Construction And Basic Application Of Wound Dressing Based On GelMA Hydrogel And Prevascularized Cell Sheets

Objective: In this study,a pre-vascularized wound dressing was constructed using cell sheet technology and methacrylic gelatin(GelMA),and then it was applied to the skin defect of nude mice,aiming at observing the healing effect of the wound dressing on wound healing.This study would provide a new treatment idea for the repair of skin wound in the clinic.Methods:1)Construction of pre-vascularized cell sheets: extraction of rabbit BMSCs for culture and amplification;determination of growth curve of rabbit BMSCs by MTT assay;detection of multi-directional differentiation of rabbit BMSCs.Human umbilical vein endothelial cells(HUVECs)were cultured,high-density inoculation of BMSCs into culture dishes for 10 days,and cultured with human umbilical vein endothelial cells for 5 days.2)Construction of pre-vascularized dressing: Preparation of GelMA and characterization of its physical and chemical properties,co-culture of the prepared gelatin with pre-vascularized gelatin.3)Pre-vascular dressing experiment with wound healing in nude mice: a full-thickness skin wound model of nude mice was constructed,and the constructed pre-vascularized dressing was applied to the wounds of nude mice.Tissue samples derived from the wound were extracted for 1,7 and 14 days respectively and H&E stained was used to study the effect of the dressing on the healing of the host wound.Results:1)The spindle shape of rabbit BMSCs could be observed under inverted light microscope.The results of MTT assay indicated that the growth curve of rabbit BMSCs was "S" shape.The results of multi-directional differentiation test verified that BMSCs had the ability to differentiate into adipogenic,osteogenic and chondroblastic cells.Immunofluorescence staining confirmed that the BMSCs sheets had high cell content,abundant extracellular matrix,cell-cell connection,and existence of a large number of type I Collagen fibers.For pre-vascularized cell sheets,HUVECs migrated both horizontally and vertically,and formed a network structure on the surface of BMSCs sheets,indicating that BMSCs sheet would provide a 3D microenvironment for the growth and migration of HUVECs.2)FTIR results showed that GelMA had no significant effect on the gelatin protein backbone structure.MTT results showed that GelMA hydrogel had low cytotoxicity and facilitated BMSCs proliferation and growth.3)The prepared prevascularized constructs were successfully transplanted to the nude mice to observe the healing effect,and the results showed that the healing rate of the transplanted group was faster than that of the control group.Immunohistochemical staining further confirmed that the pre-formed microvascular structures in the pre-vascularized construct were anastomosed with the host vasculature.Conclusions:The results of this study showed that the new wound dressing based on prevascularized stem cell sheet and GelMA hydrogel had the ability to promote the wound healing in the skin defect repair and regeneration experiment of immunodeficient mice,but its healing mechanism still needed to be further confirmed via a lot of basic experiments and clinical research.