The Regulation of Osteogenesis Using

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The Regulation of Osteogenesis Using Electroactive Polypyrrole Films Chuan Li 1,2, *, Yi-Ting Hsu 3 and Wei-Wen Hu 2,3, * 1 2 3

*

Department of Biomedical Engineering, Yang-Ming University, Taipei 11221, Taiwan Centre for Biomedical Cell Engineering, National Central University, Zhongli District, Taoyuan City 32001, Taiwan Department of Chemical and Materials Engineering, National Central University, Zhongli District, Taoyuan City 32001, Taiwan; [email protected] Correspondence: [email protected] (C.L.); [email protected] (W.-W.H.); Tel.: +886-2-2826-7397 (C.L.); +886-3-425-7163 (W.-W.H.); Fax: +886-2-2823-3887 (C.L.); +886-3-422-5258 (W.-W.H.)

Academic Editor: Changsik Song Received: 24 March 2016; Accepted: 5 July 2016; Published: 13 July 2016

Abstract: To evaluate the effect of electrical conductivity of biomaterials on osteogenesis, polypyrrole (PPy) was fabricated by oxidative chemical polymerization as substrates for cell culture. Through adjusting the concentrations of monomer and initiator, polypyrrole films with different electrical conductivities were fabricated. These fabricated polypyrrole films are transparent enough for easy optical microscopy. Fourier transform infrared spectroscopy, X-ray spectroscopy and four-point probe were used to assess the microstructures, surface chemical compositions and electrical sheet resistance of films, respectively. Results indicate that higher monomer and initiator concentration leads to highly-branched PPy chains and thus promotes the electron mobility and electrical conductivity. Selected polypyrrole films then were applied for culturing rat bone marrow stromal cells. Cell viability and mineralization assays reveal that not only these films are biocompatible, but also capable of enhancing the calcium deposition into the extra cellular matrix by the differentiated cells. Keywords: polypyrrole; osteogenesis; conductive polymer; electroactive; bone marrow stromal cells; mineralization

1. Introduction Wolff‘s law is a golden rule for bone regeneration, which states that induced mineralization makes bones more resistant to mechanical loads. It was found that stress-induced stains may generate an electrical potential along the collagen fibers in bones to stimulate osteogenesis [1]. In other words, the bone regeneration may be due to the piezoelectric effect under which the electrical cue is generated by the external mechanical loads on bones. Therefore, electric stimulations are considered as physical signals to promote osteogenesis of stem or progenitor cells [2,3]. To simulate electrical cues generated by ECM during osteogenesis, substrate-mediated direct current field (DCF) is developed, in which the substrate for cell culture is electrically conductive and electrodes are placed outside the culture media. An immediate question for substrate-mediated DCF is the selection of materials for substrates. For the purpose of electric conducive, metals are the top choice but their biocompatibility is a major concern [4–6]. Moreover, the high density of current can damage cells during stimulation and the corrosion of metals in culture media can lead to the release of cytotoxic debris [7]. The next choice therefore is averted to polymers or polymeric composites. For example, multi-walled carbon nanotubes (MWCNTs) have been added to polymers to prepare electroactive scaffolds [8,9]. Polypyrrole (PPy) doped by various materials also can demonstrate enhanced conductivity [10–12]. However, these conductive polymers were opaque and unsuitable

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for optical microscopy. Fluorescent staining and scanning electron microscopy (SEM) were employed for observation; however, continuous monitoring cellular activities become impossible by these end-point analyses. Therefore, transparent and conductive PPy films by chemical oxidation polymerization were prepared in this study. These films were biocompatible for cell culture and transparent and for optical microscopy. Through the control of monomer to initiator concentration ratio, the electrical conductivity of PPy films can be adjusted. Many studies indicate that electroactive surfaces of conductive polymers may increase intercellular communication to promote cell proliferation and adhesion [13–16]. Whether fabricated PPy films possess such capacity remains to be assessed. Our results demonstrate that the PPy films’ surface is highly electroactive, which is able to expedite the cell differentiation. These films’ properties suggest that the fabricated PPy thin films are potentially suitable for surface coating on medical implants in orthopedic applications. 2. Materials and Methods 2.1. Materials Ammonium persulfate (APS) and pyrrole (Py) were purchased from Showa (Tokyo, Japan) and Acros (Geel, Belgium), respectively. Dulbecco’s modified Eagle medium (D-MEM), fetal bovine serum (FBS), and trypsin–EDTA were obtained from Biowest (Nuaillé, France). Triton X-100, dexamethasone, 2-phospho-L-ascorbic acid trisodium salt, β-glycerophosphate disodium salt hydrate, and glutaraldehyde were purchased from Sigma-Aldrich (St. Louis, MO, USA). 2.2. The Preparation of PPy Films Polystyrene (PS) made petri dish (ϕ 35 mm in diameter, Nunc, Penfield, NY, USA) was used as the substrate for PPy films deposition. Pyrrole and APS aqueous solutions with different concentrations were gently mixed in petri dishes (2 mL) for 15 min at room temperature. The deposited films were washed by deionized water and dried in oven. 2.3. Fourier Transform Infrared (FTIR) Spectroscopy To determine the structures of the deposited PPy films, attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectrometer (Spectrum 100, Perkin Elmer, Waltham, MA, USA) was used with resolution of 1 cm´1 . The range of spectrum is set between 400 to 3600 cm´1 and transmission mode is used for detections. 2.4. Scanning Electron Microscopy (SEM) Surface morphologies of PPy films were observed by scanning electron microscopy (SEM, 3500N, Hitachi, Tokyo, Japan). Gold was plated on the films’ surface by sputtering before examination. The film thickness was measured from the cross sections by cyrofracturing of films in liquid nitrogen. 2.5. Four Point Probe Analysis Four point probe (EverBeing, Hsinchu, Taiwan) was used to determine the sheet resistance of PPy film. Twenty spots were randomly selected in each sample to measure the spatial variation of the sheet resistance. The electrical conductivity of PPy films then was calculated based on the thickness measured by SEM. 2.6. X-Ray Photon-Electron Spectrometry (XPS) Surface chemical analysis of PPy films was carried out by X-ray photon-electron spectrometry (XPS, Kα, Thermo, Waltham, MA, USA). Peaks of C1s between 280 and 292 eV was analyzed by software Magicplot (Magic systems, Saint Petersburg, Russia). The software is capable of removing background signals and iterative Gaussian/Lorentzian fitting.

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2.7. In Vitro Cell Culture of BMSCs Rat bone marrow stromal cells (BMSCs) harvested from 8-week-old Sprague-Dawley rats were cultured for determining the effect of PPy films on osteogenesis. These cells were maintained in DMEM with 10% FBS. Osteogenic supplement (100 µm ascorbic-2-phosphate, 10 mM β-glycerophosphate, and 100 nM dexamethasone) was applied to medium during the osteogenesis experiment. 2.8. Biocompatibility of PPy Films To evaluate biocompatibility of PPy films, BMSCs were seeded to PPy films with density of 17,000 cells/cm2 , and the (4,5-cimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used to quantify cell viability. After 2-day culture, 100 µL of MTT solution (5 mg/mL in PBS) and 900 µL of medium were added to each film and kept for 3 h at 37 ˝ C. The supernatant was removed and 1 mL of dimethyl sulfoxide (DMSO) was added to dissolve formazan, which then was analyzed by the spectrometer at the wavelength of 550 nm. 2.9. Quantification of Calcium Deposition in Extracellular Matrix The differentiated osteoblasts from BMSCs can deposit calcium in the extracellular matrix (ECM) as a process called mineralization [17]. To investigate this process, the calcium ortho–cresolphthalein complexone method (Ca-o-CPC) and alizarin red S staining were used to quantify the calcium deposition by BMSCs seeded on PPy films after 24 h. The Ca-o-CPC assay measures the amount of calcium deposited in ECM by analyzing the optical absorbance of purple-colored complex at wavelength of 570/660 nm. The resulting increase in optical absorbance of the mixture is directly proportional to the calcium concentration in the solution via the following reaction [18]: Ca2` paqq ` o ´ CPCpaqq Ñ Ca ´ o-CPC complex (violet)

(1)

Before the assay, osteogenic medium was washed away from the well using PBS. Then, 100 µL of 0.5 N acetic acid was added to release calcium ions. Equal volume (200 µL) of calcium binding reagent (1 g/L of o-cresolphthalein complexone, and 0.1 g/L of 8-hydroxyquinoline) and calcium buffer reagent (1.6 M of 2-Amino-2-methyl-1-propanol, pH 10.7) were added to 10 µL of calcium released sample. After 15-min incubation at room temperature, 100 µL of purple-colored Ca-o-CPC complex was transferred to 96-well multiplates to measure its optical absorbance at wavelength of 575 nm. The amount of calcium in cell lysate was calculated according to the linear calibration results of calcium chloride standard solutions. To normalize the amount of calcium deposition per cell, lactate dehydrogenase (LDH) assay was used to count the cell numbers using CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). Briefly, 100 µL of fresh medium was replaced in each well before the assay, and 15 µL of lysis buffer was added to sample and kept for 1 h at 37 ˝ C to release LDH from live cells. After transferring 50 µL of these LDH released medium to 96-well multiplates, 50 µL of LDH reagent was added and incubated for 30 min at room temperature. Finally, 50 µL of stop solution was added to each well and then were analyzed for the optical absorbance at wavelength of 490 nm. For calibration, a standard curve for lysing known cell numbers is applied to convert the optical absorbance to total cell number. For alizarin red S staining, the cultures were rinsed with PBS followed by fixation (1% glutaraldehyde in PBS) for 30 min at 37 ˝ C and then stained by 2% for alizarin red S solutions for another 20 minutes at room temperature. After PBS washes, the stained samples were examined by inverted microscope (Eclipse Ti-U, Nikon, Tokyo, Japan).

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3. Results and Discussion Polymers 2016, 8, 258 of Microstructures and Chemical Composition 3.1. Characteristics

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The with different APSAPS to pyrrole concentration ratios (APS/Py) are shown The synthesized synthesizedPPy PPyfilms films with different to pyrrole concentration ratios (APS/Py) are in Figure 1a where all films are dark but transparent. Higher ratios of of APS totoPy shown in Figure 1a where all films are dark but transparent. Higher ratios APS Pylead leadto to darker darker films. films. These films were also also inspected inspected by SEM. SEM. The The roughness roughness obviously obviously increased increased with with the the pyrrole pyrrole concentrations concentrations as well well as as APS/Py APS/Py ratios, suggesting that the accelerated accelerated polymerization polymerization at high high concentrations of monomer or initiator reduces the uniformity of films (Figure 1b). The cross section concentrations of monomer or initiator reduces the uniformity of films (Figure 1b). The cross section SEMs SEMs in Figure Figure 1c 1c show show the the dense dense structure structure of of films films and and their their thickness thickness are are estimated estimated to to be be around around 70 70 to to 100 100 µm. μm. Higher Higher pyrrole pyrrole concentration concentration and and APS/Py APS/Py ratio tend to thicken PPy films slightly. Figure Figure 22 shows shows the ATR-FTIR ATR-FTIR analyses analyses of of selected selected PPy PPy films. films. Similar spectrums spectrums are are also obtained obtained ´ 1 −1 for pyrrole concentrations. concentrations.The Thetwo twospecific specific peaks 1690 and 1360 are ascribed be for other pyrrole peaks at at 1690 and 1360 cmcmare ascribed to beto C=N ´1 are due to stretch −1 are C=N and groups, C–N groups, respectively [19].absorptions The absorptions atand 14751550 andcm 1550 cmdue and C–N respectively [19]. The at 1475 to stretch modes 1 −1´is modes C=Cpyrrole and pyrrole respectively [20,21]. In addition, absorption S=O 1050cm cm of C=Cofand ring, ring, respectively [20,21]. In addition, the the absorption of of S=O at at 1050 is attributed theinitiator initiatorAPS APS[22]. [22].These Theseresults resultsconfirmed confirmedthat that the the fabricated fabricated PPy PPy films were attributed bybythe were consistent with previous studies. consistent with previous studies.

(a)

(b) Figure 1. Cont. Figure 1. Cont.

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(c) (c) Figure 1. The fabricated polypyrrole (PPy) films: (a) PPy films were fabricated by different Figure The fabricated fabricated polypyrrole polypyrrole (PPy) films: (a) (a) PPy PPy films were fabricated Figure 1.1. The fabricated by by different different concentrations of pyrrole and APS/Py ratios; (b) surface roughness of PPy films by SEM (Scale bar = concentrations of pyrrole and APS/Py ratios; (b) surface roughness of PPy films by concentrations of pyrrole and APS/Py ratios; (b) surface roughness of PPy films by SEM (Scale SEM bar = 10 μm);bar and (c) µm); cross sections SEMsections of PPy films of (Scale = 50 μm).bar = 50 µm). (Scale = 10 (c) cross PPy bar films 10 μm); and (c) crossand sections SEM of PPySEM films (Scale bar =(Scale 50 μm).

Figure 2. The FT-IR spectra of selected PPy films. Figure 2. 2. The The FT-IR FT-IR spectra spectra of of selected selectedPPy PPyfilms. films. Figure

3.2. Electrical Resistance 3.2. Electrical Electrical Resistance Resistance 3.2. Figure 3a presents the measured sheet resistivity of PPy films by the four point probe. It turns Figure3a 3apresents presentsthe themeasured measured sheet resistivity PPy films by the four point probe. It turns Figure sheet resistivity of of PPy films by the point probe. It turns out out that higher pyrrole concentrations and APS/Py ratios result in four lower electrical resistance. out higher that higher pyrrole concentrations and APS/Py ratiosin result in lower resistance. electrical resistance. that pyrrole concentrations and APS/Py ratios result lower electrical However, However, notably, the sheet resistance of films fabricated using higher pyrrole concentration is much However, notably, the sheet resistance of films fabricated using higher pyrrole concentration is much notably, the sheet resistance of In films fabricated using higher concentration is much less less affected by the ASP/Py ratio. other words, the initiator haspyrrole a limited influence on the electrical less affected by ASP/Py the ASP/Py ratio. In other words, the initiatorhas hasa alimited limitedinfluence influenceon onthe theelectrical electrical affected by the ratio. In other words, the initiator resistance of films if pyrrole concentration is high enough. Following the resistance measure, PPy resistance of of films ifif pyrrole pyrrole concentration concentration isis high highenough. enough. Following Following the the resistance resistance measure, measure, PPy PPy resistance films preparedfilms by pyrrole concentrations of 0.1, 0.3, and 0.5 and APS/Py ratio of 0.1, 0.2, and 0.4 were films prepared by pyrrole concentrations of 0.1, 0.3, and 0.5 and APS/Py ratio of 0.1, 0.2, and 0.4 were filmsfor prepared by pyrrole concentrations of 0.1, 0.3, and 0.5 and APS/Py ratioisofthe 0.1, 0.2, and 0.4 were used the following study on cells. The corresponding conductivity, which reciprocal of sheet used for for the the following following study study on on cells. cells. The The corresponding conductivity,which whichis is the the reciprocal reciprocalof of sheet sheet used corresponding conductivity, resistance × film thickness, is shown in Figure 3b for later data analyses. resistance × film thickness, is shown in Figure 3b for later data analyses. resistance ˆ film thickness, is shown in Figure 3b for later data analyses. Theoretically, APS/Py ratio should be 1:1 for ideal electron transfer. Blinova et al. have applied 0.2 M Py solution with different concentrations of APS, and their results suggested that 0.25 M of APS, i.e., APS/Py = 1.25, demonstrated the best conductivity [23]. However, their preparation was at acidic condition (with 0.2 M HCl). In this study, oxidation is performed at neutral condition, and the results suggest that film conductivity increased with increasing APS. However, conductivities

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of the APS/Py = 0.8 group is only slightly better than those of APS/Py = 0.4, suggesting the APS effect should be saturated. We deduce that Py in neutral form are not as easily oxidized as those in acidic Polymerscondition. 2016, 8, 258 6 of 11

(a)

(b)

Figure 3. The electrical resistance of PPy films: (a) the sheet resistance; and (b) the conductivity (reciprocal of sheet resistance × film thickness).

Theoretically, APS/Py ratio should be 1:1 for ideal electron transfer. Blinova et al. have applied 0.2 M Py solution with different concentrations of APS, and their results suggested that 0.25 M of APS, i.e., APS/Py = 1.25, demonstrated the best conductivity [23]. However, their preparation was at acidic condition (with 0.2(a) M HCl). In this study, oxidation is performed at (b) neutral condition, and the results suggest that film conductivity increased with increasing APS. However, conductivities of the Figure 3. 3. The resistance of PPy films: films: (a) the sheet sheet resistance; and (b) the the conductivity conductivity Figure The electrical electrical resistance of PPy and (b) APS/Py = 0.8 group is only slightly better than those(a)ofthe APS/Py =resistance; 0.4, suggesting the APS effect should (reciprocal of sheet resistance × film thickness). (reciprocal of deduce sheet resistance thickness). be saturated. We that Pyˆinfilm neutral form are not as easily oxidized as those in acidic condition. Theoretically, APS/Py ratio should be 1:1 for ideal electron transfer. Blinova et al. have applied 3.3. XPS XPS Analysis Analysis and and Molecular Molecular Structures Structures 3.3. 0.2 M Py solution with different concentrations of APS, and their results suggested that 0.25 M of Figure 4a shows shows thedemonstrated XPS analyses analyses the on selected selected PPy films films[23]. for the the bindingtheir energy of C1s C1s orbitals. orbitals. APS,Figure i.e., APS/Py = 1.25, best conductivity However, preparation was at 4a the XPS on PPy for binding energy of In all analyses, the C1s peak can be de-convoluted by three Gaussian curves, namely CI(α), CII(β), acidic conditionthe (with M HCl). Inde-convoluted this study, oxidation is performed at neutral condition, the In all analyses, C1s0.2 peak can be by three Gaussian curves, namely CI(α),and CII(β), and CIII(disorder) peaks, which corresponds corresponds towith the α αincreasing carbons, carbons, and carbons carbons at structural results suggest thatpeaks, film conductivity increased However, conductivities of the and CIII(disorder) which to the carbons, β βAPS. carbons, and at structural disorder sites, respectively (cf.Figure Figure 4b) [24–26]. Pyrroles polymerized to form APS/Py =sites, 0.8 group is only slightly better than those of APS/Py = 0.4, suggesting theoxidation APS effect should disorder respectively (cf. 4b) [24–26]. Pyrroles areare polymerized viavia oxidation to form the + . These cationic radicals interact each other for radical-radical bond the pi-radical cation C H NH +● be saturated. We deduce that in neutral form are notinteract as easilyeach oxidized those in acidic condition. 4 . Py pi-radical cation C4H44NH These cationic radicals otherasfor radical-radical bond formation. This This process process can can be be repeated repeated many many times times to formation. to form form final final PPy PPy [23,27]. [23,27]. Following Following Pfluger’s Pfluger’s study [25], the majority of the pyrrole units are linked through the C–2,5 carbons to form linear chains 3.3. XPS Analysis and Molecular Structures study [25], the majority of the pyrrole units are linked through the C–2,5 carbons to form linear chains (α–α’ linkage). However, partial pyrroles can couple through the C–2,3 carbons (α–β’ linkages). IfIfα–β’ (α–α’Figure linkage). However, pyrroles couplePPy through carbons (α–β’ of linkages). α– 4a shows the partial XPS analyses oncan selected films the for C–2,3 the binding energy C1s orbitals. linkage branches fromfrom the α–α’ linkedlinked main chains, it leadsittoleads side chain or inter-chain link formation β’ linkage branches the α–α’ main chains, to side chain or inter-chain link In all analyses, the C1s peak can be de-convoluted by three Gaussian curves, namely CI(α), CII(β), (Figure 4c). Different from pyrroles in the α-α’ linked main chains, the hydrogen atoms atoms on theon β formation (Figure 4c). Different pyrroles in linked main chains, thecarbons hydrogen and CIII(disorder) peaks, whichfrom corresponds to the the α-α’ α carbons, β carbons, and at structural carbons of theofbranch pyrroles are changed to carbon atoms, which resulted in a disorder peakpeak in a the β carbons the branch pyrroles carbon atoms, which resulted in a disorder disorder sites, respectively (cf. Figureare 4b)changed [24–26].to Pyrroles are polymerized via oxidation to form the higher binding energy with larger width compared to α and β carbons. In this study, the electrophile in a highercation binding with larger width compared to α and β carbons. In this study, the pi-radical C4Henergy 4NH+●. These cationic radicals interact each other for radical-radical bond attack or oxidation is oxidation carried out by the initiator APS. If highAPS. concentrations of monomerofor initiator electrophile attack or carried out by the initiator If high concentrations formation. This process can be isrepeated many times to form final PPy [23,27]. Followingmonomer Pfluger’s areinitiator introduced during polymerization, the chance of the forming branched or cross-linked pyrroles would or aremajority introduced during polymerization, chance of C–2,5 forming branched or cross-linked study [25], the of the pyrrole units are linked through the carbons to form linear chains be also high and thus reduce the linear chain of PPy. pyrroles would be also high and thus reduce the linear chain of PPy. (α–α’ linkage). However, partial pyrroles can couple through the C–2,3 carbons (α–β’ linkages). If α– β’ linkage branches from the α–α’ linked main chains, it leads to side chain or inter-chain link formation (Figure 4c). Different from pyrroles in the α-α’ linked main chains, the hydrogen atoms on the β carbons of the branch pyrroles are changed to carbon atoms, which resulted in a disorder peak in a higher binding energy with larger width compared to α and β carbons. In this study, the electrophile attack or oxidation is carried out by the initiator APS. If high concentrations of monomer or initiator are introduced during polymerization, the chance of forming branched or cross-linked pyrroles would be also high and thus reduce the linear chain of PPy. (a) Figure 4. Cont. Figure 4. Cont.

(a) Figure 4. Cont.

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Oxidation

Ν

Ν +

α －

β

e-

●

Ν +

+

Ν

● － 2H+

or

α α Ν or

Ν +

Ν ●

Ν α β

(b) N

N

k

N

carbons in branched pyrroles

N

m

N N N

N

n

N

(c) Figure PPy films: (a)(a) thethe XPS analysis; (b) (b) schematic illustration of Figure4.4.The Thechemical chemicalcompositions compositionsofof PPy films: XPS analysis; schematic illustration the cationic radicals of pyrrole link may each other through the 2,5 position (α–α’ linkage) or the 2,3 of the cationic radicals of pyrrole link may each other through the 2,5 position (α–α’ linkage) or the position (α–β’ linkage); andand (c) the polymerization of of pyrrole is is mainly through 2,3 position (α–β’ linkage); (c) the polymerization pyrrole mainly throughthe theα–α’ α–α’linkage. linkage. However, if α–β’ linkage branches from the α–α’ linked main chains to form side chains or However, if α–β’ linkage branches from the α–α’ linked main chains to form side chains orinter-chain inter-chain links, links,the theC1s C1sof ofββcarbons carbonsin inbranched branchedpyrroles pyrrolesisisshifted shiftedto toform formaadisorder disorderpeak. peak.

Therefore, the area ratios of disorder peak to sum of three peaks are defined as disorder ratios Therefore, the area ratios of disorder peak to sum of three peaks are defined as disorder ratios to to quantify the extent of branching or crosslinking within PPy: quantify the extent of branching or crosslinking within PPy:

Area of CIII(disorder) Area of CIII(disorder) Disorder ratio “Area of CI(α) + Area of CII(β) + Area of CIII(disorder)

Disorder ratio =

Area of CI pαq ` Area of CII pβq ` Area of CIII(disorder)

(2) (2)

Figure 5a shows the disorder ratio as functions of the APS/Py ratio under different pyrrole Figure 5a shows the disorder ratio as functions of the APS/Py ratio under different pyrrole concentrations. As the APS/Py ratio and pyrrole concentration becomes higher, the branching or concentrations. As the APS/Py ratio and pyrrole concentration becomes higher, the branching or crosslinking also shifts to high scores. The more branches and crosslinks there are, the more possible crosslinking also shifts to high scores. The more branches and crosslinks there are, the more possible conducting paths for the electrons to move in the PPy film there will be. If we carefully correlated the conducting paths for the electrons to move in the PPy film there will be. If we carefully correlated electrical conductivity from Figure 3b with the disorder ratio, a linear function can be found in the the electrical conductivity from Figure 3b with the disorder ratio, a linear function can be found in Figure 5b. One possible 2D networking of PPy is schematically drawn in Figure 4c where the pi the Figure 5b. One possible 2D networking of PPy is schematically drawn in Figure 4c where the pi electrons can move or jump along many the inter-connection of pyrrole rings for conduction [24–26]. electrons can move or jump along many the inter-connection of pyrrole rings for conduction [24–26]. Similar effects of branching or crosslinking on conductivity have also been reported by Joo et al. Similar effects of branching or crosslinking on conductivity have also been reported by [28]. Different from our study, they varied the dopant systems for chemical or electrochemical PPy Joo et al. [28]. Different from our study, they varied the dopant systems for chemical or electrochemical synthesis. Their XPS results suggested that the levels of disorder of PPy are critical to PPy PPy synthesis. Their XPS results suggested that the levels of disorder of PPy are critical to PPy conductivity. PPy systems with lower interchain links or side chains resulted in weaker interchain conductivity. PPy systems with lower interchain links or side chains resulted in weaker interchain interaction, which lead PPy in an insulating state. To explain this phenomenon, Prigodin et al. have interaction, which lead PPy in an insulating state. To explain this phenomenon, Prigodin et al. have developed a random network model of metallic wires, which indicated the concentration of the developed a random network model of metallic wires, which indicated the concentration of the junctions, such as interchain linkage, highly impacts the insulator–metal transition in conducting junctions, such as interchain linkage, highly impacts the insulator–metal transition in conducting polymers [29,30]. polymers [29,30].

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of 11 11 88 of

12% 12%

Disorder Disorderratio ratio

11% 11% 10% 10% 0.1 M M Py Py 0.1 0.3 M M Py Py 0.3 0.5 M M Py Py 0.5

9% 9% 8% 8%

APS/Py 0.1 0.1 APS/Py APS/Py 0.2 0.2 APS/Py APS/Py 0.4 0.4 APS/Py

(a) (a)

Conductivity Conductivity(S/m) (S/m)

44

0.1 M M Py Py 0.1 0.3 M M Py Py 0.3 0.5 M M Py Py 0.5

33 22

2

R2 == 0.9057 0.9057 R

11 00

8% 8%

9% 9%

10% 11% 10% 11% Disorder ratio ratio Disorder

12% 12%

(b) (b)

Figure 5. 5. The The chemical compositions compositions of of PPy films: films: (a) (a) the branching ratio ratio calculated calculated for for different different Figure The chemical chemical compositions of PPy PPy (a) the the branching branching calculated APS/Py ratios ratios in PPy films; and (b) the branching ratio as function of of the the electrical electrical conductivity conductivity of of APS/Py APS/Py ratiosin inPPy PPyfilms; films; and and (b) (b) the the branching branching ratio ratio as as aa function PPy films. PPy films.

3.4. MTT Assay Assay for the the Biocompatibilities of of PPy Films Films 3.4. 3.4. MTT MTT Assay for for the Biocompatibilities Biocompatibilities of PPy PPy Films MTT assay in in Figure 6a 6a shows that that the average average viabilities of of rat BMSCs BMSCs on different different substrates. MTT MTT assay assay in Figure Figure 6a shows shows that the the average viabilities viabilities of rat rat BMSCs on on different substrates. substrates. Compared to cells on tissue culture polystyrene (TCPS), the PPy films yield lower viabilities but still still Compared the PPy yield lower Compared to to cells cells on on tissue tissue culture culture polystyrene polystyrene (TCPS), (TCPS), the PPy films films yield lower viabilities viabilities but but still capable of of maintaining maintaining it above above 70%. 70%. Increasing Increasing APS/Py APS/Py ratios ratios significantly significantly improved improved MTT MTT results results capable capable of maintaining ititabove 70%. Increasing APS/Py ratios significantly improved MTT results up up to 80%~90%. This enhancement may be attributed to the good electrical conductivity of underlying up to 80%~90%. This enhancement may be attributed to the good electrical conductivity of underlying to 80%~90%. This enhancement may be attributed to the good electrical conductivity of underlying PPy PPy films, which which can can directly directly promote promote the the cellular cellular activities. activities. On On the the other other hand, hand, the the overall overall PPy films,films, which can directly promote the cellular activities. On the other hand, the overall morphology of morphology of cells has no significant difference between cultured on TCPS and PPy films (Figure morphology of cells has no significant difference TCPS and PPy cells has no significant difference between culturedbetween on TCPScultured and PPyon films (Figure 6b). films These(Figure results 6b). These These results results suggested that that the the PPy PPy films films are are biocompatible. biocompatible. 6b). suggested that thesuggested PPy films are biocompatible.

(a) (a)

(b) (b)

Figure 6. 6. Biocompatibility Biocompatibility of PPy PPy films: (a) (a) MTT assay; assay; and (b) (b) cell morphologies morphologies of BMSCs BMSCs were Figure Figure 6. Biocompatibility of of PPy films: films: (a) MTT MTT assay; and and (b) cell cell morphologies of of BMSCs were were observed one day after being seeded on PPy films (Scale bar = 100 μm). observed being seeded seeded on on PPy PPy films films (Scale (Scale bar bar = = 100 observed one one day day after after being 100 μm). µm).

3.5. Osteogenesis Osteogenesis of of BMSCs BMSCs on on PPy PPy Films Films 3.5. 3.5. Osteogenesis of BMSCs on PPy Films Figure 7a 7a shows shows the the results results from from Ca-o-CPC Ca-o-CPC assay assay for for differentiated differentiated BMSCs BMSCs on on different different Figure Figure 7a shows the results from Ca-o-CPC assay for differentiated BMSCs on different substrates substrates at at different different times. times. Note Note that that there there was was no no detectable detectable calcium calcium in in all all samples samples until until Day Day 7. 7. substrates at different times. Note that there was no detectable calcium in allincreases samples whether until Dayon 7. Starting from Starting from Day 7, the calcium content in all sample groups TCPS or PPy Starting from Day 7, the calcium content in all sample groups increases whether on TCPS or PPy Day the 14 calcium content in all sample groups increases whether on TCPS or of PPy films. ratio Day 0.2 14 films.7,Day Day has the the most significant significant increase among all sample sample groups. Films APS/Py films. 14 has most increase among all groups. Films of APS/Py ratio 0.2 has most significant increase allthe sample groups. Films offaster APS/Py 0.2 andof 0.4TCPS had and the 0.4 had had differentiated differentiated BMSCsamong to boost boost deposition of calcium calcium thanratio the groups groups and 0.4 BMSCs to the deposition of faster than the of TCPS differentiated BMSCs to boost the deposition of calcium faster than the groups of TCPS and lower and lower lower APS/Py APS/Py ratio ratio 0.1. 0.1. In In particular, particular, the the films films made made of of 0.5 0.5 M M pyrrole pyrrole and and APS/Py APS/Py ratio ratio 0.4 0.4 have have and the most positive effects to promote the deposition of calcium into ECM, which almost tripled the the most positive effects to promote the deposition of calcium into ECM, which almost tripled the amount of of calcium calcium by by differentiated differentiated BMSCs BMSCs on on TCPS TCPS on on Day Day 14. 14. Another Another important important remark remark is is that that amount

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APS/Py ratio 0.1. In particular, the films made of 0.5 M pyrrole and APS/Py ratio 0.4 have the most positive2016, effects of Polymers 8, 258to promote the deposition of calcium into ECM, which almost tripled the amount 9 of 11 calcium by differentiated BMSCs on TCPS on Day 14. Another important remark is that the level of calcium its saturation point at around 28 at days. This 28 observation exemplifymay the the leveldeposition of calciumreached deposition reached its saturation point around days. Thismay observation required period for a full osteogenesis of BMSCs. exemplify the required period for a full osteogenesis of BMSCs.

(a)

(b)

(c)

(d)

Figure Figure 7. 7. The The calcium calcium deposition deposition of of differentiated differentiated BMSCs BMSCs on on PPy PPy films: films: (a) (a) the the calcium calcium measured measured by by the Ca-OCPC complex method; (b) the normalized calcium deposition in ECM using the surface cell the Ca-OCPC complex method; (b) the normalized calcium deposition in ECM using the surface cell number number determined determined by by the the LDH LDH assay; assay; (c) (c) alizarin alizarin red red SS staining staining shows shows the the level level of of mineralization mineralization on on Day 14 (Scale bar = 100 μm); and (d) the relation between normalized calcium deposition Day 14 (Scale bar = 100 µm); and (d) the relation between normalized calcium deposition and andelectrical electrical conductivity conductivity of of PPY PPY films. films.

The quantity of calcium deposition can be normalized by the average number of cells (LDH The quantity of calcium deposition can be normalized by the average number of cells (LDH assay) assay) on the surface, as shown in Figure 7b. Although it seems no new information is provided by on the surface, as shown in Figure 7b. Although it seems no new information is provided by Figure 7b, Figure 7b, as the plot presents a similar trend to Figure 7a, Figure 7b does clarify one possible doubt as the plot presents a similar trend to Figure 7a, Figure 7b does clarify one possible doubt that the that the increase of calcium deposition is not primarily due to the proliferation (high cell numbers) increase of calcium deposition is not primarily due to the proliferation (high cell numbers) but rather but rather largely to the function of mineralization. Since differentiated cells can die in a mature bone largely to the function of mineralization. Since differentiated cells can die in a mature bone matrix, the matrix, the culture until 28 days does not necessarily have a high number of cells. Thus, the culture until 28 days does not necessarily have a high number of cells. Thus, the significantly higher significantly higher calcium per unit area per cell on Day 28 evidently elucidates the suspicion. calcium per unit area per cell on Day 28 evidently elucidates the suspicion. Figure 7c presents the alizarin red S staining at Day 14. This is to confirm the results from the Figure 7c presents the alizarin red S staining at Day 14. This is to confirm the results from the Ca-o-CPC assay. Obviously, the control group of TCPS, and the group of 0.1 M Py and APS/Py 0.1 Ca-o-CPC assay. Obviously, the control group of TCPS, and the group of 0.1 M Py and APS/Py 0.1 have much less stained color than others. The higher pyrrole concentration and APS/Py ratio, the have much less stained color than others. The higher pyrrole concentration and APS/Py ratio, the denser of the color exhibits in the microscopic photos. These are all in accordance with the results in denser of the color exhibits in the microscopic photos. These are all in accordance with the results in Figure 7a. Figure 7a. The relation between normalized calcium concentration (Figure 7b) and electrical conductivity (Figure 3b) are plotted in Figure 7d where a linear regression fits the data fairly well ( R 2 = 0.9206 ). This shows a strong support for the claim mentioned earlier that the electroactive surfaces can improve the osteogenesis of differentiated BMSCs (osteoblasts). One possible explanation for this correlation is that surface charges on these electroactive films facilitate the protein adsorption to induce a cascade of intra-cellular reactions for cell differentiation [31,32].

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The relation between normalized calcium concentration (Figure 7b) and electrical conductivity (Figure 3b) are plotted in Figure 7d where a linear regression fits the data fairly well (R2 “ 0.9206). This shows a strong support for the claim mentioned earlier that the electroactive surfaces can improve the osteogenesis of differentiated BMSCs (osteoblasts). One possible explanation for this correlation is that surface charges on these electroactive films facilitate the protein adsorption to induce a cascade of intra-cellular reactions for cell differentiation [31,32]. 4. Conclusions In this study, biocompatible PPy films are fabricated by oxidative chemical polymerization using different pyrrole concentrations and initiators to regulate their conductivities. These fabricated PPy films are transparent and can be used easily for optical microscopy. For the fabrication part, XPS together with four point probe show that increasing either the higher pyrrole concentration or monomer/initiator ratio can increase the branching ratio in PPy chains, which in turn facilitates the crosslinking or networking among monomers. The high crosslinks and networks definitely enhance the overall electron mobility and electrical conductivity. The highest electrical conductivity of films measured in this study is around 3.628 S/m for films made of 0.5 M pyrrole and APS/Py ratio at 0.8. For the biological part, bone marrow stromal cells of rats were chosen to cultivate on highly conductive PPy films. The MTT assay confirms the biocompatibility of fabricated films with viability more than 80% for APS/Py ratios of 0.2 and 0.4 in the PPy films. The Ca-o-CPC assay further demonstrates that on differentiated BMSCs (osteoblasts) on PPy films had enhanced calcium deposition or mineralization in ECM and reached the saturation after 28-day culture. Lastly, a good linear correlation between the electrical conductivity and calcium deposition by differentiated BMSCs on PPy films supported the proposed hypothesis, i.e., the electroactive surfaces of electrically conductive PPy films can promote the osteogenesis of cells. Acknowledgments: This research was supported by the grant of MOST 104-2221-E-008-105- from the Ministry of Science and Technology of Taiwan and 105 CGH-NCU-A2 from the National Central University and Cathy General Hospital Joint Research Center. The authors thank Yu-Che Chen and Chih-Cheng Chien for technique support. Author Contributions: Wei-Wen Hu conceived and designed the experiments. Yi-Ting Hsu performed the experiments. Chuan Li analyzed the data and wrote the paper. All authors discussed the results, commented on, and approved the final manuscript. Conflicts of Interest: The founding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

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