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PVP-b-PCL block copolymers for enhanced cell biocompatibility
Accepted Manuscript Preparation of hydrophilic PCL nanofiber scaffolds via electrospinning of PCL/PVP-bPCL block copolymers for enhanced cell biocompatibility Sung Ju Cho, Sang Myung Jung, Munhyung Kang, Hwa Sung Shin, Ji Ho Youk PII:
S0032-3861(15)00479-6
DOI:
10.1016/j.polymer.2015.05.037
Reference:
JPOL 17875
To appear in:
Polymer
Received Date: 13 February 2015 Revised Date:
12 May 2015
Accepted Date: 19 May 2015
Please cite this article as: Cho SJ, Jung SM, Kang M, Shin HS, Youk JH, Preparation of hydrophilic PCL nanofiber scaffolds via electrospinning of PCL/PVP-b-PCL block copolymers for enhanced cell biocompatibility, Polymer (2015), doi: 10.1016/j.polymer.2015.05.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Hydrophilic PCL nanofiber scaffolds
PCL/PVP-b-PCL (90/10, w/w)
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PCL
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Preparation of hydrophilic PCL nanofiber scaffolds via electrospinning of PCL/PVP-b-PCL block copolymers for
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enhanced cell biocompatibility
Sung Ju Choa§, Sang Myung Jungb§, Munhyung Kanga, Hwa Sung Shinb, and Ji Ho
Department of Applied Organic Materials Engineering, Inha University, Incheon, 402-751,
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a
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Youka,c*
Republic of Korea b
c
Department of Biotechnology, Inha University, Incheon, 402-751, Republic of Korea
Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI
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48109, USA.
* Corresponding author. Tel.: +82 32 860 7498; Fax: +82 32 873 0181. E-mail addresses: [email protected] (J. H. Youk). §
These authors contributed equally to this work.
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ABSTRACT: The hydrophilicity of the extracellular matrix is one of the most important factors affecting
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cell adhesion in tissue engineering. In the present study, to improve the cellular biocompatibility of poly(ε-caprolactone) (PCL) nanofiber scaffolds,
their surface
hydrophilicity was enhanced by electrospinning PCL with biocompatible, amphiphilic
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poly(N-vinylpyrrolidone)-b-PCL (PVP-b-PCL) block copolymer. The PVP-b-PCL block copolymer (Mn = 26,300 g/mol, Mw/Mn = 1.14) was synthesized using 2-hydroxyethyl 2-
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(ethoxycarbonothioylthio)propanoate as a dual initiator for reversible addition-fragmentation chain transfer polymerization and ring opening polymerization in a one-pot procedure. As the content of PVP-b-PCL block copolymer increased, the surface of the PCL/PVP-b-PCL nanofiber scaffolds became more hydrophilic. The scaffolds showed no cytotoxicity, better
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cell adhesion, and improved viability of primary fibroblasts than PCL scaffolds, and did not lose their structure during cell culture. In particular, the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold produced the highest cell viability, suggesting that appropriate scaffold
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hydrophilicity is required to enhance cell activity.
ACCEPTED MANUSCRIPT 1. Introduction In tissue engineering, scaffolds serve as a template for cell adhesion, proliferation, and formation of the extracellular matrix (ECM) [1]. Nanofiber scaffolds fabricated by
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electrospinning of polymers have drawn considerable attention because they have a high surface-to-volume ratio and closely resemble the physical structure of the ECM. They can provide a good environment for cell attachment and proliferation [2,3]. Poly(ε-caprolactone)
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(PCL) is a common biodegradable polymer that has been electrospun to fabricate nanofiber scaffolds by many researchers because it has relatively good mechanical properties and can
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be processed easily, compared to other commonly used biodegradable scaffold materials, such as poly(lactide) (PLA) and poly(glycolic acid) (PGA) [4]. However, the intrinsic hydrophobicity of PCL strongly limits its scaffolding applications because the hydrophilicity of scaffold surfaces is very important for cellular behavior. Adherent cells regulate signals by
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focal adhesions to the ECM, which affect their migration and morphology [5]. Therefore, control of focal adhesion is a primary consideration in scaffold fabrication. To improve the hydrophilicity of PCL scaffolds, blend scaffolds consisting of PCL and other hydrophilic
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polymers have been developed. For example, poly(N-vinylpyrrolidone) (PVP)/PCL blend nanofiber scaffolds and poly(vinyl alcohol)/PCL nanofiber scaffolds electrospun by double
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spinnerets have been fabricated [6,7]. Recently, PLA/poly(ethylene glycol) (PEO) and PLA/PLA-b-PEO block copolymer solutions have been electrospun to produce PLA nanofibers with surface hydrophilicity; use of PLA-b-PEO block copolymers has been found to be more effective than addition of PEG homopolymer in tailoring surface hydrophilicity without losing the original nanofiber structure [8]. In the present study, to improve cell adhesion and proliferation on PCL nanofiber scaffolds by improving their hydrophilicity, PCL was electrospun with biocompatible, amphiphilic poly(N-vinylpyrrolidone)-b-PCL (PVP-b-PCL) block copolymer for the first time. PVP is a
ACCEPTED MANUSCRIPT well-known hydrophilic biocompatible polymer and has been extensively used in pharmaceutical industry [9-11]. It is expected that the hydrophilicity of PCL nanofiber scaffolds will be enhanced by phase separation of the PVP blocks into the nanofiber surface
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during the electrospinning process. The PCL blocks will prevent the PVP-b-PCL block copolymers from dissolving during cell culture. To determine the optimal hydrophilicity of PCL/PVP-b-PCL nanofiber scaffolds, in vitro cell adhesion and viability of primary
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fibroblasts were evaluated on PCL/PVP-b-PCL nanofiber scaffolds with various contents of
2. Experimental 2.1 Materials
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PVP-b-PCL block copolymers.
N-vinylpyrrolidone (VP, Aldrich, ≥99%) was passed through neutral alumina, dried over hydride,
distilled
under
reduced
pressure,
and
degassed
using
three
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calcium
freeze−pump−thaw cycles. ε-Caprolactone (CL, Aldrich, 99%) and anisole (Junsei, 98.0%) were dried over calcium hydride, distilled under reduced pressure, and degassed by three
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freeze-pump-thaw cycles. PCL (Mn = 80,000 g/mol, Aldrich), 2,2′-azobis(4-methoxy-2,4dimethylvaleronitrile) (V-70, Wako, 96%), diphenyl phosphate (DPP, Aldrich, 99%),
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tetrahydrofuran (THF, Duksan, 99.5%), dimethyl sulfoxide (DMSO, Aldrich, 99.5%), and N,N-dimethylformamide (DMF, OCI, 99.0%) were used as received, without further purification.
2-Hydroxyethyl
2-(ethoxycarbonothioylthio)propanoate
(HECP)
was
synthesized using a previously described method [12]. HECP was a viscous yellow oil (yield=57.5 %). [1H NMR (CDCl3, δ, ppm): 4.57 (q, 2H), 4.34 (q, 1H), 4.20 (t, 2H), 3.82-3.70 (m, 2H), 2.61-2.50 (broad peak, 1H, OH), 1.52 (d, 3H), 1.35 (t, 3H). 13C NMR (CDCl3, δ, ppm): 212.51, 171.63, 70.67, 67.45, 61.23, 47.37, 13.90] Cell culture supplements (Dulbecco’s modified Eagle
medium - High glucose (DMEM-H), fetal bovine serum (FBS), antibiotic-antimycotic, and
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2.2 Synthesis of PVP-b-PCL block copolymer
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The one-pot synthesis of PVP-b-PCL block copolymer was performed at a feed ratio of [CL]0/[VP]0/[HECP]0/[DPP]0/[V-70]0=200/200/1/2/0.4 as follows: CL (3.05 ml, 27 mmol), HECP (0.0328 g, 0.14 mmol), and DPP (0.0688 g, 27 mmol) were dissolved in 3 ml of
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anisole. The mixture was stirred at 30 °C for 6 h. Subsequently, a solution of V-70 (0.0170 g, 0.055 mmol) in VP (2.94 ml, 27 mmol) was injected into the reaction mixture and stirred at
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30 °C for 12 h. The resulting product was precipitated in an excess of diethyl ether, filtered, and dried under vacuum for 24 h. The conversion of the two monomers was 62%.
2.3 Electrospinning of PCL/PVP-b-PCL nanofiber scaffolds
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PCL and PVP-b-PCL block copolymer were dissolved in THF/DMF (1/1, v/v) at weight ratios of 100/0, 90/10, 85/15, 80/20, and 70/30, and their solution concentrations were adjusted to 11.0, 11.5, 12.3, 13.0, and 15.0%, respectively. The viscosities of the solutions
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were measured using a viscometer (Brookfield DV-E, Brookfield Engineering Laboratories, Inc.). The electrospinning setup used in this study consisted of a 5-ml syringe and a needle
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(ID = 0.85 mm), a syringe pump (model 100, KD Scientific Inc.), a rotating drum collector (d = 8.0 cm), and a high voltage supply (Chungpa EMT Co., Ltd.). A syringe pump connected to the syringe controlled the flow rate. All of the solutions were electrospun at room temperature at a positive voltage of 13 kV, a working distance of 13 cm (distance between the needle tip and the collecting plate), a rotating drum speed of 25 rpm, and a solution flow rate of 0.8 ml/h. The resulting PCL/PVP-b-PCL nanofiber scaffolds were dried at 30 °C for 24 h under vacuum to remove residual solvent.
ACCEPTED MANUSCRIPT 2.4 Fibroblast cell culture Fibroblasts were isolated from the skin of Sprague-Dawley rats. The rat skin was cut into 1 x 1 cm squares and treated with a 1 mg/ml dispase solution in HBSS at 4 °C for 24 h.
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Subsequently, the epidermis was removed and the remaining dermis was minced and incubated in a 1 mg/mL collagenase solution in HBSS at 4 °C for 24 h. The dermis was filtered with a 70-µm pore-size cell strainer and cultured in DMEM-H containing 10% FBS
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and 1% antibiotics-antimycotics. The fibroblasts obtained after 2 rounds of subculturing were used for all assays. The cytotoxicity, cell adhesion, and viability of the fibroblasts were
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evaluated using an MTT assay. MTT solution (0.5 mg/ml) was added to assay samples and incubated for 1 h. The MTT solution was then exchanged for DMSO at room temperature for 1 h. The optical density (OD) was measured at 540 nm using a Sunrise ELISA reader (Tecan, Austria). To test the cytotoxicity of the PCL/PVP-b-PCL nanofiber scaffolds, each was
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immersed in DMEM-H containing 1% antibiotics-antimycotics for 24 h to prepare extraction media. To prepare reference cells, fibroblasts were seeded in 24-well plates (SPL Life Sciences, Korea) at a concentration of 3.0 x 104 cells/well and incubated at 37 °C and 5%
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CO2 for 24 h to allow the cells to completely attach to the tissue culture plates. The nanofiber-extracted media were replaced with the culture media and then they were incubated
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for a further 24 h. Comparative data for cytotoxicity were obtained by performing an MTT assay. To test cell adhesion, fibroblasts (3.0 x 104 cells/well) were seeded on PCL/PVP-bPCL nanofiber scaffolds in 24-well plates and incubated at 37 °C. After 2, 8, and 24 h, the media were removed and non-adherent and loosely attached cells were removed by gently tapping the plates and washing the wells with phosphate buffered saline (PBS). The remaining cell activity was measured by performing an MTT assay, as described above. For the viability test, fibroblasts (1.0 x 104 cells/well) were seeded on PCL/PVP-b-PCL nanofiber
ACCEPTED MANUSCRIPT scaffolds in 24-well plates and incubated at 37 °C for 1, 4, and 7 days. To maintain the culture environment at a constant level, media were changed each day. After 1, 3, and 5 days, the media were removed and the wells were washed with PBS. Cell activity on the nanofiber
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scaffolds was measured by performing an MTT assay, as described above. To observe cell adhesion and morphology, fibroblasts inoculated using the method used for the viability test were cultured for 7 days and then fixed with 4% paraformaldehyde and dehydrated
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sequentially with 20%, 40%, 60%, 80%, 90%, and 100% ethanol.
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2.5 Statistical analysis
Proliferation and viability data were expressed in the bar graph as mean ± SD. Statistical analysis (Student’s t-test) was performed using SigmaPlot software and p-values are indicated
2.6 Characterization
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in given Figures.
The molecular weight (MW) and the MW distribution of PVP-b-PCL block copolymer were
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determined by gel permeation chromatography (GPC, Young Lin SP930D solvent delivery pump) coupled with an RI detector and two columns (GPC KD-806M x 2, Shodex). The
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eluent used was 0.01 M LiCl/DMF at a flow rate of 1.0 mL/min at 40 °C, and PMMA standards were used for calibration. 1H nuclear magnetic resonance (NMR) spectroscopy was performed using a Varian VXR-Unity NMR spectrometer (400 MHz) with CDCl3. The morphology and structure of the PCL/PVP-b-PCL nanofiber scaffolds were characterized by scanning electron microscopy (SEM, Hitachi S-4300) at an accelerating voltage of 15 kV. The average diameter of the electrospun nanofibers was determined by measuring the images of 20 fibers, which were randomly acquired from the SEM images. X-ray photoelectron spectroscopy (XPS) analysis was performed using a Thermo K-Alpha XPS system. A
ACCEPTED MANUSCRIPT monochromatized Al Kα X-ray source (E = 1361 eV) with a spot size of 400 µm operating at a power of 72 W was used. The contact angles of deionized water on PCL/PVP-b-PCL nanofiber scaffolds were measured at the self-produced contact angle stage, using a digital
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camera. For the water extraction test, a PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold was cut into a circular shape (φ = 19 mm), immersed in distilled water, and then placed in an oven set to 37 °C. After predetermined time intervals, the nanofiber scaffolds were taken out,
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washed with methanol, and then dried at 30 °C for 12 h under vacuum for further analysis. The morphology of the attached cells was observed using SEM (Hitachi S-4200). Before
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analysis, the cells were dehydrated and coated with Pt using a sputter coater. The cell dispersion inside scaffolds was observed using confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany). The cells on each scaffold were cultured for 4 days and treated
3. Results and discussion
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with DAPI to stain their nuclei.
3.1 Preparation of PCL/PVP-b-PCL nanofiber scaffolds
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In our previous study, a very convenient one-pot procedure for the synthesis of PVP-bPCL block copolymers at 30 °C was developed, using HECP as a dual initiator for reversible chain
transfer
(RAFT)
polymerization
and
ring
opening
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addition-fragmentation
polymerization (ROP) [13]. Fig. 1 shows the 1H NMR spectrum and the GPC trace of PVP-bPCL block copolymer. The number average MW of the PVP-b-PCL block copolymer was 26,300 g/mol and its polydispersity index was 1.14. The mole fractions of the PVP and PCL blocks in the PVP-b-PCL block copolymer were 0.57 and 0.43, respectively; these were determined by 1H NMR spectroscopy by comparing the integration ratio of the methylene peaks for PVP and PCL at 3.2 and 4.1 ppm, respectively. PCL/PVP-b-PCL nanofiber scaffolds were prepared by electrospinning of PCL/PVP-b-
ACCEPTED MANUSCRIPT PCL solutions in a mixed solvent of THF/DMF (1/1, v/v). Table 1 lists their electrospinning conditions and average fiber diameters. The solution concentrations of various PCL/PVP-bPCL blends were adjusted to almost identical viscosities, to produce nanofibers with similar
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fiber diameters. The average diameters of nanofibers are known to be strongly dependent on the viscosity of the electrospinning solution [14]. Fig. 2 shows SEM images of the PCL/PVPb-PCL nanofiber scaffolds obtained. The scaffolds were found to be well-fabricated, without
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any defects or beads, and with very similar average fiber diameters (Table 1). This result suggests that the effects of the morphology and average diameter of the PCL/PVP-b-PCL
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nanofiber scaffolds on cell adhesion and proliferation are negligible.
3.2 Surface hydrophilicity of PCL/PVP-b-PCL nanofiber scaffolds In order to evaluate the surface hydrophilicity of the PCL/PVP-b-PCL nanofiber scaffolds as
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a function of the content of PVP-b-PCL block copolymers, the water contact angles on the PCL/PVP-b-PCL nanofiber scaffolds were measured consecutively after 1 and 2 sec (Fig. 3). The PCL nanofiber scaffold showed the highest water contact angle. Interestingly, as the
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content of the PVP-b-PCL block copolymers increased, the water contact angles decreased, and the water droplets were rapidly absorbed by the scaffolds. To analyze the quantity of PVP
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blocks on the surface of the PCL/PVP-b-PCL nanofiber scaffolds, an XPS analysis was performed. Fig. 4 shows the XPS spectra of the PCL/PVP-b-PCL nanofiber scaffolds. The PCL nanofiber scaffold showed only C1s and O1s peaks, whereas as the content of PVP-bPCL block copolymers increased, a peak appeared at ~399.0 eV, attributed to N1s in the PVPb-PCL block copolymers, and increased proportionally. The quantities of PVP blocks on the surfaces of the PCL/PVP-b-PCL nanofiber scaffolds were compared by determining the area ratio of the N1s peak to the O1s peak. The area ratios of the PCL/PVP-b-PCL nanofiber scaffolds obtained by blend weight ratios of 90/10, 85/15, 80/20, and 70/30 were 0.0678,
ACCEPTED MANUSCRIPT 0.0774, 0.0906, and 0.1386, respectively. Thus, the more PVP-b-PCL block copolymers were added, the more PVP chains were phase-separated into the surface of the PCL/PVP-b-PCL nanofiber scaffolds during the electrospinning process. This result suggests that the PVP-b-
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PCL block copolymer affects the surface hydrophilicity of PCL/PVP-b-PCL nanofiber scaffolds. To confirm that PVP-b-PCL block copolymers would not be released into the aqueous phase during cell culture, an extraction test for PCL/PVP-b-PCL (70/30, w/w)
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nanofiber scaffolds was performed in water at 37 °C. To provide a stable hydrophilic surface for cell adhesion and proliferation, the PVP-b-PCL block copolymers should not be released
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into an in vitro cell culture environment. Fig. 5 shows SEM images of the PCL/PVP-b-PCL nanofiber scaffolds following 10 days of water extraction testing. Neither significant weight loss nor morphological changes were observed, suggesting that PVP-b-PCL block copolymers are not released into water, and that the surface hydrophilicity of PCL/PVP-b-
3.3 Cell Culture
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PCL nanofiber scaffolds is maintained during cell culture.
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Fig. 6a shows the cytotoxicity test results of the solutions released from PCL and PCL/PVPb-PCL nanofiber scaffolds. Cell activities in the solutions were determined by MTT assay.
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The MTT values of the PCL/PVP-b-PCL nanofiber scaffolds were higher than that of the PCL nanofiber scaffold, indicating that the PVP-b-PCL block copolymers showed no significant cytotoxicity. PCL is known to be non-cytotoxic. Primary fibroblasts were cultured on PCL/PVP-b-PCL nanofiber scaffolds. Fig. 6b shows fibroblast adhesion on various PCL/PVP-b-PCL nanofiber scaffolds at 2, 8, and 24 h after inoculation. Cell adhesion was evaluated until 24 h after inoculation because attached cells do not proceed with metabolism until completely attached to the scaffold surface [15,16]. Except for the PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold, all scaffolds showed increased cell adhesion at an early stage.
ACCEPTED MANUSCRIPT As the surface hydrophilicity of the PCL/PVP-b-PCL nanofiber scaffolds increased, the cells were better able to contact and attach to the scaffold surface, resulting in enhanced cell adhesion. The increased hydrophilicity allows some serum proteins, such as vitronectin and
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fibronectin, to aid in cell attachment and spreading on the scaffold surface [17]. However, the PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold did not show enhanced cell adhesion. It has been reported that when the surface of a scaffold is too hydrophilic, a thin layer of water can
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be formed, interfering with direct contact between the scaffold and cellular membranes, consequently preventing protein adhesion [17-20]. This result suggests that there is an
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optimal range for scaffold surface hydrophilicity. Fig. 7 shows SEM images of cells attached to PCL/PVP-b-PCL nanofiber scaffolds at 4 days after inoculation. The cells on the scaffolds spread their cytosol and filopodia along the direction of the nanofibers. This shows that fibroblasts on the scaffolds adhered well to the nanofibrous structure, even though differences
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were observed at an early stage [21,22]. However, cell viability during culture showed a different result. Fig. 8 shows the viability of rat primary fibroblasts on PCL/PVP-b-PCL nanofiber scaffolds. Cell viability was determined after 7 days of culture. PCL/PVP-b-PCL
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nanofiber scaffolds produced higher cell viability than PCL nanofiber scaffolds. The PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold produced the highest cell viability. The cell
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viability of adherent cells is known to be strongly dependent on cellular metabolism, which is activated by cell motility, ECM adsorption, proliferation, and cell density after complete adhesion [22]. The degree of hydrophilicity of the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffolds was found to be suitable for encouraging the metabolic activity and viability of fibroblasts [23-25]. Fig. 9 shows images of DAPI-stained fibroblasts on the PCL/PVP-b-PCL (90/10) nanofiber scaffold, which exhibited the highest cell viability. The cells grew and dispersed well on the scaffold. Cell density increased continuously. A high cell density promotes cell-to-cell interactions, and is the most important requirement for tissue formation
ACCEPTED MANUSCRIPT on scaffolds. Based on these results, the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold was found to provide a better environment for fibroblast growth than the PCL nanofiber
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scaffold, without loss of morphological integrity.
4. Conclusions
Amphiphilic PVP-b-PCL block copolymer (Mn = 26,300 g/mol, Mw/Mn = 1.14) was
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synthesized using HECP as a dual initiator for RAFT polymerization and ROP in a one-pot procedure. The mole fractions of the PVP and PCL blocks in the PVP-b-PCL block
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copolymer were 0.57 and 0.43, respectively. PCL/PVP-b-PCL nanofiber scaffolds were prepared by electrospinning of PCL/PVP-b-PCL solutions in a mixed solvent of THF/DMF (1/1, v/v). As the content of PVP-b-PCL block copolymers increased, the quantity of PVP blocks on the surface of PCL/PVP-b-PCL nanofiber scaffolds increased, and the surface
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consequently became more hydrophilic. PCL/PVP-b-PCL nanofiber scaffolds showed no significant weight loss or structural changes following 10 days of water extraction testing. No cytotoxicity was observed for PCL/PVP-b-PCL nanofiber scaffolds. These novel scaffolds
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showed higher fibroblast cell adhesion and viability than the PCL scaffold; in particular, the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold exhibited the highest cell viability. An
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optimally hydrophilic scaffold surface was observed to enhance cell activity.
Acknowledgments
This study was supported by INHA UNIVERSITY Research Grant, Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2006392), and NRF grant funded by the Korea government (MSIP) (NRF-2014R1A2A1A11052143).
ACCEPTED MANUSCRIPT References Chen GP, Ushida T, Tateishi T. Biomaterials 2001;18: 2563-7.
[2]
Deitzel JM, Kleinmeyer J, Harris D, Tan NCB. Polymer 2001;42:261-72.
[3]
Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. J Biomed Mater Res
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[1]
2002;60:613-21. [4]
Mei N, Chen G, Zhou P, Chen X, Shao ZZ, Pan LF, Wu CG. J Biomater Appl
SC
2005;19:323-39.
Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY. J Cell Biol 1996;134:793-9.
[6]
Kim GM, Le KH, Giannitelli SM, Lee YJ, Rainer A, Trombetta M. J Mater Sci Mater Med 2013;24:1425-42.
[7]
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[5]
Kim CH, Khil MS, Kim HY, Lee HU, Jahng KY. J Biomed Mater Res B 2006;78:28390.
Hendrick E, Frey M. J Eng Fiber Fabr 2014;9:153-64.
[9]
Forster A, Hempenstall J, Rades T. J Pharm Pharmacol 2001;53:303-15.
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[8]
[10] Kadajji VG, Betageri GV. Polymers 2011;3:1972-2009.
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[11] Mansour HM, Sohn MJ, Al-Ghananeem A, DeLuca PP. Int J Mol Sci 2010;11:3298-322. [12] Nicolaÿ R, Kwak Y, Matyjaszewski K. Chem Commun 2008:5336-8.
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[13] Kang HU, Yu YC, Shin SJ, Kim JS, Youk JH. Macromolecules 2013;46:1291-5. [14] Deitzel JM, Kleinmeyer J, Harris D, Tan NCB. Polymer 2001;42:261-72. [15] Sun X, Skorstengaard K, Mosher DF. J Cell Biol 1992;118:693-701. [16] Rebl H, Finke B, Schroeder K, Nebe JB. Int J Artif Organs 2010;33:738-48. [17] Wei J, Igarashi T, Okumori N, Igarashi T, Maetani T, Liu B, Yoshinari M. Biomed Mater 2009;4:045002. [18] Hu W, Crouch AS, Miller D, Aryal M, Luebke KJ. Nanotechnology 2010;21:385301. [19] Keefe AJ, Brault ND, Jiang S. Biomacromolecules 2012;13:1683-7.
ACCEPTED MANUSCRIPT [20] Patel P, Choi CK, Meng DD. J Lab Autom 2010;15:114-9. [21] Albuschies J, Vogel V. Sci Rep 2013;3:1658. [22] Hamilton DW, Oates CJ, Hasanzadeh A, Mittler S. PLoS One 2010;5:e15129.
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[23] Dowling DP, Miller IS, Ardhaoui M, Gallagher WM. J Biomater Appl 2011;26:327-47. [24] Valamehr B, Jonas SJ, Polleux J, Qiao R, Guo S, Gschweng EH, Stiles B, Kam K, Luo TJ, Witte ON, Liu X, Dunn B, Wu H. PNAS 2008;105:14459-64.
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[25] Geckeler K, Wacker R, Martini F, Hack A, Aicher W. Cell Physiol Biochem
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EP
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2003;13:155-64.
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Figure Captions Fig. 1. (a) 1H NMR spectrum and (b) GPC trace of the PVP-b-PCL block copolymer.
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Fig. 2. SEM images of PCL/PVP-b-PCL nanofiber scaffolds. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
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Fig. 3. Water contact angle measurement of PCL/PVP-b-PCL nanofiber scaffolds.
Fig. 4. XPS wide scan of PCL/PVP-b-PCL nanofibers. The PCL/PVP-b-PCL blend ratios
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were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
Fig. 5. SEM images of the PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold after water extraction for 10 days.
Fig. 6. (a) Cytotoxicity and (b) fibroblast adhesion on PCL/PVP-b-PCL nanofiber scaffolds at
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2, 8, and 24 h after inoculation. The asterisks denote significant differences compared with the 100/0 sample at corresponding time points (*** p < 0.001).
Fig. 7. SEM images of cells attached to PCL/PVP-b-PCL nanofiber scaffolds at 4 days after
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and (e) 70/30.
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inoculation. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20,
Fig. 8. Evaluation of cell viability using fibroblasts on PCL/PVP-b-PCL nanofiber scaffolds. The asterisks denote significant differences compared with the 100/0 sample at corresponding time points (* p < 0.05, ** p < 0.01, *** p < 0.001).
Fig. 9. Images of DAPI-stained fibroblasts on the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold at: (a) 1 day, (b) 4 days, and (c) 7 days.
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nanofiber scaffolds
concentration
(w/w)
(%)
Viscosity (cP)
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Solution
PCL/PVP-b-PCL
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Table 1. Electrospinning conditions and the average fiber diameters of PCL/PVP-b-PCL
Fiber diameter (nm)
11.0
161
476.7±31.7
90/10
11.5
148
407.8±20.4
85/15
12.3
141
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Fig. 1. (a) 1H NMR spectrum and (b) GPC trace of the PVP-b-PCL block copolymer.
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Fig. 2. SEM images of PCL/PVP-b-PCL nanofiber scaffolds. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
PCL/PVP-b-PCL (w/w)
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Fig. 3. Water contact angle measurement of PCL/PVP-b-PCL nanofiber scaffolds.
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Fig. 4. XPS wide scan of PCL/PVP-b-PCL nanofibers. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
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Fig. 5. SEM images of the PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold after water extraction for 10 days.
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Fig. 6. (a) Cytotoxicity and (b) fibroblast adhesion on PCL/PVP-b-PCL nanofiber scaffolds at 2, 8, and 24 h after inoculation. The asterisks denote significant differences compared with the 100/0 sample at corresponding time points (*** p < 0.001).
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Fig. 7. SEM images of cells attached to PCL/PVP-b-PCL nanofiber scaffolds at 4 days after inoculation. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
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Fig. 8. Evaluation of cell viability using fibroblasts on PCL/PVP-b-PCL nanofiber scaffolds. The asterisks denote significant differences compared with the 100/0 sample at corresponding
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Fig. 9. Images of DAPI-stained fibroblasts at the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold: (a) 1 day, (b) 4 days, and (c) 7 days.
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Highlights
•
PVP-b-PCL block copolymer was synthesized using HECP as a dual initiator for RAFT
•
The surface hydrophilicity of PCL nanofiber scaffolds was improved by incorporating PVP-b-PCL block copolymer.
•
PCL/PVP-b-PCL nanofiber scaffolds showed no cytotoxicity, better cell adhesion, and
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improved viability of primary fibroblasts than PCL scaffolds.
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The PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold produced the highest cell viability.
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polymerization and ROP in a one-pot procedure.
S0032-3861(15)00479-6
DOI:
10.1016/j.polymer.2015.05.037
Reference:
JPOL 17875
To appear in:
Polymer
Received Date: 13 February 2015 Revised Date:
12 May 2015
Accepted Date: 19 May 2015
Please cite this article as: Cho SJ, Jung SM, Kang M, Shin HS, Youk JH, Preparation of hydrophilic PCL nanofiber scaffolds via electrospinning of PCL/PVP-b-PCL block copolymers for enhanced cell biocompatibility, Polymer (2015), doi: 10.1016/j.polymer.2015.05.037. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Hydrophilic PCL nanofiber scaffolds
PCL/PVP-b-PCL (90/10, w/w)
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PCL
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Preparation of hydrophilic PCL nanofiber scaffolds via electrospinning of PCL/PVP-b-PCL block copolymers for
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enhanced cell biocompatibility
Sung Ju Choa§, Sang Myung Jungb§, Munhyung Kanga, Hwa Sung Shinb, and Ji Ho
Department of Applied Organic Materials Engineering, Inha University, Incheon, 402-751,
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a
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Youka,c*
Republic of Korea b
c
Department of Biotechnology, Inha University, Incheon, 402-751, Republic of Korea
Department of Materials Science and Engineering, University of Michigan, Ann Arbor, MI
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48109, USA.
* Corresponding author. Tel.: +82 32 860 7498; Fax: +82 32 873 0181. E-mail addresses: [email protected] (J. H. Youk). §
These authors contributed equally to this work.
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ABSTRACT: The hydrophilicity of the extracellular matrix is one of the most important factors affecting
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cell adhesion in tissue engineering. In the present study, to improve the cellular biocompatibility of poly(ε-caprolactone) (PCL) nanofiber scaffolds,
their surface
hydrophilicity was enhanced by electrospinning PCL with biocompatible, amphiphilic
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poly(N-vinylpyrrolidone)-b-PCL (PVP-b-PCL) block copolymer. The PVP-b-PCL block copolymer (Mn = 26,300 g/mol, Mw/Mn = 1.14) was synthesized using 2-hydroxyethyl 2-
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(ethoxycarbonothioylthio)propanoate as a dual initiator for reversible addition-fragmentation chain transfer polymerization and ring opening polymerization in a one-pot procedure. As the content of PVP-b-PCL block copolymer increased, the surface of the PCL/PVP-b-PCL nanofiber scaffolds became more hydrophilic. The scaffolds showed no cytotoxicity, better
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cell adhesion, and improved viability of primary fibroblasts than PCL scaffolds, and did not lose their structure during cell culture. In particular, the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold produced the highest cell viability, suggesting that appropriate scaffold
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hydrophilicity is required to enhance cell activity.
ACCEPTED MANUSCRIPT 1. Introduction In tissue engineering, scaffolds serve as a template for cell adhesion, proliferation, and formation of the extracellular matrix (ECM) [1]. Nanofiber scaffolds fabricated by
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electrospinning of polymers have drawn considerable attention because they have a high surface-to-volume ratio and closely resemble the physical structure of the ECM. They can provide a good environment for cell attachment and proliferation [2,3]. Poly(ε-caprolactone)
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(PCL) is a common biodegradable polymer that has been electrospun to fabricate nanofiber scaffolds by many researchers because it has relatively good mechanical properties and can
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be processed easily, compared to other commonly used biodegradable scaffold materials, such as poly(lactide) (PLA) and poly(glycolic acid) (PGA) [4]. However, the intrinsic hydrophobicity of PCL strongly limits its scaffolding applications because the hydrophilicity of scaffold surfaces is very important for cellular behavior. Adherent cells regulate signals by
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focal adhesions to the ECM, which affect their migration and morphology [5]. Therefore, control of focal adhesion is a primary consideration in scaffold fabrication. To improve the hydrophilicity of PCL scaffolds, blend scaffolds consisting of PCL and other hydrophilic
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polymers have been developed. For example, poly(N-vinylpyrrolidone) (PVP)/PCL blend nanofiber scaffolds and poly(vinyl alcohol)/PCL nanofiber scaffolds electrospun by double
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spinnerets have been fabricated [6,7]. Recently, PLA/poly(ethylene glycol) (PEO) and PLA/PLA-b-PEO block copolymer solutions have been electrospun to produce PLA nanofibers with surface hydrophilicity; use of PLA-b-PEO block copolymers has been found to be more effective than addition of PEG homopolymer in tailoring surface hydrophilicity without losing the original nanofiber structure [8]. In the present study, to improve cell adhesion and proliferation on PCL nanofiber scaffolds by improving their hydrophilicity, PCL was electrospun with biocompatible, amphiphilic poly(N-vinylpyrrolidone)-b-PCL (PVP-b-PCL) block copolymer for the first time. PVP is a
ACCEPTED MANUSCRIPT well-known hydrophilic biocompatible polymer and has been extensively used in pharmaceutical industry [9-11]. It is expected that the hydrophilicity of PCL nanofiber scaffolds will be enhanced by phase separation of the PVP blocks into the nanofiber surface
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during the electrospinning process. The PCL blocks will prevent the PVP-b-PCL block copolymers from dissolving during cell culture. To determine the optimal hydrophilicity of PCL/PVP-b-PCL nanofiber scaffolds, in vitro cell adhesion and viability of primary
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fibroblasts were evaluated on PCL/PVP-b-PCL nanofiber scaffolds with various contents of
2. Experimental 2.1 Materials
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PVP-b-PCL block copolymers.
N-vinylpyrrolidone (VP, Aldrich, ≥99%) was passed through neutral alumina, dried over hydride,
distilled
under
reduced
pressure,
and
degassed
using
three
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calcium
freeze−pump−thaw cycles. ε-Caprolactone (CL, Aldrich, 99%) and anisole (Junsei, 98.0%) were dried over calcium hydride, distilled under reduced pressure, and degassed by three
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freeze-pump-thaw cycles. PCL (Mn = 80,000 g/mol, Aldrich), 2,2′-azobis(4-methoxy-2,4dimethylvaleronitrile) (V-70, Wako, 96%), diphenyl phosphate (DPP, Aldrich, 99%),
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tetrahydrofuran (THF, Duksan, 99.5%), dimethyl sulfoxide (DMSO, Aldrich, 99.5%), and N,N-dimethylformamide (DMF, OCI, 99.0%) were used as received, without further purification.
2-Hydroxyethyl
2-(ethoxycarbonothioylthio)propanoate
(HECP)
was
synthesized using a previously described method [12]. HECP was a viscous yellow oil (yield=57.5 %). [1H NMR (CDCl3, δ, ppm): 4.57 (q, 2H), 4.34 (q, 1H), 4.20 (t, 2H), 3.82-3.70 (m, 2H), 2.61-2.50 (broad peak, 1H, OH), 1.52 (d, 3H), 1.35 (t, 3H). 13C NMR (CDCl3, δ, ppm): 212.51, 171.63, 70.67, 67.45, 61.23, 47.37, 13.90] Cell culture supplements (Dulbecco’s modified Eagle
medium - High glucose (DMEM-H), fetal bovine serum (FBS), antibiotic-antimycotic, and
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2.2 Synthesis of PVP-b-PCL block copolymer
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The one-pot synthesis of PVP-b-PCL block copolymer was performed at a feed ratio of [CL]0/[VP]0/[HECP]0/[DPP]0/[V-70]0=200/200/1/2/0.4 as follows: CL (3.05 ml, 27 mmol), HECP (0.0328 g, 0.14 mmol), and DPP (0.0688 g, 27 mmol) were dissolved in 3 ml of
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anisole. The mixture was stirred at 30 °C for 6 h. Subsequently, a solution of V-70 (0.0170 g, 0.055 mmol) in VP (2.94 ml, 27 mmol) was injected into the reaction mixture and stirred at
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30 °C for 12 h. The resulting product was precipitated in an excess of diethyl ether, filtered, and dried under vacuum for 24 h. The conversion of the two monomers was 62%.
2.3 Electrospinning of PCL/PVP-b-PCL nanofiber scaffolds
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PCL and PVP-b-PCL block copolymer were dissolved in THF/DMF (1/1, v/v) at weight ratios of 100/0, 90/10, 85/15, 80/20, and 70/30, and their solution concentrations were adjusted to 11.0, 11.5, 12.3, 13.0, and 15.0%, respectively. The viscosities of the solutions
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were measured using a viscometer (Brookfield DV-E, Brookfield Engineering Laboratories, Inc.). The electrospinning setup used in this study consisted of a 5-ml syringe and a needle
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(ID = 0.85 mm), a syringe pump (model 100, KD Scientific Inc.), a rotating drum collector (d = 8.0 cm), and a high voltage supply (Chungpa EMT Co., Ltd.). A syringe pump connected to the syringe controlled the flow rate. All of the solutions were electrospun at room temperature at a positive voltage of 13 kV, a working distance of 13 cm (distance between the needle tip and the collecting plate), a rotating drum speed of 25 rpm, and a solution flow rate of 0.8 ml/h. The resulting PCL/PVP-b-PCL nanofiber scaffolds were dried at 30 °C for 24 h under vacuum to remove residual solvent.
ACCEPTED MANUSCRIPT 2.4 Fibroblast cell culture Fibroblasts were isolated from the skin of Sprague-Dawley rats. The rat skin was cut into 1 x 1 cm squares and treated with a 1 mg/ml dispase solution in HBSS at 4 °C for 24 h.
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Subsequently, the epidermis was removed and the remaining dermis was minced and incubated in a 1 mg/mL collagenase solution in HBSS at 4 °C for 24 h. The dermis was filtered with a 70-µm pore-size cell strainer and cultured in DMEM-H containing 10% FBS
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and 1% antibiotics-antimycotics. The fibroblasts obtained after 2 rounds of subculturing were used for all assays. The cytotoxicity, cell adhesion, and viability of the fibroblasts were
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evaluated using an MTT assay. MTT solution (0.5 mg/ml) was added to assay samples and incubated for 1 h. The MTT solution was then exchanged for DMSO at room temperature for 1 h. The optical density (OD) was measured at 540 nm using a Sunrise ELISA reader (Tecan, Austria). To test the cytotoxicity of the PCL/PVP-b-PCL nanofiber scaffolds, each was
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immersed in DMEM-H containing 1% antibiotics-antimycotics for 24 h to prepare extraction media. To prepare reference cells, fibroblasts were seeded in 24-well plates (SPL Life Sciences, Korea) at a concentration of 3.0 x 104 cells/well and incubated at 37 °C and 5%
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CO2 for 24 h to allow the cells to completely attach to the tissue culture plates. The nanofiber-extracted media were replaced with the culture media and then they were incubated
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for a further 24 h. Comparative data for cytotoxicity were obtained by performing an MTT assay. To test cell adhesion, fibroblasts (3.0 x 104 cells/well) were seeded on PCL/PVP-bPCL nanofiber scaffolds in 24-well plates and incubated at 37 °C. After 2, 8, and 24 h, the media were removed and non-adherent and loosely attached cells were removed by gently tapping the plates and washing the wells with phosphate buffered saline (PBS). The remaining cell activity was measured by performing an MTT assay, as described above. For the viability test, fibroblasts (1.0 x 104 cells/well) were seeded on PCL/PVP-b-PCL nanofiber
ACCEPTED MANUSCRIPT scaffolds in 24-well plates and incubated at 37 °C for 1, 4, and 7 days. To maintain the culture environment at a constant level, media were changed each day. After 1, 3, and 5 days, the media were removed and the wells were washed with PBS. Cell activity on the nanofiber
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scaffolds was measured by performing an MTT assay, as described above. To observe cell adhesion and morphology, fibroblasts inoculated using the method used for the viability test were cultured for 7 days and then fixed with 4% paraformaldehyde and dehydrated
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2.5 Statistical analysis
Proliferation and viability data were expressed in the bar graph as mean ± SD. Statistical analysis (Student’s t-test) was performed using SigmaPlot software and p-values are indicated
2.6 Characterization
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The molecular weight (MW) and the MW distribution of PVP-b-PCL block copolymer were
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determined by gel permeation chromatography (GPC, Young Lin SP930D solvent delivery pump) coupled with an RI detector and two columns (GPC KD-806M x 2, Shodex). The
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eluent used was 0.01 M LiCl/DMF at a flow rate of 1.0 mL/min at 40 °C, and PMMA standards were used for calibration. 1H nuclear magnetic resonance (NMR) spectroscopy was performed using a Varian VXR-Unity NMR spectrometer (400 MHz) with CDCl3. The morphology and structure of the PCL/PVP-b-PCL nanofiber scaffolds were characterized by scanning electron microscopy (SEM, Hitachi S-4300) at an accelerating voltage of 15 kV. The average diameter of the electrospun nanofibers was determined by measuring the images of 20 fibers, which were randomly acquired from the SEM images. X-ray photoelectron spectroscopy (XPS) analysis was performed using a Thermo K-Alpha XPS system. A
ACCEPTED MANUSCRIPT monochromatized Al Kα X-ray source (E = 1361 eV) with a spot size of 400 µm operating at a power of 72 W was used. The contact angles of deionized water on PCL/PVP-b-PCL nanofiber scaffolds were measured at the self-produced contact angle stage, using a digital
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camera. For the water extraction test, a PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold was cut into a circular shape (φ = 19 mm), immersed in distilled water, and then placed in an oven set to 37 °C. After predetermined time intervals, the nanofiber scaffolds were taken out,
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washed with methanol, and then dried at 30 °C for 12 h under vacuum for further analysis. The morphology of the attached cells was observed using SEM (Hitachi S-4200). Before
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analysis, the cells were dehydrated and coated with Pt using a sputter coater. The cell dispersion inside scaffolds was observed using confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany). The cells on each scaffold were cultured for 4 days and treated
3. Results and discussion
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with DAPI to stain their nuclei.
3.1 Preparation of PCL/PVP-b-PCL nanofiber scaffolds
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In our previous study, a very convenient one-pot procedure for the synthesis of PVP-bPCL block copolymers at 30 °C was developed, using HECP as a dual initiator for reversible chain
transfer
(RAFT)
polymerization
and
ring
opening
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addition-fragmentation
polymerization (ROP) [13]. Fig. 1 shows the 1H NMR spectrum and the GPC trace of PVP-bPCL block copolymer. The number average MW of the PVP-b-PCL block copolymer was 26,300 g/mol and its polydispersity index was 1.14. The mole fractions of the PVP and PCL blocks in the PVP-b-PCL block copolymer were 0.57 and 0.43, respectively; these were determined by 1H NMR spectroscopy by comparing the integration ratio of the methylene peaks for PVP and PCL at 3.2 and 4.1 ppm, respectively. PCL/PVP-b-PCL nanofiber scaffolds were prepared by electrospinning of PCL/PVP-b-
ACCEPTED MANUSCRIPT PCL solutions in a mixed solvent of THF/DMF (1/1, v/v). Table 1 lists their electrospinning conditions and average fiber diameters. The solution concentrations of various PCL/PVP-bPCL blends were adjusted to almost identical viscosities, to produce nanofibers with similar
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fiber diameters. The average diameters of nanofibers are known to be strongly dependent on the viscosity of the electrospinning solution [14]. Fig. 2 shows SEM images of the PCL/PVPb-PCL nanofiber scaffolds obtained. The scaffolds were found to be well-fabricated, without
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any defects or beads, and with very similar average fiber diameters (Table 1). This result suggests that the effects of the morphology and average diameter of the PCL/PVP-b-PCL
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nanofiber scaffolds on cell adhesion and proliferation are negligible.
3.2 Surface hydrophilicity of PCL/PVP-b-PCL nanofiber scaffolds In order to evaluate the surface hydrophilicity of the PCL/PVP-b-PCL nanofiber scaffolds as
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a function of the content of PVP-b-PCL block copolymers, the water contact angles on the PCL/PVP-b-PCL nanofiber scaffolds were measured consecutively after 1 and 2 sec (Fig. 3). The PCL nanofiber scaffold showed the highest water contact angle. Interestingly, as the
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content of the PVP-b-PCL block copolymers increased, the water contact angles decreased, and the water droplets were rapidly absorbed by the scaffolds. To analyze the quantity of PVP
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blocks on the surface of the PCL/PVP-b-PCL nanofiber scaffolds, an XPS analysis was performed. Fig. 4 shows the XPS spectra of the PCL/PVP-b-PCL nanofiber scaffolds. The PCL nanofiber scaffold showed only C1s and O1s peaks, whereas as the content of PVP-bPCL block copolymers increased, a peak appeared at ~399.0 eV, attributed to N1s in the PVPb-PCL block copolymers, and increased proportionally. The quantities of PVP blocks on the surfaces of the PCL/PVP-b-PCL nanofiber scaffolds were compared by determining the area ratio of the N1s peak to the O1s peak. The area ratios of the PCL/PVP-b-PCL nanofiber scaffolds obtained by blend weight ratios of 90/10, 85/15, 80/20, and 70/30 were 0.0678,
ACCEPTED MANUSCRIPT 0.0774, 0.0906, and 0.1386, respectively. Thus, the more PVP-b-PCL block copolymers were added, the more PVP chains were phase-separated into the surface of the PCL/PVP-b-PCL nanofiber scaffolds during the electrospinning process. This result suggests that the PVP-b-
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PCL block copolymer affects the surface hydrophilicity of PCL/PVP-b-PCL nanofiber scaffolds. To confirm that PVP-b-PCL block copolymers would not be released into the aqueous phase during cell culture, an extraction test for PCL/PVP-b-PCL (70/30, w/w)
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nanofiber scaffolds was performed in water at 37 °C. To provide a stable hydrophilic surface for cell adhesion and proliferation, the PVP-b-PCL block copolymers should not be released
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into an in vitro cell culture environment. Fig. 5 shows SEM images of the PCL/PVP-b-PCL nanofiber scaffolds following 10 days of water extraction testing. Neither significant weight loss nor morphological changes were observed, suggesting that PVP-b-PCL block copolymers are not released into water, and that the surface hydrophilicity of PCL/PVP-b-
3.3 Cell Culture
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PCL nanofiber scaffolds is maintained during cell culture.
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Fig. 6a shows the cytotoxicity test results of the solutions released from PCL and PCL/PVPb-PCL nanofiber scaffolds. Cell activities in the solutions were determined by MTT assay.
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The MTT values of the PCL/PVP-b-PCL nanofiber scaffolds were higher than that of the PCL nanofiber scaffold, indicating that the PVP-b-PCL block copolymers showed no significant cytotoxicity. PCL is known to be non-cytotoxic. Primary fibroblasts were cultured on PCL/PVP-b-PCL nanofiber scaffolds. Fig. 6b shows fibroblast adhesion on various PCL/PVP-b-PCL nanofiber scaffolds at 2, 8, and 24 h after inoculation. Cell adhesion was evaluated until 24 h after inoculation because attached cells do not proceed with metabolism until completely attached to the scaffold surface [15,16]. Except for the PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold, all scaffolds showed increased cell adhesion at an early stage.
ACCEPTED MANUSCRIPT As the surface hydrophilicity of the PCL/PVP-b-PCL nanofiber scaffolds increased, the cells were better able to contact and attach to the scaffold surface, resulting in enhanced cell adhesion. The increased hydrophilicity allows some serum proteins, such as vitronectin and
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fibronectin, to aid in cell attachment and spreading on the scaffold surface [17]. However, the PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold did not show enhanced cell adhesion. It has been reported that when the surface of a scaffold is too hydrophilic, a thin layer of water can
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be formed, interfering with direct contact between the scaffold and cellular membranes, consequently preventing protein adhesion [17-20]. This result suggests that there is an
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optimal range for scaffold surface hydrophilicity. Fig. 7 shows SEM images of cells attached to PCL/PVP-b-PCL nanofiber scaffolds at 4 days after inoculation. The cells on the scaffolds spread their cytosol and filopodia along the direction of the nanofibers. This shows that fibroblasts on the scaffolds adhered well to the nanofibrous structure, even though differences
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were observed at an early stage [21,22]. However, cell viability during culture showed a different result. Fig. 8 shows the viability of rat primary fibroblasts on PCL/PVP-b-PCL nanofiber scaffolds. Cell viability was determined after 7 days of culture. PCL/PVP-b-PCL
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nanofiber scaffolds produced higher cell viability than PCL nanofiber scaffolds. The PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold produced the highest cell viability. The cell
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viability of adherent cells is known to be strongly dependent on cellular metabolism, which is activated by cell motility, ECM adsorption, proliferation, and cell density after complete adhesion [22]. The degree of hydrophilicity of the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffolds was found to be suitable for encouraging the metabolic activity and viability of fibroblasts [23-25]. Fig. 9 shows images of DAPI-stained fibroblasts on the PCL/PVP-b-PCL (90/10) nanofiber scaffold, which exhibited the highest cell viability. The cells grew and dispersed well on the scaffold. Cell density increased continuously. A high cell density promotes cell-to-cell interactions, and is the most important requirement for tissue formation
ACCEPTED MANUSCRIPT on scaffolds. Based on these results, the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold was found to provide a better environment for fibroblast growth than the PCL nanofiber
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scaffold, without loss of morphological integrity.
4. Conclusions
Amphiphilic PVP-b-PCL block copolymer (Mn = 26,300 g/mol, Mw/Mn = 1.14) was
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synthesized using HECP as a dual initiator for RAFT polymerization and ROP in a one-pot procedure. The mole fractions of the PVP and PCL blocks in the PVP-b-PCL block
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copolymer were 0.57 and 0.43, respectively. PCL/PVP-b-PCL nanofiber scaffolds were prepared by electrospinning of PCL/PVP-b-PCL solutions in a mixed solvent of THF/DMF (1/1, v/v). As the content of PVP-b-PCL block copolymers increased, the quantity of PVP blocks on the surface of PCL/PVP-b-PCL nanofiber scaffolds increased, and the surface
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consequently became more hydrophilic. PCL/PVP-b-PCL nanofiber scaffolds showed no significant weight loss or structural changes following 10 days of water extraction testing. No cytotoxicity was observed for PCL/PVP-b-PCL nanofiber scaffolds. These novel scaffolds
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showed higher fibroblast cell adhesion and viability than the PCL scaffold; in particular, the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold exhibited the highest cell viability. An
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optimally hydrophilic scaffold surface was observed to enhance cell activity.
Acknowledgments
This study was supported by INHA UNIVERSITY Research Grant, Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2013R1A1A2006392), and NRF grant funded by the Korea government (MSIP) (NRF-2014R1A2A1A11052143).
ACCEPTED MANUSCRIPT References Chen GP, Ushida T, Tateishi T. Biomaterials 2001;18: 2563-7.
[2]
Deitzel JM, Kleinmeyer J, Harris D, Tan NCB. Polymer 2001;42:261-72.
[3]
Li WJ, Laurencin CT, Caterson EJ, Tuan RS, Ko FK. J Biomed Mater Res
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[1]
2002;60:613-21. [4]
Mei N, Chen G, Zhou P, Chen X, Shao ZZ, Pan LF, Wu CG. J Biomater Appl
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2005;19:323-39.
Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY. J Cell Biol 1996;134:793-9.
[6]
Kim GM, Le KH, Giannitelli SM, Lee YJ, Rainer A, Trombetta M. J Mater Sci Mater Med 2013;24:1425-42.
[7]
M AN U
[5]
Kim CH, Khil MS, Kim HY, Lee HU, Jahng KY. J Biomed Mater Res B 2006;78:28390.
Hendrick E, Frey M. J Eng Fiber Fabr 2014;9:153-64.
[9]
Forster A, Hempenstall J, Rades T. J Pharm Pharmacol 2001;53:303-15.
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[8]
[10] Kadajji VG, Betageri GV. Polymers 2011;3:1972-2009.
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[11] Mansour HM, Sohn MJ, Al-Ghananeem A, DeLuca PP. Int J Mol Sci 2010;11:3298-322. [12] Nicolaÿ R, Kwak Y, Matyjaszewski K. Chem Commun 2008:5336-8.
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[13] Kang HU, Yu YC, Shin SJ, Kim JS, Youk JH. Macromolecules 2013;46:1291-5. [14] Deitzel JM, Kleinmeyer J, Harris D, Tan NCB. Polymer 2001;42:261-72. [15] Sun X, Skorstengaard K, Mosher DF. J Cell Biol 1992;118:693-701. [16] Rebl H, Finke B, Schroeder K, Nebe JB. Int J Artif Organs 2010;33:738-48. [17] Wei J, Igarashi T, Okumori N, Igarashi T, Maetani T, Liu B, Yoshinari M. Biomed Mater 2009;4:045002. [18] Hu W, Crouch AS, Miller D, Aryal M, Luebke KJ. Nanotechnology 2010;21:385301. [19] Keefe AJ, Brault ND, Jiang S. Biomacromolecules 2012;13:1683-7.
ACCEPTED MANUSCRIPT [20] Patel P, Choi CK, Meng DD. J Lab Autom 2010;15:114-9. [21] Albuschies J, Vogel V. Sci Rep 2013;3:1658. [22] Hamilton DW, Oates CJ, Hasanzadeh A, Mittler S. PLoS One 2010;5:e15129.
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[23] Dowling DP, Miller IS, Ardhaoui M, Gallagher WM. J Biomater Appl 2011;26:327-47. [24] Valamehr B, Jonas SJ, Polleux J, Qiao R, Guo S, Gschweng EH, Stiles B, Kam K, Luo TJ, Witte ON, Liu X, Dunn B, Wu H. PNAS 2008;105:14459-64.
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[25] Geckeler K, Wacker R, Martini F, Hack A, Aicher W. Cell Physiol Biochem
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2003;13:155-64.
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Figure Captions Fig. 1. (a) 1H NMR spectrum and (b) GPC trace of the PVP-b-PCL block copolymer.
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Fig. 2. SEM images of PCL/PVP-b-PCL nanofiber scaffolds. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
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Fig. 3. Water contact angle measurement of PCL/PVP-b-PCL nanofiber scaffolds.
Fig. 4. XPS wide scan of PCL/PVP-b-PCL nanofibers. The PCL/PVP-b-PCL blend ratios
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were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
Fig. 5. SEM images of the PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold after water extraction for 10 days.
Fig. 6. (a) Cytotoxicity and (b) fibroblast adhesion on PCL/PVP-b-PCL nanofiber scaffolds at
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2, 8, and 24 h after inoculation. The asterisks denote significant differences compared with the 100/0 sample at corresponding time points (*** p < 0.001).
Fig. 7. SEM images of cells attached to PCL/PVP-b-PCL nanofiber scaffolds at 4 days after
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and (e) 70/30.
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inoculation. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20,
Fig. 8. Evaluation of cell viability using fibroblasts on PCL/PVP-b-PCL nanofiber scaffolds. The asterisks denote significant differences compared with the 100/0 sample at corresponding time points (* p < 0.05, ** p < 0.01, *** p < 0.001).
Fig. 9. Images of DAPI-stained fibroblasts on the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold at: (a) 1 day, (b) 4 days, and (c) 7 days.
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nanofiber scaffolds
concentration
(w/w)
(%)
Viscosity (cP)
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Solution
PCL/PVP-b-PCL
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Table 1. Electrospinning conditions and the average fiber diameters of PCL/PVP-b-PCL
Fiber diameter (nm)
11.0
161
476.7±31.7
90/10
11.5
148
407.8±20.4
85/15
12.3
141
422.3±26.8
80/20
13.0
140
421.3±29.0
70/30
15.0
142
438.3±24.6
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100/0
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B S O
O
a b
S N
O
O
m
OO c d e
g g f
(a)
OH
h i
n
e, f
b, g
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h
10
d
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ac
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CDCl3
8
6
4
2
0
Chemical shift (ppm)
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(b)
16
18
20
22
Elution time (min)
Fig. 1. (a) 1H NMR spectrum and (b) GPC trace of the PVP-b-PCL block copolymer.
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(b)
20 μm
(d)
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(a)
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20 μm
Fig. 2. SEM images of PCL/PVP-b-PCL nanofiber scaffolds. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
PCL/PVP-b-PCL (w/w)
After 1 sec
After 2 sec
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90/10
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80/20
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85/15
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70/30
Fig. 3. Water contact angle measurement of PCL/PVP-b-PCL nanofiber scaffolds.
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C 1s
Intensity (a.u.)
(a)
C 1s
(b)
O 1s
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O 1s
N 1s
1200
900
600
300
1200
0
900
Binding energy (eV)
600
(d)
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N 1s
900
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Intensity (a.u.)
O 1s
1200
300
1200
0
900
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Binding energy (eV)
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C 1s
O 1s
N 1s
600
300
0
Binding energy (eV)
C 1s
(e)
1200
300
Binding energy (eV)
C 1s
(c)
600
O 1s
N 1s
900
600
300
0
Binding energy (eV)
Fig. 4. XPS wide scan of PCL/PVP-b-PCL nanofibers. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
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(a)
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5 μm
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Fig. 5. SEM images of the PCL/PVP-b-PCL (70/30, w/w) nanofiber scaffold after water extraction for 10 days.
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Fig. 6. (a) Cytotoxicity and (b) fibroblast adhesion on PCL/PVP-b-PCL nanofiber scaffolds at 2, 8, and 24 h after inoculation. The asterisks denote significant differences compared with the 100/0 sample at corresponding time points (*** p < 0.001).
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(b)
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(a)
18 μm
(d)
18 μm
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Fig. 7. SEM images of cells attached to PCL/PVP-b-PCL nanofiber scaffolds at 4 days after inoculation. The PCL/PVP-b-PCL blend ratios were: (a) 100/0, (b) 90/10, (c) 85/15, (d) 80/20, and (e) 70/30.
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Fig. 8. Evaluation of cell viability using fibroblasts on PCL/PVP-b-PCL nanofiber scaffolds. The asterisks denote significant differences compared with the 100/0 sample at corresponding
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time points (* p < 0.05, ** p < 0.01, *** p < 0.001).
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Fig. 9. Images of DAPI-stained fibroblasts at the PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold: (a) 1 day, (b) 4 days, and (c) 7 days.
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Highlights
•
PVP-b-PCL block copolymer was synthesized using HECP as a dual initiator for RAFT
•
The surface hydrophilicity of PCL nanofiber scaffolds was improved by incorporating PVP-b-PCL block copolymer.
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PCL/PVP-b-PCL nanofiber scaffolds showed no cytotoxicity, better cell adhesion, and
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improved viability of primary fibroblasts than PCL scaffolds.
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The PCL/PVP-b-PCL (90/10, w/w) nanofiber scaffold produced the highest cell viability.
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polymerization and ROP in a one-pot procedure.