Regulation of cutaneous pigmentation by titration of human melanocytes in cultured skin substitutes grafted to athymic mice

Pigmentation of healed cultured skin substitutes in burn patients is frequently irregular and unpredictable which compromises solar protection and the patient's self-image. To address these morbidities, human fibroblasts were inoculated on a collagen-glycosaminoglycan substrate followed 1 day later by the addition of keratinocytes at 1.1 x 10(6)/cm(2) combined with either 0, 1.1 x 10(2),1.1 x 10(3), or 1.1 x 10(4) melanocytes/cm(2). The skin substitutes were incubated in vitro for 3 weeks and grafted to athymic mice. In vitro, the number of L-Dopa-positive melanocytes in the skin substitutes increased proportionately to the number of melanocytes inoculated. The melanocytes localized to the basal epidermis when labeled for MEL-5. The skin substitutes with 1.1 x 10(4) melanocytes/cm(2) were significantly darker than other groups in vitro by chromameter evaluation. By 12 weeks after grafting, the cultured skin ranged from no pigment in the control group, to 75% pigmented area in the 1.1 x 10(3) melanocytes/cm(2) group, to complete pigmentation in the 1.1 x 10(4) melanocytes/cm(2) group. In vivo, the mean chromameter values were significantly darker for the grafts with 1.1 x 10(3) and 1.1 x 10(4) melanocytes/cm(2). These results suggest that complete restoration of cutaneous pigmentation can be accomplished by addition of between 0.1 and 1.0 x 10(4) melanocytes/cm(2) to skin substitutes.