeared to be poorly adherent to the basementmembrane.Interstitial fibroblasts migrated through gaps in the basement membrane into tiny clefts beneath the basalcells, where they appeared to participate in the formation of new connective tissue elements.These basalcells appearedto be involved in the synthesis of new basement membrane components

210

Allograft at 4 weeks.At severalpoints there are focal, microscopicareasof separationof the grafted epidermis from the dermis. Some dermal fibroblasts have penetrated through the epithelial basementmembrance(A) and have proliferatedin the areasof separation.(PAM stain, x 680.)

FigurelO.

(Figttre10). The remaining tissuesof the dermal allograft appeared to have been totalIy replaced by granulation tissue, which was in direct continuity with the recipient bed. PCNA-reactive cells became scarce,and the connective tissueelementsof allograft skin becameindistinguishable in areassubjacent to the new epidermis of recipient origin. Skin appendageswere not developed in theseareas.

Discussion The present study provides a characterization of the time course of the changes that develop in autografted and allografted skin in relation to the healing processof bums. Immediate activation of recipient epidermal cells The epidermal and mesenchymal cells of autografted skin showed evidence of rapid activation of their capacity to undergo migration and proliferation. The epidermal cells showed a more intense and widespread reactivity toward PCNA than did the epidermal cells of normal skin. They extended over the raw surfaces of the dermis and their cytoplasm contained coarseglycogen granuals.These cells expresseda form of keratin (LMK) that normally is found in fetal epidermis’“‘20and in normal adult glandular cell2’. These features indicate that transplanted skin autografts show characteristicsof embryonic epidermal cells,particularly in their expression of keratin and in their proliferative capacity. The proliferation and migration of epidennal cells occurred in autograft skin and in remnants of recipient skin asearly as 5 days after transplantation. The surfacesof the bum wound became covered by epithelial cells that originated exclusively from recipient epidermal elements in autograft and allograft skins. This conclusion is in agreement with that of Gibson5 and Kistler et al.9 in their studiesof allografts used for the treatment of burns. Sequestration of allografted skin Allograft skin was often detached either from the epidermal-dermal junction or from a variable depth in the allografted dermis. Only some portions of allografted dermis were incorporated into recipient tissue by becoming covered by recipient epidermalcells.Medawart3 observed that the sequestration of the transplanted skin is inversely related to the thickness of the grafted skin. Furthermore, Yang et al.‘, Namba et a1.4*8,Gibsor?, Mukae6, Lin et a1.2*and Kistler et alz5 reported that the immune rejection of allograft skin began to take place

Bums: Vol. 22, No. 3, 1996

around I week after the transplantation and that the timing of this event coincided with that of perfusion of blood into the graft. According to Li and Yangz6, the mixed leucocyte reaction with epidermal cellsis more intense than that with either fibroblasts or endothelial cells. This is consistent with the fact that epidermis is highly immunogenic and is apt to be rejected. In the present study, the allografted skin contained. a scattering of inflammatory cells near the junction with the recipient bed. This degree of inflammatory reaction is in sharp contrast to that found in the lesions associatedwith the rejection of allografted fresh skin5*25.Therefore, allografted frozen skin appeared to desquamate from the recipient skin without causing an intense immune cellular reactionz5. We assumethat the freezing and thawing treatment makes the skin less immunogenicI5. Nonetheless, the epidermal components of these allografts were in fact rejected. Further studieswill be needed to optimize the processing and storage of allografted skin’0Z’4. Incorporation of granulation tissue into recipient skin The dermal capillariesin the autografted skin contained red blood cells at 5 days after transplantation, which indicated that reperfusion of these vessels had been achieved through direct connections between the vesselsof grafted and recipient skin’8*23.This finding is in accord with our observations showing a high frequency of PCNA reactivity (indicative of cellular proliferation)” in the endothelial cells of both recipient and grafted tissues.In contrast, the capillaries in the allograft dermis became collapsed and necrotic immediately after transplantation. It became apparent that thesevesselsdid not survive the freezing and thawing procedure during pretreatment of the graft. At 5 days after transplantation, the endothelial cells along the boundary between the allograft and the recipient tissue lost their reactivity against F-VIII, This reactivity reappeared during the secondweek after transplantation. Then all endothelial cellsin the whole dermisbecameremarkably positive for F-VIII. These observations are similar to those previously reported” during the process of vascular remodelling in pulmonary fibrosis induced by intratracheal instillation of silica. Following initial damage to capillary endothelial cells by the silica, the endothelial cells of peribronchial vessels showed a frequent reaction for PCNA and migrated by extending cytoplasmic processes along damagedcapillary tubes within 2 weeks of injury. In the course of these changes, the endothelial cells transiently lost their reactivity for F-VIII until their cytoplasm contained a large number of Weibel-Palade bodies, in which von Willebrand factor is localizedz2. The dermis of the allograft was gradually replaced by new collagen fibresl”, which extended from the borders of the graft. It is possiblethat the adhesionof the grafted skin in both autografts and allografts is mediated by the formation of granulation tissue16-“. At the same time, these grafts were incorporated into recipient skin as they became covered by recipient epidermal cell?. Such an incorporation of the allografted dermis into the recipient tissue corresponds to the ‘sandwich phenomenon’ reported by Yang et al.’ in transplanted fresh allografts. The pre-existing collagen fibres in the autografts and allografts were gradually replacedby newly formed collagen*~‘*.The presenceof partial epidermization in the skin appendages coincided with the acanthotic changes in the epidermal layer. Such an expression of epidermization does not occur

Omi et al.: Morphology

of skin allografts

211

in normal skin. The factors related to these pathological changesare not understood at present. The keratinized foci were removed by the phagocytic activity of macrophages in derrnal regions of both the autografts and allografts. The healing

stage of burns Endothelial budding and sprouting

occurred

in developing

granulation tissue after 3 weeks of transplantation in allografts and after 2 weeks in autografts. These growing vessels tended to show palisading patterns arranged perpendicularly to the skin surface. In contrast, large number of fibroblasts were arranged parallel to the surface both in autografted and allografted skin. This pattern of arrangements of capillaries and fibroblasts has not been reported previously in studies of the healing of skin transplants. However, this pattern of organization of granulation tissueis known to develop at the basesof scars of skin woundsls and of gastric ulcersz8.We consider that these palisading patterns of capillaries plays an important role in providing an adequate blood supply to the connective tissue and the epidermis. It remains to be elucidated how cytokines such as basic fibroblast growth facto?’ and vascular endothelial growth factorsz9 function in the healing processesof transplanted skin. Based on the observations in the present study, we believe that the frozen-thawed

allografted

against

injection

combinedwith widely expandedautograftsin the treatment of extensivethird-degreebums.Burns 1993; 19: 142-145. 13 Horch R, Stark GB, Kopp J, SpilkerG. Colognebum centre experiences with glycerol-preserved allogeneic skin: Part 1: Clinicalexperiencesand histologicalfindings(overgraft and sandwich technique). Bwns 1994; 20: S23-26. 14 HenmiH, KawaiM, Tuzii A, KusimotoS,Gotou M, Katsumi A et al. Skin BankReview. Nessha 1994; 20: 103-117. 15 Abbott WM, Hembree JS. Absence of antigenicity in freeze-dried skin allografts. Cry&o&y 1970; 6: 416-418. 16 Klein L, RudolphR. 3H-collagen turnover in skingrafts. Strrg Gynecol Obsfef 1972; 135: 49-57. 17 RudolphR. Initial healingof skingrafts.In: RudolphR, Fisher JC,NinnenmannJL (eds)Skin Grafting. Boston:Brown Little & co, 1979; pp 107-111. 18 Walter JB,IsraelMS. Wound healing.In: General Pathology, 6th edn. Edinburgh: Churchill Livingstone, 1987; pp 117-129. 19 Moll R, FrankeWW, SchillerDL, Geiger B, Krepler R. The

catalog of human cytokeratins: patterns of expressionin normal epithelia, 1982; 31: 11-24.

dermis provides

an appropriate matrix for the epithelial relining of the skin surface.The allografted dermis also facilitated the proliferation, migration and implantation of various cellular components, including epithelial cells,endothelial cells and fibroblasts. The use of frozen allograft skin tissue can contribute to protection body water.

11 Hackett MEJ.Preparation,storageanduseof homograft.BrJ Hosp Med 1975; 13: 272-284. 12 KreisRW,VloemansAF, HoekstraMJ, Ma&e DP, Hermans RP. The use of non-viable glycerol-preserved cadaver skin

1 HansbroughJF. Current status of skin replacementsfor coverage of extensive bum wounds. J Trauma 1990; 30: S155-160. 2 PamelaH, Walter RN. Bum wound management. AACN ChinIssues Crif Care Nurs 1993; 4: 378-387. 3 JacksonD. A clinicalstudy of the useof skinhomograftsfor bums. Br ] Plasf Surg 1954; 7: 26-43. 4 NambaK, Koga Y, Mukae N. Clinical effects of allograft. Nessho 1984; 9: 145-151. 5 GibsonT. The secondsetphenomenon asfirst shownin skin allografts. A historical case which shows also the behaviour of cell free collagen. Br ] Plasf Surg 1986; 39: 96-102. 6 Mukae N. Histologic expectationsof skin allografts and clinical significance.Nihon fiiseigeka Gakkai-shi 1988; 8: 752-764. 7 Yang CC, Xizo YR, Li YY. Allografts, xenografts, and

intermingledtransplantation.In: Yang CC, HsuWS, ShihTS (eds).Treatment of Bums. Berlin:Springer-Verlag,1982; chap 111-6, pp 93.-105. 8 NambaK. Clinicaleffectsof allograftsandxenografts.fii,sei Geka 1989; 32: 693-702. 9 Kistler D, HafemannB, Hettich R. Cytogenetic investigations of the allodennisafter intermingledskingrafting. Burns 1989; 15: 82-84. 10 TsujiiA, YasudaK, KawaiM, HenmiH. Limitationof thermal

cells. Review.

Cell

20 Omi T, Kawanami0, Honda M, AkamatsuH. Humanfetal mast cells under development of the skin and airways. Arenrgie 1991; 40: 1407-1414. 21 WaseemNH, LaneDP. Monocionalantibody analysisof the

proliferatingcell nuclearantigen(PCNA). Structuralconservation and the detection of a nucleolar form. 1 Cell Sci 1990; 96: 121-129.

and the loss of

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