The efficacy and durability of wound closure was examined in a prospective randomized unbalanced clinical trial using the application of a living serum-free cultured epidermal autograft in conjunction with wound-area debridement and a four-layer compression wrap (N=10) compared with wound-area debridement and a four-layer compression wrap in patients with hard-to-heal leg ulcers arising from confirmed venous stasis (N=5). All 15 patients who presented with full-thickness venous ulceration were treated weekly for 8 weeks, with a 12-week final evaluation. The average time to wound closure for the grafted wounds was 4.1 weeks for 80% (8/10) of the cases that closed in 12 weeks compared with 12 weeks for the one closed in the control case. All of the grafted wounds remained closed at 12-month follow-up and one more healed at 30 weeks postenrollment. In the control group, one additional wound healed at 21 weeks postenrollment after the placement of an autograft. No serious adverse events were reported and subjective pain assessment was substantially reduced immediately after graft application. The graft treatment significantly improved outcome and provided durable wound closure. The data suggest that this adaption of this procedure may reduce the management costs of these wound types.
Cell culture techniques for producing a three-dimensional autologous epidermal autograft (cultured epidermal autograft) suitable for tissue grafting and wound healing procedures are described. This chapter commences with surgical biopsy of patient's skin tissue, further reduction of skin tissues to keratinocyte cells by enzymatic treatment, and recovery of viable adult keratinocytes in a new balanced buffered salt media supportive of the growth of clonally enriched isolated basal keratinocytes. Culture techniques required for the formation of a hole-free monolayer of undifferentiated basal keratinocytes without the use of an organotypic matrix substrate are accomplished with a specially designed nutrient basal media (HECK 109) that is a chemically defined and subsequent culture in this serum-free culture media supplemented with hormones and two human recombinant protein growth factors (EGF and IGF-1). Further culture techniques and media manipulations, including brief exposure to β-TGF to induce reversible G1-phase growth arrest, are followed by para-synchronous induction of a multilayered stratification and keratinizing epidermal differentiation, yielding a living three-dimensional epidermis formed entirely in cell culture. Protocols are listed for its enzymatic removal, floatation, and transfer for shipment to the clinic ready for surgical grafting to the self-same patient's debrided chronic leg ulcers. Recent clinical trial results have demonstrated the utility and efficacy of these grafts in forming durably healed chronic wounds.
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