This article is licensed under CC-BY 4.0 www.acsami.org Research Article Enhanced Proliferation and Differentiation of Human Osteoblasts by Remotely Controlled Magnetic-Field-Induced Electric Stimulation Using Flexible Substrates Oriol Careta, Aliona Nicolenco, Filippos Perdikos, Andreu Blanquer, Elena Ibañez, Eva Pellicer, Christina Stefani, Borja Sepúlveda, Josep Nogués, Jordi Sort,* and Carme Nogués* Cite This: ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 Read Online ACCESS Metrics & More Article Recommendations * sı Supporting Information ABSTRACT: With the progressive aging of the population, bone fractures are an increasing major health concern. Diverse strategies are being studied to reduce the recovery times using nonaggressive treatments. Electrical stimulation (either endogenous or externally applied electric pulses) has been found to be effective in accelerating bone cell proliferation and differentiation. However, the direct insertion of electrodes into tissues can cause undesirable inflammation or infection reactions. As an alternative, magnetoelectric heterostructures (wherein magnetic fields are applied to induce electric polarization) could be used to achieve electric stimulation without the need for implanted electrodes. Here, we develop a magnetoelectric platform based on flexible kapton/FeGa/P(VDF-TrFE) (flexible substrate/magnetostrictive layer/ferroelectric layer) heterostructures for remote magnetic- field-induced electric field stimulation of human osteoblast cells. We show that the use of flexible supports overcomes the clamping effects that typically occur when analogous magnetoelectric structures are grown onto rigid substrates (which preclude strain transfer from the magnetostrictive to the ferroelectric layers). The study of the diverse proliferation and differentiation markers evidence that in all the stages of bone formation (cell proliferation, extracellular matrix maturation, and mineralization), the electrical stimulation of the cells results in a remarkably better performance. The results pave the way for novel strategies for remote cell stimulation based on flexible platforms not only in bone regeneration but also in many other applications where electrical cell stimulation may be beneficial (e.g., neurological diseases or skin regeneration). KEYWORDS: magnetoelectric heterostructure, flexible biomaterial, magnetoelectric stimulation, wireless actuation, proliferation, differentiation, osteoblasts 1. INTRODUCTION bone regeneration.6 These electric potentials can be naturally Due to the increased aging of the population, bone fractures induced through piezoelectricity, which is attributed mainly to are a major health concern which causes an increasing collagen.7 Some authors have suggested that bone endogenous economic burden.1 To mitigate the diverse effects of bone electricity enhances the process of bone healing by the fractures, reducing the recovery time of patients is fundamental. Hence, diverse strategies to help the healing of fractured bones are being investigated. Applied or induced Received: June 30, 2023 electric charges have been shown to play a fundamental role in Revised: November 17, 2023 promoting cell differentiation and proliferation.2−4 Endoge- Accepted: November 20, 2023 nous currents related to inherent mechanical strains are Published: December 5, 2023 important for bone remodeling.2,5 In fact, during recovery, bones are capable of generating electric potentials that facilitate © 2023 The Authors. Published by American Chemical Society https://doi.org/10.1021/acsami.3c09428 58054 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article Scheme 1. Schematic Representation of the Cell Stimulation Approach by Using Magnetoelectric Materials; (a) Without Magnetic Field, No Electric Field is Induced and, Thus, the Cell is Not Stimulated; (b) When a Magnetic Field is Applied, the Magnetostrictive Layer (Black) Changes Its Shape or It Expands/Contracts, Thus Transferring Strain to the Adjacent Ferroelectric Layer (Gray) in Which Electric Charges (+) Are Created, Thus Generating an Electric Fielda a This electric field stimulates the cells to promote proliferation and differentiation, probably by the opening of the voltage-gated calcium channels (VGCCs) and the stretch-activated calcium channels (SACCs), which allow for the inward flow of Ca2+ ions. This is possible when a flexible substrate is used (in this case, kapton, depicted in orange color). MR: membrane receptor. stimulation of the calcium−calmodulin pathway, which but using, instead, the mechanical and chemical energies induces the upregulation of several promoters of bone present in the body.17 In this framework, piezoelectric/ regeneration, such as bone morphogenetic proteins, trans- flexoelectric materials are excellent candidates to assist in this forming growth factor-β, and other cytokines.8−10 Remarkably, process since they are able to generate electrical voltage in application of exogenous electricity in large bone fractures response to mechanical stress.18,19 As aforementioned, the where the bone regeneration is impaired has been demon- bone itself has inherent piezoelectric/flexoelectric properties, strated not only to enhance osteoblast proliferation but also to producing several electrical and biochemical signals that regulate proteoglycan and collagen synthesis, as well as to enhance its growth in response to mechanical activity. accelerate bone formation and repair.11−13 Similarly, mechanical stimulation of piezoelectric materials Traditionally, electrical stimulation has been applied to cells can be achieved either by the application of physiological in vitro studies through direct current stimulation using compressive loads,20 by the stress applied by cells growing on electrodes either in direct contact with a conductive substrate the material surface,21 or, most commonly, by ultrasounds on top of which the cells are grown or contacting the culture stimulation.22 medium. Even though the use of electrodes is the easiest Even though the use of piezoelectric materials stimulated by option and the most widely used method, it presents some the cells growing on their surface represents a great important drawbacks such as insufficient biocompatibility of improvement compared with traditional electrode stimulation the electrodes’ components or the conductive substrates14 or (i.e., avoiding direct contact between cells and electrodes), the hazardous changes induced in the medium such as temperature fact that both the generated electrical potential and the rise, pH variations, or the generation of harmful byproducts.15 application time cannot be controlled constitutes a drawback In addition, in vivo applications, the use of electrodes can lead for their use. For this reason, new ways to mechanically to important inflammation effects or even infections.16 stimulate piezoelectric materials remotely in a more control- Because of these disadvantages, efforts have been made to lable way, such as using ultrasounds, are being developed. develop energy harvesting materials to induce electrical However, ultrasounds are known to have several limitations for stimulation without the need of direct insertion of electrodes, their use on biological tissues, such as limited penetration, local 58055 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article temperature increase (which can be particularly significant in over, it is easy to grow and is quite resistant to oxidation. In bones), cavitation, or acoustic streaming, just to mention a addition, prior investigations within our research group have few.23,24 These can result in cell detachment, changes in confirmed its noncytotoxic nature.36 These combined proper- membrane permeability, or even cell lysis. Additionally, the ties make it ideal for biomedical applications. The choice of frequencies used in ultrasounds experiments are exceedingly ferroelectric/piezoelectric material [i.e., P(VDF-TrFE)] was high (in the MHz range, i.e., ns−μs stimulation) for the typical supported by its strong ferroelectric response despite being response of cells (which commonly occurs in the ms range).25 flexible and lead-free. Moreover, the cytocompatibility of Thus, in recent years, alternative stimulation pathways are P(VDF-TrFE) has been demonstrated in previous studies.37,38 being explored.26 Among them, magnetoelectric actua- Finally, the use of kapton is a keystone of our study, since tion,27−30 which uses rather low intensity, highly penetrating, contrarily to most of the substrates used in magnetoelectric cell magnetic fields at low frequencies, is emerging, with stimulation (e.g., silicon), it is rather flexible (see Figure S1; stimulation times that are commensurate with the cell with a Young’s modulus of about 2.5 GPa). The flexibility of response. Magnetoelectric materials are materials in which the kapton substrate was expected to improve the efficiency of electric polarization can be generated by magnetic fields or, the system compared with analogous heterostructures grown conversely, magnetization can be modulated with electric on rigid substrates (with strong clamping effects). fields. A common approach to this type of materials is to 2.1. Characterization of the FeGa/P(VDF-TrFE) Heter- combine a ferromagnetic magnetostrictive layer with a ostructures. The scanning electron microscopy (SEM) image ferroelectric or piezoelectric layer.31,32 When these structures of the sputtered FeGa layer shows its polycrystalline structure are exposed to a magnetic field, electric polarization is (Figure 1a). The energy-dispersive X-ray spectroscopy (EDX) generated by the strain induced in the magnetostrictive analysis reveals that the Fe/Ga atomic ratio is 72:28 (±5%) material, which is transferred to the adjacent ferroelectric (Figure 1c, i.e., close to the alloy composition that is known to layer (see Scheme 1). More specifically, when subject to an exhibit the highest magnetostriction coefficient for this applied magnetic field, the magnetostrictive layer deforms alloy39). The X-ray diffraction (XRD) pattern (inset in Figure because of the magnetostrictive effect. This change in shape is transferred to the adjacent ferroelectric layer since they are both mechanically coupled. In turn, the change of shape of the ferroelectric layer induces an electric polarization due to the ferroelectric effect. Nonetheless, the performance of this type of systems is often limited by clamping effects, i.e., the expansion-contraction constraints imposed by the rigid substrates on top of which these structures are typically grown.33,34 As a consequence, when growing magnetoelectric heterostructures onto rigid substrates, the effects are small even when using high magnetic fields, high frequencies, and very long stimulation times. Diverse approaches are being developed to reduce clamping effects, such as the implementation of 3D magnetoelectric structures or the use of magnetoelectric membranes.35 However, so far, magnetoelectric systems have demonstrated only a moderate improvement in bone regeneration experi- ments. Thus, novel approaches to improve the efficiency of bone cell proliferation and differentiation are highly desirable. Here, we present the use of a flexible magnetoelectric heterostructure [kapton/FeGa/P(VDF-TrFE)], which is able to produce an electrical response upon application of alternating magnetic fields. This magnetoelectric stimulation is efficient in remotely stimulating osteoblasts in vitro (Scheme 1). 2. RESULTS AND DISCUSSION The material under investigation consists of a flexible kapton substrate on which a magnetostrictive FeGa layer is grown. The top layer of the magnetoelectric heterostructure is a ferroelectric polyvinylidenefluoride-co-trifluoroethylene [P(VDF-TrFE)] layer (see Materials and Methods). The growth of adjacent ferromagnetic/ferroelectric layers in direct Figure 1. (a) SEM image of the sputtered FeGa films; (b) SEM image contact between each other allows the whole deformation of of the top P(VDF-TrFE) layer; (c) SEM image taken on one edge of the sample showing the P(VDF-TrFE) layer deposited on top of the magnetostrictive layer induced by the magnetic field to be FeGa (the sample was slightly tilted on purpose to provide the transferred to the ferroelectric layer, consequently maximizing perspective) together with a representative EDX analysis to assess the the generated electric field. Concerning the choice of materials, composition of the FeGa films; and (d) room-temperature magnetic FeGa was selected as the magnetostrictive layer for several hysteresis loops applying the magnetic field along in-plane and reasons. First, FeGa has one of the largest magnetostrictive perpendicular (out of plane) directions of the film. The inset in (d) constants of rare-earth-free magnetostrictive materials. More- shows the XRD pattern of the FeGa film. 58056 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 2. (a) XRD patterns of the P(VDF-TrFE) films grown onto kapton/FeGa (blue curve) and onto kapton (red curve)�the black curve is the XRD pattern corresponding to bare kapton with no layers grown on top (the cut in the 2θ axis is to avoid a peak from the sample holder); (b) FTIR experiments of the same samples as in panel (a), where the peaks shadowed in gray univocally correspond to the β-phase of P(VDF-TrFE); (c) PFM phase image obtained after writing two concentric squares (ferroelectric domains) with opposite polarity, with an applied ±9 DC voltage; and (d) local piezo-response phase and (e) amplitude loops obtained from the FeGa/P(VDF-TrFE) heterostructured films without (H off) and with (H on) an external magnetic field of 1000 Oe, applied along the in-plane direction of the sample. 1d) indicates that the FeGa film grows in the expected body phase is obtained by Fourier transform infrared (FTIR) centered cubic (bcc) structure, and it is textured along the measurements48 (shown in Figure 2b). Note that the inclusion (110) direction. No evidence for phase separation (i.e., of the FeGa layer has no effect on the formation of the β- occurrence of Fe-rich or Ga-rich phases) or Fe/Ga oxides phase. was observed by XRD. The magnetic characterization of the 2.2. Ferroelectric Properties of the FeGa/P(VDF-TrFE) films (Figure 1d) reveals a clear in-plane effective magnetic Heterostructures. The ferroelectric properties of shape anisotropy, with a very square and low-coercivity in- P(VDF-TrFE) were assessed by piezo-response force micros- plane hysteresis loop and a tilted out-of-plane loop with larger copy (PFM). The layer was prepoled by applying a constant coercivity. Such coercivity increase in the out-of-plane DC bias through the tip of −9 V in a 5 × 5 μm2 squared area. hysteresis loops has been observed by several authors in After the frame was completed, a consecutive, concentric FeGa layers grown by different methods, and it has been square of 2.5 × 2.5 μm2 was performed with a constant DC ascribed to the effects of competing magnetic anisotropies.40,41 bias of the opposite magnitude, +9 V (Figure 2c). The ability The SEM image of the P(VDF-TrFE) film grown onto FeGa to switch the ferroelectric polarization by applying external can be seen in Figure 1b,c. The layer grows homogeneously on electric fields leading to stable domains after electric field is FeGa and is clearly polycrystalline with a grain size larger than switched off demonstrates the good ferroelectric properties of that of FeGa. P(VDF-TrFE). Further evidence of ferroelectricity is obtained The XRD patterns of the P(VDF-TrFE) spin-coated onto from the phase versus voltage bias loop (Figure 2d), where an kapton/FeGa are shown in Figure 2a (blue curve) together electric coercive voltage of around 6 V is observed (hence with that of bare kapton with no layers on top (black curve) smaller than the DC bias strength used to switch the and P(VDF-TrFE) grown onto the kapton substrate using the polarization in Figure 2c). In addition, the amplitude versus same growth conditions (red curve). The peak at 2θ = 20.1° bias curve shows the typical “butterfly” shape dependence of a corresponds to the (110)/(200) reflections of the β-phase of ferroelectric material (Figure 2e)44 Note that the phase P(VDF-TrFE),42 and it is superimposed to the kapton switching angle is close to 180°, indicating a major diffraction background.43 Importantly, the β-phase of P- contribution of the electromechanical response over the (VDF-TrFE) is the one showing optimum ferroelectric electrostatic one. The asymmetry of the butterfly loops properties.44−47 Further evidence for the formation of the β- might originate from different factors such as a dissimilar 58057 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article work function at the bottom (FeGa) and top (PtIr-coated Si probe) electrodes or occurrence of internal electric field distribution across the P(VDF-TrFE) layer.49 2.3. Magnetoelectric Coupling of FeGa/P(VDF-TrFE) Layers. Next, the magnetoelectric coupling between the FeGa and P(VDF-TrFE) layers was directly probed by PFM at a given location of the sample, comparing the phase and amplitude curves without a magnetic field and with an applied in-plane magnetic field of 1000 Oe. Note that this magnetic field is sufficient to magnetically saturate the FeGa layer, thus ensuring a significant effect of magnetostriction on the eventual strain-mediated magnetoelectric coupling. In the absence of external magnetic field, the coercive voltages for the FeGa/ P(VDF-TrFE) heterostructure are −5.8 and +6.6 V, giving an average coercive electric field of 62 MV m−1, which is a typical value for P(VDF-TrFE).47 Application of the magnetic field Figure 3. AFM topological images (top) and contact angle results in an overall decrease of the coercive electric field to a measurements (bottom) of (a) the as-grown and (b) the oxygen value around 55 MV m−1. This reduction of the coercive plasma-treated FeGa/P(VDF-TrFE) heterostructures. electric field is in agreement with previous results from the literature44 and it indicates a decrease of the energy barrier to 2.5. Effect of the Magnetic-Field-Induced Electrical switch the ferroelectric polarization. This suggests that the Stimulation on Osteoblast Viability, Morphology, and induced magnetostrictive strain in the FeGa film is transferred Adhesion. Human osteoblast cells (hOBs) were seeded on to P(VDF-TrFE), facilitating voltage-driven reorientation of top of the magnetoelectric heterostructures and kept under the ferroelectric domains. In other words, a net electric field is standard culture conditions for 24 h to allow cell adhesion. generated in the system by the application of external magnetic Then, the heterostructures with the adhered hOBs were fields (direct magnetoelectric effect). The applied magnetic transferred to the cuvettes of the magnetic stimulation setup field also causes a change in the ferroelectric amplitude versus (see Materials and Methods) and cultured daily either under a voltage butterfly loops, which is another sign of magneto- magnetic field of 400 Oe@100 Hz for 1 h to induce an electric coupling. One way to estimate the local magneto- electrical stimulation (electrically stimulated; ES) or without a electric coupling coefficient is to assess the change in the magnetic field (not electrically stimulated; n-ES). asymmetry of the piezo-response loops when the magnetic A live/dead kit was used to determine the viability of the field is applied, i.e., αE = ΔE/ΔH, where ΔH is the increment cells grown on the surface of the heterostructures. As shown in in the applied magnetic field and ΔE the induced change in Figure 4a, cells were able to grow on the heterostructures in electric coercive voltage.49 Using this method, the local both conditions (n-ES and ES). In both cases, a high number magnetoelectric coefficient in our case is 50 V cm−1 Oe−1. of live cells were observed after 3 days in culture (95 ± 3% and 97 ± 3% of live cells for the n-ES and ES conditions, This value is comparable to other magnetoelectric hetero- respectively). Nonetheless, the images revealed a significantly structures using P(VDF-TrFE) as their ferroelectric counter- higher number of cells on top of the heterostructures under part.50 This quantification should be taken with some caution. magnetoelectric stimulation (32,000 ± 5000 cells/hetero- Namely, since PFM is a local technique, the electric field structure) than without applying the magnetic field (14,000 ± generated by the tip is very inhomogeneous and it cannot be 3000 cells/heterostructure). easily quantified, since it depends on many factors (e.g., the The magnetoelectric heterostructure showed remarkable shape of the tip, the conductivity of the layers, the roughness of cytocompatibility. Neither the P(PVDF-TrFE) component nor the films, among others). the FeGa or kapton materials exhibit any toxicity toward the 2.4. Wettability Characterization. To facilitate cell cells. This outcome aligns with other studies, where adhesion and proliferation, it is important to have hydrophilic P(VDF-TrFE),37,38 FeGa,36,53,54 and kapton55,56 have been surfaces; otherwise, cells easily detach and die during their shown to have no adverse effects on cell proliferation. growth. One of the drawbacks of P(VDF-TrFE) and other After the viability assessment, cell morphology was studied similar ferroelectric polymers is that they are hydrophobic.51,52 through SEM analysis of hOBs grown on the heterostructures This is evidenced in Figure 3, where a contact angle of around under the two conditions. In both cases, cells were evenly 90° is obtained for the as-grown (i.e., untreated) hetero- distributed on the surface after 3 days of culture (Figure 4b). structure. Interestingly, the contact angle significantly Overall, hOBs under ES and n-ES conditions presented a flat decreases to 40° after exposure of the outer P(VDF-TrFE) and polygonal morphology, with some thin filopodia layer to an oxygen plasma treatment. Topological images protruding from the membrane surface in different directions, obtained by atomic force microscopy (AFM) indicate that showing that both conditions allowed good cellular adhesion. oxygen plasma induces some changes in the surface top- Cell adhesion to the heterostructure surface was also ography. In particular, the P(VDF-TrFE) grains become more confirmed through the analysis of the distribution of actin visible, although the overall surface roughness remains below stress fibers after 3 days in culture. This analysis showed that 20 nm for both the as-grown and treated samples. When the the hOBs cytoplasm was crossed by well-defined stress fibers, P(VDF-TrFE) is exposed to oxygen plasma, highly reactive most of them found in parallel orientation, an indicator of a oxygen species are generated, resulting in the formation of structured cell disposition and complete adhesion (Figure 4c), oxygen-containing functional groups on the surface which in agreement with the SEM observations. These results are enhance the hydrophilic character of the material.37 consistent with previous studies which have shown that 58058 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article the magnetic field was not applied, and consequently, the electric field on the heterostructures was zero. This analysis was conducted at 1, 3, and 7 days after osteoblast seeding on top of the magnetoelectric heterostructure. Under both conditions, the number of cells significantly increased over time. Interestingly, significant differences were observed between stimulated and nonstimulated heterostructures after 7 days in culture (Figure 5b). Namely, the number of cells was higher when the FeGa/P(VDF-TrFE) heterostructures were electrically actuated with the magnetic field, as confirmed by the images of the nuclei of the cells on day 7 (Figure 5c). In addition to proliferation, electrical stimulation has also been described to enhance osteoblast differentiation. Osteo- blast differentiation is usually divided in three stages: (i) cell proliferation, (ii) extracellular matrix (ECM) maturation, and (iii) ECM mineralization.59,60 During the proliferation phase, several ECM proteins, such as collagen I (COLI) and fibronectin, can be detected. The matrix maturation phase is characterized by the expression of specific genes needed for the synthesis and maturation of the ECM, like alkaline phosphatase (ALPL), COLI, and bone sialoprotein (IBSP). Finally, during matrix mineralization, genes encoding proteins such as osteocalcin (BGLAP) and osteonectin (SPARC) are expressed. Once mineralization is completed, calcium deposi- tion can be visualized using different staining methods. The ability of the magnetic-field-induced electrical stim- ulation to enhance the differentiation of hOBs grown on top of Figure 4. Viability, morphology, and adhesion of osteoblasts cultured the FeGa/P(VDF-TrFE) heterostructures was assessed by on top of the FeGa/P(VDF-TrFE) heterostructures either under analyzing the gene expression of several early and late magnetic-field-induced electrical stimulation (ES) or without osteoblast marker genes, production of both early and late stimulation (n-ES). (a) Cell viability of osteoblasts after 3 days in culture. Live and dead cells appear in green and red, respectively. (b) osteoblast protein markers, activity of alkaline phosphatase SEM images of the osteoblasts cultured under ES and n-ES conditions (ALP), and formation of extracellular calcium deposits. Gene for 3 days. (c) Cytoskeleton (actin, green) and nuclei (blue) of expression was analyzed through the whole culture period osteoblasts cultured in ES and n-ES conditions for 3 days. (proliferation and differentiation phases), whereas protein expression, ALP activity, and calcium deposits were only analyzed during the differentiation phase (7 and 14 days). P(VDF-TrFE) is biocompatible and facilitates good cell First, we analyzed the expression of the three early adhesion, as well as cell−material interactions.27,37,57 osteogenic marker genes COLI, ALPL and IBSP, and the two 2.6. Effect of the Magnetic-Field-Induced Electrical late osteogenic marker genes BGLAP and SPARC in hOBs after Stimulation on Osteoblast Proliferation and Differ- 3, 7, and 14 days in culture under both conditions (Figure 6a). entiation. Previous studies have suggested that external magnetic stimulation can induce additional physical effects When compared with unstimulated cells, expression levels of on cells.58 Accordingly, to verify that the magnetic field used in COLI were drastically higher at all time-points in stimulated our study did not impact cell proliferation, we conducted a cells (more than four times for each time-point), and those of control cell culture experiment. Osteoblasts were cultured on ALPL and SPARC were significantly increased only after 14 glass coverslips, excluding the presence of magnetoelectric days of culture (more than 2.5-fold and more than 4-fold, layers, and exposed (or not exposed) to the same daily respectively). Due to the electrical stimulation, the initial magnetoelectric stimulation regime for 7 days. When using expression of ALPL during proliferation was nearly nonexistent glass coverslips under the applied magnetic field conditions, no but increased during matrix maturation. For the other two discernible effects on cell proliferation were observed, neither genes (IBSP and BGLAP), expression levels were similar at all in stimulated (ES) nor the nonstimulated (n-ES) hOBs cells time-points between stimulated and nonstimulated cells. (Figure 5a). The results demonstrate that this stimulation had Next, protein levels were assessed after 7 and 14 days in neither detrimental nor beneficial effects on the cells, thus culture through immunofluorescence detection of COLI, confirming the innocuous/neutral nature of the magnetic characteristic of the first stages of osteoblast differentiation actuation under these parameters (400 Oe@100 Hz for 1 h and ECM maturation, and osteocalcin (OCN), a protein that daily). is more abundant during the mineralization phase. COLI After confirming that remote stimulation of osteoblasts does protein levels were significantly higher in stimulated than in not compromise cell adhesion or viability and that the applied nonstimulated hOBs at both time-points, whereas the levels of magnetic field does not interfere with the osteoblast the OCN were similar (Figure 6b). These results are in proliferation when they are cultured on glass coverslips, we agreement with the gene expression results. proceeded to analyze the effects of magnetic-field-induced In addition, ALP activity was quantified after 7 and 14 days electrical stimulation on osteoblast proliferation. Note that in culture. After 7 days, no significant differences were during the seeding of the cells and the 24 h adhesion period, observed between the two conditions tested. However, after 58059 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 5. Proliferation of hOBs cultured on top of (a) glass coverslips or (b) FeGa/P(VDF-TrFE) heterostructures under magnetic-field-induced electrical stimulation (ES) and without it (n-ES) for 1, 3, and 7 days in culture. No significant differences were found when growing on the glass coverslip at any time-points analyzed, but statistically significant differences were found at 7 days in osteoblasts grown on the heterostructures. Asterisks indicate significant differences (p < 0.05) among both conditions at the same time-point. (c) Images of cell nuclei (blue) of hOBs grown on top of the FeGa/P(VDF-TrFE) heterostructures in the two conditions at day 7. ES was started 24 h after cell seeding (day 1). 14 days, ALP activity strongly increased in both conditions, In previous studies where a piezoelectric material was used being significantly higher in the stimulated cells (Figure 6c). to electrically stimulate osteoblast, we demonstrated that when Finally, to demonstrate that hOBs showed a mature osteoblast adhere to a network of ZnO nanosheets21 or to a differentiation state in the presence of the magnetoelectric PVDF scaffold,37 the resulting adhesion forces caused the ZnO stimulation, the capacity to mineralize the ECM was analyzed nanosheets or the PVDF fibers to bend, generating a local after 7 and 14 days in culture. A significant increase in electric field of sufficient magnitude to stimulate the cells and extracellular calcium deposits was detected in the stimulated modulate their activity. In both studies, calcium content within cells at day 14, but not at day 7 (Figure 6c). the cells was monitored over time, revealing high- or low- The four experiments performed to quantify the osteoblast amplitude Ca2+ transients that can be correlated with the differentiation (gene expression, protein detection, ALP opening of voltage or stretch calcium channels (VGCCs or activity, and calcium deposits quantification) were all SACCs), or with the reorganization of plasma membrane concordant and demonstrated that stimulation using the receptors, respectively. These findings suggest that a similar flexible magnetoelectric heterostructures significantly increased mechanistic process may also occur when using the FeGa/ and possibly accelerated hOBs differentiation. P(VDF-TrFE) heterostructure since the upper layer of this As demonstrated by the magnetoelectric characterization heterostructure is piezoelectric and thus it induces an electrical (Figure 2d,e), when a magnetic field is applied to the FeGa/ field when a magnetic field is applied. P(VDF-TrFE) heterostructures, an electric field is generated. Note that several authors have described an increase in cell This generated electric field should be capable of stimulating proliferation when cells are remotely subjected to an electrical osteoblast cells (Scheme 1), consequently promoting cell stimulus using different approaches based on piezoelectric/ proliferation and differentiation.8,9 ferroelectric materials. For example, an increase in proliferation The proposed mechanism of this electrical field enhanced has been observed when using different piezoelectric layers proliferation and differentiation is based on the insights of activated by ultrasounds63,64 or different magnetoelectric different authors. It is suggested that the proximity of the systems activated magnetically.27−29 In addition, Liu et al.29 electric field to the plasma membrane triggers the opening of have analyzed osteoblast differentiation using magnetoelectric voltage-gated calcium channels. This, in turn, allows a stimulation, reporting an enhanced differentiation of bone cells controlled influx of calcium ions into the cells, activating exposed to 2300 Oe DC magnetic field during 12 h daily for 14 signaling pathways such as the calcium−calmodulin pathway, days. In fact, when compared with other studies using ultimately contributing to bone regeneration.8,9 Within this magnetoelectric approaches, the conditions used in our study framework, the ES condition would also trigger the opening of (400 Oe@100 Hz, for 1 h daily) are rather mild. Studies using stretch-activated calcium channels and activate membrane considerably longer times, higher fields or higher frequencies receptors coupled to phospholipase C enzyme, which is can be found in the literature.22−24,30 involved in calcium release from the reticulum calcium The good performance of the kapton/FeGa/P(VDF-TrFE) storages.61,62 heterostructures probably stems from the planar interface 58060 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article fields compared with those of other types of structures. Second, the flexible character of the kapton substrate should also play a key role in the enhanced hOBs proliferation and differentiation. Namely, common magnetoelectric systems grown on rigid substrates are prone to clamping effects,65 where the mechanical attachment of the magnetoelectric layers to the substrate impedes the proper transmission of the strain between the magnetostrictive and ferroelectric layers, thus hampering the production of electric fields. Although there are several strategies to minimize clamping effects,44,66,67 growing the magnetoelectric layers on flexible kapton layer appears to be an efficient and relatively straightforward strategy to reduce the clamping effect, and consequently induce sufficient electric fields to stimulate hOBs. Another factor that may have a positive influence on hOBs is the stimulation frequency. In our case, we used 100 Hz, which is commensurate with the typical response time of many of the cell processes.68 Note that the magnetoelectric heterostructures did not present any obvious sign of deterioration after the biological experiments. In the long term, after in vivo studies, given its flexibility, this type of heterostructure might be suitable as a remotely actuated conformal graft for bone regeneration (in a “band- aid”-like approach).69−71 3. CONCLUSIONS In this work, the potential use of flexible magnetoelectric heterostructures [i.e., kapton/FeGa/P(VDF-TrFE)] to pro- mote cell proliferation, ECM maturation, and mineralization has been demonstrated. After characterizing the magnetic and ferroelectric properties of FeGa and P(VDF-TrFE) separately, evidence for strain-mediated magnetoelectric coupling between the two layers was obtained by PFM. The flexible character of the substrate, minimizing clamping effects, is probably crucial to induce such a coupling. Next, hOBs have been cultured onto the magnetoelectric heterostructures, after a prior treatment with O2 plasma to enhance hydrophilicity, and thus cell adhesion. By subjecting the whole structure (material + cells) to an alternating magnetic field (daily for a duration of 1 h), clear evidence of efficient magnetoelectric cell stimulation is obtained. The results may have an impact in advanced healthcare technologies based on wireless electrical cell stimulation, not only to accelerate the healing of bone fractures but also in other areas, such as muscle stimulation or neural tissue engineering. 4. MATERIALS AND METHODS 4.1. Fabrication of the FeGa/P(VDF-TrFE) Heterostructures. Figure 6. Differentiation of osteoblasts cultured on top of the FeGa/ Fe72Ga28 (at. %) alloy films (100 nm-thick) were grown by DC P(VDF-TrFE) heterostructures under magnetic-field-induced elec- sputtering using Fe75Ga25 targets onto kapton foils (50 μm thick, HN trical stimulation (ES) and without stimulation (n-ES) for 3, 7, and 14 200 from Isovolta S.A.U.) previously metallized with a 10 nm thick days. (a) Expression of osteoblast differentiation marker genes COLI, sputtered Ti adhesion layer. Sputtering of both layers was performed ALPL, IBSP, BGLAP, and SPARC. The proteins encoded by these using an AJA International magnetron system with a base pressure of genes are indicated below the names of the genes. The mRNA levels 1 × 10−8 Torr and setting the working pressure at 3 × 10−3 Torr. The are expressed as the ratio of the target gene expression to that of the P(VDF-TrFE) copolymer top layer (250 nm thick) was grown by reference genes (TBP and HPRT1). (b) Relative fluorescent intensity spin coating. P(VDF-TrFE) powders in a 70/30 wt ratio were first of the COLI and the osteocalcin (OCN) osteoblast differentiation dissolved in diethyl ketone to form a transparent solution with a protein; (c) ALP activity and extracellular calcium deposits of the concentration of 4 wt/vol %. The solution was then spin-coated at ECM. Asterisks indicate significant differences (p < 0.05) among the 3000 rpm onto the sputtered FeGa film, for 60 s, using a Suss conditions at the same time-point. Microtech spinner. These conditions were selected based on our previous studies (see Figure S2) to obtain relatively thick P(VDF- TrFE) films without compromising their roughness. This facilitates between the magnetostrictive and the ferroelectric layers, the characterization of the structure and the ferroelectric properties of which maximizes the strain transfer between the FeGa and the the polymer. Then, the samples were annealed at 125 °C for 6 h in air P(VDF-TrFE), and should, in turn, produce larger electric using a DZF-6020-ZZKD oven to promote the crystallization of the 58061 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article Figure 7. (a) Schematic representation of the toroidal actuation setup; (b) photograph of the experimental system. (c) Photograph of the cuvettes. ferroelectric β-phase of P(VDF-TrFE). In order to increase the into pieces of 3−5 mm in diameter. Finally, bone fragments were hydrophilicity of the naturally hydrophobic surface of P(VDF-TrFE), vortex-mixed to remove hematopoietic cells. The bone fragments the samples were treated for 1 min with oxygen plasma using the were cultured as explants in 75 cm2 flasks for at least 14 days. hOBS PlasmaPro Cobra 100 ICP Etching System (HF 5 W, ICP 300 W, 17 were used because they are considered to have a physiological mTorr, O2�50 sccm). phenotype of an osteoblast differentiating into an osteocyte.73 4.2. Structural and Morphological Characterization. The The bone explants, as well as the isolated hOBs, were cultured in morphology of the different layers was investigated by SEM using a Dulbecco’s modified Eagles medium (DMEM; Gibco, Thermo Fisher Zeiss MERLIN field emission SEM at 5 keV. The elemental Scientific, Waltham, MA, USA) supplemented with 20% fetal bovine composition was assessed by EDX using SEM with an acceleration serum (FBS; Gibco) and 2% penicillin−streptomycin (P/S; Biowest, voltage of 20 keV. The XRD patterns were acquired on a Philips Riverside, MO, USA), under standard conditions (37 °C, 5% CO2). X’Pert diffractometer using Cu Kα radiation in θ−2θ geometry. FTIR The experiments were performed by using hOBs between passages spectroscopy was recorded using a Hyperion 2000 microspectrometer 2 and 10. Cells were maintained at 37 °C in a humidified atmosphere (Bruker) with an attenuated total reflectance (ATR) objective and a of 5% CO2 (standard conditions). The FeGa/P(VDF-TrFE) mercury−cadmium−telluride (MCT) detector cooled with liquid heterostructures were sterilized using a UV-C led sterilization nitrogen. The spectra were registered for 132 scans with a 4 cm−1 chamber (59S, Shenzhen, China) and individually introduced into a resolution. four-well plate. Then, different numbers of cells, according to the 4.3. Magnetic Characterization. Magnetic hysteresis loops were experiment (see the following sections), were seeded into each well. recorded at room temperature along in-plane and perpendicular-to- Cells were cultured with DMEM supplemented with 20% FBS and 2% plane directions using a vibrating sample magnetometer (VSM) from P/S for 24 h to allow cell adhesion to the heterostructures. Afterward, MicroSense. the heterostructures with the adhered hOBs were transferred to the 4.4. Atomic Force Microscopy and Piezo-Response Force cuvettes and cultured daily either under a magnetic field of 400 Oe at Microscopy Characterization. All AFM measurements were 100 Hz for 1 h to induce an electrical stimulation (electrically conducted on an MFP-3D Asylum AFM (Asylum Research, Oxford stimulated; ES) or without magnetic field (not electrically stimulated; Instruments). The cantilevers used for PFM experiments were PPP- n-ES). EFM tips (Nano sensors; Schaffhausen, Switzerland) with a stiffness For viability, morphology, adhesion, and proliferation experiments, constant of k = 2 N/m and coated with Ptlr. PFM lithography was cells were cultured with DMEM supplemented with 20% FBS and 2% done by applying −9 V on a scan area of 5 × 5 μm2 and +9 V on a P/S. For the differentiation experiments (gene expression, osteogenic scan area of 2.5 × 2.5 μm2 through the tip. The tip was in contact with protein markers immunodetection, quantification of ALP activity, and the sample applying a force of 60 nN. To record the ferroelectric mineralization), the serum content was reduced to 10% FBS. hysteresis loops, we used the same cantilevers and the same applied 4.8. Cell Viability Assay. Cell viability was assessed by the force. detection of the intracellular esterase’s activity using the live/dead 4.5. Contact Angle Measurements. Contact angle measure- viability/cytotoxicity kit for mammalian cells (Invitrogen, Thermo ments were conducted to assess the hydrophilicity of the films using Fisher Scientific, Waltham, MA, USA) according to the manufac- the sessile-drop technique (DSA 100 system from Krüss). Micro- turer’s protocol. To perform the assay, 20,000 cells were seeded on droplets of 10 μL of milli-Q water were employed for such the heterostructures and cultured for 3 days under ES and n-ES measurements. Contact angle values were obtained by averaging conditions. The cells were incubated with the kit reagents for 15 min several measurements in different locations on the films’ surfaces. and immediately observed. Live cells showed green color because 4.6. Magnetic Stimulation Setup. The magnetic actuation their esterase activity converts nonfluorescent calcein AM into device used to stimulate the cells consisted of a ferrite toroid (60 mm fluorescent, whereas dead cells showed red color because of the in diameter with a 13 mm gap), with 65 turns of 1 mm laminated permeability of their damaged plasma membrane to propidium iodide. copper wire wound around it. The actuator was powered by a Siglent Samples were observed at 40× magnification with an Olympus IX71 SDG-1025 signal generator and an Accel TS200 power supply and Inverted Fluorescence Microscope (Olympus, Shinjuku, Japan) was able to generate magnetic field in the gap of the electromagnet equipped with a 10MP CMOS Camera. Representative images from ranging between 100 Oe (@110 kHz) and 1000 Oe (@1 Hz). In the different regions of each sample were captured, and the percentage of current experiments, a field of 400 Oe at 100 Hz was used. The viable cells of a minimum of 2500 cells in five fields was calculated for samples were placed in the gap of the electromagnet by using a each condition. homemade poly methyl-methacrylate (PMMA) cuvette (Figure 7). 4.9. Cell Morphology Analysis. After the cell viability assay, the 4.7. Cell Culture and Stimulation Conditions. hOBs were same samples were processed to be analyzed by SEM. Briefly, cells isolated from explanted trabecular bone obtained from a healthy were washed in 0.1 M cacodylate buffer saline (CBS), fixed in 2.5% donor after a removal surgery with informed consent. The procedure glutaraldehyde 0.1 M in CBS for 45 min at room temperature (RT) to obtain primary cell cultures from human bones was approved by and rinsed again twice in CBS. Cell dehydration was performed in a the Ethics Committee (Comissió d’È tica en Experimentació Animal i series of increasing ethanol concentrations (50, 70, 90, and twice Humana) of the Universitat Autònoma de Barcelona (CCEAH-2672). 100%) for 8 min each. Finally, the samples were dried using The isolation procedure was performed according to the method hexamethyldisilazane (Electron Microscopy Sciences, Hatfield, PA, detailed by Gallagher.72 Briefly, the trabecular bone was isolated from USA) for 15 min. The samples were then mounted on special stubs the cortex and connective tissues. Then, the trabecular bone and metalized by using a E5000 Sputter Coater (Emitech, France) for fragments were washed in phosphate-buffered saline (PBS) and cut 2 min. Finally, they were analyzed using a MERLIN FE-SEM (Zeiss, 58062 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article Oberkochen, Germany) in order to observe cell morphology at PrimePCR SYBR Green Assays (BioRad) for COLI different magnifications. Representative images from different regions (qHsaCED0043248), ALPL (qHsaCID0010031), IBSP of each culture sample were obtained. (qHsaCED0002933), BGLAP (qHsaCED0038437), SPARC (qHsa- 4.10. Actin Cytoskeleton Distribution. The analysis of the cell CID0010332), TBP (qHsaCID0007122), and HPRT1 (qHsa- cytoskeleton was performed by staining of the actin filaments. In these CID0016375) were used. studies, 20,000 cells were seeded onto the heterostructures, and after 4.13. Immunodetection of Osteogenic Markers. Quantifica- 72 h, the samples were washed twice in PBS and cells fixed in 4% tion of the osteogenic markers was performed by immunofluorescence paraformaldehyde (PFA) in PBS for 20 min at RT. After washing detection of two different proteins involved in osteoblast differ- again twice in PBS, the cells were permeabilized with 1% Tween-20 in entiation: type I collagen (COLI) as an early differentiation marker PBS for 20 min. The samples were then incubated with a mixture of and osteocalcin (OCN) as a late differentiation marker. To perform Phalloidin-Atto 488 (Sigma-Aldrich, Merck, Burlington, MA, USA), immunodetection, 100,000 hOBs were seeded onto the hetero- and Hoechst 33258 (Sigma-Aldrich) for 60 min in the dark at RT. structures and cultured for 7 and 14 days in both ES and n-ES Finally, cells were washed in PBS, air-dried, and mounted on 35 mm conditions. Then, the cells were fixed in 4% PFA in PBS for 20 min at glass bottom dishes (Ibidi, Gräfelfing, Germany) using ProLong RT, permeabilized with PBS containing 1% Tween-20 for 20 min, and Antifade mounting solution (Life Technologies, Carlsbad, CA, USA). blocked with 5% bovine serum albumin (BSA; Sigma) in PBS for 20 The cytoskeleton evaluation was done at 630× in a Confocal TCS min at RT. The samples were then incubated overnight at 4 °C with SP5 (Leica, Heerbrugg, Switzerland) by using a PL APO 63× primary antibodies: rabbit antiosteocalcin T-4743 (1:200; Peninsula objective. Representative images from different regions of each sample Laboratories, San Carlos, CA, USA) or mouse monoclonal were obtained. anticollagen I LB-1197 (1:200; Cosmo Bio, Tokyo, Japan). After 4.11. Cell Proliferation Assay. The proliferation of hOBs was being rinsed twice with 1% BSA in PBS, the cells were incubated for 1 determined by the quantification of the cell activity using alamar blue h at RT with the secondary antibody Alexa Fluor594-conjugated goat reagent (Thermo Fisher Scientific) at days 1, 3, and 7 of culture. antirabbit (Thermo Fisher Scientific) or Alexa Fluor 488-conjugated Briefly, 20,000 cells were seeded into each well of a four-well plate goat antimouse (Thermo Fisher Scientific) diluted 1:400 in PBS containing the heterostructure. After 24 h to allow cell adhesion (day together with the nuclear dye Hoechst 33258 (Sigma-Aldrich). 1), heterostructures with adhered cells were moved to a new well to Images from randomly selected regions were obtained, and the discard cells growing outside them. Then, fresh DMEM without fluorescence intensity was measured. To measure fluorescence phenol red (Gibco) and with 10% alamar blue was added. Cells were intensity, at least five different captures from each time-point and incubated for 4 h in the dark and under standard conditions. After condition were taken with the same exposure time, laser power, and incubation, the supernatant was collected, and its fluorescence was detector voltage and analyzed using FIJI (ver. 1.53u, 2022, available measured at a wavelength of 585 nm after excitation at 560 nm on a from http://imagej.nih.gov/ij). In each 8 bit image from the specific Spark multimode microplate reader (Tecan, Männedorf, Switzerland). protein channel, the integrated density (sum of pixel values in a After supernatant collection, samples were transferred to the plastic selected area multiplied by the escalated area of one pixel, where the buckets, and fresh medium was added to the cultures for the assay to selected area is determined by using a threshold) normalized to the be repeated at 3 and 7 days under ES or n-ES conditions. The number of cells (number of cell nuclei on the blue channel) was experiments were performed in triplicate. After the last test at day 7, calculated. the samples were washed twice in PBS and the cells were fixed in 4% 4.14. Quantification of the ALP Activity. The differentiation of PFA in PBS for 20 min at RT. After washing again twice in PBS, cells hOBs was also studied by measuring the ALP activity. For this assay, were incubated with Hoechst 33258 (Sigma-Aldrich) for 15 min in 100,000 cells were seeded onto the heterostructures and cultured the dark at RT. Finally, cells were washed in PBS and observed at 40× during 7 and 14 days. Then, the ALP activity was determined by the magnification with an Olympus IX71 Inverted Fluorescence Micro- hydrolysis of p-nitrophenyl phosphate (pNPP), which produces p- scope equipped with a 10MP CMOS Camera. Representative images nitro-phenol (pNP). Briefly, 300 μL of 1-step pNPP (Thermo Fisher from different regions of each culture sample were obtained. Scientific) was added to the cells and, after 30 min incubation at RT, 4.12. Expression of Osteogenic Marker Genes. The the supernatant was collected. The absorbance was measured at 405 expression of osteogenic marker genes encoding type I collagen nm using a Spark multimode microplate reader (Tecan) and (COLI), alkaline phosphatase (ALPL), bone sialoprotein (IBSP), normalized using Alamar Blue results performed on the same samples osteocalcin (BGLAP), and osteonectin (SPARC) was analyzed by prior to ALP. Three independent experiments were used for each real-time quantitative polymerase chain reaction (qPCR). For gene time-point and condition. expression analysis, 100,000 hOBs were seeded onto the hetero- 4.15. Mineralization Assay. The mineralization of the ECM by structures and the total RNA was extracted from the cell cultures at 3, differentiated osteoblasts was evaluated through the detection of 7, and 14 days using the Maxwell RSC simplyRNA tissue kit calcium deposits. Specifically, calcium deposition was determined by (Promega, Madison, WI, USA) according to the manufacturer’s alizarin red staining (ARS, Sigma-Aldrich). Briefly, 100,000 hOBs protocol. Then, the RNA concentration and purity were determined were seeded on the heterostructures for 7 and 14 days. Later, the cells using a Nanodrop spectrophotometer (Nanodrop 1000, Thermo were rinsed twice in PBS, fixed in 4% PFA in PBS for 20 min at RT Scientific, Thermo Fisher Scientific). Reverse transcription was and then washed twice with distilled water. Then, the samples were performed with 500 ng of total RNA using the iScript cDNA incubated with 2% ARS in distilled water at pH 4.2 for 45 min. synthesis kit (BioRad, Hercules, CA, USA) according to the Finally, the samples were washed four times with distilled water to manufacturer’s instructions. The mRNA levels were assayed in clear any nonbound alizarin red. To measure the absorbance, the triplicate in CFX384 arrays (BioRad) using 5 μL of iTaq Universal incorporated ARS dye was extracted from the cell cultures with 10% SYBR Green Supermix (BioRad), 0.5 μL of PrimePCR Assays cetylpiridinium chloride (CPC, Sigma-Aldrich) in 10 mM sodium (BioRad), and 20 ng of cDNA in a total volume of 10 μL. Then, a phosphate (Fluka, Honeywell, Charlotte, NC, USA) at pH 7 for 15 PCR amplification was performed following the next steps: initial min on a shaker at RT. The extracted product was transferred to a 96- heating at 95 °C for 3 min, followed by 40 cycles at 95 °C for 10 s, 60 well plate, and the absorbance was measured at 540 nm using a Spark °C for 30 s, and a final melt curve from 65 to 95 °C, in 0.5 °C multimode microplate reader (Tecan) and normalized using alamar increment each 5 s in a C1000 Touch Thermal Cycler (BioRad). The blue results performed on the same samples prior to ARS. The expression values were obtained from cycle quantification (Cq) values experiments were performed in triplicate. determined with BioRad CFX Maestro Software. The target gene 4.16. Statistical Analysis. All quantitative data were analyzed levels are expressed as a relative value: the ratio of the target gene with GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, expression to that of the reference TATA-box binding protein (TBP) USA) and presented as the mean ± standard error of the mean. and hypoxanthine phosphoribosyl transferase (HPRT1) genes. The Statistical comparisons were performed using one-way analysis of relative gene expression was calculated as 2-ΔCq. Validated variance (ANOVA) with Bonferroni correction for multiple 58063 https://doi.org/10.1021/acsami.3c09428 ACS Appl. Mater. Interfaces 2023, 15, 58054−58066 ACS Applied Materials & Interfaces www.acsami.org Research Article comparison tests for cell proliferation, qPCR assays, osteogenic https://pubs.acs.org/10.1021/acsami.3c09428 protein markers intensity, ALP activity, and ARS absorbance. A value of p < 0.05 was considered to be significant. Author Contributions ■ ASSOCIATED CONTENT Data Availability Statement O. Careta: conceptualization, investigation, data curation, formal analysis, validation, visualization, and writing�original draft; A. Nicolenco: conceptualization, investigation, data The data sets and Supporting Information can be acquired curation, formal analysis, validation, and visualization; F. online or by contacting the corresponding authors. Perdikos: conceptualization, investigation, data curation, * sı Supporting Information formal analysis, validation, and visualization; A. Blanquer: The Supporting Information is available free of charge at conceptualization, investigation, and writing�review and https://pubs.acs.org/doi/10.1021/acsami.3c09428. editing; E. Ibañez: conceptualization, methodology resources, Photograph of a piece of kapton/FeGa/P(VDF-TrFE) supervision, and writing�review and editing; E. Pellicer: sample during manual bending and dependence of the conceptualization, methodology, resources, supervision, and thickness and roughness of the P(VDF-TrFE) films writing�review and editing; C. Stefani: investigation, data depending on synthesis conditions (PDF) curation, formal analysis, validation, and visualization; B. Sepúlveda: conceptualization, methodology resources, super- ■ AUTHOR INFORMATION Corresponding Authors vision, and writing�review and editing; J. Nogués: resources, supervision, writing�review and editing, project administra- tion, funding acquisition, and project administration; J. Sort: Jordi Sort − Departament de Física, Universitat Autònoma de conceptualization, methodology, resources, supervision, writ- Barcelona, Bellaterra, Cerdanyola del Vallès E-08193, Spain; ing�review and editing, project administration, and funding Institució Catalana de Recerca i Estudis Avançats (ICREA), acquisition; and C. Nogués: conceptualization, methodology, Barcelona E-08010, Spain; orcid.org/0000-0003-1213- resources, supervision, writing�review and editing, and 3639; Email: jordi.sort@uab.cat project administration. Carme Nogués − Departament de Biologia Cellular, Fisiologia Notes i Immunologia, Universitat Autònoma de Barcelona, The authors declare no competing financial interest. Bellaterra, Cerdanyola del Vallès E-08193, Spain; orcid.org/0000-0002-6361-8559; Email: carme.nogues@uab.cat ■ ACKNOWLEDGMENTS This project has received funding from the European Union’s Authors Horizon 2020 research and innovation programme under the Oriol Careta − Departament de Biologia Cellular, Fisiologia i Marie Skłodowska-Curie grant agreements no. 861046 and no. Immunologia, Universitat Autònoma de Barcelona, 861145 (Bioremia and BeMAGIC European Training Net- Bellaterra, Cerdanyola del Vallès E-08193, Spain works), grants PID2020-116844RB-C21, PID2020 116844RB- Aliona Nicolenco − Departament de Física, Universitat C22, and PID2019-106229RB-I00 funded by MCIN/AEI/ Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès E- 10.13039/501100011033 and the Generalitat de Catalunya 08193, Spain; CIDETEC, Parque Científico y Tecnológico de (2021-SGR-00651, 2021-SGR-00122). We also acknowledge Gipuzkoa, San Sebastián 20014, Spain the European Research Council (2021-ERC-Advanced RE- Filippos Perdikos − Catalan Institute of Nanoscience and MINDS grant no. 101054687). A.N. acknowledges funding Nanotechnology (ICN2), CSIC and BIST, Bellaterra, from the European Union’s Horizon 2020 research and Barcelona E-08193, Spain; orcid.org/0000-0002-3268- innovation programme under the Marie Skłodowska-Curie 6356 grant agreement no. 892661-MAGNUS. A.B. has received Andreu Blanquer − Departament de Biologia Cellular, funding from a postdoctoral fellowship within the Beatriu de Fisiologia i Immunologia, Universitat Autònoma de Pinós programme, funded by the Secretary of Universities and Barcelona, Bellaterra, Cerdanyola del Vallès E-08193, Spain Research (Government of Catalonia) and by the Horizon 2020 Elena Ibañez − Departament de Biologia Cellular, Fisiologia i programme of research and innovation of the European Union Immunologia, Universitat Autònoma de Barcelona, under Marie Sklodowska-Curie grant agreement no. 801370. Bellaterra, Cerdanyola del Vallès E-08193, Spain ICN2 is funded by the CERCA programme/Generalitat de Eva Pellicer − Departament de Física, Universitat Autònoma Catalunya. The ICN2 is supported by the Severo Ochoa de Barcelona, Bellaterra, Cerdanyola del Vallès E-08193, Centres of Excellence programme, Grant CEX2021-001214-S, Spain; orcid.org/0000-0002-8901-0998 funded by MCIN/AEI/10.13039.501100011033. The authors Christina Stefani − Departament de Física, Universitat would like to thank the staff from the Servei de Microscòpia of Autònoma de Barcelona, Bellaterra, Cerdanyola del Vallès E- Universitat Autònoma de Barcelona. The authors also want to 08193, Spain thank Iris Nogués for preparing Scheme 1 and Nicolau López- Borja Sepúlveda − Instituto de Microelectronica de Barcelona Pintó for his help in growing some of the samples. (IMB-CNM, CSIC), Bellaterra, Barcelona E-08193, Spain; orcid.org/0000-0002-1562-7602 Josep Nogués − Catalan Institute of Nanoscience and Nanotechnology (ICN2), CSIC and BIST, Bellaterra, ■ REFERENCES (1) Wu, A. M.; Bisignano, C.; James, S. L.; Abady, G. G.; Abedi, A.; Barcelona E-08193, Spain; Institució Catalana de Recerca i Abu-Gharbieh, E.; Alhassan, R. K.; Alipour, V.; Arabloo, J.; Asaad, M.; Estudis Avançats (ICREA), Barcelona E-08010, Spain; Asmare, W. N.; Awedew, A. F.; Banach, M.; Banerjee, S. K.; Bijani, A.; Birhanu, T. T. M.; Bolla, S. 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