Three-dimensional Bio-printed Micro-architecture Involves In The Behavior Of Epithelial Stem Cells In Tissue Engineering Epidermis

Background:The skin is the largest organ in the body.Large scale of burn wounds and skin injuries can lead to local anomalies,skin dysfunction,the failure of maintaining internal hemostasis,and even death.Therefore,it poses a furious challenge on repair and regeneration of skin wounds in clinical trial.The skin substitutes based on tissue engineering were once considered to be potential in skin restitution.However,the skin substitutes lack skin appendages,such as sweat glands,hair follicle and sebaceous,which exhibits problems on restoring physiological function of skin.However,in contrast to relatively well characterized hair-follicle or other epidermal derivatives,much less is known about the development of sweat glands.Functionalized sweat glands can help to improve the adaptation to foreign environment and restoring sweat production.As skin and other appendages are all derived from epithelial stem cells,epithelial stem become very promising candidates for regenerating sweat glands.In the preliminary research,our team has proved that the three-dimensional micro-architecture can induce the differentiation of epithelial stem cells to sweat gland cells.However,the inductive effect was not uniform based on different constitution of the three-dimensional architecture,which lays difficulties on how to choose the appropriate three-dimensional bio-printed architecture in sweat regeneration research.Thus,it is necessary to explore the behavior of epithelial stem cells responding to different three-dimensional bio-printed architecture,and to build a reliable three-dimensional architecture with high inductive quality,which will become a solid foundation for the future research.Purpose:Explore how different architectures involved in three-dimensional bio-printing technique influences the behavior of epithelial stem cells.To build a reliable three-dimensional micro-architecture with high inductive activity.To provide theoretic basis for the repairing and regenerating the injured skin.Methods:1:Epidermal Stem Cell Isolation:mice,C57BL/6,with embryonic age of 12.5 days were chosen,which were green fluorescent protein(GFP)modified,and their skin was cut and cultured using the epidermal stem cell culture method.2:The toe of the dermal matrix homogenate to obtain:wild-type C57BL/6 mice with age of 0.5 days were chosen,cut its toe grinding into dermal matrix homogenate(PD).3:3D Bio-printing:Based on the 3D bio-printing platform,three different diameters of nozzle(210μm,340μm and 420pm)were chosen to construct microstructures.4:Epidermal stem cells behavioral change observation and detection:fluorescence microscopy and live cell staining technique were used to display 3D microstructures of epidermal stem cell proliferation and cell viability;immunofluorescence staining technique were used to inspect how the epidermal stem cells differentiate to sweat gland cells on 3D microstructure level.5:statistical analysis:ANOVA and two sample t test were used to analyze all data.Results:All of the 3D cell-laden micro-architectures(210μm,340μm and 420μm)promote cell proliferation;the viability of the cells in the 210μm-printed micro-architecture was obviously reduced compared with that of the cells in the 340μm-printed or 420μm-printed micro-architecture;live cells in the 340μm-printed micro-architecture were more than the others.Conclusion:The group using 340μm head for three-dimensional bio-printing showed the highest inductive activity.This study can provide foundation for the future research on regeneration of sweat glands involving three-dimensional bio-printing technology,and for building a tissue engineering epithelial cell model with good bio-compatibility.