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

Abstract

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)/cm2 combined with either 0, 1.1 x 10(2), 1.1 x 10(3), or 1.1 x 10(4) melanocytes/cm2. 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/cm2 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/cm2 group, to complete pigmentation in the 1.1 x 10(4) melanocytes/cm2 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/cm2. 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/cm2 to skin substitutes.

Wound Healing on Athymic Mice With Engineered Skin Substitutes Fabricated with Keratinocytes Harvested from an Automated Bioreactor

“…The athymic mouse has been the preferred model to study the wound healing capability of human skin substitutes (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33), because of its immunologic tolerance of xenograft and the limited scarring that occurs during healing. ESS grafts fabricated with keratinocytes harvested from the Kerator had well keratinized and desquamating surfaces after two weeks following surgery (Figure 3).…”

Dermis Graft for Wound Coverage

“…3,4 Because melanocytes are localized to the basal cell layer of the epidermis, the origin of the color mismatch has been assumed to be the epidermal melanin. 1,2 It was hypothesized that the problems of color mismatch could be reduced if the composition of the epidermal portion of grafts was similar to that of surrounding skin. This was the reason for using the dermis graft.…”

“…This is largely due to the differences in the degree and extent of melanization in the donor and recipient sites, which are dependent on the number and activity of melanocytes in the basal layer of the epidermis. [1][2][3][4][5] Because regular skin grafts transferred to new locations maintain their epidermal specificity, a pigmentation difference with the surrounding skin is unavoidable.…”

Efficacy of Autogenous Dermis Graft for Wound Coverage

“…These include the amount of melanin, the degree of transfer of melanosomes to keratinocytes, and the number of melanocytes (Carlson et al, 2002;Velangi & Rees, 2001). Because the melanocytes are localized to the basal cell layer of the epidermis, the origin of the color mismatch has been assumed to be in the epidermal melanin (Tyack et al, 1997;Swope et al, 2002). Because regular skin grafts transferred to new locations maintain their epidermal specificity, a pigmentation difference with the surrounding skin is unavoidable.…”