| Currently,cardiovascular diseases are threatening human health and life.Blockage of blood vessels is a typical symptom of cardiovascular disease.Once a blood vessel encounters blockage,there is an urgent need to re-establish blood flow to prevent ischemia in downstream tissues.Tissue-engineered vascular grafts are a potential solution for reconstructing blood flow.Given the multilayered characteristic structure of natural vessels,grafts should allow for the formation of a monolayer of luminal endothelial cells(EC),thus mimicking the intima.The accumulation and orientation of vascular smooth muscle cells(SMC)should be considered when attempting to mimic the intima.However,current small-diameter vascular grafts still remain challenges such as insufficient endothelialization,poor smooth muscle cells colonization,and failure to mimic vascular structures well.Our group has successfully prepared small-diameter tissue-engineered vascular grafts with bilayer dual-orientation.However,the scaffold cannot mimic the trilayered characteristic structure of natural blood vessels well and promote the migration of SMC to the scaffold poorly.Therefore,this study will optimize and improve this in two aspects: one is to prepare a trilayered dual-orientation tubular scaffold by multi-layer electrospinning combined with rotating,coiling and folding methods to mimic the multilayer characteristic structure of natural blood vessels;the other is to prepare a large pore size bilayered dual-orientation tubular scaffold with hierarchical pore size by increasing the fiber diameter to better promote the migration of SMC to the inside of the scaffold.The specific studies are as follows.A trilayered tubular vascular scaffold with axial inner layer,circumferential middle layer and random outer layer was prepared by co-blending polycaprolactone(PCL),polylactic acid glycolic acid copolymer(PLGA)and gelatin(Gel)in the ratio of 1:1:1 through electrospinning combined with rotating,coiling and folding.The fiber orientation,fiber diameters and pore sizes of the three fibrous membranes,as well as the growth states and contact-guiding effects of SMC,EC and fibroblasts(NIH3T3)on the different layers were investigated respectively.The results showed that the inner and middle layer fibers were highly aligned,the outer layer fibers were random.The inner and middle aligned fibers could guide the aligned growth of EC and SMC,while the outer layer random fibers were in the non-oriented growth state.Although the trilayered scaffold well mimics the characteristic structure of natural vascular multilayers,its dense fiber is not conducive to cell infiltration,which will cause smooth muscle cells to not migrate into the scaffold faster and better.Therefore,it is necessary to further improve the pore size of the middle layer fibers to facilitate faster and better migration of SMC into the scaffold for functional reconstruction.Based on this,we further prepared bilayered scaffolds with hierarchical pore size constructed from nano and microfibers by electrospinning PCL-PEG-PCL(PCE)and a blend of PCE and gelatin(PCEG)sequentially.The structural features of nano and microfibers were tuned by the concentration of PCE and the proportion of PCE/Gel in electrospun solution respectively.The results demonstrated the best nanofiber morphology and relatively high mechanical properties were achieved in 18%(w/v)PCE(PCE18)and PCE/Gel(w/w)= 7:3(P7G3)electrospun membranes.The in vitro co-culturing studies of cells and membranes indicated all the PCE membranes supported the proliferation and spreading of endothelial cells and the further endothelialization of the membranous surface,while PCEG membranes facilitated the migration inward of SMC.Taking the porosity and mechanical properties into consideration,PCE18 and P7G3 were chosen to construct the inner and outer layers of the bilayered scaffold with hierarchical pore size respectively.After cells were co-cultured with this bilayered scaffold for 7 days,the results manifested a continuous endothelial monolayer has formed on the luminal surface and the SMC have started to colonized from outer layers,indicating the vast potential of this bilayered scaffold in vascular remodeling and regeneration.We further introduced the orientation structure and prepared a bilayered dual-orientation scaffold.SEM results showed that a bilayered scaffold with vertical orientation of the inner and outer layers was successfully prepared.The scaffold had higher burst pressure and suture retention than coronary arteries and saphenous veins to meet in vivo implantation requirements.In vitro cell experiments showed that both the inner and outer aligned fibers could guide the aligned cell growth,and the cell on the membrane proliferation was higher than TCP,showed good biocompatibility of the fibers.In co-culture of cells and scaffolds,the inner layer can support the formation of endothelial monolayer while the outer layer supports SMC infiltration and directs their circumferential arrangement.After 1 and 2 months of dynamic in vitro culture,HE staining revealed a large number of collagen fibers in the scaffold,indicating the formation of a natural ECM-like structure.In rabbit carotid artery in situ replacement,a large number of cells grown inside the scaffold after one month implantation.All these results indicate that b PCEG scaffolds have promising applications in the repair and regeneration of small-diameter vascular tissue engineering. |
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