SCENCNNEWS www.advancedsciencenews.com ww.afm-journalde a「co10 SEMCO10E[coc CFOCFOCFO RK CFO 10-E b 20〓cFo10-/Mmco10E■ CFO O-E■ 平 Day 3 ALP Day 3 RUNX2 Day 14 Collagen Day 14RUNX2 cFo10E/McFo10E■cFo0E■Nc MAPKI MAPK3 Figure 3. Osteoinductive potential of the CFO/P(VDF-TrFE) magnetoelectric nanocomposite membrane and the potential mechanisms by which induces osteogenesis in vitro. a)Immunofluorescence images showed the upregulated expression of RUNX2 in the CFo 10-E/M group 100 um). b)RT-qPCR revealed the upregulation of osteogenic markers(ALP, RUNX2, and Collagen I)in the CFO 10-E/M the upregulation of Itas, FN, MAPK], MAPK3, FAK, and YAP in the CFO 10-E/M group. (*VS CFO 10-E/M, VS NC, p<0.05)d) Western bl indicated that the CFO 10-E/M group exhibited upregulated expression of integrin as/ERK1/2 cascade- related proteins and increased YAP activation e)Immunofluorescence staining showed enhanced nuclear localization of YAP in the CFO 10-E/M group(Scale bars: 50 um) cell downstream signaling proteins such as ERK and nuclear phosphorylated- YAP(Figure 3d), which were consistent with localization mechanosensitive transcription regulators like the immunostaining results. Therefore, we propose that th YAP/TAZ. 565) Next, we evaluated the activation of ERK signaling magnetoelectric microenvironment provided by CFO 10-E/M and YAP nuclear localization to characterize the intracellular induces BM-MSC osteogenic differentiation by increasing RGD signaling pathways. The CFO 10-E/M membrane markedly exposure and initiating sensors of biomechanical signaling, upregulated the gene expression levels of ERK1/2, which were which in turn promotes FA maturation and trigger a series of consistent with increased protein expression(Figure 3c, d ). mechanotransduction-related molecular processes, involving Immunofluorescence staining showed that YAP is concen- the activation of ERK signaling and nuclear localization of YAP trated within the cell nuclei on CFO 10-E/M(Figure 3e). The statistical analysis of fluorescence intensity ratios between the cell's nucleus and cytoplasm demonstrated that nuclear locali- 2.5. Biomimetic Magnetoelectric Microenvironment Modulates zation of YAP is significantly higher on CFO 10-E/M versus the Osteoimmunomodulatory Responses In Vitro other membrane groups(Figure S7b, Supporting Information The protein expression results showed that BM-MSCs cul- Bone regeneration is a highly complex process which involves tured on CFo 10-E/M displayed increased YAP but decreased various different cell types such as ne cells, progenitor Adw. Funct Mater. 2020. 2006226 2006226(6of1) g 2020 Wiley-VCH GmbHwww.advancedsciencenews.com www.afm-journal.de 2006226 (6 of 11) © 2020 Wiley-VCH GmbH cell downstream signaling proteins such as ERK and nuclear localization mechanosensitive transcription regulators like YAP/TAZ.[36] Next, we evaluated the activation of ERK signaling and YAP nuclear localization to characterize the intracellular signaling pathways. The CFO 10-E/M membrane markedly upregulated the gene expression levels of ERK1/2, which were consistent with increased protein expression (Figure  3c,d). Immunofluorescence staining showed that YAP is concen￾trated within the cell nuclei on CFO 10-E/M (Figure  3e). The statistical analysis of fluorescence intensity ratios between the cell’s nucleus and cytoplasm demonstrated that nuclear locali￾zation of YAP is significantly higher on CFO 10-E/M versus the other membrane groups (Figure S7b, Supporting Information). The protein expression results showed that BM-MSCs cul￾tured on CFO 10-E/M displayed increased YAP but decreased phosphorylated-YAP (Figure  3d), which were consistent with the immunostaining results. Therefore, we propose that the magnetoelectric microenvironment provided by CFO 10-E/M induces BM-MSC osteogenic differentiation by increasing RGD exposure and initiating sensors of biomechanical signaling, which in turn promotes FA maturation and trigger a series of mechanotransduction-related molecular processes, involving the activation of ERK signaling and nuclear localization of YAP. 2.5. Biomimetic Magnetoelectric Microenvironment Modulates Osteoimmunomodulatory Responses In Vitro Bone regeneration is a highly complex process which involves various different cell types such as immune cells, progenitor Figure 3. Osteoinductive potential of the CFO/P(VDF-TrFE) magnetoelectric nanocomposite membrane and the potential mechanisms by which it induces osteogenesis in vitro. a) Immunofluorescence images showed the upregulated expression of RUNX2 in the CFO 10-E/M group (Scale bars: 100 µm). b) RT-qPCR revealed the upregulation of osteogenic markers (ALP, RUNX2, and Collagen I) in the CFO 10-E/M group. c) RT-qPCR revealed the upregulation of Itgα5, FN, MAPK1, MAPK3, FAK, and YAP in the CFO 10-E/M group. (* VS CFO 10-E/M, # VS NC, p < 0.05) d) Western blot analysis indicated that the CFO 10-E/M group exhibited upregulated expression of integrin α5/ERK1/2 cascade-related proteins and increased YAP activation. e) Immunofluorescence staining showed enhanced nuclear localization of YAP in the CFO 10-E/M group (Scale bars: 50 µm). Adv. Funct. Mater. 2020, 2006226