Effects Of Macrophage-Mediated 3D-Printed Cu-Bearing Titanium Alloy On The Biological Behavior Of MC3T3-E1 Cells

ObjectiveThe long-term therapeutic effect of medical titanium/titanium alloy implants depends not only on the physical and chemical properties of the implants,but also on the host immune response after implantation.The role of macrophages in tissue regeneration has attracted extensive attention and research.In view of the effect of macrophages on bone tissue regeneration,the effect of 3D-printed Cu-bearing Titanium Alloy on the biological behavior of RAW264.7 cells was investigated in vitro.Secondly,the mouse embryonic osteoblast precursor cells(MC3T3-E1)were cultured by collecting the extracts of Ti6Al4 V alloy materials and macrophage conditioned medium to study the effect of immune microenvironment on MC3T3-E1 cells.Methods1.The porous Ti6Al4V-6Cu alloy,Ti6Al4V-6Cu alloy,porous Ti6Al4 V alloy and Ti6Al4 V alloy were prepared by Selective Laser Melting(SLM)technology,and the surface of the materials was treated with hydrofluoric acid solution.The surface morphology and elemental composition of the alloy materials were analyzed by Scanning Electron Microscope(SEM)and Energy Dispersive Spectroscopy(EDS).2.The square titanium alloys with size of 1 cm × 1 cm × 0.2 cm was prepared for cell experiments in vitro.Macrophage RAW264.7 was co-cultured with porous Ti6Al4V-6Cu alloy,Ti6Al4V-6Cu alloy,porous Ti6Al4 V alloy and Ti6Al4 V alloy.The proliferation,adhesion,cell morphology and cell polarization of RAW264.7 macrophages were detected.3.The sample extracts of each group(RAW-group)and macrophage conditioned medium(RAW+ group)were collected to culture MC3T3-E1 cells.The proliferation of MC3T3-E1 cells under the two conditions was detected by CCK-8 kit;the cytoskeleton was labeled by rhodamine-labeled ghost pen cyclic peptide staining;the osteogenic differentiation ability of MC3T3-E1 cells was determined by alkaline phosphatase(ALP)activity assay and alizarin red staining of each group of sample extracts and conditioned medium.Results1.CCK-8 results showed that all titanium alloy materials had no toxicity to RAW264.7 cells and MC3T3-E1 cells in vitro.2.pTC4-6Cu and pTC4 promoted the early adhesion of RAW264.7 cells.3.The results of flow cytometry experiments demonstrated that 3D-printed porous copper-bearing titanium alloy(pTC4-6Cu)facilitated the expression of CD206 in RAW264.7 cells.4.Compared with other groups,the pTC4-6Cu group extract had a significant advantage in MC3T3-E1 cell proliferation and osteogenic differentiation.ConclusionsThe results of this study are as follows: all titanium alloy materials have good compatibility with MC3T3-E1 and RAW264.7 cells in vitro;Compared with the dense titanium alloy,the porous titanium alloy was more conducive to the early adhesion of RAW264.7.Cu promoted the expression of CD206 in RAW264.7 cells on the surface of pTC4-6Cu alloy,resulting in a favorable microenvironment and enhanced osteogenic differentiation ability.Compared with the extract of each alloy material,macrophage conditioned medium could promote the proliferation level and osteogenic ability of MC3T3-E1 cells.