Surface And Interface Engineering Of The Biomimetic Aligned Fibrous Substrates To Modulate The Functional Expression Of Vascular Cells

Vascular diseases(e.g.,vascular injury,ischemic disease and aneurysm),frequently occurred in the clinical setting,lead to a huge demand for vascular grafts every year.Tissue-engineered vascular grafts(TEVGs)have been proposed as the most promising ‘ideal' vascular substitutes for the treatment of various vascular diseases.However,the low long-term patency(one of the primary reasons noted)post-implantation seriously affects the clinical application efficacy of the TEVGs,especially for the case of small-diameter TEVGs(<6 mm).This mainly attributes to the insufficient biomimicry of TEVGs to the native vascular extracellular matrix(ECM),as well as the lack of a comprehensive understanding on the mechanisms involved in cell-matrix interactions in vascular regeneration.Electrospun biomimetic fibers have drawn a great deal of attention in the field of vascular tissue engineering.Given the recognition of the anisotropic architecture of native blood vessels,there are increasing efforts in using electrospun aligned fibers for constructing TEVGs.To realize the construction of highly biomimetic TEVGs for long-term patency in vivo and accelerate transformation of clinical applications,an insightful understanding on the role and mechanisms of some representative surface and interface signals(i.e.,topological,biomechanical and biochemical cues)engineered to the electrospun aligned fibrous substrates in regulating the functional expression of vascular cells is critical.To this end,current dissereation research was designed and carried out as follows:(1)From the perspective of engineering the aligned fiber surface and interface with topological signals,first of all four groups of aligned poly(lactide-co-caprolactone)(PLCL)fibers with different fineness(0.49,1.76,3.06 and 4.15 ?m)were fabricated through varying solution concentrations and injection rates in electrospinning,followed by an evaluation on the response behavior of human umbilical vein endothelial cells(huvECs)cultured onto the fibrous PLCL substrates.Results showed that too thin and too thick fibers both weakened the contact guiding effect of the oriented topological structure on cell growth.While thinner fibers reduced the cell-matrix interactions,thicker fibers decreased cell-cell interactions.Only fibers with ca.2 ?m diameter gave rise to abalance between the cell-cell and cell-matrix interactions,thereby promoting the functional expression of the regenerated endothelium.Then,a comparison on the difference of response behavior of huvECs to directional grooves and aligned fibers was performed.Directional grooves(20 ?m in width and spacing)induced the cells to orient mainly through inhibiting the cell growth space,while cell orientation on aligned fibers was achieved by topological guidance of the substrate surface and stress fibers distribution affected by focal adhesions.Rho/ROCK signaling pathway had no significant influence on cell spreading,but regulated cell adhesion rate and cell morphology.Nucleus orientation of cells on directional grooves was mainly regulated by the Rho/ROCK signaling.Differently,this was jointly governed by Rho/ROCK signaling and matrix topology of the aligned fibers.Overall,the topography of aligned fibers is more beneficial to enhance the functional expression of ECs than that of oriented grooves.Finally,fibrous mats with directional grooves were prepared through directly electrospinning randomly-oriented nanofibers onto silicon mold with the designed grooves.As such a multiscale topology structure combines the advantages of the nanofibers and the directional grooves,it allowed to promote functional expression and enhance the endothelium integrity and antithrombotic ability of the regenerated ECs.The study also confirmed that the regulation pathway of cell function by the multiscale topological structure is closely related to the Rho/ROCK signaling pathway.(2)From the perspective of engineering the aligned fiber surface and interface with different stiffness(rigidity),by judiciously choosing the elastic PLCL as the shell and the rigid poly(L-lactic acid)(PLLA)as the core,highly-aligned shell-core structured PLCL/PLLA fibers with varying stiffness(14.68-2141.72 MPa)while keeping the same surface chemistry and topographic features,were prepared by stable jet coaxial electrospinning(SJCES).Subsequently,effects of the aligned fiber stiffness on the response behavior of human umbilical artery smooth muscle cells(huaSMCs)and huvECs were explored.Results confirmed that increasing stiffness of the aligned fibers had no significant effect on cell morphology but modestly enhanced the density of internal F-actin fibers assembled in huaSMCs.This consequently gave rise to improved capacity in cell proliferation and migration,as well as phenotypic alteration to synthetic and pathological(e.g.,macrophage-like phenotype)in huaSMCs,as evidenced by secreting inflammatory factors.Secretion of inflammatory factors would enable to recruit inflammatory cells,thus leading to disrupt endothelial cell-cell junctions and excessive proliferation of SMCs with the risk of causing thrombosis and neointimal hyperplasia.In the case of huvECs,higher fiber stiffness rendered the cytoskeletal organization in huvECs in a state of high-tension,which thereby enhanced the cell contractility and weakened the endothelial cell-cell junctions(promoting the formation of endothelial gap).The loss of cell-cell junctions consequently undermined the intercellular interactions allowing the cells to actively proliferate and migrate,and impaired the structural integrity,antithrombotic function and remodeling ability in the endothelial monolayer,with the risk of developing vascular diseases such as atherosclerosis and local inflammation.(3)From the perspective of engineering the aligned fiber surface and interface with biochemical signals,after verifying the promoting effect of lysine(Lys)on function expression of vascular cells(ECs and SMCs)and dopamine(DA)polymerization,a novel surface modification method to form a Lys-mediated polydopamine(PDA)coating(i.e.,PDA-Lys)atop the aligned PLCL fiber surface was developed for the purpose of promoting endothelialization.A series of surface characterization demonstrated that Lys was covalently cross-linked with PDA through the Schiff-base reaction and the Michael addition reaction,which thereby significantly reduced the formation of non-covalent bonds(e.g.,?-? superposition and hydrogen bond)in the PDA coating,leading to the formation of smooth,stable,super-hydrophilic and pro-protein adsorption coatings.Presence of the PDA-Lys coating was proved to significantly promote the adhesion and spreading of huvECs and enhance both the cell-matrix and cell-cell interactions,in favor of regenerating endothelium and maintaining the structural and functional integrity of the oriented huvEC monolayer.To summarize,this study systematically explored the effects of topological,mechanical(stiffness)and biochemical signals,engineered onto the surface and interface of electrospun aligned fibers,on the functional expression of vascular cells.Topologically,it revealed the regulation mechanism of the aligned fiber fineness on the behavior of huvECs,compared the differences in growth and functional expression of the huvECs induced by directional grooves and aligned fibers,and demonstrated the potential of applying multiscale topological structure for endothelialization.Mechanically,it established an effective approach for generating aligned fibers with the stiffness varied in a large range without involving topological and chemical interferences,and demonstrated that formation of a fully covered endothelial monolayer in ‘correct' cell shape doesn't necessarily mean ‘correct' functionality in ECs and SMCs,and an improper aligned fiber stiffness may give risk to cause functional disorder of the regenerated endothelium and SMCs for vascular complications.Biochemically,it developed a novel surface modification method(PDA-Lys coating)and demonstrated its potential in accelerating healthy endothelial regeneration on the surface of aligned fibers.This also offers a possibility for improving the biocompatibility of other inert synthetic materials.All the results achieved will not only enrich our understanding on the underlying mechanisms of those surface and interface cues of the biomimetic aligned fibers in modulating the functional expression of vascular cells,but also offer technical methodologies for the pathological analysis of cardiovascular diseases,and provide a momentum for the construction of aligned fibrous TEVGs for efficacious clinical applications.