Annals of Burns and Fire Disasters - vol. XII - n° 4 - December 1999

Smirnov S.V.,¹ Vasiliev A.V.,² Paramonov B.A.,³ Loginov L.,¹ Kiseliov I.V.,² Danilova T.I.,² Terskikh V.V.²

¹ Sklifosovsky Research Institute of Emergency Care, Moscow, Russia
² Institute of Development Biology, Russian Academy of Sciences, Moscow
³ Military Medical Academy, St Petersburg

SUMMARY. This study was conducted over a period of seven years (1992-98) in burn units at the Sklifosofsky Research Institute of Emergency Care (Moscow) and the Military Medical Academy (St Petersburg). Sixty transplants were performed in 48 patients with second- and third-degree burns and donor sites. Allotransplantation was found to have a beneficial effect on wound healing. We suggest that, after grafting, allogenic keratinocytes activated by in vitro cultivation may produce a reparative environment necessary for the proliferation and migration of recipient keratinocytes.

Rapid closure of burn wounds significantly improves the treatment of thermal injuries, but successful wound coverage is often limited by a lack of suitable autografts.
Transplantation of cultured keratinocyte sheets is a promising approach in the restoration of skin lesions after severe burns. Cultured keratinocyte allografts were first used as a replacement of missing skin because it was thought that, in culture, keratinocytes did not express HLADR. Later it was shown that cultured allografts are replaced by the recipient's own cells. However, they offer certain advantages - immediate graft availability, an almost unlimited supply, and bulk culture of keratinocytes. It is now evident that allografts may function not only to replace skin but also to stimulate re-epithelialization in the recipient's wound.
We first treated burns with cultured autologous grafts, after which we used cultured allografts. We now report on our seven years of experience from 1992 to 1998 in allograft treatment of burns.

Materials and methods

Wound preparation and treatment
In serious burns necrectomy was performed in one or several steps. Wounds were treated with antiseptics and water-soluble ointments. All patients were treated with antibiotics. Chlorhexidine, hydrogen peroxide, and potassium permanganate were also used when necessary. In the event of excessive granulation, hydrocortisone ointments were applied (two cases). In two cases prednisolone was included in the treatment procedure in order to suppress the immune reaction before and after grafting. Beginning 3-5 days before grafting the wounds were bandaged daily.
Before transplantation the wounds were treated with gentamicin solution (0.16 g/1). In three patients chlorhexidine solution was applied before gentarnicin treatment.
The wound bed was considered ready for grafting on the basis of the following criteria:

1. presence of clean fine-granular reddish granulation not elevated above the level of healthy tissues
2. absence of Staphylococcus and Streptococcus contaruination
3. adhesiveness of wound bed
4. marginal epithelialization

Keratinocyte culture
Allogenic keratinocytes were isolated from skin fragments taken from healthy patients undergoing plastic surgery and cultured according to the method described by Rheinwald and Green (with some modifications). In brief, epidermal keratinocytes were isolated by overnight trypsinization at 4° C followed by additional treatment with diluted trypsin. The complete medium consisted of DMEM:F12 (1:1) mixture supplemented with 10% foetal calf serum (FCS), 10 ng/rnI epidennal growth factor (EGF) (Sigma), 5 ng/ml insulin (Sigma), 10⁶ M isoproterenol (Sigma), and 5 ng/ml sodium selenite (Sigma). Keratinocytes were cultivated without feeder layer in 75 cm² flasks (Costar) coated with collagen. The medium was changed every other day and cultures were kept at 37° C in a humid atmosphere incubator containing 3% CO,. The cultures became confluent within 3 to 4 weeks.

Transportation of cultured sheets to hospital
Confluent epithelial sheets were either used instantly or stored at 26 'C until use. The day before transplantation the culture medium was changed for the complete medium without FCS. The culture flasks filled with Eagle MEM were delivered to the hospitals in thermostatic containers.

Keratinocyte grafting
Keratinocyte cultures suitable for grafting were treated with 1.2 U/ml Dispase 11 (Sigma) in serum-free medium. Dislodged sheets, fixed on Paranett or Jelonet dressing (Smith and Nephew), were grafted on prepared wounds pre-treated with gentarnicin in normal saline solution (0. 16 gJ). The dressing was covered over with gauze moistened with the same solution.
The first change of dressing depended on wound conditions. Usually it was performed on day 3-5 postgraft. Further dressings were done every second or third day. In cases where the grafted epithelial sheet was visible on the wound surface, the wound was dressed with Paranett or Jelonet and the gauze was moistened with gentamicin solution. When the epidermal sheet was not seen on the wound surface but pronounced marginal epithelialization was observed, dressings with antiseptics and water-soluble ointments were applied to prevent bacterial contamination. In cases where visual inspection did not reveal either the grafted epithelial sheet or marginal epithelialization we repeated grafting with allogenic cultured sheets or with split-thickness transplants.

Histological analysis
Small biopsies obtained from grafted sites were fixed in alcohol-acetic acid and embedded in paraffin, after which 5 [tm-thick sections were stained with haematoxylin and eosin.

Sixty transplantations were performed in 48 patients at the Sklifosovsky Research Institute of Emergency Care and the Military Medical Academy. The transplantations were carried out two weeks to three months after injury in second- and third-degree burns and in donor sites.
Thirty allogenic grafts were performed in 25 patients at the Sklifosovsky Research Institute of Emergency Care (SRIEC). In one patient the same wound was grafted twice. In four patients the transplantations were performed in two successive stages. Twenty-three patients were grafted with allogenic cultured epithelial sheets at the Military Medical Academy (MMA). In two patients grafting was performed in three stages, and in three patients in two stages.
The presence of the epidermal sheet on the wound bed during the first dressing change provided considerable support for the success of the grafting. In practice, epidermal sheet lysis was usually observed during the first rebandaging. We rarely observed total rejection of the sheet after temporary take. Fig. I shows the distribution of results according to these criteria. At SRIEC sheets were visible on the wound in 15 cases (50% ) during the first rebandaging.
Lysis occurred later in three cases. Partial lysis of the sheets was observed in six cases (20%) and total lysis in nine cases (30%). At MMA these parameters corresponded respectively to 13 cases (43%), 14 cases (47%), and 3 cases (10%).

Lysis of grafted epithelial sheets after the first dressing was observed more frequently in third-degree than in second-degree burns or donor sites (Table 1).

At SRIEC two allogenic transplantations were performed on donor sites and six on partial-thickness burns. Epithelial sheets were visible during the first dressing change in all cases. Out of 22 allogenic sheets grafted onto full-thickness burns the transplants were visible during the first change of dressing in only seven cases (32%).
We found that the main causes of grafting failures were manipulation errors during the procedure of grafting and further treatment of patients, the unsatisfactory condition of granulation tissues, and the development of wound contamination.
The application of allogenic sheets in patients with second-degree burns was intended to decrease the duration of treatment. When correct grafting was performed soon after burn trauma on prepared wounds, restoration of the epidermis occurred 3-12 days earlier than with routine treatment.
The maximum area of restored epidermis using allogenic sheets was 450 cm² at SRIEC and 450 cm² at MMA.
In patients with full-thickness burns, grafting of allogenic keratinocyte sheets can take the place of routine split~thickness skin autografting in some cases. Table II summarizes our data when full or partial skin restoration after allogenic keratinocyte grafting onto limited areas of full-thickness burns was observed.

In patients with third-degree burns 22 transplantations were performed at SRIEC. Complete skin restoration without split-thickness skin autografting was achieved in seven cases (32%). It is interesting to note that in two patients the epidermal sheet was invisible during the first rebandaging and that take of the graft was temporary in one patient. Restoration of skin in these three patients was complete due to strongly induced marginal epithelialization. The maximum area of restored skin was 300 cm². Partial skin restoration was observed in six cases, while in one case the transplant sheet could not be observed during the first rebandaging. In eight cases (36%) skin was not restored. At MMA complete skin restoration in patients with full-thickness burn was achieved in two cases (33.3%) without split-thickness autografting and partial restoration in two cases, with failure in two cases. The maximum area of restored skin was 70 cm².

Case 1. A 69-yr-old female arrived at SRIEC with flame burns in the back, the right buttock and part of the hip, and the right lateral part of the body. She had sustained 20% TBSA burns (9% full-thickness). She was considered to be in a bad condition because of her age, burn area, and associated diseases (mitral insufficiency, cholelithiasis, chronic pancreatitis, psoriasis, frequent pneumonia). She was given appropriate medical therapy. By day 33 of treatment the area of granulation tissue was about 4% TBSA. Successive necrectomies were performed. The burn wounds were treated conservatively because the patient refused surgery. On day 50 post-trauma granulations remained in 1.5% TBSA. The granulation tissue was fine granular, pale pink, and with scant wound fluid. By day 51 1.5% TBSA was covered with allogenic cultured sheets. The first wound inspection was performed three days post-graft. Pronounced marginal epithelialization was recorded. The wound area decreased by one-third, but grafted sheets could not be seen. By day 6 post-graft we observed progressive wound decrease owing to marginal epithelialization. By day 10 post-graft the wound area amounted to 3 cm². By day 15 post-graft (day 66 post-burn) the skin was completely restored.

Case 2. A 38-yr-old male was hospitalized in the MMA Burn Unit with burns in the head, neck, trunk, and limbs. The patient had sustained 50% TBSA flame burns (20% full-thickness skin loss). The patient was given appropriate proper fluid and antibiotic therapy. The wounds were prepared for autograffing intensively by necrolytic drugs. By day 16 post-injury mesh split-thickness autografting onto full-thickness burns was performed. Partial-thickness wounds of the trunk (300 cm²) and arms (150 cm²) were covered with allogenic cultured keratinocyte sheets. Restored skin was observed on day 6 post-graft on the Dearly complete graft area during the first dressing change. The grafted epithelium looked like a dim film. Biopsy samples of reconstructed skin were taken on day 7 post-graft. The epidermis contained 10-13 cell layers and was loosely connected with underlying granulation tissue. The basal cells exhibited destructive alterations, while the prickly cells appeared normal. The partial-thickness wounds not treated with keratinocytes healed 7-12 days later. ne patient was discharged from hospital on day 54 post-lesion. Skin biopsies were made in the allograft area two years after treatment. The histological examination revealed a well-developed epidermis with an almost straight border between dermis and epidermis. The deep skin appendages were preserved.

The use of cultured allografts has significantly extended current knowledge of the regenerative biology of skin grafts. Allogenic keratinocyte sheets have been shown to promote healing in a wide variety of clinical instances, such as burns, leg ulcers, Lyell's syndrome, recessive dystrophic epidermolysis bullosa, chronic otorrhoea, and donor sites.
Originally it was thought that cultured allografts acted as a "biological" dressing and skin replacement. Today it is known that cultured grafts can also work as a pharmacological agent.
The grafting of allogenic keratinocyte sheets leads not to permanent but to slightly prolonged graft survival.. The use of specific probe for Y chromosome and DNA fingerprinting of the wound covering epidermal cells taken after successful allografting showed that keratinocytes originated from the recipient and not the donor cells. Zhao et al. employed two methods to identify the presence of allografted keratinocytes on the wounds: an indirect enzyme-conjugated Staphylococcus protein A to detect A or B blood groups and a polymerase chain reaction to amplify the Y chromosome-specific DNA sequence. It was concluded that the survival time of cultured allografts was prolonged up to 35 days and that recipient tissue and graft tissue combined to provide coverage of mixed host and donor cells. Moll et al. used a skin organ culture model for epidermal healing to investigate the fate of transplanted keratinocytes. It was shown that autologous as well as allogenic transplanted keratinocytes were integrated into the regenerating epidermis.
Relatively late rejection of allogenic keratinocyte grafts with no clinically visible inflammation reaction suggests a temporary take of allogenic keratinocytes. There are data suggesting that the effects of cultured allografts on wound healing may be growth-factor-mediated as keratinocytes produce TGF-a and TGF-b. Cultured keratinocytes synthesize fibronectin and collagen IV which stimulate the spreading and proliferation of keratinocytes. Allogenic keratinocytes also produce interleukins.
It has been shown that allogenic grafts produce healthylooking granulation tissue, induce the edge effect, and accelerate the shift of the epidermis from an inflammatory to a regenerative state. Cultured keratinocytes also have antibacterial activity. It is therefore evident that allografts exert different influences on the process of wound healing that induce the proliferation and migration of recipient keratinocytes and stimulate assembling of the basal membrane before they are rejected. Compton et al. studied the reconstruction of skin in patients treated with cultured autologous keratinocytes. Six days after grafting the process of de novo fon-nation of the dermal-epidermal junction had begun and within 3-4 weeks the formation of mature hemidesmosomes and the dermal-epidermal junction was complete. Cultured keratinocytes undergo "activation", which includes increased cell attachment, spreading, and migration as well as metabolic changes. We therefore suggest that culture-activated allogenic keratinocytes stimulate the restoration of skin after transplantation onto the wound bed.
Besides immediate graft availability and unlimited supply, allogenic keratinocytes accelerate wound healing and produce excellent cosmetic results. The great advantage of cultured allografts is the possibility of storing them for long periods of time in skin banks.
Since the classical method of keratinocyte cultivation was described by Rheinwald and Green, various other methods have been proposed. To facilitate grafting, keratinocytes were cultivated on a polymer film. The growing of cells on micro-carriers is the most promising method of cultivation that is suitable for the storage of cultured keratinocytes in skin banks.

Although the role of keratinocyte allografts in the acceleration of wound healing is not completely understood, the use of allografts has led to a fascinating variety of medical applications. Allografts are replaced by the recipient's keratinocytes but they stimulate wound healing. We suggest that, after grafting, the allogenic keratinocytes activated by in vitro cultivation produce the reparative environment necessary for the proliferation and migration of recipient keratinocytes. The most significant advantage of using allografts is their ready availability and the possibility of cryopreservation in skin banks.

RESUME. Cette étude a été effectuée pendant une période de sept années (1992-1998) dans les Unités de Brûlures à l'Institut de Recherches de la Thérapie d'Urgence Sklifosovsky (Moscou) et à l'Académie Médicale Militaire (Saint-Pétersbourg). Soixante transplantations ont été effectuées dans 48 patients atteints de brûlures de deuxième degré et dans les sites donneurs. L'effet positif de l'allotransplantation sur la guérison des lésions a été observé. Selon les Auteurs, après la greffe, les kératinocytes activés par la culture in vitro créent l'environnement réparateur nécessaire pour la prolifération et la migration des kératinocytes receveurs.

BIBLIOGRAPHY

1. Terskikh V.V., Vasiliev A.V.: Cultivation and transplantation of epidermal keratinocytes. Intern. Rev. Cytol., 188: 41-72, 1999.
2. Morherm V.13., Benike C., Cox AJ. et al.: Cultured human epidermal cells do not synthesize HLA-DR. J. Invest. Dermatol., 78: 32-7, 1982.
3. Hefton LM., Amberson LB., Biozes D.G., Weksler M.E.: Loss of HLA-DR expression of human epidermal cells after growth in culture. J. Invest. Dermatol., 83: 48-50, 1984.
4. Aubbck J., Irschick E., Romani N. et al.: Rejection, after a slightly prolonged survival time, of Langerhans cell-free allogenic cultured epidermis used for wound coverage in humans. Transplantation, 45:730-7, 1988.
5. Oliver A.M., Kaawach W., Mithoff E.M. et al.: The differentiation and proliferation of newly formed epidermis on wounds treated with cultured epithelial allografts. Br. J. Dermatol., 25: 147-54, 1991.
6. Malakhov S.V., Vasiliev A.V., Paramonov B.A., Bautin E.A., Terskikh V.V.: Autotransplantation of keratinocytes grown in vitro for treatment of extensive burns. Vestnik Khirurgii im. I.I. Grekova, 4: 59-62, 1993 (Russian).
7. Malakhov S.V., Paramonov B.A., Vasiliev A.V., Terskikh V.V.: Preliminary report of clinical use of cultured allogenic keratinocytes. Burns, 20: 463-6, 1994.
8. Rheinwald J.G., Green H.: Serial cultivation of strains of human epidermal keratinocytes: The formation of keratinizing colonies from single cells. Cell, 6: 331-44, 1975.
9. De Luca M., Albanese E., Bondanza S., Megna M., Ugozzoli L., Molina F. et al.: Multicentre experience in the treatment of burns with autologous and allogerric cultured epithelium fresh or preserved in a frozen state. Burns, 15: 303-10. 1989.
10. Madden MR., Finkelstein J.L., Staiano-Coico L. et al.: Grafting of cultured allogenic epidermis in second- and third-degree burn wounds on 26 patients. J. Trauma, 26: 955-62, 1986.
11. Phillips T.J., Gilchrest B.A.: Clinical application of cultured epithelium. Epith. Cell. Biol., 1: 39-46, 1992.
12. De Luca M., Albanese E., Cancedda R., Viacava A., Faggioni A., Zan Bruno G. et al.: Treatment of leg ulcers with cryopreserved allogenic cultured epithelium: A multicentre study. Arch. Dermatol.: 128: 633-8, 1992.
13. Teepe R.G.C., Roseeuw D.I., Hermans J., Koebrugge E.J., Altena T., De Coninck A. et al.: Randomized trial comparing cryopreserved cultured epidermal allografts with hydrocolloid dressing in healing chronic venous ulcers. J. Am. Acad. Dermatol., 29: 982-9, 1993.
14. Napoli B., D'Arpa N., Masellis M. et al.: Use of cultured homologous keratinocytes in the local treatment of Lyell's syndrome. Ann. Burns and Fire Disasters, 9: 163-7, 1996.
15. McGuire J., Birchall N., Cuono C., Moellman G., Kuklinska E., Langdon R.: Successful engraftment of allogenic keratinocyte cultures in recessive dystrophic epidermolysis bullosa. J. Invest. Dermatol., 88: 506A, 1987.
16. Somers T., Verbeken G., Vanhalle S. et a].: Treatment of chronic post-operative otorrhoea with cultured keratirocyte sheets. Ann. Orel. Rhinol. Laryngol., 106: 15-21, 1997.
17. Teepe R.G.C., Koch R., Haeseker B.: Randomized trial comparing cryopreserved cultured epidermal allografts with tulle gras in the treatment of split-thickriess skin graft donor sites. J. Trauma, 35: 850-4, 1993.
18. Duinslaeger L.A.Y., Verbeken G., Vanhalle S., Vanderkelen A.: Cultured allogenic keratinocyte sheets accelerate healing compared to Op-Site treatment of donor sites in burns. J. Burns Care Rehabil., 18: 545-51, 1997.
19. Kirsner R.S., Falanga V., Kerdel F.A., Katz M.H., Eaglstein W.H.: Skin grafts as pharmacological agents: Pre-wounding of the donor site. Br. J. Dermatol., 135: 292-6, 1996.
20. Rouabhia M.: Skin regeneration after heterologous epidermal substitute grafting. Ann. Burns and Fire Disasters, 10: 98-104, 1997.
21. Brain A., Purkis P., Coates P. et al..: Survival of cultured allogenic keiatinocytes transplanted to deep dermal bed assessed with probe specific for Y chromosome. Br. Med. J., 298: 917-9, 1989.
22. Van der Merve A.E., Mattheyse F.J., Bedford M. et al.: Allografted keratinocytes used to accelerate the treatment of burn wounds are replaced by recipient cells. Burns, 16: 193-7, 1990.
23. Zhao Y.B., Zhao X.-F., Li A. (Ngao) et al.: Clinical observations and methods for identifying the existence of cultured epidermal allografts. Burns, 18: 4-8, 1992.
24. Moll L, Houdek P., Schmidt H., Moll R.: Characterization of epidermal wound healing in a human skin organ culture model: Acceleration by transplanted keratinocytes. J. Invest. Dermatol., 111: 251-8, 1998.
25. Coffey R.J., Derynck R., Wilcox J.N. et a].: Produduction and autoinduction of transforming growth factor a in human keratinocytes. Nature (Lond.), 328: 817-20, 1987.
26. Partridge M., Green M.R., Langdon I.D., Feldman M.: Production of TGF-a and TGF-P by cultured keratinocytes, skin, and oral squamous cell carcinomas - potential autocrine regulation of normal and epithelial cell proliferation. Brit. J. Cancer, 60: 542-8, 1989.
27. O' Keefe E.J., Woodley D.T., Castello G. et al.: Production of soluble and cell-associated fibronectin by cultured keratinocytes. J. Invest. Dermatol., 82: 150-5, 1984.
28. Ohkura K., Fujii T., Konishi R., Terada H.: Increased attachment and confluence of skin epidennal cells in culture induced by ascorbic acid: Detection by permeation of trypan blue across cultured cell layers. Cell Struct. and Funct., 15: 142-50, 1990.
29. Woodley D.T., Wynn K.C., O'Keefe E.J.: Type IV collagen and fibronectin enhance human keratinocyte thymidine incorporation and spreading in the absence of soluble growth factors. J. Invest. Dermatol., 94: 139-43, 1990.
30. Partridge M., Chantry D., Turner M. et al.: Production of interleukin- I and interleukin-6 by human keratinocytes and squamous cell carcinoma cell line. J. Invest. Dermatol., 96: 771-6, 1991.
31. Moll L, Schonfeld M., Jung E.C.: Applikation von Kerafinozyten in der Therapic von Ulcera crumm. Hautarzt, 105: 548-52, 1995.
32. Lang E., Schaefer E.M., Eickhoff U., Hohl H.-P., Kramer M.D.: Rapid normalization of epidermal integrin expression after allografting of human keratinocytes. J. Invest., Dermatol. 107: 422-7, 1996.
33. Maier K., Ehrhard G., Frevert J.: Antibacterial activity of cultured human keratinocytes. Arch. Dermatol. Res., 284: 119-21, 1992.
34. Grinnell F.: The activated keratinocyte. Upregulation of cell adhesion and migration during wound healing. J. Trauma, 30: 144-9, 1990.
35. Burt A.M., Clarke J.A.: Transfer of autologous keratinocytes grown on a polymer reduces time taken from biopsy to graft. Ann. Burns and Fire Disasters, 10: 215-8, 1997.

36. Vasiliev A.V., Smimov S.V., Ermolinsky I.I., Zaikonnikova A.P., Samarova A.V., Kiseliov LV., Terskikh V.V.: Restoration of epithelia] lesions by transplantation of human allogenic keratinocytes. Intern. European A.I.R.R. Conference, Cologne, Germany, 44, 1997.