A. Live/dead assay

Cell viability is an important parameter for any in vitro cell assay. Culture conditions and experimental treatments can affect cell viability by directly or indirectly inducing cytotoxicity, apoptosis, and/or necrosis.3232. W.-S. Yeow, A. Baras, A. Chua, D. M. Nguyen, S. S. Sehgal, D. S. Schrump, and D. M. Nguyen, J. Thorac. Cardiovasc. Surg. 132, 1356 (2006). https://doi.org/10.1016/j.jtcvs.2006.07.025 A hallmark of viable cells is an intact plasma membrane and cellular metabolic activity.3333. D. Fischer, Y. Li, B. Ahlemeyer, J. Krieglstein, and T. Kissel, Biomaterials 24, 1121 (2003). https://doi.org/10.1016/S0142-9612(02)00445-3 These two features form the basis of this live and dead cell assay. Live cells are identified on the basis of intracellular esterase activity that generates green fluorescence.3434. E. J. Pavlik et al., Cancer Invest. 3, 413 (1985). https://doi.org/10.3109/07357908509039802 Dead cells are identified by the lack of esterase activity and nonintact plasma membrane that allows red dye staining of the cell nucleus. Live/dead assays for 1st, 7th, and 14th days in the absence [Fig. 2(a)] and presence [Fig. 2(b)] of CES were performed. These results demonstrated that copolymers showed good cell viability and proliferation for all studied surfaces. Figure 2(a) shows an increasing spread of cells onto all surfaces with time, indicating cell proliferation throughout the studied period in the absence of CES. On the other side, EF caused a reduction in the cell coverage over all surfaces after seeding for 24 h, and the same was observed at 7 and 14 days [Fig. 2(b)]. Moreover, hMSC-BM under CES was disconnected with elongated cell morphology [Fig. 2(b), 14th day], indicating a reduced cell-cell interaction, which was in sharp contrast to what was observed in the absence of EF [Fig. 2(a)]. The results showed that the cellular distribution was decreased even though the mineralization of the extracellular matrix over the copolymers with higher PEDOT content had enhanced. To understand the relationship between cell differentiation and their proliferation behavior under CES, an analysis of cell metabolic behavior and DNA expressions levels are also very important.

B. Cell metabolic behavior

In order to evaluate the influence of EF on hMSC-BM metabolism, AlamarBlue reduction was measured in the absence [Fig. 3(a)] and presence [Fig. 3(b)] of the CES from the 1st to the 14th day. Figure 3 shows that the fluorescence intensity of AlamarBlue reduction increased steadily with time for both conditions. Figure 3(a) shows that at 1st day, the metabolic activity of hMSC in the absence of CES over the studied materials presented comparable behavior (i.e., there was no significant difference in fluorescence intensity between them at this time point). However, from the 3rd day, the stem cell metabolic activity over the copolymer 1:50 overtake all the others, whereas for EDOT-PDLLA and copolymers 1:25 and 1:5, the metabolic activity was comparable between them until 14th. On the other hand, when the cells’ metabolic behavior in the presence of CES is analyzed in detail [Fig. 3(b)], a higher metabolic activity over copolymers 1:25 and 1:5 from the 1st to the 5th day than for EDOT-PDLLA and copolymer 1:50 is observed. Moreover, from the 7th day, the cells over copolymers 1:25 and 1:50 had higher metabolic activity than 1:5. It is also observed that from the 7th day, there was a constant increase in the cell’s metabolism over the copolymer 1:50, which overtakes all the others only at the 14th day. For EDOT-PDLLA, it is observed that the cells’ metabolic activity over this material was lower in the whole analyzed range, which overtakes all the others only at the 11th day. EF effects are not neutralized after the exposition of 2 h per day, causing a decrease in cell metabolic activity if compared with the absence of CES. Furthermore, there are differences to the outcomes showed off for the spread cell over biomaterials as much in the absence as in the presence of the CES. The same is observed in DNA expression. In contrast, the ARS assay showed off over the copolymers with a higher conductive backbone structure (PEDOT-co-PDLLA 1:25 and 1:5) presented higher calcium deposition levels, indicating that intrinsic conductivity of the copolymers plays a key role function in promoting the osteogenic differentiation of hMSC-BM, since in the insulating polymer (EDOT), the calcium levels were smaller. In this context, Khatib and co-workers3535. L. Khatib, D. E. Golan, and M. Cho, FASEB J. 18, 1903 (2004). https://doi.org/10.1096/fj.04-1814fje observed that ES promoted enhanced intracellular calcium increase mediated by phospholipase C activation in human osteoblasts. The same was observed by Leppik,3636. L. Leppik, K. M. C. Oliveira, M. B. Bhavsar, and J. H. Barker, Eur. J. Trauma Emerg. Surg. 46, 231 (2020). https://doi.org/10.1007/s00068-020-01324-1 where ES has been shown to promote bone healing and regeneration in both animal experiments and clinical treatments. On the other hand, what da Silva et al.2525. A. C. Da Silva, A. T. S. Semeano, A. H. B. Dourado, H. Ulrich, and S. I. Cordoba De Torresi, ACS Omega 3, 5593 (2018). https://doi.org/10.1021/acsomega.8b00510 proposed is based on the surface charge density promoted electrostatic interactions between the scaffold of the conductive copolymer and the anchoring proteins of cells that trigger cell signaling that models cell spreading, migration, and differentiation. The orientation and adsorption rate of serum proteins in the substrate are also affected by their surface charge, interfering in the activation of anchoring protein. Although it is not clear yet, some explanations were proposed on how electrical stimulation could alter calcium ion concentration in the cell. Based on data reported in the literature, a discussion much more deeply was done in the section about the osteogenesis metabolic cell process.

C. DNA expression levels

The effect of cellular stress on hMSC-BM adhesion and proliferation on different surfaces submitted to the CES were assessed through the quantification of the DNA content with PicoGreen, as shown in Fig. 4. These results were obtained after 21 days of culture in the growth medium under the CES with a regime of a 28 mV/mm electric field for 2 h per day. For comparison, the samples without CES were also included in the study.Interestingly, in the absence of the CES [Fig. 4(a)], the hMSC DNA content (correlating to total cell number) on PEDOT-co-PDLLA 1:25 was higher than on all the other substrates. The expressed values for DNA content were 46.00, 91.00, 69.00, and 63.00 ng  ml−1 for PEDOT-co-PDLLA 1:50, 1:25, and 1:5, and EDOT-PDLLA, respectively. In contrast, under CES [Fig. 4(b)], reduced DNA expression levels were observed in all samples, with values of the DNA content of 19.00, 18.00, 21.00, and 24.00 ng  ml−1 for the respective PEDOT-co-PDLLA 1:50, 1:25, and 1:5, and EDOT-PDLLA. Several cellular effects are understood to be mediated by the EF applied in the CES through an electro-coupling mechanism.3737. M. R. Cho, IEEE Trans. Plasma Sci. 30, 1504 (2002). https://doi.org/10.1109/TPS.2002.804200 The basis of invoking such an indirect effect emerges from the high resistance imparted by the plasma membrane, which prevents the penetration of electric stimuli, regardless of the conducting nature of the cytoplasm.3838. P. W. Brandt and A. R. Freeman, Science 155, 582 (1967). https://doi.org/10.1126/science.155.3762.582 One of the possible electro-coupling mechanisms involves asymmetric redistribution/diffusion of electrically charged cell-surface receptors in response to an EF applied, which further activates numerous downstream signaling cascades.39,4039. F. Ke et al., Stem Cells 32, 2799 (2014). https://doi.org/10.1002/stem.176340. D. Liu, C. Yi, C.-C. Fong, Q. Jin, Z. Wang, W.-K. Yu, D. Sun, J. Zhao, and M. Yang, Biol. Med. 10, 1153 (2014). https://doi.org/10.1016/j.nano.2014.02.003 Another possible mode is via the voltage-gated calcium channel activation (VGCC) by cell membrane depolarization, which leads to the most consistently occurring cellular response to electric stimuli, i.e., the elevation of intracellular calcium ion concentration.3939. F. Ke et al., Stem Cells 32, 2799 (2014). https://doi.org/10.1002/stem.1763 It is very interesting because the efficacy of substrate conductivity and capacitive electrical stimulation acting in synergy could direct hMSC-BM differentiation toward osteogenic lineage.4141. G. Thrivikraman, S. K. Boda, and B. Basu, Biomaterials 150, 60 (2018). https://doi.org/10.1016/j.biomaterials.2017.10.003

D. Osteogenesis cell differentiation

The growth of the new bone tissue on a biocompatible material in regenerative medicine requires various factors that need to be optimized and controlled. Utilizing the piezoelectric nature of bone,5,75. R. Balint, N. J. Cassidy, and S. H. Cartmell, Acta Biomater. 10, 2341 (2014). https://doi.org/10.1016/j.actbio.2014.02.0157. C. Ribeiro, D. M. Correia, I. Rodrigues, L. Guardão, S. Guimarães, R. Soares, and S. Lanceros-Méndez, Mater. Lett. 209, 118 (2017). https://doi.org/10.1016/j.matlet.2017.07.099 CES can be an additional tool to control osteogenesis. In order to evaluate the hMSC-BM differentiation toward osteogenic lineage under CES, Alizarin Red stained images were obtained to identify the calcified nodules [Figs. 5(a) and 5(b)]. In the absence of CES [Fig. 5(a)], lightly stained areas that were likely caused by the cellular metabolism of calcium and magnesium were observed; in contrast, under CES [Fig. 5(b)], significantly more calcified nodules were seen, indicating the initiation of mineralization of the extracellular matrix.In order to better assess the osteogenic effect of CES, ARS was extracted by cetylpyridinium chloride and subsequently quantified [Figs. 5(c) and 5(d)]. Figure 5(c) shows that the copolymers (PEDOT-co-PDLLA) and the macromonomer (EDOT-PDLLA) without CES had comparable amounts of extracted dye. In contrast [Fig. 5(d)], the thin films of copolymers under capacitive electrical stimulation presented higher calcium deposition levels, especially for the copolymers with the higher molar ratios of PEDOT (PEDOT-co-PDLLA 1:25 and 1:5). As already reported in a previous publication, cyclic voltammetry confirmed the electroactive character of the materials.2525. A. C. Da Silva, A. T. S. Semeano, A. H. B. Dourado, H. Ulrich, and S. I. Cordoba De Torresi, ACS Omega 3, 5593 (2018). https://doi.org/10.1021/acsomega.8b00510 Conductivity measurements were performed via electrochemical impedance spectroscopy, and the bulk conductivities of PEDOT-co-PDLLA 1:5, 1:25, and 1:50 were 5.35 × 10−5, 2.07 × 10−5, and 4.19 × 10−8 S  cm−1, respectively.2525. A. C. Da Silva, A. T. S. Semeano, A. H. B. Dourado, H. Ulrich, and S. I. Cordoba De Torresi, ACS Omega 3, 5593 (2018). https://doi.org/10.1021/acsomega.8b00510 This feature perhaps indicates that the intrinsic conductivity of the copolymers plays a key role in the differentiation process from hMSC-BM to bone cells under CES. It should be noted that the trends in normalized calcium deposition were similar to those of total deposition on day 21, suggesting that improvements in the calcium levels were not affected by the number of cells (Fig. S4).7373. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001435 for a scheme of the biorector and a computational modelling of the electric field distribution inside it. Interestingly, because the mature bone matrix resulted in differentiated cell death, a lower number of cells led to higher normalized calcium deposition. These results supported that the electroactive surfaces improved osteogenesis.The use of an electroactive and partially biodegradable polymer with this experimental setup is a novelty and little work has been reported in the literature. Data reported in our previous publication2323. J. G. Hardy, J. Y. Lee, and C. E. Schmidt, Curr. Opin. Biotechnol. 24, 847 (2013). https://doi.org/10.1016/j.copbio.2013.03.011 showed that in vitro biodegradability of PEDOT-co-PDLLA in the molar proportions mentioned above was evaluated using proteinase K. After 35 days, 1:5, 1:25, and 1:50 copolymers reached 45.9 ± 4.1, 35.6 ± 2.0, and 29.2 ± 2.0% weight losses, respectively.2323. J. G. Hardy, J. Y. Lee, and C. E. Schmidt, Curr. Opin. Biotechnol. 24, 847 (2013). https://doi.org/10.1016/j.copbio.2013.03.011 Noncytotoxicity was assessed by adhesion, migration, and proliferation assays using embryonic stem cells (E14.tg2a); excellent neuronal differentiation was observed. PEDOT-co-PDLLA presented surface chemistry and charge density properties that make them potentially useful as scaffold materials in different fields of applications, especially for tissue engineering.2525. A. C. Da Silva, A. T. S. Semeano, A. H. B. Dourado, H. Ulrich, and S. I. Cordoba De Torresi, ACS Omega 3, 5593 (2018). https://doi.org/10.1021/acsomega.8b00510 With regard to the electroactive properties of copolymers, some studies indicated that electroactive surfaces may promote cell differentiation,42,4342. M.-T. Tsai, W.-J. Li, R. S. Tuan, and W. H. Chang, J. Orthop. Res. 27, 1169 (2009). https://doi.org/10.1002/jor.2086243. W.-W. Hu, Y.-T. Hsu, Y.-C. Cheng, C. Li, R.-C. Ruaan, C.-C. Chien, C.-A. Chung, and C.-W. Tsao, Mater. Sci. Eng. C 37, 28 (2014). https://doi.org/10.1016/j.msec.2013.12.019 and experimental evidence suggested one plausible mechanism that involves an induced electric field in the cell membrane causing alterations in the transmembrane potential.44–4644. P. Marszalek, D. S. Liu, and T. Y. Tsong, Biophys. J. 58, 1053 (1990). https://doi.org/10.1016/S0006-3495(90)82447-445. B. Valic, M. Golzio, M. Pavlin, A. Schatz, C. Faurie, B. Gabriel, J. Teissié, M.-P. Rols, and D. Miklavcic, Eur. Biophys. J. 32, 519 (2003). https://doi.org/10.1007/s00249-003-0296-946. D. K.-L. Cheng, L. Tung, and E. A. Sobie, Am. J. Physiol. Circ. Physiol. 277, H351 (1999). https://doi.org/10.1152/ajpheart.1999.277.1.H351 The potential difference across the cell membrane originates from the interaction between the intra- and extracellular ionic concentration,3939. F. Ke et al., Stem Cells 32, 2799 (2014). https://doi.org/10.1002/stem.1763 and it is regulated through ion channels, pumps, and transporter proteins.4747. M. Kayahara, X. Wang, and C. Tournier, Mol. Cell. Biol. 25, 3784 (2005). https://doi.org/10.1128/MCB.25.9.3784-3792.2005 When an external EF is applied, the induced electric field in the cell membrane can cause modification of its potential, which in turn can increase the activity of sodium, potassium, or calcium channels and alter the enzyme activity of phosphates containing the voltage-sensor domain.48,4948. M. Sasaki, Science 312, 589 (2006). https://doi.org/10.1126/science.112235249. S. A. Pless, J. D. Galpin, A. P. Niciforovic, and C. A. Ahern, Nat. Chem. Biol. 7, 617 (2011). https://doi.org/10.1038/nchembio.622 Therefore, the EF produced by CES may also induce the differentiation process of stem cells to other tissue types. The intrinsic conductivity of the copolymers, mainly those with higher conducting segments in their backbone structure (1:25 and 1:5), could potentialize the EF effect on ion channels and enzyme activity in the hMSC-BM, based on calcium deposition observed on ARS results. Our results supported the osteogenesis of hMSC-BM from CES, which can be caused by the induced EF that may trigger a variety of intracellular signaling events involving the charge redistribution and the ion flow, including signal propagation via Ca2+, gap junctions, or even protein-protein interactions,39,47,4939. F. Ke et al., Stem Cells 32, 2799 (2014). https://doi.org/10.1002/stem.176347. M. Kayahara, X. Wang, and C. Tournier, Mol. Cell. Biol. 25, 3784 (2005). https://doi.org/10.1128/MCB.25.9.3784-3792.200549. S. A. Pless, J. D. Galpin, A. P. Niciforovic, and C. A. Ahern, Nat. Chem. Biol. 7, 617 (2011). https://doi.org/10.1038/nchembio.622 inducing calcium deposition in the extracellular matrix.43,5043. W.-W. Hu, Y.-T. Hsu, Y.-C. Cheng, C. Li, R.-C. Ruaan, C.-C. Chien, C.-A. Chung, and C.-W. Tsao, Mater. Sci. Eng. C 37, 28 (2014). https://doi.org/10.1016/j.msec.2013.12.01950. H. Yoshimoto, Y. M. Shin, H. Terai, and J. P. Vacanti, Biomaterials. 24, 2077 (2003). https://doi.org/10.1016/S0142-9612(02)00635-X These data demonstrated that the conductive copolymers’ thin films promoted the osteogenic differentiation of hMSC-BM through elevated ARS and further suggested the great potential of PEDOT-co-PDLLA for bone tissue engineering applications.

E. Osteogenesis metabolic cell process

It is widely known that electrical signals are sensed and converted into biochemical cues by multiple pathways within the cells, resulting in various biological responses.51–5351. A. Martínez-Ruiz, S. Cadenas, and S. Lamas, Free Radic. Biol. Med. 51, 17 (2011). https://doi.org/10.1016/j.freeradbiomed.2011.04.01052. A. Lacy-hulbert, J. C. Metcalfe, and R. Hesketh, FASEB J. 12, 395 (1998). https://doi.org/10.1096/fasebj.12.6.39553. G. Chan and D. J. Mooney, Trends Biotechnol. 26, 382 (2008). https://doi.org/10.1016/j.tibtech.2008.03.011 The activation of signal transduction pathways is considered as the possible mechanism by which the CES applied may express biological responses and exert control over cellular functions.40,4140. D. Liu, C. Yi, C.-C. Fong, Q. Jin, Z. Wang, W.-K. Yu, D. Sun, J. Zhao, and M. Yang, Biol. Med. 10, 1153 (2014). https://doi.org/10.1016/j.nano.2014.02.00341. G. Thrivikraman, S. K. Boda, and B. Basu, Biomaterials 150, 60 (2018). https://doi.org/10.1016/j.biomaterials.2017.10.003 A major cellular signal transduction biological pathway, which governs the transcription of specific mRNAs in response to external stimulation, such as ES, is the activation of the mitogen-activated protein kinase (MAPK) cascades.5454. K. R. Mahtani, M. Brook, J. L. E. Dean, G. Sully, J. Saklatvala, and A. R. Clark, Mol. Cell. Biol. 21, 6461 (2001). https://doi.org/10.1128/MCB.21.9.6461-6469.2001 The MAPKs are proteins of serine/threonine kinases that control intracellular metabolism events in response to extracellular stimulations.5454. K. R. Mahtani, M. Brook, J. L. E. Dean, G. Sully, J. Saklatvala, and A. R. Clark, Mol. Cell. Biol. 21, 6461 (2001). https://doi.org/10.1128/MCB.21.9.6461-6469.2001 The sequential activation of protein kinases within these cascades (extracellular signal-regulated kinases: ERK1/2 and ERK5, Jun amino-terminal kinases: JNK, p38MAPK) mediates numerous important cellular biological responses, including proliferation, differentiation metabolism, cell cycle progression, and apoptosis relying on the time of ES and the type of cell.4747. M. Kayahara, X. Wang, and C. Tournier, Mol. Cell. Biol. 25, 3784 (2005). https://doi.org/10.1128/MCB.25.9.3784-3792.2005 The ES inducing MAPK activation was documented in endothelial angiogenic response and in HL-60 (human promyelocytic leukemia cell line) differentiation.41,4741. G. Thrivikraman, S. K. Boda, and B. Basu, Biomaterials 150, 60 (2018). https://doi.org/10.1016/j.biomaterials.2017.10.00347. M. Kayahara, X. Wang, and C. Tournier, Mol. Cell. Biol. 25, 3784 (2005). https://doi.org/10.1128/MCB.25.9.3784-3792.2005 Mechanistically, cell motility and wound healing responses elicited by electrical current gradients take place through the dynamic mediation of PI (3)Kγ (phosphoinositide 3-kinase) and PTEN (phosphate and tensin homolog) signaling.5555. J. P. Crow and J. S. Beckman, “The importance of superoxide in nitric oxide-dependent toxicity,” in Biological Reactive Intermediates V. Advances in Experimental Medicine and Biology, edited by Snyder R. (Springer, Boston, MA, 1996), Vol. 387, pp. 147–161, see https://doi.org/10.1007/978-1-4757-9480-9_21 An accelerated and progressive enhancement in the phosphorylation of extracellular-signal-regulated kinase, p38 MAPK, Src, and Akt on Ser 473 site was distinctly observed in cells undergoing electrotaxis.4141. G. Thrivikraman, S. K. Boda, and B. Basu, Biomaterials 150, 60 (2018). https://doi.org/10.1016/j.biomaterials.2017.10.003 It also was reported that the applied low-intensity 0.1 ms electrical current could induce a transient and low-level activation of the p38-p53 pathway, which is implicated to play significant roles in the annihilation of malignant tumors and in the downregulation of inflammatory cytokine responses and metabolism. The cellular orientation and movement of adult stromal cells in response to ES were observed to be linked to the activation of PI3K and ROCK signaling pathways.5656. M. C. Ruth, Y. Xu, I. H. Maxwell, N. G. Ahn, D. A. Norris, and Y. G. Shellman, J. Invest. Dermatol. 126, 862 (2006). https://doi.org/10.1038/sj.jid.5700211 However, the orientation and movement of hMSC-BM over EF due to CES applied did not take place in the present study. A schematic of the (a) EF applied at 28 mV/mm on the bioreactor with the seeded cell, (b) enlargement of the selected area with attached hMSC-BM over PEDOT-co-PDLLA, and (c) intracellular metabolic activity behavior under CES, respectively, is presented in Fig. 6.Although a possible trend to osteogenic lineage under CES, some studies demonstrate that the EF applied could induce an increased intracellular Ca2+ produced by VGCC. This activation may lead to multiple regulatory responses, including the increased nitric oxide levels produced through the action of the two Ca2+/calmodulin-dependent nitric oxide synthases (NOS), neuronal NOS, and endothelian NOS. Increased nitric oxide levels typically act in a physiological context through an increased synthesis of cyclic guanosine 3′, 5′-monophosphate (cGMP) and the subsequent activation of the protein kinase G.51,57,5851. A. Martínez-Ruiz, S. Cadenas, and S. Lamas, Free Radic. Biol. 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Biophys. 322, 500 (1995). https://doi.org/10.1006/abbi.1995.149365. S. Burney, J. L. Caulfield, J. C. Niles, J. S. Wishnok, and S. R. Tannenbaum, Mutat. Res. Mol. Mech. Mutagen. 424, 37 (1999). https://doi.org/10.1016/S0027-5107(99)00006-866. M. G. Salgo and W. A. Pryor, Arch. Biochem. Biophys. 333, 482 (1996). https://doi.org/10.1006/abbi.1996.0418 producing a reduced spread over conductive copolymers. In order to better understand DNA content results, the cellular metabolic behavior in the absence and presence of the CES must be further analyzed.The EF effects caused by CES on cell proliferation were widely reported and it included both inhibitory and stimulating effects, depending on the cell type and exposure conditions.67–6967. M. H. Ryu, H. M. Park, J. Chung, C. H. Lee, and H. R. Park, Biochem. Biophys. Res. Commun. 393, 11 (2010). https://doi.org/10.1016/j.bbrc.2010.01.06068. M. Simko, Curr. Med. Chem. 14, 1141 (2007). https://doi.org/10.2174/09298670778036283569. M. Cappelletti, I. Zampaglione, G. Rizzuto, G. Ciliberto, N. La Monica, and E. Fattori, J. Gene Med. 5, 324 (2003). https://doi.org/10.1002/jgm.352 Low-frequency EF could actuate on the morphogenesis of mammalian embryo, wound healing, or tumor.4141. G. Thrivikraman, S. K. Boda, and B. Basu, Biomaterials 150, 60 (2018). https://doi.org/10.1016/j.biomaterials.2017.10.003 For in vitro cultures, proliferation was observed for various cell types (HL-60 leukemia cells, Rat-1 fibroblasts, and WI-38 diploid fibroblasts) when also exposed to low-frequency EF.4141. G. Thrivikraman, S. K. Boda, and B. Basu, Biomaterials 150, 60 (2018). https://doi.org/10.1016/j.biomaterials.2017.10.003 Nevertheless, under identical exposure conditions, unexpected DNA strand breaks in later hours were noticed suggesting that the EF exposure caused a temporary mitogenic effect, followed by a loss of DNA integrity.6969. M. Cappelletti, I. Zampaglione, G. Rizzuto, G. Ciliberto, N. La Monica, and E. Fattori, J. Gene Med. 5, 324 (2003). https://doi.org/10.1002/jgm.352 In agreement with the mitogenic effect mentioned above, the cell coverage analysis showed a decreased spread over time under CES (Fig. S3),7373. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001435 for a scheme of the biorector and a computational modelling of the electric field distribution inside it. even though a positive effect on osteogenesis was observed. Furthermore, reductions in the number of cells were observed at the same EF applied (Fig. S4).7373. See supplementary material at https://www.scitation.org/doi/suppl/10.1116/6.0001435 for a scheme of the biorector and a computational modelling of the electric field distribution inside it. Hence, from a pathogenic point of view, this study showed that short-term exposures induced differentiation, whereas prolonged exposures could interrupt the cell cycle by causing DNA damage.6969. M. Cappelletti, I. Zampaglione, G. Rizzuto, G. Ciliberto, N. La Monica, and E. Fattori, J. Gene Med. 5, 324 (2003). https://doi.org/10.1002/jgm.352 Thus, exposing cells to the EF for a shorter time is a viable way to enhance proliferation and differentiation without inducing undesired DNA damage. Therefore, how DNA damage occurs and how cell differentiation from hMSC-BM to bone cells takes place need to be further investigated. Reported data in the literature show that, in osteogenic stimulation, voltage-gated calcium channel stimulation leads to an increase in intracellular Ca2+, acting, in turn, to stimulate the two calcium/calmodulin-dependent nitric oxide synthases, and an increase in nitric oxide.7070. A. C. Gadano, P. Sogni, S. Yang, S. Cailmail, R. Moreau, P. Nepveux, D. Couturier, and D. Lebrec, J. Hepatol. 26, 678 (1997). https://doi.org/10.1016/S0168-8278(97)80435-7 An enhancement of the intracellular Ca2+ level can drive to osteogenic differentiation.5,715. R. Balint, N. J. Cassidy, and S. H. Cartmell, Acta Biomater. 10, 2341 (2014). https://doi.org/10.1016/j.actbio.2014.02.01571. M. K. Shin, M.-K. Kim, Y.-S. Bae, I. Jo, S.-J. Lee, C.-P. Chung, Y.-J. Park, and D. S. Min, Cell. Signal 20, 613 (2008). https://doi.org/10.1016/j.cellsig.2007.11.012 Nevertheless, bone differentiation of hMSC-BM needs other biological responses to take place. In the case of cell proliferation, nitric oxide may act in pathophysiological responses to CES exposure, by acting as a precursor of peroxynitrite, producing both oxidative stress and free radical breakdown products.6666. M. G. Salgo and W. A. Pryor, Arch. Biochem. Biophys. 333, 482 (1996). https://doi.org/10.1006/abbi.1996.0418 The biologicals signaling mentioned above seems to induce single-stranded DNA breaks,7272. R. Hess, A. Jaeschke, H. Neubert, V. Hintze, S. Moeller, M. Schnabelrauch, H.-P. Wiesmann, D. A. Hart, and D. Scharnweber, Biomaterials 33, 8975 (2012). https://doi.org/10.1016/j.biomaterials.2012.08.056 which could influence cell spread over studied materials at the EF applied.