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http://journals.tubitak.gov.tr/biology/

Turkish Journal of Biology Turk J Biol(2016) 40: 462-472© TÜBİTAKdoi:10.3906/biy-1505-114

Osteogenic differentiation of electrostimulated human mesenchymal stem cells seeded on silk-fibroin films

Anıl S. ÇAKMAK1,2, Soner ÇAKMAK1,3, James D. WHITE1, Waseem K. RAJA1,David L. KAPLAN1, Menemşe GÜMÜŞDERELİOĞLU2,3,4,*

1Department of Biomedical Engineering, Tufts University, Medford, MA, USA2Bioengineering Division, Graduate School of Science and Engineering, Hacettepe University, Beytepe, Ankara, Turkey

3Nanotechnology and Nanomedicine Division, Graduate School of Science and Engineering, Hacettepe University, Beytepe, Ankara, Turkey4Department of Chemical Engineering, Hacettepe University, Beytepe, Ankara, Turkey

* Correspondence: menemse@hacettepe.edu.tr

1. IntroductionIn vivo bone regeneration is a complex and tightly regulated process affected by various biochemical and biophysical factors (Hess et al., 2012). Low intensity pulsed ultrasound (LIPU) (Yang et al., 1996), low intensity high frequency vibration (LIHFV) (Srinivasan et al., 2002), low level laser therapy (LLLT) (Stein et al., 2005), and electrostimulation (Tsai et al., 2009) are used as important biophysical factors to manipulate bone regeneration. The regenerative role of electric fields has been studied since the discovery of the piezoelectric properties of bone by Fukada and Yasuda (Castano et al., 2004). In this context, it is manifested that dynamic energy applied on bones is transformed into new tissue formation (Chao, 2003).

In bone tissue, streaming potentials, currents, and piezoelectricity of collagen molecules are directly related to bioelectricity (Brighton et al., 2001). There have been many in vitro studies indicating enhanced messenger RNA (mRNA) expression, protein synthesis, and differentiation of hMSCs in response to electric fields (Hronik-Tupaj

et al., 2011). For example, osteogenic differentiation of hMSCs was investigated in the presence of an electric field. The results showed that alkaline phosphatase (ALP) expression was enhanced under the electric field compared to the control group and calcium mineralization was also higher in the stimulated group (Sun et al., 2009). In another example, hMSCs were exposed to an electric field during osteogenic differentiation. It was observed that the electric field increased ALP and type I collagen expressions (Hronik-Tupaj and Kaplan, 2012). It has also been shown that electric fields enhanced mineralization via upregulating the expression of genes, e.g., bone morphogenetic proteins (BMP-2, BMP-4), transforming growth factor (TGF-β1), ALP, and prostaglandin E. In addition, extracellular matrix (ECM) deposition of decorin, osteocalcin, osteopontin, type I collagen, and type III collagen was also increased in bone tissue according to the molecular effect of electric fields (Lohmann et al., 2003; Fassina et al., 2006).

Electrostimulation can be applied as direct current (DC) or alternative current (AC). Uniform and continuous

Abstract: Electric field is known as an important regulator to guide the development and regeneration of many tissues. The aim of this study was to investigate the osteogenic differentiation potential of human mesenchymal stem cells (hMSCs) cultivated on silk-fibroin films in response to different parameters, i.e. frequency, voltage, distance between electrodes, and/or culture conditions (growth medium or osteogenic medium). Silk films were prepared in the presence of platinum wires to study the impact of exogenous electrostimulation on the cells for up to 14 days. The experimental groups can be defined as high voltage in osteogenic differentiation medium, low voltage in osteogenic differentiation medium, and low voltage in growth medium in this study. Compared to the unstimulated controls (silk films without platinum wires), low voltage (10 mV) did not influence proliferation, while it enhanced osteogenic differentiation according to early and late osteogenic markers in osteogenic differentiation medium. In growth medium, low voltage increased cell proliferation in contrast to osteogenic medium. On the other hand, high voltage (500 mV) stimulated cell proliferation and only late osteogenic markers in osteogenic medium. The results suggest the potential to exploit exogenous biophysical control of cell functions towards tissue-specific goals.

Key words: Silk films, electrostimulation, osteogenic differentiation, human mesenchymal stem cells

Received: 03.06.2015 Accepted/Published Online: 28.09.2015 Final Version: 23.02.2016

Research Article

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electric fields are provided by DC devices and they are generally used to manipulate cardiomyocyte migration and alignment (Tandon et al., 2006). AC devices supply bidirectional electric fields and are classified as capacitively coupled (CC) and inductively coupled (IC). AC devices are mainly used for functional tissue engineering studies such as contraction in cardiac or skeletal muscle cells, communication within neural networks, and cellular differentiation (Hronik-Tupaj and Kaplan, 2012). Electric fields alter osteoblast activities via the calcium/calmodulin pathway and basically increase the intracellular Ca+2 level due to the applied field type (Kim et al., 2006). DC and CC stimuli affect voltage-gated Ca+2 channels in the epithelial cells, human keratinocytes, and hMSCs membrane and increase Ca+2 influx. Furthermore, Ca+2 is released from intracellular storage such as endoplasmic reticulum as a result of IC induction (Nuccitelli, 2003; Titushkin and Cho, 2009). The enhanced calcium level in hMSCs may elicit changes in the expression of proliferative and differentiative genes by altering downstream processes (Kim et al., 2008).

Electrostimulation is shown to initiate signaling pathways and provides a strategy to enhance osteogenic differentiation of stem and/or progenitor cells (McCullen et al., 2010). In the literature, external electric fields have been applied on cells cultured in tissue culture polystyrene flasks and well plates via electrodes and coins (Sun et al., 2009; Jansen et al., 2010; McCullen et al., 2010). The aim of those studies was to investigate the effect of electric fields on osteogenic differentiation of cells rather than to suggest a functional system for bone fracture healing. On the other hand, implantable devices incorporating electric fields need to be optimized for tissue specific goals in research and in the clinic. This is why, combination of scaffolds and electrostimulation is a challenging field that can provide guided bone tissue mineralization and, later on, regeneration. Silk is a suitable biomaterial for bone tissue due to its unique properties such as mechanical strength, biocompatibility, and slow degradation rate (Sofia et al., 2001). Silk has already been used as an implantable device with electrodes for neural tissue engineering (Kim et al., 2010). This is the first study to evaluate the effect of electric fields on osteogenic differentiation of hMSCs seeded on silk films. For this purpose, electrode incorporation was achieved in silk films and the effects of different system parameters such as frequency, electrode distance and voltage, and culture conditions on cellular activities were demonstrated based on cellular activities of hMSCs. The advantages of electrode embedded silk films are that cell responses can be easily monitored in this system, electrostimulation can be applied on multiple samples at the same time, and a potential use of implantable silk-based electrostimulation model system is suggested to induce osteogenic differentiation of human MSCs for bone regeneration.

2. Materials and methods2.1. Fabrication of silk and platinum wire embedded silk (e-silk) filmsAqueous solution of silk fibroin was obtained from B. mori silkworm cocoons (Tajima Shoji Co. LTD, Yokohama, Japan) according to our previously reported methods (Nazarov et al., 2004). The 7% (w/v) silk solution was diluted to 3% (w/v) with ultrapure water. Then 250 µL of silk solution was cast upon flat polydimethylsiloxane (PDMS) round molds (11 mm in diameter) to obtain 50-µm-thick films. The films were covered with a venting lid and allowed to dry overnight. In order to embed platinum wires into the silk films, at first platinum wires (A-M Systems, USA) 127 µm in diameter were placed on PDMS molds with distances between them of 2.5 or 5 mm. Then 250 µL of 3% (w/v) silk solution was cast upon flat PDMS molds and the films were dried overnight. These platinum wire embedded silk films are referred to as e-silk films. Once the silk and e-silk films were dried, water annealing (Hu et al., 2011) was performed to generate water stable features, by placing the films within a water-filled desiccator, a 24 mmHg vacuum was pulled, and then the films were left within the water vapor environment for 4 h. The films were then removed from their PDMS substrates.2.2. GRGDS coupling to silk films Glycine-arginine-glycine-aspartic acid-serine (GRGDS) was coupled to silk films to improve cellular attachment as we have previously reported (Sofia et al., 2001). First, silk films were equilibrated in 2-(N-Morpholino) ethanesulfonic acid hemisodium salt (MES buffer) (Thermo Scientific, Waltham, MA, USA) for 30 min at room temperature. Then they were incubated with N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride/N-Hydroxysuccinimide (EDC/NHS) (Thermo Scientific, Waltham, MA, USA) solution (50 mg of EDC/70 mg of NHS in 100 mL of MES buffer) for 30 min. After incubation they were washed with MES buffer twice and placed into 0.2 mg mL–1 GRGDS solution (Bachem, Torrance, CA, USA) in MES buffer (pH 6.0) for 2 h at room temperature. Finally, after washing with Dulbecco’s phosphate buffer saline (DPBS; Life Technologies, Grand Island, NY, USA), the films were sterilized in 70% ethanol for 30 min and washed with DPBS 7–8 times. 2.3. Electrostimulation system designDelrin blocks, 30.5 × 30.5 cm, 1.25 cm in thickness (McMaster Carr, Chicago, IL, USA) were used to design electrostimulation chambers. Each block was divided into two sections and each section had two columns and 5 rows with holes suited to 35 mm cell culture petri dishes (35 mm in diameter, BD Biosciences, CA, USA). Two lines of holes for the wires, one around the outside and one inside, were

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placed around the petri dish holes in order to accomplish the electrical connections of the samples. With this design, 10 e-silk films could be stimulated concurrently.

Each e-silk film was placed in a petri dish in sterile conditions and put inside the electrostimulation chamber. Then platinum wires were connected to the positive wire line and the other was connected to the ground wire line. Soldering was used to make connections of the e-silk samples to the electrical wire line (Figures 1a and 1b) and different parameters such as the electrode distance, frequency, and applied voltage were studied.

A TENMA Universal Test Center 72-1005 Function Generator (TENMA Test Equipment, Springboro, OH, USA) was used as a voltage source generating sinusoidal-wave electric signals for all electrostimulation experiments. Applied voltage and frequency were confirmed using a Tektronix TDS 2024 Digital Oscilloscope (Tektronix, Beaverton, OR, USA).2.4. Cell culture2.4.1. Human mesenchymal stem cell (hMSC) isolation and expansionhMSCs were isolated from bone marrow aspirate (Lonza, Gaithersburg, MD, USA) as reported before (Altman et al., 2002). Basically, bone marrow aspirate was diluted to 56 mL with expansion medium including Dulbecco’s modified Eagle medium (DMEM, Life Technologies) supplemented with 10% (v/v) fetal bovine serum (FBS, Life Technologies), 1% (v/v) antibiotic/antimycotic (penicillin, streptomycin and fungizone, Life Technologies) and 1 ng mL–1 basic fibroblast growth factor (bFGF, Life Technologies). Sixteen milliliters of expansion medium and 4 mL of bone marrow were added to each T-175 flask (Corning, MA, USA). Twenty milliliters of expansion medium was added twice a week until the cells reached 50% confluence. Above 50% confluence, nonadherent hematopoietic cells were removed by washing with DPBS and the cells were cultured in expansion medium until either passaged or frozen. 2.4.2. Cell seeding and electrostimulationhMSCs were trypsinized upon confluency and suspended in osteogenic differentiation medium containing α-MEM (Life Technologies) supplemented with 10% (v/v) FBS, 1% (v/v) antibiotic/antimycotic, 100 nM dexamethasone (Sigma Aldrich, St. Louis, MO, USA), 10 mM β-glycerol phosphate (Sigma Aldrich), and 0.05 mM ascorbic acid (Sigma Aldrich) with a cell density of 3 × 106 cells mL–1. Both silk and e-silk films were placed in 35-mm cell culture petri dishes (Corning, MA, USA) and each film was conditioned in a growth medium containing α-MEM supplemented with 10% (v/v) FBS and 1% (v/v) antibiotic/antimycotic for 2 h before cell seeding. Petri dishes containing e-silk films were positioned in the electrostimulation chamber and they were connected to a copper wire via soldering.

Growth medium was removed from each dish and 30 µL of cell suspension was added onto each silk and e-silk film. Cells were allowed to adhere for 30 min and then 30 µL of differentiation medium was added every 30 min to each dish. After 3 h, the petri dishes were filled with 3 mL of differentiation medium. Electrostimulation was applied to the cells seeded on e-silk films the following day after seeding for 1 h per day up to 14 days in order to determine the effect of the electric field on osteogenic differentiation of hMSCs. The distance between electrodes was changed from 2.5 mm to 5 mm, frequency was applied as 20 Hz or 60 kHz, and the applied voltage was varied in the range of 10–500 mV. Stimulation was also carried out with the controls, i.e. silk films without platinum wires.2.4.3. Cell proliferationThe Alamar Blue assay was used for the evaluation of cell proliferation and viability according to the manufacturer’s instructions. Seeded films in growth or differentiation medium were incubated in culture medium supplemented with 10% (v/v) Alamar Blue reagent for 3 h at 37 ° C with 5% CO2. Then 200 µL of culture medium was transferred to a 96-well plate and fluorescence intensity was measured by a microplate reader (Spectramax Gemini XS, Molecular Devices, USA) with an excitation wavelength of 550 nm and emission wavelength of 590 nm. The same medium without cells was analyzed similarly as a blank solution. 2.4.4. Real-time polymerase chain reaction (RT-PCR) analysisSilk films were first washed with DPBS before the analysis and transferred to new RNase, DNase free Eppendorf tubes. Two hundred microliters of Trizol (Life Technologies) was added to each Eppendorf tube and cell seeded samples were chopped with micro-scissors. Total RNA was extracted using an RNeasy mini kit (Qiagen, Valencia, CA, USA) following the supplier’s instructions and the RNA concentration was determined by measuring optical density at 260 nm using a Nanodrop 2000 (Thermo Scientific, Wilmington, DE, USA). cDNA was synthesized using a high-capacity cDNA kit (Applied Biosystems, Foster City, CA, USA) and real-time PCR was carried out with Taqman Gene Expression assay kit (Applied Biosystems) to detect transcript levels of runt-related transcription factor 2 (RUNX-2; Hs00231692_m1), collagen 1 alpha 1 (COLL1A1; Hs00164004_m1), alkaline phosphatase (ALP; Hs01029144_m1), bone sialoprotein (BSP; Hs00173720_m1), and osteopontin (OPN; Hs00167093_m1). Expression levels of genes were quantified using a Stratagene MX30009 QPCR System (Stratagene, La Jolla, CA, USA) and they were normalized to the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Hs99999905_m1). Then the data were expressed as fold change relative to silk films alone according to the first day of analysis using the 2-(ΔΔCt) method.

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2.4.5. Scanning electron microscope (SEM) analysisMorphology of the cells on silk films on day 14 was visualized by Zeiss Ultra-55 Field Emission SEM (Germany). After the culture medium was removed, samples were gently washed with DPBS (pH 7.4) twice and cells were fixed with 2.5% (v/v) glutaraldehyde (Electron Microscopy Sciences, Hatfield, PA, USA) in 0.1 M DPBS (pH 7.4) for 30 min at 4 °C. Then they were dehydrated in a series of ethanols (30%, 50%, 70%, 90%, and 100% v/v) and treated with hexamethyldisilazane (HMDS; Electron Microscopy Sciences, Hatfield, PA, USA) before being dried. After the samples were completely dry, they were coated with a 5-nm gold layer prior to SEM analysis.2.4.6. Alizarin Red stainingCalcium-rich extracellular matrix mineralization was visualized with Alizarin Red staining. After fixation, monolayer cells were covered with Alizarin Red solution and incubated for 5 min at room temperature. After that, Alizarin Red solution was removed and samples were washed with distilled water three times (Camassola et al., 2012). 2.5. Statistical analysisAll data were expressed as mean ± standard deviations of a representative of three parallel samples. Statistical analyses were performed with GraphPad InStat software (Graphpad Software, Inc., La Jolla, CA, USA). One-way ANOVA was used to determine the significant differences among the groups and statistical significance was assigned as *P < 0.05, **P < 0.01, and ***P < 0.001, respectively.

3. Results 3.1. Selection of electrostimulation system parametersFor the in vitro studies, electric fields were applied to hMSCs using platinum wire embedded silk (e-silk) films (Figure 1). Previously reported in vitro studies indicated that CC electric fields induced osteoblast proliferation,

maturation, and ECM protein synthesis. In most of these studies, 60 kHz frequency and 20 mV/cm electric field strength were used and the results showed increased aggrecan and collagen gene expression, and BMP and ALP activities (Wang et al., 2006; Clark et al., 2014). 3.1.1. Frequency and electrode distanceAll in vitro studies are summarized in the Table. By taking into account the reported values (Hartig et al., 2000; Ercan and Webster, 2010), electric field parameters were varied based on frequency and electrode distances with low voltage, 10 mV, for the electrostimulation. High frequency (60 kHz) and low frequency (20 Hz) waves with 10 mV were applied to the hMSCs with electrode distances of 2.5 mm (0.004 V/mm) and 5 mm (0.002 V/mm).

Cellular activity was evaluated by Alamar Blue assay in all study groups over the 7-day stimulation (Figure 2a). The results showed that electrostimulation did not affect cellular proliferation at high or low frequency stimulation. Samples with 2.5-mm electrode distances under both high and low frequency stimulation conditions had the same or higher fluorescence intensities than at 5.0 mm electrode distances; therefore, the closer samples were chosen for further RT-PCR analysis (Figure 2b).

The expression of ALP was 3 times higher in the high frequency stimulated samples than the hMSCs alone and low frequency stimulated samples (P < 0.001). Expression of COLL1A1 was 1.6 times higher in high frequency stimulated samples than in the control group (P < 0.05). There was no significant difference in terms of RUNX-2 expression between each condition. Expression of late osteogenic marker BSP was 2 times higher in the high frequency stimulation (P < 0.05). 3.1.2. Voltage In the electrostimulation system, the applied voltage was changed in the range of 10–500 mV to optimize outcomes. Sixty kilohertz and 2.5 mm were chosen as frequency

Figure 1. Platinum wire embedded silk films (a) and electrostimulation system (b).

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Frequency(2.5 mm, 10 mV, OM)

Cell culture medium(2.5 mm, 60 kHz, 10 mV)

Table. Cellular responses of hMSCs seeded on platinum wire embedded silk films under the electric fields with different conditions compared to unstimulated groups.

Differentiation

Parameters Proliferation RUNX2 ALP COLL1A1 OPN BSP

20 Hz - - - - - -

60 kHz - - + + - +

50 mV - - - - + -

100 mV - - - - + -

250 mV + - - - - +

500 mV + - - - - -

Differentiation medium (OM) - - + + - +

Growth medium (GM) + - - - - -

{

{

{

Voltage(2.5 mm, 60 kHz, OM)

Figure 2. Electrostimulation studies performed in osteogenic differentiation medium; (a) cell proliferation obtained by Alamar Blue assay with respect to different frequencies and electrode distances, (b) quantitative gene expression profiles in low and high frequency stimulation, (c) cell proliferation, and (d) gene expression profiles at different voltages at day 7. The results are represented as the mean ± standard deviation for n = 3. Statistically significant differences are denoted by symbols: * P < 0.05, ** P < 0.01 and *** P < 0.001.

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and electrode distance, respectively, based on the results obtained from Figure 2b. In contrast to low voltage (10 mV), the higher voltages (250 and 500 mV) increased cell proliferation at day 7. Cell proliferation reached the highest levels at 250 mV and 500 mV (Figure 2c).

The RT-PCR results (Figure 2d), showed the expression of early osteogenic genes such as RUNX-2, ALP, and COLL1A1 did not change under high voltage (50–500 mV). However, the expression of late osteogenic markers, BSP and OPN, increased under electric fields. Expression of BSP reached the highest level at 250 mV. Fifty millivolts and 100 mV enhanced expression of OPN compared to the control group (unstimulated samples). Five hundred millvolts suppressed the expression of OPN while it stimulated cell proliferation. 3.1.3. Culture conditionsIn order to investigate the effect of electrostimulation in different culture conditions, electric fields were also applied to hMSCs seeded on silk films in growth medium in addition to the differentiation medium described before. For this purpose, electrostimulation parameters were chosen from previous studies as 60 kHz frequency, 10 mV voltage, and 2.5 mm electrode distance. According to Alamar Blue analysis, electric fields enhanced cell proliferation (Figure 3a) and did not affect gene expression (Figure 3b) in growth medium at day 7, in contrast to hMSCs cultured in the differentiation medium (Figures 2a and 2b). 3.2. Cell culture studies at the conditions of 10 mV, 60 kHz, 2.5 mmThis group of studies was carried out at predetermined electrostimulation conditions (10 mV, 60 kHz, 2.5 mm).

The effect of osteogenic differentiation on cell morphology was studied after 2 weeks. The cells spread on the silk film surfaces and formed a thin uniform layer. There were no differences in cell morphology between the stimulated groups and control unstimulated groups in growth medium (Figures 4a–4c). In addition, cellular extensions toward the platinum wires were observed under the electric field (Figure 4d). In osteogenic differentiation medium, cells fully covered the silk film surfaces in all samples (Figures 4e–4h), indicating that the cells strongly attached to the electrode (platinum wire) surfaces and produced ECM in the presence of the electric field (Figures 4g and 4h). Moreover, osteoblastic matrix structure was characterized by a mineral-like formation in stimulated samples (Figures 4i and 4j).

Alizarin Red staining was performed to determine matrix mineralization in both growth and osteogenic differentiation media. Although cells were not stained densely in growth medium, some local staining was observed in the stimulated group at day 14 (data not shown). In osteogenic differentiation medium, cells were stained in the stimulated samples at very early stages of cultivation (day 3) compared to the control group (Figures 5a and 5b). Mineralization localized especially near the electrodes (Figures 5c and 5d).

4. DiscussionWe exposed CC electric fields to hMSCs cultivated on platinum wire embedded silk films. Clinically, electric fields are used to enhance bone fracture healing via DC, CC, or IC methods (Isaacson and Bloebaum, 2010; Hronik-Tupaj

Figure 3. Electrostimulation studies performed in growth medium; the effect of electrostimulation on (a) cell proliferation and (b) gene expression profiles of hMSCs at day 7. Electrostimulation was carried out at 60 kHz and 10 mV, 2.5 mm electrode distance. The results are represented as the mean ± standard deviation for n = 3. Statistically significant differences are denoted by symbols: * P < 0.05, ** P < 0.01 and *** P < 0.001.

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Figure 4. SEM images of hMSCs on (a), (b) silk films in growth medium, (c), (d) electrically stimulated silk films in growth medium, (e), (f) silk films in osteogenic medium, (g), (h) electrically stimulated silk films in osteogenic medium. (i) and (j) show mineralized areas on electrically stimulated silk films. All images were acquired at day 14. Electrostimulation was carried out at 60 kHz and 10 mV, 2.5 mm electrode distance. Left column indicates low magnification whereas right column shows high magnification images.

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and Kaplan, 2012). DC methods have some disadvantages such as toxicity due to the electrodes, changes in pH, reduced levels of molecular oxygen, and the formation of Faradic products inside culture medium (Merrill et al., 2005). To avoid these problems, we used CC. In the CC system, homogeneous electric fields are generated between two parallel layers of noncorrosive platinum electrodes and electric current can be transferred to ionic current at

the electrode–electrolyte interface (Pedrotty et al., 2005; Isaacson and Bloebaum, 2010; Hronik-Tupaj et al., 2011).

Cellular activity was evaluated by Alamar Blue assay and osteogenic differentiation was determined by RT-PCR in all study groups. The results showed that electrostimulation with a low voltage (10 mV) did not affect cell proliferation but stimulated expression of both early (RUNX-2, ALP, COLL1A1) and late osteogenic genes

Figure 5. Alizarin Red staining images of hMSCs on (a), (b) silk films at day 3, (c), (d) electrically stimulated silk films at day 3, (e) silk films at day 14, and (f) electrically stimulated silk films at day 14. Small images on top right of (e) and (f) show the general view of silk films after staining. Electrostimulation was carried out at 60 kHz and 10 mV, 2.5 mm electrode distance in osteogenic medium.

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(BSP, OPN). On the other hand, expression of only late osteogenic genes was enhanced at high voltage (50, 100, and 250 mV) in addition to increasing cell proliferation.

Electric fields were also applied to hMSCs seeded on silk films in growth medium in addition to the osteogenic differentiation medium. In growth medium, low voltage (10 mV) enhanced cell proliferation in contrast to osteogenic differentiation medium. Control of cells’ mechanical parameters as a result of modulation of cytoskeleton features, via external electric fields, has been shown previously. When 2 V/cm direct current was applied to hMSCs and osteoblasts, the results indicated reduced cell elasticity due to substantial actin cytoskeleton reorganization during exposure to an external electric field (Titushkin and Cho, 2009). Actin reorganization could be related to increased intracellular Ca2+ levels and elevated ATP consumption. In hMSCs, a lower membrane tension may better facilitate endo- and exocytosis and contribute to a higher sensitivity of these cells to various soluble factors in the cell culture medium. In the present study, improvement in the proliferation of hMSCs in growth medium and enhanced differentiation of hMSCs in osteogenic medium supported this hypothesis.

SEM images indicated that hMSCs strongly attached onto silk film surfaces under the electric field and matrix mineralization was characterized by a mineral-like structure. Electric fields can affect ECM components such as soluble ions and charged groups in glucosaminoglycans (GAGs) and proteins. Cell membrane binding proteins extend along electric fields to reach binding sites on cell surface receptors (Adey, 1993). Adsorption of serum proteins, specifically fibronectin (FN), was induced by electric fields and facilitated cell attachment onto electrodes (Kotwal and Schmidt, 2001). Electric fields of 15 Hz and 9 mV/cm on SAOS-2 cells resulted in significantly increased mineral nodule formation after electrostimulation (Martino et al., 2008). In another study, mineral deposition increased in stimulated groups relative to control groups under electric fields of 1 Hz, 1 V/cm (McCullen et al., 2010). The SEM images in the present study are consistent with this prior literature, as are the RT-PCR results.

Matrix mineralization was also confirmed by Alizarin Red staining. It was shown that electric field enhanced mineralization. Electrostimulation has been previously reported to promote earlier calcium deposition with respect to control samples (Ercan and Webster, 2010). At day 14, the stimulated samples showed more intense staining than the control group (Figures 5e and 5f). In earlier studies, it was found that alternating current of 10 Hz and 10 µA on osteoblasts resulted in a 307% increase in calcium deposition by osteoblasts after 21 days with

respect to unstimulated samples (Supronowicz et al., 2002). A dose-dependent increase in cytoplasmic mineralized calcium was also observed in response to electric fields of 1 Hz and 1 V/cm (McCullen et al., 2010).

In vitro studies indicated that the electric field enhances osteoblast proliferation, maturation, and ECM protein production by inducing synthesis of various cytokines (BMP-2, IGF-1, and VEGF) (Jin and Kim, 2013). For example, an electric field was applied with a CC of 12–54 V/cm and 3–100 Hz on cultured rat embryo calvarial bone cells. Radioactively labeled calcium was added to culture medium to follow calcium uptake in cells and it was observed that calcium influx was enhanced during electric stimulation (Brighton et al., 2006). In another study, it was found that electrostimulation accelerated osteoblast mineralization (Meng et al., 2011). Wiesmann et al. (2001) reported that an electric field of 6–21 kV/m with 16 Hz frequency stimulated the formation of mineral crystals of osteoblast-like cells in vitro. Sixty kilohertz frequency and 20 mV/cm electric field strength have been used as parameters for several studies investigating the effect of CC on bone and cartilage cells. Results showed increased ECM production according to aggrecan and collagen gene expression, and BMP and ALP activity (Wang et al., 2004; Brighton et al., 2006; Wang et al., 2006). In these studies an electric field was applied on cells seeded on tissue culture plates by using media conduction or cells seeded on conducting polymers were exposed to an electric field. In our study, silk films were preferred because of its unique properties for bone tissue regeneration and an implantable, electrode embedded system was suggested to enhance bone fracture healing.

In conclusion, the results indicated that CC electric fields enhanced the expression of BSP, which is important for bone matrix mineralization, when the cells were cultivated on silk films. Based on Alizarin Red staining, matrix mineralization started early during cell culture and proceeded during the cultivation as an indication of BSP expression. We concluded that an electric field can be used as a biophysical stimulant for in vitro studies and cellular response can be manipulated under the electric field by altering process conditions such as frequency, voltage, or composition of cell culture medium.

Acknowledgments We thank the NIH (R01 AR061988, R01 AR005593) for support of this work. Anıl S. Çakmak thanks the Scientific and Technological Research Council of Turkey (TÜBİTAK) for providing an International Research Fellowship. Soner Çakmak was supported by the Hacettepe University, Scientific Resarch Projects Coordination Unit, BAB 6091–International Scientific Cooperation Development Support.

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