Cultured Epithelial Autograft

A 21-year-old male presented with a full-thickness flame burn that affected 90% of his body surface area. Immediate medical attention was focused on airway management, fluid resuscitation, and prevention of organ failure, and multiple escharotomies were performed. He subsequently underwent staged excision of his burns, and the wounds were temporarily covered with human cadaver allograft. A 2-cm² skin biopsy specimen was procured from the abdominal wall at the time of the initial excision on the third postburn day. This specimen was expanded in the laboratory over 3 weeks. Autograft was harvested from an area on his unburned thigh, and the allograft was removed. The wounds were treated with cultured epithelial autograft (CEA) and laid over a 4:1 meshed autologous split-thickness skin graft. This technology played a vital role in allowing the patient to survive a massive burn injury and achieve a reasonable quality of life (Figure 1).¹

CEA are cohesive sheets of autologous keratinocytes grown in tissue culture and transferred to the surface of a wound. The successful culture was described by Rheinwald and Green in the mid-1970s.² Keratinocytes were isolated from a full-thickness skin biopsy using enzymatic digestion and expanded in vitro by serial subcultivation in a medium containing fetal bovine serum overlying a feeder layer of lethally irradiated mouse 3T3 fibroblasts.³ Epidermal growth factor and cholera toxin, a stimulant of cellular cyclic adenosine monophosphate (AMP) formation, were also added to accelerate colony formation. The role of the fibroblasts is unclear but is thought to aid keratinocyte growth and inhibit fibroblast growth. There is a concern, however, that transplanting a xenobiotic cell line carries a risk of disease transmission and immunological rejection. Improved culture media have since allowed keratinocytes to be cultured without the fibroblast feeder layer.⁴ 

Delicate and confluent sheets of epithelial cells can be detached from the culture dish by the enzyme dispase and then clipped to a backing of petrolatum-impregnated gauze before transfer to the wound bed. Within 3 to 4 weeks, sheets of keratinocytes can be grown 8 to10 cells thick and can exceed expansion ratios of 1:10,000, producing sufficient cultured epithelium to cover the entire surface of an average-sized adult. An 8-year study by Compton et al, performing serial biopsies on patients treated with CEA, reported that at transplantation the grafts appeared as unevenly stratified sheets of keratinocytes lacking both granular and cornified cell layers. However, by 6 days post-grafting, CEA differentiated into all normal epidermal strata, and Langerhans cells, melanocytes, and Merkel cells repopulated the CEA during the first year.⁵

Since the first clinical application was reported in 1981, the use of CEA has had a considerable impact on the treatment of patients with massive burns.^6,7,8 The FDA has approved Epicel in the United States for use on patients with burns greater than 30% total body surface area under a Humanitarian Device Exemption. Several reports indicate the use of CEA for covering large wounds, including those caused by venous insufficiency, sickle cell anemia, surgical wounds,⁹ and excision of giant congenital nevi.¹⁰ More recently, the retroviral transduction of the LAMB3 gene into autologous keratinocytes has been successfully used to correct junctional epidermolysis bullosa, a blistering disease due to a mutation of LAM5, a gene that codes for the basement membrane component laminin 332.¹¹

Reports regarding the use of CEA for permanent coverage of burns have shown inconsistent and often disappointing results. Blight et al reported suboptimal engraftment rates with graft take ranging from 0 to 98% with a mean value of 15%. Notably, high values were observed on wounds prepared with allograft, presumably due to enhanced vascularization.¹² CEA is also friable and easily susceptible to shearing forces, with poor durability and spontaneous blistering or avulsion. These limitations can be attributed to the absence of dermis, basement membrane, mature hemidesmosomes, and anchoring fibrils at the attachment face until 3 to 4 weeks after grafting, as well as an absence of rete ridges for a further 5 to 18 months.⁵ Keratinocyte basement membrane proteins are also lost during the enzymatic detachment of the CEA sheets from the culture flasks by dispase. Late results have been unsatisfactory with substantial graft contraction, hypertrophic scarring, and hypopigmentation.

CEA is susceptible to infection by bacteria and fungi due to its vulnerability to proteases and cytotoxins during the first week of maturation. Staphylococcus, Pseudomonas, Candida, Acinetobacter, Enterococcus, Proteus, Serratia, and Aspergillus are particularly detrimental to the adherence and viability of cultured keratinocytes. 

Other disadvantages include a 3- to 4-week delay in obtaining grafts, high costs, and increased hospitalizations compared with the use of split-thickness autografts.¹³ 

The stability of CEA can be improved by the provision of a dermal bed. One technique involves applying CEA to a mechanically de-epithelialized allogeneic skin transplant, which improves bonding by creating a non-antigenic allodermis for CEA engraftment.¹⁴ However, this technique necessitates a 2-stage procedure and may be complicated by the loss of allograft and infection. An alternative method is to place the CEA over widely meshed autograft (Figure 1E). Careful handling is required to reduce the shearing of the delicate grafts, which places a huge burden on the patient and staff.¹ They are also best confined to the anterior surfaces of the body. The lower limbs can be grafted circumferentially with the aid of external fixation to elevate them off the bed. Postoperatively the wound should remain undisturbed for 7 to 10 days.

The susceptibility of grafts to infection demands strict sterile precautions during dressing changes. Before CEA is applied, wound infections must be adequately treated. The use of antibiotics is guided by quantitative cultures and prescribed even for bacterial counts lower than 10³/mm³. In the case described, the CEA was treated with gentamycin spray and left open to the air.