Objective To construct tissue-engineered skin containing melanin with mixed culture of human keratinocytes (KCs) and melanocytes (MCs) on de-epidermized dermis (DED) in vitro. Methods Single-cell suspension was obtained by digestion of isolated preputial epidermis with pancreatin. Keratinocyte serum-free medium (K-SFM) and modified M254 culture medium were used to culture KCs and MCs respectively. Third-passage KCs were seeded into cell culture flasks and cultured for 48 hours; then, third-passage MCs were seeded into the same cell culture flasks with the MC/KC ratio being 1: 10 followed by a 5-day coculture. The suspension of third-passage KCs and MCs with the MC/KC ratio of 10:1 were seeded onto the surface of a prepared DED and maintained at the air-liquid interface for 11 days following a 4-day submerged culture.Subsequently, the constructed tissue-engineered skin was examined with HE staining, immunohistochemical staining for keratin and Masson-Fontana staining. Results After coculture in flasks for 5 days, KCs exhibited a typical paving-stone appearance, MCs with projected dendrites were scattered in the extracellular space between KCs. HE staining revealed 3 to 6 layers of cells with the formation of stratum corneum after mixed culture on DED for 15 days. Keratin protein was positive throughout the artificial epidermis, and melanin pigments were located in the basal layer of the epidermis as Masson-Fontana staining showed. Conclusions The co-culture of MCs and KCs can form single-cell layers with the contact between MCs and KCs in flasks, and construct tissue-engineered skin with melanin component on DED in vitro.

Objective To evaluate the feasibility of repair of full-thickness skin defects in nude mice with tissue-engineered skin which was constructed by culture of human amniotic epithelial cells (hAECs) and fibroblasts on human de-epidermized dermis (DED).Methods Healthy human amniotic tissues were treated with trypsin at a low concentration in multi-steps to prepare hAECs,and a two-step collagenase digestion was used to treat healthy children's prepuce tissues to prepare fibroblast suspensions.When fibroblasts were cultured in vitro up to passage 3-5 and hAECs up to passage 2,they were seeded on the reticular dermal surface and basement membrane surface of the DED respectively to construct the tissueengineered skin.A total of 20 heahhy male nude mice aged 3-4 weeks were enrolled into this experiment,and full-thickness skin defects were made on the middle of the back of mice.Then,these mice were randomly divided into 2 groups by using a lottery method,and reconstructed full-thickness tissueengineered skin grafts and vaseline oil gauze were used to cover the wounds in the tissue-engineered skin group and control group respectively.The whole body and transplantation sites of the nude mice were observed on day 7,14,21 and 28 after transplantation,the wound healing time and rate were compared between the above two groups,and skin tissues at the transplantation site were harvested at 4 weeks after transplantation and subjected to histological examination.Results HAECs had stem-cell characteristics and expressed octamer-binding protein-4 (OCT-4) and embryonic marker stage-specific embryonic antigen4 (SSEA-4).After 2-week organ culture,the in vitro reconstructed tissue-engineered skin showed 4-9 continuous layers of stratified epithelium,and the histological structure of the epidermis was similar to that of the normal human skin.Compared with the control group,the tissue-engineered skin group showed significantly higher wound healing rates on day 7,14 and 21 after transplantation (57.49％ ± 6.11％ vs.22.93％ ± 4.26％,92.80％ ± 3.10％ vs.54.57％ ± 7.94％,98.83％ ± 0.25％ vs.91.16％ ± 4.79％,respectively;n =10,t =27.36,32.23,11.80,respectively,all P ＜ 0.001),shorter wound healing time [(21.51 ± 1.51) d vs.(28.80 ± 1.14) d,n =10,t =42.23,P ＜ 0.001],with the color of skin grafts closer to that of autologous skin on day 28 after transplantation.Histological examination revealed distinct stratification of the epithelium,obvious keratinization and favorable growth of cells in the dermis in the tissue-engineered skin group,but thin epithelium with some defects,indistinct stratification of the dermis,and inflammatory cell infiltration in the control group.Condusion Tissue-engineered skin constructed by the culture of hAECs and fibroblasts on human DED can survive in nude mice after transplantation,resulting in a more favorable healing of wounds,and is expected to serve as a kind of ideal tissue-engineered skin.