A Stem-derived Skin Substitute Based On The DOPA-EGF Modified PLGA Microfiber Membrane To Repair The Full-thickness Back Skin Defect

Large area of full-thickness skin injury caused by burns,diabetes,long-term bed compression and other reasons is still a major problem in clinical treatment.Although autologous or allogeneic skin grafts have certain therapeutic effects,there are problems of limited source of grafts and immune rejection.With the development of tissue engineering,more attention has been paid to the research and application of artificial skin.As we know,biomaterials,seed cells and growth factors related to skin repair are the three major elements of tissue engineering skin,among which,biomaterials and seed cells jointly constitute the main framework of tissue engineering skin.Artificial skin based on biodegradable synthetic macromolecular materials is a new kind of artificial skin in recent years.The biodegradable synthetic macromolecular materials are not only widely used,but also have great biocompatibility and degradability.It is an ideal tissue engineering skin material.The selection of seed cells is particularly important for the construction of tissue-engineered skin.The selected cells should have good proliferation ability and dermal differentiation ability,and be easily obtained in large quantities.In a large number of existing studies,researchers have applied a variety of stem cells as seed cells to the construction of tissue-engineered skin,and achieved good results,paving the way for the application of stem cells in skin tissue engineering.Unlike the other two factors,the role of growth factors in tissue engineering is mainly functional.The introduction of growth factors can greatly enhance the biological activity of tissue engineering materials,so that seed cells planted on the surface of the materials can grow better.Growth factor belongs to the substance secreted by normal cells,so it has the advantages of low toxicity,low immunogenicity and high efficiency.However,due to the shortcoming of short half-life and easy to be diffused and lost with blood,the growth factor cannot act on the lesion in a long-term and stable way,which hinders the development of its clinical application.The research objective of this study is to prepare a tissue engineering skin derived from stem cells with excellent properties for the application of full-thickness skin damage.First of all,through genetic engineering,fermentation engineering and enzymatic reaction,the epidermal cell growth factor with the adhesion ability to material(DOPA-Lysine-DOPA-Lysine-DOPA-EGF,DOPA-EGF)was prepared,and adhered to Poly lactic acid/glycolic acid copolymer(PLGA)electrostatic spinning membrane.Then,human umbilical cord derived mesenchymal stem cells(hUCMSC)were planted on the modified electrostatic spinning membrane to construct a stem-cell-derived skin substitute.Finally,the skin substitute was used to repair full-thickness skin defects on the back of rats to evaluate the repair effect.The specific research content was as follows:1.gene engineering was applied to integrate the short peptide gene sequence containing tyrosine-lysine-tyrosine-tyrosine(YKYKY)at the end of the EGF gene sequence and loaded into the plasmid vector pET15 b.The recombinant plasmid vector was then transformed into E.coli BL21(DE3)to construct a prokaryotic expression vector for precursor protein of DOPA-EGF(YKYKY-EGF).After a series of expression and purification processes,YKYKY-EGF was successfully obtained.After that,YKYKY-EGF was further converted into DOPA-EGF by enzymatic hydroxylation of tyrosinase.The results of circular dichroism(CD)and biological activity showed that the secondary spatial structure and biological activity of both YKYKY-EGF and DOPA-EGF were consistent with the natural EGF.2.PLGA electrostatic spinning fiber film was successfully prepared by using electrostatic spinning technology.The results of scanning electron microscope showed that the PLGA electrostatic spinning fiber membrane was composed of a large number of micron-level fibers with a porous structure.The diameter of a single fiber was about 5~10 ?m,and the pore diameter between the fibers was about 20~50 ?m.Furthermore,the adhesion ability of DOPA-EGF on PLGA electrostatic spinning film was qualitatively tested by ELISA,FITR and XPS.The results showed that the adhesion of d DOPA-EGF to PLGA electrostatic spinning film was significantly higher than that of EGF and YKYKY-EGF.After that,we tested the hydrophilicity of the modified PLGA electrostatic spinning film and found that DOPA-EGF modification could greatly improve the hydrophilicity of PLGA electrostatic spinning film.3.after inoculating the hUCMSC on modified PLGA electrostatic spinning membrane,a stem-cell-derived skin substitute was successfully prepared.The results of cell adhesion and proliferation test showed that DOPA-EGF-modified PLGA electrostatic spinning film was more favorable for the adhesion and proliferation of hUCMSC on the surface.Furthermore,immunofluorescence staining and real-time quantitative PCR results showed that,under the induction of EGF,hUCMSC gradually differentiated into skin stem cells(SSCs),and secreted a large amount of extracellular matrix components(COLI)and vascular endothelial growth factor(VEGF)that were beneficial to skin repair.4.the repair effect of tissue engineered skin was evaluated by using the full-thickness skin defect model of rat back.The results showed that the DOPA-EGF group was significantly superior to the other experimental groups and the blank control group in terms of wound closure,wound formation,new and old tissue fusion and vascularization.To sum up,a stem-cell derived tissue engineering skin based on DOPA-EGF modified PLGA electrospun membrane was successfully prepared.This tissue engineered skin can effectively promote the repair of full-thickness skin defects on the back of rats,which has a very bright clinical application prospect.