Study On The Controllable Surface Morphology Of Tissue Engineering Scaffold To Coordinate BMSCs And Macrophage In Osteogenesis

Surface microstructures,as one of the physical characteristics of material surface,have the biomimetic properties of simulating extracellular matrix and have an important role in regulating cell behavior,which has been widely used to study cell behavior for realizing tissue regeneration.At present,most studies on cell behavior are based on two-dimensional surface morphology,reckon without the fact that almost of natural extracellular matrixes are a three-dimensional topological structure.Although a few studies have made corresponding attempts,and provides some references for bone tissue engineering scaffolds to apply relevant technical to multicellular synergy in bone regeneration.However,due to technical limitations,there are still major challenges to the integration of micropatterned microstructures into three-dimensional(3D)scaffolds.In this study,under the combination with soft lithography,3D printing and layer-to-layer stacking,we used soft lithography to prepare controllable surface topological morphology on3 D printed scaffold lamellas,and then obtained internal controllable surface topology scaffolds by scaffolds lamellas stacking.To obtain a bioactive bone repair scaffold with synergistic regulation of osteoblast differentiation of mouse bone marrow mesenchymal stem cells(m BMSCs)and immune differentiation of macrophages(RAW264.7),we fabricated the structure substrates respectively consisted of the pit array of 25.19±0.40μm、34.81±0.63μm、45.04±0.65μm and the groove array of 21.60±0.56μm、30.76±0.72μm、39.47±0.90μm,and investigated the regulation of surface microstructure on the immune of RAW264.7 cells and the osteogenic differentiation of m BMSCs.The results showed that the size about 25μm pit group and 40μm groove group had better immune regulation ability to macrophages,which were mainly manifested in the up-regulation of the M2 marker gene(Arginase,IL-10)in M2 RAW264.7 cells,down-regulated the M1 marker gene(TNF-α,i NOS)in M1 RAW264.7 cells.Similarly,the size about 25μm pit group and 40μm groove group also were up-regulation the expression of osteoblastic differentiation related genes(ALP COLI and Runx2)of m BMSCs,promoted calcium nodule formation and alkaline phosphatase activity.Among them,the size about 25μm pit group had the best regulation effect on RAW264.7 cells and m BMSCs.In addition,the size about 25μm pit group still significantly promoted the osteogenic differentiation of m BMSCs in the direct/indirect co-culture constructing with M2RAW264.7 cells.Finally,rat skull defects repair experiments showed that the bone repair performance of the size about 25μm pit micropattern scaffold was better than that of the Flat scaffold.In this study,the scaffold with controllable surface topography was successfully fabricated by stacking scaffolds lamellas preparing by the combination of 3D printing and soft lithography.And it was confirmed that the micropattern regulating RAW264.7 cell immunophenotype and m BMSCs osteoblast differentiation on the two-dimensional film transferred to scaffold internal surface could also promote the bone repair ability of the scaffold.These studies are expected to contribute to the potential application of three-dimensional scaffold design based on internal surface topology in bone tissue regeneration.