Structure Control And Bio-functionalization Of Scaffolds Made Of Electrospun Nanofibers For Applications In Soft Tissue Engineering

Electrospinning has been widely used to fabricate nano-/micro-fibers from various kinds of synthetic polymers,natural polymers,or their composites.Tissue-engineered scaffolds made by electrospinning can recapitulate the component,length scale,and architecture typically of native extracellular matrix(ECM).Furthermore,the mechanical and biodegrdable properties of the scaffolds can be tuned to the expected performance for use in regenerating different types of tissues by selecting the apporatiate raw materias and/or adjusting the weight ratio of the materials in a composite.Most importantly,scaffolds made of nanofibers with a diameter in the range of 50-500 nm can be easily obtained by tuning the parameters during electrospinning.As such,the scaffolds made of electrospun nanofibers play critical roles in tissue engineering.In addition to the solid nanofibers made by traditional electrospinning,the nanofibers with specific topography have also been developed by several modified electrospinning methods.For example,nanofiber yarns can be fabricated by dynamic liquid electrospinning,conjugated electrospinning,or commercial electrospinning equipment.The nanofiber yarns possess higher porosity and greater pore size when compared with dense nanofibers,which can manipulate the morphology,phenotype,migration,and infiltration of cells.However,the bare scaffolds without any biological guindance are insufficient in promoting cell growth and tissue regeneration in most of the cases,where bio-functionalization is needed to provide a biomimetic scaffold.The stratergies for the bio-functionalization on tissueengineered scaffolds mainly involve: i)generating biological cues on the nanofiber surface by chemical modification or physical adsorption;and ii)incorporating biological cues into the core of nanofibers by coaxial electrospinning.The biological cues can be either in a homogenous or a gradient fashion.Based on the stratergies of bio-functionalization on tissue-engineered scaffolds and the potential application for renegerating different kinds of tissues,the major contents in this thesis are presented as follows:(1)Poly(lactide-co-glycolide)(PLGA)nanofibers and nanofiber yarns were fabricated by traditional electrospinning and electrospinning equipment for fiber yarns,respectively.Then,laminin was uniformly coated on the surfaces of PLGA nanofiber and nanofiber yarns through covalent binding for bio-functionalization.The results from scanning electron microscope(SEM),X-ray photoelectron spectrometry(XPS),and MicroBCA protein assay confirmed the existence of laminin on the surfaces on PLGA nanofibers and nanofiber yarns.Mechanical tests showed the enhanced mechanical strength and Young's modulus after laminin coating on the nanofibers and nanofiber yarns.Water contact measurements also indicated the improvement of hydrophilicity after laminin coating.When culturing Schwann cells(SCs)on PLGA nanofibers and nanofiber yarns,the proliferation of SCs on the nanofiber yarns was significantly better than that on dense nanofibers,owing to the three-dimensional and porous structure of nanofiber yarns.After laminin coating,the proliferation of SCs on the nanofiber yarns was further promoted,showing the promising potential of laminin coating along with the nanofiber yarns for peripheral nerve repair.Based on the above results,a laminin-coated and yarn-encapsulated PLGA nerve guidance conduit(LC-YE-PLGA NGC)was designed and fabricated to biomimetic the fascicle structures in a native nerve.The measurement of the compressive behavior showed that the LC-YE-PLGA NGC presented good resilience properties upon compression both in the radial and axial directions.After seeding SCs in the LC-YE-PLGA NGC,the images of H&E and immunofluorescent staining showed that the SCs grew around every single yarn and filled the spaces between the adjacent yarns in the lumen of the NGC.Furthermore,both SCs production and migration along the interior nanofiber yarns were observed from the LC-YE-PLGA NGC,attributing to the guidance of arrayed nanofiber yarn and laminin coating.Therefore,the bio-functionalization provided by laminin coating along with the yarn-encapsulated NGCs provided a promising alternative for regulating SCs proliferation and migration in nerve tissue engineering.(2)By changing the collector to a metallic ring with a sharp needle in the center during electrospinning,radially aligned nanofibers of polycaprolactone(PCL)were fabricated.Then,we report a general method for generating circular gradients of active proteins on scaffolds composed of radially aligned nanofibers for bio-functionalization.In a typical process,the scaffold,with its central portion raised using a copper wire to take a cone shape,was placed in a container(upright or up-side-down),followed by dropwise addition of bovine serum albumin(BSA)solution into the container.As such,a circular gradient of BSA was generated along each nanofiber.The bare regions uncovered by BSA were then filled with an active protein of interest.To visualize the gradients,we used FITC-BSA as a model protein and performed the above procedures to obtain nanofiber scaffolds covered with a graded or uniform coating of FITC-BSA.Along the radial direction,a continuous change in the fluorescence intensity was clearly observed for the nanofiber scaffold coated with FITC-BSA in a gradient either from the periphery to the center or from the center to the periphery.In demonstrating their potential applications,we used different model systems to examine the effects of two types of protein gradients.While the gradient of laminin and epidermal growth factor accelerated the migration of fibroblasts and keratinocytes,respectively,from the periphery toward the center of the scaffold,the gradient of nerve growth factor promoted the radial extension of neurites from the embryonic chick dorsal root ganglion.This method for generating circular gradients of active proteins can be readily extended to different types of scaffolds to suit wound closure and related applications that involve cell migration and/or neurite extension in a radial fashion.(3)Coaxial electrospinning was used to fabricate poly(L-lactide-co-caprolactone)/collagen(PLCL/COL)nanofibers incorporated with heparin and anti-CD133 antibody(PLCL/COLHEP/CD133)for the bio-functionalization.Measurements of platelet adhesion and hemolysis rates indicated the good blood-compatibility of the bio-functional nanofibers.Furthermore,when culturing with endothelial progenitor cells(EPCs),the PLCL/COL-HEP/CD133 nanofibers showed improvement in EPCs recruitment and proliferation.Fluorescent micrographs showed superior intercellular interaction and a continuous cell monolayer on the PLCL/COL-HEP/CD133 nanofibers,in comparison to the PLCL/COL-HEP and PLCL/COL nanofibers.The results indicate that the PLCL/COL-HEP/CD133 nanofibers are advantageous in promoting EPCs recruitment and maturation as the lumen surface of a vascular scaffold.In addition,PLCL/COL nanofiber yarns were fabricated by dynamic liquid electrospinning.SEM images showed the porous and puff structures of the nanofiber yarns.When culturing with smooth muscle cells(SMCs),the PLCL/COL nanofiber yarns showed significantly better SMCs proliferation relative to the case of dense PLCL/COL nanofibers.SEM images displayed that the SMCs spread along the direction of PLCL/COL nanofiber yarns.After SMCs were cultured for 4 days,they infiltrated into the PLCL/COL nanofiber yarns due to the larger porosity and pore size of the nanofiber yarns.Based on these results,we further designed and fabricated a bilayer vascular scaffold with PLCL/COL-HEP/CD133 nanofibers as the inner layer and the PLCL/COL nanofiber yarns as the outer layer.The bilayer scaffolds demonstrated a compliance comparable to the human saphenous vein and improved over commercially available e-PTFE grafts.The incorporated components(HEP/ CD133)were released over a period of nearly 40 days,during which the nanofibers and nanofiber yarns maintained their structures.Moreover,the released heparin contributed to lumen anticoagulation functionality initially,and the incorporated anti-CD133 antibody promoted the development of a neo-intima.In addition,SMCs proliferated and penetrated throughout the entire nanofiber yarn outer structure.In vivo evaluations demonstrated that a monolayer of endothelial cells(CD31 positive),as well as the infiltrated smooth muscle tissues(?-SMA positive),were regenerated on the inner and outer layers of the vascular scaffold,respectively,demonstrating the capacity in mimicking the structure of a native blood vessel.In conclusion,the functionalized bilayer scaffold can be viewed as a promising candidate for vascular tissue regeneration.(4)Coaxial electrospinning was used to fabricate PLCL/COL nanofibers incorporated with heparin and vascular endothelial growth factor(PLCL/COL-HEP/VEGF)for the biofunctionalization.In the previous chapter,we have demonstrated that the heparin-incorporated nanofibers are advantageous in blood compatibility.Furthermore,when culturing EPCs on the PLCL/COL-HEP/VEGF nanofibers,both EPCs recruitment and proliferation were improved.Fluorescent micrographs showed better interaction between cells relative to the cases of PLCL/COL-HEP and PLCL/COL nanofibers.A continuous cell monolayer on the PLCL/COLHEP/VEGF was also observed.When culturing human umbilical vein endothelial cells(HUVECs)in the release medium of the PLCL/COL-HEP/VEGF nanofibers,both the adhesion,proliferation,and cell-cell interaction of HUVECs were promoted owing to the release of VEGF.Atrributed to the good performance in EPCs recruitment and ECs proliferation,the PLCL/COL-HEP/VEGF nanofibers are advantageous in anticoagulation and accelerating endothelialization as the lumen surface of a vascular scaffold.In addition,aligned and porous PLCL/COL nanofiber yarns incorporated with threedimensional gradient of platelet derived growth factor(PDGF)were fabricated by combining the conjugated electrospinning and coaxial electrospinnig methods.Form the in vitro release curves,the release of PDGF from the nanofiber yarns was slower relative to the case of nanofibers.When culturing SMCs on the nanofiber yarns incorporated with graded PDGF,the proliferation,alignment,and infiltration of SMCs were all promoted,owing the the contact guidance provided by the aligned,porous nanofiber yarns and the chemotaxis offered by the PDGF gradient.Based on these results,we further designed and fabricated a bilayer vascular scaffold with PLCL/COL-HEP/VEGF nanofibers as the inner layer and the PLCL/COL nanofiber yarns incorporated with graded PDGF as the outer layer.The mechanical property of the bilayer vascular scaffold was comparable with that of a native porcine coronary artery,and its compliance was comparable to a human saphenous vein.The release curves of heparin,VEGF,and PDGF showed a time-sequence behavior,approaching the ideal profiles for vascular reconstruction.The incorporated heparin,VEGF,and PDGF were released over a period of nearly 40 days.The released heparin contributed to maintain the patency and inhibit thrombus in lumen initially,and the incorporated VEGF accelarated endothelialization on lumen surface.In addition,SMCs showed high alignment,enhanced proliferation and infiltration on the nanofiber yarns incorporated with PDGF gradient.After implanation into a rat abdominal aorta over a period of two month,a continuous monolayer of endothelial cells and aligned,infiltrated smooth muscle tissues were regenerated on the inner and outer layers,respectively.Furthermore,the production of muscle fibers and collagen were both observed by the H&E and Masson trichromic staining images.The overall results demonstrated the promising potential of this dual-functional vascular scaffolds in mimicking the components and architecture of a native blood vessel,which is worthy being viewed as a promising candidate for in situ vascular tissue regeneration in large animals over a long-term period.In conclusion,in this study,we developed four kinds of bio-functionalized,tissue-engineered scaffolds based on electrospun nanofibers and/or nanofiber yarns.Owing to the different architecture and bio-functions,the scaffolds were explored for applications involving peripheral nerve repair,wound closure,and vascular tissue regeneration,respectively.Taken together,these studies provide valuable information for designing bio-functionalized scaffolds made of electrospun nanofibers and/or nanofiber yarns for potential use in the regeneration of both two-dimensional and three-dimensional tissues.