Cytotoxicity Of TC4 Porous Scaffolds On Mouse Fibroblasts And Mouse Embryonic Fibroblasts

Objective:Objective to study the cytotoxicity of porous TC4（Ti-6Al-4V）scaffold prepared by selective laser melting（SLM）technology on the activity of mouse fibroblasts and mouse embryonic fibroblast cell.Method:1.Culture mouse fibroblasts and mouse embryonic fibroblast primary cells in vitro,select well-differentiated 3^rd cells,and use immunofluorescence（IFC）chemical staining for cell phenotype identification;2.TC4 metallic scaffold preparation,three-dimensional structure and surface topography analysis:1)To prepare square porous cube scaffolds with pore size of 0.6mm（φ0.6）,0.8mm（φ0.8）,and1.0mm（φ1.0）respectively by SLM;2)By micro computed tomography（Micro-CT）to quantitatively analyze the three-dimensional structural parameters of the TC4 scaffold material,including pore size,porosity etc;3)To observe the surface morphology of three groups of different apertures by scanning electron microscope（SEM）.3.In vitro cell experiment of titanium alloy porous scaffoldThe 3^rdgeneration mouse fibroblasts and mouse embryonic fibroblasts were respectively inoculated on the surface of TC4 scaffolds with different pore diameters:1)Use SEM to observe the morphology and adhesion of cells attached to the scaffold 48h after cell inoculation2)Use live/dead cell staining kit to stain,and observe the cell adhesion on the scaffold 48h after the scaffold is inoculated with cells by a Confocal Laser Scanning Microscope（CLSM）;3)Use the CCK-8 kit（Cell Counting Kit-8,CCK-8）to detect the OD values of cells and scaffolds co-cultured for 1,3,5 and 7 days,and analyze the cytotoxicity of TC4 scaffolds with different pore diameters;4)Using fluorescence real-time quantitative reverse transcription polymerase chain reaction（quantitative Reverse-Transcription polymerase chain reaction,q RT-PCR）to detect and co-culture with the scaffold for 7 days,the two types of fibroblasts contained Type III Collagenα（Co LIIIa1）and relative of fibronection（FN）m RNA;5)Using inductively coupled plasma mass spectrometry（Inductively Coupled Plasma Massspectrometry,ICP-MS）and inductively coupled plasma emission spectrometry（Inductively Coupled Plasma Optical Emission Spectrometry,ICP-OES）to detect two groups of cells co-cultured with the scaffold 1,3,5.The precipitation content of titanium（Ti）,aluminum（Al）and vanadium（V）metal elements in the complete medium for 7 days.Result:1.ICF results showed that fibroblasts originated from mesenchymal cells in the embryonic stage,and highly expressed mesenchymal markers vimentin and fibroblast specific protein 1（FPS1）.After ICF,the total cell count in the field of vision was checked by DAPI staining,and the rate of vimentin and FPS1 positive expression cells was calculated to be close to 100%,indicating that the purity of the fibroblasts extracted in this experiment was good.2.The analysis results of the three-dimensional structure and surface topography of the TC4scaffold:1)Micro-CT results show:After data analysis,the pore size of scaffolds are consistent with the theoretical design parameters.The specific surface area of the three sets of scaffold isφ0.8>φ1.0>φ0.6 stents;The porosity ofφ0.6,φ0.8 andφ1.0 is 73.67%,84.47%and 87.29%respectively;2)SEM results show that the pore size distribution of the three groups of scaffolds prepared by SLM can be seen under the microscope;a large number of spherical powder solidified structures can be seen on the surface of the three groups of stents.The powder has high sphericity and uniform particle size,without obvious needle-like solidified structures.Meet the requirements of porous scaffolds for surface morphology.3.In vitro cell viability experiment of TC4 porous scaffold1)SEM results show that three groups of TC4 porous scaffolds have cell adhesion under the microscope.Among them,theφ0.6 scaffold has good cell adhesion,more extended cell morphology,and pseudopodia sticking out,indicating that the smaller pore size is more It is conducive to cell adhesion;Cells tended to be in the micropores and gully of the scaffold.The indirect bridging exhibits cell migration behavior.2)CLSM showed the results of cell attachment:two kinds of cells were co-cultured with the three sets of scaffolds for 48 hours,and the three sets of scaffolds all had cell adhesion,and theφ0.6 had the highest cell density,showing obvious cell adhesion ability.φ0.8 is the next,which means that with the decrease of pore size in this experimental grouping,the porous scaffold shows better cell adhesion ability;3)The CCK-8 method test results show that the three groups of scaffolds all have the effect of promoting cell proliferation;compared with the first and third groups,the OD value of theφ0.6is significantly higher,However,at 5 days and 7 days,the OD value ofφ0.6 increased insignificantly.In contrast,the increase of OD value ofφ0.8 showed a very significant difference（P<0.01）;it means that in this experimental grouping,small pore size stents show an early stage of cell valuation.Superior cell adhesion ability,but with the passage of time,the cells adhered to the small pore size scaffolds tend to differentiate and mature earlier,and the growth of the OD value slows down.The largest specific surface area onφ0.8,the proliferation of cells is still more obvious after the fifth day;4)The results of q RT-PCR show that the difference in pore size can affect the relative expression Co LIIIa1,and FN m RN of mouse fibroblasts and mouse embryonic fibroblast.In the group of scaffolds in this experiment,the results of both cells were that theφ0.6 scaffold showed the highest m RNA expression level（P<0.01）.5)The results of ICP-MS and IC-OES showed that the three groups of scaffolds were immersed in the culture and co-cultured with the cells within 1,3,5,and 7 days.There was no significant change in the amount of Ti,Al,and V ions,and there was no significant difference in the amount of ions.It shows that the porous TC4 scaffold designed in this experiment,with the difference of the pore size,the surface area changes,and there is no obvious change in the dissolution and release of metal ions.Conclusion:1)The TC4 porous scaffold prepared by the SLM process has no cytotoxicity to mouse fibroblasts and mouse embryonic fibroblast,and is good for cell adhesion.The TC4 porous scaffold material with a pore size of 0.8 mm is beneficial to cellst.The TC4 porous scaffold material with a pore size of 0.6 mm is conducive to the adhesion and aggregation of cells,and promotes the relative expression of Co LIIIa1 and FN m RNA.2)In the grouping of TC4 porous scaffolds prepared by SLM in this experiment,the surface area changes with the pore diameter within 1 week,and there is no obvious change in the dissolution and release of metal ions.