Electrical Stimulation Modulates the Expression of Multiple Wound Healing Genes in Primary Human Dermal Fibroblasts.

Tissue Engineering Part A Electrical stimulation modulates the expression of multiple wound healing genes in primary human dermal fibroblasts (doi: 10.1089/ten.TEA.2014.0687) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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1 Electrical stimulation modulates the expression of multiple wound healing genes in primary human dermal fibroblasts

Hyun Jin Park, PhD,1,2 Mahmoud Rouabhia, PhD,1 Denis Lavertu, MD,2 and Ze Zhang, PhD2

1

Groupe de Recherche en Écologie Buccale, Facultéde Médecine Dentaire, UniversitéLaval,

2

Axe médecine régénératrice, Centre de recherche du CHU de Québec; Département de chirurgie, Facultéde médecine, UniversitéLaval, QC G1V 0A6, Canada

Running title: Effect of electrical stimulation on wound healing genes.

Correspondence to: Dr. Mahmoud Rouabhia, E-mail: [email protected]

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2 Abstract This study profiled multiple human dermal fibroblast wound-healing genes in response to electrical stimulation (ES) by using RT2profiler PCR-Array system. Primary human skin fibroblasts were seeded on heparin (HE)-bioactivated polypyrrole (PPy)/poly(L-lactic acid) (PLLA) conductive membranes, cultured, and subsequently exposed to ES of 50 or 200 mV/mm for 6 h. Following ES, the cells were used to extract RNA for gene profiling and culture supernatants were used to measure the level of the different wound healing mediators. A total of 57 genes were affected (activated/repressed) by ES; among these, 49 were up-regulated and 8 were down-regulated. ES intensities at 50 and 200 mV/mm activated/repressed different genes. The ES modulated genes are involved in cell adhesion, remodeling and spreading, cytoskeletal activity, extracellular matrix metabolism, production of inflammatory cytokines/chemokines and growth factors, as well as signal transduction. The expression of several genes was supported by protein production. Protein analyses showed that ES increased CCL7, KGF and TIMP2 but reduced MMP2. This study demonstrated that ES modulates the expression of a variety of genes involved in the wound healing process, confirming that ES is a useful tool in regenerative medicine. Keywords: Electrical stimulation, conductive material, fibroblasts, gene profile, wound healing

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3 Introduction Skin wound healing is a complex multi-stage process that orchestrates the reconstitution of the dermal and epidermal layers of the skin.1 Fibroblasts are an important cell type during wound healing by participating in inflammation, proliferation and remodeling.1 In addition, different growth factors, cytokines and chemokines are released by a variety of cells, including fibroblasts, to direct this process.1,2 Inflammation, angiogenesis, the formation of granulation tissue and extracellular matrix (ECM) synthesis are thus key steps in the wound healing sequence.3 Along with cytokines and chemokines, growth factors play an important role in controlling these key steps and also in mediating gene expression involved in cellular migration, differentiation and proliferation.4 Wound healing genes are grouped into different but complementary control pathways for collagen production, cell adhesion and spreading, tissue remodelling, inflammatory cytokines and chemokines, and for growth factors and signal transduction.5 These genes can be modulated by various conditions such as diabetes4 or an exposure to exogenous stimuli like ultraviolet light6 and electrical stimulation (ES).7 ES in its various forms has been shown to promote wound healing through increasing the migration of keratinocytes and macrophages7, enhancing angiogenesis8 and stimulating dermal fibroblasts.9,10 It has been reported that the human body generates trans-epithelial electrical potential (TEP) ranging between 10 and 60 mV in various locations11 and contributing to wound healing.12 Injured epidermis is characterized by a TEP short circuit that gives rise to a measurable DC current efflux between 1 and 10 µA/cm2 and an estimated current up to 300 µA/cm2 near the edge of the wound.13 This wound current corresponds to a relatively steady local EF between 40 and

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4 200 mV/mm which persists until complete wound re-epithelialization is achieved.14 EF was also shown to guide the migration of fibroblasts and keratinocytes14,15 and to promote angiogenic responses by enhancing endothelial cell elongation and directional migration.8 Thus, ES offers a rational and potentially highly efficient approach to promoting wound healing by modulating fibroblast activities. Pioneering in the field, the group of Bayat established a non-invasive ES device called the Fenzian treatment system in the management of chronic scars, pain and itch.16 With such ES-device Sebastian et al. were able to successfully alter the expression of collagen I in keloid fibroblasts.17 Additionally, the same group demonstrated that cutaneous wounds receiving degenerate wave (DW) of ES displayed accelerated healing as ascertained by reduced inflammation, enhanced angiogenesis and advanced remodeling phase.18 We previously developed a biodegradable conductor made of 5% polypyrrole (PPy) and 95% polylactide (PLA)19 as well as an electronic system that enables cells to be cultured on the surface of the conductors then electrically stimulated.9 When the PPy-containing scaffold is connected to an electrical power source, only the cells cultured on the scaffold surface are exposed to the electrical field, thus, allowing for a spatially confined ES.10,20,21 We also improved the cell adhesion and electrical stability of the conductive material in culture medium by adding heparin (HE) as dopant to PPy.21 Using this conductive material and ES, we have been able to augment the proliferation and extracellular matrix deposition of fibroblasts10 and osteoblasts,20 and demonstrated that such wound healing related cellular activities were coordinated by relevant cytokines and growth factors. Considering the variety of cellular activities reportedly modified by ES, we are very interested in knowing the big picture of gene activation upon ES. Such knowledge will help us to target the genes that are most likely to be affected through ES. Taking advantage of our ES model, the goal of this study was to determine the effect of ES on the 4


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5 expression profiles of the genes involved in the wound healing process in normal human dermal fibroblasts using electrically conductive PPy/HE/PLLA membranes.

Materials and Methods Human dermal fibroblast extraction and culture: Human skin biopsies were collected from patients following their informed consent and the approval of the UniversitéLaval-CHU Ethics Committee. The biopsies were treated with thermolysin (500 μg/mL) to separate the epidermis from the dermis. The extracted fibroblasts were seeded in 75-cm2 flasks and grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) in a humidified incubator at 37C with 5% CO2. The medium was changed three times a week. The fibroblasts were used once the cultures had reached 90% confluence. Conductive membrane: Freshly vacuum-distilled pyrrole monomers (98%, Sigma-Aldrich) were slowly added to a water-in-oil (chloroform) (3:7) emulsion system containing 0.5 mg of HE (MW: 13,500–15,000; EMD Biosciences) and Fenton’s reagent made with H2O2 and FeCl3 under nitrogen protection.21 The emulsifier was 1%-dodecylbenzenesulfonic acid sodium salt (DBS; Sigma-Aldrich). Polymerization was maintained at room temperature for 24 h under vigorous stirring, after which time the HE-doped PPy particles (PPy/HE) were precipitated with methanol and washed thoroughly with a methanol water solution (1:1) to remove the emulsifier and impurities. After drying, the PPy/HE particles were kept under vacuum and room temperature. The PPy/HE/PLLA membranes were prepared by first adding the PPy/HE particles into the PLLA (η = 1.3 dL/g) solution in chloroform under magnetic stirring and then casting the solution onto a polytetrafluoroethylene plate to dry. The thickness of the conductive membranes was 5


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6 approximately 0.5 mm. The final concentration of HE in the PPy/HE/PLLA membrane was approximately 0.9 mg/cm2. To avoid moisture, the prepared membranes were kept in a desiccator until use. Electrical stimulation device: The multi-well electrical cell culture plate was designed to avoid any electrochemical reaction that may release cytotoxic products into the culture medium and consequently interfere with the effect of electrical field. As illustrated in Figure 1, the culture plate was constructed by a top well sitting on a bottom plate, between them the conductive PPy/HE/PLLA membrane was sandwiched in such a way that no culture medium would be leaked out of the well. The two ends of the membrane outside the culture well were connected to a constant power source with the current automatically recorded. Compared with the ES system using electrodes where cells are either seeded on the working electrode or exposed between two electrodes that generate ionic current in culture medium, the electrode-free setup completely ruled out the controversial interference of either the redox activity at the electrode/medium interface or the ionic current in culture medium. Therefore this electrode-free design ensured that the effect of electrical field on cells would not be complicated by other factors. Electrical stimulation of human skin fibroblasts: The PPy/HE/PLLA membranes were sterilized and cut to fit into the electric cell culture plates as showed in Figure 1. Prior to cell seeding, membranes were pre-incubated for 48 h with Dulbecco's Modified Eagle Medium (DMEM) to wash out any possible leachables, with the medium changed every 24 h. The culture medium used in the pre-incubation was tested in standard culture conditions and found noncytotoxic. Cell (0.5 x 106) in 3 ml of medium were seeded on each PPy/HE/PLLA membrane (4 cm2) and subsequently incubated under 5% CO2 at 37°C for 24 h prior to ES. Cells were electrically stimulated by connecting the PPy/HE/PLLA membranes to a DC constant potential 6


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7 source operated at 50 and 200 mV/mm across the membrane. We previously showed that ES intensity ranging between 50 and 200 mV/mm was not toxic to the cells and increase cell viability.9,10 A high dose of ES such as 500 mV/mmm on the other hand, was found negatively affected cell morphology.9 According to previously established protocols,19-21 the cells were stimulated for 6 h and further cultured for 18 h prior to analysis. Sham control groups (0 mV/mm) followed the same protocol except for ES. A minimum of four experiments were performed for each condition. Quantitative real-time PCR array: Total RNA was extracted from the fibroblasts by means of an RNAspin mini kit (GE, Canada). RNA concentrations and qualities were determined with the Experion automated electrophoresis station from BioRad (Mississauga, Canada). RNA quality was considered optimal in any sample with 28S/18S rRNA ratios above 1.8. Real-time PCR arrays were performed using the RT2 Profiler PCR Array System (Bioscience). Briefly, total RNA (1000 ng) was used to prepare cDNA with the RT2 first strand kit (QIAGEN). The PCR arrays contained 84 gene-specific primers related to wound healing. Since the manufactured RT2 first strand kit did not contain TIMP2 gene, we then used the cDNA extracted from our experiment samples and performed a separate qPCR. Results were analyzed using the

ΔΔ

Ct

method and the fold changes between the non-stimulated and ES-stimulated samples were calculated. All experiments have been performed three separate times; analyses were performed in triplicates for each condition. To eliminate feebly expressed genes, the latter were selected if a fold change above 1.4 was observed and if baseline cycle thresholds were below 30 (n = 3). ELISA: Immediately after exposure to ES, the culture medium was collected for further use, and then new medium was added to each well to be cultured for an additional 24 h. Both supernatants collected immediately after ES exposure, and those collected 24 h post ES-exposure were used to 7


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8 measure different mediators (KGF, CCL7, MMP2, TIMP1 and TIMP2) by means of ELISA (R&D Systems) assay. The ELISA plates were read at 450 nm and analyzed using a Microplate Reader Model 680 (Bio-Rad) (n = 4). Statistical Analysis: All quantitative data are presented as mean ±SD. To compare the statistic difference between any pair of data, t-test was used to calculate the p value that is considered significant when it is less than or equal to 0.05.

Results and discussion The gene profiling results revealed that multiple genes were either up- or down-regulated following ES. These genes are involved in all phases of wound repair and are indicated in many signaling pathways crucial to the wound healing process, such as ECM production and cell adhesion, inflammatory cytokines and chemokines, growth factors and signal transduction. Only those with fold changes  1.4 or  0.8 were taken into account in our analyses. Among the 12 genes involved in cell adhesion pathway only ITGA2 and ITGB5 were found upregulated at 200 mV/mm (Fig. 2a). ITGA2 encodes alpha 2 integrin subunit that contributes to 21 of CD49b helping cell adhesion to collagen and laminin of extracellular matrix (ECM). ITGB5 encoding for beta 5 integrin subunit was activated with 200 mV/mm. ITG-A3, -A4, -A6 and -AV were down regulated showing a folded change ranging from ≤ 0.8 to 0.6. Finally several genes such as CDH1, ITGA1, ITGA5, ITGB1, ITGB3 and ITGB6 were unchanged or marginally over expressed (fold change  1.2), suggesting that these genes might also be involved in the ES promoted cell migration and inflammatory processes. Cell interactions with ECM play critical

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9 roles in normal cell adhesion and migration and also in inflammatory diseases.22 By modulating ITGA2 (1.7 folds expression) and possibly other adhesion genes, ES modulates cell migration and wound closure. Indeed, the involvement of α2β1 integrin in wound healing is now known.23 This integrin is moved from the basal cell area to concentrate at the forward leading edge of migrating keratinocytes, bringing it in contact with type I collagen.24 Thus, ES is expected to modulate wound healing through α2β1 and may also be through other integrin subunits (αV, 5) as demonstrated in this study (Fig. 2a). Elevated expression of specific integrins, such as α2β1 and α4β1, has been known to play a role in the preparation of cells for the re-stratification process.25 Studies reported that different subunits α, β and γ are forming laminin isoforms.26 Laminin, a major component of human skin, is located in the dermo-epidermal junction and is secreted by keratinocytes when interacting with dermal fibroblasts. The increased expression of ITGA2 following ES can explain the presence of a high amount of laminin after the ES aided skin healing, an observation in our skin wound healing experiment and those in the literature.27 Evidently, further investigations are required to confirm this. ES also increased the expression of ACTA2, ACTA1 and RAC1 genes of cytoskeleton pathway (Fig. 2b). ACTA2 and ACTA1 encoding smooth muscle alpha 2 actin and alpha 1 actin and RAC1 (encoding for GTPase belonging to the RAS superfamily of small GTP-binding proteins) mRNA expression increased following cell exposure to either 50 or 200 mV/mm, with a more significant expression recorded at 200mV/mm. ACTA2 is expressed in different cells and in vascular smooth muscle cells in particular.28 Smooth muscle α-actin (α-SMA) is also a major player in wound healing.29 The phenotypic changes involved in the transition of fibroblasts to myofibroblasts are well defined and characterized by the expression of specific markers such as SMA encoded by 9


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10 ACTA230 and the acquisition of contractile and tissue remodeling capacity.31 During skin wound healing SMA augments the contractile activity of fibroblastic cells and assures the wound remodeling.29,30 We previously reported the up-regulation of α-SMA in the electrically stimulated fibroblasts showing myofibroblast phenotype.10 Another activated cytoskeleton gene was RAC1, a member of the Rho GTPase family, which also includes RHO and CDC42. These proteins are key players in controlling the assembly/disassembly of cytoskeletal elements.32 RAC1 activity is critically involved in normal cellular processes, including adhesion and differentiation of multiple cell types.33 ES was shown to promote fibroblast adhesion and growth,9 which is in consistent with the fact that RAC1 mRNA increased when exposed to 200 mV/mm (Fig. 2b). Overall, the data demonstrated that ES upregulated the cytoskeleton genes in favour of fibroblast adhesion, growth and transdifferentiation to myofibroblasts, which is also in favour of wound healing. Seven out of ten ECM associated genes, namely, COL14A1 (This gene encodes the alpha chain of type XIV collagen), COL1A1 (encodes the pro-alpha1 chains of type I collagen), COL3A1 (encodes the pro-alpha-1 chains of type III collagen), COL4A1 (encodes the pro-alpha1 chains of type IV collagen), COL4A3 (encodes the alpha3 (IV) chain of type IV collagen), COL5A3 (encodes the pro-alpha1 chains of type V collagen) and VTN (encodes vitronectin a cell adhesion and spreading factor) were significantly activated following exposure to either 50 or 200 mV/mm of ES (Fig. 2c). Other genes, such as COL1A2, COL5A1 and COL5A2, were also marginally activated. This suggests that the fibroblasts were activated by ES to secrete ECM proteins, as previously reported under other experimental conditions.34 Supportive to our study, the expression of COL1A1 and COL3A1 genes were reported to be an initiator of wound healing processes.35 Among the three significantly activated genes, VTN is a key gene playing an active role in epithelial adhesion and outgrowth.36 It also enhances the motility of cultured human 10


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11 keratinocytes37 and provides an essential component of the temporary wound bed matrix.38 This adhesive protein is also crucial to fibroblast adhesion and growth, explaining the ES enhanced cell proliferation.9,10 Fibroblasts are vital cells, as they secrete extracellular matrix, and cooperate with other cells to ensure tissue structure and functionality.39 ECM production and deposition by fibroblasts lies under the control of various remodeling enzyme genes,40 which are modulated by various factors/agents41 possibly including ES.7 In this study, we demonstrated that dermal human fibroblasts exposed to either 50 or 200 mV/mm expressed variable levels of mRNA of the remodelling enzyme genes (Fig. 2d). Among the 17 genes analyzed, 9 of them (CTSL2, F3, MMP2, PLAU, PLAUR, PLG, SERPINE1, TIMP1 and TIMP2) were up-regulated. But 2 genes (CTSG and MMP9) were down-regulated in response to ES. The other genes were unchanged. Although there are no similar ES studies reported in the literature, other agents such as ultraviolet radiation were reported having a comparable effect on remodeling enzyme mRNA expression.6 Inflammatory cytokines and chemokines are important signaling molecules that facilitate communication between cells.42 This intercellular communication constitutes a key activity in the wound healing process.43 Expression and production of cytokines and chemokines may be promoted by various agents.6,44 Among the 13 genes analyzed, 9 genes (CCL2, CCL7, CD40LG, CXCL1, CXCL2, CXCL5, IL-10, IL-1B and IL-4) were significantly up-regulated and another’s such as IFNG and IL-6 were reduced (Fig. 3a). This up-regulation was found more significant at 200 mV/mm than that at 50 mV/mm, pointing to potential dose effect. It has been reported that dermal fibroblasts exposed to burn wound expressed high level of CXCL1, CXCL8, CCL2 and CCL20, and also secreted angiogenic factor VEGF and IL-6.45 The increasing levels of

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12 inflammatory cytokines and chemokine by ES suggest its potential role in promoting granulation tissue formation and angiogenesis.1 During wound healing, various growth factors stimulate an array of cell proliferation and differentiation processes.46 Following ES, among the 17 growth factor encoding genes analyzed, 11 genes (CSF2, CSF3, EGF, FGF10, FGF2, FGF7, HGF, IGF1, MIF, TGFA and VEGFA) were significantly up-regulated. But only 2 genes were significantly down-regulated (CTGF and HBEGF) (Fig. 3b). EGF and FGF7 are potent growth factors involved in wound healing and contribute particularly to epithelialisation. HGF and VEGFA on the other hand are important for tissue regeneration and angiogenesis. While ES has been reported to accelerate wound healing,1618

this work is the first study to demonstrate the involvement of ES in the wound healing process

through growth factor gene expressions. The results also support a study on cultured retinal Müller cells, in which ES up-regulated the transcriptional induction of brain-derived neurotrophic factor.47 The effect of ES on growth factors expression was supported by protein secretion as we demonstrated that ES promotes FGF2 secretion by primary human dermal fibroblasts after 6 h exposure to 50 or 200 mV/mm. These data are supportive to those previously published showing increased FGF2 secretion by ES exposed fibroblasts then cultured for 24h.10 This suggests that the effect of ES on FGF2 secretion starts as early as 6 h and was maintained up to 24 h. The effect of growth factors takes place through signal transductions,48 a key process through which external agents stimulate cell membrane receptors leading to the information transfer into the cell where second messengers relay the information via the activation of protein kinases.49 In the present study, several signal transduction genes (TGFBR3, WISP1, MAPK1, MAPK3, EGFR, IL6ST and PTGS2) in the ES-exposed dermal fibroblasts recorded a significantly up-regulated expression, and other genes (STAT3, CTNNB1, WNT5A and PTEN) were unchanged (Fig. 4). In 12


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13 general, gene activations were obtained at both 50 and 200 mV/mm, with greater effect at either intensity. Among the pathways, EGFR has been significantly indicated in wound healing through EGF, which was also found up-regulated in this work. MAPK1 is a member of the MAPK family that is widely involved in various stimulating signals and regulate many cellular activities, such as mitosis and differentiation. IL6ST and PTGS2 play roles in inflammation. The up-regulated expression of these genes is found associated with the pathways all relevant to wound healing. This is consistent with previously reported findings using different cell types and stimulation agents.50 The two ES intensities (50 and 200 mV/mm) we used showed some times opposite effect on a specific gene expression. As an example, COL4A3 was expressed more under 50 mV/mm than under 200 mV/mm (Fig. 2c). This suggest that this gene is more sensitive to low but not to high ES intensities. This can be supported by one previous study reporting that cell proliferation and spreading on the surface of biomaterials were enhanced within a narrow window of voltage/frequency of ES.51

Gene expressions of MMP2, TIMP1, TIMP2, CCL7 and KGF were supported by protein production. The ELISA analyses show that ES modulated remodeling enzyme proteins MMP2 and TIMP2 (Fig. 5a and Fig. 5b). The effects were obtained with the cultures 24 h post ES as compared to the supernatant collected immediately after ES-exposure. Although TIMP1 secretion did not change, MMP2 and TIMP2 secretions were significantly modulated by ES. When the primary dermal fibroblasts were exposed to ES, MMP2 decreased three to four times. In contrast, TIMP2 significantly increased, particularly at 200 mV/mm. While TIMPs can inhibit all active MMPs, TIMP2 is a more effective inhibitor of MMP2.52 In the gene expression profile, ES 13


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14 increased both MMP2 and TIMP2 gene expressions, while protein analysis showed some differences. The decrease of MMP2 in wound healing, as supported in one study,53 suggests the need by fibroblasts to produce more ECM rather than degrade it through MMPs, such as MMP2. During the wound healing process, MMP inhibitors may be needed to overcome MMP activity that degrades the newly produced ECM.54 Thus, the increase in TIMP2, as shown in the present study, supports the involvement of ES in the promotion of healing by activating fibroblasts to produce less MMP2 and more TIMP2. Further investigations will be required, however, to confirm this hypothesis. ES also modulated chemokine production by the dermal fibroblasts. CCL7 concentration was measured using the supernatant collected immediately or 24h post-ES exposure, showing that ES at 50 and 200 mV/mm significantly increased CCL7 secretion (Fig. 6a). This supports our gene expression data relative to CCL7. While our study confirms the involvement of CCL7 in wound healing,55 it demonstrates for the first time the contribution of ES in the wound healing process through CCL7. CCL7 participates wound healing through ECM protein and chemokine modulation. Also CCL7 plays a critical role in inflammation phase during wound healing process. CCL7 is an important leukocyte chemoattractant and a critical mediator for monocyte mobilization.56 The ELISA analyses also revealed high levels of KGF following ES stimulation, compared to the non-stimulated cells (Fig. 6b). KGF is an important contributing factor in cell growth, as it stimulates the growth and differentiation of different cell types including fibroblasts.57 It also contributes to the chemotaxis of microvascular endothelial cells and to tissue neovascularization.58 Thus, the up-regulated KGF production supports previous results showing increased fibroblast proliferation9 and differentiation10 following exposure to ES.

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15 There have been numerous studies about the mechanisms of ES to cells. Among them the voltage gated ion channels and the subsequent signalling pathway activations are the most frequently studied. Cho et al. in 2002 reported that ES between 1 to 10 Hz at an intensity of 2V/cm increased 6 folds Ca2+ in human fibroblasts, and demonstrated that both depletion of extracellular calcium in culture medium or using verapamil (L-type calcium channel blocker) inhibited calcium ion increase.59 Zhao et al. also suggested that ion exchanger NHE1 was responsible to couple ES and intracellular signaling.13 EF as low as 10 mV/mm was reported to induce human fibroblast movement which was integrin- and Ca2+-dependent.60 Shanley et al. showed that the electrotaxis of Dictyostelium cells requires the influx of extracellular calcium ions.61 A depletion of Ca2+ completely abolished the directional migration of Dictyostelium cells in EF. In 2008, Sato et al. reported that retinal Müller cells of rats secreted significantly increased amount of insulin growth factor 1 (IGF-1) mRNA under the stimulation of biphasic pulses (duration, 1 ms; frequency, 20 Hz; current, 0-10 mA) for 30 min, and that this increase is associated with the increase of intracellular free Ca2+ through L-type calcium channels.47 A long standing controversy with respect to the effect of EF or ES to cellular activities is about the side effects or byproducts that may be generated through electrode activity. Electrodes when used in direct contact with culture medium may generate oxidative products that are known to activate cells through, for example, TRP channels.62 Jennings et al. reported the gene profiling following ES to dermal fibroblasts.63 While they identified 126 transcripts at a level of 1.4 fold of changes, there is little overlap between their report and this work. In fact, the activation of many important genes such as that of MMP, TIMP, COL, EGF, FGF and many others are first time reported in this work.

63

Moreover, the ES system used in that experiment was agar bridged

electrodes, which may have ion exchange between culture medium and agar bridges and may also 15


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16 disturb the distribution of the charged molecules in the culture medium. The system used in our work did not contain any electrode and therefore excluded such potential controversy. Overall, this study demonstrated that ES promotes fibroblast activities by regulating a wide spectrum of gene expression. This may help developing a new therapeutic approach regenerating skin through ES and conductive scaffold.

Conclusion This study is the first to demonstrate the effects of electrical stimulation at 50 and 200 mV/mm on the expression of wound healing genes in primary human dermal fibroblasts. The expression of some of these genes was confirmed by protein production. This study presents clear evidence that conductive polymer-based electrical stimulation can modulate multiple genes involved in the wound healing process.

Funding: This work was supported by The Canadian Institutes of Health Research, Grant no. 106555.

Author Disclosure Statement: The authors declare that no conflict of interests exists.

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18 12. Zhao, M. Electrical fields in wound healing-an overriding signal that directs cell migration. Semin Cell Dev Biol 20, 674, 2009. 13. Zhao, M., et al. Electrical signals control wound healing through phosphatidylinositol-3-OH kinase-gamma and PTEN. Nature 442, 457, 2006. 14. Sta Iglesia, D.D., and Vanable, J.W. Jr. Endogenous lateral electric fields around bovine corneal lesions are necessary for and can enhance normal rates of wound healing. Wound Repair Regen 6, 531, 1998. 15. Song, B., et al. Electrical cues regulate the orientation and frequency of cell division and the rate of wound healing in vivo. Proc Natl Acad Sci USA 99, 13577, 2000. 16. Perry, D., et al. Treatment of symptomatic abnormal skin scars with electrical stimulation. J Wound Care 19, 447, 2010. 17. Sebastian, A., et al. A novel in vitro assay for electrophysiological research on human skin fibroblasts: degenerate electrical waves downregulate collagen I expression in keloid fibroblasts. Exp Dermatol 20, 64, 2011. 18. Sebastian, A., et al. Acceleration of cutaneous healing by electrical stimulation: degenerate electrical waveform down-regulates inflammation, up-regulates angiogenesis and advances remodeling in temporal punch biopsies in a human volunteer study. Wound Repair Regen 19, 693, 2011. 19. Shi, GX., et al. A novel electrically conductive and biodegradable composite made of polypyrrole nanoparticles and polylactide. Biomaterials 25, 2477, 2004. 20. Meng, S., Rouabhia, M., and Zhang, Z. Electrical stimulation modulates osteoblast proliferation and bone protein production through heparin-bioactivated conductive scaffolds. Bioelectromagnetics 34, 189, 2013.

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19 21. Meng, S., et al. Heparin dopant increases the electrical stability, cell adhesion, and growth of conducting polypyrrole/poly(L,L-lactide) composites. J Biomed Mater Res A 87, 332, 2008. 22. de Fougerolles, A.R., and Koteliansky, V.E. Regulation of monocyte gene expression by the extracellular matrix and its functional implications. Immunol Rev 186, 208, 2002. 23. Dumin, J.A., et al. Pro-collagenase-1 (matrix metalloproteinase-1) binds the alpha(2)beta(1) integrin upon release from keratinocytes migrating on type I collagen. J Biol Chem 276, 29368, 2001. 24. Martin, P. Wound healing – aiming for perfect skin regeneration. Science 276, 75, 1997. 25. Stepp, M.A., Zhu, L., and Cranfill, R. Changes in beta 4 integrin expression and localization in vivo in response to corneal epithelial injury. Invest Ophthalmol Vis Sci 37, 1593, 1996. 26. Spinardi, L., et al. A recombinant tail-less integrin beta 4 subunit disrupts hemidesmosomes, but does not suppress alpha 6 beta 4-mediated cell adhesion to laminins. J Cell Biol 129, 473, 1995. 27. Cheli, Y., et al. Transcriptional and epigenetic regulation of the integrin collagen receptor locus ITGA1-PELO-ITGA2. Biochim Biophys Acta 1769, 546, 2007. 28. Solouki, A.M., et al. A genome-wide association study identifies a susceptibility locus for refractive errors and myopia at 15q14. Nat Genet 42, 897, 2010. 29. Hinz, B., et al. Recent developments in myofibroblast biology: paradigms for connective tissue remodelin. Am J Pathol 180, 1340, 2012. 30. Rockey, D.C., Weymouth, N., and Shi, Z. Smooth muscle α actin (Acta2) and myofibroblast function during hepatic wound healing. PloS One 8, e77166, 2013. 31. Hinz, B. Formation and function of the myofibroblast during tissue repair. J Invest Dermatol 127, 526, 2007.

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20 32. Bustelo, X.R., Sauzeau, V., and Berenjeno, I.M. GTP-binding proteins of the Rho/Rac family: regulation, effectors and functions in vivo. Bioessays 29, 356, 2007. 33. Heasman, S.J., and Ridley, A.J. Mammalian Rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9, 690, 2008. 34. Schiller, M., Javelaud, D., and Mauviel, A. TGF-β-induced SMAD signaling and gene regulation: consequences for extracellular matrix remodeling and wound healing. J Dermatol Sci 35, 83, 2004. 35. Chan, K.M., et al. Expression of transforming growth factor beta isoforms and their roles in tendon healing. Wound Repair Regen 16, 399, 2008. 36. Stenn, K.S. Epibolin: A protein of human plasma that supports epithelial cell movement. Proc Natl Acad Sci USA 78, 6907, 1981. 37. Brown, C., et al. Vitronectin: effects on keratinocyte motility and inhibition of collageninduced motility. J Invest Dermatol 96, 724, 1991. 38. Preissner, K.T. Structure and biological role of vitronectin. Annu Rev Cell Biol 7, 275, 1991. 39. Wang, J.H., et al. Mechanoregulation of gene expression in fibroblasts. Gene 391, 1, 2007. 40. Kennedy, L., et al. Fibroblast adhesion results in the induction of a matrix remodeling gene expression program. Matrix Biol 27, 274, 2008. 41. Gilkes, D.M., et al. Hypoxia-inducible factor 1 (HIF-1) promotes extracellular matrix remodeling under hypoxic conditions by inducing P4HA1, P4HA2, and PLOD2 expression in fibroblasts. J Biol Chem 288, 10819, 2013. 42. O'Hayre, M., et al. Chemokines and cancer: migration, intracellular signalling and intercellular communication in the microenvironment. Biochem J 409, 635, 2008.

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21 43. Stappenbeck, T.S., and Miyoshi, H. The role of stromal stem cells in tissue regeneration and wound repair. Science 324, 1666, 2009. 44. Semlali, A., et al. Whole cigarette smoke increased the expression of TLRs, HBDs, and proinflammory cytokines by human gingival epithelial cells through different signaling pathways. PLoS One 7, e52614, 2012. 45. van den Broek, L.J., et al. Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27. Tissue Eng Part A 20, 197, 2014. 46. Chiara Barsotti, M., et al. Effect of platelet lysate on human cells involved in different phases of wound healing. PLoS One 8, e84753, 2013. 47. Sato, T., et al. Direct effect of electrical stimulation on induction of brain-derived neurotrophic factor from cultured retinal Müller cells. Invest Ophthalmol Vis Sci 49, 4641, 2008. 48. Kiwanuka, E., et al. CCN2 is transiently expressed by keratinocytes during reepithelialization

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22 51. Dubey, A.K., Gupta, S.D., and Basu, B. Optimization of electrical stimulation parameters for enhanced cell proliferation on biomaterial surfaces. J Biomed Mater Res B Appl Biomater 98, 18, 2011. 52. Itoh, Y., et al. Plasma membrane-bound tissue inhibitor of metalloproteinases (TIMP)-2 specifically inhibits matrix metalloproteinase 2 (Gelatinase A) activated on the cell surface. J Biol Chem 273, 24360, 1998. 53. Howard, E.W., et al. MMP-2 expression by fibroblasts is suppressed by the myofibroblast phenotype. Exp Cell Res 318, 1542, 2012. 54. Letra, A., et al. MMP-7 and TIMP-1, new targets in predicting poor wound healing in apical periodontitis. J Endod 39, 1141, 2013. 55. Ploeger, D.T., et al. Cell plasticity in wound healing: paracrine factors of M1/ M2 polarized macrophages influence the phenotypical state of dermal fibroblasts. Cell Commun Signal 11, 29, 2013. 56. Tsou, C.L., et al. Critical roles for CCR2 and MCP-3 in monocyte mobilization from bone marrow and recruitment to inflammatory sites. J Clin Inves 117, 902, 2007. 57. Jettanacheawchankit, S., et al. Acemannan stimulates gingival fibroblast proliferation; expressions of keratinocyte growth factor-1, vascular endothelial growth factor, and type I collagen; and wound healing. J Pharmacol Sci 109, 525, 2009. 58. Nakamura, T., et al. Signals via FGF receptor 2 regulate migration of endothelial cells. Biochem Biophys Res Commun 289, 801, 2001. 59. Cho, M.R., et al. Control of calcium entry in human fibroblasts by frequency-dependent electrical stimulation. Front Biosci 7, a1, 2002.

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23 60. Pu, J., et al. EGF receptor signalling is essential for electric-field directed migration of breast cancer cells. J Cell Sci 120, 3395, 2008. 61. Shanley, L.J., et al. Influx of extracellular Ca2+ is necessary for electrotaxis in Dictyostelium. J Cell Sci 119, 4741, 2006. 62. Yue, Z., et al. Role of TRP channels in the cardiovascular system. Am J Physiol Heart Circ Physiol ajpheart.00457.2014. doi: 10.1152/ajpheart.00457.2014. 63. Jennings, J., Chen, D., and Feldman, D. Transcriptional response of dermal fibroblasts in direct current electric fields. Bioelcectromagnetics 29, 394, 2008.

Figures’ legends

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Tissue Engineering Part A Electrical stimulation modulates the expression of multiple wound healing genes in primary human dermal fibroblasts (doi: 10.1089/ten.TEA.2014.0687) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 25 of 51

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26 Figure 1: Diagram representing the electrical stimulation culture system. The conductive membranes were cut into rectangular specimens and assembled tightly into a homemade electrical cell culture plate to prevent culture medium from leaking. Fibroblasts were seeded into the culture well adhering to the membrane. The edges of the conductive membrane were linked to a DC power source through two metal plates. The metal plates were not in contact with the culture medium preventing any redox reaction and ionic current formation.

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35 Figure 2: Effect of electrical stimulation on cell adhesion and extracellular matrix genes expression by primary human dermal fibroblasts. Cell were exposed or not to ES at 50 or 200 mV/mm for 6 h then cell were collected and used to extract total RNAs. These were used to evaluate genes ‘expressions/repressions by mean of RT2 Profiler PCR Array. Panel (a) shows cell adhesion pathway, panel (b) shows cytoskeleton pathway, panel (c) shows ECM components pathway, and panel (d) shows remodeling enzymes’ pathway.

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40 Figure 3: Effect of electrical stimulation on inflammatory cytokines/chemokines and growth factors ‘genes expressions by primary human dermal fibroblasts. Cell were exposed or not to ES at 50 or 200 mV/mm for 6 h then cell were collected and used to extract total RNAs. These were used to evaluate genes ‘expressions/repressions by mean of RT2 Profiler PCR Array. Panel (a) shows inflammatory cytokines/chemokines pathway, panel (b) shows growth factors pathway.

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Figure 4: Effect of electrical stimulation on signal transductions ‘genes expressions by primary human dermal fibroblasts. Cell were exposed or not to ES at 50 or 200 mV/mm for 6

h then cell were collected and used to extract total RNAs. These were used to evaluate

genes ‘expressions/repressions by mean of RT2 Profiler PCR Array.

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48 Figure 5: Electrical stimulation reduced MMP2 but increased TIMP2 secretions by primary human dermal fibroblasts. Following the exposure of fibroblasts to ES at either 50 or 200 mV/mm, the culture supernatant was collected either immediately or 24 h postexposure and assayed to measure MMP2 (A), TIMP1 and TIMP2 (B) by means of ELISA (n = 4).

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Figure 6: Electrical stimulation increased CCL7 and KGF secretions by primary human dermal fibroblasts. Following the exposure of fibroblasts to ES at either 50 or 200 mV/mm,

the culture supernatant was collected either immediately or 24 h post-exposure and

assayed to measure CCL7 (b) or KGF (b) by means of ELISA (n = 4).

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Electrical Stimulation Technologies for Wound Healing.

Wound healing properties of ethyl acetate fraction of Moringa oleifera in normal human dermal fibroblasts.

Chronic dermal ulcer healing enhanced with monophasic pulsed electrical stimulation.

Electrical Stimulation Therapy and Wound Healing: Where Are We Now?

Honey exposure stimulates wound repair of human dermal fibroblasts.

Angiogenesis is induced and wound size is reduced by electrical stimulation in an acute wound healing model in human skin.

Panx1 regulates cellular properties of keratinocytes and dermal fibroblasts in skin development and wound healing.

Stimulation of human dermal fibroblasts with interleukin 2.

Stem cell therapy for dermal wound healing.

The efficacy of electrical stimulation in lower extremity cutaneous wound healing: A systematic review.

Human dermal fibroblasts express multiple bFGF and aFGF proteins.

Electrical Stimulation for Wound-Healing: Simulation on the Effect of Electrode Configurations.

Piper betle L. Modulates Senescence-Associated Genes Expression in Replicative Senescent Human Diploid Fibroblasts.

Electrical Stimulation and Cutaneous Wound Healing: A Review of Clinical Evidence.

Biology of fetal wound healing: collagen biosynthesis during dermal repair.

Electrical stimulation in multiple sclerosis.

Transcutaneous electrical nerve stimulation (TENS) accelerates cutaneous wound healing and inhibits pro-inflammatory cytokines.

The Electrical Response to Injury: Molecular Mechanisms and Wound Healing.

Hyaluronidase modulates inflammatory response and accelerates the cutaneous wound healing.

Acidic fibroblast growth factor accelerates dermal wound healing.

Nanofeatured silk fibroin membranes for dermal wound healing applications.

PKCδ inhibition normalizes the wound-healing capacity of diabetic human fibroblasts.

Effects of fibroblasts and basic fibroblast growth factor on facilitation of dermal wound healing by type I collagen matrices.

Surgical diverticulitis is not associated with defects in the expression of wound healing genes.