In Vitro Studies on Regulation of Osteogenic ... - ACS Publications

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In Vitro Studies on Regulation of Osteogenic Activities by Electrical Stimulus on Biodegradable Electroactive Polyelectrolyte Multilayers Haitao Cui,†,‡ Yu Wang,† Liguo Cui,† Peibiao Zhang,† Xianhong Wang,† Yen Wei,*,§ and Xuesi Chen*,† †

Key Laboratory of Polymer Ecomaterials, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun 130022, People’s Republic of China ‡ University of Chinese Academy of Sciences, Beijing 100039, People’s Republic of China § Department of Chemistry, Tsinghua University, Beijing 100084, People’s Republic of China S Supporting Information *

ABSTRACT: In this study, a novel electroactive tetreanilinecontaining degradable polyelectrolyte multilayer film (PEM) coating [(poly(L-glutamic acid)-graf t-tetreaniline/poly(L-lysine)-graf t-tetreaniline)n, (PGA-g-TA/PLL-g-TA)n] was designed and fabricated by layer-by-layer (LbL) assembly method. Compared with the nongrafted PEMs, the tetreaniline-grafted PEMs showed higher roughness and stiffness in micro/nanoscale structures. The special surface characteristics and the typical electroconductive properties were more beneficial for adhesion, proliferation, and differentiation of preosteoblast MC3T3-E1 cells. Moreover, the enhanced effects were observed on the modulation of MC3T3-E1 cells that differentiated into maturing osteoblasts, when the electroactive PEMs were coupled with electrical stimulus (ES), especially in the early phase of the osteoblast differentiation. The alkaline phosphatase (ALP) activity, calcium deposition, immunofluorescence staining, and RT-qPCR were evaluated on the differentiation of preosteoblast. These data indicate that the comprehensive effects through coupling electroactive scaffolds with electrical stimulus are better to develop bioelectric strategies to control cell functions for bone regeneration.

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INTRODUCTION Bioelectricity plays an essential role in the functioning of all living organisms, not just in the action potentials of nerves and muscles, but also controlling cellular functions. Therefore, external electrical stimulus (ES) is considered to be an attractive guiding signal for controlling cell behavior and promoting tissue regeneration.1 In vivo and in vitro studies, ES has been proved as an effective way to improve the cardiac contractility, to increase the cellular alignment and the length of neurite outgrowth, to promote the bone fracture healing, and to enhance the proliferation and differentiation of precursor cells or stem cells.2−5 The capacitively coupled electric field has been found to enhance proliferation of preosteoblastic MC3T3-E1 cells and increase levels of transforming growth factor-beta 1 (TGF-β1).6 The biphasic current stimulation system has been proved to enhance mesenchymal stem cell (MSC) proliferation, alkaline phosphatase (ALP) activity, and expression of vascular endothelial growth factor (VEGF) and bone morphogenetic protein-2 (BMP-2).7 In clinical treatments, a wide range of electrical therapeutic devices have also been developed to treat specific ailments and tissue types.8,9 For example, the implantable and external bone growth stimulators have been successful in treating osteoporosis, osteoarthrosis, normal, and nonunion fractures.10,11 In recent years, the electroactive materials such as metal,12 carbon nanotube,13,14 graphene,15,16 and conductive polymers17−19 have been developed to fabricate new functional © 2014 American Chemical Society

scaffolds to deliver the ES through an evenly distributed and well-controlled manner. They could permit the external control over the intensity and position to better regulate the cellular activities both on the surface and within the biomaterial scaffold. Among them, the conductive polymers such as polypyrrole (PPy) and polyaniline (PANi) have been widely used and studied due to their convenient and profitable synthesis, controllable electrochemical activity, and excellent biocompatibility.19−22 Studies have shown that the conductive polymers are beneficial to modulating osteoblast proliferation and promoting osteogenesis.23−25 However, some drawbacks have limited their further application such as poor solubility, processability, and especially nondegradability. The prolonged residence time of the conducting polymer in vivo may cause the local inflammation and lead to the need for surgical removal.26,27 To solve these problems, a concept of the electroactive degradable polymers containing conductive oligomers has been proposed to meet biomedical applications, especially the tissue engineering field.26,28−31 Similar to the conductive polymers, their oligomers characteristically have a conjugated backbone with a certain degree of p-orbital overlap. Through a doping process, the neutral chain becomes positively charged with Received: May 27, 2014 Revised: July 1, 2014 Published: July 4, 2014 3146

dx.doi.org/10.1021/bm5007695 | Biomacromolecules 2014, 15, 3146−3157

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Scheme 1. (A) Synthetic Procedure of PGA-g-TA and PLL-g-TA Copolymers; (B) Schematic Illustration of the Process of Preparing Polyelectrolyte Multilayer Films: (a) Glass; (b) (PGA/PLL)10; (c) EM (PGA-g-TA/PLL-g-TA)10; (d) EMS (PGA-gTA/PLL-g-TA)10; (C) Scheme of Electrical Stimulus Apparatus

polarons and bipolarons as the charge carriers for electrical conduction. These oligomers show good electroactivity and biodegradability in vitro and in vivo. Therefore, a few different electroactive polymers containing conductive oligoanilines have been synthesized to prepare their scaffolds and hydrogels for nerve, cardiac, and bone tissue engineering.27,32−35 In a previous study, we successfully synthesized a triblock copolymer PLA-AP-PLA (PAP) of polylactide (PLA) and aniline pentamer (AP). Later, we found the PAP/hydroxyapatite/poly(lactide-co-glycolide) nanocomposites to be beneficial to enhance the osteoblasts adhesion and to increase the gene expression levels of BMP-2 and osteonectin.36 We also synthesized the tetraaniline (TA) grafted poly(ester amide)s and found that it could serve to enhance the ALP activity and calcium level of MC3T3-E1 cells stimulated by pulsed electrical signal.37 Although the application of various approaches for incorporating electroactive segments within the systems have been applied, a straightforward strategy with a fabricated electroactive material still remains a challenge for tissue engineering application. Moreover, despite the beneficial effect of combining the conducting polymer with ES on tissue

regeneration has been reported, there is rare work on investigating the cellular functions in depth and in detail on the electroactive oligomer scaffolds. In fact, the interactions between host cell and matrix mainly reflect in the interface of various synthetic or natural scaffolds.38,39 Thus, the surface coating has emerged as a means of modulating cellular events at the implant−tissue interface,40−42 and the advantage of the functionalized surface method is that it affected least the bulk properties of the scaffolds or implants.43,44 Compared with the conventional implant coating methods, the layer-by-layer (LbL) thin film assembly is an inexpensive, convenient, and efficient coating technique based on physical interactions that can be used to modify the implants or scaffolds and construct a controllable release system so that it greatly enhances integration with host tissue and regulation on cell behavior.45−48 Among LbL assembly methods, polyelectrolyte multilayer film (PEM) is easy to be fabricated by the alternating deposition of polymers with opposite charges. Hence, we designed and fabricated the electroactive tetreaniline-containing degradable PEMs [(poly(Lglutamic acid)-graf t-tetreaniline/poly(L-lysine)-graf t-tetreanili3147


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7.77(s, 1H), 7.70−7.60 (d, 2H), 7.44−7.33 (d, 2H), 7.30−7.21 (s, 1H), 7.20−7.08 (m, 2H), 7.08−6.78 (m, 8H), 6.67 (m, 1H), 2.73 (m, 2H), 2.35−2.27 (m, 2H). MALDI/TOF-MS (m/e [MH]+): 465.2. PGA, EDC·HCl, and NHS were dissolved in DMSO. After the mixture was stirred at room temperature for 24 h, TA in DMSO solution was added into the above mixture and the stirring was continued for 24 h at 50 °C. After the reaction, the solution was cooled to room temperature and poured into diethyl ether to precipitate the product. The precipitate was collected by filtration and dialyzed with distilled water, and then freeze-dried to give the PGA-gTA product (yield: 84%). The PLL-g-TA has similar synthetic procedure (yield: 83%). Polyanionic sodium salt (PGA or PGA-g-TA) and polycationic hydrobromide (PLL or PLL-g-TA) were prepared and used in the following experiments. Characterization. 1H NMR spectra were recorded on a Bruker AV 400 MHz spectrometer in deuterium oxide (D2O). Fourier transform infrared (FT-IR) spectra were recorded on a Bio-Rad Win-IR instrument in the range of 4000−500 cm−1. Gel permeation chromatography (GPC) was carried out with a Waters GPC instrument (using Waters 515 HPLC pump, with DAWN EOS 18 Angles Laser Light Scattering Instrument and OPTILAB DSP Interferometric Refractometer (Wyatt Technology) as the detector), and water (phosphate buffer, pH 7.4) was used as an eluent. Poly(ethylene glycol) with different molecular weights was used as standard samples. Matrix-assisted laser desorption/ionization time-offlight (MALDI-TOF) mass spectra were performed on an AXIMACFR laser desorption ionization time-of-flight spectrometer (COMPACT). The surface potential (ζ) of the sample was measured on the Zeta-Potential-Analyzer (Brookhaven). The ellipticity of polymer aqueous solution (0.05 wt %) was obtained on a JASCO J-810 spectrometer at room temperature. Electrochemical Properties. To investigate the electrochemical properties of polymers, (PGA-g-TA/PLL-g-TA) complex solution was prepared by mixing oppositely charged PGA-g-TA solution with PLLg-TA solution (mole ratio 1/2). The ultraviolet−visible (UV−vis) spectra were recorded on a UV-2401PC spectrophotometer. Cyclic voltammetry (CV) was conducted on a CHI660 electrochemistry system (CHI, U.S.A.) using Ag/AgCl and Pt as the reference and counter electrodes, respectively. The indium tin oxide (ITO) electrode was used as the working electrode, and the scan rate was 100 mV s−1. Fabrication of PEMs. LbL film assembly was performed using the repeated sequential dipping of a substrate into dilute polycation and polyanion solutions with rinsing between each deposition step (Scheme 1B). In brief, the substrates (glass, quartz or silicon slides) were cleaned sequentially in piranha (H2SO4/H2O2(aq), 7:3 (v/v)) for 30 min, rinsed in water, and then immersed in RCA (H2O2(aq)/ ammonia/water, 1:1:5 (v/v)) for 20 min at 75 °C, rinsed in water, and finally dried with N2. The multilayer films were fabricated on the substrate for different characterization methods. The substrate was first immersed in the PEI solution for 4 h, then dipped in polyanionic PGA or PGA-g-TA solution (1 mg mL−1 in 0.1 M sodium chloride solution) for 30 min, and subsequently rinsed with water. After the deposition of polyanion, the substrates were then dipped into polycationic PLL or PLL-g-TA solution (1 mg mL−1 in 0.1 M sodium chloride solution) for 30 min, followed by the same rinsing procedure. This dipping protocol was repeated n times to produce the final PEM designated (PGA/ PLL)n or (PGA-g-TA/PLL-g-TA)n, where n represents the number of bilayers deposited. Monitoring the LbL Deposition Process. UV−vis spectroscopy was used to determine the accumulation of PLL (or PLL-g-TA) and PGA (or PGA-g-TA) on quartz slides during the PEM growth process. Quartz Crystal Microbalance with Dissipation (QCM-D) was used to monitor the in situ deposition of the polyelectrolytes took place at the fifth overtones (25 MHz) on gold-coated quartz crystals (fundamental frequency 5 MHz). The changes in resonance frequencies (Δf) and in dissipation (ΔD) of the crystals were monitored on Q-Sense E4 (Qsense, Sweden). All measurements were stabilized at 20 °C. Data was analyzed with the aid of Q-Tools software.

ne)n, (PGA-g-TA/PLL-g-TA)n], and using MC3T3-E1 cells as in vitro model studied their potential to support osteoblast function and promote the osteogenic differentiation coupled with ES. Interestingly, in this system, the introduction of tetreaniline increased roughness and stiffness of the PEMs, so the appropriate biomechanical properties of films were more beneficial to support the osteoblast function. In addition, the cellular behaviors were highly influenced by the electroconductive property of the PEMs surface. Therefore, it is meaningful research to study the interactions between the eletroactive surface and the osteoblast. Up to now, few studies have explored the use of the degradable electroactive polymers as the osteoconductive coatings for scaffolds or implants to investigate systematically the biological activities of cells.

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EXPERIMENTAL SECTION

Materials. Poly(ethylenimine) (PEI, MW = 25, 000), ammonium persulfate (APS), N-phenyl-1,4-phenylenediamine, 1-ethyl-3-(3-dimethyllaminopropyl) carbodiimide hydrochloride (EDC·HCl), 4-dimethylaminopyridine (DMAP), and succinic anhydride were purchased from Sigma-Aldrich. n-Hexylamine (HA, 99%) was purchased from Aldrich without further purification. γ-Benzyl-L-glutamate (BLG) and ε-benzyloxycarbonyl-L-lysine (ZLL) were purchased from GL Biochem Co., Ltd. γ-Benzyl-L-glutamate-N-carboxyanhydride (BLGNCA) and ε-benzyloxycarbonyl-L-lysine-N-carboxyanhydride (ZLLNCA) were synthesized as described in previous work.49,50 N,NDimethylformamide (DMF) and dimethyl sulfoxide (DMSO) were dried over calcium hydride (CaH2) before vacuum distillation. All the other reagents and solvents were purchased from Sinopharm Chemical Reagent Co. Ltd. and used as received. All chemicals were of analytical grade or higher. Deionized water (18.2 MΩ, Milli-Q Ultrapure Water System, Millipore) was utilized in experiments. Synthesis of Poly(L-glutamic acid)-graft-tetreaniline (PGA-gTA) and Poly(L-lysine)-graft-tetreaniline) (PLL-g-TA) Copolymers. As shown in Scheme 1A, poly(L-glutamic acid) PGA and poly(L-lysine) PLL were synthesized through the ring-opening polymerization (ROP) of BLG-NCA and ZLL-NCA in DMF using HA as initiator and followed by deprotection reaction, respectively. Typically, 1 g BLG-NCA was first dissolved in anhydrous DMF, then a certain amount of HA was added. After stirring for 3 d at room temperature, the solution was precipitated into excess amount of diethyl ether. Subsequently, the precipitate was dissolved in dichloroacetic acid and HBr/acetic acid (33 wt %) was added. The deprotection reaction was conducted at 30 °C for 2 h and then the mixture was precipitated into excessive diethyl ether. After drying under vacuum, the precipitate was dialyzed with distilled water and freeze-dried to give the PGA product. (yield: 74%). The PLL has similar synthetic procedure except for trifluoroacetic acid as solvent in deprotection reaction (yield: 51%). Tetraaniline (TA) and carboxylcapped tetraaniline (CTA) were synthesized according to the similar procedure reported in the literature.33 First, N-pheny-1,4-phenylenediamine (0.02 mol) was dissolved in a mixture solution of hydrochloric acid (HCl), acetone, and water (vol. ratio 1/4/4). The emeraldine (EM) base form of TA was obtained upon addition of ammonium persulfate (0.02 mol) as the oxidant at 0 °C under stirring for 3 h. The mixture was filtered to collect the TA, and the cake was then washed with 1 M HCl and distilled water. The TA was dedoped in 1 M NH4OH, followed by filtration and washing until the filtrate was neutral. Finally, the TA was lyophilized (yield: 83%). 1H NMR (400 MHz, DMSO-d6, ppm): 8.38 (s, 1H), 7.24 (t, 2H), 7.09 (s, 4H), 7.03−6.96 (m, 5H), 6.91−6.80 (m, 2H), 6.82−6.79 (m, 2H), 6.61−6.60 (m, 2H), 5.53 (s, 2H). MALDI/ TOF-MS (m/e [MH]+): 366.6. The CTA was synthesized from the carboxylation reaction of TA and succinic anhydride in CH2Cl2, and the crude product was washed with distilled water, followed by washing in a Soxhlet extractor with CH2Cl2 until the filtrate became colorless. The product was dried under vacuum for 48 h (yield: 77%). 1 H NMR (400 MHz, DMSO-d6, ppm): 12.11 (s, 1H), 9.71 (s, 1H), 3148


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Table 1. Sequences of Primers for the RT-qPCR gene

forward primer sequence

reverse primer sequence

RUNX 2 BMP 2 OPN GAPDH

5′-GCCCTCATCCTTCACTCCAAG-3′ 5′-GCTCCACAAACGAGAAAAGC-3′ 5′-TCAGGACAACAACGGAAAGGG-3′ 5′-CAACCTGGTCCTCAGTGTAGC-3′

5′-GGTCAGTCAGTGCCTTTCCTC-3′ 5′-AGCAAGGGGAAAAGGACACT-3′ 5′-GGAACTTGCTTGACTATCGATCAC-3′ 5′-CGTGCCGCCTGGAGAAACCTGCC-3′

Contact Angle and Surface Morphology. Static air−water contact angle measurements of (PLL-g-TA/PGA-g-TA)n PEMs were obtained using the sessile drop method on a contact angle system (VCA 2000, AST). Environmental scanning electron microscopy (ESEM) was performed on an XL 30 scanning electron microscope (Micrion FEI PHILIPS). ESEM was used to study film morphology on dried samples. Preosteoblast Culture on PEMs. Mouse preosteoblastic MC3T3-E1 cell line was used for electoactive PEM−cell interaction studies with or without electrical stimulus (ES). MC3T3-E1 cells were cultured in a growth medium (DMEM; Dulbecco’s Modified Eagle Medium (Gibco) supplemented with 10% fetal calf serum (Gibco) and 100 U mL−1 penicillin-streptomycin (Sigma)) and incubated under standard conditions (a humidified incubator, 37 °C, 5% CO2). Culture medium was changed every 2−3 days. MC3T3-E1 cells were subcultured when near 90% confluence with the use of 0.05% trypsine/EDTA solution. Before the cells were seeded on the PEMs, the PEMs were sterilized under UV radiation for 1 h, then immersed in 75% ethanol for 24 h, and finally rinsed with PBS. Electrical Stimulation (ES) Device and Parameters. As shown in Scheme 1C, a self-made ES device was designed to perform the ES experiments, which were conducted after cells were seeded on the substrates for 2 day. Cells were stimulated for 2 h per day. The ES were applied by the signal generator (Rigol DG1022 Function/ Arbitrary Waveform Generator), and the signals were displayed and checked on the wave inspector (Rigol DS1022C Digital Oscilloscope). The square wave, frequency of 100 Hz, 50% duty cycle, and electrical potential of 0.5 V were adopted in the experiment. The electrical potential was added directly on the PEMs through two microwire platinum electrodes. In the following experiment, MC3T3-E1 cells were cultured under four experimental conditions: (1) growth medium with ES, (2) growth medium without ES, (3) differentiation medium (growth medium supplemented with 50 mg mL−1 L-ascorbic acid and 10 mM β-glycerol phosphate) with ES, and (4) differentiation medium without ES. Cell Adhesion and Proliferation Assays. MC3T3-E1 cells were seeded onto various substrates at an initial seeding density of 2 × 104 cells cm−2 and incubated for 3, 6, and 12 h, respectively. The cells were washed three times with phosphate buffered saline (PBS), fixed with 4% paraformaldehyde (PFA) at room temperature for 10 min, dyed with 2% fluoresceinisothiocyanate (FITC) DMSO/H2O solution for 10 min, and then washed with PBS three times. Cell attachment was observed qualitatively under a reverse microscope (TE2000U, Nikon). The fluorescence pictures were taken by Digital Camera DXM1200F (Nikon) and analyzed with “NIH ImageJ” software (>20 per sample). To investigate the effect of surface features and ES on the cell adhesion and spreading, the organization of actin filaments of adherent cells cultured on various substrates was evaluated after 48 h with or without ES. MC3T3-E1 cells were cultured on substrates at a density of 5 × 103 cells cm−2. Cytoskeletons were identified following double stain of actin staining (green) using Alexa Fluor 488 phalloidin and nuclei staining (blue) using 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) (Invitrogen). Cells were harvested and fixed using 4% PFA for 15 min, followed by washes in PBS for three times. Cell membranes were permeabilized in 0.1% Triton X-100 and blocked in 1% BSA, followed by washes in PBS three times. Cells were incubated with phalloidin for 15 min and DAPI for 3 min and then washed in PBS three times. Samples were observed and imaged using confocal microscope (Zeiss LSM 780). To investigate the cell proliferation on PEMs with or without ES, MC3T3-E1 cells were cultured on the substrates at a density of 1 × 104 cells cm−2. After the indicated

incubation times, the medium was changed by 2-(2-methoxy-4nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt (WST-8) solution (10% v/v in medium; Cell Counting Kit-8). After 3 h of incubation, the absorbance values at 450 and 600 nm were measured on multifunction microplate scanner (Tecan Infinite M200). Cell Differentiation Assays. Alkaline phosphatase (ALP) activity was determined after 5 and 10 days after the initiation of MC3T3-E1 osteogenic differentiation by quantitation of the enzyme activity. The medium of each well was carefully removed. Then MC3T3-E1 cells were washed three times with PBS, lysed in RIPA buffer freezing at −80 °C for 30 min, and thawing at 37 °C. Then p-nitrophenol phosphate substrate (pNPP) solution was added and incubated in the dark for 30 min at 37 °C. The reaction was terminated with 3 M NaOH and ALP activity read on multifunction microplate scanner at 405 nm. Measurements were compared to p-nitrophenol standards and normalized by the number of cells. After 10 and 20 days of exposure to both growth medium and differentiation medium with or without ES, MC3T3-E1 cells were assayed for calcium deposition or mineralization nodules using ARS and von Kossa staining. The cells were washed with PBS and fixed with 4% PFA for 10 min. After three rinses of 5 min in water, ARS stain solution (0.1% ARS in Tris HCl buffer, pH 8.0, Sigma) was incubated with cells for 30 min at room temperature. The cells were then washed in distilled water 3 times for 5 min each. The ARS stained cultures were imaged by phase contrast microscopy. For von Kossa staining, cells were washed with PBS, fixed with 4% PFA for 10 min, and then washed in distilled water. The cells were incubated in a 5% aqueous silver nitrate solution for 30 min. The silver nitrate stained cultures were then treated under UV light at 254 nm for 30 min. The cultures were washed with water and then neutralized with 5% sodium thiosulfate for 10 min at room temperature. After washing with water, the cultures were stained in neutral red for 10 min and imaged by phase contrast microscopy. For immunofluorescence staining, the cells on each substrate were fixed with 4% PFA in PBS for 15 min at room temperature. Cell membranes were permeabilized in 0.1% Triton X-100 for 10 min, washed three times with PBS, blocked in 1% BSA in TBST (0.2% Tween 20 in PBS) for 30 min, and washed three times with PBS. The cells were incubated with primary antibodies for 1 h at room temperature. The following primary antibodies were used for staining: mouse monoclonal anti-Runt-related transcription factor 2 (RUNX 2) antibodies (1:500; Abcam) and rabbit polyclonal anticollagen type I (COL I) antibodies (1:500; Abcam). After incubation with primary antibodies, the cells were washed three times with PBS for 5 min each. Alexa Fluor-488 goat antimouse IgG (1:500; Abcam) and Alexa Fluor488 goat antirabbit IgG (1:500; Abcam) as secondary antibodies were added and incubated 1 h at room temperature, respectively. The cell nuclei were counterstained using DAPI for 3 min. Fluorescence images were observed using confocal microscope (Zeiss LSM 780). MC3T3-E1 cells cultured on various substrates with or without ES were incubated at 10 and 20 days and the expression of osteogenesisrelated genes was quantitatively assessed using RT-qPCR technique. The total RNA was extracted using TRIzol Reagent (Invitrogen) according to the manufacturer’s protocol. Total RNA concentration and purity were estimated using Nanodrop Plates (Tecan Infinite M200) and reverse transcribed as described in M-MLV manual (Promega). RNA was added to a 20 μL reverse transcription reaction mixture containing 5 × M-MLV buffer, dNTP Mixture, RNase inhibitor, RTase M-MLV, RNase free dH2O and oligo (dT) primer. The expression of osteogenic markers was quantified by qPCR SYBR 3149


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Green Mix Kit (Stratagene). Gene-specific primers including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), anti-Runtrelated transcription factor 2 (RUNX 2), bone morphogenetic protein-2 (BMP 2), and osteopontin (OPN) were designed using the primer design software of beacon 5.0 (Table 1). Specificity of listed oligonucleotides was checked by BLASTN (Basic Local Alignment Search Tool) against the mouse RefSeq RNA database at NCBI. The qPCR amplification was performed as follows: initial heating at 95 °C for 10 min, followed by 40 cycles at 95 °C for 30 s, 58 °C for 60 s, and 72 °C for 60 s. Expression levels were obtained using threshold cycles (Ct) that were determined by the iCycler iQ Detection System software. Relative transcript quantities were calculated using the ΔΔCt method. The gene GAPDH was used as a reference gene and was amplified along with the target genes from the same cDNA samples. The difference in Ct of the sample mRNA relative to GAPDH mRNA was defined as the ΔCt. The difference between the ΔCt of the control cells and the ΔCt of the cells grown on substrates was defined as the ΔΔCt. The fold change in mRNA expression was expressed as 2−ΔΔCt. Statistical Analysis. The data presented are the mean (standard deviation ± SD). Independent and replicated experiments were used to analyze the statistical variability of the data using Student’s test, with p < 0.05 being statistically significant (*p < 0.05; **p < 0.01; ***p < 0.001).

methylene proton of the side group of the glutamate and lysine units, with the signal at 0.8 ppm assigned to the methylene proton of hexylamine. The proton signal at 8.0−6.5 ppm corresponding to aromatic protons further confirmed the introduction of TA to the side chains of PGA and PLL. It should be noted that Mn measured by GPC was relatively higher than that measured by 1H NMR, due to the structural differences between the resultant copolymer and poly(ethylene glycol) as standard samples in GPC analyses. The successful synthesis of copolymers was further confirmed by FT-IR and scale-expanded IR spectra were shown in Figure 1B. The results also demonstrated the generation of PGA and PLL based on the absorption at 1716 cm−1 (νCO) corresponded to the carboxyl group of PGA, while at 1685 cm −1 (ν NH ) corresponded to the amino group of PLL. In addition, the absorption at 1650 cm−1 (νCO) and 1550 cm−1 (νC(O)−NH) were attributed to the amide bond in the backbone of PGA and PLL. In the spectra of PGA-g-TA and PLL-g-TA, the typical absorption peaks of TA at 1510 cm−1 (s, −N−B−N−) and 1601 cm−1 (s, −NQN−) were assigned to the benzenoid unit and the quinoid unit of the TA segments, respectively. These data showed the successful synthesis of PGA-g-TA and PLL-g-TA copolymers. The ζ potential of polyelectrolyte solution was determined and shown in Figure 1C. The ζ potential of G0 was negative (about −45 mV), whereas L0 produced positive ζ potential (about 35 mV). When TA was linked to the side chains of PGA and PLL, a decrease of the value of the ζ potential was observed with the increase of TA contents, suggesting that charge density of the TA modified polyelectrolyte decreased. To investigate the solution behavior of the polyelectrolytes, circular dichroism (CD) was applied to test the copolymer conformation. As shown in Figure 1D, the CD spectra of G0 and L0 aqueous solution showed a negative Cotton band at 197 nm, which was characteristic Cotton band corresponding to a random structure.51 The same results were also found in the TA modified polymers. However, minimum values of intensities were different, suggesting that the addition of TA generated less random structure in the aqueous solution. This was because the rigid conjugated TA side chain weakened freemotion and random coil of the polyelectrolytes. This fact showed that the TA-modified ionic polypeptides were still random conformation in aqueous solution. It is well-known that polyaniline and its oligomers have different oxidation states (that is, leucoemeraldine (LM), emeraldine base (EMB or EM), and pernigraniline (PN)) when they are treated by different voltages or oxidating and reducing agents.27 The electrochemical property of (G1/L1) complex solution was investigated by UV−vis and CV spectra. The UV−vis absorption spectra of the complex solution oxidized by ammonium persulfate (APS) are shown in Figure 2A. The UV−vis spectra of the sample exhibited a stepwise oxidation process from the LM state to the EM state. The LM sample showed only one peak at 310 nm, which was associated with the π−π* transition of the aromatic benzene ring. Further oxidation caused blue-shift of the peak at 307 nm and the appearance of a new peak at 576 nm which were attributed to the excitonic transition from benzene ring to quinoid ring (πb−πq). As shown in Figure 2B, when (G1/L1) complex solution was doped with the 1 M HCl, the peak at 576 nm decreased and almost disappeared. At the same time, the π−π* transition peak at about 305 nm persisted and decreased in its intensity. In addition, the new polaron peaks at 410 nm and the

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RESULTS AND DISCUSSION The procedures for the synthesis of PGA-g-TA and PLL-g-TA are shown in Scheme 1A. Briefly, PBLG and PZLL were synthesized through ROP of BLG-NCA and ZLL-NCA initiated by hexylamine and followed by acidolysis deprotection and then coupled with TA. The characteristics and properties of the copolymers, prepared with different feed weight percentages of TA (in order to investigate the effect of different TA content), are summarized in Table 2 (e.g., PGA is Table 2. Characterization of the Copolymers TA yelid mol %a

Mn (dgn.) g/mol

Mn (cal.) g/molb

Mn (GPC) g/mol

PDI

5

4.1

10000 11400

7931 9030

9800 11000

1.1 1.3

10

7.4

12800

10020

11500

1.4

5

3.4

10000 11700

5906 7110

8300 9500

1.2 1.4

10

6.5

13500

8230

10500

1.4

TA feed mol % PGA (G0) PGA-gTA1 (G1) PGA-gTA2 (G2) PLL (L0) PLL-gTA1 (L1) PLL-gTA2 (L2) a

1

Determined by UV−vis spectra. bCalculated from the integration of H NMR signals.

defined as G0, PGA-g-TA1 is defined as G1, etc.). The content of TA was measured using the same method in our privious work.37 And the TA content in the products increased with the increase of TA feed weight fraction, but the grafting degree is somewhat lower than the theoretical value. The structures of the resulting copolymers were determined by 1H NMR spectra and FT-IR. The typical 1H NMR spectra of PGA-g-TA and PLL-g-TA are shown in Figure 1A, and all peaks have been well assigned. The degree of polymerization (DP) of the polypeptide backbone was calculated by comparing the integrated area of the peak at 4.2 ppm, attributed to the 3150


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Figure 1. (A) 1H NMR spectra of the copolymers in D2O. (B) FT-IR spectra of the copolymers. (C) The surface potential (ζ) of samples. (D) Circular dichroism spectra of the sample aqueous solutions (a) L2, (b) L0, (c) G2, and (d) G0.

Figure 2. (A) UV−vis spectra of the (G1/L1) complex oxidized by APS. (B) UV−vis spectra of the (G1/L1) complex in the leucoemeraldine (LM), emeraldine base (EM), and emeraldine salt (EMS) states (doped with 1 M HCl aqueous solution). (C) Cyclic voltammogram of the (G1/L1) complex in 1 M HCl aqueous solution.

localized polaron peak at ∼800 nm confirmed the generation of emeraldine salts (EMS) and the ability of conducting electrons of the copolymer. Figure 2C shows CV of the (G1/L1) complex solution obtained by directly diluting into HCl electrolyte. The complex solution showed one pair of obvious reversible redox peaks, and the mean peak potential E1/2 was 450 mV (E1/2 = (Epa + Epc)/2), which was due to the transition from LM state to EM state. The results of UV−vis and CV demonstrated that our copolymers maintained a good level of electroactivity.

The accumulation of each layer deposited on quartz substrates during PEM growth was monitored by UV−vis spectroscopy. This allowed serial monitoring of the increase in (G0/L0)n (amide bond absorption peak of polypeptide backbone at 200 nm) and (G1/L1)n (aromatic ring and πbπq absorption peak of TA at 300 and 600 nm, respectively). With the increasing of bilayer number, the progressive increases in absorption intensity were observed at the 200 nm wavelengths for (G0/L0)n, whereas at the 300 and 600 nm wavelengths for (G1/L1)n and (G2/L2)n, as shown in Figure 3151


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S1A. The rates of increase in the film components were determined from the slopes of lines in Figure S1B and showed that the growth of (G/L)n film was exponential. This exponential growth of the polyelectrolyte multilayers have been explained by polyelectrolyte diffusion mechanism.52,53 In addition, the various films showed different growth behaviors, which the rates of increase in (G0/L0)n film were higher than in (G1/L1)n and (G2/L2)n film. It was mainly because that the assembly of the multilayer films was mainly affected by the charge density of polyelectrolyte. On one hand, the grafted TA made the charge density of polyelectrolyte decrease; on the other hand, the steric hindrance of π-conjugated side chains also affected the assembly of polyelectrolytes. The stepwise build-up process of the multilayer films was real-time monitored by QCM-D. Figure 3 represents the evolution of resonant frequency change (Δf) and energy

dissipation change (ΔD). Δf is related to the mass of polymer deposited on the crystal (Δm), and ΔD is related to the viscoelasticity of the deposited polymer. The observed Δf decrease after each G and L solution injection was representative of the film build-up and was associated with an increase in the adsorbed mass. And an exponential rise in Δf shift was observed during PEM growth; however, the rates of Δf shift were different in various PEMs. The reason was the graft copolymer through chemical-modified TA had different surface affinities from unmodified ones. In addition to the electrostatic interaction, hydrophobic aggregation, hydrogen bonding, and π−π stacking also affected the assembly of the copolymer and the properties of PEMs. The ΔD of (G0/L0)10 film rose up with the progressive increase of the mass, whereas the ΔD of (G1/L1)10 and (G2/L2)10 film resulted in a slight reduction in the dissipation. The results showed the PEMs became more rigid, when TA was linked to the polypeptide, due to the introduction of the conjugated aromatic ring. Owing to the relatively large changes in ΔD, the (G/L)n films were not in accordance with the Sauerbrey relation.48 The dates suggested that the polyelectrolyte conformation in PEMs could be affected by the hydrophobicity and hydrophilicity of copolymers, chain stiffness and charge density. These QCM-D results were consistent with the exponential increases in G and L absorbance monitored via UV−vis and also showed markedly different in the viscoelastic properties of the various films. Contact angle measurements were performed in order to assess the wettability of (G/L)n PEMs in emeraldine (EM) and emeraldine salts (EMS) states, as shown in Figure S2A. The contact angles of the (G1/L1)n and (G2/L2)n films in EM state were much higher than those of the nongrafted (G0/L0)n films. The contact angle of (G/L)n film slightly increased with increasing number of bilayers from 5th to 10th. The results could be explained that the grafted TA segments increased hydrophobicity of PEMs. After the (G/L)n film were doped in acidic solution (pH 4.0), the contact angle of the EMS (G/L)n film decreased obviously compared to that of the EM (G/L)n film, because the hydrophilicity of TA segments in the EMS state was higher than those in the EM state. As is well-known, the segments with higher surface affinity were mainly located on the outer layer of film, whereas the hydrophobic units were embedded into the inner layer. Therefore, with the increasing hydrophilicity of TA segments in PEMs, the surface conformation of the EMS film changed correspondingly. To investigate the microstructure of the films, the surface morphology was characterized by SEM. Figure S2B shows a notable difference between the nongrafted PEMs and grafted ones. The surface of the (G0/L0)n film showed a smooth and featureless morphology. However, the surface of the (G1/L1)n and (G2/L2)n films became rough and formed the hump structure. In addition, the film was relatively smooth at lower n, but became much rougher at higher n. Compared to the (G2/ L2)n film, much broader distribution of islands and smoother surface features were seen in the (G1/L1)n film. Thus, the PEMs grafted higher content of TA demonstrated the significant increases in surface roughness. The roughness of the outermost layer significantly changed, due to the stacked TA segments bulged onto the surface of PEMs. Because of the hydrogen bonding and the π−π stacking between the conjugated aromatic ring chains, the TA segments are inclined to aggregate the close packing to form the microphase separated morphology in the assembly process, so the segments acted as rigid and stiff support in PEMs. When doped in acidic

Figure 3. QCM-D measurement of frequency shifts (Δf; black line) and energy dissipation change (ΔD; gray line) during alternate deposition in G and L layers: (a) (G0/L0)n; (b) (G1/L1)n; and (c) (G2/L2)n. 3152


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average area compared with others. It indicated that the higher grafted density of TA resulted in the weak cell adhesion due to the changes of millimeter-scale structure and wettability. In addition, there was generally no statistical difference in the MC3T3-E1 cell adhesion between on the (G/L)5 and (G/L)10 films, indicating the negligible effect of changes in the nanometer-scale roughness on the cell adhesion (not shown). The results suggested that the cell adhesion was related to the surface characteristics of materials, including stiffness, roughness, charge density, and hydrophilicity. Cytoskeleton staining showed that the MC3T3-E1 cells are well spread and organized, as shown in Figure 4B. F-actin staining showed the presence of polymerized actin fibers lying parallel one to each other. Overall, the MC3T3-E1 cells maintained their normal polygonal morphology with multiple cellular projections both on glass and on all PEMs. The MC3T3-E1 cells showed a cuboidal morphology on glass and the (G0/L0)10 films, whereas these cells showed an elongated, spindle-like morphology on the electroactive PEMs. Moreover, when the MC3T3-E1 cells were employed with electrical stimulus (ES), the morphology of cells showed the more connected actin fibers and the distinct cell pseudopodium, which were beneficial to enhance the cell−cell communication. After the MC3T3-E1 cells become attached to an osteoconductive surface, they enter a rapid proliferative growth phase in order to establish critical cell−cell interactions essential for the subsequent postconfluent differentiation growth phase. Therefore, the electroactive PEMs were studied on their ability to promote cell proliferation in growth medium. The MC3T3-E1 cell proliferation was monitored quantitatively via the use of the WST-8 assay to measure the metabolic activity of the total population of cells for 5 and 10 days (Figure 5A,B). In the initial days, the MC3T3-E1 cell proliferation rate was most rapid on TCPs. In contrast, the MC3T3-E1 cells proliferated at slower and similar rates on the glass and the (G0/L0)10 film. The cell proliferation in the electroactive PEMs groups obviously increased when the cells were applied with ES. After 10 days of the cell seeding, the metabolic activity of the MC3T3-E1 cells was higher than that at 5 day in culture. The proliferation of the MC3T3-E1 cells on the EMS (G1/ L1)10 film revealed the similar values as on TCPs. Over all, the higher osteoblast proliferation was generally seen on the (G1/ L1)10 film than on glass and pure PEMs. The results suggested that, the increased roughness enhanced the cell−substrate interaction and surface charge accelerated the cell−cell communication on the electroactive substrate, leading to an

solution, the PEMs became smoother over extended periods. It was suggested that the TA segments might be sufficiently mobile within the multilayer to allow significant rearrangement of the PEMs during their construction in EMS state. The assembly of the electroactive PEMs not only affected the wettability of the substrate, but also changed the surface morphology. It is expected that, through grafting the TA segments onto the polyelectrolytes, this way could provide special functional groups for the surface modification to promote cell−substrate interaction. MC3T3-E1 cell behavior on various (G/L)n films was investigated compared to a control substrate (glass). The cells were seeded and cultured for 3, 6, and 12 h on different substrates to observe the cell adhesion (Figure 4A). After 6 h,

Figure 4. (A) Cell adhesion on different substrates for 3, 6, and 12 h (scale: 100 μm). (B) Cytoskeleton staining of MC3T3-E1 cells on different substrates for 3 days with or without electrical stimulus (ES; scale: 20 μm).

the cell average areas cultured on the different substrates showed the different results. With the increase of culture time, the cell average area of the EMS (G1/L1)10 film was the highest. However, the EM (G2/L2)10 film showed lower cell

Figure 5. MC3T3-E1 cell proliferation on different substrates with and without electrical stimulus (ES) for (A) 5 and (B) 10 days: (a) TCPs; (b) glass; (c) (G0/L0)10; (d) EM (G1/L1)10; (e) EMS (G1/L1)10; (f) EM (G2/L2)10; and (g) EMS (G2/L2)10. 3153


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Figure 6. ALP activity of MC3T3-E1 cells on different substrates with and without electrical stimulus (ES) at 5 and 10 days (A) in differentiation medium and (B) in growth medium: (a) TCPs; (b) glass; (c) (G0/L0)10; (d) EM (G1/L1)10; (e) EM (G2/L2)10; and (f) EMS (G1/L1)10.

increase in the osteoblast cell proliferation. As previously known,37 the grafted TA segments could improve the cell adhesion, but the contents was a main concern; thus, to be useful as biomaterial, the percentage of TA segments would need to be at an optimal level to preserve the good performance and electroactivity while avoiding undesirable side effects. Therefore, the preosteoblast differentiation was investigated on the (G1/L1)10 film with and without ES in following experiments. In addition to an osteoconductive surface to support the cell growth in bone formation, the osteoinductive signal to stimulate the differentiation of osteogenic stem and/or progenitor cells along an osteoblastic pathway is a critical progress. The electroactive PEMs applied with or without ES were investigated for their ability to support the differentiation of preosteoblast cells. After seeded in growth medium for a 48 h culture stabilization period, the cells were cultured for a subsequent 3 weeks in osteogenic differentiation medium. ALP enzyme activity is an early marker of osteogenic differentiation. The significant increase in ALP activity of the MC3T3-E1 cells on all substrates with ES was observed at 5 days in differentiation medium, as shown in Figure 6A, while there was no significant difference of ALP activity at 10 days. At 5 days, ALP activities of cells on the electroactive (G/L)n film both with ES and without ES were significantly higher than those of cells on the glass and the (G0/L0)10 film, suggesting that the cell differentiation toward osteogenesis was better on the electroactive film than other substrates. Especially, the value of ALP activity on the EMS (G1/L1)10 film was about 1.4 times as that on glass and 1.2 times as that on TCPs. The ALP activity of cells on these substrates were also studied in growth medium, that is, in the absence of any stimulating exogenous biochemical molecules to evaluate the intrinsic osteoconductivity of the substrates (Figure 6B). Although the mean values on the electroactive PEMs were greater than those of control groups without ES, the statistical analysis showed that there was no significant difference (P > 0.05). Interestingly, when the cells were applied with ES, ALP activity significantly increased on the electroactive PEMs both at 5 and 10 days. The result suggested the electroactive substrate with ES could effectively enhance the osteogenic differentiation. The capacity to deposit minerals is a marker for mature osteoblasts, which can be used to confirm that MC3T3-E1 cell differentiate and enter into the mineralization phase to deposit mineralize extracellular matrix (ECM). The osteogenic differ-

entiation of the MC3T3-E1 cells was induced on the different substrates (glass, (G0/L0)10 film, and EMS (G1/L1)10 film) under the osteogenic medium conditions, as shown in Figure S3A,B. After 10 and 20 days of culture in differentiation medium, the cells were stained with alizarin red S (ARS) staining and von Kossa staining to examine calcium precipitation and mineralization. Compared to no distinct staining in control groups, the deposited minerals on the electroactive PEMs were observed after ARS staining at 10 day. After 20 days, all the substrates had considerable calcium phosphate deposition due to the presence of differentiation medium. On the electroactive PEMs, larger area of continuous ARS staining was displayed. Especially with ES, the intensity of the staining and the size of the deposit increased on the electroactive PEMs. The von Kossa staining had the same result as those depicted in the ARS images and the deposition was also enhanced on the electroactive PEMs. The ARS and von Kossa staining of the long-term MC3T3-E1 cell cultures strongly suggested that the electroactive PEMs possessed particular physiochemical characteristics and intrinsic osteoconductive properties that favored the differentiation of the osteoblastic progenitors in the early stage and the mineralization of the ECM. Although the electroactive PEMs with ES enhanced the osteogenic differentiation in the experiments, the presence of the medium that supplemented with the osteogenic factors complicated the assessment of the effect of electroactive PEMs on the cell differentiation. Therefore, the differentiation experiment was further conducted under the nonosteogenic culture conditions. However, there is nearly no calcium deposition after 10 days of culture (not shown). Calcium deposition at 20 day was much less extensive in growth medium than in differentiation medium condition on all substrates because of the absence of osteogenic soluble factors (Figure S3C). The staining showed that all the substrates with ES had positive effect, and the electroactive PEMs enhanced the effect. This result further indicated that the cells on the electroactive PEMs differentiated into the osteogenic cells better than those on other substrates. Although it was reported that ES of the osteoblasts enhanced ALP synthesis and calcium deposition, this research for the first time showed that the electroactive substrate possessed the particular physiochemical characteristics combining with ES to promote earlier differentiation and larger calcium deposition. To study the enhanced differentiation effect of the electroactive PEMs, the immunofluorescence staining of the 3154


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osteoblasts on the electroactive PEMs led to COL I secretion over a wider area, and the morphological differences in RUNX 2 expression on the electroactive PEMs were most pronounced. These results confirmed that the electroactive PEMs with ES enhanced the commitment of MC3T3-E1 cells to the osteogenic lineage in the early phase of the osteoblast differentiation. Quantitative analysis of the osteogenesis-related gene expression (RUNX 2, BMP 2, and OPN) using RT-qPCR supported the results from the calcium deposition and the immunocytochemical staining. The RT-qPCR analysis after 10 days of culture in differentiation medium demonstrated that the expression of osteogenic genes (RUNX 2, BMP 2, and OPN) increased, when the MC3T3-E1 cells were cultured on all the substrates with ES, indicating that ES could enhance the osteogenic differentiation. The expression of RUNX 2 was obviously up-regulated on the electroactive PEMs, especially after being applied with ES, the values were about 1.5-fold higher than that on glasses (Figure 8A). However, BMP 2 (belongs to the transforming growth factor-beta (TGF-β) superfamily, can induce bone formation) expression was independent of the substrates, and no significant difference was observed (Figure 8B). OPN is an extracellular matrix protein, known to be expressed during maturation of osteoblasts. When the cells were cultured on the electroactive PEMs with ES, OPN was up-regulated to 5-fold at 10 days (p < 0.001; Figure 8C). The expression levels further increased up to 20 days, at which the cells cultured on the electroactive PEMs showed the similar expression levels (Figure 8D−F). Although the cells cultured on glass without ES also exhibited the increased OPN expression, but the levels were 2-fold lower (p < 0.001) compared to those of the cells cultured on the EMS (G1/L1)10 film. The experiments determining the response of cells with or without ES were also performed on all substrates

osteogenic markers was conducted in the early phase of osteoblast differentiation. Immunofluorescence staining of osteogenic markers collagen type I (COL I; main component of the organic part of bone) and runt-related transcription factor 2 (RUNX 2; a key transcription factor for osteogenesis) after the 10 days of culture revealed that the electroactive PEMs increased the expression of COL I and RUNX 2 in the differentiated MC3T3-E1 cells (Figure 7), although the

Figure 7. Immunofluorescence staining of osteogenic markers COL I and RUNX 2 in MC3T3-E1 cell with and without electrical stimulus (ES) for 10 days in differentiation medium.

increase was not significantly notable. However, when the cells were applied with ES, a noticeable difference was observed in the osteogenic marker expression between the electroactive PEMs group and the control group. The greater spreading of

Figure 8. Quantitative analysis of osteogenesis-related gene expression (RUNX 2, BMP 2, and OPN) with and without electrical stimulus (ES) for 10 (A−C) and 20 days (D−F) in differentiation medium: (a) TCPs; (b) glass; (c) (G0/L0)10; (d) EM (G1/L1)10; (e) EM (G2/L2)10; and (f) EMS (G1/L1)10. 3155


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Notes

in growth medium (Figure S4 A−F). However, compared with previous experiments, the expression of the osteogenic genes increased when the cells were applied without ES in growth medium, but there was no remarkable difference between the electroactive PEMs and the control groups. Therefore, the different substrates without ES have no significant effect on the osteogenic activity in cells. Moreover, the gene-expression of RUNX 2 was not regulated by ES. In contrast, ES resulted in higher increases in BMP2 and OPN activity. In particular, OPN was up-regulated to 30% on the EMS (G1/L1)10 film, showing the highest values after 20 days. Overall, the gene-expression analysis showed the increases in the osteogenic activity when ES were applied. As determined in previous experiments, the electroactive PEMs alone could promote the osteogenic expression compared with other substrates. This elevated expression was further enhanced both in the growth medium and the differentiation medium, if the cells were applied with ES. The results could be explained that on one hand, the positively charged surface may serve as a binding site for acidic phospholipids and is critical for the nucleation of mineralization; on the other hand, the electroactive substrate combining with ES enhances the intercellular signal transduction. Of cause, the material surface chemistry and topography are also key regulators of the osteogenic differentiation at the cell-implant interface. The combination of high nanometer-scale roughness and matrix stiffness resulted in the greatest enhancement of the osteogenic differentiation.

The authors declare no competing financial interest.

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REFERENCES

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CONCLUSIONS In this study, the tetreaniline-based polyelectrolyte multilayer films (PEMs) were fabricated using layer-by-layer assembly method. The electroactive PEMs possessed good electroactivity, stiffness, roughness and biocompatibility. Because of the introduction of tetreaniline segments, this electroactive PEM system did not require harsh postfabrication cross-linking treatments to increase PEM stiffness, thus greatly facilitating osteoblast cell−substrate interactions. The results showed the excellent osteoconductivity that had the ability to act as a scaffold coating to support the robust adhesion, proliferation, and differentiation of preosteoblast cells. Especially, the electroactive PEMs with ES enhanced the commitment of MC3T3-E1 cells to the osteogenic lineage in the early phase of the osteoblast differentiation, which was proved by immunofluorescence staining of the osteogenic markers and osteogenesis-related gene expression using RT-qPCR. Hence, this work represents a new approach for the electroactive surface functionalization of implants to improving osteogenesis. ASSOCIATED CONTENT

S Supporting Information *

The growth of PEMs monitored by UV−vis spectra (Figure S1), contact angle data and the surface morphology of PEMs (Figure S2), calcium deposition on different substrates with and without electrical stimulus (Figure S3), and quantitative analysis of osteogenesis-related gene expression in growth medium (Figure S4). This material is available free of charge via the Internet at http://pubs.acs.org.

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ACKNOWLEDGMENTS

This research was financially supported by National Natural Science Foundation of China (Projects 51103149, 51233004, 51203152, 51390484, and 51321062), Ministry of Science and Technology of China (International Cooperation and Communication Program 2011DFR51090).

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. 3156


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