| The biomimetic tissue engineering scaffold simulates the micro-nano microstructure and proteoglycan composition of the extracellular matrix,which provides biochemical and physiological stimuli to promote adherent cell proliferation and differentiation,and then affect the formation of new tissue after implantation.Therefore,it is of great significance to select appropriate materials and appropriate techniques to construct tissue engineering scaffolds that mimic the structure and composition of extracellular matrix.This paper intends to use gelatin and chitosan as raw materials to simulate extracellular matrix proteins and polysaccharides.And prepare gelatin/chitosan electrospun nanofiber composite membrane by electrospinning technique to mimic the nanofiber network structure of extracellular matrix,which purpose is to obtain tissue-engineered electrospinning membranes with a biomimetic extracellular microenvironment both in terms of composition and structure.(1)Preparation of electrospinning nanofibrous membranes using gelatin as raw material,and the effects of solvent types,gelatin concentration,cross-linking conditions and other factors on the fiber membranes were studied.Using acetic acid and acetic acid/ethylene glycol mixed solution as solvent,spinning under different concentration gradients.The results showed that the spinning membrane prepared by 13% gelatin solution in mixed solvent had the best spinning effect,the fiber membrane was smoother and more uniform,fiber diameter up to 125 nm.With 2%(w/v)genipin as the cross-linking agent,the results showed that the optimal cross-linking effect was achieved by genipin cross-linking for 2 days in the presence of 80% ethanol as the solvent.Infrared spectroscopy shows the cross-linking reaction between genipin and gelatin.Thermog-ravimetric analysis showed that the decomposition temperature of gelatin membrane after cross-linking increased from 311 ℃ before cross-linking to 315 ℃,its thermal stability was enhanced.The gelatin membrane cannot exist stably in the water system before cross-linking,but can be stable after cross-linking,and the tensile strength was5.3 MPa.In vitro degradation indicated that the cross-linked gelatin membrane was completely dissolved after 8 days in the water system and could not exist stably.In conclusion,gelatin alone can prepare electrospinning nanomembranes,and genipin can crosslink and stabilize the gelatin membrane to a certain extent,but the water stability is still poor.(2)The gelatin/chitosan/PVA electrospinning composite membranes with gelatin,chitosan and PVA as raw materials were prepared by single needle electrospinning technology,and the effects of different component ratios and cross-linking conditions on the spinning membrane were investigated.When the volume ratio of 13 wt% gelatin:(2 wt% chitosan / 8 wt% PVA)is 1:3,the average fiber diameter of the spinning membrane is 149 nm and the tensile strength after crosslinking is 14.5 MPa;When the volume ratio is 1:2,the average diameter of the fiber is 135 nm.When the fixed volume ratio is 1:3,the electrospun membrane is crosslinked with genipin.When the concentration of genipin is 2%(w/v),the crosslinking time is 2 days,and the crosslinking is stable.The infrared results showed that genipin undergoes cross-linking reaction with gelatin and chitosan,and the thermal weight loss results showed that the thermal stability of the composite membrane was better than that of the pure gelatin and chitosan electrospun membranes.The water absorption of the composite membrane decreased from552.6±49.6% before crosslinking to 339±18.6%.In addition,the mechanical test results showed that the chitosan/PVA component can enhance the mechanical strength of the spun membrane,and the tensile strength of the composite membrane after cross-linking increased from 8.5 MPa before cross-linking to 14.5 MPa,with a strain of 5.4%.The in vitro degradation results showed that the weight loss rate of the cross-linked composite membrane reached 43% after 6 weeks of degradation,and scanning electron microscope observation found that a large number of concave holes appeared along the fiber direction after the composite membrane was degraded.(3)Using gelatin,chitosan,polyvinyl alcohol and dialdehyde starch as raw materials,the in-situ crosslinked gelatin/chitosan/PVA/DAS electrospun nanofibers were prepared by double needle spinning.Scanning electron microscope test found that the surface of the electrospun nanofiber composite membrane was smooth,the fibers were evenly distributed,and the average diameter was about 221 nm.Infrared spectrum analysis showed that dialdehyde starch,gelatin and chitosan had cross-linking reaction.The thermal analysis results show that the decomposition temperature of the composite membrane is 330 ℃,which is higher than the thermal decomposition temperature of the composite membrane,and the thermal stability is enhanced.The tensile test results showed that the mechanical strength of the composite membrane was 38.3 MPa,which was higher than that of the single-component electrospinning.Comparative analysis of water absorption and shrinkage of Gelatin/chitosan/PVA,PVA/DAS and Gelatin/chitosan/PVA/DAS composite membrane,the results showed that the water absorption rate of Gelatin/ chitosan/PVA/DAS was 402.2±25.3% and the shrinkage rate was 9.2±2.1%,which was lower than the water absorption rate of its two single-spun components.After six weeks of degradation of the composite membrane,it was found that the weight loss rate reached 41%,and scanning electron microscope showed that there were large-scale degraded concave pores on the surface of the composite membrane.(4)Adipose-derived mesenchymal stem cells were seeded on Gelatin/chitosan/PVA and Gelatin/chitosan/PVA/DAS composite membranes,respectively,and the system cytocompatibility of the composite membrane was investigated.The CCK-8 method was used to measure the change of absorbance after cell inoculation.The results showed that the absorbance increased positively within 14 days after the cells were inoculated on the composite membrane,indicating that the cells could adhere and proliferate on the composite membrane.The cells on the composite membrane were qualitatively analyzed using Dio staining fluorescent labeling,the results of fluorescence microscopy showed that the number of cells gradually increased with the prolongation of culture time,which was consistent with the quantitative analysis of CCK-8.Scanning electron microscope was used to observe the cell/composite membrane complex,and it was found that after 1,3,5,and 7 days of in vitro culture,the number of cells on the composite membrane gradually increased,and the cells maintained a long spindle cell phenotype.In conclusion,Gelatin/chitosan/PVA and Gelatin/chitosan/PVA/DAS composite membranes have good cell compatibility,can promote the adhesion and proliferation of adipose-derived mesenchymal stem cells,and maintain the cell phenotype. |
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