Harnessing Cell Dynamic Responses on Magnetoelectric

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Harnessing Cell Dynamic Responses on Magnetoelectric Nanocomposite Films to Promote Osteogenic Differentiation Bolin Tang, Junjun Zhuang, Liming Wang, Bo Zhang, Suya Lin, Fei Jia, Lingqing Dong, Qi Wang, Kui Cheng, and Wen-Jian Weng ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.7b19385 • Publication Date (Web): 07 Feb 2018 Downloaded from http://pubs.acs.org on February 16, 2018

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ACS Applied Materials & Interfaces

Harnessing Cell Dynamic Responses on Magnetoelectric Nanocomposite Films to Promote Osteogenic Differentiation Bolin Tang, † Junjun Zhuang, † Liming Wang, † Bo Zhang, § Suya Lin, † Fei Jia, † Lingqing Dong,*,†, ‡ Qi Wang,§ Kui Cheng,† and Wenjian Weng*,† †

School of Materials Science and Engineering, State Key Laboratory of Silicon

Materials, Zhejiang University, Hangzhou 310027, China ‡

The Affiliated Stomatologic Hospital, School of Medicine, Zhejiang University,

Hangzhou 310003, China §

Soft Matter Research Center and Department of Chemistry, Zhejiang University,

Hangzhou 310027, China

Corresponding Authors *E-mail: [email protected] (W.W.). *E-mail: [email protected] (L.D.).

Keywords: magnetoelectric, biomaterial, nanocomposite film, dynamic regulation, osteogenic differentiation

Abstract The binding of cell integrins to proteins adsorbed on materials surface is a highly dynamic process and also critical for guiding cellular responses. However, temporal dynamic regulation of adsorbed proteins to meet the spatial conformation requirement of integrins for a certain cellular response remains a great challenge. Here, an active CoFe2O4

(CFO)/poly(vinylidene

fluoride-trifluoroethylene)

(P(VDF-TrFE))

nanocomposite film, which was demonstrated an obvious surface potential variation (∆V≈93 mV) in response to the applied magnetic field intensity (0~3000Oe), was designed to harness the dynamic binding of integrins-adsorbed proteins by in-situ controlling the conformation of adsorbed proteins. Experimental investigation and

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molecular dynamic (MD) simulation confirmed the surface potential-induced conformational change in the adsorbed proteins. Cells cultured on nanocomposite film indicated that cellular responses in different time periods (adhesion, proliferation and differentiation) required distinct magnetic field intensity, and synthetically programming the preferred magnetic field intensity of each time period could further enhance the osteogenic differentiation through FAK/ERK signaling pathway. This work therefore provides a distinct concept that dynamic controllable modulation of material surface property fitting the binding requirement of different cell time periods would be more conducive to achieving desired osteogenic differentiation.

Introduction Cells are able to sense the microenvironment of materials surface through the binding of cell transmembrane integrins with proteins adsorbed on material surface.1-3 This binding is highly dynamic, in which, integrins incessantly probe and respond to surface protein cues by adjusting conformation of integrins ectodomain, resulting in the distinct binding states, which governs different cellular responses.4,5 As a major integrin-binding anchor protein on material surface, the adsorbed status of fibronectin (FN), including density, distribution, especially, conformation, is critical for the binding of integrins.6-8 Different FN conformation on material surface exposes different active binding sites for integrins, such as RGD, PHSRN, LDV sequence, producing the distinct binding states of integrin-FN, and then mediating downstream signals

that

control

cellular

responses,

such

as

adhesion,

proliferation,

differentiation.9,10 Some studies showed that the binding of integrin to only RGD-exposed FN presented a low affinity integrin-FN complex, which controlled the cell adhesion to material surface. With both RGD and PHSRN exposed on FN surface, the binding affinity of integrin-FN was significantly enhanced, which further improve cell proliferation, differentiation.11,12 These findings indicate that specific cell response requires a distinct binding states of integrin-FN. Therefore, harnessing the binding between integrin and FN in specific time period may control cellular responses, through temporal dynamic regulation of FN conformation. ACS Paragon Plus Environment

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As a soft protein, FN adsorbed on materials surface can adjust itself conformation by perceiving the change of material surface properties, such as composition, surface topography, surface potential.13-15 However, it is a great challenge to dynamically and precisely regulate adsorbed FN conformation in the time required, once material is implanted into the body. Maybe, the dynamic changes of material surface properties triggered by external stimulus give a new idea to address this issue. In recent years, some

publications

indicated

that

stimulus

responsive

materials,

such

as

piezoelectricity-responsive,16-19 temperature-responsive,20,21 photo-responsive,22 could obviously affect cell-material interaction and enhance cell adhesion, proliferation or differentiation under dynamic condition. However, the interfacial interactions between cells and material, i.e. integrin-adsorbed protein interactions, is not understood yet. Fortunately, the magnetoelectric composite materials composed of piezoelectric and magnetostrictive phase maybe bring a new hope for understanding the interactions of cell-material. In the presence of an applied magnetic field, the electrical polarization of piezoelectric phase will be changed arising from deformation of magnetostrictive phase, which leads to the variation of surface potential.23,24 Among various magnetoelectric materials, the polymer-based magnetoelectric composite materials attracted extensive attention, due to its easy formability.25 S. Lanceros-Mendez’ groups developed a large number of PVDF-based magnetoelectric composite materials, especially, ferrite/P(VDF-TrFE) and also made a lot of efforts to understand the magnetoelectric effect of polymer-based composite materials.26-29 Further, they found that magnetoelectric stimulus, i.e. electrical potential induced by intermittent application

of

magnetic

field

could

promote

cell

proliferation

on

PVDF-TrFE/Terfenol-D composite films.30 This indicated that magnetoelectric composite materials maybe provided an ideal platform to explore and dynamically control cellular responses. Nevertheless, the precise control of surface potential of magnetoelectric composite materials to meet the microenvironmental requirement of cell-material interaction is sought. In this work, the CFO/P(VDF-TrFE) nanocomposite film was designed, through the combination of magnetostrictive CFO nanoparticles with piezoelectric ACS Paragon Plus Environment


P(VDF-TrFE) matrix. The cellular responses on nanocomposite film in different time periods were investigated by temporal dynamic regulation of magnetic field. Moreover, this work shed light on the underlying molecular mechanism of cellular osteogenic differentiation on dynamic material surface, and provided a novel strategy for controlling cell fate through intelligently harnessing the interactions between cell and material in specific cell time period.

Results and discussion Preparation and characterization of CFO/P(VDF-TrFE) nanocomposite films The nanocomposite films were fabricated with a tape casting method (Figure 1A). With natural spreading, drying of CFO/P(VDF-TrFE) mixed solutions and annealing process in sequence, the unpoled nanocomposite films were prepared. After subjected to an applied electric field for reorientating electric dipole of piezoelectric P(VDF-TrFE), the poled nanocomposite films were obtained. Among the nanocomposite films with different CFO content, 10% nanocomposite film exhibited the homogeneous dispersion of CFO nanoparticles (Figure S1), good biocompatibility (Figure S2), especially, the best magnetoelectric performance (Figure S3). Thus, the 10% nanocomposite film was selected for subsequent studies. The surface morphology of 10% nanocomposite film was investigated by field-emission scanning electron microscope (FE-SEM) and atomic force microscope (AFM). A large number of streak-like whisker was observed on nanocomposite film (Figure 1B,C), indicating the crystalline nature of nanocomposite film. Before and after electric poling, no obvious change was observed on the surface morphology of nanocomposite film, as well as average surface roughness (Ra) (Figure S4). This sequesters the influences of surface roughness on cell responses. The thickness of nanocomposite film was measured to about 50 µm. A linear scanning of Fe, Co, O elements along the cross-section of nanocomposite film showed a homogeneous dispersion of CFO nanoparticles in the longitudinal direction (Figure 1D). The elemental mappings of Co, Fe and O further indicated a transverse uniform dispersion of CFO nanoparticles in P(VDF-TrFE) matrix (Figure 1E). The C 1s X-ray ACS Paragon Plus Environment

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photoelectron spectroscopy (XPS) of CFO/ P(VDF-TrFE) nanocomposite film (Figure S5) present four different C states, corresponding to -CF2-, -CFH-, -CH2- and organic contaminants, respectively.31 The atomic ratio of -CF2-/-CH2- with 0.76 for unpoled nanocomposite film and 0.78 for poled nanocomposite film (Table S1) indicated electrical poling produced negligible variation on surface chemical composition of nanocomposite film.

Figure 1. Preparation and characterization of CFO/P(VDF-TrFE) nanocomposite films. (A) Illustration for preparation of CFO/P(VDF-TrFE) nanocomposite films. (B,C) SEM images of 10% unpoled and poled nanocomposite film. (D) SEM images of cross-section of 10% poled nanocomposite film. (E) Elemental mappings of Co,



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Fe, O of 10% poled nanocomposite film.

The X-ray diffraction (XRD) patterns of nanocomposite film was recorded at room temperature (Figure 2A). The diffraction peaks at 2θ = 30.2° and 35.6° were indexed to (104) and (113) planes of rhombohedral CFO (JCPDS No. 79-1744). The strong diffraction peak at 2θ = 19.9° was assigned to the reflections of (110) and (200) planes of piezoelectric β phase P(VDF-TrFE).32 It is noteworthy that the stronger diffraction peak intensity at 2θ = 19.9° in poled nanocomposite film implied the increased β phase crystallinity of P(VDF-TrFE), ascribed to the rearrangement of electric dipole of β phase along electric field direction.31 Fourier transform infrared spectroscopy (FTIR) of nanocomposite film (Figure 2B) displays three β phase associated absorption bands, with 1288 and 840 cm-1 corresponding to the CF2 symmetric stretching with the dipole moments parallel to polar b axis, and the absorption band at 1400 cm-1 ascribed to the CH2 wagging vibration with the dipole moment along c axis.33 The more fraction of β crystalline phase in poled nanocomposite film (75.9%) than that in unpoled nanocomposite film (71.0%) confirmed that electrical poling enhanced piezoelectric β phase content of P(VDF-TrFE). For evaluating magnetic property, the magnetization loops of CFO and 10% nanocomposite film were investigated (Figure 2C). CFO powders and nanocomposite film developed a hysteresis loops with coercivity (Hc) of ~1900 Oe. The maximum magnetization with 61 emu·g-1 for pure CFO powders and 6.5 emu·g-1 for 10% nanocomposite film revealed that the maximum magnetization value of 10% nanocomposite film was approximately proportional to the amount of CFO nanoparticles in the P(VDF-TrFE) matrix. The original surface potential was determined to -5 ± 3 mV for unpoled nanocomposite film and 97 ± 12 mV for poled nanocomposite

film

through

scanning

kelvin

probe

microscopy

(SKPM).

Magnetoelectric coupling tests (Figure 2D) showed that the magnetoelectric coupling coefficient α of the unpoled nanocomposite film was absent (0 mV·cm-1·Oe-1), due to the unordered arrangement of electric dipole of β phase P(VDF-TrFE). However, obvious magnetoelectric coupling effect with a maximum value of 6.5 mV·cm-1·Oe-1 ACS Paragon Plus Environment

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was observed on the poled nanocomposite film. With further increasing the magnetic field, the decreased α was obtained, arising from the saturation of magnetostriction coefficient of CFO nanoparticles.34 The magnetic-field-induced surface potential of the poled nanocomposite film could be modulated from 0 to 93 mV. Herein, four different magnetic field (0, 1400, 2000 and 2600 Oe, corresponding to distinct surface potential) were adopted for subsequent biological assays.

Figure 2. Characterization of 10% CFO/P(VDF-TrFE) nanocomposite film. (A) XRD patterns and (B) FT-IR spectrum of nanocomposite film. (C) Hysteresis loops of CFO powders and nanocomposite film. (D) Magnetoelectric properties of nanocomposite film.

Cellular responses on poled nanocomposite film under different magnetic field The effects of nanocomposite film with different magnetic field on adhesion, proliferation and differentiation of MC3T3-E1 cells were investigated. Herein, cell culture time was divided into three different stages, mainly corresponding to cell adhesion (0~1 day), proliferation (2~4 days) and differentiation (5~7 days) periods, respectively (Figure 3A). The cell adhesion, proliferation and differentiation showed



no obvious difference on unpoled nanocomposite film under different magnetic field (Figure S6), suggesting the insignificant effect of magnetic field on cell responses. When cells were cultured on the poled nanocomposite film, the best cell adhesion (Figure 3B) and proliferation (Figure 3C) was observed at 2000 and 2600 Oe, respectively. In other words, the poled nanocomposite film created a favorable surface for cell adhesion at 2000 Oe and cell proliferation at 2600 Oe, also verified by Figure S7. As an important mark of osteogenic differentiation, the ALP activity of MC3T3-E1 cells showed no significant difference (Figure 3D), when magnetic fields were applied at only 5~7 days. This suggested that magnetic field applied at only cell differentiation period had a negligible influence on cellular osteogenic differentiation. Since the favorable magnetic field in different cell time periods are distinct, and the optimal magnetic field for cell adhesion and proliferation was 2000 and 2600 Oe, respectively, the combination of optimal magnetic field was carried out to evaluate the ALP activity. A significant enhancement of ALP activity on the 2000/2600/0 (Figure 3E) indicated that the combination of best adhesion and proliferation could dramatically enhance cellular osteogenic differentiation. It is noteworthy that the 0/2600/0 presented a higher ALP activity than 2000/0/0, which implied the cell proliferation period played a more important role in cellular osteogenic differentiation. Meanwhile, the higher ALP activity on the 2000/2600/0 than that on 2000/2000/0 and 2600/2600/0 further demonstrated that the temporal dynamic regulation of magnetic field was necessary for further enhancing osteogenic differentiation (Figure 3F). Furthermore, the quantified expression of osteogenesis-related gene including Runx2, Col-I and OCN was analyzed by real-time polymerase chain reaction (RT-PCR) (Figure 3G-I). The highest gene expression of Runx2, Col-I and OCN was observed on the 2000/2600/0 at 1 day, 4 days, 7 days and 14 days, also indicated the temporal dynamic regulation of magnetic field could enhance osteogenic differentiation of MC3T3-E1 cells.


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Figure 3. Cell responses of MC3T3-E1 cells on 10% poled CFO/P(VDF-TrFE) nanocomposite film under different magnetic field. (A) Cell culture time was divided into three different stages, mainly corresponding to cell adhesion (0~1 day), proliferation (2~4 days) and differentiation (5~7 days), respectively. (B-D) The effects of the nanocomposite film with different magnetic field at only cell adhesion (B), proliferation (C) or differentiation (D) periods (MF: magnetic field; ×: without cell culture). (E) The effects of combination of optimal magnetic field of cell adhesion and proliferation on cellular ALP activity. (F) ALP activity on the nanocomposite film with temporal dynamic regulation of magnetic field. (G-I) The gene expression of Runx2, Col-I and OCN on the nanocomposite film with temporal dynamic regulation of magnetic field. β-actin was used as housekeeping gene.

Cell morphologies on poled nanocomposite film under different magnetic field Cellular spreading morphologies on material surface have an important influence on cell differentiation. To study the effect of temporal dynamic regulation of magnetic field on cell morphologies, the cell nucleus and F-actin of MC3T3-E1 cells was ACS Paragon Plus Environment


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stained after seeded on poled nanocomposite film for 4 days. As observed in Figure 4A,B, cells attached well on all the poled nanocomposite film. Quantitative analysis showed that cells on the 2000/2600/0 achieved the largest cell area, perimeter, feret’s diameter, together with the lowest nuclear/cytoplasmic ratio (Figure 4C-F), indicated that the temporal dynamic regulation of magnetic field facilitated cell spreading.

Figure

4.

Cell

morphologies

of

MC3T3-E1

cells

on

the

10%

poled

CFO/P(VDF-TrFE) nanocomposite film under different magnetic field. (A) Confocal microscope images and (B) magnified images of MC3T3-E1 cells on the nanocomposite film with temporal dynamic regulation of magnetic field after 4 days of cell culture. F-actin was stained by Alexa-Fluors 594 phalloidin (red), and cell nuclei were stained by DAPI (blue). (C-F) Quantitative analysis of cell area, perimeter,


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feret’s diameter and nuclear/cytoplasmic ratio.

Interactions of cells with poled nanocomposite film under different magnetic field and MD simulation Surface potential of materials can strongly affect adsorbed status of proteins, such as adsorbed fibronectin, distribution, especially conformation.35 When cells contact with materials, the binding of cell surface integrin to protein adsorbed on materials surface play a key role in cell responses.36 As the major integrin-binding anchor protein, FN can be recognized by many different integrins, such as αVβ1, αVβ3, α5β1, and α8β1.37 A large number of studies demonstrated that the binding between integrin α5β1 and FN dramatically influenced cell adhesion, subsequent proliferation and differentiation.10,38,39 Thus, the gene expression of α5 and β1 subunits of cellular α5β1 integrin and absorbed FN density on poled nanocomposite film were tested. The results showed that the gene expression of α5 and β1 subunits was always the highest on the 2000/2600/0 (Figure 5A,B). For FN adsorption, a comparable adsorbed density was observed on the poled nanocomposite film with 2000 and 2600 Oe (Figure 5C), maybe ascribing to the saturation adsorption of FN. FN conformation was further investigated by enzyme-linked immunosorbent assay (ELISA). The specific monoclonal antibodies were able to probe conformational change and activity of FN, with HFN7.1 directed to the flexible linker between the 9th and 10th type III repeats of FN involved with cell adhesion domain (RGD) and mAb1937 bound to the 8th type III domain closer to the synergy domain (PHSRN) of FN.13 A higher HFN7.1 value on the poled nanocomposite film with 2000 Oe indicated the higher RGD availability. However, the poled nanocomposite film with 2600 Oe exhibited a higher mAb1937 value, suggesting the higher PHSRN availability (Figure 5D). The initial binding of integrin α5β1 to FN only depends on the interaction of β1 subunit with RGD sequence, resulting in a low affinity binding state of α5β1-FN (RGD), which decides cell adhesion. Afterwards, the further binding of α5 subunit to synergy PHSRN sequence switchs low affinity binding of α5β1-FN (RGD) to high affinity binding of α5β1-FN (RGD and PHSRN), activating subsequent cell responses such as proliferation, ACS Paragon Plus Environment


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differentiation.12,40,41 When magnetic field was changed to 2600 Oe, therefore, the increased PHSRN availability induced the up-regulated gene expression of α5 and β1 was observed on the 2000/2600/0. In addition, no significant difference was observed on the poled nanocomposite film with 2600 Oe and 2000/2600 Oe, which confirmed FN conformation was dependent on final magnetic field exposure. To understand the conformational change of FN on the poled nanocomposite film with different magnetic field at molecular level, MD simulation was carried out (Figure 5E). After obtaining the equilibrated structure of FN-Ⅲ7-10, a better RGD exposure was observed on the surface with 53 mV (corresponding to 2000 Oe). When surface potential was increased to 88 mV (corresponding to 2600 Oe), the RGD almost decreased to the same height as the PHSRN, which enhanced the PHSRN exposure, to a certain extent. More importantly, the distance between RGD and PHSRN significantly decreased, from 41 Å on 53 mV to 33 Å on 88 mV. It is reported that a ~35 Å of distance between RGD and synergy PHSRN strongly favor the binding of integrin α5β1 to FN. However, increasing distance of RGD-PHSRN would reduce the binding affinity of α5β1-FN.11,42,43

The

interaction

energies

indicated

the

driving

force

for

conformational change of FN-Ⅲ7-10 mainly originated form the van der Waals interaction between FN-Ⅲ7-10 and nanocomposite film. MD simulation further confirmed the conformational change of FN on poled nanocomposite film under different surface potential.


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Figure 5. Investigation of interactions of MC3T3-E1 cells with 10% poled CFO/P(VDF-TrFE) nanocomposite film. (A,B) Gene expression of α5 and β1 subunits of cellular α5β1 integrin on the nanocomposite film with temporal dynamic regulation of magnetic field. β-actin was used as housekeeping gene. (C) Adsorbed FN density and (D) conformation on the nanocomposite film with different magnetic field (2600 Oe represented FN was adsorbed at 2600 Oe for 12 h; 2000/2600 Oe ACS Paragon Plus Environment


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represented FN was adsorbed at 2000 and 2600 Oe for respective 6 h). (E) MD simulation for adsorbed conformation of FN-Ⅲ7-10 on the nanocomposite film with 53 and 88 mV after 10 ns MD simulation. Blue represented the same adsorbed residues. The interaction energy (Eint) between FN-Ⅲ7-10 and nanocomposite film included electrostatic interaction energy (Ees) and van der Waals interaction energy (EvdW).

Proteins expression assays and possible molecular mechanism The binding states of α5β1-FN govern cell differentiation through integrin-mediated FAK/ERK

signaling

pathway.44,45

Thus,

proteins

expression

of

integrin

α5β1-midiated FAK/ERK signaling pathway was investigated to understand the molecular mechanism of osteogenic differentiation on poled CFO/P(VDF-TrFE) nanocomposite film with the temporal dynamic regulation of magnetic field. As shown in Figure 6A-F, the expression level of all the protein including α5, β1, p-FAK and p-ERK were significantly up-regulated on the 2600/2600/0 at 4 days, compared with the 2000/2000/0 and 2600/2600/0. This is attributed that a transformation from low affinity binding state of α5β1-FN to high affinity binding state of α5β1-FN strengthen integrin-mediated FAK/ERK signaling pathway, when the temporal dynamic regulation of magnetic field was performed on poled nanocomposite film. Based on the results and discussion above, a mechanism including recognition and strengthening process between integrin α5β1 and FN is proposed for enhancing cellular osteogenic differentiation based on integrin-mediated FAK/ERK signaling pathway, as illustrated in Figure 6G. After cells are seeded on the poled nanocomposite film, a specific recognition between integrin α5β1 and FN adsorbed on the poled nanocomposite film will be followed. This recognition process is only depended on RGD availability of FN, coming into being a low affinity binding of α5β1-FN, which causes an insufficient activation of signaling pathway for cell differentiation. When FN conformation is adjusted to a higher PHSRN availability by altering magnetic field, the binding state of α5β1-FN significantly strengthens, forming a high affinity binding state of α5β1-FN. The high affinity binding state up-regulates expression of downstream p-FAK and p-ERK, which enhances ACS Paragon Plus Environment

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osteogenesis-related gene expression and cellular osteogenic differentiation.

Figure 6. Proteins expression of integrin-mediated FAK/ERK signaling pathway on 10% poled CFO/P(VDF-TrFE) nanocomposite film. (A-F) The relative expression level of α5, β1, p-FAK and p-ERK on the nanocomposite film with temporal dynamic regulation of magnetic field, normalized to β-actin. (G) Schematic diagram of cellular osteogenic

differentiation

on

magnetically

responsive

CFO/P(VDF-TrFE)

nanocomposite film with temporal dynamic regulation of magnetic field.

Conclusions In summary, we engineered an active CFO/P(VDF-TrFE) magnetoelectric nanocomposite film to harness temporal dynamic responses of MC3T3-E1 cells during different cell time periods. The surface potential of nanocomposite film could ACS Paragon Plus Environment


be modulated with applied magnetic field on demand. The optimal magnetic fields for cell adhesion and proliferation were 2000 and 2600 Oe, respectively. Moreover, synthetically programming the preferred magnetic field of cell adhesion and proliferation periods during cell growth could significantly enhance cellular osteogenic differentiation. The underlying mechanism of multi-period optimization to achieve desired osteogenic differentiation could be attributed to the temporal dynamic control of FN conformations, corresponding to the optimal ones for integrin α5β1 recognition (adhesion) and strengthening (proliferation and differentiation), which further activated integrin α5β1-mediated FAK/ERK osteogenic differentiation signaling pathway. This work provides a smart platform and new strategy for regulating cell responses by harnessing the dynamic binding between integrins and adsorbed proteins, which presents a promising potential for cell fate decision.

Methods Materials. CFO nanoparticles with the dimension of about 40 nm were purchased from Aladdin (Shanghai, China). P(VDF-TrFE) (70/30) powders were supplied by Piezotech (France). N,N-dimethylformamide (DMF), HF, concentrated HNO3, acetone and absolute ethanol were purchased from Sinopharm Chemical Reagent Co. Ltd. (Shanghai, China). Ti substrates (10 × 10 × 0.5 mm in dimensions) were obtained from Taizhou Huihuang new materials Co. Ltd. (Taizhou, China). Before using, Ti substrates were firstly treated in mixed solution of HF, HNO3 and H2O (1:2:4 in volume) for about 40 seconds. Then the treated Ti substrates were ultrasonically cleaned in acetone, absolute ethanol, and deionized water in sequence, followed by drying in ambient air. Preparation of CFO/P(VDF-TrFE) nanocomposite films. The desired amount of CFO nanoparticles was added to DMF and placed in an ultrasound bath for 6 h in order to obtain a good dispersion of CFO nanoparticles. P(VDF-TrFE) powders were then added into CFO solution and stirred mechanically for 2 h with supersonic vibration to avoid the agglomeration of CFO nanoparticles during the mixing process. After that, the mixed solution was spread on a clean Ti substrate and DMF ACS Paragon Plus Environment

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evaporation was completed at room temperature (RT). Crystallization of nanocomposite film was performed inside an oven at 210 °C for 1 h. After cooling down to room temperature, the CFO/P(VDF-TrFE) nanocomposite film was obtained on the Ti substrate. Herein, 5%, 10%, 20% and 30% (CFO weight content) was designed for selecting the nanocomposite film with optimal magnetoelectric effect. The poled nanocomposite film was obtained through electric poling of the unpoled nanocomposite film, with using an applied electric field for 5 min at RT in a home-made device with oil bath. Afterwards, the nanocomposite films were cleaned with Extran (Merck, Darmstadt, Germany) and distilled water to remove the silicone oil on the surface. Surface topographies. Surface topography of the CFO/P(VDF-TrFE) nanocomposite film was investigated by atomic force microscope (AFM, NTEGRA Spectra C) with a gold coated silicon nitride cantilevers (NSG013) and field-emission scanning electron microscope (FE-SEM, Hitachi SU-70) equipped with an energy dispersive X-ray spectrum (EDS, Inca Energy-200) at an accelerating voltage of 10 kV. The slice (a thickness of ~80 nm) of CFO/P(VDF-TrFE) nanocomposite films were obtained by cryosection system (Ultracut UC7, Leica), and the slice was observed by transmission electron microscopy (TEM, JEM-1230). X-ray diffraction (XRD) analysis. Phase structure of the nanocomposite film was analyzed by X-ray diffractometer (Thermo ARL X’TRA) using Cu Kα radiation source at 35 kV with a scan rate of 2° min-1 in the 2θ range of 10~80°. Fourier transform infrared spectroscopy (FTIR) analysis. FTIR were recorded by an infrared spectrometer (Nicolet 5700) with an attenuated total refraction (ATR) accessory (ART-FTIR). The fraction of the β crystalline phase can be estimated from the absorbance of the characteristic absorption bands of all crystalline forms and their absorption coefficients, as follows:

Fβ =

⁄

× 100%

(1)

Where F(β) represented the β phase content, Aα and AβⅢwere the absorbance at 766 and 840 cm-1; Kα and Kβ were the absorption coefficients at the respective



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wavenumber, which values were 6.1×104 and 7.7 ×104 cm2 mol-1, respectively.46 X-ray photoelectron spectroscopy (XPS) analysis. Surface chemical composition of the nanocomposite film was investigated with XPS (Escalab 250Xi) using Mg Kα (1253.6 eV) source at a base pressure of 3.5 × 10-9 Torr. Magnetic and magnetoelectric properties. The piezoelectric response (d33) of the nanocomposite film was analyzed with a quasi-state d33 meter (ZJ-3A, Institute of Acoustics, Chinese Academy of Science). Magnetic hysteresis loops of samples were measured using a vibrating sample magnetometer (VSM, Cryogenic J3426) with a step size of 1000 Oe from 0 to15000 Oe. The magnetoelectric coupling coefficient α was measured by a multiferroic materials magnetoelectric measure system (SuperME-II, Quantum Design). Silver electrode was coated on surface of nanocomposite films with room temperature-dried silver paste (Ted Pella) and then copper conductor was used to connect sample and magnetoelectric measure system. A dc (0~3000 Oe) and ac (0.82 Oe, 5 kHz) magnetic field were applied along the direction parallel to the surface of nanocomposite film. In magnetoelectric composites, α and magnetic-field-induced surface potential V could be determined by the following equations: α = ∆X / (Hac × d)

(2)

V = α × d × Hdc

(3)

Where ∆X, Hac, d and Hdc were the magnetoelectric signal voltage generated in the composite, ac magnetic field intensity, thickness of the magnetoelectric composite and dc magnetic field intensity, respectively.23-25 Fibronectin adsorption and conformation. In order to investigate the adsorbing capacity and conformation of cell adhesion-related protein, human plasma fibronectin (FN) was used as a model protein in this study. 10 µg/mL FN solution was prepared by using phosphate buffered saline (PBS, pH = 7.4) to dilute human fibronectin (200 µg/mL, Sigma, U.S.A.). FN adsorption was performed by immersing samples in 10 µg/mL FN solution at 37 °C for 12 h or 6 h/6 h. After adsorption, samples were washed twice with PBS by gentle agitation to remove non-adsorbed proteins. The adsorbed proteins on samples were then extracted with a 2 mg/mL SDS elution buffer. ACS Paragon Plus Environment

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The concentration of adsorbed fibronectin was analyzed by using a micro BCA Protein Assay Kit (Beyotime Biotechnology, China). Optical density was recorded at 562 nm using a microplate reader (Multiskan MK3, Thermo Scientific, U.S.A.). FN conformation was investigated by enzyme-linked immunosorbent assay (ELISA). The primary monoclonal antibody HFN7.1 (Developmental Studies Hybridoma bank, U.S.A.) directed against the flexible linker between the 9th and 10th type III repeat of FN and mAb1937 (Millipore, Billerica, MA) directed against the 8th type III repeat of FN was used.13 The samples were immersed in 10 µg/mL FN solution for 12 h or 6 h/6 h at 37 Ⅲ. The FN-adsorbed samples were then washed with PBS to remove non-adsorbed FN, and non-specific antibody binding was blocked in 1% BSA/PBS blocking buffer for 30 min at 37 Ⅲ. The sample was then incubated with HFN7.1 (1:4000 in blocking buffer) and mAb1937 (1:1000 in blocking buffer) for 1 h at 37 Ⅲ. After rinsed with 0.1% Tween 20/PBS, all samples were incubated with a secondary antibody (alkaline phosphatase-conjugated anti-mouse IgG, Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) for 1 h at 37 Ⅲ. After rinsed again, the 4-methylumbelliferyl phosphate (4MUP, Sigma) was added to the samples for 45 min at 37 Ⅲ. The reaction products were quantified by using a fluorescence microplate reader (Gemini XPS, Molecular Devices, U.S.A) at 365 nm excitation/460 nm emission. Molecular dynamic (MD) simulation. The initial structure for the simulations was obtained from refined X-ray crystal structures of FN-III7-10 by Protein Data Bank (PDB entry: 1FNF). The parameter of P(VDF-TrFE) was obtained from the general AMBER force field (GAFF) using AmberTools.47 The HF 6-31G+d restrained electrostatic potential (RESP) charge was calculated by Gaussian 03, which was used for the charge method of GAFF.48 A 3 layers of P(VDF-TrFE) bulk model (VDF:TrFE =7:3) with a dimension of 10.02 × 7.65 × 2.45 nm3 was constructed and the interlayer spacing was set to 0.38 nm, and the in-plane spacing between the polymer chains was 0.46 nm, according to these references.49,50 The of the resulting P(VDF-TrFE) structure was optimized under an electric filed (56V/µm, simulating poling electric field). After 10 ns MD simulations (Gromacs 5.0.7), the optimized configuration of ACS Paragon Plus Environment


P(VDF-TrFE) was obtained. Visual molecular dynamics (VMD) graphics software was used to inspect trajectory and view molecules. FN-III7-10 was pulled close to the substrates using the method of steered molecular dynamics (SMD) at a constant force of 100 kJ mol-1 nm-2. The conformations of different contact area between FN-III7-10 and P(VDF-TrFE) substrate were selected. After that, the system was solvated using the SPC water molecule models and neutralized by several sodium ions (Na+). Along with energy minimization, an NVT MD simulation of 10 ns was performed to make sure that the FN-III7-10 contacted with P(VDF-TrFE) substrate appropriately by restraining backbone of the P(VDF-TrFE). The most stable conformation was selected according to lowest binding energy and least conformation transformation. Then, a relatively reliable structure was achieved. Periodic boundary conditions were applied in NVT MD and SMD. These simulations were carried out with a time step of 2 fs. The electrostatic interactions were calculated using particle-mesh Ewald (PME),51 and the Lennard-Jones potential was calculated by Lorentz-Berthelot rule for non-bonded interaction of molecules, i.e. van der Waals interaction.52 The cut-off of electrostatic and van der Waals interaction were both set as 1.2 nm. The LINCS algorithm was used for constraining the bond length.53 The temperature of the system was kept at 310 K by Nose-Hoover thermostat method.54 After simulation, the obtained every state was observed using VMD. The interaction energy (Eint) between FN-III7-10 and P(VDF-TrFE) was summed by electrostatic interaction energy (Ees) and van der Waals interaction energy (EvdW). Both of them were calculated by Gromacs. Cell culture. MC3T3- E1 cells (preosteoblastic cells, CRL-2594, ATCC, Manassas, VA) were cultured with α-modified Minimum Essential Medium (α-MEM, Gibco) supplemented with 10% fetal bovine serum (PAA Laboratories Pty Ltd.), 1% sodium pyruvate (Gibco), 1% MEM nonessential amino acids (Gibco) and 1% antibiotic solution containing 1×104 units/mL penicillin and 10 mg/mL streptomycin (Gibco) under a humidified atmosphere of 5.0% CO2 at 37 °C. MC3T3-E1 cells in culture polystyrene were trypsinized with 0.25% trypsin containting 1 mM ethylenediamine triacetic acid (Gibco) and were subcultured on samples. During cell culture, the magnetic field was applied by a pair of NdFeB permanent magnet (Yantai Magnet Co. ACS Paragon Plus Environment

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Ltd., Shanghai, China) placed parallelly in polymethylmethacrylate (PMMA) support (self-designed) and an 8-well polytetrafluoroethylene (PTFE) plates was placed in the middle of PMMA support, as shown in Figure S8. Magnetic field in different position was measured by a HT 100G digital tesla meter (Lichun Precise Instrument Co. Ltd., Shanghai, China). The magnetic field intensity generated by different thickness of NdFeB permanent magnet was listed in Table S2. Cell attachment and proliferation assays. The MC3T3- E1 cells were seeded on samples placed in 8-well PTFE plates at a density of 3 × 104 cells/well. After culturing for 1, 4, and 7 days, the cell counting kit-8 (CCK-8, Dojindo Laboratories) assay was used to assess cell attachment and proliferation. In brief, the samples were transferred to new 24-well plates and washed at the designed time points. 500 µL of medium and 50 µL of CCK-8 solutions were then added and incubated at 37 °C for 2 h. Finally, the absorbance was detected at 450 nm. Alkaline phosphatase assay. To demonstrate the primary osteogenic effect, alkaline phosphatase (ALP) activity was tested. The MC3T3-E1 cells (3.0 × 104 cells/well) were seeded on samples (three replicates). After incubation for 7 days, the samples were transferred to new 24-well plates and were rinsed three times (PBS solution). The cells on samples were lysed with CelLytic Buffer (Sigma). The supernatants of cell lysate were collected for ALP activity measurement at a wavelength of 405 nm. The ALP activity was expressed as U (nmol/µL) per mg of protein. The total protein content was analyzed using a BCA protein assay kit (Thermo Scientific, U.S.A.). Immunofluorescent staining. The MC3T3-E1 cells (1.0 × 104 cells/well) were seeded on samples. After 4-day cell culture, the samples were fixed with 4% paraformaldehyde for 15 min, permeabilized with 0.05% Triton X 100 in PBS, and then blocked in PBS solution. The F-actin were stained by Alexa-Fluors 594 phalloidin (Sigma) and nuclei were stained by DAPI (Sigma) according to the manufacturer’s protocols. After staining completed, samples were visualized by a confocal laser scanning microscopy (LSM780, ZEISS, Germany). ImageJ software were used for quantitative analysis of cell morphologies. Quantitative Real-Time polymerase chain reaction (RT-PCR) assays. The ACS Paragon Plus Environment


expression of integrin and osteogenesis-related genes was measured through RT-PCR assay. The MC3T3-E1 cells (3.0 × 104 cells/well) were seeded on each sample (three replicates). The total RNA was extracted using miRNeasy Mini Kit (Qiagen, 217004) and the cDNA was reverse transcribed from 1.0 µg of RNA. The primers for the target genes were listed in Table S3. RT-PCR analysis of genes was performed using the Roche LightCycler480 system with a SYBR Green I matermix. The ∆∆cT method was used to calculate relative gene expression with β-actin as reference gene (housekeeping gene). Western blotting assays. Western blotting was performed to analyze integrin α5, β1, p-FAK and p-ERK. Briefly, MC3T3-E1 cells (3 × 104 cells/well) were seeded on each sample. After 1 day, 4 days of culture, the total protein of cells were extracted by protein extraction reagent (RIPA lysis buffer) including 1 mM phenylmethanesulfonyl fluoride (PMSF) (Kangchen, China). Protein samples were then subjected to SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocked in by 5% skim milk powder for 1 h at room temperature, the membranes were incubated with primary antibodies overnight at 4 Ⅲ and horseradish peroxidase (HRP)-conjugated secondary goat-anti-rabbit antibody (Thermo Pierce) for 2 h at room temperature. These primary antibodies included anti-integrin α5, anti-integrin β1 antibodies (Abcam), anti-p-FAK, anti-p-ERK antibodies (Cell Signaling Technology) and anti-β-actin antibody (Santa Cruz). Immunodetection was performed with chemiluminescence through using ECL plus reagents (Thermo Scientific) and quantified with Image J software. Statistical analysis. All data were expressed as mean ± standard deviation (triplicate). Statistical analysis was performed by one-way ANOVA with Tukey’s post hoc test. A value of p < 0.05 was considered statistically significant (*p < 0.05, **p < 0.01, ***p < 0.001).

Supporting Information Additional Table (Table S1-S3) and Figures (Figure S1-S8). TEM images (Figure S1), biocompatibility (Figure S2), magnetoelectric properties ACS Paragon Plus Environment

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(Figure S3), AFM images (Figure S4) and XPS spectra (Figure S5) of the nanocomposite films; Cell responses on the nanocomposite films (Figure S6 and S7); Photograph of cell culture device (Figure S8); Summary of XPS spectra (Table S1); The correlation of magnet thickness with magnetic field intensity (Table S2); Primers used for RT-PCR (Table S3).

Acknowledgements This work is financially supported by National Natural Science Foundation of China (51772273, 51472216, 51502262), the 111 Project under Grant No. B16042, and the Postdoctoral Science Foundation of China (Grant No. 2017M621923).

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