| Background and aims: Stem cells are undifferentiated cells with the ability of self-renewal,which mainly comprises embryonic stem cells,induced pluripotent stem cells and mesenchymal stem cells.Mesenchymal stem cells are extensively used in various human diseases such as anti-aging,organ transplantation,repair from tissue damage because of their advantages of extensive sources,easy acquisition,low immunogenicity and good differential potential of multiple cell lineage.However,their wider clinical application is limited because of a series of problems including the easy loss of stemness genes during the process of expansion in vitro,as well as the defeat to differentiate into cells with specific functions after their transplantion into the body resulting from the changes of the microenvironment of stem cells.The microenvironment in which stem cells live determines the fate of stem cells such as quiescence,self-renewal,or differentiation,includes components such as extracellular matrix,mechanical stress,topology,and soluble cytokines.Although there are currently models of extracellular matrix simulated the in vivo nanotopography(nanopillars,nanoscale grooves,grooved surfaces,graded nanopillars or grooved surfaces,and nanofibers)and models simulated in vivo mechanical stress(1D,2D,and 3D),but the methods to fabricate the traditional models modulating nanotopography are complex,costly,time-consuming,and unfavorable for scale-up production,which limits its application.Electrospun nanofibers can be used as bio-manufacturing materials because their acquisition is simple and convenient,capable of scaling up to industrial level,and cost-effective.At present,the effects of the electrospinning nanofiber models with different structures and different sizes or the model of simulating in vitro 3D mechanical stress on stem cell behavior have not been reported.This study aims to prepare electrospun nanofibers with different structure and size,as well as the cell culture device with a substrate of electrospun nanofibrous membrane undergoing periodic three-dimensional stretching,namely as the joint models combined with the electrospinning nanofiber and 3D mechanical stress,then explore their regulatory roles in stem cell fate,respectively.This project should provide the theoretical and experimental basis for the design and application of a new cell fermentation tank and organ chip with simulation of the stem cell microenvironment.Methods:1.Preparation of electrospun nanofiber membranes with different alignments and pore sizes: 1)First,an electrospinning preparation platform was built,and then the electric field in the electrospinning device was simulated using Sim4 Life V5.0.1 software;2)In order to fabricate electrospun nanofiber membranes with different alignments and different pore sizes,the collector for preparing randomly arranged and vertically crossed electrospun nanofiber membranes was designed,respectively;3)Observation,classification and screening of electrospun nanofiber membranes under ordinary optical microscope,identification by SEM,image analysis by Image J,statistics by Graph Pad Prism.2.The effects of electrospun nanofiber membranes with different alignments and pore sizes on bone marrow mesenchymal stem cells: 1)First,frbricate upper and lower rings of PDMS,and then stick them with the prepared electrospun nanofiber membranes with different alignments and different pore sizes.Then,a sandwichlike cell culture device was obtained,which was hydrophilized by an oxygen plasma-vacuum plasma cleaner,immersed in 75% ethanol and sterilized by UV light for 30 min,and finally coated with 50 μg/m L type I Collagen overnight at 37°C;2)MTT was used to detect cell proliferation;3)q PCR was used to detect the expression of stemness genes,differentiation genes and PIEZO1 of stem cells;4)Oil red O staining was used to detect the adipogenic differentiation of hMSCs;5)The osteogenic differentiation of hMSCs was detected by ALP staining;6)The cell morphology was detected by immunofluorescence.3.The effect of 3D periodic stretching of electrospun nanofiber membrane substrate on bone marrow mesenchymal stem cells: 1)First,3D printing was used to prepare the molds of the PDMS module of the bottom pneumatic chamber layer,the middle soft matrix chamber layer and the top cell culture chamber layer,The PDMS solution was then poured on the corresponding molds,and then cured at 75°C for 2 hours to prepare the corresponding PDMS modules.Then,the PDMS film,soft matrix PDMS,and electrospun nanofiber membrane were combined with the corresponding PDMS modules to prepare the underlying pneumatic chamber layer,the middle soft matrix chamber layer and the top cell culture chamber layer.Lastly,three layers are reversibly combined and assembled into a 3D periodic stretch electrospun nanofiber membrane substrate culture device;2)MTT detects cell proliferation;3)q PCR detects the gene expression of stemness,differentiation and PIEZO1;4)Oil red O staining to detect the adipogenic differentiation of hMSCs;5)ALP staining to detect the osteogenic differentiation of hMSCs;6)Immunofluorescence to detect cell morphology and the distribution of YAP within nucleus and cytoplasm of hMSCs.Results:1.Electrospun nanofiber membrane with random alignment of small pores and medium pores and vertical cross alignment of medium pores and large pores : 1)Sim4Life V5.0.1 software simulated the electric field in the electrospinning device to obtain the top view of the electric field vector on the surface of the collector electrode,which indicates the distribution of electric field in the electrospinning device is related to the shape of the surface electrode of designed collector;2)The designed surface electrode of collector composed of 10-layer stacked aluminum foil tapes preparing randomly arranged electrospun nanofiber membranes and surface electrodes of collector composed of two pairs of parallelarranged copper strips preparing vertically cross-arranged electrospun nanofiber membranes;3)Electrospun nanofiber membranes with different alignments and pore sizes were obtained,and the average pore area of RS was 8.54 ± 1.82 μm2,the average pore area of RM is 23.33 ± 3.28 μm2,that of MM is 20.97 ± 4.56 μm2,and that of ML is 62.59 ± 39.72 μm2.2.Electrospun nanofiber membranes with different alignments and pore sizes regulated the fate of hMSCs: 1)MTT and q PCR assays showed that combined the ranking of the promotion of cell proliferation and the maintenance of related stemness genes of hMSCs cultured on five different substrates respectively,the comprehensive ranking of expansion of hMSCs in vitro(promoting cell proliferation as much as possible on the premise of maintaining stemness genes)is: random alignment with small pores(RS),random alignment with medium pores(RM),PCL film(PCL film),vertically intersecting alignment with medium pores(MM),and vertically intersecting alignment with large pores(ML);2)Oil red O staining showed that the electrospun nanofiber membranes with vertically intersecting medium pores and large pores inhibited the adipogenic differentiation of hMSCs;3)ALP staining showed that electrospun nanofibrous membranes with randomly arranged small pores and medium pores promoted the osteogenic differentiation of hMSCs;4)Immunofluorescence staining showed that based on cell spreading of hMSCs on 5 different substrates from large to small and from easy to difficult,the ranking is: PCL film,random alignment with small pores(RS),random alignment with medium pores(RM),vertical cross alignment with large pores(ML),vertical cross alignment with medium pores(MM);5)q PCR detection showed that the gene expression of PIEZO1 in hMSCs was decreased to varying degrees along with the increase of culture time.The change was reversed with the increasing number of cell proliferation of hMSCs.3.3D periodic stretching of electrospun nanofibrous membrane substrates regulated the fate of hMSCs: 1)MTT and q PCR assays showed that 3D periodic stretching of electrospun nanofibrous membrane substrates could maintain the stemness of hMSCs and promote cell proliferation;2)Oil red O staining and ALP staining showed that 3D cyclically stretched substrates of electrospun nanofibrous membrane promoted adipogenic differentiation of hMSCs and inhibited their osteogenic differentiation;3)Immunofluorescence staining showed that 3D cyclically stretched substrate of electrospun nanofibrous membranes can promote cell spreading from anisotropy in multiple directions to isotropy,and the cell spreading is easier,and so as to cell proliferation;4)Immunofluorescence staining showed that the 3D periodic stretched substrate of electrospun nanofibers membrane induced the translocation of YAP from nucleus to cytoplasm;5)q PCR detection showed that the expression of PIEZO1 gene of hMSCs on the 3D periodically stretched substrate of electrospun nanofiber membrane was increased,and the difference is identically significant after 7 days and 14 days.Conclusions:1.Electrospun nanofibrous membranes with the random alignment of small pores or medium pores as well as the vertical cross alignment of medium pores or large pores were obtained by using a stack of ten-layer of cohesive aluminum foil tape and two symmetrically arranged electrodes of copper strip with different deposition time of electrospinning,respectively.2.The alignment and pore size of electrospun nanofibrous membrane can regulate the fate of hMSCs.Comparison of 5 different substrates cultured with hMSCs,namely,randomly aligned electrospun nanofibrous membranes with small or medium pores(RS/RM),and vertically crossed aligned electrospun nanofibrous membranes with medium or large pores(MM/ML)and PCL film: on the experiments of expansion in vitro after 9 or 14 days,the random alignment with small pores(RS)can better promote the in vitro expansion of hMSCs.On the experiments of differentiation in vitro after 14 days,vertically crossed aligned electrospun nanofibrous membranes with medium or large pores inhibited the adipogenic differentiation of hMSCs.Nevertheless,the randomly aligned electrospun nanofibrous membranes with small or medium pores(RS/RM)promote the cellular osteogenic differentiation of hMSCs.3.The cell culture device with a substrate of electrospun nanofibrous membrane undergone periodic three-dimensional stretching,that is,the joint models combined with the electrospinning nanofibers and 3D mechanical stress,can regulate the fate of hMSCs.Compared with randomly aligned electrospun nanofibrous membranes with medium pores,on the experiments of expansion in vitro,with the periodic three-dimensional stretching for 7 days,the stemness of hMSCs can be further maintained and their cell spreading were induced,followed with the significantly increased number of cell proliferation after 14 days;on the experiments of differentiation in vitro,with the periodic three-dimensional stretching for 14 days,it can promote adipogenic differentiation and inhibit osteogenic differentiation. |
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