| The topological morphology of the substrate surface can significantly affect cell behavior.Understanding the different responses of cells to the topological structure of the substrate,is of great significance to tissue engineering and regenerative medicine.With the rapid development of micro-processing technology,it has been able to fabricate topological structures in micron level or even nano level.Which studied more are micro-groove,micro-column,micro-well,etc.Since the micro structures of the horizontal cylindrical surfaces are more difficult to fabricate than other structures,the existing studies on the effects of cylindrical micro-topographic surfaces on cell behavior are rather limited.In practice,however,there are a large number of circular concave and convex structures present in vivo,typically like various sizes of blood vessels.Systematic study of this structure can provide new directions for the study of angiogenesis in vitro and the design of vascular stents.In this study,cylindrical PDMS concave and convex substrates were prepared by negative pressure forming method.Four types of cells,human umbilical vein endothelial cells(HUVEC),A7r5 smooth muscle cells,HFF-1 fibroblasts and MDA-MB-231 breast cancer cells,were then used to systematically evaluate these substrates.On the other hand,the MEMS-based microfluidic chip developed in recent years has gradually become a conventional research platform for in vitro cell three-dimensional culture.And because it can provide extracellular matrix,controllable biochemical factor concentration distribution and mechanical loading,etc.,it is significantly better in the area of in vitro tissue simulation and micro-environment construction than the traditional method.In particular,it shows a unique advantage in the construction of in vitro angiogenesis models and the study of angiogenesis microenvironment.In this study,a novel coverslip molding method was used to rapidly fabricate the PDMS microfluidic device.Due to the characteristics of HUVEC in the concave substrate,the cylindrical microhole array with concave surface was combined with the microfluidic device to construct the in vitro angiogenesis model,with a view to providing a good platform for angiogenesis-related research and drug screening.The main contents of this dissertation are as follow:(1)Four kinds of cylindrical concave and convex substrates with different widths were prepared and these substrates were evaluated by MDA-MB-231 cells.We fabricated four different semi-cylindrical concave and convex PDMS substrates with a width of 25,50,100 and 200?m using a negative pressure molding method.Due to the innovative use of double-layer PDMS membranes to assist the preparation of the substrate,so that the substrate surface could be more smooth.Using phase-contrast microscopy and scanning electron microscopy,we found that the surfaces of the concave and convex substrate were regular,clear,no edges and corners,their characteristics were basically the same with the planar PDMS substrate.MDA-MB-231 cells were seeded on concave and convex substrates of different widths.It was found that in a larger width structure,because of its small curvature radius,cells would be more susceptible to structural effects and grow along the axial direction.To obtain a larger observation area,we selected the substrates with a width of 200?m for subsequent experiments.(2)Observations on polarization growth behavior of four different cells on concave and convex substrates.HUVEC,A7r5,HFF-1 and MDA-MB-231 cells were seeded on the substrates,and the polarization behaviors of the cells in different regions were quantitatively analyzed from the ratio of polarized cells and the direction of polarization.The results showed that the polarization ratios of all four types of cells cultured on the concave and convex substrates were increased.HUVEC,A7r5 and HFF-1 cells grew in the circumferential direction of the concave substrate,whereas cells tended to grow in the axial direction of the convex substrate.Scanning electron microscopy and immunofluorescence results of the cells on the substrates were consistent with those observed by optical microscopy.F-actin immunefluorescence observation of planar cells showed that F-actin was bundled in HUVEC,A7r5 and HFF-1 cells and circular in MDA-MB-231 cells.This may be one of the reasons why MDA-MB-231 cells grew more randomly.In addition,the study also found that the expression of p-cofilin and integrin?1 in HUVECs induced by tumor supernatant increased,correspondingly,their polarization abilities on the concave and convex substrate were enhanced.(3)Observation of migration behavior of HUVECs in concave and convex substrates.We observed the migration behavior of HUVECs by using time-lapse photography.The cells migrated faster on the concave substrate,and along the same direction as the polarization,migrated slower in the planar substrate,the slowest in the convex substrate.Further analysis showed that HUVECs gradually migrated from the planar region to the concave region and did not like to grow on the convex region.Therefore,we consider HUVEC has concave-taxis and convex-avoidance.(4)The finite element simulation of adhesion behavior of cells on the concave and convex surfaces.In order to simulate and analyze the mechanical behaviors of cells and cell-substrate adhesion,the standard linear solid model and the bilinear cohesive zone model were used to model the cells adhering to the concave and convex substrates.After prestressing the cells,the differences in stress distribution of the cell adhesion on different substrates were observed.The results showed that the simulation results of HUVEC,A7r5and MDA-MB-231 cells were basically consistent with the experimental observations.The outer periphery of the cell adhesion layer in the circumferential direction was subject to greater traction from the concave substrate.Through the simulation of the cell adhesion behavior on the concave-planar and convex-planar substrates,the concave-taxis and convex-avoidance of HUVEC were well explained.Further study also found that the effect of the cell instantaneous elastic modulus(E_C),the adhesion interface elastic modulus(E_A)and the substrate radius on cell adhesion.(5)Fabrication of the microfluidic device combining cylindrical microhole arrays.In this study,a five-chamber PDMS microfluidic device was prepared conveniently and quickly by a coverslip molding method.Since HUVECs could grow circularly in the circumferential direction of the cylinder concave surface,PDMS barrier structure with cylindrical microhole array was added to the vertical surface between the adjacent chambers of the device for constructing an in vitro angiogenesis model.After perfusion of collagen in the middle of the device,we used FITC-dextran to simulate the diffusion behavior of soluble factors in the device.The results showed that FITC-dextran could form a good linear gradient between peripheral chamber and middle chamber.The diffusion behavior of VEGF in the device was further simulated by finite element method,and the results were basically consistent with the experimental results of FITC-dextran.(6)Construction of an in vitro angiogenesis model based on microfluidic device.HUVECs were seeded into microfluidic device and then VEGF was added into the middle chamber to induce the cells to migrate and sprout into the collagen.After observation using confocal laser scanning microscopy,it was found that HUVECs grew circularly along the inner wall of the microhole and formed annular cell clusters on the collagen surface,then invaded into the 3D collagen gel and migrated to the region of high VEGF concentration and formed sprouts and tube-like structures.The sprouts on the side of the device with the microhole barrier were significantly longer than the control group(no microhole barrier),and the number of the pores formed between the sprouts were also significantly larger than that of the control group.These results suggesting that HUVECs in the device with barrier could form longer sprouts and more well-interconnected tube-like structures,thus were more consistent with the distribution of microvasculature in vivo,and would provide a good model for angiogenesis related research.In summary,we prepared a smooth,cylindrical concave and convex substrates with the aid of double-layer PDMS membranes.We found that the cells in the concave surface easily grew in the circumferential direction,while in the convex surface easily grew in the axial direction,and also found that HUVEC has concave-taxis and convex-avoidance.These findings would help us to understand and regulate the growth behavior of cells on topological structure.In this study,a microfluidic device based on cylindrical microhole array was constructed to make the HUVECs grow annularly and sprout in the microholes and form well-interconnected tube-like structures,which can be used for angiogenesis related research or drug screening. |
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