Engineering Highly-aligned Ultrafine Biomimetic Fibers For Potential Uses In Anisotropic Tissue Regeneration

In the past decade,electrospun ultrafine fibers,able to biologically mimic the nano-/micro-scale fineness of the fibrous constituents within the extracellular matrix(ECM),has drawn a great deal of interest in the field of tissue engineering & regenerative medicine(TERM).However,it is still a huge technological challenge in achieving aligned fibers for engineering those structurally anisotropic tissues(e.g.,blood,bone and tendon)due to the inherent chaotic motion of an electrospinning jet.This limits to precisely explore,at the molecular,cellular and tissue levels,the regulation effects of the constituents,structure,and properties of a biomimetic fibrous scaffold on cellular function,working mechanism and tissue regeneration efficacy.We have previously developed a novel spinning approach termed stable jet electrospinning(SJES)to prepare continuous and aligned ultrafine fibers by formulating viscoelasticity of a spinning dope.This not only enables to address some crucial problems(e.g.,cell infiltration)relating to electrospun fibers for TERM applications,but also provides a platform for the biomimetic construction of anisotropic tissues.In this study,highly-aligned ultrafine fibers from SJES are employed to afford effective solutions in addressing the noted issue of cellular infiltration with respect to electrospun scaffolds,modulation of vascular smooth muscle cells(v SMCs)phenotype for blood vessel regeneration,and promoting human-derived mesenchymal stem cells(h MSCs)to differentiate into bone or tendon through structural design,ECM-components modification or varying stiffness with the produced ultrafine fibers.The main contents are detailed as follows:(1)Both SJES and parallel electrodes(as collector)electrospinning(PES)are used to prepare aligned poly-L-lactic acid(PLLA)fiber membranes,from which a comparative study on the performances of thus produced aligned fibers can be carried out.Through a systematic characterization including scanning electron microscopy(SEM),differential scanning calorimetry(DSC),X-ray diffraction(XRD),polarized FTIR spectroscopy(P-FTIR),wide-angle X-ray diffraction(WAXD)and mechanical testing,it is evidenced that the as-electrospun fibers via SJES are not only macroscopically highly-aligned,but also molecularly well-oriented,accordingly leading to enhanced mechanical performance as compared to those by PES.Thus,aligned ultrafine fibers from SJES can meet the requirements for engineering anisotropic tissues in terms of the degree of fiber alignment and mechanical attributes.(2)Conventional electrospinning processes are currently beset by the limitation in producing fibrous scaffolds to allow for cellular ingrowth.Based on the SJES principle,we propose a novel strategy to print 3D fibrous scaffolds with the well-aligned PLLA fibers in a diameter of approximately 2 ?m by combining the SJES method with an X-Y stage technique.Our approach allows linearly deposited electrospun aligned-fibers to assemble into 3D structures with tunable pore sizes and desired patterns.Process variables(e.g.,plotting speed,feeding rate,and collecting distance)are optimized in order to achieve stable jet printing of ultrafine PLLA fibers.Lastly,the printed 3D scaffolds are successfully used for biological assays in terms of v SMCs' adhesion,spread,oriented growth along the fiber direction,and penetration ability.This study demonstrates the feasibility and great potential in engineering aligned fibers and patterned architectures for constructing 3D tissues.(3)Highly aligned HA/PLLA nanofibers in core-shell(Shell: hyaluronic acid,HA;Core: PLLA)structure are prepared by a modified SJES approach towards regulation of v SMCs' contractile phenotype for vascular tissue engineering.In vitro results show that the aligned HA/PLLA nanofibers significantly promote v SMCs to elongate,orientate,and proliferate,and also up-regulate expressions of contractile genes/proteins(e.g.,?-SMA,SM-MHC)as well as synthesis of elastin.Moreover,six weeks of in vivo replacement of rabbit carotid artery shows that vascular conduits(diameter: 3 mm)made of circumferentially aligned HA/PLLA nanofibers(resembling the tunica media structure of a blood vessel)could maintain patency and promote oriented v SMCs regeneration,lumen endothelialization,and capillary formation.This study demonstrates the synergistic effect of nanotopographical and biochemical cues in one biomimetic scaffold design for efficacious vascular regeneration.(4)Annealing treatment is applied to the SJES-produced aligned PLLA fibers for the purpose of altering fiber stiffness,from which the impact of fiber stiffness on h MSCs differentiation can be explored.It is found that higher fiber stiffness is beneficial to cell spreading,proliferation,upregulated m RNA expression of RUNX2 and downregulated m RNA expression of SCX.This suggests that increasing fiber stiffness favorably drives h MSCs to differentiate into the osteogenic lineage.Cells on stiffer substrates show activated AKT(Serine/threonine Kinase,AKT)and YAP(Yes-associated protein)as well as upregulated transcript expression of YAP target genes(ANKRD1,CTGF).Conversely,inhibition of AKT leads to decreased expression of YAP and RUNX2.Furthermore,macrophage migration inhibitory factor(MIF)is increasingly produced by the cells on stiffer substrates,and knocking down MIF by si RNA results in decreased AKT phosphorylation.Collectively,we successfully demonstrate that simply using the annealing approach can manipulate stiffness of an aligned fibrous substrate without altering the material chemistry,and substrate stiffness dictates h MSC differentiation through the MIF-mediated AKT/YAP/RUNX2 pathway.(5)Likewise,SJES is used to fabricate highly-aligned PLLA fibers with surfaces decorated by type 1 collagen(COL1)and chondroitin sulfate(CS),which are two of the crucial ECM components in the tendon tissue.Effect of the biomimetic COL1-CS(shell)/PLLA(core)fibers on h MSCs' tenogenic differentiation is investigated.The results show that the highest rates of cell spread and proliferation are observed on the aligned COL1-CS/PLLA fibers compared to that on the controls COL1/PLLA and plain PLLA fibers.The expression of tendon-associated genes SCX,TN-C,and COL1 as well as protein tenomodulin(TNMD)are significantly increased,indicative of the promoting role of the COL1-CS/PLLA fibers in directing h MSCs' differentiation toward tenogenic lineage.Introduction of mechanical signal gives rise to synergistic effect on tenogenic differentiation of h MSCs.Furthermore,TGF?2,TGF?3,TGFBRII,and Smad3 are increasingly produced by the cells on the COL1-CS/PLLA fiber substrates.This confirms that COL1-CS/PLLA ultrafine fibers dictate the h MSC tenogenic differentiation through the TGF?/Smad3/SCX pathway.Taken together,it is concluded that SJES can easily fabricate continuous,aligned,ultrafine,high-strength,and functionalized fibers to cater for the increasing demands of constructing anisotropic tissues.Direct printing of patterned three-dimensional ultrafine fibrous scaffolds by combining an SJES method and an X-Y stage technique allows for cellular ingrowth and 3D tissue regeneration.Highly-aligned HA/PLLA nanofibers play a promoting role in modulating v SMC regeneration in vitro and in vivo.Fiber stiffness dictates h MSC osteogenic differentiation through the MIF-mediated AKT/YAP/RUNX2 pathway.COL1-CS/PLLA ultrafine fibers dictate h MSC tenogenic differentiation through the TGF?/Smad3/SCX pathway.Thus,this thesis demonstrates that SJES-produced aligned ultrafine fibers can not only regulate cell differentiation for regeneration of anisotropic tissues,but also provide a platform to explore the underlying mechanisms that govern the cell behavior.Highly-aligned biomimetic fibers are of great values in constructing anisotropic tissues and pinpointing the underlying mechanisms of anisotropic tissue regeneration.