Cellular artificial skin substitute produced by short period simultaneous culture of fibroblasts and keratinocytes.

We developed a novel artificial skin substitute consisting of two collagen sponge layers with different pore sizes and cross-link densities. Fibroblasts suspended in 0.5 ml Dulbecco-modified Eagle's medium (DMEM) + 10% fetal bovine serum (FBS) were seeded on the lower dermal sponge layer, then epidermal collagen sponge and 0.1 ml suspension of keratinocytes in KGM were layered in this order. After a few hours, the medium was changed to DMEM + 5% FBS. These processes were carried out in one day, and the composite layers were then cultured by the air-liquid interface culture method. Three to five days after seeding, keratinocytes had grown to about ten layers, and fibroblasts had grown three-dimensionally into the lower dermal sponge layer. This novel cellular artificial skin substitute was grafted onto nude mice and took in 4 weeks. This skin substitute has the advantage of a shorter culturing period than previously cultured skins, and may be clinically useful for grafting that is urgently required in patients with severe generalised burns.

Review of acellular and cellular artificial skins.

Following the development of a bilayer acellular artificial skin by Yannas et al. which seemed unique with respect to spontaneous conversion of the inner collagen sponge into a dermis-like connective tissue layer, we started to develop an alternative acellular artificial skin and extended the indications for the material. Since reporting our early results of experimental and clinical use of the original version of the acellular artificial skin, several improvements have been made in stages to eliminate some drawbacks related to disinfection and preservation and to reduce the primary cost of manufacture. We used this material on 51 skin defects in 39 patients with success. The latest version of the material was also evaluated in multicenter clinical trials involving 80 cases. Separately, a material capable of sustained release of an antibiotic was developed and used in 6 wounds prone to infection with success. To solve the problem of two-stage surgery, a cellular artificial skin composed of an outer keratinocytic layer and an inner collagen sponge containing fibroblasts was produced using cell culture method by modifying the technique proposed by Boyce et al. This report reviews our acellular and cellular artificial skins.

Further applications of "bilayer artificial skin".

A "bilayer artificial skin", composed of an inner layer of collagen sponge and an outer silicone layer, was developed by modifying the material reported by Yannas and Burke. Since our early results from experimental and clinical use of the original version of the "bilayer artificial skin" were reported, several improvements have been made in stages to eliminate some drawbacks related to disinfection and preservation and to reduce the primary cost of manufacture. The latest version of the material was successfully used in 27 sites on 23 patients. In this paper, the improvements in the material and the clinical results are described.

A new skin equivalent: keratinocytes proliferated and differentiated on collagen sponge containing fibroblasts.

Three types of artificial skin containing keratinocytic components were prepared and tested for comparison. Keratinocytes were cultured on the artificial skin dermis (collagen sponge) by the air-liquid interface culture method. In order to create continuous keratinocytic layers on the artificial skin dermis, pores of its uppermost layer were filled beforehand with type I collagen gel, Matrigel, or fibroblasts. A band of keratinocytes consisting of two to six cell layers was formed on collagen gel-coated artificial skin dermis. On Matrigel-coated artificial skin dermis, keratinocytes were piled up into about 20 cell layers, but cell differentiation was incomplete; cornified material was not fully developed, and the proportion of cuboidal cells was very high compared with normal epidermis. Keratinocytes formed continuous layers on the fibroblasts-artificial skin dermis complex without gel coating. Keratinocytes proliferated well and differentiated properly on this matrix, and their histologic appearance was similar to that of normal epidermis. Thus keratinocytes cultured on the fibroblast-artificial skin dermis complex seem to be a good skin equivalent.

Ultrastructural and immunohistochemical characterization of basal cells in three-dimensional culture models of the skin.

Keratinocytes were cultured on fibroblast-free dermal substitutes made of type I collagen film (collagen dermal substitute) and an extracellular matrix gel film (matrix dermal substitute), each of which was laid on a lyophilized type I collagen sponge. The morphology of the basal keratinocytes in these three-dimensional culture models of the skin was studied ultrastructurally and immunohistochemically to assess their differentiation to basal cells. The basal keratinocytes in the artificial epidermis cultured on the collagen dermal substitute showed poorly organized tonofibril networks and desmosomes. Neither the tonofibril-hemidesmosome complex nor the lamina densa were detected along the interface, where many cytoplasmic projections of basal keratinocytes were noted. There were no detectable antigens of type IV or VII collagen, LDA-1, or laminin in the interface. Bullous pemphigoid (BP) and 1-2B7B antigens and integrins were expressed along the cytoplasmic membrane and the projections of the basal keratinocytes. A high molecular weight keratin (keratin 1, 68 kDa, 34 beta B4) was detected only in part of the uppermost layers of this artificial epidermis. In contrast, basal keratinocytes in the artificial epidermis on the matrix dermal substitute developed tonofibril networks radiating to desmosomes and hemidesmosomes, under which a primitive lamina densa was present. Basement membrane zone antigens, such as type IV and VII collagens, LDA-1 and laminin were noted along the interface as were 1-2B7B and BP antigens and integrins. Laminin and type VII collagen were also detected along or in the membrane of the endoplasmic reticulum of basal keratinocytes.(ABSTRACT TRUNCATED AT 250 WORDS)