Ann. Medit. Burns Club - vol. V71 - n. 4 - December 1994


Brychta R*, Adler J.**, Rihovd R*, Suchdnek V, Komdrkovd J.**

* Burns Centre, Brno, Czech Republic
** Tissue Culture Laboratory, Masaryk University, Brno

SUMMARY. After considering the history of research into the cultivation in vitro of skin cells, the authors describe their experience at the Brno Burns Centre with this technique in the treatment of burn patients. So far about 10,000 sq cm of cultured epidermal grafts have been applied. The results and costs are described. Keratinocytes cultured in vitro can also be used to test the cytotoxicity of various synthetic bandages. The importance of in vitro cultured skin cells is destined to increase, both on the theoretical level and in clinical practice.

The history of in vitro skin cell cultivation is not long. In the 1950s Billingharn succeeded in dividing the epidermis from the dermis enzymatically using trypsin, without destroying the vitality of the epithelial cells. The next important milestones were in the early 1960s, when Karasek demonstrated that keratinocytes could survive in tissue culture, and in 1975, when Rheinwald and Green published their work (1) on the growth and proliferation of dermal cells in vitro up to the point at which a larninar, keratinizing, transplantable skin epithelium was achieved. Finally O'Connor et al. (2), in 1981, reported the first success with the autotransplantation of cultured epidermal grafts in man.
Many other researchers and institutions soon realized the importance of this method, which allows an expansion of the epithelium of up to several thousand times for use in the treatment of burns, and the cultivation of keratinocytes and their application began to be intensively performed. In the Brno Burns Centre we have also followed with interest developments in in vitro cultivation of skin cells, and since 1991 we have used the method ourselves. We have so far applied about 10,000 sq cm of cultured epidermal grafts. About twice as much was actually cultivated. We use a slightly modified version of the Rheinwald-Green method using 3T3, sublethally inhibited with mitomycin. In cases of extensive bums we take a skin sample 2 to 6 cm square at full thickness, or using a Watson knife in the form of a split-thickness skin graft. Disinfection is carried out using 76% ethanol or 3% iodonal. Any area not affected by bums can be used. We also use skin fragments obtained during plastic surgery, the best being skin from the eyelids or from the back of the ears. Samples are rinsed in Ringer solution, placed in transfer medium and immediately taken to the laboratory for cultivation.
In the laboratory the skin is cleaned of the subcutis and placed in 0.2% trypsin solution, which separates the epidermis from the dermis in 16 to 20 hours. The keratinocytes are mechanically loosened, flushed, centrifuged and resuspended in a nutrient medium. The primary culture is established from cells prepared in this fashion. Each plastic flask (capacity 80 sq cm) is inoculated with about 2 X 106 cells. Subcultures from the primary culture are established on about day 10, and on about day 18 to 21 growth of multilayers can be observed on the base of the flasks. The upper parts are then sliced off using thin wire. The grafts are released from the flasks using Dispase, prefixed to the carrier, which is usually Grasolind, and then either transferred in a transfer medium back to the operating theatre or cryopreserved for later use.
In the operating theatre the grafts are applied to the wound after disinfection with antibiotic solution (not normal antiseptics) according to sensitivity, and flushed with Ringer solution. They are fixed with metal clamps, or bandages if the area is a small one. We apply cultured keratinocytes to areas of second- and third-degree burns, either as autografts or as allografts.
All donors for keratinocyte cultivation are screened according to a standard protocol to eliminate the possibility-of infection from HIV 1/2, HBsAg, BWR and CMV a~lografts. The cost of cultured epidermal grafts is 193 crowns per sq cm and 208 crowns per sq cm in the case of cryoconservation (i.e. US$ 6.45 and 6.93 respectively). In third-degree burns we achieved only 18% take, which is relatively very low. The cause in all cases was bacterial contamination of the burn wounds and insufficient use of cadaver dermoepidermal allografts. Conversely, with the application of cultured epidermal grafts as allotransplants in very deep second-degree bums, we recorded excellent results. The patients experienced alleviation of pain within a few hours and on rebandaging after 48 hours the wound exhibited a dry, pain-free, epithelialized surface. It had already been proved that these allografts were also subject to rejection and that the best results were achieved by maximum stimulation of the remaining cell elements through apocrine production of various cytokines and hormones in the donor keratinocytes and fibroblasts, of which about 30 have so far been identified.
Treatment of deep dermal bums using cultured epidermal allografts is of great importance in infants, as these account for over 90% of burn cases in hospitalized children. These injuries are usually 5-40% TBSA in extent, and treatment is a major problem. A comparison was made between the cost of typical child burn treatment, i.e. second-degree scalds in about 10% BSA, with application of cultured epidermal allografts and with conservative treatment using Flamazine. The conservatively treated group consisted of ten infants and the cultured allograft group of three typical patients. The cost of conservative treatment was US$ 1672 (average hospital stay: 18.4 days per patient). With cultured allografts the cost was US$ 1037 (average hospital stay: 7.3 days).
A totally different application of the use of keratinocytes cultured in vitro is the testing of cytotoxicity of various synthetic bandages or temporary skin covers. We use the elution method and we have so far tested 15 synthetic materials in the context of HeLa and LEP (human embryonic lung) cells. It is interesting that some commercially available synthetic bandages were cytotoxic for human keratinocytes.
In order to follow current trends in the study of cultured skin cells we adopted three basic lines in our practical experience. Substitution of the dermal component of skin and its recombination with cultured human keratinocytes in vitro. The best material in vitro appears to be cadaver dermis, trypsinized and meshed in the ratio of 1 to 1.5. Surface-cultured keratinocytes form a good multilayer and dermoepidermal junction. Another suitable dermal substitute is the collagen lattice, contracted by human fibroblasts and subsequently set with cultured human keratinocytes. In this case, however, a problem arises because the cultivation time is rather long and the price comparatively high. It is also difficult to control continuing collagen contraction.
In an animal experiment we created a circular fullthickness skin defect on the back of rats (diameter 3 cm), using a plastic ring which prevented spontaneous healing from the edges of the wound. The rats were non-inbred, and we exchanged their corium with epidermis removed by trypsination. To this corium we applied the keratinocytes themselves, which had meanwhile been cultivated in vitro to a multilayer. Wound healing took 25 days, while in the control group there was no healing before removal of the ring.
We have achieved very well formed recombined skin, consisting of allogenous dermis and native cultured keratinocytes, including a very well formed structure of the dermis and dermoepidermal junction.
A similar approach was used in the clinical experiment. In a male child with extensive burns to whom classical dermoepidermal allografts were applied, taken from the father, we covered part of the burn wound with the socalled second-cut-dermis, while the epidermis was returned to the donor site of his father. The healing of the donor site was naturally very fast and less painful than in the case of other areas, and the cosmetic effect was better.
There was likewise no problem in the take of the meshed dermis on the burn wound. However, subsequent application of autologous cultured keratinocytes was not successful owing to secondary rejection caused by massive contamination of all wound surfaces with methicillin-resistant Staphyloccus aureus.

This is a modification of the method presented by Hickerson (3), who after the dermoepidermal allografts have taken removes the epidermis with a dermatome and substitutes it with autologous cultured keratinocytes on day 7- 10 after transplantation.
Take is over 90%, but removal of the epidermis is difficult and it cannot be returned to the donor. At present we are trying to compare the two methods. Another direction of our studies is the use of the above-mentioned effects of cultured epidermal allografts to prepare so-called active wound dressing, in which the optimum effects of synthetic cover is recombined with the secretory effects of cultured skin cells. In this case the cells need not be applied as a multilayer, but on a suitable car~ rier. We are trying various forms of collagen and silicone.
It is very interesting to compare keratinocytes and fibroblasts taken from adults with the saine cells from embryonic and fetal sources. The generally higher metabolic activity of embryonic and fetal keratinocytes can be seen in the faster confluence and the formation of multilayer (13-16 days), and there is a striking increase in the speed of contraction of the collagen lattice by embryonic fibroblasts (4-7 days).
Morphology in the electron microscope shot shows a thick, reticular nucleolus, very well developed mitochondria and multiple keratin fibrils. This indicates the high metabolic activity of embryonic and fetal skin cells.
In the clinical experiment there.was also rapid epithelialization of the wound as compared to use of the classical bandage. In conclusion we would express the conviction that the importance of cultured skin cells in vitro will increase, both on the theoretical level and in clinical practice.

RESUME. Après avoir considéré l'histoire des recherches dans le champ de la cultivation in vitro des cellules cutanées, les auteurs décrivent leur expérience au Centre des Brûlés de Brno avec cette technique dans le traitement des patients brûlés. Jusqu'ici ils ont appliqué environ 10.000 cm' de greffes épidermiques cultivées. Les résultats et les coûts sont discutés. On peut aussi utiliser les kératinocytes cultivés in vitro pour vérifier la cytoxicité de divers pansements synthétiques. L'importance des cellules cutanées cultivées in vitro est destinée à augmenter non seulement sur le plan théorique mais aussi dans la pratique clinique.


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