Clinical And Experimental Study Of HF-SCs Combined With 3D Printing Skin To Repair Skin Defects Under The Intervention Of Astragalus Membranaceus And Its Extract

[Objective]To observe the effect of Astragalus membranaceus on clinical skin repair,and further explore the role of Astragaloside Ⅳ（AIV）and rat hair follicle stem cells（rHFSCs）in skin repair by isolating and culturing rHFSCs in vitro,preparing AIV sustained-release microspheres and 3D printing skin model,so as to establish a new and efficient tissueengineered skin to guide clinical skin repair.[Methods]Thirty cases of skin defects in Jiangnan Hospital Affiliated with Zhejiang University of Traditional Chinese Medicine were collected and divided into treatment group and control group.The wound healing time and wound healing rate of each group were observed and compared.The effect of Astragalus on wound healing was studied by scar condition,safety observation index and adverse reaction events after wound healing.RHFSCs were cultured in vitro to obtain stable,high purity and strong value-added rHFSCs.RHFSCs were identified by flow cytometry and immunofluorescence assay.In order to further clarify the effect of active components of AIV on wound repair,AIV sustained-release microspheres were prepared.The entrapment efficiency,drug loading rate and in vitro sustained-release rate were measured,and the cell proliferation rate was observed by gradient intervention of rHFSCs;Three-dimensional skin model was made by 3D printing technology.RHFSCs were implanted on the surface of the model.The biocompatibility between rHFSCs and skin model was observed and evaluated under the microscope.Twelve 6-week-old nude mice were divided into group A（blank group）,group B（empty 3D model group）and group C（rHFSCs/AIV/3D printing skin model system group）.RHFSCs were labeled with EDU.The wound repair of nude mice was observed macroscopically and histologically on days 7,14 and 21 to explore the process of rHFSCs in wound repair.[Results]In the clinical experiment,the average wound healing time in the experimental group was 31±7.32 days and that in the control group was 43±6.79 days,P<0.05;The wound healing rate of the two groups was insignificant at 1 week（P>0.05）,and there was a significant difference between the two groups at 2 and 4 weeks（P<0.05）.The average scar score in the treatment group was 4.07±1.27 and that in the control group was 6.60±1.76,P<0.05.The isolated,cultured and purified rHFSCs had good cloning ability and strong vitality,and the specific markers of hair follicle stem cells were CK15,P63,Intergrin α6 and Integrinβ1 was highly expressed;The entrapment efficiency of AIV sustained-release microspheres was 72.677%,the drug loading rate was 1.245%,the release amount of microspheres in 12 hours was 5.78%,and the cumulative release rate in 21 days was as high as 81.86%;AIV microspheres at concentrations of 5μg/mL and 10μg/mL promoted the value-added and activity of rHFSCs in varying degrees;Under the microscope,it was observed that rHFSCs grew in clusters close to the surface of the skin model,indicating that rHFSCs had good compatibility with 3D printing skin model;In the animal experiment,there was no significant difference in the wound healing rate between the groups on the 7th day（P>0.05）;On the 14th and 21st days,the rHFSCs/AIV/3D printing skin model system group was better than the empty printed skin model group,better than the blank group,and the new epidermis of this group was close to the normal skin.The results of wound HE staining,CD31 immunohistochemistry,immunofluorescence staining and EDU cell tracking showed that the skin composite system group formed more hair follicle like tissues and had the strongest ability of neovascularization.[Conclusion]It is observed that Astragalus membranaceus can accelerate the rate of wound healing and make the wound closer to normal skin tissue;The laboratory results show that rHFSCs with high purity and strong value-added can be stably obtained by using mature hair follicle stem cell culture technology in vitro;3D printing skin scaffold has good biocompatibility;AIV of astragalus extract can promote the value-added of rHFSCs and jointly participate in the formation of hair follicles and blood vessels during wound repair,so as to promote wound repair;The new tissue-engineered skin model can promote wound healing in nude mice.