SCENCENEWS m-journal de potential could be sustained for as long as required by applying of the cell-binding domain of RGD on adsorbed Fn was fur- a magnetic field in a non-contact manner. For instance, the ther evaluated. On CFO 10-E/M, the RGD site combined with nanocomposite membrane with 10 wt% of CFO content has the HFNZ1 antibody 52 displayed the highest intensity and big appropriate concentration for optimal magnetoelectric effect. gest area among all groups( Figure S4d, Supporting Informa The surface potential of CFO/P(VDF-TrFE)membranes were tion). These results thus showed that CFO 10-E/M can enhance decreased after 14 days immersion in culture medium without the FN adsorption and RGD site exposure. The enhanced FN application of a remote DC magnetic field( Figure 2d). The adsorption and RGD exposure could promote cell adhesion, surface potential of 10 wt% CFO content membranes could migration, and spreading. 245) Consistent with the prediction be tuned to around 54 mV with a remote DC 23000e mag. of MD simulation, the magnetoelectric microenvironment pre netic field, which were utilized for subsequent biological vided by CFo 10-E/M can modulate cellular behavior assays(Figure 2c). Upon application of a remote DC mag. The biological performance of the CFO/P(VDF-TrFE)mag- netic field, the surface potential of the membranes were main- netoelectric nanocomposite membranes was further investi tained throughout 14 days of immersion in culture medium gated. Assessment of proliferation through the cell counting Figure 2d). Since the diurnal geomagnetic variation has a pro. kit-8(CCK-8 )assay demonstrated that there were no significar nounced circadian rhythmicity that exerts biological effects, 4 differences between groups(Figure S4e, f, Supporting Infor a remote DC magnetic field mimicking the natural magnetic mation). However, the cytoskeleton immunostaining images field with 12 h shifting was selected to control the magneto- showed that BM-MSCs on CFO 10-E/M exhibited the largest electric microenvironment in this study. The surface rough- spreading area and the most abundant FA formation among all ness and water contact angle measurements yielded different groups( Figure S5a-f, Supporting Information). The increased values according to the different nanoparticle contents of the FA and spreading area are correlated with enhanced RGD expo- various groups( Figure S3a, b, Supporting Information). Some sure, and might also be correlated to the osteogenic potential of researchers have reported that roughness difference, which is BM-MSCs 1.3 a microroughness level of around 100-500 nm, exert negligible effects on the cell adhesion and biological functions of larger cell types, such as BM-MSCs, osteoblasts, and neurons. 2 2.4. Biomimetic Magnetoelectric Microenvironment Enhances The surface wettability study also showed that there is no dif- Osteogenesis of BM-MSCs through RGD Exposure-Mediated ference in cell adhesion when the surface contact angles are Cellular Mechanotransduction round 60 to 80 degrees after 180 min of incubation. ol Hence, the roughness and contact angle values differences between The pro-osteogenic effects of the magnetoelectric microen- groups should have no biological effects in this study. Tensile vironment provided by CFO/P(VDF-TrFE) membranes were strength and the elastic modulus of membranes decreased evaluated with BM-MSCs, under exposure to a remote DC along with increased CFO concentrations. However, based on magnetic field. The quantitative analysis of alizarin red staining our previous study, changes to the elastic modulus and tensile demonstrated significantly higher mineralization values strength in this scale will have negligible effects on osteogen- CFO 10-E/M compared to membranes with different CFO s31(Figure S3c, d, Supporting Information). Moreover, the contents(Figure S6a, Supporting Information). CFO 10-E/M tensile strength and elastic modulus values showed that the exhibited the strongest fluorescence intensity of RUNX2 immu- fexibility of the membranes could be suitable for clinical appli- nostaining after 3 days of incubation(Figure 3a; Figure S6d, cations, such as being utilized as non-resorbable membranes Supporting Information). Osteogenic gene expression and pro- to prevent the ingrowth of non-osteogenic tissues and main- tein production were further evaluated and the results showed tain the capacity for cell occlusion 13,31 After corona poling, that CFO 10-E/M upregulated the expression of osteogenesis 10 wt% CFO content membranes combined with application of related genes(RUNX2, ALP, Collagen I, OCN, OSX)and a remote DC magnetic field can provide an optimal controllable proteins(OPN, BMP2, RUNX2) in the absence of osteogenic magnetoelectric microenvironment. supplements( Figure 3b: Figure S6b, c, f, Supporting Informa tion). After 7 days of culture without osteogenic supplements. higher ALP activity was detected on CFO 10-E/M(Figure S6e 2.3. Biomimetic Magnetoelectric Microenvironment Promotes Supporting Information). These results thus demonstrated that FN Adsorption and RGD Exposure That Leads to Enhanced CFO 10-E/M provides an optimal magnetoelectric microenv Cell Adhesion ronment for BM-MSC osteogenic differentiation To explore the mechanisms by which osteogenesis of BM In biological assays, the membranes were grouped as follows: MSCs is promoted by enhanced FN adsorption and RGD expo CFO 0, CFO 5, CFO 10, CFO 15, and CFO 20, which represent sure in the CFO 10-E/M group, the expression of integrin was 0, 5, 10, 15, and 20 wt% of CFO content within membranes, analyzed and it was found that the gene expression and pro- represents group exposed to up with polarization treatment. M tein production levels of integrin as were significantly high a remote DC magnetic field. Nc in CFO 10-E/M versus other groups(Figure 3c, d; Figure S7a, represents blank group without membrane Supporting Information). Integrin a5 has been reported to par To assay FN adsorption in vitro, quantitative evaluation and ticipate in MSC osteogenesis. 242, 34 Integrins connect the cell immunofluorescence images were used. The results dem. cytoskeleton to the microenvironment and serve as sensors of onstrated that CFO 10-E/M had the greatest FN adsorption mechanical signals. 35 Increased integrin clustering can lead capacity(Figure S4a-C, Supporting Information). The exposure to increased adhesion maturation, which in turn can modulate Adv Funct. Mater. 2020. 2006226 20062265of1) o 2020 Wiley-VCH GmbHwww.advancedsciencenews.com www.afm-journal.de 2006226 (5 of 11) © 2020 Wiley-VCH GmbH potential could be sustained for as long as required by applying a magnetic field in a non-contact manner. For instance, the nanocomposite membrane with 10 wt% of CFO content has the appropriate concentration for optimal magnetoelectric effect. The surface potential of CFO/P(VDF-TrFE) membranes were decreased after 14 days immersion in culture medium without application of a remote DC magnetic field (Figure  2d). The surface potential of 10 wt% CFO content membranes could be tuned to around 54  mV  with a remote DC 2300Oe  mag￾netic field, which were utilized for subsequent biological assays (Figure  2c). Upon application of a remote DC mag￾netic field, the surface potential of the membranes were main￾tained throughout 14 days of immersion in culture medium (Figure 2d). Since the diurnal geomagnetic variation has a pro￾nounced circadian rhythmicity that exerts biological effects,[14] a remote DC magnetic field mimicking the natural magnetic field with 12  h shifting was selected to control the magneto￾electric microenvironment in this study. The surface rough￾ness and water contact angle measurements yielded different values according to the different nanoparticle contents of the various groups (Figure S3a,b, Supporting Information). Some researchers have reported that roughness difference, which is a microroughness level of around 100–500 nm, exert negligible effects on the cell adhesion and biological functions of larger cell types, such as BM-MSCs, osteoblasts, and neurons.[29] The surface wettability study also showed that there is no dif￾ference in cell adhesion when the surface contact angles are around 60 to 80 degrees after 180 min of incubation.[30] Hence, the roughness and contact angle values differences between groups should have no biological effects in this study. Tensile strength and the elastic modulus of membranes decreased along with increased CFO concentrations. However, based on our previous study, changes to the elastic modulus and tensile strength in this scale will have negligible effects on osteogen￾esis[13] (Figure S3c,d, Supporting Information). Moreover, the tensile strength and elastic modulus values showed that the flexibility of the membranes could be suitable for clinical appli￾cations, such as being utilized as non-resorbable membranes to prevent the ingrowth of non-osteogenic tissues and main￾tain the capacity for cell occlusion.[13,31] After corona poling, 10 wt% CFO content membranes combined with application of a remote DC magnetic field can provide an optimal controllable magnetoelectric microenvironment. 2.3. Biomimetic Magnetoelectric Microenvironment Promotes FN Adsorption and RGD Exposure That Leads to Enhanced Cell Adhesion In biological assays, the membranes were grouped as follows: CFO 0, CFO 5, CFO 10, CFO 15, and CFO 20, which represent 0, 5, 10, 15, and 20 wt% of CFO content within membranes, respectively. E represents group with polarization treatment. M represents group exposed to a remote DC magnetic field. NC represents blank group without membrane. To assay FN adsorption in vitro, quantitative evaluation and immunofluorescence images were used. The results dem￾onstrated that CFO 10-E/M had the greatest FN adsorption capacity (Figure S4a–c, Supporting Information). The exposure of the cell-binding domain of RGD on adsorbed FN was fur￾ther evaluated. On CFO 10-E/M, the RGD site combined with HFN7.1 antibody[32] displayed the highest intensity and big￾gest area among all groups (Figure S4d, Supporting Informa￾tion). These results thus showed that CFO 10-E/M can enhance the FN adsorption and RGD site exposure. The enhanced FN adsorption and RGD exposure could promote cell adhesion, migration, and spreading.[24b] Consistent with the prediction of MD simulation, the magnetoelectric microenvironment pro￾vided by CFO 10-E/M can modulate cellular behavior. The biological performance of the CFO/P(VDF-TrFE) mag￾netoelectric nanocomposite membranes was further investi￾gated. Assessment of proliferation through the cell counting kit-8 (CCK-8) assay demonstrated that there were no significant differences between groups (Figure S4e,f, Supporting Infor￾mation). However, the cytoskeleton immunostaining images showed that BM-MSCs on CFO 10-E/M exhibited the largest spreading area and the most abundant FA formation among all groups (Figure S5a–f, Supporting Information). The increased FA and spreading area are correlated with enhanced RGD expo￾sure, and might also be correlated to the osteogenic potential of BM-MSCs.[13,33] 2.4. Biomimetic Magnetoelectric Microenvironment Enhances Osteogenesis of BM-MSCs through RGD Exposure-Mediated Cellular Mechanotransduction The pro-osteogenic effects of the magnetoelectric microen￾vironment provided by CFO/P(VDF-TrFE) membranes were evaluated with BM-MSCs, under exposure to a remote DC magnetic field. The quantitative analysis of alizarin red staining demonstrated significantly higher mineralization values in CFO 10-E/M compared to membranes with different CFO contents (Figure S6a, Supporting Information). CFO 10-E/M exhibited the strongest fluorescence intensity of RUNX2 immu￾nostaining after 3 days of incubation (Figure 3a; Figure S6d, Supporting Information). Osteogenic gene expression and pro￾tein production were further evaluated and the results showed that CFO 10-E/M upregulated the expression of osteogenesis￾related genes (RUNX2, ALP, Collagen I, OCN, OSX) and proteins (OPN, BMP2, RUNX2) in the absence of osteogenic supplements (Figure  3b; Figure S6b,c,f, Supporting Informa￾tion). After 7 days of culture without osteogenic supplements, higher ALP activity was detected on CFO 10-E/M (Figure S6e, Supporting Information). These results thus demonstrated that CFO 10-E/M provides an optimal magnetoelectric microenvi￾ronment for BM-MSC osteogenic differentiation. To explore the mechanisms by which osteogenesis of BM￾MSCs is promoted by enhanced FN adsorption and RGD expo￾sure in the CFO 10-E/M group, the expression of integrin was analyzed and it was found that the gene expression and pro￾tein production levels of integrin α5 were significantly higher in CFO 10-E/M versus other groups (Figure  3c,d; Figure S7a, Supporting Information). Integrin α5 has been reported to par￾ticipate in MSC osteogenesis.[24a,34] Integrins connect the cell cytoskeleton to the microenvironment and serve as sensors of mechanical signals.[35] Increased integrin clustering can lead to increased adhesion maturation, which in turn can modulate Adv. Funct. Mater. 2020, 2006226