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Egypt. J. Plast. Reconstr. Surg., Vol. 26, No. 2, 2002: 161 - 165

Cultured Allogenic Keratinocyte Grafts in The Treatment of Burns: Preliminary Report.

ABD EL-A IZ HANAFY A. AHMAD, M.D.; MOSTAFA HEMIEDA, M.D.; HASSAN A. BADRAN, M.D., F.R.C.S. and IKRAM I. SEIF, M.D.
The Department of Plastic and Reconstructive Surgery, Faculty of Medicine, Ain Shams University.

ABSTRACT

The treatment of burns represents an important clinical and economic problem. Advances in tissue culture techniques allowed preparation of confluent epidermal sheets in vitro. Cultured epidermal autografts had been used in the coverage of extensive burn wounds. The epidermal cells separated from the skin biopsy could be serially cultured and the surface area expanded in 3-4 weeks. We are reporting on our early experience in the use of cultured allogenic keratinocyte grafts in the treatment of limited burn !wounds and split-thickness skin graft donor sites. Normal human keratinocytes were grown on the serum-free MCDB-153 culture medium into stratified sheets. The sheets were applied directly onto split-thickness skin graft donor sites, tangentially excised deep partial-thickness burns and 1:4 widely meshed split thickness autograft in excised full-thickness burn wounds. We could successfully prepare the culture medium and grow normal human epidermal cells into sheets suitable for grafting. The results of ur early clinical attempts are presented.

INTRODUCTION

Advances in the tissue culture techniques aver the past 2 decades enabled research works to grow confluent sheets of epidermis in vio [Il. Both autologous and allogenic sheets of ltured keratinocytes were used as grafts in anal studies [2,3] and humans with burn wounds [4,5] and chronic skin ulcers [6]. Several Egypan plastic surgeons performed research work on cultured keratinocytes in the United States in attempts to master the technique and introduce in Egypt [7,8,9]. In Ain Shams Plastic Surgery department, we established a tissue culture rearch laboratory and started keratinocyte culre work in 1997. In this study, we are reporting on our experience in cultivation of ratinocytes in vitro and the use of cultured alto promote healing of burn wounds and split thickness skin graft (STSG) donor sites.

MATERIAL AND METHODS

 In vitro cultivation of human epidermal cells:

Epidermal cell cultures were done in the tissue culture laboratory, Plastic Surgery Department, Ain Shams University. The source of epidermal cells was excised skin pieces obtained from abdominoplasty and reduction mammaplasty or extra pieces of split-thickness skin taken during grafting procedures. Primary keratinocyte cultures were established, using MCDB153 (Molecular, Cellular and Developmental Biology), culture medium, supplemented with epidermal growth factor, hydrocortisone, insulin and bovine pituitary extract [10,11,12]. When the primary cultures were about 70% confluent, the cultured cells were trypsinized and passaged [13]. Cultured keratinocyte grafts were prepared from P2 keratinocytes 19-21 days after starting the primary cultures. When these cultures were fully confluent, stratification was promoted by increasing calcium chloride concentration from 0.15 to 1.2 mM. After 5 days, the stratified sheets are separated from the culture dishes by 0.25% dispase II enzyme [14].

Patient population:

Four patients were grafted in the Burn Unit, Plastic Surgery Department, Ain Shams University Hospitals. Two were females and two were males. Their ages ranged from 2-38 years. The extent of burn ranged from 5-54% TBSA. The cultured allogenic keratinocyte grafts were applied to STSG donor sites, granulating full thickness burn wounds on top of 1:4 meshed split-thickness skin autograft, on top of deep partial-thickness burn wound and on top of localized full-thickness burn. The wounds covered by the cultured grafts represented only limited parts of the burned areas. Control areas of the STSG donor sites, the partial-thickness burn wound or the full-thickness burn covered by meshed skin grafts were treated by conventional dressing in each case.

Grafting technique:

The cultured epidermal grafts were transferred to cover the wounds backed by vaseline gauze. They were applied either onto STSG donor site or to the burn wound. The burn wound was considered ready for grafting if it was free of infection and necrotic tissue. The wound was prepared by betadine antiseptic followed by sterile normal saline solution. The grafts were applied with the basal layer directed to the Vol. 26, No. 2 /Cultured Allogenic Keratinocyte Grafts wound bed and fixed with few stitches. The number of sheets of cultured epidermal grafts applied per patient ranged from 2-28. The size of each sheet was slightly less than 75 cm, which is the surface area of the culture dish because the sheet retracts after separation. After transfer, the cultured grafts were covered by sterile dressing and crepe bandage. The dressing was observed for evidence of excessive discharge, which was considered a sign of infection and cultured graft loss. If the dressing remained dry, it was left undisturbed for 3 days.. At that time, the outer dressing was carefully . removed till the layer of vaseline gauze. This layer was left for 2 days more to be removed at the fifth postoperative day. Documentation of the clinical cases was done by photographing before and immediately after the cultured graft application, after the first dressing change and after complete healing. The clinical data of these patients are summarized in table (1).

 
Case # Age Sex Recipient wound

Number of shetts applied

1 38 Yrs. Female Full-thickness chemical burn in the thigh 20
      STSG donor sites 8
2 22 Yrs. Female Full-thickness chemical burn in the abdomen 2
      STSG donor sites 4
3 2 Yrs. Male Deep partial thickness scald burn in the chest 2
4 7 Yrs. Male Full-thickness burn in the hand 2
Table (1): The clinical data of cases trated with cultured allogenic keratinocyte grafts.

 

RESULTS

In this study, cultured allogenic keratinocyte grafts were prepared and used to treat 4 cases of burn wounds and STSG donor sites. Of the 12 experiments done in the laboratory, 8 experiments failed because of contamination or premature degenerative changes in the cultured cells. Contamination, whether bacterial or fungal was more common with skin specimens taken from burned patients despite careful preparation (Fig. 1, a&b). Cultured allogenic grafts were found to pro- mote healing provided that the recipient area was well prepared. In case #1, complete closure of the interstices of the meshed graft occurree in 5 days (Fig. 2, a&b) and full epithelializatio of the STSG donor site was observed in 6 day (Fig. 2, a&b). Control areas in the same pane required 9 and 11 days respectively for co plete healing. In the second case, cultured all genic keratinocyte grafts reduced the areas full-thickness burn by 70% in 5 days and pr moted healing of a rather deep STSG don site. Graft loss occurred in case 3 due to imp fect immobilization and in case 4 due to infection.

Fig. (1-A): Bacterial contamination of keratincoyte culture. Inverted phase micrograph magnified 100 X

Fig. (1-B): Fungal contamination of keratinocyte culture. Inverted phase micrograph magnified 100 X.
Fig. (1-A): Bacterial contamination of keratincoyte culture. Inverted phase micrograph magnified 100 X Fig. (1-B): Fungal contamination of keratinocyte culture. Inverted phase micrograph magnified 100 X.

Fig. (2-A) :Cultured allogenic keratinocyte gafts applied onto meshed skin autograft.

Fig. (2-B): The same case showing complete closure of the interstices of the meshed autograft by the 5th postoperative day.

Fig. (2-A) :Cultured allogenic keratinocyte gafts applied onto meshed skin autograft. Fig. (2-B): The same case showing complete closure of the interstices of the meshed autograft by the 5th postoperative day.

Fig. (3-A) Split-thickness skin gaft donor site covered by cultured allogenic keratinocyte grafts, intraoperative view

Fig. (3-B): The same donor site, 7 days postoperatively. See delayed healing of the periphery in aereas not covered by cultured allograft.
Fig. (3-A) Split-thickness skin gaft donor site covered by cultured allogenic keratinocyte grafts, intraoperative view Fig. (3-B): The same donor site, 7 days postoperatively. See delayed healing of the periphery in aereas not covered by cultured allograft.

DISCUSSION

The clinical applications of tissue culture have been increasing since the development of reproducible culture techniques. A recent method that had been developed for wound closure is the use of cultured autologous keratinocyte grafts that had been used to close burn wounds [4] and leg ulcers [15].

Cultured autologous keratinocytes can permanently attach [5]. However, they require the use of a biopsy from the skin of the patient and there is a delay corresponding to the time required for cultivation of the epidermal cells in vitro [6]. Another problem is the slow growth and reduced culture life span for cells from old donors [16]. The epidermis produced by cultured autologous keratinocyte grafts is unstable [17,18] and 5 years are needed for a neodermis to form [19]. Therefore, attention was focused on the use of cultured autologous keratinocytes with dermal equivalent [20,21]

. On the other hand, cultured allogenic keratinocyte grafts could be immediately available, especially when cryopreserved without the need for skin biopsy from the recipient [22,23]. It is also possible to growth cells from young donors, which have a higher growth potential and better wound healing power than cells from old cell donors [6,24]. Cultured allogenic keratinocyte grafts were thought to take permanently because they do not express the class II HAL-DR antigen [25,26]. However, further investigations by several authors showed that cultured allogenic grafts undergo gradual replacement by cells from the recipient without detectable rejection [27,28].

In our experimental work, we were able to prepare the MCDB-153 culture medium. This medium was used to grow and propagate normal human adult keratinocytes for 5 passages without the need of the special murine 3T3 feeder layer of cells [14]. We also could promote differentiation and stratification of PI and P2 cultures to prepare epidermal sheets in vitro suitable for grafting. Contamination was responsible for culture failure during the in vitro stage. This was common when the biopsy specimen was taken from burned patients. The skin of these patients may be colonized by resistant bacteria from the septic burn wounds, a problem that is not existing in non burned patients and cases of elective aesthetic surgical procedures. This is why we decided to start our tissue culture work by allogenic cells.

In this study, burn wounds and STSG donor sites were treated with cultured allogenic keratinocyte grafts. Wound healing was accelerated as evidenced by rapid closure of the interstices of the meshed STSG autograft (case 1), prompt healing of residual deep burn wound (case 2) and early epithelialization of STSG donor sites (cases 1 and 2). Promotion of healing would minimize the period of hospital stay and allow reharvesting of the same STSG donor sites in burned patients. Vol. 26, No. 2 / Cultured Allogenic Keratinocyte Grafts

Considered as an indicator for promotion of burn wound healing rather than permanent coverage. Cultured allogenic keratinocyte grafts probably exert their effects on wound healing through the release of cytokines and growth factors [29]. These factors act by stimulating the epidermal cells from the margins of the raw area or from the meshed STSG in full-thickness burn wounds and by stimulating dermal appendages in STSG donor sites and partialthickness burn wounds. The attached allogenic cells are gradually replaced by autologous cells from the recipient .without observable rejection [27,28]. Failure occurred in two cases due to shearing and infection (cases 3 and 4).

We should emphasize that although successful attempts are reported in this study, the areas treated by the cultured allografts represented only localized parts of the expensive bum wounds. A similar observation was reported by authors with cultured keratinocyte autografts [30,31): This was true especially in case 2 in our study. The cost effectiveness is not studied for this research procedure.

Summary:

This study represents the first Egyptian report on the in vitro cultivation of normal human keratinocytes into stratified sheets suitable for grafting. We also described the clinical application of these grafts to treat a limited number of cases of burn wounds and STSG donor sites. The procedure requires strict aseptic laboratory technique and good preparation of the cultured graft recipient sites. The ability to grow normal. human keratinocytes in vitro opens the door for laboratory research on skin cells, including work on composite skin substitutes propriate dermal equivalents with the epidermal cells.

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