Comprehensive Evaluation of Surface Potential Characteristics on

Research Article Cite This: ACS Appl. Mater. Interfaces 2019, 11, 22218−22227

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Comprehensive Evaluation of Surface Potential Characteristics on Mesenchymal Stem Cells’ Osteogenic Differentiation Fei Jia,† Suya Lin,† Xuzhao He,† Jiamin Zhang,† Shuxian Shen,† Zhiying Wang,† Bolin Tang,†,‡ Cheng Li,† Yongjun Wu,† Lingqing Dong,†,§ Kui Cheng,† and Wenjian Weng*,† †

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School of Materials Science and Engineering, State Key Laboratory of Silicon Materials, Zhejiang University, Hangzhou 310027, China ‡ College of Materials and Textile Engineering, Jiaxing University, Jiaxing 314001, China § The Stomatologic Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China S Supporting Information *

ABSTRACT: The surface electric potential of biomaterials has been extensively proven to play a critical role in stem cells’ fate. However, there are ambiguous reports on the relation of stem cells’ osteogenic capacity to surface potential characteristics (potential polarity and intensity). To address this, we adopted a surface with a wide potential range and both positive/negative polarity in a comprehensive view to get insight into surface potential-regulating cellular osteogenic differentiation. TbxDy1−xFe2 alloy/poly(vinylidene fluoride-trifluoroethylene) magnetoelectric films were prepared, and the film could provide controllable surface potential characteristics with positive or negative polarity and potential (ϕME) intensity variation from 0 to ±120 mV as well as keep the surface chemical composition and microstructure unchanged. Cell culture results showed that osteogenic differentiation of mesenchymal stem cells on both positive and negative potential films was obviously upregulated when the /ϕME/ intensities were set from 0−55 mV. Differently, the highest upregulated osteogenic differentiation on the positive potential films corresponded to the /ϕME/ intensity from 35−55 mV and was better than that on the negative potential films whereas the highest on the negative potential films corresponded to the /ϕME/ intensity from 0−35 mV and was better than that on the positive potential films. This fact could illustrate why previous reports appeared ambiguously; i.e., the comparative result in osteogenic differentiation between the positive and negative potential films strongly depends on the selection of surface potential intensity. On the basis of assaying of the exposed functional sites (RGD and PHSRN) of the adsorbed fibronectin (FN) and the expression of cellular integrin α5 and β1 subunits, the difference in the behavior between the positive and negative potential films was attributed to the distinct conformation of adsorbed fibronectin (FN) and the opposite changing trend with /ϕME/ for the two films, which triggers the osteogenesis-related FAK/ERK signaling pathway to a different extent. This study could provide new cognition for the in-depth understanding of the regulation mechanism underlying surface potential characteristics in cell behaviors. KEYWORDS: magnetoelectric film, positive and negative potential films, fibronectin conformation, mesenchymal stem cells, osteogenic differentiation

1. INTRODUCTION

osteogenic differentiation, whereas negatively polarized BaTiO3/PVDF nanocomposite membranes that mimic the endogenous negative potential were proved to promote the recruitment of stem cells to the bone defect area and effectively enhance regeneration.8 Both positively and negatively polarized ferroelectric crystal surfaces (LiNbO3, BiFeO3) could upregulate osteogenic differentiation of mesenchymal stem cells (MSCs), and the positive surface showed a better effect.9,10 For positively and negatively polarized hydroxyapa-

The osseointegration of metal implants determines the efficacy and longevity of bone repair1 and is closely related to the surface characteristics of the implant. The surface modifications of the implants in physical and chemical properties are extensively studied to promote osteogenic differentiation and bone tissue regeneration.2−4 Among various surface properties, surface electric potential has been shown to be able to upregulate cellular osteogenic differentiation and promote the osseointegration.5 Positively polarized poly(vinylidene fluoride) (PVDF) or poly(vinylidene fluoride-trifluoroethylene) (P(VDF-TrFE)) films6,7 demonstrated to be able to upregulate cellular © 2019 American Chemical Society

Received: April 24, 2019 Accepted: June 4, 2019 Published: June 4, 2019 22218

DOI: 10.1021/acsami.9b07161 ACS Appl. Mater. Interfaces 2019, 11, 22218−22227

Research Article

ACS Applied Materials & Interfaces tite, the positive surface was stronger in promoting MSC differentiation than the negative surface under surface charge density of 5.2 μC cm−2; however, the comparison result was reversed at charge density of 0.05 μC cm−2; the early bone consolidation of Wistar rats on the negative surface was better than that on the positive surface.11,12 Although the surface potential is well documented to have an ability to promote osteogenesis, the above comparative results9−13 (see Table S1) between positive and negative potential surfaces show a controversy; the ambiguity needs to be clarified under defined surface potential characteristics (positive or negative polarity as well as potential intensity). Here, we adopted a strategy to utilize a surface with wideranged potential variation and positive/negative polarity in an attempt to get insight into the understanding of surface potential-regulating cellular osteogenic differentiation under comprehensive consideration. In our previous work, P(VDFTrFE) piezoelectric films were easily polarized to have positive or negative surface potential and positively polarized P(VDFTrFE) films were used to evaluate the role of surface potential in promoting osteogenic differentiation of preosteoblast cells; CoFe2O4/P(VDF-TrFE) magnetoelectric films could give wide potential variation manipulated by applying magnetic filed,6,14 which was employed to evaluate the role of surface potential change in promoting osteogenic differentiation of osteoblast cells during the cell culture. In this work, we selected a stronger magnetostrictive TbxDy1−xFe2 alloy (Terfenol-D, TD) to substitute for CoFe2O4 to form TD/P(VDF-TrFE) magnetoelectric films, trying to create desired potential characteristics in wider potential intensity variation under an applied magnetic field and easily obtained positive/negative polarity as well as unchanged surface chemical composition and microstructure for different surfaces studied. The mesenchymal stem cells (MSCs) were used to evaluate the cellular responses to the two surfaces, and adsorbed protein status and osteogenesis-related FAK/ERK signaling pathway were also assayed. The results were discussed and the underlying mechanism was proposed.

Figure 1. Preparation method and microstructure of TD/P(VDFTrFE) magnetoelectric film. Preparation method of the film (A); scanning electron microscopy (SEM) images of the nonpoled film, positive potential film, and negative potential film (B−D); SEM images of the cross section of the nonpoled film (E); cross-sectional SEM images of the film (F), the mappings of Fe (G1), Tb (G2), Dy (G3), and C (G4) of the film.

2. RESULTS 2.1. Preparation and Characterization of TD/P(VDFTrFE) Magnetoelectric Film. The TD/P(VDF-TrFE) magnetoelectric films were prepared as reported in our previous study with minor modifications (Figure 1A). First, we explored the effects of TD weight content on the magnetoelectric properties of TD/P(VDF-TrFE) magnetoelectric film. The results indicated that the film with 13% TD showed the optimal magnetoelectric properties and could be stably polarized (Figure S2 and Table S2). Therefore, the 13% TD films were selected for the subsequent characterizations and cell culture evaluations. The surface morphology of the film showed a streaklike roughness, suggesting a good crystallization nature (Figure 1B). After electric poling, the surface morphology (Figure 1C,D) was observed with unchanged surface roughness (Ra) (Figure S3) and surface atomic ratio (Figure S4 and Table S3). The thickness of the film was estimated at 100 μm in the SEM image (Figure 1E). The cross-sectional SEM images and elemental mappings further showed a homogeneous dispersion of TD particles in the film (Figure 1E−G). In the X-ray diffraction (XRD) patterns (Figure 2A), the peaks were well matched to TD and P(VDF-TrFE) and the sharp peak at 19.9° in the nonpoled film, positive potential film

(P-film), and negative potential film (N-film) indicated that the films were crystallized well to the β crystalline phase of P(VDF-TrFE).15 The Fourier transform infrared (FTIR) spectrum of P(VDF-TrFE) (Figure 2B) was used to estimate the β phase content using eq 1, on the basis of the symmetric stretching of CF2 at the 840 and 1285 cm−1 bands and the CH2 rocking vibration at the 1400 cm−1 band, respectively.16 The β phase contents in the nonpoled film, P-film, and N-film were 67, 70, and 69%, respectively. The magnetization loops (Figure 2C) showed that the maximum magnetization of pure TD and the film was 55 and 6.8 emu·g−1, respectively, indicating TD in P(VDF-TrFE) matrix functioned well on basis of its fraction in the film. Magnetoelectric coupling measurements showed that the magnetic-field-induced surface potential (ϕME) of the P-film and N-film were almost symmetrical and the variation range was from 0 to ±120 mV (Figure 2D). On the basis of the magnetoelectric curve, the ϕME of 0, ±20, ±35, ±55, and ±120 mV could be obtained on the P-film or N-film under 0, 1400, 2100, 2800, and 3200 Oe magnetic-field intensity supplied by NdFeB magnet (Table S4), respectively. 2.2. Cellular Growth in Response to Surface Potentials on the Positive and Negative Potential Films. 2.2.1. Cellular Adhesion, Morphology, and Prolifer22219


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Figure 2. Characterization of the TD/P(VDF-TrFE) magnetoelectric film. XRD patterns (A), FTIR spectra (B), M−H loops (C), surface potential/ϕME (D).

Figure 3. Evaluation of the effects of a static magnetic field on adhesion (A) and proliferation (B) of MSCs. Cell adhesion (C) and proliferation (D) of MSCs on P-films and N-films.

ation. Because the surface potential in this work was controlled by applying a magnetic field, the influence of the magnetic field itself on the cellular growth was first evaluated (Figure 3A,B). The results indicated that the magnetic field with the intensity range of 0−2800 Oe had no significant influence on the cellular growth. Therefore, the safe range of magnetic field intensity in this work was set as 0−2800 Oe. Then, we explored the effect of surface potential characteristics on cellular viability. As shown in Figure S5, both P-films and N-films could enhance the cellular viability compared to that of the nonpoled films. However, the N-films seemed stronger in upregulating cellular viability than the P-films.

Furthermore, the optimal potential /ϕME/ for cellular adhesion and proliferation on both P-films and N-films was 20 mV (Figure 3C,D). For the cellular morphology after 1 and 3 day culture (Figure 4A,B), the cells grew in an elliptical shape on the Pfilms whereas they were rod-shaped on the N-films. There was an obvious difference in the cell area (Figure 4C,E) and the length−diameter ratio (Figure 4D,F) of the two films. For the cell spreading area, the cell area of P-films was larger than the cell area of N-films. On the P-film, the cell area showed a parabolic trend with /ϕME/ increasing and the area was the largest under /ϕME/ of 55 mV. On the N-film, the cell 22220


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Figure 4. Cellular morphology of MSCs on P-films and N-films. The nucleus is stained blue with 4′,6-diamidino-2-phenylindole (DAPI), and the cytoskeleton is stained red with F-actin. The confocal laser scanning microscopy (CLSM) images of MSCs cultured for 1 day (A) and 3 days (B), cytomorphometric evaluations of cell perimeter area for 1 day (C) and 3 day culture (E), and the length−diameter ratio for 1 day (D) and 3 day (F) culture.

spreading area has no significant change with /ϕME/. Compared with the MSCs cultured for 1 day, both the cell area on the P-films and the N-films increased after culturing for 3 days and the difference between the P-films and the N-films was relatively reduced. For the cell length−diameter ratio, the length−diameter ratio of N-films was larger than that of Pfilms. On the N-film, the cell length−diameter ratio showed a parabolic trend with /ϕME/ increasing and the length− diameter ratio was the largest under the /ϕME/ of 20 mV. On the P-film, the cell length−diameter ratio has no significant change with the /ϕME/. Compared with the MSCs cultured for 1 day, the cell length−diameter ratio on the N-films was further increased after being cultured for 3 days but there is no significant change on the P-films. 2.2.2. Cellular Osteogenic Differentiation. The result in Figure S5 showed that P-films and N-films with the initially

polarized potential could promote the osteogenic differentiation of MSCs. We further compared the MSCs osteogenic differentiation between P-films and N-films by evaluating the alkaline phosphatase (ALP) activities after culture for 7 and 14 days (Figure 5A,B). At /ϕME/ of 0−35 mV, the ALP expression of MSCs on the N-films was higher than on the P-films. When /ϕME/ reached 55 mV, the ALP expression on the P-films became higher. The expression of osteogenesis-related genes was also evaluated by reverse transcription polymerase chain reaction (RT-PCR). For the N-film, the osteogenic gene expressions (Runx2 and OCN) were significantly upregulated and showed a parabolic-like changing trend with /ϕME/ change and got the biggest expression at −20 mV. For the P-film, the osteogenic gene expression was only significantly upregulated at ϕME of +20 and +55 mV and the maximum value was obtained at +55 22221


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Figure 5. ALP activities and osteogenesis-related gene (OCN and Runx2) expressions of MSCs on P-films and N-films. The ALP activity of MSCs after culture for 7 days (A) and 14 days (B), the OCN relative expression of MSCs after culture for 7 days (C) and 14 days (D), the Runx2 expression of MSCs after culture for 7 days (E) and 14 days (F).

Figure 6. Assay of the exposed FN functional sites (HFN7.1 and mAb1937) at different /ϕME/. Exposed HFN7.1 sites (A) and exposed mAb1937 sites (B). k− or k+ indicates the slope of the curves for the number of the exposed site response to /ϕME/ on the N-films or P-films.

N-films was of no significant difference and higher than that of nonpoled films (Figure S7). Enzyme-linked immunosorbent assay (ELISA) assay results (Figure 6A,B) showed the amount of the exposed HFN7.1 (corresponding to the integrin recognition site RGD) and mAB1937 (corresponding to the integrin recognition site synergy PHSRN) sites of FN adsorbed on both P-films, and N-films exhibited a parabolic-

mV (Figure 5C−F). At low /ϕME/ (below 35 mV), the N-films showed a better osteogenic differentiation. However, at high /ϕME/ (more than 35 mV), the P-films become better than the N-films. These results were consistent with the ALP expression. 2.3. Adsorbed FN Conformation in Response to Surface Potentials on the Positive and Negative Potential Films. The FN adsorption density on P-films and 22222


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Figure 7. Western blotting assays of expressions of integrin (α5β1) and osteogenic signaling pathway of MSCs at different /ϕME/ (culture for 4 days, normalized to β-actin). The image of protein electrophoresis on the P-films (A), the image of protein electrophoresis on N-films (B), integrin β1 (C), integrin α5 (D), and osteogenesis-related signal p-FAK (E) and p-ERK (F).

the P-films at /ϕME/ of more than 35 mV and reversed when /ϕME/ increased to 55 mV.

like trend with /ϕME/, and the maximum value appeared at /ϕME/ of 20 mV. The amount of the exposed HFN7.1 and mAb1937 sites on the N-films was higher than that on the P-films, the difference in the amount of the exposed mAb1937 sites becomes small at /ϕME/ of more than 35 mV. The slope of the curves for the number of the exposed site response to /ϕME/ on the N-films (k−) was larger than that on the P-films (k+), suggesting that FN protein structure was more sensitive to negative potentials.17 2.4. Expressions of Integrinα5β1 and OsteogenesisRelated Signaling Pathway Protein FAK/ERK in Response to Surface Potentials on the Positive and Negative Potential Films. We evaluated the expression of integrinα5β1 and osteogenesis-related signaling pathway FAK/ ERK in response to ϕME. For the N-film, the relationship between the expressions of integrin α5β1 or FAK/ERK and ϕME behaved almost in a parabolic-like trend and the highest expression happened at ϕME of −20 mV (Figure 7C−F). In terms of the P-films, the relationship between the expressions of integrin α5β1 or FAK/ERK protein and ϕME behaved in a zigzag trend and the highest expression happened at ϕME of +55 mV (Figure 7C−F). The expressions of integrin β1 or FAK/ERK protein on the N-films were higher than those on

3. DISCUSSION It is believed that positive and negative potentials can effectively upregulate cellular osteogenic differentiation by promoting cell adhesion plaque formation and the expression of osteogenesis-related genes.8,18 However, the results were always controversial on the osteogenic capacity of positive/ negative potentials.9,11,13,19 We therefore designed such a surface with both positive and negative potentials and adjustable potential intensity. The TD/P(VDF-TrFE) magnetoelectric films after positive or negative polarization (Figure 1A) produced the positive or negative potential surfaces. Both surfaces showed similar surface morphology to exclude the possible effects of different surface geometry topologies on cellular responses. The homogenous distribution of TD particles in P(VDF-TrFE) (Figure 1G) also ensured a stable magnetoelectric behavior based on the integration of piezoelectric P(VDF-TrFE) with magnetostrictive TD under a magnetic field (Table S2 and Figure S1). The magnetic-fieldinduced surface potential (ϕME) of the films varied from 0 to ±120 mV (Figure 2D) in response to the applied 0−3200 Oe 22223


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Figure 8. Schematic diagram of spatial distribution of integrin recognition site (RGD and PHSRN) on FN regulated by surface potential.

magnetic field,14,20 providing a platform to investigate the effect of ϕ on cellular behaviors. Upon applying a magnetic field in the safe range of 0−2800 Oe (Figure 3A,B), the osteogenic differentiation level of MSCs on both P-films and N-films was upregulated and showed a similar trend and a parabolic-like curve with /ϕME/. However, there was a significant difference in /ϕME/ corresponding to the maximum value in the curve: 20 mV for the N-films and 55 mV for the P-films. The comparative result in MSCs osteogenic differentiation between the P-films and N-films strongly depended on the observing range of /ϕ/ (Figure 5). The cell-material interaction is controlled by the adsorption of protein on material surfaces.21 FN is considered as a major integrin-binding ankyrin that plays a significant role in cellular recognition and integrin-binding establishment.22 The conformation of FN significantly affects its binding to integrins. When more functional sites of HFN7.1 (RGD) and mAb1937 (synergistic PHSRN) are exposed and the distance of the two sites was close to 3.5 nm,23 a strong binding of cellular integrins with the adsorbed proteins could be established. As a result, the activation of the osteogenic-related signaling pathways or integrin-mediated osteogenic differentiation would be strengthened.24,25 The different cell morphologies on the P-films and N-films shown in Figure 4 implies that there is a significant difference in the FN adsorption conformation between the P-films and N-films.6,26 As shown in Figure 6, the amount of the exposed functional sites (RGD and PHSRN) on P-films and N-films showed a parabolic trend with /ϕME/ and both reached a maximum at 20 mV but the amount on N-films was significantly higher than that on P-films. This again indicates that the upregulation of the integrin-mediated osteogenic

differentiation does not depend only on the amount of the exposed functional sites. It has been demonstrated that the spatial distribution of the two sites of adsorbed FN also plays a critical role in establishing a full integrin binding, which well triggers the osteogenesis-related signaling pathway to upregulate integrinmediated osteogenic differentiation. However, the changing trend of the spatial distribution of the two sites of adsorbed FN with /ϕME/ was different between P-films and N-films.14,27 As in the schematic image shown in Figure 8, the FN structure on P-films tended to be “tight” with increasing /ϕME/; as a result, the spatial distance decreased, whereas the FN structure on Nfilms became “loose” with increasing /ϕME/ and the spatial distance correspondingly increased. When the spatial distribution approached a distance of 3.5 nm, full binding of integrin could be established to produce the strongest integrinmediated osteogenic differentiation. Hence, it is suggested that the full binding appeared at +55 mV for the P-films and −20 mV for the N-films according to the relations of osteogenic differentiation with /ϕME/ (Figure 7). For FAK/ERK as the main protein of the integrin-mediated osteogenic differentiation signaling pathway,28,29 expressions of the cells on P-films or N-films were well consistent with the expressions of integrin β1 and α5 with /ϕME/ (Figure 7), evidencing that the integrin-mediated signaling pathway upregulates the expressions of osteogenic-related genes and proteins. Comprehensively considering the effects of surface potential characteristics on osteogenic differentiation, as shown in Figure 9, the binding of adsorbed FN with integrins at relatively low potentials (0−20 mV) demonstrates a full binding status on Nfilms whereas there is a weaker binding status on P-films, 22224


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210 °C for 2 h; after natural cooling, crystallized films were obtained. The positive and negative potential films were obtained through electric poling of the nonpoled films by the homemade device for 3 min (Figure S8). Then, the films were first cleaned with Extran cleaner, followed by anhydrous ethanol and pure water. The TD weight contents of 10, 13, and 15% films were prepared to select the films with the available and optimal magnetoelectric effect. 5.3. TD/P(VDF-TrFE) Magnetoelectric Film Characterization. 5.3.1. Microstructure, TD Particle Distribution, and Roughness. The surface microstructure and the TD particle distribution of the film were analyzed by the field-emission scanning electron microscope (FE-SEM, Hitachi SU-70) equipped with an EDS module. An atomic force microscope (NTEGRA Spectra C) was employed to investigate the roughness of the films. 5.3.2. X-ray Diffraction (XRD) Analysis. An X-ray diffractometer (Thermo ARL X’TRA) was used to analyze the phase structure of the films; the test parameters were as follows: 2θ range of 10−60°; scanning rate of 2° min−1; and Cu Kα radiation source at 35 kV. 5.3.3. Fourier-Transformed Infrared Spectroscopy (FTIR) Analysis. The β crystalline phase content of films was analyzed by an attenuated total refraction infrared spectrometer (ART-FTIR, Nicolet 5700) according to all crystalline forms’ characteristic absorption bands and coefficients reported in ref 29, using the equation

Figure 9. Schematic diagram of MSCs’ osteogenic differentiation on P-films and N-films.

resulting in a higher osteogenic differentiation level on N-films than on P-films. As /ϕME/ increased (35−55 mV), the binding became a full binding status on P-films whereas it was weaker on N-films, leading to a reversal in the comparative result of osteogenic differentiation between N-films and P-films. Hence, the ambiguous appearance of the previous reports on the role of P-films and N-films could be ascribed to the difference in the selection of potential intensity /ϕ/ during the comparison.

F(β) =

Aα × 100% (Kα /Kβ)Aα + Aβ

(1)

The percentage of the β crystalline phase is shown by F(β), the absorbance and absorption coefficients of 766 and 840 cm−1 are represented by Aα/Aβ, and Kα/Kβ, and 6.1 × 104 and 7.7 × 104 cm2 mol−1 are the values of Kα and Kβ. 5.3.4. X-ray Photoelectron Spectroscopy (XPS) Analysis. XPS (Escalab 250Xi) was used to analyze the film surface chemical composition; the test parameters are base pressure (3.5 × 10−9 Torr) and Mg Kα (1253.6 eV) source. 5.3.5. Magnetic and Magnetoelectric Properties. The ZJ-3A quasi-state d33 meter (Acoustics Institute of Chinese Science Academy) was used to analyze the piezoelectric performance of the positive and negative potential films. A vibrating sample magnetometer (Cryogenic J3426) was used to analyze the magnetic susceptibility of the films. A multiferroic material magnetoelectric measure system (Super ME-II, Quantum Design) was used to analyze the magnetic-field-induced surface potential (ϕME). The test method has been reported in the literature.30−32 First, the copper wire was connected to the sample surface and the magnetoelectric test system by silver paste (Ted Pella); then, an alternating magnetic field (the direct current (dc)) was applied in the range of 0−3500 Oe and an alternating current (ac) with the intensity of 0.82 Oe and frequency of 5 kHz. The potential V was measured by the following equations

4. CONCLUSIONS The work demonstrated that the surface potential characteristics (positive or negative polarity and intensity) played a crucial role on upregulating the osteogenic differentiation of MSCs. Although both P-films and N-films could upregulate the osteogenic differentiation level in the potential intensity (/ϕME/) range of 0−55 mV, a higher osteogenic differentiation level occurred on the N-films at 0−35 mV, whereas it shifted to the P-films at 35−55 mV. Hence, the comparative result in upregulating osteogenic differentiation between P-films and Nfilms strongly depends on the selection of a specific potential intensity during the comparison. This work might be able to deepen our understanding on the role of material surface potential on cell behaviors. However, the differences in protein adsorption status and enhanced osteogenesis between P-films and N-films need to further be confirmed by computational simulation and in vivo evaluation, respectively. 5. EXPERIMENTAL SECTION 5.1. Materials. Terfenol-D (TD) particles were supplied by ETREMA Products, Inc. (Ames, IA); the mean size of the particles was ∼4 μm. N,N-Dimethylformamide (DMF) was supplied by Shanghai Aladdin biochemical technology Co. Ltd. (Shanghai, China). P(VDF-TrFE) was purchased from Piezotech (France). Ti plates were purchased from Baoji Hongshen titanium products Co. Ltd. (Baoji, China) with the size of 10 × 10 × 0.5 mm3 and cleaned by homemade mixed acid according to the method described before.14 5.2. TD/P(VDF-TrFE) Magnetoelectric Film Preparation. The preparation of the magnetoelectric film drew on the methods reported on.14,20 In short, the required amounts of TD particles and DMF were weighed and added to a suitable container and the container was placed in an ultrasonic generator to fully ultrasonically disperse the TD particles. Then, the P(VDF-TrFE) powers were added to the container and a Teflon stir bar was used to stir for 2 h under an ultrasound bath. Thereafter, the TD/P(VDF-TrFE) mixture was aspirated from the container and the mixture was spread on the cleaned Ti plates. Then, a muffle heat treatment was carried out at

α = ΔX /(Hac × d)

(2)

V = α × d × Hdc

(3)

The magnetoelectric signal voltage, thickness of film, and ac and dc magnetic field intensity were represented by ΔX, Hac, d, and Hdc, respectively. 5.4. Density and Conformation of FN. Human plasma FN was chosen to investigate the effect of the surface potential on the density and conformation of adsorbed proteins in this study. The micro BCA Protein Assay Kit (Beyotime Biotechnology, China) was used to measure the density of FN adsorbed on the films. First, the 200 μg mL−1 human FN (Sigma) was diluted to 10 μg mL−1 FN solution by phosphate-buffered saline (PBS, pH = 7.4). Then, the samples were immersed in a sufficient amount of 10 μg mL−1 FN solution at 37 °C for 1 day. Thereafter, the nonadsorbed FN was cleaned for the film by PBS and the adsorbed FN was dissolved by 2 mg mL−1 sodium dodecyl sulfate solution. The microplate reader was used to measure the optical density at 562 nm. The enzyme-linked immunosorbent assay (ELISA) was used to investigate the effect of surface potential on the conformation of 22225


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ACS Applied Materials & Interfaces adsorbed FN. Typically, the films were immersed in a sufficient amount of bovine serum albumin (BSA) solution at 37 °C for 1 day. Then, the films were kept in 10 μg mL−1 FN protein solution at 37 °C for 1 day. Next, the films were washed with PBS and 1% BSA/PBS blocking buffer was used to block the films at 37 °C for 30 min. The mAb1937 (Millipore) and Primary monoclonal antibodies HFN7.1 (Developmental Studies Hybridoma Bank) directed against the 8th type III repeat and the flexible linker between the 9th and 10th type III repeat, respectively.33 The specific experimental method was based on the report.14 5.5. Cell Culture. The Sprague-Dawley (SD) rats were used to extract the mesenchymal stem cells (MSCs). The culture of MSCs was carried out on the basis of previous reports.34 The minimum essential medium (MEM Alpha, Cellmax) was used to culture the MSCs at 37 °C with the CO2 content of 5% and a humid atmosphere, and 1% MEM nonessential amino acids, 10% fetal bovine serum (Cellmax), 10 000 μg mL−1 streptomycin, 1% sodium pyruvate, and 1% antibiotic solution containing 10 000 units mL−1 penicillin (all from Gibco) were added into the Medium. The homemade equipments were used to culture cell, as shown in Figure S9. The NdFeB permanent magnet provided the magnetic field, and the intensity is listed in Table S4. 5.6. Cell Vitality Assays. The 500 μL MSC suspension was seeded in each of the samples with a density of 1 × 105 cells mL−1 and cultured for 1 and 4 days in an incubator. The cell viability of the films was measured by the cell counting kit-8 (CCK-8, Dojindo Laboratories) essay. In short, the films were transferred to new plates and the 500 μL PBS was used to wash the films. Then, each well of the plate was added into a suitable quantity of cell counting kit-8 (CCK-8, Dojindo Laboratories, Kumamoto, Japan) solution and fresh culture media (ratio of 1:10) and incubated for 3 h (37 °C) in the incubator. Finally, the microplate reader was used to analyze the absorbance of samples at 450 nm. 5.7. Cell Immunofluorescent Staining. The 200 μL MSC suspension was seeded on each of the samples with a density of 1 × 105 cells mL−1 and cultured in an incubator for 1, 3, and 7 days, respectively. Then, the DAPI (Sigma) and Alexa-Fluor 594 phalloidin (Sigma) were employed to stain the nuclei and F-actin, respectively. LSM780 CLSM (ZEISS, Germany) was employed to visualize the samples after staining. The cell morphology data was analyzed by the software ImageJ. 5.8. ALP Assay. The 500 μL MSC suspension was seeded on each sample with a density of 1 × 105 cells mL−1 and cultured for 7 and 14 days in an incubator and the medium was changed every 2 days. The osteogenic induction medium was used to replace the ordinary medium, at the fourth day culture. When the training finished, the cells were lysed and proteins extracted. The LabAssay ALP purchased from Wako Pure Chemical Industries, Ltd (Japan) via was used to measure the ALP quantity by analyzing the OD value (at 405 nm), and Thermo Scientific BCA protein assay kit was used to assay the total protein content of the samples. 5.9. Quantitative RT-PCR Assays. The RT-PCR assay was used to measure the expressions of osteogenesis-related genes. The 500 μL MSC suspension was seeded on each sample with a density of 1 × 105 cells mL−1 and cultured for 7 and 14 days in an incubator. The miRNeasy Mini Kit (Qiagen, 217004) was used to collect the total RNA, and 1.0 μg of extracted RNA was used to obtain the cDNA by reverse transcribing. The Table S5 showed the primers for the target genes. The Mastercycler ep realplex equipped a SYBR Green I matrix was employed to implement RT-PCR analysis of the genes. 5.10. Western Blotting Assays. The expressions of integrin β1/ α5 and osteogenic signaling pathway protein p-FAK/p-ERK were analyzed by Western blotting. The 500 μL MSC suspension was seeded on each sample with a density of 1 × 105 cells mL−1 and cultured for 4 days in an incubator. After the training, the RIPA lysis buffer including 1 mM PMSF (Kangchen, China) was used to collect the total protein. The electrophoresis analysis was performed to analyze the extracted protein samples. The ECL plus reagents (Thermo Scientific) were used to analyze immunodetection, and ImageJ software was used to measure the intensity.

5.11. Statistical Analysis. Statistical analysis used Scheffe’s post hoc test and a one-way analysis of variance. All values are expressed as mean ± standard deviation. SPSS software was used to analyze the data. Differences were statistically significant when *p < 0.05, **p < 0.01, ***p < 0.001.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.9b07161. Magnetoelectric coupling schematic diagram and magnetoelectric properties of TD/P(VDF-TrFE) magnetoelectric film; piezoelectric properties of 13% TD/ P(VDF-TrFE) magnetoelectric film; high-voltage polarization device; surface average roughness (Ra) and surface chemical composition analysis (XPS) of nonpoled films and positive and negative potential films; homemade equipments for cell culture under a magnetic field; magnetic field intensity supplied by NdFeB magnet with different thicknesses equipped on the homemade equipment; RT-PCR primers; CLSM images of MSCs (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Lingqing Dong: 0000-0002-2203-3212 Kui Cheng: 0000-0003-4828-6450 Wenjian Weng: 0000-0002-9373-7284 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is financially supported by National Natural Science Foundation of China (51772273, 51872259, and 51472216) and the 111 Project under Grant No. B16042.

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