Repair of skin defect with 3D-bioprinted organoid artificial skin derived from adult stem cells in mice
Objective To construct 3D-bioprinted organoid artificial skin derived from adult stem cells and investigate their effects on repair of skin defect in mice.Methods The cell suspension mixture was prepared with human skin keratinocytes,fibroblasts and vascular endothelial cells with a ratio of 2∶1∶1 and cultured in ultra-low attachment plates,and morphological changes of cell spheres were observed with an inverted phase contrast microscope.After 7 days of culture,cell spheres were collected and immunofluorescence staining was performed to characterize the expression and structural distribution of the epidermis,dermis and blood vessels.The artificial skin composed of skin organoids were printed through 3D printing and morphology of printed artificial skin and dressing was observed.Ten immunodeficient balb/c female mice were divided into hydrogel group and organoid group,with 5 mice in each group with the method of random number table.The full-thickness skin defect model with a diameter of 1 cm was established in all mice,and the wound was covered with the hydrogel dressings in hydrogel group and with 3D-printed skin organoids of the same size in organoid group.Wound healing and healing rate of the two groups were observed at 0,4,8,12 and 16 days after modeling.At 16 days after modeling,HE staining was performed on wound skin samples to observe the epidermal keratosis and dermal epidermal junction of the wound surface and Masson staining to observe the density of collagen fibers and dermal fiber thickness of the wound surface.Results(1)The cell suspension mixture of keratinocytes,fibroblasts and vascular endothelial cells could self-aggregate into cell spheres in the ultra-low attachment plates,and it was observed with the inverted phase contrast microscope that the volume of cell spheres gradually increased with the extension of culture time.(2)Immunofluorescence staining of the cell spheres showed that epidermal markers such as keratin(K)l,K10,and K14 were expressed in the outer layer of the cell spheres,and dermal markers such as vimentin(VIM)and vascular markers CD31 were expressed in the core,which indicated the epidermis was located in the outer layer of the sphere,and the dermis and blood vessels were located in the core of the sphere,with the same structural characteristics of the skin organoids.(3)The 3D-printed organoid artificial skin and hydrogel dressing were round and transparent,with a diameter of 10 mm and a thickness of 1 mm.(4)As shown in the general observation of the wound surface,the wound area of both groups decreased with the extension of treatment time.The wound of the organoid group healed faster,which showed obvious epithelization at 4 days after modeling and basic wound healing at 16 days after modeling.At 0 day after modeling,there was no obvious difference in the appearance of wound surface between the two groups.At 4 and 8 days after modeling,the wound healing rates were(31.7±1.0)％and(52.4±5.4)％in the organoid group,and(24.3±6.8)％and(45.4±7.0)％in the hydrogel group(P＞0.05).At 12 and 16 days after modeling,the wound healing rates were(78.6±8.0)％and(91.1±5.6)％in the organoid group,and were(58.5±5.4)％and(71.9±7.8)％in the hydrogel group(P＜0.01).(5)HE staining showed that in the organoid group epidermal keratinization was found better,with the epidermis being more intact and well attached to the dermis.Epidermal keratinization was not complete in hydrogel group and the epidermis and dermis were obviously separated.Masson staining showed the formation of collagen fiber structures in the wound surface of both groups,which were blue and reticular.The collagen fiber structure was more compact and the dermal fiber thickness was smaller in the organoid group,while the collagen fiber structure was loose and the dermal fiber thickness was greater in the hydrogel group.Conclusions Adult stem cells of skin can successfully form skin organoids in 3D culture conditions and organoid artificial skin can be constructed with 3D bioprinting technology.Compared with hydrogel dressing,3D-bioprinted organoid artificial skin can significantly improve the healing rate in mice,with better epidermal keratinization and closer attachment of the epidermis to the dermis.Moreover,the collagen fiber structure of the wound is more compact,with smaller dermal fiber in thickness.
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维普
