ECM Sheet And EIF4E Gene Therapy For Skin Wound Healing

Background:Wound healing is a complex and dynamic biological process that often occurs during human daily life.And skin tissues had evolved to develop interaction and coordination of varieties of cellular and molecular activities to repair or regenerate after being injuried.However,with improper treatment or under certain pathological conditions such as diabetes,acute wounds often transform into chronic wounds,which not only seriously affects the appearance and function of skin,but also results in immense economic burden for patients.Therefore,it has an important significance and social needs to explore novel materials and effective strategies for promoting wound healing and avoiding chronic wound.Object:1.To explore the effects of chemical conditioning on biological properties of MSC sheet derived ECM sheet;2.To explore the therapeutic effects of ECM sheet derived from chemical-conditioned MSC sheet on mice full-thickness skin defect and diabetic wound healing;3.To investigate the function and molecular mechanism of eIF4F complex on mouse skin wound healing;4.To investigate the efficiency of eIF4E1 and its mimic phosphorylation mutants gene therapy on mouse skin wound healing.Methods:1.Rabbit bone marrow mesenchymal stem cells(MSCs)were isolated and cultured in DMEM/F12 medium with 10%fetal bovine serum and 100?g/mL Vitamin C to form MSC sheets.The MSC sheets were further cultured in DMEM/F12 medium with 10%fetal bovine serum supplemented with LiCl or/and CoCl2 in vitro.After another 7 days,decellularization was conducted to fabricate extracellular matrix(ECM)sheets.The morphology of MSC sheets and their derived ECM sheets were observed by picrosirius staining;the ultrastructure of ECM sheets were analyzed using scanning electron microscope;the surface topography and 3-dimensional structure of ECM sheets were observed using atomic force microscope;the collagens and growth factors expression in MSC sheets and their derived ECM sheets were evaluated by western blot;the tensile strength of ECM sheets were measured using electronic universal material machine.2.Full-thickness skin defect models were created on both normal Balb/C mice and STZ-induced diabetic C57BL mice.The efficiency of MSC sheet derived ECM sheets were evaluated on cutaneous wounds and diabetic wounds through analysis of wound healing area percentages,granulation tissue thickness,re-epithelialization closure percentages,and new vessel numbers.3.pSecX-4EBP1,pSecX-K100,and pSecX-eIF4E1 were constructed and transfected into HaCaT cells.Their effects on the expression levels of wound healing related proteins expression and cell migration were evaluated by western blot and in vitro wound healing assays.The in vivo effects of 4EBP1,K100,and eIF4E1 topical gene therapy on wound healing were evaluated via analysis of wound healing area percentages,granulation tissue thickness,re-epithelialization closure percentages,and new vessel numbers.4.The effects of eIF4El and its mimic phosphorylation mutants on the expression levels of wound healing related proteins and cell migration in high glucose condition were evaluated by western blot and in vitro wound healing assays.And topical gene therapy experiments were carried out to compare the efficiency of eIF4El and its mimic phosphorylation mutants on diabetic wound healing.Results:1.Chemical conditioning MSC sheets with LiCl and CoCl2 activated(3-catenin and HIF-la,and led to homogeneous morphology and compact ultrastructure in MSC sheet-derived ECM sheets.CoCl2 alone or combined with LiCl increased the amount of collagen fibers and high tensile strength in ECM sheets.Chemical conditioning,especially LiCl together with CoCl2 significantly upregulated the expression level of collagens and growth factors in MSC sheets,and increased the content of collagens and growth factors in ECM sheets.2.ECM sheets made from non-conditioned MSC sheets promoted skin wound healing,but their effects were limited.Conditioning MSC sheets with LiCl or CoCl2 significantly improved the wound healing efficacy of their derived ECM sheets.And ECM sheets fabricated from LiCl and CoCl2 co-conditioned MSC sheets resulted in the best wound healing outcomes in both cutaneous wounds and diabetic wounds through enhanced granulation tissue growth,rapid re-epithelialization,and augmented angiogenesis.3.Overexpression of eIF4E1 upregulated the expression levels of many wound healing related proteins,promoted HaCaT cells migration,and accelerated cutaneous wound healing through increasing eIF4F complex activity.On the contrary,overexpression of 4EBP1 and K100 remarkably downregulated the expression levels of wound healing related proteins,inhibited HaCaT cell migration and impaired in vivo wound repair via blocking eIF4F complex assembly or inhibiting eIF4El phosphorylation,respectively.4.Topical gene therapy with eIF4El and its mimic phosphorylation mutants significantly accelerated diabetic wound healing through promoting granulation tissue formation,accelerating re-epithelialization,and enhancing angiogenesis.Conclusions:1.Chemical conditioning MSC sheets with LiCl and CoCl2 significantly improved the structural and biological properties of their derived ECM sheets with increased amounts of collagen and growth factors.And the resulted ECM sheets exhibited improved wound repair properties in both cutaneous and diabetic wound healing.2.Chemical conditioning provided a novel and simple strategy to fabricate efficient 3-D biomaterial for wound healing.3.eIF4F complex played pivotal roles in regulating wound healing.4.Up-regulation of eIF4F activity significantly promoted diabetic wound healing.