SCENCENEWS m-journal de ells, and MSC. I/ Macrophages are the first cell type to arrive at Supporting Information). These elevated integrin expression the bone defect site, and have long been thought to contribute levels on CFO 10-E/M indicated that the magnetoelectric micro- to the initial inflammation and debridement of the injury loca- environment could enhance macrophage polarization through zation is a key regulator of bone regeneration. /g o phage polari- integrin-related pathways. The western blot results showed that the protein expression levels of integrin pl, phosphoryla To further investigate the osteoimmunomodulatory effects tion levels of phosphoinositide 3-kinase(PI3K) and serine/ of the magnetoelectric microenvironment, we evaluated the threonine kinase Akt(Akt) were increased in the CFO 10-E/M polarization of macrophages and osteogenic differentiation group(Figure S12a, Supporting Information). The CFO 10-E/M of BM-MSCs within a co-culture system. Macrophages were group exhibited the strongest fluorescence intensity of phos eeded on the nanocomposite membranes, while BM-Msc phorylated Akt immunostaining( Figure S12b, Supporting were seeded in the upper chambers of transwell culture dishes. Information). Furthermore, the western blot results showed Macrophages cultured on CFO 10-E/M expressed high expres- that the CFO 10-E/M group had significantly reduced nuclear sion levels of CD206, which is the M2 marker(Figure 4a, b; Fig. factor kappa B(NF-xB/p65)levels compared to other groups ures S8a and S1a, Supporting Information). Meanwhile, there (Figure S12a, Supporting Information). Protein adsorption and is no significant change in the expression of the MI marker integrin binding interactions have been demonstrated to modu CCR7(Figure S8b, c, Supporting Information) by macrophages late inflammation. 24C.32. 4) On CFO 10-E/M, the enhanced FN cultured on CFO 10-E/M, as compared with the other groups. adsorption resulted in adoption of an active conformation that The results proved that CFO 10-E/M could promote M2 polari- led to more RGD binding site exposure and increased macro- zation of macrophages. The immunofluorescence staining phage integrin Bl binding Increased integrin Bl binding acti- showed that expression levels of integrin a5, integrin Bl, and vates PI3K/Akt signaling, leading to the inhibition of NF-xB vinculin in macrophages were increased( Figures S9-S11, activation and subsequent anti-inflammatory polarization(M2) CFO 0- cFo10E/M‖cFo10E CFO O-E 目喝 叫 CFO 10-E 56.0 38.2 35.4 =:o10E1=e Day14 RUNX2 , o10E/M■co0E CFO 10-E Macrophage polarization and osteogenesis of BM-MSC on CFO/P(VDF-TrFE) magnetoelectric nanocomposite membranes in vitro. a)Immu- #f VS NC, p<0.05) Adv Funct. Mater. 2020. 2006226 2006226卩7of1) o 2020 Wiley-VCH GmbHwww.advancedsciencenews.com www.afm-journal.de 2006226 (7 of 11) © 2020 Wiley-VCH GmbH cells, and MSC.[37] Macrophages are the first cell type to arrive at the bone defect site, and have long been thought to contribute to the initial inflammation and debridement of the injury loca￾tion.[38] Mounting evidence has shown that macrophage polari￾zation is a key regulator of bone regeneration.[39] To further investigate the osteoimmunomodulatory effects of the magnetoelectric microenvironment, we evaluated the polarization of macrophages and osteogenic differentiation of BM-MSCs within a co-culture system. Macrophages were seeded on the nanocomposite membranes, while BM-MSC were seeded in the upper chambers of transwell culture dishes. Macrophages cultured on CFO 10-E/M expressed high expres￾sion levels of CD206, which is the M2 marker (Figure 4a,b; Fig￾ures S8a and S13a, Supporting Information). Meanwhile, there is no significant change in the expression of the M1 marker CCR7 (Figure S8b,c, Supporting Information) by macrophages cultured on CFO 10-E/M, as compared with the other groups. The results proved that CFO 10-E/M could promote M2 polari￾zation of macrophages. The immunofluorescence staining showed that expression levels of integrin α5, integrin β1, and vinculin in macrophages were increased (Figures S9–S11, Supporting Information). These elevated integrin expression levels on CFO 10-E/M indicated that the magnetoelectric micro￾environment could enhance macrophage polarization through integrin-related pathways. The western blot results showed that the protein expression levels of integrin β1, phosphoryla￾tion levels of phosphoinositide 3-kinase (PI3K) and serine/ threonine kinase Akt (Akt) were increased in the CFO 10-E/M group (Figure S12a, Supporting Information). The CFO 10-E/M group exhibited the strongest fluorescence intensity of phos￾phorylated Akt immunostaining (Figure S12b, Supporting Information). Furthermore, the western blot results showed that the CFO 10-E/M group had significantly reduced nuclear factor kappa B (NF-κB/p65) levels compared to other groups (Figure S12a, Supporting Information). Protein adsorption and integrin binding interactions have been demonstrated to modu￾late inflammation.[24c,32,40] On CFO 10-E/M, the enhanced FN adsorption resulted in adoption of an active conformation that led to more RGD binding site exposure and increased macro￾phage integrin β1 binding. Increased integrin β1 binding acti￾vates PI3K/Akt signaling, leading to the inhibition of NF-κB activation and subsequent anti-inflammatory polarization (M2) Figure 4. Macrophage polarization and osteogenesis of BM-MSC on CFO/P(VDF-TrFE) magnetoelectric nanocomposite membranes in vitro. a) Immu￾nostaining images and b) FC analysis of CD206 expression, which indicated that the CFO 10-E/M group promoted macrophage M2 polarization in the co-culture system (Scale bars: 25 µm). c) Alizarin red staining (Scale bars: 200 µm), d) immunostaining images of RUNX2 expression (scale bars: 100 µm) and e,f) RT-qPCR analysis showing that the CFO 10-E/M group enhanced BM-MSC osteogenesis in the co-culture system. (* VS CFO 10-E/M, # VS NC, p < 0.05). Adv. Funct. Mater. 2020, 2006226