| With the tissues and organs damaged or functionally loss due to some injury or disease,human health and life safety will be influenced by some serious problems.The timely repair and functional recovery of diseased tissues have become an important part of the diseases treatment.Up to now,it is probably the best solution to repair the lesion parts by replacing or rebuilding with autologous,allograft or artificial organs.However,there are still many major issues need to be solved,urgently.These problems can be summarized as the following two aspects.Firstly,the number of donated organs are obviously insufficient,which is difficult to meet clinical needs.Secondly,the artificial tissues and organs have poor homology,and it is difficult to have a good biological compatibility.Therefore,maximizing the biocompatibility of artificial biomaterials and shortening the development cycle of biomaterials is an important guarantee for the successful clinical application of artificial tissues and organs.At present,biocompatibility evaluation of biomaterials mainly consists of two parts.In vitro evaluation is based on cytology experiments established in vitro to assess the compatibility of materials with cells or blood.While,in vivo evaluation is to directly implant materials into experimental animals to evaluate the compatibility of materials and tissues.Compared with animal experiments,in vitro evaluation is more convenient and faster,and it can save a lot of time,money and manpower,and it has been widely used in the design and evaluation of biocompatibility of biomedical materials.However,the current in vitro evaluation has certain limitations.First,qualitative analysis needs to be supported by quantitative analysis.Second,there is a fatal flaw in both methods,which is difficult to reveal the intrinsic factors and regulatory mechanisms of cell growth.Therefore,in the in vitro evaluation system,it is of great significance to introduce a suitable index and reasonable method that can both reflect the state of cell growth and reveal the mechanism cell growth.In view of the defects existing in the current in vitro evaluation of biocompatibility,this study focuses on the influence of micro-and nano-structures of fiber aggregates on cell adhesion,focusing on the following four aspects.(1)The intercellular adhesion molecule-1(ICAM-1)was introduced as an marker to reveal the regulatory mechanism between the material structure and cell adhesion.The PCL porous membranes with micropore size distributions of 0-38 ?m,38-75 ?m,and 75-100 ?m were prepared by selecting sieves with different pore sizes to control the size of the pore-foaming agent.The water contact angle was measured to evaluate the hydrophilicity and hydrophobicity of the materials with different pore size.Subsequently,the material was co-cultured with human umbilical vein endothelial cells(HUVECs),and enzyme-linked immunosorbent assay(ELISA)were used to detect whether the pore size distribution could affected the cell membrane intercellular adhesion molecule(Membrane ICAM-1,mICAM-1)and soluble ICAM-1(sICAM-1)expression,and analyze the relationship between mICAM-1 and sICAM-1 expression in cells,exploring the feasibility,reliability,or accuracy of using ICAM-1 as a marker to explore the adhesion of cells on the surface of the material.(2)Through the fabrication of homogeneous microporous plain weave structure,the relationship between different micron-sized pore size and cell growth characteristics was studied the optimal structure was selected.Select 30D polyester monofilaments and multifilament as basic materials,chose 30D polyester monofilament as warp yarm,control warp density as 850/10 cm,use 30D polyester monofilament or multifilament as weft and change weft density(monofilament 600,700 and 800/10 cm,multifilament 500,600 and 700/10 cm),weaving polyester plain woven structures with different pore size in mircon range.Characterizing the porosity,tightness,and hydrophilicity and hydrophobicity of the surface of the material;evaluating the cell growth characteristics by CCK-8,scanning electron microscopy,confocal laser scanning,and ICAM-1 expression,and optimize the most suitable structure for cell adhesion and proliferation.(3)The woven fabric was treated with low-temperature plasma etching to form a topological structure with nano-trenches on the surface.The effect of surface nanostructure changes on the glycomics was investigated.Argon plasma was used to treat 5 minutes at a pressure of 20 Pa and the etching power(150 W,200 W,250 W,and 300 W)was changed.Polyester plain weave fabrics were subjected to low-temperature plasma etching,and four different nano-grooves were constructed on the monofilaments surface.Using CCK-8,scanning electron microscopy and laser confocal technology,the activity and proliferation of cells adhered on the surface of the material were detected.The expression levels of mICAM-1 and sICAM-1 were detected by immunofluorescence and ELISA to evaluate the effect of the nano-grooves topology on cell compatibility.The latest technology cellular O-Glycome Reporter/Amplification(CORA),Western Blot,in-gel digestion and mass spectrometry analysis were introduced to the biocompatibility evaluation system of biomaterials.On these basics,the mechanisms for cell growth on different structures could be revealed by exploring the influence of micro-nanostructures on the expression of N-glycans and O-glycans,that is,changes in cell glycomics.(4)Exploring the strength of adhesion of endothelial cells on the surfaces of different micro/nanostructures at different flow speed.By using a in vitro dynamic culture system,and control the flow rate of the fluid in the system(1 m/s,2 m/s,and 3 m/s).Detecting and analyzing the quaintities of adhered cells on per unit area of the material surface under different flow speed,and finding out he relationship between the fluid shear force and cells adhesion strength on different surfaces.The experimental results show that:(1)Within a certain range(0-100 ?m),the hydrophilicity of the PCL porous membrane increases with the pore size increases.At the same time,the adhesion per unit area of the type I collagen also increases,correspondingly.The pore size distribution not only affects the hydrophilicity and hydrophobicity of the material surface,but also influences the adhesion of the extracellular matrix on the surface of the material and the cells adhesion and proliferation of material surfaces.Human Umbilical Vein Endothelial Cell(HUVEC)was grown on the surface of PCL membrane with a poresize of 0-38 ?m for 7 days to form a single-cell endothelial layer.The cell surface coverage area of PCL membrane with a poresize of 38-75 ?m was at 60%,while the cell coverage area on a PCL membrane with a diameter of 75-100 ?m was 80%.Indeed,adhesion molecules play an important role in regulating the HUVECs' adhesion and proliferation,as indicated by our results demonstrating that a higher expression of ICAM-1 on the cell plasma membrane(mICAM-1)was associated with better cell adhesion and proliferation,while high levels of soluble ICAM-1(sICAM-1)in the culture medium were consistently associated with weaker cell adhesion and proliferation.The expression level of ICAM-1 can be used as an important marker to evaluate the adhesion and proliferation of cells on different materials.(2)Controlling the type of warp yarn as monofilament and keeping the warp density and changing the weft density only and selecting monofilament or multifilament as weft yarm.Six kinds of woven plain weave structures were successfully constructed with different porosity and tightness.The contact angles were all within 30°-45°,and there was no significant difference in chemical structure.When the weft yarn are monofilaments,the hydrophilicity improves with the increase of the weft density.When the weft yarn is a multifilament,the hydrophilicity decreases with the increase of the warp density.The adhesion of cells on the weft multifilament is better than that of the monofilament.This is because there is a natural parallel groove structure on the surface of the multifilament,and the surface at the interlacing is relatively flat,and there are many sites where cells can attach to.The initial adhesion of cells to the surface of the woven microporous material was mostly concentrated on the interlacing.The smaller the pore size at the interlacing,the higher the level of mICAM-1 and the lower the concentration of sICAM-1 were detected.The best design density is warp(monofiliments)× weft(multifilament)850 ×700/10 cm,and the pore size less than 20 ?m will promote the endothelialization process.(3)Argon plasma is used to etch the preferred woven polyester plain weave material(E:850X700 yarns per 10 cm),by changing the etching power(150W,200W,250W and 300W).Four types of parallel grooves with different widths and depths(A,B,C,D in order)were constructed and their chemical structures were unchanged.Compared with an untreated one(E),the level of sICAM-1 on the surface of the plasma-treated material was significantly reduced.Compared with the blank control sample,the expression of Core 1(955,1003 Da),Core 2(1317,1404,1766 Da)on the surface of non-plasma-treated material was reduced,and no significant change was observed in N-glycans.Thus,plasma-treated strutcture changes cannot affect the expression of N-glycans on the cell plamsa membrane,but can affect the expression of O-glycans.A-structured material can only increase the expression of the cells Core 1(1003 Da)and Core 2(1404 Da).B,C and D structural materials can increase the expression of Core 1 and Core 2 structure.(4)The results of in vitro dynamic culture experiments show that the micro-nano structure constructed by plasma treatment can enhance the adhesion of cells on the surface of the material.When the material is used for making an artificial blood vessel,a suitable material can be selected according to the shear force of the blood vessel wall and the amount of remaining adherent cells on the surface of the material under the effect of different shear forces.According to different plasma etch powers,the experimental materials were numbered as A(150W),B(200W),C(250W),D(300W),and E(untreated sample).When the shear force was 5.4dyn(in vitro culture flow rate was 1 m/s),the selection sequence was D>B=C>A=E.When the shearing force was 7.0dyn(in vitro culture flow rate was 2 m/s),the order of the materials was D>A>C=B>E.When the shear force was 11.2dyn(in vitro culture flow rate was 3 m/s),the selection sequence was D=A>B>C=E.In summary,this study constructed woven plain weave surfaces with different micro-nano topological structures,which not only confirmed that micro-nano topology can affect cell adhesion,but also successfully introduced adhesion molecules into the evaluation system of material biocompatibility.The basic mechanism of the effect of materials on the adhesion of endothelial cells was revealed for the first time by investigating the influence of the material structure on the expression of the glycomics on the plasma membrane of adherent cells.On this basis,with the help of dynamic in vitro culture,the effects of different fluid shear forces on the adherent cells on the surface of the material were explored,and the in vitro biocompatibility evaluation system was further perfected,laying the foundation for the structural design and biocompatibility evaluation of the materials. |
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