| Most of the human tissue cells live in extracellular matrix(ECM)with a fibrous network structure.Natural ECM is a complex of proteins and polysaccharides,which not only provides structural support for tissues and organs,but also regulates many physiological behaviors of cells including adhesion,proliferation,migration,differentiation,and so on.In the process of tissue development and regeneration,the interactions between cells and ECM are highly dynamic and mutually regulated.In response to external stimuli,cells degrade the matrix by secreting degradation enzymes(e.g.,matrix metalloproteinases MMPs,collagenase,serine protease,etc.),adjust the matrix structure through the contractile force of the cells,and constantly synthesize new extracellular matrix components,all of these constitute the cells'remodeling behavior to the ECM.The purpose of tissue engineering scaffolds is to simulate the ECM characteristics of a target tissue so as to promote the tissue regeneration and repair process.The biomimetic fibrous scaffolds prepared by electrospinning are of advantage in structurally mimicking the ECM microenvironment,but many previous studies focused mainly on the influences of those densely packed fibrous scaffolds,which are difficult to disclose the ability of cells in degrading matrix and reshaping their residing microenvironment.To explore how the variation in electrospun fiber density affects cells'capacity in degrading and reshaping the matrix environment,in this study four groups of electrospun nonwoven gelatin/polycaprolactone(GT/PCL)fiber substrates with different densities were collected on silanized glass slides by varying the collection time during the process of electrospinning.Thereafter,a variety of experimental methods including CCK-8,cytoskeleton staining,SEM,q RT-PCR,ELISA,immunofluorescence staining and so on were employed to study the effects of fiber density on the proliferation,cytomorphology,degradation and production of new extracellular matrix in the mouse embryonic fibroblast stem cells(C3H/10T1/2),followed by examining the underlying mechanism associated with the Rho/ROCK signaling pathway by using the Y-27632 inhibitor.On the basis of the above endeavor,the effects of fiber density on the proliferation,morphology,degradability and polarization phenotype of mouse macrophages(RAW 264.7)were determined as well.The obtained results are summarized as follows.In the first part of this thesis research,the water contact angle test confirmed that the surface of the glass slides was silanized successfully.The results of CCK-8,cytoskeleton staining and live cell staining showed that compared with non-silanized slides,the viability of cells on silanized slides was weaker with desired poor spreading and adhesion ability.The fiber densities of the four groups collected onto the silanized slides were 39.1±12.1?g/cm~2(10 s,G1),103.0±13.2?g/cm~2(50 s,G2),208.0±12.6?g/cm~2(100 s,G3),and 471.8±33.6?g/cm~2(10 min,G4),respectively.The results of CCK-8,cytoskeleton and immunofluorescence staining showed that there were significant differences in the proliferation,cytomorphology and contractility of C3H/10T1/2 cultured on the four groups of fibrous substrates.Cells on low-density fiber substrates showed the"rejuvenated"state of growth activity.Moreover,the low-density fiber substrates also supported enhanced capability of cells in secreting MMPs and new extracellular matrix,which was also confirmed by examining the degradation degree of the fibrous scaffolds/substrates.However,the so-called"low-density"is not the absolute"lowest"density,but an optimal range of low-density.It was found that the cells showed the best responses on the fiber substrates with a fiber density of 103.0±13.2?g/cm~2(G2).Rho/ROCK signaling pathway,as one of the well-identified cellular mechanotransduction mechanism,was verified by using the Y-27632 inhibitor to treat cells cultured on the fibrous substrates of low-density G2 and high-density G4.Prior to being treated,the cells were observed to grow in a manner conforming with the fiber matrix,showing an orderly state with clear profile and discernible stress fibers within the cell-body.After being treated with the Y-27632 inhibitor,the cell profile became blurred and the formation of stress fibers was not obvious,generally showing a"lazy"state;in addition,the expression of MMPs decreased significantly,but not much on the matrix secretion.It was thereby inferred that different fiber densities with varied physical and mechanical properties could have affected the expression of Myosin IIA and contractile force of cells through the Rho/ROCK signaling pathway,eventually regulated the expression of MMPs.In the second part of this thesis research,unlike the C3H/10T1/2 cells,morphologically the RAW 264.7 macrophages tended to grow in a"clump"state and were observed to vary with the fiber density.The cells on G1 substrates were more three-dimensional and did not spread completely.Large-scale clustering was noted for the cells on G2 substrates and the clump state of cells on G3substrates was on a smaller scale.Whereas cells on G4 substrates displayed a scattered agglomeration morphology.In terms of the secretion of degrading enzymes,except for the specific expression of MMP-8 and MMP-10 in macrophages,the MMPs expression of RAW 264.7 in response to the fiber-density variation was not as obvious as C3H/10T1/2.Apart from degrading enzymes,macrophages also secrete reactive oxygen species(ROS)for degrading the matrix,which was found to be fiber-density dependent as well.Amongst,the G2 fiber-density outperformed the other counterparts in mediating the expression of ROS,which was also verified by examining the degradation degree of fiber matrix.Furthermore,the effect of fiber density on the macrophage polarity was determined by performing q RT-PCR,immunofluorescence staining and ELISA assays.It was found that the change of fiber density affected the responsive behavior of the macrophages,and the expression of inflammatory/anti-inflammatory cytokines was different.An appropriate density of fiber scaffolds induced macrophages to express more inflammatory cytokines.This promoted the secretion of ROS and some MMPs to participate in the degradation process,and induced macrophages to express more anti-inflammatory cytokines to maintain the balance of the system.However,the fiber density had no significant effect on the phenotypic polarization of M1/M2,and the difference in the degradation kinetics of scaffolds had nothing to do with the typical M1/M2 phenotype of macrophages.In summary,for the first time this study explored the effects of varying the electrospun GT/PCL fiber density on the response behavior of C3H/10T1/2 and RAW 264.7 cells,in which particular attention was on the cellular capability in degrading fiber matrix,producing ECM components and polarization characteristics of the macrophages.The overall results show that compared with high-density fiber substrates,the identified fiber matrix at a suitable low-density of 103.0±13.2?g/cm~2gave rise to the best outcomes in inducing cells to remodel the fiber matrix effectively.This study paves the way to pinpoint the unique role of the electrospun biomimetic fibers in promoting the matrix-remodeling effect of cells and encourages to use the electrospun biomimetic fibers in a rational manner. |
PDF Full Text Request