JanezŠicŠ T.

Burns Unit, Ljubljana Clinical Centre, Ljubljana, Slovenia

SUMMARY. This paper presents a single-centre experience with cultured epithelial autografts (CEAs). Six patients with critical thermal injuries (mean burn area, 73% TBSA; mean full-thickness burns, 64% TBSA) were treated in the period 1993-1999. The full-thickness burn wounds were closed by early and sequential excisions, split-thickness skin autografts, and CEAs. CEAs were applied to a mean area of 18% TBSA. Three patients died in the course of treatment. The mean CEA take 30 days post-transplantation was 45% (min., 10%; max., 80%) and the mean area closed by CEAs was 7% TBSA (min., 2% TBSA; max.: 11% TBSA). CEAs were found to be a very important part of the life-saving treatment of critically injured thermal patients with extensive full-thickness burn wounds.

Introduction

The first report of keeping human skin in culture was published in 1898 when skin fragments were kept for long periods in ascitic liquid and then grafted back to the donors.1

Studies performed in 1948 indicated that cultured epithelium could be used as a graft but this technique was limited by the extent of expansion of the epithelial cell population.2 The first well-documented transplantation of keratinocytes in rabbits was published in 1968.3

The method of culturing epidermal cells was improved in the 1970s by Rheinwald and Green, who cultivated human epidermal skin cells serially.4 This serial cultivation technique made it possible to use the patient’s keratinocytes to promote closure of the wounds. The possibilities of this technique in the treatment of burn patients was quickly recognized.5 Later the cultured autologous epidermis was used and evaluated in a wide range of burn centres.6-12 This paper presents the data of six critically ill thermally injured patients with extensive full-thickness burns treated also with cultured autologous epidermis. The patients in this series were all treated in the Ljubljana Burns Unit in the period 1993-1999.

Patients and methods

The burns unit in the Ljubljana Clinical Centre is one of the two burn centres in the Republic of Slovenia, which has a population of two million inhabitants. It is a 28-bed facility with seven intensive care beds and treats 150-200 burn patients per year. In the period 1993-1999 six patients with extensive full-thickness thermal injuries were treated using CEA in the Ljubljana Burns Unit. Their mean age was 32 yr (range 14-53 yr); two patients were females and four males. They had sustained burns in a mean burn area of 73% TBSA (range 55-84% TBSA) with a mean full-thickness burn area of 64% TBSA (range 30-60% TBSA) (Table I).

Closure of the burn wounds was achieved mainly by split-thickness skin autografts, and a minor portion of the burn wounds was closed by means of CEA. The first thermally injured patient in whom CEA was used was treated in 1993. The patient died when nearly healed and the CEA take was not satisfactory. In the light of the experience gained in the treatment of this patient, a clinical protocol for the treatment with CEA of critically ill thermally injured patients suffering from full-thickness burn wounds was later established. The protocol covers ten fields, namely:

* definition of procedures upon admission

* plan of treatment

* selection of patients (inclusion criteria)

* excision of burn wound

* skin biopsy for culturing

* cadaver skin used

* surgical procedure of CEA transplantation

* dressings

* control of local microbial colonization

* additional CEA or skin autografts

Selection of patients (inclusion criteria)

Patients aged 0-65 yr with full-thickness burn wounds large enough to present a clinically important lack of donor areas for split-thickness skin autografts.

Admission

Standard admission protocol for critically injured burn patients. Burn wounds dressed with absorptive dressings of saline soaked gauzes over vaseline gauze.

Plan of treatment

The plan of treatment of the burn wounds is made at the time of admission or on day 1 and is crucial for the treatment of such patients. A decision is made regarding which body areas will be treated with CEA. The preferred area is the anterior trunk. The timing of skin biopsy, CEA transplantation, and all burn wound excisions is determined with respect to body areas, method of definite closure, and the temporary cover of burn wounds and donor site areas.

Skin biopsy

The skin biopsy for skin culturing is taken as soon as possible, not later than 5 days post-injury.

Excision of burn wound

Excision of the burn wound is started as soon as possible and not later than day 5 post-injury. The excision of the burn wound, in one surgical session, is limited to 30% TBSA. The excision of the burn wound to be treated with CEA is performed as soon as possible and depending on the timing of CEA delivery from the laboratory (we consider 2-3 weeks before the availability of skin cultures to be the optimum period owing to the behaviour of the cadaver skin with which the bed is prepared.) The excision is performed tangentially or fascially. The excision is performed under tourniquets on the extremities, while elsewhere epinephrine solution is injected under the burn eschar.13,14 The area to be treated by CEA is covered immediately after the excision with cadaver skin meshed 1:1.5. Other areas are covered with meshed cadaver skin, or closed with sandwich grafts.

Cadaver skin

The cadaver skin used to prepare the wound bed for CEA transplantation was glycerolized and taken from the Ljubljana Burns Unit skin bank.15

Surgical bed preparation for CEA transplantation

Preparation of the surgical bed is performed during the same session as the CEA transplantation. The cadaver skin, often with epidermolysis, and the whole area to be transplanted are shaved using a Watson knife, dermabrader, scalpel, brush, or dermatome. The small bleeding points are exceptionally coagulated with electrocautery, and haemostasis is performed with adrenaline-soaked gauzes. CEAs mounted on a fibrin carrier are then transplanted using forceps.

Dressings

Cadaver skin is overlaid with vaseline gauze and dry gauze. From the second week the wound bed is treated with iodine solution and vaseline gauze is mixed with iodine cream; unexcised areas are treated with silver sulphadiazine cream. Immediately post-transplantation, the CEAs are dressed with Adaptic® (Johnson & Johnson, Arlington, USA) and a bridal veil on the top. Vaseline gauze mixed with iodine cream is applied around the grafted areas. A final dressing is made by applying dry gauze to the top. After transplantation the dry gauze is changed daily, and iodine cream is used around the grafted areas. In the event of bacterial contamination the dry gauze is exchanged for gauze soaked in saline containing antibiotics depending on the antibiogram (vancomycin, polymyxin, amphotericin B). Take-down is performed at day 8-10. The grafted area is dressed with N-A Ultra® (Johnson & Johnson, Arlington, USA). This dressing is changed daily for the next ten days.

Control of local microbial colonization

This is achieved by taking swabs, quantitative swabs, and quantitative biopsies. For preparation of the wound bed, iodine solution, vancomycin, polymyxin, and amphotericin B are used, taking into account the cytotoxicity of certain chemotherapeutics used locally.16 Iodine cream is used around the grafted area. Unexcised areas are treated with silver sulphadiazine cream. Prophylactic use of systemic antibiotics is made per-operatively and therapeutically if the patient’s clinical condition so requires.

Additional CEA or skin autografts

Secondary grafting of areas of CEA with non-take of CEA is not performed. Areas of non-take are closed by skin autografts.

Patients

In the period 1993-1999 all patients admitted to Ljubljana Burns Unit who satisfied the treatment protocol inclusion criteria were treated using CEA. The six patients were admitted to the our burns centre immediately after injury. None presented any injury other than thermal injury. Inhalation injury was diagnosed by bronchoscopy in one patient. The average abbreviated burn severity index (ABSI)17 was 11 (range, 9-13). Three patient in this series died. Patient 1 died on day 87 post-burn owing to multi-organ failure and signs of systemic immune response syndrome. She had a few small unhealed spots. Patient 3 died of pneumonia on day 75 post-injury. Patient 6 died on day 28 post burn after 19 days of veno-venous ECMO therapy. The cause of death was respiratory failure as a consequence of inhalation injury. All the patients were treated according to the protocol described (Tables I, II). CEA take was estimated clinically by observing the typical appearance of the epidermis after 10 minutes of exposure to air. The areas of take were marked on a transparent plastic foil that was laid on the body area to be inspected and measured.

Results

Patient 6 died on day 28 post-burn and 6 days after CEA transplantation and it was therefore not possible to evaluate CEA take. The mean area treated by CEA was 18% TBSA (min., 9%; max., 33%). The mean epithelialized area of CEA grafts in the five patients on day 8 post-graft was 12% of the area covered with CEA (min., 5%; max., 20%). The mean epithelialized area of CEA grafts on day 30 post-graft was 45% (min., 10%; max., 80%). The mean area finally closed at the end of treatment was 7% TBSA (min., 2% TBSA; max., 11% TBSA) (Table III).

The mean number of surgical procedures on burn wounds in this series was 7 operations per patient (min., 2; max., 10). Tracheotomy was performed in all but one patient. The mean number of days of ventilatory support was 45 (min., 1 day; max., 87 days). The mean hospitalization time was 84 days (min., 28 days; max., 185 days) (Table II). Excision of the burn eschar on areas to be treated by CEA was performed on average on day 5 post-injury (range, 2-9).

The three patients that survived were rehabilitated in the rehabilitation centre for 3-4 weeks after discharge from the burns centre, after which they were discharged home. Bacteriology of the burn wounds showed Pseudomonas spp. and Staphylococcus spp. in the first two weeks, while later MRSA, Acinetobacter spp., Escherichia coli, and enterobacteria were isolated. In patients 1 and 5 Candida albicans was also isolated after the first month of treatment.

Discussion

In this series CEAs were applied to six patients who had sustained median full-thickness thermal injuries in 70% TBSA (range, 30-80% TBSA). The median ABSI score was 12 (range, 9-13), with a mortality rate of 50%. The median area treated with CEA was 16% TBSA (range, 9-33%), which is sirnilar to some previous reports18,19 but less than that reported Carsin et al.20 The median area definitively closed with CEA was 7% TBSA (range, 2-11% TBSA), which is comparable to some reports18-20 but less than that reported by others.20,22-24 It is difficult to compare take rates in different studies because the epithelialized area is often estimated at different times after CEA transplantation. The evaluation of the area definitively closed with CEA is a more reliable guide. The CEA take rate in different studies is also difficult to compare because of the different surgical techniques, protocols, and wound bed preparation methods. A solution to this problem would be to establish a strict clinical protocol for patients treated with CEA in one or several burn centres - this would make the evaluation of such treatment much more reliable. It was for this reason that such a protocol was established at the Ljubljana Burns Centre. Another variable that changes between different studies or even within a single study of the treatment of thermal injuries with CEA is the role played by the various tissue laboratories that culture the skin. In this series all the CEAs were cultured in one laboratory.

The median range of the area definitively closed with CEA in this series was 7% TBSA. That means that CEAs were useful in the treatment of critical thermally injured patients with extensive full-thickness burns in this series and substantially contributed to closure of the burn wounds. Considering the serious lack of donor sites in the patients in this series, even 7% TBSA is a relatively very important area to be definitively closed with CEA. However, CEAs were only a supplement to split-skin autografts (SSAs) in the closing of the burn wounds. This means that SSAs were primarily used for this purpose and that CEAs were used only when there was a lack of donor sites for SSAs.

The mean time of CEA transplantation was 23 days post-burn (SD, 5 days; min., 19 days; max., 32 days [first patient]), which is relatively short and means that in that time 2-3 crops of split-skin autografts were harvested. To reduce the time of CEA transplantation all procedures for culturing had to be systematized since in this series the laboratory for culturing epithelial autografts was abroad and all transport was performed by commercial airlines.

In this series, CEA take was also very dependent on the areas treated. Clinically speaking, take on the anterior trunk was much superior to that on the extremities. The cause of the poorer take on the extremities was mechanical damage to the CEAs. This might also be the reason for the different take rates in other series.

Conclusion

In this series cultured epithelial autografts were found to be very important in the treatment of thermally injured patients with extensive full-thickness burns in the definitive closure of burn wounds. This method proved to be a very important part of the treatment of such patients and constitutes a life-saving procedure. The use of CEAs requires extra work and organizational skills on the part of the burns unit staff but does not involve any additional employment of staff. The cost of CEAs is itself very high and in our case was covered by the Slovenia Medical Insurance Company, but presented only a minor portion of the overall costs of treating the patients in this series.

RESUME. L’Auteur décrit l’expérience d’un centre des brûlures avec l’emploi des autogreffes épithéliales cultivées (sigle anglais, CEA). Pendant la période 1993-1999) il a traité six patients critiquement malades atteints de brûlures (superficie brûlée moyenne, 73%; superficie moyenne de brûlures à toute épaisseur, 64%). Les brûlures à toute épaisseur ont été fermées avec l’emploi des excisions précoces et séquentielles, des autogreffes à epaisseur variable et des CEA. Les CEA ont été appliquées dans une zone moyenne de 18% de la superficie corporelle totale brûlée (SCTB). Trois patients sont décédés pendant le traitement. La prise moyenne des CEA 30 jours après la greffe était de 45% (min., 10%; max., 80%) et l’aire moyenne couverte par les CEA était de 7% de la SCTB (min., 2%; max., 11%). Les CEA se sont révélées tres importantes pour sauver la vie des patients critiquement malades atteints de brûlures étendues à toute épaisseur.

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