Annals of Burns and Fire Disasters - vol. X - n. 1 - March 1997

USE OF A BIOLOGICAL FILM AS A CULTURED EPIDERMAL AUTOGRAFT SUPPORT IN THE TREATMENT OF BURNS: PRELIMINARY REPORT OF A NEW TECHNIQUE

Gueugniaud Fabreguette A. Oddou L, Petit P, Collombel Damour

Lyons Burn Centre, Edouard Herriot Hospital, Claude Bernard University, Lyons, France
Skin Substitute Laboratory, CNRS, 1341 URA, Lyons

SUMMARY. The long-term outcome of the most critically burned patients is now in part linked to the successful use of cultured human epidermal sheets. This technique however presents certain disadvantages: a long delay before the sheets are available, the fragility and difficult handling of the grafts, and the variability of the graft take rate, which is often poor, particularly in some difficult areas (e.g. joints, back, neck), where the gauze used to transfer the epidermal sheet impedes adherence of the sheet to the prepared wound. The aim of this preliminary study is to assess a biological film for supporting keratinocyte growth and transferring cultured epithelium to the wound without gauze transfer. The film consists of 72% collagen, 20% chitosan and 8% glycosaminoglycans. The technique was used in three patients following the surgical methods routinely used in our Centre, i.e. either "combined" graft with conventional large mesh amograft covered with autologous cultured sheets, or cultured epidermal autografts covering remaining allodermis after early homograft. The culture time was shortened by several days because perfect confluence of epidermal cells to achieve a multilayered epidermis was no longer necessary: cultured grafts were available 14-16 days post-biopsy. The in vitro manipulation before grafting allowed a saving of several hours by eliminating transfer on to a gauze. The areas thus covered were the shoulder, flank, trunk, and forearm (range, 600 to 960 CM2). Clinically, the seeded biological film was easy to handle and more adhesive to irregular wounds than any classical dry or vaseline gauze. When the dressing was removed for graft verification, the film disintegrated spontaneously, without detrimental effects to initial take. The success rate ranged from 85 to 98% after the first grafting procedure. In conclusion, the film seems to be useful as it accelerates keratinocyte cutture. It permits good co-ordination between the biological and the medical teams, and it therefore appears to be a valid technical alternative to conventional cultured epidermis, especially in mobile zones.

The prognosis for the most critically burned patients has considerably improved in the last few years thanks to the progress achieved in resuscitation techniques. Fluid expansion was the first step in this progress.' The better understanding of the initial haemodynamic disorders of patients with major thermal burns has improved vital prognosis  The full recovery of these critically burned patients who are today successfully resuscitated cannot be achieved without the use of skin substitutes.
First described twenty years ago,' cultured epithelium allows complete coverage of the skin, which is impossible to achieve with traditional grafts because of the limited donor sites.' The main hope for the future regarding the treatment of very severely burned patients lies in the success of cultured epithelium, the production of which is now fully under the biologists' control. As regards burn coverage, Gallico was one of the first surgeons to use epidermal sheets obtained by Rheinwald and Green's rnethod.` Within three weeks, this technique enables medical teams to have at their disposal a considerable quantity of cultured epidermal autografts (CEA) which can be grafted on to an area previously prepared by surgical excision of burned tissues. The most effective method of protecting the wound bed, pending grafting with CEA, is considered to be the use of engrafted allodermis via early homografts.
However, the clinical success of cultured epithelium remains variable: the literature reports an average success rate ranging from 30 to 70%. Several factors affect the success of CEA take, among which we may schematically distinguish clinical factors related to the patient and biological factors related to the culture technique and the transfer of the sheets.
With regard to clinical factors, the quality of the patient's receiving site is of prime importance for the success of CEA: the preliminary surgical excision must prepare a clean and well-vascularized wound to receive CEA. Today, after excision, the temporary application of homografts (when available) allows a better preparation of these areas pending permanent coverage. This preparation is however usually disturbed during the first three weeks by deterioration of the patient's general condition. The success of CEA is also compromised by initial haemodynamic disturbances, decreased immunity, denutrition and, above all, problems of local or general infection.
Thus, as far as clinical factors are concerned, improvement of the technique of cultured epithelium requires a reduction in the time taken to create the sheets. This would prevent serious sepsis, which is often responsible for premature non-take of the temporary homograft or permanent autograft.
With regard to biological factors, the culture technique of the epidermal cells and the phase of the transfer of the sheets are both necessary to achieve a successful graft. Today, in addition to confluence of the keratinocytes, it is necessary to achieve good cohesion between the cells and to dispose of a strong sheet that can be be transferred on dry petrolatum gauze: it thus takes about 21 days to obtain CEA. This period could be reduced if confluence ceased to be a necessity and if cell proliferation could continue after transfer on to the receiving site itself.
An ideal support would have other requirements and could be defined according to certain biological and clinical characteristics. Biologically, the culture could be produced directly on the support, where the cells could adhere and proliferate. This support would be biocompatible and easy to handle, and it would allow a shorter transfer time. Clinically, the support would have to be: a) flexible, to allow total contact between CEA and the receiving site; b) thin and permeable, to prevent any risk of complete occlusion of the wound and to facilitate the escape of exudates from the underlying tissues; and c) biodegradable, for easy removal of the biornaterial when the graft is checked.
Following the initial collagen sponge model proposed by Yannas, the first biological films to be developed were collagen films, which are now successfully used for burn coverage. Subsequently, other films were used, composed of materials such as hyaluronane, chitin, fibronectin, and fibrin molecules. Fibrin offers a number of possibilities: the cells can be seeded on a fibrin film or incorporated into the fibrin to be sprayed on to the wound.
We present here a film made of collagen, glycosamino-glycans (GAG), and chitosan. The aim of our study is to assess this biological filtr as a support for the development of keratinocytes cultured in vitro and for the transfer of CEA on to the receiving site, without the use of any artificial support. Compatibly with the condition of the patient, the surgical techniques adopted in our Centre (after early excision down to the adipose tissue) consist either of homografts when available, followed two weeks later by CEA covering any remaining allodermis, or - as soon as possible - "combined" grafts, with a large mesh autograft covered with cultured epidermal sheets: for the clinical reports, the same techniques are tested in this preliminary study.

The cells
The keratinocytes were isolated from the patient's healthy skin biopsy by trypsinization overnight at 4 'C. The keratinocyte suspension was seeded on a feeder layer made of human irradiated foreskin fibroblasts, in a medium containing Dulbecco's modified Eagle's medium (DMEM) and HAM F-12 (SIGMA Laboratories), supplemented with 10% of foetal calf serum (SIGMA Laboratories) and 0.4 mg/ml hydrocortisone, 5 mg/ml insulin, epidermal growth factor (EGF), 5 mg/ml transferrin, 2 x 10-1 mol/ml tri-iodothyronine, 10-11 mol/ml choleratoxin, and 8 x 10` mol/ml adenine (SIGMA Laboratories). The cultures were incubated at 37 'C in a humidified atmosphere containing 5% C02, and the medium was changed three times weekly. After three days, 10 ng/ml EGF was added to the medium. When the primary culture was nearly confluent, the keratinocytes were resuspended by trypsinization for secondary culture or film seeding.

The components of thefilm
Our film was made of collagen, glycosaminoglycans (Chondroitin-2 and 4-Sulphate) (GAG) and chitosan, all purchased from SADUC (Lyons, France). The biosafety of the components was checked in conformity with CEE recommendation number 111/3298/91 (Guidelines for minimizing the risk of transmissible agents causing spongiform encephalopathy via medical products).

Preparation of thefilm
As we found previouSly,2' the optimal composition for the preparation of the film is 72% collagen, 20% chitosan and 8% GAG. This is prepared in large sheets and left to dry. The films are sterilized by gamma irradiation (25 kG).

Culture conditions
Before use, the film is washed twice with sterile phosphate buffered saline, then equilibrated with DMEM. At the optimal pH of 7 the cells are seeded on the film in rectangular metal frames at a density of 101 cells per CM2 . The frames are removed three hours after seeding, when the cells are attached to the film. This cell density (used in the three cases described) is near subconfluence and allows grafting of the film one day after seeding. A lower density can be used if the graft is expected later, allowing modulation.

Controls
Our film is not transparent enough to allow a lightmicroscope evaluation of the cells on the film. We therefore looked for a macroscopic method that would permit appreciation of the cell density we required. We used a colorimetric method known as the MTT test which is normally employed to evaluate cell viability: a yellow tetrazolium salt is reduced by the viable cells to an insoluble blue formazan product. Before the graft, a small portion of the film was taken and incubated in NITT solution. After 30 minutes' incubation, direct observation of the blue area allowed evaluation of viability and confluence.

The seeded film was tested for partial skin covering in three patients, with the approval of the Lyons University Hospital Ethics Committee.
In our Centre, virus-free donor skin is applied, if available, after early scar excision in order to prepare wounds for CEA. However, so long as donor sites remain valid, we use CEA to cover conventional large mesh autografts, especially in difficult areas, e.g. joints and the back.
These techniques were applied for seeded film in the following three case reports (Table 1).

Case 1
An I I -year-old boy, a fire casualty, was admitted to our Burn Centre with burns of 80% T13SA (60% full skinthickness and 20% deep partial). His state of health required over two months' stay in the intensive care unit, with artificial ventilation for 45 days. A skin biopsy for keratinocyte culture was harvested on day 4 post-burn. During the first two weeks, local treatment consisted of complete surgical excision of the burned areas in three weekly surgical sessions, with initial covering by collagen dressing,` because no homograft was available. The excised wounds rapidly appeared favourable for grafting and a first graft session was planned at the end of the second week of treatment to cover the superior limbs and the left hemitrunk. At this point, traditional CEA was not yet available as the keratinocytes were not yet confluent, and we therefore decided to seed some biological films two days before the graft session. As the donor sites were extremely limited, "combined" grafts with mesh 6:1 autograft covered with eight seeded films (12 x 10 cm) were layered on to the side of the left hemitrunk 14 days after biopsy. When the graft was checked on day 8, the result of this "combined" autograft on the left half of the trunk was a 98% success; also, the large openings of the 6:1 mesh under the films had completely disappeared in the same short period. The successive grafts were produced using conventional cultured keratinocytes available on dry gauze. The patient was discharged alive on day 98 postburn.

A 40-year-old man was burned by blazing gas when attempting to take his life, suffering 35% TBSA full skin-thickness burns and 10% deep partial burns. The areas concerned (only partially) were the head, the front and back of the trunk, and the upper limbs. The patient also presented an important primary respiratory lesion, and his initial state necessitated artificial respiration after tracheotomy for three weeks. The haemodynamic condition remained unstable, requiring pulmonary artery monitoring and the use of catecholamines. Prolonged circulatory shock prevented surgical debridement during the first week, and initial local wound therapy consisted of silver sulphadiazine dressings. Owing to the delay in excision, and considering the size and depth of the wounds, a skin biopsy was taken on day 7 post-admission.During the second week of treatment, three excisiongraft sessions were performed. From the surgical point of view, the burns quickly reached a satisfactory state, and the quality of the autografts permitted excellent coverage before the end of the third week. However, one last surgical session proved necessary in order to complete the grafts as some areas had not completely healed: at that moment, the confluence of the keratinocytes was not perfect, and a conventional sheet was not possible: some films (600 cm') with autologous keratinocytes were made on day 22 post-burn (corresponding to day 15 post-biopsy) and used the following day both on welldefined spots to complete the traditional graft (left flank) and on the left shoulder to cover a dermo-epidermal graft in wide mesh (mesh M) according to the "combined" technique.
The initial results obtained with this technique on the patient were satisfactory, with healing in approximately 85% of the zones covered (90% in the shoulder). We present here the photographs of the graft performed on the left shoulder, with a 3:1 mesh dermo-epidermal graft, with half the external area covered by the seeded film (the "combined" technique). The internal part (near the neck) was grafted only with autografts, and can thus be regarded as a control area. Fig. ]a shows the aspect of the wound bed before grafting. Although the zone is irregular and not flat, it is possible to observe perfect adhesion to the film to the wound (Fig. lb). As the zone was in a mobile joint, a dry gauze was spread over the graft to keep it adherent to the wound. The result of this graft on day 6 is presented in Fig. ]c and on day 10 in Fig. M. Fig. ]c shows the dry brown aspect of the film which partially hides the underlying dermo-epidermal graft. Fig. ]d highlights the excellent rate of take, with complete healing of the mesh holes under the seeded film, unlike the control zones (Fig. M). There was partial take of the autograft on the film-free area. The patient was discharged on day 45.

Case 3

A 13-year-old child suffered burns in a brushwood fire involving 85% T13SA (75% full skin-thickness). On day 2 post-burn a culture biopsy was harvested from the unhurried scalp, during the first escharectomy session. Further excisions down to the adipose tissue were performed in the anterior side of the trunk on day 5 and the upper limbs on day 8, with coverage by fresh meshed cadaver skin. When the allogfaft was checked seven days later, the clinical result was particularly good in the left forearm. At this early stage of treatment the patient was free of local or general infection. We therefore decided to cover the forearm with autologous keratinocytes, and six biological films were immediately seeded at a density of 101 cells per cral. On day 15 post-biopsy, seeded films were applied on the left forearm and elbow after surgical alloepidermis removal. At the first dressing change seven days later, the primary take rate was only 85% because the sides of the graft were destroyed. However, one week later, the appearance of the healed skin was excellent. The patient was discharged on day 82 after 65 days in the intensive care unit.

The successful use of cultured epithelium depends on both biological and clinical factors. The biological aspect is now perfectly codified. However, in vitro development can still be improved, and two points seem to be of particular importance.
First of all, clinicians generally find that it takes too long to produce CEA. A reduction of a few days would mean great progress, especially when homografts are unavailable. The use of the film presents numerous advantages: it obviates the need of a transfer step on petrolatum gauze, and the time taken for culture is shortened as perfect confluence of the cells is no longer needed to make the graft possible, and cell differentiation can also be avoided. The time taken to produce an epidermal culture before its clinical application can thus be shortened by four to seven days. In Case 1, the early "combined" graft permitted a spectacular result on a very large mesh. In Case 2, we cl obtained good healing of the joint, but only under the film. Joints are normally considered unfavourable areas, unsuitable for conventional grafts. In Case 3, the early cultured cells autografted on the remaining allodermis of the forearm were easier to graft and more efficient than the later traditional sheet autografts performed during a persistent and resistant sepsis syndrome (due to Pseudomonas) that occurred one week after the seeded film graft.
Secondly, it is difficult to predict the general state of health of burn patients: sometimes a graft session has to be advanced a few days. As the culture time is shortened, the cells are not sufficiently confluent to allow the production of conventional sheets. They can therefore be trypsinized and seeded for one night on the film. On the other hand, in the event of a contaminated wound or severe deterioration of the patient's general conditions, a graft may have to be postponed. CEA cannot however be used too long after confluence, because the keratinocytes lose their proliferative power. To solve this problem, the cultures can be trypsinized before confluence and their differentiation. They can be seeded on the film, with a density relative to the delay of the graft. From the end of week 2 post-burn, seeded film grafts can be planned even one day before surgery.
It is also important that the biological film preserves the epidermal cells in an active proliferating state. Here we support Kaiser et al., who proposed the application to burn wounds of non- confluent cultured autologous keratinocytes in a suspension of fibrin glue.

Fig. 1c - Result of graft on day 6: brown aspect of dried film partially concealing wound healing. Fig. ld - Result of graft on day 10: complete healing under film. Control area presents incomplete healing.

With regard to biological requirements, the film is ideally biocompatible. The MTT test, which is basically a cytocompatibility test, confirmed the absence of toxicity, and the fact that the cells proliferated proves that our film is an excellent support for in vitro cell culture. The film is easy to handle in vitro, and the adherence of cultured keratinocytes is excellent. With regard to clinical conditions, the film is easy to use as a support. Its flexibility is very useful because it adheres perfectly to the receiving site, achieving total contact with the seeded film (e.g. Case 2) and partial contact with gauze in "difficult" sites. Its permeability also allows exudates to pass through from the receiving site, and this reduces soaking. The film completely disintegrates within five to eight days - a period corresponding to the take of epidermal grafts. It is thus possible to avoid removal of the support, which is always particularly critical and often deleterious (due to adhesion to the wourid and/or haernorrhagic removal).

The preliminary results of the use of this biological film as a support to grow keratinocytes in subculture and as a transfer support look very promising as regards the ranges of application that have been tested, especially in mobile zones and joints. This new approach is promising both biologically and clinically, and justifies further research with a view to improving its use as a technical alternative to cultured autologous epithelium in the care of the most critically burned patients.

RESUME. La survie des brûlés les plus sévères passe par l'utilisation des feuillets d'épiderme humain cultivé. Les principaux inconvénients de cette technique sont les délais avant l'obtention des feuillets, leur fragilité ainsi que leur faible maniabilité. De plus, les pourcentages de prise de greffe sont variables et parfois faibles, particulièrement pour les zones difficiles, comme les articulations, le dos et le cou, où les gazes de transfert des feuillets ne sont pas suffisamment adhérentes au lit de la plaie. Cette étude préliminaire décrit l'utilisation d'un film biologique permettant la culture de kératinocytes et le transfert des cellules directement sur la plaie sans gaze de transfert. Le film (composé de 72% de collagène, 20% de chitosane et 8% de glycosaminoglycanes) a été utilisé chez trois patients qui avaient été traités par les méthodes chirurgicales habituellement utilisées dans notre Centre: soit la méthode "combinée" avec des feuillets épidermiques cultivés recouvrant une autogreffe dermo-épidermique largement meshée, soit les feuillets d'épiderme cultivé recouvrant une allogreffe après excision de l'épiderme. Les temps de culture ont pu être raccourcis de quelques jours puisque la parfaite confluence cellulaire ainsi que l'obtention d'un épiderme pluristratifié ne sont plus nécessaires: ainsi les cellules épidermiques ont pu être greffées entre le 14e et le 16e jour après la biopsie. Les manipulations nécessaires au transfert du feuillet sur la gaze, qui prenaient quelques heures, ne sont également plus nécessaires. Les zones ainsi recouvertes sont: une épaule, des flancs, un dos et un avant-bras, avec des surfaces allant de 600 à 960 cm'. D'un point de vue clinique, le film est facile à manipuler et beaucoup plus adhérent à la plaie que les pansements classiques ou vaselinés. Quand le pansement est enlevé lors de la vérification de la greffe, le film se désintègre spontanément sans effet délétère pour la prise de greffe, qui etait de 85 à 98% en moyenne après la première greffe. En conclusion, le film en accélerant la croissance des kératinocytes permet une bonne coordination entre l'équipe médicale et les biologistes. Il semble être une alternative technique possible à l'utilisation des feuillets épidermiques, plus spécialement pour les zones mobiles.

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