Critical Toxicity Study Of Ionic Liquids On Cell/Artificial Tissue Constructs

The ionic liquid is composed of an anion and a cation,a salt which is in a liquid state at100?or lower.It is liquid at room temperature,so it is also called a room temperature molten salt.In recent years,ionic liquids have become a hotspot of research and application due to their own superiority,especially it is widely used in the industrial field as a"green substitute"for traditional organic solvents.However,the"risk"of ionic liquids has attracted more and more attention.An important part of ionic liquid environmental risk assessment is its biological toxicity measurement,but the toxicity of ionic liquids varies according to the target.Only by obtaining the toxic liquid toxicity data of various organisms and cell lines,filling in the ionic liquid toxicity data blank,and clarifying its toxicity mechanism,can we better determine its overall ecological environment and human health.The impact of this will lay the foundation for the future design of a new type of ionic liquid that is truly non-toxic,environmentally friendly and meets functional requirements.In this experiment,1-ethyl-3-imidazole diethylphosphate?[Emim]DEP?was prepared using 1-methylimidazole and triethyl phosphate as raw materials and configured[Emim]DEP-basal medium mixture for cytotoxicity assay.On the two-dimensional level,it can be seen from the results of MTT test that[Emim]DEP ionic liquid has a proliferation inhibitory effect on MC3T3-E1 cells,and this effect has time correlation and concentration correlation,and the action time increases and the concentration increases.The inhibition is enhanced.According to the inhibition rate-concentration curve of[Emim]DEP ionic liquid,the IC₅₀ values of[Emim]DEP type ionic liquid were 3959?g/mL,2226?g/mL and1884?g/mL after 24h,48h and 72h,respectively.The fluorescence of MC3T3-E1 cells treated with[Emim]DEP ionic liquid was observed by fluorescence staining,hematoxylin-eosin staining and scanning electron microscopy.The number of MC3T3-E1 viable cells decreased with the increase of ionic liquid concentration.The morphology of the cells changed significantly,the cells were shrunk into spheres by full spreading,and the spreading area of single cells was significantly reduced.The morphology of the cells was more stereoscopic under scanning electron microscopy.The cells were ruptured and the morphology was incomplete with increasing ionic liquid concentration.Cell differentiation of MC3T3-E1 cells treated with a lower concentration?1000?g/mL?of[Emim]DEP ionic liquid showed that the expression of ALP in MC3T3-E1 cells treated with trace ionic liquid was significantly reduced.The ability to differentiate into osteoblasts is disrupted by ionic liquids.On the basis of previous experiments,the toxic effects of[Emim]DEP-type ionic liquids on tissue constructs were studied by constructing a three-dimensional?3D?cell model.In this experiment,CS/Gel composite scaffold,20%nHAp/CS-Gel composite scaffold,40%nHAp/CS-Gel composite scaffold and 60%nHAp/CS-Gel composite scaffolds were prepared by physical mixing-freeze drying method.The microstructure,porosity,water absorption,mechanical properties and biocompatibility were characterized and analyzed.The scaffold pore size is between 90?m and 200?m,which provides sufficient space for the adhesion,proliferation and differentiation of MC3T3-E1 cells.The composite scaffold material has a large number of microporous structures embedded in the pore walls,ranging from 20?m to50?m.The structural characteristics of the large hole set of small holes can further improve the mass transfer ability between the holes and the holes,between the inside and the outside of the support,facilitate the transfer of metabolic waste and nutrients,and provide a suitable growth environment for cell proliferation and differentiation.The porosity of each group is between 81.17%and 93.69%,which provides sufficient space for cell growth and adhesion.Among them,20%nHAp/CS-Gel stent has the highest porosity.The water absorption rate of the composite scaffolding material first increased and then decreased,and the water absorption of the composite scaffold was the highest when it reached 20%incorporation,which was 1300±2.67%.The elastic modulus of the composite scaffold materials are960±2.41%MPa,1002±1.37%MPa,776±1.29%MPa and 379±2.38%MPa,respectively.With the increase of nHAp,the elastic modulus of the composite scaffold material first increases and then decreases,when the nHAp incorporation reaches 20%,the elastic modulus is the highest.MC3T3-E1 cells were seeded on the composite scaffold material,and the cell-scaffold complex was stained for life and death after three days of culture.Compared with the other three groups,the cell proliferation on the 20%nHAp/CS-Gel composite scaffold material is most obvious,and the stretch is good and a large amount of extracellular matrix is secreted into one piece with other cells.In summary,the 20%nHAp/CS-Gel composite scaffold was selected as the best scaffold material,and the scaffold material was used to construct an in vitro three-dimensional cell model for subsequent detection toxity of[Emim]DEP type ionic liquid on tissue construct Toxic effects.From the inhibition rate-concentration fitting curve,the IC₅₀ values of[Emim]DEP ionic liquid on MC3T3-E1 cells at three-dimensional level for 24h,48h and 72h were 12566?g/mL,9015?g/mL and 7896?g/mL,respectively.Compared with the IC₅₀ value of ionic liquid cytotoxicity on the two-dimensional level,the IC₅₀ value on the three-dimensional level was significantly increased by 3-4 times,indicating that the tolerance of cells on the three-dimensional level was significantly enhanced.Fluorescence staining analysis showed that MC3T3-E1 cells that had not been treated with ionic liquid adhered to the scaffold material,and the cell stretched well,and the cells were connected to each other to form a cell network.Ionic liquids have a significant inhibitory effect on the proliferation of MC3T3-E1 cells.Scanning electron microscopy showed that cells treated without ionic liquid adhered to each other on the scaffold,spreading the surface of the scaffold and the pores.In the cell-scaffold complex after ionic liquid treatment,the number of cells was significantly reduced with the increase of the concentration of ionic liquid.Increasing,the cells in the pores of the scaffold material grow from agglomeration to the presence of a single living cell.Finally,there are no intact living cells,occasionally cell debris is found.And the cell morphology has changed significantly,from stretching to shrinking into a sphere,cells are no obvious interconnection,and even some have no intact cell morphology,only in the form of cell debris.In this study,MC3T3-E1 cells were used as the research object.The cytotoxicity of[Emim]DEP ionic liquid was studied from the cell and in vitro 3D tissue constructs by MTT assay,flow cytometry,complex fluorescent staining,HE staining and SEM.It was found that the ionic liquid inhibited the proliferation of cells and 3D tissue constructs,and the effect was correlated with time and concentration.And the IC₅₀ value was obtained by inhibition rate-concentration curve.The ionic liquid also has a certain influence on the cell morphology,which results in a decrease in the spreading area of a single cell on the 2D level,a decrease in the cell adhesion ability on the 3D level,and even cell breakage.In addition,ionic liquids also have a certain destructive effect on the differentiation ability of MC3T3-E1 cells.