| A healthy dynamic remodeling of the extracellular matrix（ECM）by cells is essential in regulating cellular behavior,tissue development/regeneration and homeostasis.It is therefore important in engineering tissues to develop biomimetic model systems and investigate the cell-matrix（scaffold）interactions,especially the matrix-remodeling process by cells,towards effectively constructing tissue substitutes.Nano-and micro-scale fibers from electrospinning are known to possess the fineness of fibers found in native ECM.In view of the mechanically‘adequate’softness endowed by the ultrafine fineness,electrospun fibers not only allow for engineering ECM-like scaffolds,but also offer possibility to explore the matrix-remodeling behavior of cells in vitro.However,it is difficult for the cells to actively response（e.g.,remodeling its locally surrounding fibers by exerting actions of stretching and compaction）in contacting with those tightly packed fibrous mats produced from conventional electrospinning.While the topological and bulk mechanical effects of the electrospun fibrous mats on cell behavior have been well-studied previously,the matrix-remodeling capacity of cells from the perspective of mechanical compliance in electrospun fibers remains to be understood.To this end,this dissertation proposes a strategy of using minute-quantity and low-density electrospun fiber network to unravel the underlying mechanobiological mechanisms of the biomimetic fibers in promoting cell-mediated matrix-remodeling,especially concentrating on the role of mechanical compliance played in the electrospun fibers.The details are outlined as follows:（1）From the perspective of reducing fiber-density to weaken fiber entanglements,randomly oriented polycaprolactone（PCL）fiber networks with different densities,namely,37.7±16.3μg cm-2（D1）,103.8±16.3μg cm-2（D2）,198.2±40.0μg cm-2（D3）,and 471.8±32.7μg cm-2（D4）,were firstly prepared by controlling the collection durations（i.e.,10 s,50 s,100 s,and 10 min,respectively）during electrospinning.By examining the responsive behavior of the human induced pluripotent stem cell-derived mesenchymal stem cells（hi PS-MSCs）cultured on these fibrous substrates,we showed that hi PS-MSCs had different response characteristics with varying the fiber-density.The fiber network with a moderate fiber-density（i.e.,D2）was found to be beneficial to cell spreading,actin polymerization and focal adhesion turnover,and facilitate mechanotransduction by showing up-regulated Rho A signaling,increased nuclear localization of the Yes-associated protein（YAP）and subsequent activation of the YAP responsive gene transcription,ultimately leading to enhanced cell proliferation,migration and collagen synthesis.However,a higer fiber-density（e.g.,D3,D4）gave rise to attentuated inducing effect on the cellular responses.These results suggest that the cellular responsive behavior is fiber-density dependent,and there was an appropriate fiber-density range（e.g.,D2）that can favorably activate the cell machine to function well and promote the cell-fiber interactions.（2）Based on the derived conclusion in（1）,electrospun fibrous network of PCL with the previously optimized low fiber-density was transferred onto custom-made polydimethylsiloxane（PDMS）rings for performing suspended cell culture with the C3H/10T1/2 fibroblasts,and effects of the low-density fiber network on fibroblast activation and cell-mediated matrix-remodeling as well as the underlying mechanobiological mechanisms were investigated.For the purpose of comparison,dense electrospun fibrous mat and flat film by casting were also prepared as controls.Similar to the previous observation,the low-density fiber network promoted cell proliferation and spreading,reaching the state of full spread in about 6 hours,roughly 4 hours faster than that on the dense fibrous mat.Compared with the two controls,cells on the low-density fiber network were more responsively active,as evidenced by up-regulated expressions in phenotypic genes/proteins,enhanced transcription and synthesis of ECM-related proteins,and activated capability in reshaping the fibrous structure,thus showcasing a stronger capacity in matrix-remodeling.A detection of mechanotransduction signaling pathway revealed activation of the Rho A-ROCK pathway and enhanced cytoskeleton assembly,cell contractility,and YAP nuclear localization in cells on the low-density fiber network.While ROCK was inhibited,cytoskeleton assembly was blocked with cells showing dendritic morphology,weakened phenotype activation and YAP nuclear translocation,and decreased synthesis of ECM components.In addition to the Rho-ROCK signaling pathway,the low-density fiber network also activated the cationic signaling pathway mediated by Piezo 1,a mechanosensitive ion channel protein.Expression of the Piezo 1 protein was down-regulated if having ROCK inhibited.These results suggest that the low-density biomimetic fibers induced cellular matrix-remodeling could be regulated by the Rho-ROCK signaling pathway and the Piezo 1 ion pathway,and the two pathways were positively correlated.（3）For the sake of applying the low-density fiber network induced promoting effect in cellular matrix-remodeling to tissue formation,we proposed to develop a tissue-like cell sheet termed“cell-fiber integrated sheet（CFIS）”,within which the ECM-biomimicking fibrous network with the optimized low-density was introduced to have cells homogeneously distributed.Using rat bone marrow mesenchymal stem cells（BMSCs）as a model cell and dense fibous mat as control,it was found that cells on the low-density fibrous network（L-G）exhibited improved capacities in spreading,proliferation,stemness maintenance and matrix-remodeling during the process of CFIS formation.Structurally,the CFIS constructs revealed strong integration between the cells and the fibrous network,thus providing excellent cohesion and physical integrity to enable strengthening the formed cell sheet.By contrast,the cell sheet formed on the dense fiber mat（D-G）showed a two-layer（biphasic）structure due to the limitation on cellular invading.Morevoer,such engineered CFIS was identified with immunomodulatory effect by down-regulating the LPS-stimulated macrophages towards a M1 phenotype and enhanced pre-vascularization by promoting vessel-like tube formation in vitro.These results suggest that CFIS could provide a new form of“cell sheet”for the application of sheet-based tissue engineering method to tissue repair and regeneration.To summarize,based on the strategy of reducing fiber density to weaken fiber entanglments,first of all effect of fiber density on cell response was studied for screening an optimal fiber density that mostly activated the cellular function and promoted cell–fiber interaction.Then,based on the obtained optimal fiber density range,the beneficial effect of biomimetic electrospun fibers in promoting cell-mediated matrix-remodeling was verified,and the underlying mechanotransduction mechanisms were revealed.Finally,the optimized low-density fiber network was introduced into cell sheet structure to form a tissue-like“cell sheet”termed CFIS,in which a positive role of the electrospun biomimetic fibers in promoting cell sheet formation was demonstrated.Results derived from this study will help to understand the important role of electrospun fibers in directing cell-mediated matrix-remodeling and the underlying mechanotransduction mechanisms.This study will pave the way to precisely design biomimetic fibrous scaffolds for achieving enhanced cell-scaffold interactions,and accelerate transforming the pro-remodeling electrospun fibers into clinical settings in the future. |
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