10 Burns J, Chan VTW, Jonasson JA, Fleming KA, Taylor S, McGee JO'D. ... Anne Brain, Patricia Purkis, Philip Coates, Michael Hackett, Harshad Navsaria, Irene ...
promoting factors into the area of the wound. If this is the case then short term biological dressings incorporating suitable human cell types, free from known viral pathogens, on a substratum compatible with the wound may provide a valuable adjunct to conventional grafting procedures, with the added advantage that such material can survive cryopreservation and be reconstituted for use in large amounts and at short notice. The Institute of Cancer Research is supported by funds from the Cancer Research Campaign and the Medical Research Council. 1 Green H, Kehinde 0, Thomas J. Growth of cultured human epidermal cells into multiple epithelium suitable for grafting. Proc Natl Acad Sci USA
1979;76:5665-8. 2 Clarke JA, Burt AM, Eldad A. Cultured skin for burn injury. Lancet
1986;ii:809. 3 Eisinger M, Monden M, Raaf JH, Fortner JG. Wound coverage by a sheet of epidermal cells grown in vitro. Surgery 1980;88:287-93. 4 Gallico GG III, O'Connor NE, Compton CC, Kehinde 0, Green H.
Permanent coverage of large burn wounds with autologous cultured human epithelium. N EnglJ Med 1984;311:448-5 1. 5 Hefton JM, Madden MR, Finkelstein JL, Shires GT. Grafting of bum patients with allografts of cultured epidermal cells. Lancet 1983;iu:428-30. 6 Leigh IM, Purkis PE. Culture grafted leg ulcers. Clin Exp Dermatol 1986;11:650-2. 7 Leigh IM, Purkis PE, Navsaria HA, Phillips TJ. Treatment of chronic venous ulcers with sheets of cultured allogenic keratinocytes. -Br J Dennatol 1987;117:591-7. 8 Morhenn VB, Benike CJ, Cox AJ, Charron DJ, Engleman EG. Cultured human epidermal cells do not synthesize HLA-DR. J Invest Dermatol 1982;78:32-7. 9 Thivolet J, Faure M, Demidem A, Mauduit G. Cultured human epidermal allografts are not rejected for a long period. Arch Dermatpl Res 1986;278: 252-4. 10 Burns J, Chan VTW, Jonasson JA, Fleming KA, Taylor S, McGee JO'D. Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination on intact cells. J7 Clin Pathol 1986;38:1085-92. 1 1 Cooke HJ, Schmidtke J, Gosden JR. Characterisation of a human Y chromosome repeated sequence and related sequences in higher primates. Chromosomea 1982;87:491-502. 12 Cordell JL, Falini B, Erber WN, et al. Immunoenzymatic labelling of monoclonal antibodies using immune complexes of aLkaline phosphatase and monoclonal anti-alkaline phosphatase (APAAP complexes).J7 Histochem Cytochem 1984;32:219-29. (Accepted 30anuary 1989)
Survival of cultured allogeneic keratinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome Anne Brain, Patricia Purkis, Philip Coates, Michael Hackett, Harshad Navsaria, Irene Leigh
North East Thames Regional Plastic Unit, St Andrew's Hospital, Billericay, Essex Anne Brain, FRCS, registrar in plastic surgery Michael Hackett, FRCS, consultant plastic surgeon
London Hospital, London El 1BB Patricia Purkis, chief technician Harshad Navsaria, MSC, technician Irene Leigh, MRCP, senior lecturer in dermatology Department of Histopathology, St Bartholomew's Hospital, London EClA 7BE Philip Coates, PHD, research scientist
Correspondence and requests for reprints to: Ms Anne Brain, Department of Dermatology, The London Hospital, London El 1BB. BrMedJf 1989;298:917-9
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determined by the expression of HLA-DR antigens.4 The mixed epidermal cell lymphocyte reaction in lost in parallel, which seems to confirm the loss of the Langerhans cells.5 Other cells with the potential ability to express class II antigens, such as endothelial cells, which are particularly important in rejection reactions,6 are also lost during the culture of keratinocytes from mixed suspensions. Sheets of cultured autologous keratinocytes have been used as skin grafts in patients with severe burns,7 leg ulcers,8 and congenital skin diseases such as epidermolysis bullosa.9 The delay of three to four weeks in producing expanded sheets of cells from a donor specimen, however, remains a practical problem, especially for patients with burns. Thus cultured allogeneic keratinocytes were used as grafts in patients with burns and clinically the allograft seemed to be successful (to "take").'0 This failure of rejection was attributed to the loss of the passenger leucocytesthat is, Langerhans cells. In previous studies of transplantation the survival of skin grafts was prolonged when skin that had been irradiated with ultraviolet light was used; such irradiation also reduces the numbers of Langerhans cells. " Subsequent studies of the fate of cultured allogeneic Introduction Organs transplanted across a histocompatibility keratinocytes have been controversial, with little proof barrier are usually rejected within 10-14 days unless that the allograft took rather than accelerated wound the host's immune system is suppressed. Transplanted healing. To distinguish the two processes requires a organs contain passenger leucocytes, which include the biological technique giving unequivocal results-for antigen presenting cells that present antigen to helper example, HLA analysis, DNA fingerprinting, or T cells and may initiate the rejection of a transplant by detecting the sex of cells. In 1982 Cooke et al characterexpressing class II histocompatibility molecules and ised a sequence that was repeated in tandem with 2000 secreting cytokines. Eliminating such cells from allo- copies on the Y chromosome.'2 This constitutes a fifth grafts may reduce the graft's immunogenicity and of the DNA and is located on the tip of the long arm. A prolong its survival. Culturing endocrine tissues such simple reproducible method to detect this multiple as thyroid' and pancreatic islet cells2 both eliminates copy human gene has been established.'3 To establish whether cultured allografts take antigen presenting cells and prolongs the survival of the cultured cells when they are transplanted as a biologically we used in situ hybridisation with a biotinylated probe to the Y chromosome to detect cultured allograft. In skin grafts the main antigen presenting cells are the sex of cells in skin biopsy specimens taken from Langerhans cells, which form 5% of the total mixed deep clean surgical wounds that had been grafted epidermal cell population. Seven to 10 days after a with sheets of cultured allogeneic keratinocytes single cell suspension of mixed epidermal cells is from unrelated donors of the opposite sex to the cultured in vitro the Langerhans cells are lost,3 as patients.
Abstract To determine the survival of cultured allogeneic keratinocytes transplanted to a deep dermal bed 24 tattoos that had been removed by deep shave excision in 19 patients were grafted with sheets of cultured aliogeneic keratinocytes from donors of the opposite sex. Celis carrying the Y chromosome were identified in biopsy specimens taken from the graft site by in situ DNA hybridisation with a biotinylated Y probe (pHY 2.1) and visualised with a technique using immunoperoxidase. The cultured allograft sites were biopsied one, two, and three weeks after transplantation. No male cells were identified in any biopsy specimen from female patients who were given transplants of male cultured keratinocytes, and all biopsy specimens from male patients, who received female cultured keratinocytes, showed percentages of male cells within the normal range for male skin. The beneficial effects of cultivated allogeneic keratinocytes result from effects on wound healing other than forming a successful graft that "takes."
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Methods Redundant normal skin from female patients having plastic surgery (who had been screened after counselling for infection with HIV and hepatitis B) or from infants having circumcision was the source of the cultured allogeneic keratinocytes. The skin samples were prepared and cultured as previously described.'4 Cultures were confluent at 10-14 days and left to stratify for two to three weeks, when multilayered sheets of poorly differentiated layers of keratinocytes were obtained. Sheets were separated by treatment with 0-1% dispase for one to two hours and then backed with a dressing (Johnson's NA (non-adherent dressing)) and separated from the plastic substrate with a rubber policeman, ensuring that the graft was oriented with basal cells next to the wound bed. Nineteen patients (six men, 13 women) asking for the removal of decorative tattoos of a size that required grafting (n=24) gave their informed consent to be included in the study. The patients were treated as outpatients. The tattoos were injected with local anaesthetic (0 5% lignocaine and adrenaline 1/200 000) and removed by serial shave excision until all the pigment was removed. Thus the depth of the wound varied according to clinical need from the deep dermis to the borders of subcutaneous fat (five to six shaves of 1 mm). After haemostasis the wound was covered with a sheet of cultured allogeneic keratinocytes from an unrelated donor of the opposite sex. The wound was dressed and the arm splinted. Dressings were performed weekly until the wound was completely healed, and each time the wound was dressed a punch biopsy specimen (3 mm) was taken from its centre. The biopsy specimens were snap frozen in liquid nitrogen. Sections (4 rim) were cut, mounted on multiwell slides, and fixed in Carnoy's fixative for 15 minutes. Cells carrying the Y chromosome were identified by in situ hybridisation with a biotinylated Y probe (pHY 2.1)12 and visualised by a technique using immunoperoxidase staining.'3 The samples were processed in duplicate, each with two concentrations of probe (0- 1 and 0-2 ng/I0 jt hybridisation medium) and two negative controls (hybridisation medium alone and pBR322). 3 The sections were counterstained with haematoxylin. The number of cells containing the Y chromosome, as detected with the Y probe, was counted as a percentage of the epidermal nuclei seen under high magnification (x 1000) and the mean of two to five high power fields per section was taken to cover the whole 3 mm punch biopsy specimen.
Biopsy specimen from male patient given female donor keratinocytes two weeks after grafting. Labelling of cells with Y probe (dot in nucleus) confirms that cells are frmn male recipient
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Control samples of normal male skin and sheets of male keratinocytes were processed and counted. Skin biopsy specimens were studied after routine staining with haematoxylin and eosin, and the lowest shave of the excision was studied to assess the presence of appendageal epithelium in later biopsy specimens. Results Clinical-Three out of 21 grafts were clinically healed at one week and 13 by two weeks; all grafts were healed by three weeks. Three patients could not be assessed because of non-compliance with the changing of dressings. Analysis ofbiopsy specimens-No cells carrying the Y chromosome were seen in any biopsy specimens after male keratinocytes were transplanted to female recipients (table). When female cells were transplanted Mean percentages of cells carrying Y chromosome in cultured allogeneic keratinocytes transplanted to receipient of opposite sex to donor Female donor, male recipient Male donor, female recipient Week after No of biopsy % Cells carrying No of biopsy % Cells carrying transplant specimens Y chromosome specimens Y chromosome 1 2 3
9 7 10
52 45 59
7 5 5
0 0 0
to male recipients the percentage of cells with a Y chromosome was within the normal range for male skin in the control samples and sheets of keratinocytes (5070% of cells labelled) (figure). Histopathological results-Epidermal cells were present in 10 out of 16 biopsy specimens at one week, in 11 out of 12 at two weeks, and in all 13 at three weeks. In the early biopsy specimens the epidermis was poorly cornified and there was no evidence of a normal stratum corneum. The epidermis was hyperplastic with poor rete peg formation. Extensive immunohistochemical studies will be reported elsewhere. There were no follicular remnants seen in any of the biopsy specimens, and sweat glands were seen in only two specimens. There were no remnants of the appendage-that is, sweat glands and hair follicles-in the deep shave sections. Biopsy specimens at two and three weeks showed increasing morphological differentiation with the development of an orthokeratotic stratum corneum. A mixed cell chronic inflammatory infiltrate was seen in the dermis with no evidence of intraepidermal lymphocytes.
Discussion We found that all donor cells were lost from wounds treated with cultured allogeneic keratinocytes by seven days after grafting despite considerable epithelialisation (fig 1). We therefore have no evidence of these cells taking on to a deep surgical wound. This contrasts with reports from Mauduit et al, who used cultured allogeneic keratinocytes to graft 22 surgical wounds and claimed that they took completely on to superficial donor sites, which were clinically healed after a week. 1' The recipient's failure to express his or her blood group antigen was used as a cell marker of the allograft's survival, but the expression of blood group antigens is altered during wound healing. Another study using HLA antigens as markers in four patients showed progressive replacement of the allograft from recipient cells on grafting in the appendages from 14 days onwards.'6 The controls for HLA markers should also be examined during normal wound healing because of modulation of membrane proteins during the normal healing process. An assay system based on DNA, such
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as one using a Y probe or DNA fingerprinting, is therefore better than one based on membrane antigens. We treated wounds at a deep level of the dermis with no remaining hair follicles and few sweat glands; therefore the epithelial keratinocytes largely arose and migrated from the edge of the wound and there was little if any contribution from the appendages. Studies in animals by Hammond et al using cultured allogeneic keratinocytes in a transplantation chamber that ensured the physical separation of donor and recipient cells showed survival of donor keratinocytes (free of Langerhans cells) for 70 days compared with 14 days for intact allografts.'7 This also supports the concept that donor keratinocytes are tolerated after they have been cultured. In this system, however, there are no competing host epithelial cells to replace the allograft. In contrast, our results are supported by Aubock et al, who found rejection of cultured allogeneic keratinocytes in 11 patients 14 days after they had received the allograft.'8 Levick and Clarke et al were also unable to find convincing evidence that the allograft had taken.9 120 The loss of keratinocytes so soon after transplantation could be explained by mechanisms other than hyperacute rejection, and certainly we saw no lysis of the allograft clinically or intraepidermal infiltration by lymphocytes histologically (although in rejection, the response of lymphocytes is mixed and diagnostic markers difficult to define2). Mackenzie and Hill also used the transplantation chamber system with autologous mouse keratinocytes,22 and Mackenzie has recently shown differences between the ability of superficial and deep connective tissue to support epidermal differentiation-epidermal cells retain a simple epithelial phenotype when transplanted on to deep connective tissue (unpublished data). Thus our results could reflect the failure of deep connective tissue to support allogeneic keratinocytes. Clinical reports of autologous cells grafted on to deep fascia have, however, claimed clinical take, although the wounds were unstable with blistering.23 Our early biopsy specimens from the allograft did show substantial epithelialisation that was poorly differentiated but certainly multilayered. These features suggest rapid wound healing, which may mean that there is physical replacement of the allograft rather than rejection, and accelerated wound healing could account for the main clinical benefits reported previously.' 13 Controlled trials of cultured allogeneic keratinocytes against simple dressings in leg ulcers and against hydrocolloid dressings in donor sites are in progress that will address the question of accelerated wound healing. Activated keratinocytes in culture have multiple biological effects, which could enhance wound healing. These include the production of basement membrane proteins (collagen types IV-VII, laminin, fibronectin, basic protein antigen) to enhance cell migration; physical scaffolding; protection; and the production of growth promoting agents. Cultured keratinocytes have recently been shown to produce transforming growth factor alpha,24 which accelerates wound healing in pigs.25 As biological take does not seem to occur after transplantation from sheets of cultured allogeneic keratinocytes on to deep surgical wounds further studies are needed to examine the effects of the depth of the wound, the state of
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keratinocyte culture, the immunosuppression of the recipient, and the provision of a substrate on the success of allografts. Controlled studies of wound healing are also required to establish that wound healing is accelerated and whether it occurs because of a diffusible product or a different biological effect requiring contact with live cells. The possibility exists of producing a keratinocyte product or products by recombinant technology in a form that could help wound healing without live cells. This work was supported by the Billericay Burn Research Fund and the Skin Disease Research Fund. 1 Lafferty KJ, Bootes A, Dart G, et al. Effect of organ culture on the survival of thyroid allografts in mice. Transplantation 1976;22:138-49. 2 Kedinger M, Haffen K, Grenier J, Eloy R. In vitro culture reduces immunogenicity of pancreatic endocrine islet cells. Nature 1977;270:736-8. 3 Rheinwald JC, Green H. Serial cultivation of strains of keratinocytes: the formation of keratinising colonies from single cells. Cell 1975;6:331-43. 4 Morhenn VB, Benike CJ, Cox AJ, Charron DJ, Engelman EG. Cultured human epidermal cells do not synthesise HLA-DR. J Invest Dermatol
1982;78:32-7. 5 Hefton JM, Amberson JB, Biozes DG, Weksler ME. Loss of HLA-DR expression by human epidermal cells after growth in culture. J Invest Dermatol 1984;83:48-50. 6 Fabre JW, Milton AD, Spencer S, Settaf A, Houssin D. Regulation of alloantigen expression in different tissues. Transplant Proc 1987;19:45-9. 7 Gallico GG 3d, O'Connor NE, Cofiipton CC, Kehinde N, Green H. Permanent coverage of large bum wounds with cultured autologous human epithelium. N Engl' Med 1984;311:448-5 1. 8 Leigh IM, Purkis PE. Culture grafted leg ulcers. Clin Exp Dermatol 1986;11:650-2. 9 Carter DM, Lin AN, Varghese MC, Caldwell D, Pratt LA, Eisenger M. Treatment of junctional epidermolysis bullosa with epidermal autografts. J7 Am Acad Dermatol 1987;172:246-50. 10 Hefton JM, Madden MR, Finkelstein JL, Shires GT. Grafting of burns patients with allografts of cultured epidermal cells. Lancet 1983;ii:428-9. 11 Kripke ML. Immunological unresponsiveness induced by ultraviolet radiation. Immunol Rev 1984;80:87-102. 12 Cooke HJ, Schmidtke J, Gosden JR. Characterisation of a human Y repeated sequence and related sequences in higher primates. Chromosoma 1982;87: 49 1-502. 13 Bums J, Chan VTW, Jonasson JA, Fleming KA, Taylor S, McGee JO'D. Sensitive system for visualising biotinylated DNA probes hybridised in situ: rapid sex determination of intact cells. J Clin Pathol 1985;38:1085-92. 14 Leigh IM, Purkis PE, Navsaria HA, Phillips TJ. Treatment of chronic venous ulcers with sheets of cultured allogenic keratinocytes. Br J Dermatol 1987;117:591-7. 15 Mauduit G, Faure M, Demiderm A, Kanitakis J, Thivolet J. Cultured human epidermis used as allografts-studies on their differentiation. J7 Invest Dermatol 1986;87:1546. 16 Gielen V, Faure M, Mauduit G, Thivolet J. Progressive replacement of human cultured epithelial allografts by recipient cells as evidenced by HLA class I antigen expression. Dermatologica 1987;175:166-70. 17 Hammond EJ, Ng R, Stanley MA. Prolonged survival of allogenic cultured keratinocyte sheets in the mouse without imunosuppression. J Invest Dermatol 1986;87:143. 18 Aubock J, Fritsch P. Autologous versus allogenic cultivated epidermis for wound grafting. In: Teepe R, ed. Proceedings symposium on "clinical use of cultured epithelium in surgery and dermatology," Leiden. Wheathampstead: Medical and Scientific Conferences Ltd, 1987:59-64. 19 Levick PL. Generation of autografts-current techniques and results. In: Teepe R, ed. Proceedings symposium on "clinical use of cultured epithelium in surgery and dermatology," Leiden. Wheathampstead: Medical and Scientific Conferences Ltd, 1987:15-8. 20 Clarke JA, Burt AM, Eldad A, Yardeni P, Gusterson B. Allograft cultured epithelium and meshed allograft skin. In: Teepe R, ed. Proceedings symposium on "clinical use of cultured epithelium in surgery and dermatology, Leiden. Wheathampstead: Medical and Scientific Conferences Ltd, 1987: 33-6. 21 Hall BM. Cellular infiltrates in allografts. Transplant Proc 1987;19:50-6. 22 Mackenzie IC, Hill MW. Connective tissue influences on patterns of epithelial architecture and keratinisation in skin and oral mucosa of adult mouse. Cell Tissue Res 1984;235:551-9. 23 Gallico GG, O'Connor NE, Briggs SM, Compton CC, Kehinde 0, Green H. Cultured epithelial autografts: indications and long term results. In: Teepe R, ed. Proceedings symposium on "clinical use of cultured epithelium in surgery and dermatology," Leiden. Wheathampstead: Medical and Scientific Conferences Ltd, 1987:19-24. 24 Coffey RJ, Derynck R, Wilcox JN, et al. Production and autoinduction of transforming growth factor alpha in human keratinocytes. Nature 1987;328: 817-20. 25 Schultz GS, White M, Mitchell R, et al. Epithelial wound healing enhanced by transforming growth factor alpha and vaccinia growth factor. Science
1987;235:350-2. (Accepted 303anuary 1989)
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