International Journal of Molecular Sciences Article Biocompatibility and Electrical Stimulation of Skeletal and Smooth Muscle Cells Cultured on Piezoelectric Nanogenerators Andreu Blanquer 1 , Oriol Careta 1 , Laura Anido-Varela 1 , Aida Aranda 2 , Elena Ibáñez 1 , Jaume Esteve 2 , Carme Nogués 1, * and Gonzalo Murillo 2, * 1 Departament de Biologia Cel.lular, Fisiologia i Immunologia, Facultat de Biociencies, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain; Andreu.BlanquerJerez@fgu.cas.cz (A.B.); oriol.careta@uab.cat (O.C.); laura.anidovarela67@gmail.com (L.A.-V.); elena.ibanez@uab.cat (E.I.) 2 Instituto de Microelectrónica de Barcelona, IMB-CNM (CSIC), Til·lers s/n, Campus UAB, 08193 Bellaterra, Barcelona, Spain; aida.aranda@e-campus.uab.cat (A.A.); jaume.esteve@imb-cnm.csic.es (J.E.) * Correspondence: carme.nogues@uab.cat (C.N.); gonzalo.murillo@csic.es (G.M.); Tel.: +34-93-594-7700 (G.M.) Abstract: Nanogenerators are interesting for biomedical applications, with a great potential for electrical stimulation of excitable cells. Piezoelectric ZnO nanosheets present unique properties for tissue engineering. In this study, nanogenerator arrays based on ZnO nanosheets are fabricated on transparent coverslips to analyse the biocompatibility and the electromechanical interaction with two types of muscle cells, smooth and skeletal. Both cell types adhere, proliferate and differentiate on the ZnO nanogenerators. Interestingly, the amount of Zn ions released over time from the nanogenerators does not interfere with cell viability and does not trigger the associated inflammatory response, which is not triggered by the nanogenerators themselves either. The local electric field generated by the electromechanical nanogenerator–cell interaction stimulates smooth muscle cells by   increasing cytosolic calcium ions, whereas no stimulation effect is observed on skeletal muscle cells. Citation: Blanquer, A.; Careta, O.; The random orientation of the ZnO nanogenerators, avoiding an overall action potential aligned Anido-Varela, L.; Aranda, A.; Ibáñez, along the muscle fibre, is hypothesised to be the cause of the cell-type dependent response. This E.; Esteve, J.; Nogués, C.; Murillo, G. demonstrates the need of optimizing the nanogenerator morphology, orientation and distribution Biocompatibility and Electrical according to the potential biomedical use. Thus, this study demonstrates the cell-scale stimulation Stimulation of Skeletal and Smooth triggered by biocompatible piezoelectric nanogenerators without using an external source on smooth Muscle Cells Cultured on muscle cells, although it remarks the cell type-dependent response. Piezoelectric Nanogenerators. Int. J. Mol. Sci. 2022, 23, 432. https:// Keywords: stimulation; nanogenerators; muscle cells; piezoelectric; ZnO; electroceutical; bioelectron- doi.org/10.3390/ijms23010432 ics; nanosheets; biomedical; biocompatibility Academic Editor: Agata Przekora Received: 17 November 2021 Accepted: 28 December 2021 1. Introduction Published: 31 December 2021 The development of smart materials for biomedical applications has become one of the Publisher’s Note: MDPI stays neutral most interesting research fields in recent years. Smart materials can be defined as materials with regard to jurisdictional claims in that have one or more properties that can be changed in a controlled fashion by external published maps and institutional affil- stimuli, such as stress, temperature, moisture, pH, and electric, magnetic or ultrasonic iations. fields [1–5]. Piezoelectric materials are smart materials that are able to generate a voltage when mechanical stress is applied, and vice-versa. Among all piezoelectric materials known, ZnO has attracted much interest because of its versatility, unique properties and multiple Copyright: © 2021 by the authors. applications, including microelectronics, biosensors and tissue engineering [6–8]. The Licensee MDPI, Basel, Switzerland. potential use of ZnO in the biomedical field is wide, with a high range of structures, from This article is an open access article bulk material to different types of nanoparticles. In fact, it is already used in healthcare distributed under the terms and and cosmetic applications [8]. ZnO has been classified as a generally recognized safe conditions of the Creative Commons material by the Food and Drug Administration (FDA) of the United States. However, there Attribution (CC BY) license (https:// is still controversy about the cytotoxic effect of ZnO nanostructures, with different results creativecommons.org/licenses/by/ depending on cell types and nanostructures, which should be elucidated before their use in 4.0/). biomedical applications. Int. J. Mol. Sci. 2022, 23, 432. https://doi.org/10.3390/ijms23010432 https://www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2022, 23, 432 2 of 15 Zinc is an essential micronutrient and a vital cofactor that plays an important role in tissue metabolism. It is indispensable in numerous Zn-dependent proteins involved in transcriptional regulation, DNA repair and extracellular matrix regulation, among others [9]. Controlled low concentrations of Zn are present in the cytosol as free ions that have been identified as secondary messengers capable of interacting with proteins to regulate intracellular pathways. Therefore, the presence of low concentrations of Zn is beneficial to cells, although high concentrations could be harmful [10]. In this regard, the use of Zn-based biodegradable materials is considered a good choice for tissue regeneration. ZnO has been demonstrated to be a considerably good antibacterial material, which is an important property for very different kinds of biomedical applications. Several authors have investigated this antibacterial effect on Staphylococcus epidermidis and Staphilococcus aureus. Both types of bacteria could infect the patient during surgery when present in implants and medical devices. The infection and the consequent immunological response could result in implant failure, causing serious problems for the patient. Several authors have demonstrated a decrease in bacterial adhesion and a significant decrease in bacterial viability on ZnO samples [11,12]. Controversial results on mammalian cells’ interaction with ZnO nanostructures and nanoparticles have been reported depending on the specific shape of ZnO structures and the cell type. Hydroxyapatite-ZnO composites with polished surfaces allowed cell attachment, spreading and proliferation of osteoblasts and fibroblasts [13]. Results for ZnO nanorods showed that adhesion and, thus, viability of anchorage-dependent cells was reduced, indicating that these substrates cannot be used for biomedical implants when cell adhesion is needed [14,15]. In contrast, ZnO nanoflowers improved mouse MC3T3-E1 osteoblast adhesion, proliferation and differentiation when compared with ZnO films, although cell adhesion and differentiation were higher on polystyrene control [16]. All these results suggest that ZnO biocompatibility may depend on its structure and, also, the specific cell type. In this regard, it is necessary to analyse each nanostructure with the cell type of choice, according to the potential application, before considering its use as an implant or device candidate. Nowadays, nanostructured ZnO is very popular for its application as piezoelectric nanogenerators (NGs). Millimetre-scale NGs have already been used for biological appli- cations, including sensors and generators. However, NGs have also a great potential for electrical stimulation, at the cell scale, of excitable cells like osteoblasts, neurons and muscle cells [17]. In a previous paper, we demonstrated the cytocompatibility of ZnO nanosheets (NSs) on osteoblasts and macrophages, and their use as NGs to induce a cellular response to the local electric fields generated by the piezoelectric NSs. The inherent forces produced by the cells were able to induce a piezopotential without the use of external stimulation [18]. Electric stimulation of different cell types has an important effect on cell regeneration and metabolism. Muscle cells respond to electric fields and induce the contraction of muscle tissue. Thus, the local electric field generated by ZnO NSs would potentially improve the regeneration and rehabilitation of muscle tissue, with several future applications in chronic illnesses such as muscle atrophy, wasting and aging [19,20]. In this study, we analyse the potential application of ZnO NSs on two muscle cell lines, one derived from mouse skeletal muscle and the other from rat smooth muscle. Both cell types are electrically excitable cells. We studied the cytocompatibility of ZnO NSs and the NGs–cells interaction by analysing cell viability, morphology, cytoskeleton distribution and proliferation over time. In addition, we studied the effect of electrical fields generated by the NGs on cell response by measuring intracellular calcium changes. 2. Results 2.1. NG Characterization and Zn Ions Release over Time The grown ZnO NSs, which form the NG array on the AlN-coated coverslip, showed an optimized aspect ratio to be used for cell stimulation, with a mean thickness of 23 ± 7 nm (Figures 1A–C and S1). Due to the reduced thickness of the AlN layer and the ZnO NSs, 2. Results 2.1. NG Characterization and Zn Ions Release over Time The grown ZnO NSs, which form the NG array on the AlN-coated coverslip, showed Int. J. Mol. Sci. 2022, 23, 432 an optimized aspect ratio to be used for cell stimulation, with a mean thickness3 ofof1523 ± 7 nm (Figures 1A–C and S1). Due to the reduced thickness of the AlN layer and the ZnO NSs, the resulting glass coverslips, once covered with the NG array, are translucent, facil- itating the optical the resulting glassmicroscope inspection. coverslips, once In addition, covered with the wurtzite the NG array, crystalfacilitating are translucent, structure and preferential the optical growth microscope along (002) orientation inspection. In addition,were validated the wurtzite with crystal X-ray diffraction structure and preferen-(XRD) tial growth analysis along (Figure (002) 1D). Anorientation were validated energy-dispersive with X-ray(EDS) spectroscopy diffraction (XRD)confirmed analysis analysis the (Figure of presence 1D). ZnAnandenergy-dispersive spectroscopy (EDS) O on the NS composition before analysis confirmed immersion theculture in cell presencemedium of Zn and O on the NS composition before immersion in cell culture medium (Figure 1E). The amount of Zn ions released from the NSs after immersion in the cell cul- (Figure 1E). The amount of Zn ions released from the NSs after immersion in the cell culture medium is ture medium is shown in Figure 1F. Zn ions quantification using inductively coupled shown in Figure 1F. Zn ions quantification using inductively coupled plasma mass spec- plasma mass spectrometry (ICP-MS) indicated a time-dependent dissolution, with a pro- trometry (ICP-MS) indicated a time-dependent dissolution, with a progressive increase over gressive increase time. The quickest over time. release wasThe quickest observed afterrelease 3 days inwas observed culture, reachingafter 3 days in culture, a concentration of reaching a concentration − 1 of 4.5 µg ml −1 of Zn in a volume of 1 mL. Zn concentration in the 4.5 µg mL of Zn in a volume of 1 mL. Zn concentration in the culture medium increased culture µg mL−1 increased to 5.4 medium after 7 days,toreaching 5.4 µg ml 6.3−1µg mL− after 7 1days, reaching after 21 days. 6.3 µg ml−1 after 21 days. Figure 1. 1. Figure Characterization CharacterizationofofZnO ZnONG NG arrays. arrays. SEM SEM images images of ofNSs NSsgrown grownforfor9 h9h at at ◦ C°C 80 80 over an over AlN anlayer AlN of 100ofnm layer 100atnmdifferent amplification at different amplificationmagnitudes: magnitudes: 1 KX, 5 KX 1 KX, 5 KX and and2020KX KX(A–C); (A–C);XRDXRDmeas- urement showing measurement the high showing crystallinity the high of of crystallinity thetheNSs NSsand thepreferential and the preferential orientation orientation along along (002) (002) (D); (D); Measurement MeasurementEDS EDSat at the surfaceofofthe the surface the NSs, NSs, showing showing the presence the presence of Zn,of O Zn, and OAl and Al (E). Concentra- (E). Concentration tionofof ZnZn ions ions released released fromfrom the ZnO the ZnO NSs array NSs array overintime over time in medium culture culture medium (F). (F). 2.2.2.2. ZnO ZnO Cytocompatibility for Cytocompatibility for Smooth Smooth Muscle MuscleCells Cells Cytocompatibilityof Cytocompatibility of ZnO ZnO NG NGarrays arraysforforsmooth smooth muscle muscle(A7r5) cellscells (A7r5) was was analysed in analysed in terms of several biological parameters such as cell viability, adhesion, morphology and terms of several biological parameters such as cell viability, adhesion, morphology and proliferation. Cell viability was analysed by quantifying the number of live cells using proliferation. the Live/DeadCellkit, viability waslive presenting analysed by quantifying cells with the in esterase activity number of live green and cells dead using cells in the Live/Dead red (Figure 2A). The percentage of live cells was higher than 88%, without significantin red kit, presenting live cells with esterase activity in green and dead cells (Figure 2A). The differences whenpercentage compared withof live the cells same was cells higher grown onthan 88%, glass without coverslips significant (control sample) differ- ences (Figure 2B). In addition, no significant differences were found in the initial number of cells (Fig- when compared with the same cells grown on glass coverslips (control sample) ureadhered 2B). Inand the mean addition, nospreading significant area of the cellswere differences after 24 h in culture found in the between A7r5 cells initial number of cells growing adhered onthe and ZnOmean NG arrays and glass spreading areacoverslips (Figure of the cells after2C,D). 24 h in culture between A7r5 cells growing on ZnO NG arrays and glass coverslips (Figure 2C,D). Int. J. Mol. Sci. 2022, 23,Int. 432J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 4 of 15 4 of 16 Figure 2. SmoothFigure muscle 2. Smooth (A7r5) muscle (A7r5)on cells grown cells ZnOgrown NGon ZnO NG arrays. (A)arrays. (A) Liveand Live (green) (green) deadand dead (red) (red) A7r5 cells cultured on ZnO NG array. (B) Percentage of viable A7r5 cells after 24 h in culture on A7r5 cells cultured ZnO onNGZnO NGand arrays array. glass(B) Percentage coverslip (Ctrl), of viable A7r5 evaluated using cells after viability/cytotoxicity Live/Dead 24 h in culture on kit. (C) ZnO NG arrays Number and glass of adhered cells and (D) mean spreading area of cells after 24 h in culture onkit. coverslip (Ctrl), evaluated using Live/Dead viability/cytotoxicity ZnO NG arrays and (C) Number of adhered glass cells andcoverslip (D) mean (Ctrl). Confocalarea spreading images of α-smooth of cells after 24muscle actin immunodetection h in culture on ZnO (green) NG arrays and glass and nuclei coverslip (blue) (Ctrl). of cells images Confocal growingof onα-smooth glass coverslip (E) actin muscle and ZnO NG arrays (F) for 24 h. immunodetection SEM images of well-spread A7r5 cells on glass coverslip (G) and ZnO NG arrays (H,I) for 24 h. 2.3. (green) and nuclei (blue) of cells growing on glass coverslip (E) and ZnO NG arrays (F) for 24 h. SEM ZnO cytocompatibility for skeletal myoblasts. images of well-spread A7r5 cells on glass coverslip (G) and ZnO NG arrays (H,I) for 24 h. 2.3. ZnO cytocompatibility for skeletal A7r5 cells’myoblasts. interaction with ZnO NG arrays and the morphology of A7r5 growing on ZnO NG array were analysed after 24 h in culture using confocal laser scanning micro- A7r5 cells’scope interaction (CLSM)with and ZnO NGelectron scanning arrays and the morphology microscope of A7r5 growinganalysis (SEM). Immunofluorescence on of ZnO NG array were analysed α-smooth muscle after 24showed actin h in culture using different confocal laser distribution scanning of actin fibres inmicroscope A7r5 cells grown (CLSM) and scanning on ZnO NG electron arrays and microscope (SEM). glass coverslip. AsImmunofluorescence shown in Figure 2E,F, cells analysis grown of α- on coverslip smooth musclepresented actin showed well-defined different actin fibres crossing distribution the cell of actin from in fibres end to end, A7r5 whereas cells growncellson grown ZnO NG arraysonand a ZnO NGcoverslip. glass array presented shorter in As shown actin bundles Figure adapted 2E,F, cells to the topography grown on coverslip of the NSs. presented well-defined actin fibres crossing the cell from end to end, whereas cells grown dis- However, cell spreading indicated that cells were well adhered to ZnO NSs without on a ZnO NG arrayturbingpresented cell growth, allowing shorter a close actin interaction bundles adaptedbetween cells to the and NGs. Aof topography similar the well- spread morphology was observed for cells growing on the ZnO NG arrays using SEM. NSs. However, cell spreading indicated that cells were well adhered to ZnO NSs without Cell morphology analysis by SEM indicated that cells grown on a ZnO NG array and glass disturbing cell growth, allowing a close interaction between cells and NGs. A similar coverslip displayed a polygonal shape and a flattened morphology (Figure 2G,H), in close well-spread morphology contact with wastheobserved NGs (Figure for 2I). cells growing on the ZnO NG arrays using SEM. Cell morphology analysis by SEM indicated The effect of ZnO NGs on skeletal that cells grown muscle on a ZnO (C2C12) NG analysed cells was array and in glass terms of cell coverslip displayed viability, adhesion, morphology and proliferation, as for the smooth muscleclose a polygonal shape and a flattened morphology (Figure 2G,H), in cells. In ad- contact with thedition, NGs we (Figure analysed2I). myotubes formation by C2C12 cells fusion on the ZnO NG array. The effect of ZnO NGs on skeletal muscle (C2C12) cells was analysed in terms of cell viability, adhesion, morphology and proliferation, as for the smooth muscle cells. In addition, we analysed myotubes formation by C2C12 cells fusion on the ZnO NG array. Quantification of live cells using the Live/Dead kit showed that the viability of C2C12 cells growing on ZnO NG arrays was higher than 98% (Figure 3A), and no significant Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 5 of 16 Int. J. Mol. Sci. 2022, 23, 432 5 of 15 Quantification of live cells using the Live/Dead kit showed that the viability of C2C12 cells growing on ZnO NG arrays was higher than 98% (Figure 3A), and no significant differences differences were were observed between betweenZnO ZnOsamples samplesand andglass glasscoverslip coverslip (Figure (Figure 3B). 3B). Statis- Statisti- tically significantdifferences cally significant differenceswere werefound foundininthe theinitial initialnumber numberofofcells cellsadhered adheredafterafter2424h,h, being (64 ±±4 4× × 3 cells/cm 2 ) compared to glass coverslip beinghigher higher on on the the ZnO NG arrays arrays (64 10310cells/cm 2) compared to glass coverslip (43 3 2 ± 4± (43 4× × 10 10 cells/cm 3 cells/cm ) (Figure 2) (Figure 3C). However, 3C). However, the mean thespreading mean spreading area was area of cells of cells was signifi- 2 ), when compared significantly cantly reducedreduced for cells for cells growing growing on ZnO on ZnO NG NG arrays arrays ± 30±µm (351(351 30 2µm ), when compared to to cells growing ± µm 2 ) (Figure 3D). cells growing onon glass glass coverslip coverslip (591 (591 ± 49 49 µm2) (Figure 3D). Figure 3. Skeletal myoblasts (C2C12) cells grown on ZnO NG arrays. (A) Live (green) and dead (red) Figure 3. Skeletal myoblasts (C2C12) cells grown on ZnO NG arrays. (A) Live (green) and dead (red) C2C12 cells cultured on ZnO NG array. (B) Percentage of live cells after 24 h growing on ZnO NG C2C12 cells cultured on ZnO NG array. (B) Percentage of live cells after 24 h growing on ZnO NG arrays and glass coverslips (Ctrl), evaluated using Live/Dead viability/cytotoxicity kit. (C) Number arrays of adheredglass and cellscoverslips (Ctrl), and (D) mean evaluatedarea spreading using Live/Dead of cells after 24viability/cytotoxicity h in culture on ZnO kit. NG (C) Number arrays and of adhered glass cells(Ctrl). coverslip and (D) mean spreading Statistically areadifferences significant of cells after are24 h in culture marked by twoonasterisks ZnO NG (p arrays < 0.01)and or threecoverslip glass asterisks (Ctrl). (p < 0.001). SEM images Statistically of C2C12 significant cells morphology differences are marked on by glass twocoverslip asterisks(E) (p and ZnOor < 0.01) NG arrays three (F) after asterisks 24 h in SEM (p < 0.001). culture. images of C2C12 cells morphology on glass coverslip (E) and ZnO NG arrays (F) after 24 h in culture. 2.3. Cell Morphology 2.3. Cell Morphology Cell morphology was evaluated using SEM after 24 h and 7 days in culture. After 24 h, individualized Cell morphology C2C12 wascells showed using evaluated a well-spread SEM aftermorphology 24 h and 7ondaysbothinZnO NG array culture. After and 24 h, glass coverslip, although individualized C2C12 cellscellsshowed growinga on control glass well-spread seemed toon morphology beboth wider,ZnOas ex- NG pected for the spreading area measurements (Figure 3E,F). Cytoplasmic array and glass coverslip, although cells growing on control glass seemed to be wider, as extensions were visible, with expected for thea close interaction spreading with ZnO NG (Figure area measurements arrays. 3E,F). After 7Cytoplasmic days, a monolayer of cells extensions were was observed visible, covering with a close the whole interaction ZnO with ZnONGNG array and differentiated arrays. After 7 days, myotubes a monolayerwereof also cells observed was observed(Figure 4A). the whole ZnO NG array and differentiated myotubes were also covering Stress observed fibres4A). (Figure distribution after 7 days in culture was analysed using CLSM. C2C12 cellsStress showed cleardistribution fibres stress fibres,after without 7 daysevident differences in culture between using was analysed ZnO NG arrays CLSM. and C2C12 coverslip cells showed samples (Figurefibres, clear stress 4B,C).without evident differences between ZnO NG arrays and Aftersamples coverslip 7 days (Figure in culture with the differentiation medium, it was possible to observe 4B,C). the fusion After 7ofdaysseveral cells forming in culture with themyotubes on both differentiation samples. medium, To confirm it was possiblethat the struc- to observe the tures observed fusion of severalwerecellsmyotubes, the presence forming myotubes of sarcomeric on both samples. Toα-actinin, confirmathat component of Z- the structures disk in differentiated observed were myotubes, skeletal themuscle myotubes, presence was analysed. of sarcomeric α-actinin,Images showed that a component α-ac- of Z-disk tinin in were evident differentiated in differentiated skeletal myotubeswas muscle myotubes, in both substrates, analysed. Images without showed clear differences that α-actinin between were them.inLongitudinal evident differentiated myofilament myotubes bundles in both crossing the without substrates, whole cell weredifferences clear detected between them. Longitudinal myofilament bundles crossing the whole cell were detected (Figure 4D,E). In addition, a monolayer of undifferentiated myoblasts without α-actinin labelling or diffused signal were visible. Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW Int. J. Mol. Sci. 2022, 23, 432 (Figure 4D,E). In addition, a monolayer of undifferentiated myoblasts6 ofwithout 15 α labelling or diffused signal were visible. Figure4. 4. Figure SEMSEM images images of differentiated of differentiated C2C12 cell C2C12 cellgrowing after 7 days after 7ondays ZnO NGgrowing onCLSM arrays (A). ZnO NG ar CLSM of images images skeletalof skeletal(C2C12) myoblasts myoblasts (C2C12) cells cells differentiated differentiated after after 7 days in culture on 7glass days in culture on coverslip (B,D) and(B,D) verslip ZnO NGandarrays ZnO(C,E). Stress fibres NG arrays immunodetection (C,E). is shown in red (B,C) is Stress fibres immunodetection and α-actinin shown in red (B,C immunodetection is shown in green (D,E). Skeletal muscle myotubes (arrows) actinin immunodetection is shown in green (D,E). Skeletal muscle myotubes (arrows) were formed after wer 7 days in culture on both samples. after 7 days in culture on both samples. 2.4. Immunological Response 2.4. To Immunological Response find out whether the ZnO NG arrays activate the secretion of inflammatory cy- Towe tokines, find out whether analysed the ZnO the presence of six NG arrays cytokines activate involved in the secretionusing inflammation of inflammato the cytometric bead array (CBA) test. Although all six cytokines quantified kines, we analysed the presence of six cytokines involved in inflammation are important in the using inflammatory response, each one plays a different role and the secretion by macrophages tometric bead array (CBA) test. Although all six cytokines quantified are importan could be different. In general, IL-8, IL-12p70, IL-6, IL-1β and TNF are considered pro- inflammatory inflammatory response, cytokines, each whereas oneis plays IL-10 a different considered role and the an anti-inflammatory secretion cytokine. by macro Quan- could beresults tification different. showed In that general, IL-8, IL-12p70, no significant increase inIL-6, IL-1βconcentration cytokines and TNF are wasconsidered ob- served after 5 h and 24 h of macrophages culture on the ZnO NG array flammatory cytokines, whereas IL-10 is considered an anti-inflammatory cytokine (Figure 5). On the contrary, the presence of lipopolysaccharide (LPS) in the cell culture medium stimulated tification results showed that no significant increase in cytokines concentration the secretion of all cytokines tested. served after 5 h and 24 h of macrophages culture on the ZnO NG array (Figure 5) contrary, the presence of lipopolysaccharide (LPS) in the cell culture medium stim the secretion of all cytokines tested. Int. J. Mol. Sci. 2022, 23, 432 7 of 15 Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW 7 of 16 Figure 5.by Figure 5. Cytokine release Cytokine release by macrophages macrophages analysed analysed by the by the CBACBA test after test 5 and524and after h of culture. 24 h Sixof culture. types of cytokines involved in inflammation, IL-8 (A), IL-12p70 (B), IL-10 (C), IL-6 (D), IL-1β (E) and Six types of cytokines TNF involved in inflammation, (F), are shown in the presence ofIL-8 (A), IL-12p70 lipopolysaccharide (LPS, (B), IL-10 positive (C),ZnO control), IL-6NG(D), array,IL-1β (E) and a negative control. and TNF (F), are shown in the presence of lipopolysaccharide (LPS, positive control), ZnO NG array, and a negative control.2.5. Intracellular Calcium Changes Calcium is a second messenger involved in several intracellular pathways, such as muscleChanges 2.5. Intracellular Calcium contraction. Skeletal muscle cells and smooth muscle cells exposed to electrical stimulation undergo changes in intracellular calcium levels (Figure 6A,B), which induces Calcium is a second messenger involved in several intracellular pathways, such as muscle contraction. Results in smooth muscle cells after 24 h in culture showed that A7r5 cells grown on muscle contraction.theSkeletal muscle cells and smooth muscle cells exposed to electrical ZnO NG array presented 58% of cells with calcium transients, whereas only 7% of cells stimulation undergo changes in intracellular presented calcium Int. J. Mol. Sci. 2022, 23, x FOR PEER REVIEW calcium transients on glass coverslip levels (Figure (Figure 6C). 6A,B), By contrast, no which significant 8 of 16 induces muscle contraction.differences in intracellular calcium changes were detected between C2C12 cells grown for 7 days on ZnO NG arrays and glass coverslip (Video S1 in Supporting Information). Figure 6. Frames of time-lapse video showing florescence due to calcium influxes in smooth muscle Figure 6. Frames of time-lapse video showing florescence due to calcium influxes in smooth muscle (A7r5) cells (A). Intensity of fluorescence indicating calcium influx in a certain A7r5 cell at (A7r5) cells (A). Intensity timepoints:of5 fluorescence min, 6 min and 7indicating calcium min (B). Percentage influx in of activated a certain smooth muscle A7r5 (A7r5) cell at timepoints: cells which 5 min, 6 min andundergo 7 min changes in intracellular calcium concentration (C); statistically significant differences are (B). Percentage of activated smooth muscle (A7r5) cells marked by three asterisks (p < 0.001). COMSOL simulations with the piezopotentials generated by which undergo changes in intracellular a smoothcalcium muscle cellconcentration (C); (D) and cylinder-like cellstatistically significant (E), corresponding differences to a sarcomere of a skeletalare marked by muscle fibre, when exerting a force of 1 µN. Simulation of smooth muscle cell shape and electric potential three asterisks (p < 0.001). COMSOL simulations with the piezopotentials generated by a smooth distribution (F) and values of the potential at the membrane (G) for different muscle forces. muscle cell (D) and cylinder-like cell (E), corresponding to a sarcomere of a skeletal muscle fibre, when exerting a force of 2.6. 1 µN.Finite Element Modelling Simulation of smooth muscle cell shape and electric potential distribution (F) Finite element modelling (FEM) has been used to estimate the piezopotential that the and values of the potential at the membrane (G) for different muscle forces. NG array produces when cells adhere to the top of the material (Figure 6D). The range of forces which cells exert when they attach and move goes from 0.1 nN to 100 nN [21–23]. In case of muscle cells, the forces produced can be in the µN range [24]. As a representative value, a force of 1 µN perpendicular to the top of the cell surface has been applied for a smooth muscle cell already adhered (flattened shape, e.g., day 7 of culture). A variety of values of piezopotentials for each NSs positioned not perpendicular to the membrane can be observed, being the maximum voltages around +200 µV and −200 µV in the cell mem- Int. J. Mol. Sci. 2022, 23, 432 8 of 15 Results in smooth muscle cells after 24 h in culture showed that A7r5 cells grown on the ZnO NG array presented 58% of cells with calcium transients, whereas only 7% of cells presented calcium transients on glass coverslip (Figure 6C). By contrast, no significant differences in intracellular calcium changes were detected between C2C12 cells grown for 7 days on ZnO NG arrays and glass coverslip (Video S1 in Supporting Information). 2.6. Finite Element Modelling Finite element modelling (FEM) has been used to estimate the piezopotential that the NG array produces when cells adhere to the top of the material (Figure 6D). The range of forces which cells exert when they attach and move goes from 0.1 nN to 100 nN [21–23]. In case of muscle cells, the forces produced can be in the µN range [24]. As a representative value, a force of 1 µN perpendicular to the top of the cell surface has been applied for a smooth muscle cell already adhered (flattened shape, e.g., day 7 of culture). A variety of values of piezopotentials for each NSs positioned not perpendicular to the membrane can be observed, being the maximum voltages around +200 µV and −200 µV in the cell membrane. Considering the absolute value of the piezopotential generated for each NG, there are no important differences in magnitude. This voltage depends on the cell force, and the NS size. The size of the NSs has been optimized for cell stimulation by inherent cell forces, obtaining the best result for 9 h of growth at 80 ◦ C using an AlN seed layer of 100 nm (unpublished work). In addition, a model of a myotube with a stretching force of 1 µN, perpendicular to the cross sections of the cylinder, has been simulated. Figure 6E shows the voltage in a specific region that represents a sarcomere as unit of contraction of a skeletal muscle fibre. As can be shown, the force in that direction generates similar voltages in the cell membrane, ranging from +200 µV to −200 µV, although higher voltages can be obtained along the NSs. However, due to the overall charge cancelation, the transversal potential difference between both cross-sectional faces is lower than 1.13 µV. This potential is inferior to the local potentials found for the smooth cells, where the overall direction of the potential difference seems not to be critical for the cell activation. Figure 6F,G shows the simulations of a smooth muscle cell with the electric potential distribution and the values of the electric potential at the membrane for different muscle forces. The electric potential values go from around −300 mV to 300 mV at the cell membrane, for normal cell forces in the range of the 100 nN to 1500 nN. 3. Discussion ZnO nanostructures have attracted remarkable attention as smart piezoelectric mate- rials for biomedical applications. However, several authors have reported controversial results on the biocompatibility of different ZnO nanostructures and nanoparticles with different cell types. In this regard, the morphology of ZnO plays an important role for cytotoxicity. In the present work, ZnO NG arrays based on ZnO NSs were fabricated for biomedical purposes. The hydrothermal method to grow NSs was optimized to be per- formed on glass coverslips and to obtain a thin layer of NSs with the optimal NS thickness to ensure the piezoelectric effect. All the samples obtained presented the same area and were translucent enough to visualize the cells under an optical microscope. On the other hand, biodegradability is another important property of ZnO. New biodegradable devices have a great interest in tissue engineering, although degradable products should not cause undesirable effects. Su et al. (2018) reported that cytotoxicity of ZnO nanoparticles is mainly due to increased intracellular Zn ions as a result of ZnO solubility [10]. For this reason, we decided to quantify the concentration of Zn ions released from the ZnO NSs array over time. The results obtained showed that concentrations reached 5.4 µg mL−1 and 6.2 µg mL−1 after 7 and 21 days, respectively. It has been stated that concentrations lower than 27 µg mL−1 of Zn ions allowed human neuroblastoma cells proliferation [25]. Specific results for smooth muscle cells demonstrated that less than 5.3 µg mL−1 of Zn ions have beneficial effects on human aorta smooth muscle cell adhesion, spreading, viability, proliferation and migration [26]. However, different smooth muscle Int. J. Mol. Sci. 2022, 23, 432 9 of 15 cells from different species respond differently, showing inhibited proliferation of smooth muscle cells from carotid artery of Wistar rats at 2.5 µg mL−1 of Zn ions or significantly reduction of human prostatic smooth muscle cells proliferation above 16.7 µg mL−1 [27]. Taking into account these previous results, we expected that the concentration of Zn ions release from ZnO NSs array would not reduce the viability of smooth muscle cells and skeletal muscle cells. To elucidate this point, we analysed the viability, adhesion, proliferation and differentiation of the two muscle cell types on ZnO NG arrays up to 7 days in culture. In both cell types, cells presented high viability and were able to proliferate and differentiate on ZnO NG arrays. These results are in agreement with our previous results when analysing cytotoxicity of ZnO NG arrays for Saos-2 cells and macrophages [18]. Moreover, the Zn ions released from the ZnO NG arrays did not have any negative effect either on smooth muscle cells or on skeletal muscle cells. To go further in the analysis of biocompatibility, we analysed the immunogenic re- sponse to ZnO NG arrays. The biological response to corrosion products from the de- vices can be responsible for inflammatory reactions and can induce the formation of a non-adherent fibrous capsule surrounding the device, which could end in chronic in- flammation [28]. We analysed the release of six cytokines involved in inflammation by macrophages when cultured in the presence of ZnO NG arrays and controls. Results indi- cated that after an incubation of 5 and 24 h with ZnO NG arrays, no significant amounts of inflammatory cytokines were secreted compared with the negative control. However, the secretion of cytokines was high when macrophages were incubated with LPS as positive control. It has been reported that the inflammatory response activation depends on the particle and ion concentration. In this regard, we can correlate the ions’ release after 24 h with the inflammatory response and assume that a concentration of 3.1 µg mL−1 of Zn ions does not activate it. Surface topography can modulate cell adhesion, morphology and the interaction between cells and material. In addition, topography and stiffness could influence the differentiation of cells on biomaterials and devices. The results showed that both types of muscle cells adhered well to ZnO NSs, with the cytoskeleton adapted to the topography and cytoplasmic prolongations connected to NSs. The close interaction between cells and the NGs is necessary for the induction of a piezoelectric effect. In the case of C2C12 cells, we found an increase in the number of cells initially adhered to ZnO NG arrays compared to glass coverslips, although the spreading area of the adhered cells was lower in ZnO NG arrays. The C2C12 cells are able to proliferate as soon as they adhere to a surface and the population doubling time is very short. Therefore, due to the proliferative rate of C2C12 cells, no differences were observed after 7 days in culture. Besides the proliferation, skeletal muscle cells need to differentiate to create functional myotubes. After 4 days, myoblasts fuse in response to the differentiation medium, producing syncytia with several nuclei [29]. Ciofani et al. (2012) analysed the differentiation of H9c2 cells on ZnO nanowires as a model for skeletal muscle cells, and observed that cells displayed a disordered arrangement, without showing the typical tubular shape of H9c2 myotubes [30]. They suggested that the stiffness or the nanotopography of ZnO nanowires was cause for the prevention of myotubes formation. By contrast, our results showed that C2C12 cells were able to differentiate and form myotubes on ZnO NGs, similar to those formed when grown on control samples. The formation of myotubes is a necessary step for further functionality of muscle cells, including myotubes contraction. Both smooth muscle cells and skeletal muscle cells are able to respond to electric stimulation by voltage-gated calcium channels (VGCC). Plasma membrane depolarization caused by the electric fields opens the VGCC and, subsequently, increases the intracellular calcium concentration. In our study, we analysed the intracellular calcium changes in cells grown on the ZnO NG arrays and found different results depending on the cell type tested. Intracellular calcium transients were detected in smooth muscle cells, showing a significant increase on ZnO NG arrays (i.e., 58% of activated cells compared to 7% in the control), whereas no differences were detected in skeletal muscle cells. Results suggest Int. J. Mol. Sci. 2022, 23, 432 10 of 15 that piezoelectric ZnO NG arrays are able to generate a local electric field that activates the smooth muscle cells. These results are in agreement with our previous results on osteoblasts, where we hypothesized that the mechanical stress produced by cell adhesion is responsible for their own electromechanical stimulation [18]. Similar results were also observed in piezoelectric PVDF nanofiber scaffolds [31]. In addition, FEM simulations also validate the piezoelectric stimulation of smooth muscle cells, that is, the NGs can create local piezopotential distributions, allowing a local activation of the VGCC and the corresponding calcium influx. The simulated voltages generated by the NGs range from around −300 mV to 300 mV at the cell membrane, which are values theoretically high enough to activate the VGCCs at the cell membrane. By contrast, skeletal muscle cells did not show any effect of the ZnO NG arrays regarding intracellular calcium changes. These results were unexpected, but several factors may be responsible for the lack of cell stimulation. In the literature, several authors have studied the effect of electrical stimulation on C2C12 cells by analysing a wide range of electric fields, frequency and timing of the stimulation. All of them agree that electrical pulses with certain periodicity are necessary to mimic the physiological stimulation in vitro, and that they allow the creation of functional skeletal muscle tissues [20,32]. Indeed, single myoblasts respond to electromagnetic fields by modulation of the intracellular calcium [33]. The effect of electromagnetic fields to myoblasts could be responsible for the creation of functional myotubes. On the other hand, multinucleated myotubes in physiological tissues present a highly unidirectional orientation, which is necessary to produce contracting forces. In vitro, myotube formation loses the natural organization and the myotubes appear randomly distributed. It seems that the lack of cell alignment may alter the functionality of skeletal muscle [34]. In our study, we hypothesized that adherent cell forces would be able to mechanically stress the NGs and induce a local electric field to stimulate muscle cells. Therefore, no external energy sources were used and the potential electric stimulation was generated randomly by the different ZnO NGs of the array. In addition, a single myotube could be stimulated by several NGs at the same time and at different regions of the plasma membrane, depending on the area of the cell pressing on the ZnO NG array during adhesion or movement. This random stimulation in different directions depending on the orientation of each NS could alter the contraction because of the lack of unidirectional electrical stimulation. The randomly generated electric fields by ZnO NGs was validated with the COMSOL simulations performed with a random NG array. As has been reported, the electric field direction is crucial for correct muscle contraction when an electric field is applied [35,36]. The myotube contraction is controlled by the direction of the electric field due to the highly anisotropic electrical properties of skeletal muscle. Moreover, electrical parameters such as frequency, pulse width and voltage amplitude should be controlled to stimulate the skeletal muscle contraction without electrochemical damage or toxicity. Therefore, to allow for a specific stimulation for this type of cells, we should use an NG array with aligned NSs along the perpendicular direction of the myotubes. Unfortunately, the hydrothermal method used for this work does not allow for this alignment. However, research using a micromachined piezoelectric material, such as PVDF, with aligned patterns at the nanoscale (e.g., using stamp or soft-lithography or reactive ion etching) is currently being conducted, with promising potential. In addition, it is possible to use ultrasound signals to wirelessly actuate the NGs in order to electrically stimulate the cells. The results provided by this work allow the study of a family of groundbreaking developments for a personalized and minimally invasive nanomedicine. 4. Materials and Methods 4.1. NG Synthesis and Optimization NG arrays based on ZnO NSs were synthesised using a hydrothermal growth at mild conditions. In order to allow growth on the NS shape, a thin-film layer of 100 nm of AlN was deposited by using radio-frequency (RF) sputtering on top of glass coverslips. An aqueous solution containing hexamethylenetetramine (HMTA) and Zn(NO3 )2 was Int. J. Mol. Sci. 2022, 23, 432 11 of 15 introduced in an amber wide-mouth jar. The coated glass coverslips were placed in the jar, floating on the solution, with the AlN layer facing down. The jar was sealed and introduced in an oven at 80 ◦ C for 9 h. Then, the coverslips were collected, rinsed in deionized water and ethanol and left to dry at room temperature (RT). The growth parameters (i.e., 80 ◦ C for 9 h) were optimized in order to improve the activation effect. 4.2. NG Characterization The grown ZnO NSs on an AlN-coated coverslip were characterized using an SEM Zeiss Auriga at 3 kV and a secondary electrons detector together with an energy-dispersive X-ray spectroscopy (EDS/EDX) detector. EDX measurements were obtained by applying a voltage of 5 kV. In addition, XRD analysis was obtained on a Bruker D8 Advance diffrac- tometer (CuKα, λ = 1.5418 Å) with a bidimensional general area detector diffraction system (GADDS) detector. For this measurement, a 2θ angle ranging from 30◦ to 60◦ , a voltage of 40 KV and a current of 40 mA were used. 4.3. COMSOL Simulation We used the piezoelectric physics of COMSOL to simulate the piezopotential generated by NGs based on hexagonal ZnO NSs. The ZnO NSs were created with a thickness of 30 nm and a height of 1000 nm. A layer of 100 nm of AlN was used in the bottom surface. Mechanically, all the substrate was anchored. A force of 1 µN was applied normally to the cell membrane. A tetrahedral mesh with adaptive size (i.e., coarse for cell, medium for substrate and fine for NSs) was used (Figure S2). The piezoelectric origin axes were carefully chosen for each NS, so that they were aligned along their respective c-axis. 4.4. Zinc Ions Released from NGs In order to analyse the biodegradability of the ZnO NG arrays, the amount of zinc ions released from the NGs was quantified. ZnO NG arrays were sterilized in ethanol for 30 min and incubated with 1 mL of DMEM cell culture medium at standard conditions (37 ◦ C and 5% CO2 ). The culture medium was withdrawn after 24 h and after 3, 7 and 21 days, and the zinc concentration was analysed by ICP-MS on an Agilent ICP-MS 7500ce equipment (Agilent Technologies, Santa Clara, CA, USA). 4.5. Cell Lines Two different muscle cell lines were used to test the cytocompatibility of the ZnO NG arrays and to analyse the piezoelectric effect generated by them: C2C12 mouse skeletal myoblasts and A7r5 rat smooth muscle cells (both from ATCC). C2C12 cells were cultured under standard conditions in Dulbecco’s modified Eagle medium (DMEM; Invitrogen) supplemented with 10% foetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA). For myoblast differentiation, the growth medium was replaced by a differentiation medium containing DMEM supplemented with 10% horse serum (Thermo Fisher Scientific) after 24 h of cells seeding. The differentiation medium was replaced each 2 days for 7 days. A7r5 cells were grown in DMEM supplemented with 10% FBS under standard conditions. THP-1 monocyte cells were used to analyse the immunological response to the ZnO NG array. Monocytes were grown in an RPMI 1640 medium (Gibco) supplemented with 25% FBS in standard conditions. To differentiate monocytes into macrophages, 400,000 THP-1 cells were seeded into 24-well plates and treated with 0.16 µM phorbol-12-myristate- 13-acetate (Sigma) for 72 h. Then, the cells were washed and incubated in a fresh medium for 24 h before carrying out the experiments. 4.6. Cell Viability Assay Direct cytotoxicity was evaluated by detecting the activity of intracellular esterases using the Life/Dead Viability/Cytotoxicity kit for mammalian cells (Invitrogen), according to the manufacturer’s protocol. Briefly, ZnO NG arrays were cleaned and sterilized with absolute ethanol for at least 1 h. Once sterilized, samples were transferred into 4-well Int. J. Mol. Sci. 2022, 23, 432 12 of 15 culture plates and 1 × 105 C2C12 cells or 3 × 104 A7r5 cells were seeded on top of each ZnO NG array and cultured for 24 h. In parallel, the same number of cells were seeded on glass coverslips as control substrates. After 24 h, cells were incubated with the kit and images from different regions of the samples were captured on an inverted epifluorescence microscope (Olympus IX7). Each experiment was performed in triplicate and a minimum of 300 cells were analysed per sample. 4.7. Initial Cell Adhesion and Spreading Area of Cells To quantify the number of cells initially adhered on samples and the mean spreading area per cell, images previously captured for cell viability assay were used. The cell number was quantified using the cell counter plugin of Image J software. The spreading area was measured analysing fluorescent calcein using Image J software. For each experiment, at least 15 regions from 3 replicates were analysed. 4.8. Cell Morphology Assay At the end of the cell viability assay, the same samples were prepared for SEM visual- ization. Cells grown on samples were rinsed twice in phosphate buffered saline (PBS), fixed in 4% paraformaldehyde (PFA; Sigma) in PBS for 15 min at RT and rinsed twice in PBS. Then, the cells were dehydrated by ethanol at increasing concentrations (50%, 70%, 90% and twice 100%) for 8 min each and dried using hexamethyl disilazane (Electron Microscopy Science) for 15 min. Finally, samples were mounted on special stubs and analysed using SEM Zeiss Auriga. 4.9. Immunofluorescence Detection of Smooth Muscle Actin on A7r5 Cells Cytoskeleton distribution in A7r5 cells was analysed by detecting α-smooth muscle actin (α-SMA), a specific protein of smooth muscle cells. A total of 6 × 104 cells were seeded on ZnO NG arrays and glass coverslip controls. After 7 days in culture, the cells were fixed in 4% PFA in PBS for 15 min at RT, and permeabilised and blocked with 0.5% Triton X-100, 0.2% sodium azide and 3% goat serum in PBS for 30 min at RT. Then, the cells were incubated with mouse anti-α-SMA IgG monoclonal-antibody (1:500; Sigma) overnight at 4 ◦ C. The next day, the cells were incubated with Alexa Fluor 488 goat anti- mouse IgG (1:500; Thermo Fisher Scientific) for 2 h at RT in the dark. Finally, samples were incubated with Hoechst 33258 (5 µg/mL; Sigma) for 15 min at RT. ZnO NG arrays and control glass coverslips with stained cells were mounted using ProLong Antifade (Thermo Fisher Scientific) and evaluated with a CLSM (Olympus). 4.10. Immunofluorescence Detection of Sarcomeric α-Actinin on C2C12 Cells Myoblast differentiation was determined by an immunodetection of sarcomeric α- actinin after 7 days in a differentiation culture medium. A total of 1 × 105 C2C12 cells were seeded on ZnO NG arrays and glass coverslip controls. After 7 days in culture, the cells were fixed in 4% PFA in PBS for 15 min at RT, permeabilised with 0.1% Triton X-100 (Sigma) in PBS for 15 min and blocked for 20 min with 1% bovine serum albumin (BSA; Sigma) in PBS at RT. Samples were then incubated with mouse anti-sarcomeric α-actinin IgG monoclonal-antibody (1:500; Abcam) for 1 h at RT and washed with 1% BSA-PBS. Then, the samples were incubated with Alexa Fluor 488 goat anti-mouse IgG (1:500) for 45 min at RT in the dark. Finally, the samples were incubated with Hoechst 33258 (5 µg/mL) for 15 min at RT. ZnO NG arrays and control glass coverslips with stained cells were mounted using ProLong Antifade and evaluated with a CLSM. 4.11. Quantification of Inflammatory Cytokines Secretion THP-1 cells differentiated into macrophages were used to conduct this analysis. ZnO NG arrays were sterilized and placed over a culture of macrophages and maintained in contact with the cells for 5 and 24 h. After each time-point, supernatants were removed and used to quantify cytokines secretion. Six inflammatory cytokines, IL-8, IL-12p70, IL-10, IL-6, Int. J. Mol. Sci. 2022, 23, 432 13 of 15 IL-1β and TNF, were evaluated by flow cytometry using a CBA (Becton Dickinson, Franklin Lakes, NJ, USA). Cytokine concentration in the supernatant was analysed according to manufacturer’s protocol. A positive control with 1 µg/mL of LPS (Sigma) and a negative control were performed together with samples analysis. 4.12. Intracellular Calcium Quantification A7r5 cells were cultured on samples for 24 h and C2C12 cells were cultured on samples for 7 days in differentiation medium. Then, the cells were loaded with 2 µM Fluo-4 AM (Life Technologies, Carlsbad, CA, USA) in serum free DMEM for 30 min in the dark at RT. The samples were washed with serum-free DMEM and transferred to MatTek dishes with a fresh medium without phenol red. Images were captured using an Olympus IX71 inverted microscope equipped with epifluorescence and heating plate. A time-lapse every 1 s was recorded for 15 min for each sample (Video S1 in Supporting Information). Changes in fluorescent intensity during the time of monitoring were processed and quantified using Image J software (NIH). 4.13. Statistical Analysis The quantitative data were presented as the mean with the standard error of the mean. Cell viability and percentage of activated cells were analysed using Fisher’s exact test. The number of cells adhered and the spreading area of the cells were analysed using a t-test. Statistical comparisons of cytokine release were performed using the one-way analysis of variance (ANOVA) with Tukey’s multiple comparisons test. All statistical analyses were performed with the GraphPad PRISM software (6.01). 5. Conclusions In this study, we demonstrated the biocompatibility of ZnO NGs for smooth muscle cells and skeletal muscle cells. Both cell types were able to adhere, proliferate and differen- tiate on ZnO NGs. In addition, the ions released over time from the NGs did not affect the viability of muscle cells and did not induce an in vitro inflammatory response. Regarding the piezoelectric stimulation, the electromechanical interaction of NGs and cells was able to stimulate smooth muscle cells by inducing intracellular calcium transients, but no effect was observed on skeletal muscle cells. In single skeletal myotubes, the ZnO NGs simulta- neously stimulate different regions of the cell randomly, and the lack of a unidirectional electric field interferes with contraction. The different response of two muscle cell types to the same ZnO NG indicates that the NG distribution and morphology should be custom designed and optimized according to the potential tissue of application. Although the use of electrical stimulation on muscle cells has been extensively studied, the effect of local electric fields at a cell-scale without applying external stimulation has a high upcoming potential. The work herein paves the way for future NG development able to stimulate muscle cells by local electrical impulses. Supplementary Materials: The following are available online at https://www.mdpi.com/article/10 .3390/ijms23010432/s1. Author Contributions: Conceptualization, G.M. and A.B.; methodology, G.M., J.E., E.I. and A.B.; software, G.M.; validation, G.M., O.C., L.A.-V., A.A. and A.B.; formal analysis, L.A.-V. and A.A.; investigation, G.M., O.C., L.A.-V., A.A. and A.B.; resources, G.M. and C.N.; data curation, G.M., O.C., L.A.-V., A.A. and A.B.; writing—original draft preparation, G.M., A.B. and C.N.; writing—review and editing, G.M., A.B., E.I. and C.N.; supervision, G.M. and C.N.; project administration, G.M.; funding acquisition, G.M. and C.N. All authors have read and agreed to the published version of the manuscript. Funding: This study was supported by the Spanish Government (MAT2017-86357-C3-3-R, PID2020- 118644RB-C21, PID2020-119350RA-I00 and EUR2020-112082), La Caixa Foundation under the Junior Leader Retaining program (LCF/BQ/PR19/11700010) and the Generalitat de Catalunya (2017-SGR- 503 and 2017-SGR-1420). Int. J. Mol. Sci. 2022, 23, 432 14 of 15 Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data is contained within the article or Supplementary Material. Acknowledgments: The authors would like to thank the staff from the Servei de Microscòpia of Universitat Autònoma de Barcelona and the cleanroom at the Institute of Microelectronics of Barcelona (IMB-CNM, CSIC). Conflicts of Interest: The authors declare no conflict of interest. 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