| Purpose: Cross-linking reaction was used to improve the stability of the structure of natural substance collagen,improving the anti-coagulation and endothelialization function of the mixed polycaprolactone(PCL)/collagen,and make it a suitable candidate material for the intima of artificial vascular scaffolds.Meanwhile,a double-layer composite artificial vascular scaffold was constructed to explore the tissue reconstruction effect of polycaprolactone material with large pore size,to obtain the material structure that can promote the regeneration of smooth muscle cells and extracellular matrix in a short term,and finally to evaluate the effect of in vivo orthotopic transplantationMethods: Firstly,collagen polycaprolactone(PCL/COL)fiber membrane was prepared by electrospinning technology,and different cross-linking methods were used,namely glutaraldehyde(GA)steam method,n-hydroxysuccinimide /1-(3-dimethylaminopropyl)-3-ethyl carbodiimine hydrochloride(EDC/NHS)soaking method and Genipin soaking method.Then the physical and chemical properties and biological properties of the cross-linked materials were analyzed.The hemolysis rate,platelet adhesion and other blood compatibility evaluation as well as the mobility of endothelial cells on the surface of the materials were tested.Finally,cross-linked materials were implanted under the skin of the animals and removed at 3 days,7 days,10 days,20 days and 30 days for histological identification and inflammatory response measurement.Then,prepared large pore electrospun PCL fiber was wound around the outer layer of PCL/COL artificial vascular scaffold after cross-linking to prepare double-layer composite artificial blood vessels.After 2 weeks of subcutaneous implantation,materials were removed for in situ abdominal aorta transplantation,during which hemodynamics was monitored by ultrasound.After 4 weeks,scaffolds were taken out for histological identification was performed.Results: Genipin and EDC/NHS cross-linked PCL/COL fiber membranes have good mechanical properties,lower hemolysis rate and platelet adhesion rate,and can promote the rapid migration of endothelial cells on the material surface.Large pore size PCL material is conducive to cell growth and tissue regeneration,and histological identification indicates that the production of contractile smooth muscle cells can be observed in a short period.Ultrasound indicated that the patency rate of double-layer composite scaffold was relatively high,and no dilation and stenosis.Conclusion: Genipin and EDC/NHS cross-linked PCL/COL materials are used as the intimal layer of vascular scaffolds,while large-pore PCL materials constitute the outer membrane layer of vascular scaffolds.Double-layer composite artificial vascular scaffolds can improve the patency rate of small-diameter vascular scaffolds,rapidly promote the regeneration of endothelial cells and contraction type smooth muscle cells,and achieve the biosimulation in a short term.Part Ⅰ Study on the properties of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and genipin crosslinked polycaprolactone/collagen fiberPurpose: A promising vascular scaffold must possess satisfying mechanical properties,great hemocompatibility,and foster tissue regeneration.Combining natural with synthetical materials is a popular method of creating/enhancing such scaffolds.However,the effect of additional modification on the materials requires further exploration.Methods: We selected polycaprolactone(PCL),which has excellent mechanical properties and biocompatibility and can be combined with collagen.Electrospun fibers created using a PCL/collagen solution were used to fashion mixed nanofibers,while separate syringes of PCL and collagen were used to create separated nanofibers,resulting in different pore sizes.Mixed and separated nanofibers were crosslinked with glutaraldehyde(GA),1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC),and genipin,hence we named them as MGA(mixed glutaraldehydge),ME(mixed EDC),MG(mixed genipin),SGA(separated glutaraldehyde),SE(separated EDC),and SG(separated genipin).Results: Fourier transform infrared spectroscopy(FTIR)and X-ray diffraction(XRD)showed that crosslinking did not affect the main functional groups of fibers in all groups.ME,MG,SE,and SG met the requisite mechanical properties,and they also resisted collagenase degradation.In hemocompatibility assays,only ME and MG demonstrated ideal safety.Furthermore,ME and MG presented the greatest cytocompatibility.For vascular scaffolds,rapid endothelialization helps to prevent thrombosis.According to HUVEC migration on different nanofibers,ME and MG also are successful in promoting cell migration.Conclusion: ME and MG may be promising candidates for vascular tissue engineering.Significance: The study suggests that collagen crosslinked by EDC/NHS or genipin facilitates endothelial cells regeneration,which could be of great benefit in tissue engineering of vascular scaffolds.Part Ⅱ Study on the electrospun polycaprolactone/ollagen vascular scaffoldPurpose: Large diameter vascular scaffolds(diameter ≥ 6mm)are widely used in congenital heart surgery.However,small diameter vascular scaffolds(diameter<6mm)often have thrombosis,poor patency rate at long term,and lake tissue regeneration potential.The present study aim to construct a bilayer porous vascular scaffold based on PCL and Collagen with eligible mechanical property,and to evaluate its regeneration potential and biocompatibility after in situ implantation.Methods: The vascular scaffolds were electrospun with 10% PCL/Collagen solution,and crosslinked with EDC/NHS and Genipin respectively.The diameter of scaffolds was set as 1.6 mm.The large pore membranes were electrospun with 30% PCL solution.Then the PCL membranes were winded around above crosslinked PCL/Collagen vascular scaffolds,obtaining bilayer vascular scaffolds.They were named as EDC-PCL and Genipin-PCL.The scaffolds without enclosing the PCL membrane were named as EDC-0 and Genipin-0 as the control group.All scaffolds were subcutaneously implanted in SD rats for 2 weeks.Afterwards,explants were autotransplanted as infrarenal aortic interposition grafts.After 4 weeks,ultrasound was used to detect the blood flow in the scaffolds.Then rats were euthanized to evaluate neotissue formation.Results: According to grass view and SEM images,the inner layer of PCL/Collagen fibers were tight,and outer layer of PCL membrane was fluffy.The diameter of PCL membrane was 13.46±4.47μm and pore size was 98.64±18.92μm.After 2 weeks,there were a large number of cells infiltration in the outer layer of EDC-PCL and Genipin-PCL,and the outer layer wall of scaffolds were adhesion tightly.However,cells infiltrated in EDC-0 and Genipin-0 were rare.Four weeks after in suit implantation,EDC-PCL and Genipin-PCL showed favorable patency with ultrasonic spectrum,and the spectrum amplitudes were similar to that in autologous arteries.Nevertheless,no blood flow signal was detected in EDC-0 and Genipin-0.The lumen was covered by a layer of endothelial in Genipin-PCL and Genipi-0,while less was found in those scaffolds treated by EDC/NHS.Porous PCL membrane facilitates tissue regeneration,and α-SMA(+)myofibroblasts were detected in the outer layer of EDC-PCL and Genipin-PCL as well as Desmin(+)contractile smooth muscle cells.However,the extracellular matrix(ECM)deposition of EDC-0 and Genipin-0 was very low,and tissue regeneration was not evident.Conclusion: Crosslinked PCL/Collagen fibers guarantee the mechanical properties of vascular scaffolds,especially Genpin crosslinking scaffolds could promote rapid growth and migration of endothelial cells.For outer layer,large pore PCL membrane increased cells infiltration and ECM deposition efficiency,which was conducive to tissue regeneration.As a conclude,the bilayer vascular scaffolds showed good patency in animals. |
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