In Vivo And In Vitro Study Of In Situ Nano Gelatin Fiber Membrane Promoting Angiogenesis

ObjectiveEarly angiogenesis is one of the key steps in the process of tissue regeneration.There are some defects in autologous tissue transplantation.At present,many studies focus on tissue engineering technology to promote tissue repair.In terms of the physical properties of tissue engineering materials,the crosslinking mode of film-forming materials and fiber diameter can affect the growth of cells to a certain extent.Improving the properties of membrane materials and promoting the early formation of blood vessels is of great significance for tissue repair.However,it is unclear whether and how these two factors of gelatin fiber membrane affect early angiogenesis.PurposeTo explore the best crosslinking mode and fiber diameter of gelatin fiber scaffold to promote early angiogenesis in vivo and in vitro.Methods1.Glyoxal was used as crosslinking agent and gelatin was used as raw material to prepare in-situ crosslinked and post crosslinked gelatin fiber films respectively.The physicochemical properties of gelatin fiber membrane were tested by scanning electron microscope,water antennae experiment,roughness test and fiber diameter measurement.2.Umbilical vein endothelial cells(Huvecs)were co cultured with 700 nm fiber diameter in situ crosslinked gelatin fiber membrane and 700 nm diameter post crosslinked gelatin fiber membrane.The effects of the two materials on the proliferation of umbilical vein endothelial cells were detected by CCK-8(cell counting kit-8).He staining was used to observe the adhesion of cells on the two membranes.3.In situ crosslinked gelatin fiber films with three different fiber diameters were prepared by electrospinning with pinholes of 500 nm,700 nm and 1000 nm.After the physical and chemical properties of the materials were tested,HUVECs was co cultured with membrane materials.CCK-8 proliferation experiment was used to compare the activity of cells on the three groups of materials.F-actin cytoskeleton staining was used to compare the adhesion ability of cells.The ability of HUVECs to construct tubules in vitro was tested by in vitro tubulogenesis experiment.The growth of cells on the three materials was observed by scanning electron microscope.Western blot(WB)was used to detect angiogenesis related proteins and adhesion related proteins:angiopoietin-1(Ang-1),vascular endothelial growth factor(VEGF),integrin β1(Integrin-β1)To explore the related mechanism.4.Male SD rats aged 35～41 days were used for the experiment.Under sterile conditions,each rat prepared two incisions with the midline of the back as the baseline.One side was implanted with membrane and the other side was sham operated.Three diameters of in-situ crosslinked gelatin fiber films and 700 nm post crosslinked gelatin fiber films were implanted respectively.Tissue was obtained 7 days after operation,and angiogenesis and tissue repair were observed by HE staining.The distribution of vascular cells was observed by CD31 immunofluorescence staining.Angiogenic and adhesion related proteins Ang-1,VEGF and integrin were detected-β1 expression.Results1.Compared with 700 nm diameter post crosslinked gelatin fiber membrane,700 nm diameter in-situ crosslinked gelatin fiber membrane has smaller roughness and stronger hydrophilicity.Huvecs had stronger proliferation and adhesion on it.2.1000 nm diameter in situ crosslinked gelatin film has more significant advantages in cell proliferation,adhesion and tubule formation in vitro than the other two diameters in early angiogenesis.This may be through up regulation of Angl,advantages in cell proliferation,adhesion and tubule formation in vitro than the other two diameters in early angiogenesis.This may be through up regulation of Angl,VEGF and integrin-β1 expression.3.The above results were also verified by in vivo experiments in rats.Conclusions1.The gelatin fiber membrane prepared by in-situ crosslinking mode promotes angiogenesis more than the gelatin fiber membrane prepared by post crosslinking mode.The mechanism may be related to angiogenesis related factors and adhesion related factors VEGF,Ang-1 and integrin-β1 relevant.2.In situ crosslinked gelatin fiber membrane with a diameter of 1000 nm may solve the problem of insufficient blood supply in the early stage of soft tissue regeneration,and has important clinical application prospects in promoting tissue regeneration.