SUMMARY. The accessibility of suitable temporary covers plays the key role in the treatment of severe skin losses. Biological covers have got the longest tradition in the wound healing. Skin banks are engaged in their production and distribution. Already in 1973 J. Moserova developed the methodology of harvesting pig xenografts. Later on, the shortterm and the long-term method of storage were verified Bohm, Konicková, Vogtová). In 1986, the Skin Bank in the Prague Burn Centre was established. In Prague Burn Centre the allografts are used very rarely, usually from the living donors, family members of the patients. Therefore, in our bank, we specialized in harvesting porcine xenografts. They are produced in three different forms - fresh, deep frozen in vapours of liquid nitrogen, and glycerolized. Porcine xenografts serve as a biological cover; they make barrier against infection and evaporation and protect the wound against desiccation. They are used namely for the treatment of superficial burn wounds, as a temporary coverage of excised wounds and as a dressing on release incision. Every year more than 10,000 strips have been used in our Burn Centre, it represents the area 200 m2. Since 1991 cultivation laboratory has been a part of our Skin Bank. We are interested in cultivation of human epidermal cells keratinocytes. Cultured epidermal grafts became the first human to vitro prepared tissue, which was successfully transplanted to the patient. For the treatment of deep dermal skin losses we use either autologous keratinocytes, which can create permanent cover, or allogeneic cells, which stimulate spontaneous healing. Cultured keratinocytes are used in the treatment of burnt patients as well as in the trophic defects.
Biologische Deckung spielt eine wichtige Rolle in der Therapie von Verbrennungen. Für die Vorbereitung and Distribution solcher Hautdeckung sorgen die Hautbanken. Moserovä etwickelte im Jahre 1973 eine Abnahme Methode der Transplantate vom Schwein. Auf Grundlage extensiver Forschungen wurde im Fakultatskrankenhaus Královské Vinohrady im Jahre 1986 eine Hautbank eingerichtet. An der Klinik für Verbrennungen werden die Allotransplantate nur selten verwendet. Es handelt sich meistens um die Transplantate, die von den lebenden Spendern (Familienmitglieder der Patienten) gewonnen werden. Unsere Hautbank spezialisiert sich vornehmlich fur die Herstellung der Xenotransplantate. Die werden in drei verschiedenen Sorten vorbereitet: frische, tiefgekühlte and mit dem Glycerin konserwierte. Das Xenotransplantat vom Schwein dient als eine biologische Deckung, die die Infektion. Austrocknen des Wundes and Verlust der Flüssigkeiten verhindert. Xenotransplantate werden hauptsächlich zur Behandlung von Verbrennungen der II. Stufe, zur Deckung der Entlassungsescharotomie and zur Deckung des Wundes nach einer Escharektomie verwendet. An unserer Klinik wird jährlich mehr als 10.000 Transplantate, d. h. die Fläche um 200 Quadratmeter, verbraucht. Seit 1991 ist in der Hautbank auch ein spezialisiertes Labortätig, das sich mit einer Kultivation von menschlichen epidermalen Zellen beschäftigt. Im Therapievorgang werden sowohl die Zellen das Patienten, die eine dauerhafte Hautdeckung bilden kennen. als auch die Zellen des Spenders. die nur den Heilungsprozess beschleunigen, ausgenutzt. Kultivierte Keratinzellen werden nicht nur im Rahmen einer Therapie von schweren Verbrennungen, sondern auch bei der Behandlung von trophischen Defekte verwendet.
Key words: skin loss, skin substitute, biological cover, xenograft, cultured epithelium, keratinocytes
Speaking about the problems of treatment of severe skin losses we must strictly differentiate the terms wound closure and wound coverage. Permanent skin closure is possible by spontaneous epithelization, autologous skin graft or permanent skin substitute (autologous cultured cells). On the other hand, wound covers function as temporary substitutes which do not biodegrade and which have to be replaced with patient's own skin. They have got three main functions. The first one is the protective function as a barrier to infection. The second one is the metabolic function; the cover ought to reduce evaporative heat losses and losses of body liquids. The last but not least is the comfort function. It means the cover protects the wound from mechanical trauma and eliminates air currents and in superficial wounds minimizes pain.
We recognize three basic types of wound covers:
Conventional dressing has got the longest tradition but it does not fulfil any of the functions in comparison with synthetic and biological dressings. The last decades brought a wide expansion of synthetic dressings. According to the type of wound bed there were developed special synthetic covers. Conforming to their structure we can speak about films, gels, foams and combined laminates, which are made from two or three different layers (1).
The first mention about the use of "biological cover" we can find on an Old Egypt papyrus, where they recommend rubbing a burn wound with a frog treated with warm oil (2). Split skin grafts were used in India to close defects of nose and ears. In the 19`" century J. L. Reverdin started to use autologous and later on allogeneic grafts for the treatment of skin losses. According to our current knowledge we insert among biological covers not only allografts and xenografts but also all materials that adhere to the wound bed and stimulate wound healing (c. g. collagen sponge) (3). Their importance grew up especially during last twenty years, when experiments and clinical practice confirmed the effect of biological covers:
The use of biological covers brings also some disadvantages - they can be destroyed by enzymatic activity of the wound bed, there is a problem of their rejection (2-3 weeks after allografting), or if they are fully revascularized the surgical excision is necessary (blood loss).
At present four main types of biological covers are used in clinical practice.
Allografts from living donors are used very rarely and they are harvested usually from family members.
Allografts from cadavers are used already more than one hundred years and they play an important role in the treatment of severely burnt patients, unfortunately their amount is rather limited. Thanks to necessity of very detailed testing, they are usually produced in a form of frozen or glycerolized sheets.
Amniotic membranes are used in the treatment of skin losses thanks to their attainability and their price. After their application the decrease of evaporation is only 15%, in the case of xenograft it is 83% and allograft even 91%c. In addition they must be changed every two days.
Xenografrs from different animals (cow, dog or sheep) were tested already in 19`" century. Now the sheets of porcine skin are the most frequently used biological cover in our country and they appear in other countries as well. Thanks to immunological similarity of man and pig, there is a wide research concerning the utilization of porcine dermis for dermal substitute (5).
In the sixties and seventies the allografts from cadavers or even from living donors were used in Prague Burn Centre. Due to the development of burn medicine their quantity was not able to fulfill increasing demand for them. In 1973, J. Moserova developed the methodology of harvesting porcine xenografts (6). Later on, the short-term (at +4'C) and the long-term (at -170°C) method of storage were designed. The viability of skin grafts stored in these manners was tested using the measurement of glucose consumption and lactate production. In 1986 the Skin Bank was established as a part of Prague Burn Centre. We specialized on harvest of porcine xenografts, which have got following advantages:
The xenografts are harvested from fresh, nonscalded croupous of veterinary controlled fatted pigs in the form of dermo-epidermal grafts; their thickness is approximately 0.3 mm (Fig. 1). The grafts are treated with antibiotics and chemotherapeutics according to the microbiological testing of skin of fatted pigs. First there is used the solution of Furantoin, second the mixture of Gentamycin and Chloramphenicol. In each lavage the sheets are fifteen minutes. Their sterility is verified by microbiological testing.
<% immagine "Fig. 1","gr0000015.jpg","Porcine xenografts are prepared as thin dermo-epidermal sheets 30 x 7 cm",230 %>At present we produce xenografts in three different forms (Fig. 2).
<% immagine "Fig. 2","gr0000016.jpg","Porcine xenografts are produced in three different forms-fresh (Petri dish), frozen (aluminium tin) and glycerolized (plastic tube)",230 %>Fresh xenograft is spread on a strip of wet gauze and in a form of a scroll put into a Petri dish. They can be stored at plus 4°C for ten days. They function as fully viable temporary skin covers.
Frozen xenograft is spread on a strip of tulle gras and rolled on aluminium stand. Then it is inserted into aluminium tin and filled with cryoprotective medium (polyionic solution containing 15% of glycerol). They are stored in vapors of liquid nitrogen and the time of storage is not limited. The testing of their viability was made using the measurement of consumption of glucose and production of lactate. Although the process of freezing and thawing is optimized, their viability reaches approximately 50-70% of viability of the fresh one. They have to be applied to the wound bed during 24 hours after thawing. These grafts serve as a reserve for the case of unexpected immediate high demand for xenografts.
Glycerolized xenograft is nonviable graft. We started with their production only two years ago using the methodology of Euroskin Bank. After the harvest the sheets of porcine skin are successively transferred into 85% of glycerol (50%, 70%, 85% of glycerol) and they can be stored, scrolled in 85°l0 of glycerol, at plus 4°C for five years. Glycerolized skin grafts are widely used in the whole Europe especially thanks to their safety eliminating any infection that might be transferred to a patient. Also the preparation and handling with the graft prior to its application to the wound is easier and less time consuming than in the case of frozen one.
Xenografts seem to be very useful cover for the treatment of burn wounds and other skin losses. In 1986 we produced 4,000 xenografts and their number grew up to 14,000 pieces in the year 2003 (Fig. 3). This number represents area approximately 280 m2 More than 90% of these grafts have been used in the Prague Burn Centre, the rest has been transported to other surgical departments all over Bohemia.
<% immagine "Fig. 3","gr0000017.gif","The increase of xenograft production during the existence of Prague Skin Bank",230 %>The application of xenografts is rather comfortable, they can be easily molded and they adhere to the wound bed. First of all, xenografts are used to cover clean superficial burn wound (Fig. 4), where they produce an optimal microclimate and stimulate spontaneous healing. The second indication is to cover the wounds after escharectomy. Collagen in the dermal structures of xenografts minimizes capillary bleeding and viable cells stimulate formation of granulation tissue. Xenografts can be also used as a cover in release incisions. Further indication is so called intermingled transplantation used in severely burnt patients. The wound is grafted with widely meshed autografts and recovered with xenografts, which support the take of autograft and subsequent epithelization.
<% immagine "Fig. 4","gr0000018.jpg","Porcine xenografts are used to corer clean superficial burns. Their application is r-atheu easy and they adhere well to wound bed",230 %>However xenografts can be used also in other branches of medicine for treatment of skin losses of different origin and for the treatment of trophic and non-healing defects.
One of the biggest problems in the treatment of extensive burns is the permanent closure of wounds. Different expansion methods were developed for this purpose. At the beginning of eighties cultured epidermal grafts were used in clinical practice for the first time (7). The use of cultured epidermal grafts represents an example of tissue engineering in medical practice. These grafts enable surviving of severely burnt patients and make a good model for further tissue studies. Dr. Matousková (Institute of Molecular Genetics, CSAV) introduced the methodology of keratinocyte cultivation (8). In 1991 the cultivation laboratory was established in Prague Burn Centre and since 1993 cultured keratinocytes have been used for treatment of seriously burnt patients. Cultured epidermal grafts are prepared from a small sample (3-5 cm2) of patient's skin. Keratinocytes are cultured on subconfluent growth of feeder cells, mice fibroblasts treated with Mitomycin C. Confluent growth of keratinocytes is enzymatically detached from the bottom of culture flask (Fig. 5) and attached to textile, it is transferred to patient's wound bed. During three weeks we can get even thousands em' of cultured keratinocyte sheets. We produce more than 10,000 CM2 of cultured epidermal grafts per year. Autologous keratinocytes are used for permanent closure of deep dermal burn wounds in extensive burns, especially combined with widely meshed autografts (Fig. 6). Stimulation effect of allogeneic keratinocytes is utilized in the treatment of nonhealing residual wounds, non-healing donor sites or fresh superficial burn wounds and during the last five years for the healing of trophic defects (syndrome of "diabetic foot") as well. The possibility of deep freezing (vapours of liquid nitrogen) of cultured grafts (10% DMSO) was tested using the metabolism of glucose (9). As the results were not satisfactory, we prefer to store the cells in a form of suspension.
<% immagine "Fig. 5","gr0000019.jpg","Porcine xenografts are used to cover raw meal after necrectomy",230 %> <% immagine "Fig. 6","gr0000020.jpg","Confluent growth of cultured ker ul inocytes t, enzymatically detached from culture flask and mechanically at tached to a sheet of vazeline gauze",230 %> <% immagine "Fig. 7","gr0000021.jpg","Application of autologous cultured keratinocytes attached to vazeline gauze over ř idely meshed autograft. This procedure is used in extensive burns and brings short healing time and better cosmetic effect",230 %>Handling epidermal grafts on textile is complicated and their enzymatic digestion attenuates the viability of cells. To solve these problems, we developed the procedure of direct grafting of cultured epidermal cells on the cultivation support at the stage of non-confluent monolayer to the wound (10). We have used keratinocytes cultured on hydrogel support for the treatment of more than forty patients with skin losses of different origin (11).
Even cultured keratinocytes have not fulfilled surgeons' expectations, they represent a completely new approach to treatment of skin losses and defects. Close cooperation of research associates and surgeons is indispensable for future development of tissue engineering. However any of multiple synthetic covers can not replace biological material, particularly porcine xenografts.
They offer maximal comfort not only to patients but also to surgeons. Clinical results are a significant proof as well as the growing number of produced xenografts. From this point of view, skin bank should be an integral part of any specialized burn centre.
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