人工皮肤2

Artificial skin, split-thickness autograft and cultured autologous keratinocytes combined to treat a severe burn injury of 93% of TBSA

M. Loss, , V. Wedler, W. Künzi, C. Meuli-Simmen and V. E. Meyer

Division of Hand, Plastic and Reconstructive Surgery, University Hospital, CH-Zurich, Switzerland

Accepted 25 January 2000. Available online 31 July 2000.

Abstract
Despite refinements in burn shock resuscitation, improvements in surgical techniques, advances in intensive care medicine and the presence of very expert surgeons, the treatement of patients with severe burns exceeding 60% TBSA remains a big challenge. A major problem in the treatment of severe burn injuries is the lack of autologous skin. In selected cases cultured epidermal autograft (CEA) may be used. However, they are available only 2–3 weeks after biopsy, thus requiring a temporary wound closure after necrosectomy. A new option is IntegraTM, an artificial skin consisting of a bilayer membrane system. The three-dimensional porous matrix from bovine tendon collagen and a glycosaminoglycan layer is covered by a silicon sheet. The latter prevents fluid loss from the wounds and serves as a barrier against germ invasion. After adequate vascularisation of the dermal template, the silicon layer is removed and replaced by a thin autograft. We present a 26-year old male who sustained a 93% TBSA burn injury (60% full-thickness burn, 33% partial-thickness burn). He was treated with artificial skin, split-thickness autograft and CEA in combination. The clinical history and the follow-up of approx. 1 year are presented and the results discussed. We consider the survival of this patient being a result of the therapeutic progress of the recent decades.

Author Keywords: Burns; Wound repair; Graft; Keratinocytes; Artificial skin


Article Outline
1. Introduction
2. Case report
2.1. Surgical procedures and results
2.2. Dressing
2.3. Intensive care
2.4. Follow up
3. Discussion
Acknowledgements
References


1. Introduction
Before 1940, most patients with burns covering more than 40% of the total body surface area (TBSA) died. The high mortality rate is mostly related to the limited availability of autologous donor sites [1 and 2]. In the 1970s Janzekovic [3] recognized the advantages of early excision and immediate grafting. However, in patients with extensive burns of more than 50% TBSA, the limited availability of autograft donor sites becomes a factor limiting rapid wound closure and survival rate.

In 1975 Rheinwald and Green [4] began investigating the cultivation of human epithelial cells in the laboratory. Over the years the coverage after burn excision by CEA became an established and standardized method, practised in several centers over the world. Today, from a single biopsy measuring 2×3 cm, enough keratinocytes can be cultivated to cover the entire body of an adult in 3–4 weeks. But there is not a satisfying temporary wound coverage until the CEA are ready. The current temporary human skin substitutes — allografts — have a number of disadvantages, including antigenicity, limited availability, high cost and a limited shelf life. If fresh allografts are used, the risk of transmission of life threatening diseases such as HIV and hepatitis from the donor has to be considered.

In 1981, Burke and Yannas published preliminary clinical results reporting the use of a bilayer artificial skin consisting of a dermal template and an overlaying silicon sheet for permanent wound closure when autologous skin is unavailable [5]. But this technique requires a second operation to replace the silicon sheet which serves as a type of temporary epidermal coverage.

This report presents the case of a 26-year-old male who sustained a 93% scald of the TBSA. Artificial skin (IntegraTM), split-thickness autograft and keratinocytes were used for treatment. The clinical and functional follow-up is presented and discussed the results.

2. Case report
A 26-year old man was admitted to our hospital with 93% burn of TBSA. The accident occurred at work when a boiler exploded. The patient sustained full-thickness burns of 60% TBSA and partial-thickness burns of 33% TBSA. Helicopter transfer of the patient to our clinic was only possible 24 h after the accident due to the bad weather conditions.

At admission in our clinic the patient was sedated and ventilated and his cardio-pulmonary situation was stable. Immediate fluid resuscitation had occurred using crystalloids (Ringer lactate) and colloids (Hydroxy Ethyl Starch). Fluid input in the first 24 h totalled 30 litres, achieving a diuresis of 80–100 ml/h. The patient was cleaned and debrided in our special bath tub. He showed a mixed pattern of deep partial-thickness (back, face, chest) and full-thickness (left arm and lower extremities) burns. The right arm and some other areas had suffered superficial partial-thickness burns. A few areas such as the rima ani, the soles of the feet, the axillae and parts of the head were unharmed. Escharotomies were performed on both lower extremities and the left arm (Fig. 1).


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Fig. 1.

2.1. Surgical procedures and results
It was difficult to plan the surgical steps to close all the wounds as soon as possible without endangering the vulnerable balance of the patient. We first excised the clearly full-thickness burned lower extremities and the left arm at day 3 down to the subcutaneous fat, a few small areas were excised intradermally. The wounds were covered with artificial skin (Fig. 2 and Fig. 3). At the same time autologous skin from an unaffected part of the right arm was harvested for CEA. The artificial skin was checked daily. Complications with artificial skin were seen in the few small areas excised intradermally: hematomas had developed under the artificial skin and were evacuated immediately. A few localized pus spots were evacuated through a small cut within the artificial skin (Fig. 4).


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Fig. 2. IntegraTM-grafted left arm.

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Fig. 3. Additional fixation of the artificial skin with an elastic net (black in the picture because of the silver nitrate).

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Fig. 4. Localized accumulation of pus under IntegraTM at evacuation through a small incision.

Three weeks later we received the CEA which were used to cover the back (Fig. 5). The CEA were put on the deep dermal layer, which was debrided by scrubbing it before applying the CEA. The artificial dermis became vascularized and thus, the silicone sheet ready to be removed 6 weeks postoperatively (Fig. 6). Parts of the left thigh, the left flank and the upper part of the left arm were covered with second-generation CEA. The lower left leg, part of the left thigh, the right leg and most of the left arm were covered with thin split-thickness autografts from spontaneously healed areas (Fig. 7). In the 7th week, a local infection destroyed a small area of CEA on the upper half of the left arm which had been covered by artificial skin. Pseudomonas aeruginosa was identified, requiring local debridement and antibiotic treatment. The CEA on IntegraTM on the left thigh did not take as well for unknown reasons. So we had no CEA growing on the artificial skin. Residual defects were covered with split-thickness autografts step by step. The last surgery took place in the 13th week. After this procedure all wounds were closed (Fig. 8).


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Fig. 5. The back of the patient with the CEA sheets on it.

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Fig. 6. Removal of the silicone sheet before grafting with split-thickness skin.

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Fig. 7. Ultrathin split-thickness graft on the neodermis after removal of silicon sheet.

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Fig. 8. IntegraTM grafted sites (blue). The CEA on the IntegraTM did not take and these areas were covered with split-thickness skin later.

2.2. Dressing
The fresh burns were dressed with silver sulfadiazine 1%. We covered the artificial skin grafts with gauzes wetted every 4 h with 0.5% silver nitrate solution until the silicon sheets were removed and replaced by autologous skin.

The split-thickness autografts and CEA were dressed with paraffin gauze.

2.3. Intensive care
The clinical course was complicated by two temporary septic exacerbations caused by a respiratory infection and an infection originating from a central venous catheter. They were successfully treated with antibiotics after catheter removal. There were no further complications requiring special intensive care procedures, therefore the intensive care aspects are not discussed any further.

2.4. Follow up
In the 4 month follow-up the spontaneously healed areas showed some significant hypertrophic scars (right arm and hand). Both legs healed completely without leaving excessive scarring (Fig. 9 and Fig. 4 months, and Fig. 10 and Fig. 8 months post injury). The left arm showed mild hypertrophic scarring and no cubital contractures at all (Fig. 11) (IntegraTM grafted site), while the anterior axillary fold, not covered with artificial skin, but directly with mesh-graft, developed significant contracture. The "skin" of the IntegraTM covered sites seemed to be thicker and was more elastic (Fig. 12). The back and the left half of the anterior trunk covered with CEA showed a stable skin after uniform healing. In summary the cosmetic results were satisfactory.


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Fig. 9. Results 4 months post injury.

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Fig. 10. Results 9 months post injury.

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Fig. 11. Left cubital fold.

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Fig. 12. Visible elasticity of the skin in the left cubital fold.

3. Discussion
The 26-year old man was admitted to our hospital with 93% scald of TBSA. Because of lack of donor areas, we used artificial skin after tangential excision of the full-thickness burned extremities, in combination with other conventional techniques. The ideal skin replacement should have the following characteristics: immediate availability, functional wound coverage, barrier function against micro-organisms, no antigenicity, enhance natural healing processes and provide long term stability without contraction or hypertrophic scarring.

IntegraTM is an artificial skin consisting of a dermal and an epidermal portion. The dermal portion is made of bovine hide collagen and chondroitin 6-sulfate obtained from shark cartilage. This portion is sterilized in manufacture utilizing the two bactericidal steps of heating to 105°C followed in a subsequent step by immersion in glutaraldehyde solution, which produces a bacterial free membrane. The dermal portion serves as a template for ingrowth of host cells and vessels synthesizing a "neodermis" while the original artificial dermal scaffolding is slowly biodegraded and replaced by the patient’s own tissue. The epidermal substitute consists of silastic. This material controls the water flux from the dermis to approximately equal than of normal skin [6]. The immediate availability of artificial skin allows the surgeon to excise early all the burned areas. The silicon sheet can be left in place for 3–6 weeks or until split-thickness autografts from the meanwhile spontaneously healed areas or CEA are available. Although this requires an additional operation, the procedure takes place at a time when the patient’s conditions are relatively stable.

In 1981, Burke and Yannas [6] published their first clinical results regarding the use of artificial skin (IntegraTM). They had a 100% take of artificial skin on the excised wound bed in ten patients burned up to 50–95% of TBSA. The take of split-thickness skin graft on artificial skin ranged from 85 to 95%.

The artificial skin is prone to infections underneath the silastic layer [5]. A daily check of the artificial skin is necessary to prevent the spreading of infections by emptying localized fluid collections, hematomas or excise and even replace parts of the artificial skin if necessary [7]. Also the gauze wound dressings were changed on a daily basis. Silver nitrate was added every 4 h by pouring it over the dressing. In our case there were only little signs of pus collections which were emptied immediately as mentioned before. After removing the silicon sheet of the artificial skin, we resurfaced the neodermis on the legs with split-thickness autografts, except the upper part of the left thigh and a small part of the left arm which were grafted with CEA on artificial skin.

Burke advises the use of a very thin layer of split-thickness autograft on artificial skin to get the best results [5], which was also best in our case. We found the mechanical resistance and cosmetic aspects of the new skin which grew on the artificial dermis to be satisfactory compared to autograft applied directly to subcutaneous fat or to the muscle fascia. We subsequently found more elasticity of the artificial skin covered sites than of the other "non-IntegraTM sites" (Fig. 12) and up to now there were no contractures. A permanent skin replacement requires a dermal component to ensure adequate long-term graft stability and to prevent wound contraction. The more dermis grafted, the less wound contracture and scarring, and the more functional and rapid the healing will be [6, 8 and 9].

We believe a localized Pseudomonas aeruginosa infection on the keratinocyte-grafted site of the left arm was the main reason for graft take failure. The CEA on artificial skin of the left thigh did not take for unknown reasons.

There is not much literature on the use of CEA on artificial skin. Pandya [10] published a case report in 1997 describing the use of CEA together with artificial skin for the treatment of a 15 year old boy with deep burns up to 60% TBSA. He resurfaced the excised deep burns with IntegraTM. In the third week he removed the silicon sheet and resurfaced the anterior torso in two mirror-imaged halves with ultrathin split-thickness autografts on one side and CEA on the other: CEA and split-thickness autografts both grew and consolidated without any problems.

Artificial skin is considered to get the best ingrowth on subcutaneous fat or muscle fascia. In the few areas where we were able to excise intradermally, small hematomas developed below the artificial skin and were evacuated immediately.

In 1998, Clayton and Bishop emphasized in their paper the following specific details to be addressed for the successful use of artificial skin [7]: the excision must include all the burned tissue to avoid infections that may result in the loss of artificial skin. There must be meticulous hemostasis, otherwise the artificial skin will separate from the wound. The material must be handled carefully and the sheets must border each other properly; each split is a potential entry for germs. The artificial skin must be firmly fixed to the tissue bed. We prefer staples to sutures, to reduce the danger of damaging the artificial skin.

However, the artificial skin is not a true skin replacement, because of the missing living dermal structures. Recent advances in biotechnology have led to the development of a biosynthetic skin graft which contains living fibroblasts and epidermal cells — a so called "living skin equivalent" (LSE) [11]. Cultured fibroblasts from neonatal foreskin are mixed with a solution of bovine collagen and allowed to form a gel and so the fibroblasts remain metabolically active and begin to reorganize the bovine collagen as well as to produce their own collagen and matrix proteins. The surface of this dermal equivalent is seeded with cultured allogeneic keratinocytes. The fibroblasts in the "dermis" and the keratinocytes in the "epidermis" are viable, reproducing cells, making one of the most advanced tissue constructs to date [12, 13 and 14]. After proliferation they form an epidermal covering. It all finally results in a living skin equivalent, ApligraftTM, first described by Bell and colleagues [11] in 1981. This skin substitute, though of allogeneic origin, should not be rejected, because the Langerhans cells, which play a major immunologic role, are lost during serial culture of keratinocytes [14]. ApligraftTM may become an important tool in managing severely burned patients in the future [15]. We found no literature describing the treatement of a major burn injury with ApligraftTM and we do not have any personal clinical experiences yet.

In conclusion, the development of a near normal living skin substitute, which is available any time in unlimited quantities remains the goal and challenge to secure the survival of severely burned patients in the future.


Acknowledgements
We would like to thank Integra™ (Johnson & Johnson) for covering the cost for the coloured pictures.


References
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