[Experimental study on dermal papillary cells improving vascularization of tissue engineered skin substitutes]

To investigate the impact of dermal papillary cells on vascularization of tissue engineered skin substitutes consisting of epidermal stem cells and allogeneic acellular dermal matrix.

Human foreskins from routine circumcisions were collected to separate epidermal cells by using dispase with trypsinogen. Collagen type IV was used to isolate epidermal stem cells from the 2nd and 3rd passage keratinocytes. Dermal papilla was isolated by the digestion method of collagenase I from fetus scalp and cultured in routine fibroblast medium. Tissue engineered skin substitutes were reconstructed by seeding epidermal stem cells on the papillary side of allogeneic acellular dermis with (the experimental group) or without (the control group) seeding dermal papillary cells on the reticular side. The two kinds of composite skin substitutes were employed to cover skin defects (1 cm x 1 cm in size) on the back of the BALB/C-nu nude mice (n=30). The grafting survival rate was recorded 2 weeks after grafting. HE staining and immunohistochemistry method were employed to determine the expression of CD31 and calculate the microvessel density at 2 and 4 weeks after grafting.

Those adhesion cells by collagen type IV coexpressed Keratin 19 and beta1 integrin, indicating that the cells were epidermal stem cells. The cultivated dermal papillary cells were identified by expressing high levels of a-smooth muscle actin. The grafting survival rate was significantly higher in experimental group (28/30, 93.3%), than that in control group (24/30, 80.0%). HE staining showed that the epithelial layer in experimental group was 12-layered with large epithelial cells in the grafted composite skin, and that the epithelial layer in control group was 4-6-layered with small epithelial cells. At 2 and 4 weeks after grafting, the microvessel density was (38.56 +/- 2.49)/mm2 and (49.12 +/- 2.39)/mm2 in experimental group and was (25.16 +/- 3.73)/mm2 and (36.26 +/- 3.24)/mm2 in control group respectively, showing significant differences between 2 groups (P < 0.01).

Addition of dermal papillary cells to the tissue engineered skin substitutes can enhance vascularization, which promotes epidermis formation and improves the grafting survival rate.