PVDF electrospun scaffolds for wound healing applications

PVDF electrospun scaffolds for wound healing applications

Colloids and Surfaces B: Biointerfaces 96 (2012) 29–36 Contents lists available at SciVerse ScienceDirect Colloids and Surfaces B: Biointerfaces jou...

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Colloids and Surfaces B: Biointerfaces 96 (2012) 29–36

Contents lists available at SciVerse ScienceDirect

Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb

Piezoelectric PU/PVDF electrospun scaffolds for wound healing applications Hong-Feng Guo a , Zhen-Sheng Li b , Shi-Wu Dong a , Wei-Jun Chen a , Ling Deng b , Yu-Fei Wang a , Da-Jun Ying a,∗ a b

Department of Anatomy, Key Lab for Biomechanics of Chongqing, Third Military Medical University, Gao Tan Yan Street, Sha Ping Ba District, Chongqing 400038, China Department of Physics, Third Military Medical University, Gao Tan Yan Street, Sha Ping Ba District, Chongqing 400038, China

a r t i c l e

i n f o

Article history: Received 12 January 2012 Received in revised form 17 March 2012 Accepted 21 March 2012 Available online 29 March 2012 Keywords: Electrospinning Fibroblast Piezoelectric Polymer Scaffold Wound healing

a b s t r a c t Previous studies have shown that piezoelectric materials may be used to prepare bioactive electrically charged surfaces. In the current study, polyurethane/polyvinylidene fluoride (PU/PVDF) scaffolds were prepared by electrospinning. The mechanical property and piezoelectric property of the scaffolds were evaluated. The crystalline phase of PVDF in the scaffolds was characterised by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). In vitro cell culture was performed to investigate cytocompatibility of the scaffolds. Wound-healing assay, cell-adhesion assay, quantitative RT-PCR and Western blot analyses were performed to investigate piezoelectric effect of the scaffolds on fibroblast activities. Further, the scaffolds were subcutaneously implanted in Sprague-Dawley (SD) rats to investigate their biocompatibility and the piezoelectric effect on fibrosis in vivo. The results indicated that the electrospinning process had changed PVDF crystalline phase from the nonpiezoelectric ␣ phase to the piezoelectric ␤ phase. The fibroblasts cultured on the scaffolds showed normal morphology and proliferation. The fibroblasts cultured on the piezoelectric-excited scaffolds showed enhanced migration, adhesion and secretion. The scaffolds that were subcutaneously implanted in SD rats showed higher fibrosis level due to the piezoelectrical stimulation, which was caused by random animal movements followed by mechanical deformation of the scaffolds. The scaffolds are potential candidates for wound healing applications. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Wound healing is a process in which the skin (or other tissues) repairs itself after injury. The process can be accelerated and enhanced by the use of wound dressings. Thus far, different types of wound dressings have been developed based on the specific material, structure and drug that are favourable for wound healing [1–4]. However, wound dressings based on electrical stimulation have not been reported yet. Electrical stimulation influences cell behaviours such as proliferation, differentiation and regeneration. A number of previous studies have shown that piezoelectric materials that generate electrical charges in response to mechanical strain may be used to prepare bioactive electrically charged surfaces [5–9]. Polyvinylidene fluoride (PVDF) (Fig. 1) is a piezoelectric polymer that exists in at least three regular phases, depending on whether the chain conformations are trans (T) or gauche (G) linkages: ␣ (TG+TG−), ␤ (TTTT), ␥ (TTTG+TTTG−) phases, etc. [10]. Common free radical polymerisation-processed PVDF is typically in the ␣ phase, in which

∗ Corresponding author. Tel.: +86 23 68752226; fax: +86 23 68752226. E-mail address: [email protected] (D.-J. Ying). 0927-7765/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.colsurfb.2012.03.014

the chains stack with their respective polarisations in alternating directions resulting in nonpiezoelectric behaviour. To obtain the piezoelectric ␤ phase, the most polar phase in which the polymer chains are stacked with their respective polarisations aligned in the same direction, the ␣ phase PVDF needs to be mechanically stretched to orient the molecular chains and then poled (placed under a strong electric field to induce a net dipole moment) under tension [11]. Reports have indicated that electrospinning, which allows for solution processing under an applied electric field, combining solution casting and electric field poling into one step, is a simple technique to form the piezoelectric ␤ phase of PVDF directly from solution [12–15]. Electrospinning is an attractive approach for the fabrication of fibres with diameters ranging from a few nanometers to several micrometers by creating an electrically charged jet of polymer solution or melt. This technique has been recently introduced as the most promising technique to manufacture scaffolds for tissue engineering applications. The scaffolds could partially mimic the structure and function of natural extracellular matrices (ECM), thereby enhancing cell adhesion via (1) the interconnectivity of voids favourable for cell in-growth and (2) the high surface area to volume ratio, which enlarges cell-scaffold interface [16,17]. Thus far, a number of natural and synthetic polymers have

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Fig. 1. Chemical structures of PVDF and PU.

been electrospun into bioactive scaffolds for different applications [18–22]. A recent study has reported the preparation and in vitro cytocompatibility of poly(vinylidene fluoride-trifluoroethylene) (PVDF-TrFE) electrospun scaffolds and argued that there is tremendous potential of the scaffolds for tissue engineering applications [23]. However, the exact piezoelectric effect on the cultured cells was not investigated. In the current study, PU/PVDF scaffolds are prepared by electrospinning. Polyurethane (PU) (Fig. 1) is a thermoplastic elastomer that has been widely used as prostheses [24–26]. It is co-electrospun with PVDF because of the improved elasticity of the resulting scaffolds. The crystalline phase of PVDF in the scaffolds is characterised, and the piezoelectric effect on fibroblast activities in vitro and in vivo are thoroughly investigated with the aim of demonstrating the possible application for wound healing. 2. Materials and methods 2.1. Electrospinning of PU/PVDF scaffolds PVDF powder (Arkema, Kynar 761A, France) and PU grains (Great Eastern Resins Industrial Co., Ltd., TPU 1085AF, Taiwan) were dissolved in tetrahydrofuran/dimethylformamide (v/v = 1/1) at a concentration of 12% (w/v, g/ml), respectively. Before electrospinning, the two solutions were blended at different composition ratios ranging from 1/3 to 3/1 (v/v). The blended solution was transferred into a 10 ml glass syringe fitted with a blunt-ended 23G stainless steel needle. The syringe was connected to a metering pump (LongerPump, LSP02-1B, China) to maintain a constant flow rate of 0.8–1.0 ml/h. A positive high voltage power supply (BMEI Co., Ltd., DC High-Voltage Generator, China) was attached to the needle, and a voltage of 12–18 kV was applied between the needle tip and the stainless steel collecting plate (grounding) at a distance of 15–20 cm. The syringe was placed at a 30◦ angle, tilted downward from horizontal, and the needle was perpendicular to the collecting plate. The parameters (flow rate, applied voltage and collecting distance) were regulated to produce bead-free scaffolds. The electrospinning time was regulated to keep the thickness of the scaffolds to about 200 ␮m. 2.2. Scaffolds morphology, mechanical property and piezoelectric property

Capillary Flow Porometer, USA) according to the manufacturer’s instructions. The tensile strength and elongation at break of the scaffolds were tested using an electronic universal testing machine (REGER, RG T-5A, China). All samples were of the same size (1.0 cm × 3.0 cm). For the tensile test, ends of the specimens were mounted vertically on the machine with a 100-N load cell, leaving a 10 mm gauge length for mechanical loading. After three preloads, load-deformation data were recorded at a deforming speed of 20 mm/min. Stress–strain curves were constructed from the loaddeformation data. The tensile strength and elongation at break were calculated using a computer program in accordance with accepted formulas. The piezoelectric coefficient (d33 ) of the scaffolds was tested using Thin/Thick film Piezoelectric Analyzer (GiantForce Technology Co., Ltd., China) according to the manufacturer’s instructions. 2.3. Crystalline phase characterisation Samples were the PVDF powder, the PU/PVDF electrospun scaffolds and PU electrospun scaffolds. Characterisation of the PU scaffolds was performed to exclude possible interference of the PU component on the crystalline phase characterisation of PVDF in the blended scaffolds. Sample preparations were performed according to the instrument manufacturers’ instructions. X-ray diffraction (XRD) patterns were recorded on an X-ray diffractometer (BPGI Co., Ltd., XD2/3, China). The samples were irra˚ X-ray source diated using a monochromatised Cu K␣ (1.54056 A) with a step size (2) of 0.01◦ and a scan step time of 1.0 s. The operating voltage and current used were 36 kV and 30 mA, respectively, and the scanning range was 5–55◦ (2). Infrared spectra were obtained using a Fourier transform infrared spectrometer (FTIR, Nicolet, 5DXC FT-IR, USA) in the range of 500–900 cm−1 . The thermal analysis was conducted by differential scanning calorimetry (DSC) with a thermogravimetric analyser (Mettler Toledo, TGA/DSC 1, Switzerland) using a heating rate of 10 ◦ C/min under nitrogen atmosphere. 2.4. Cell culture The PU/PVDF scaffolds were cut into round pieces with diameters of 15 and 35 mm (fitted to the pore size of 24- and 6-well culture plates, respectively). The scaffolds were sterilised by soaking in 70% ethanol for 10 min, and then washed with PBS and soaked in PBS for at least 30 min. The latter step was repeated twice to ensure removal of the residual ethanol. Before cell inoculation, the scaffolds were further soaked in cell culture medium for 1 h. NIH 3T3 cells (mouse embryo fibroblasts) were obtained from American Type Culture Collection (ATCC). The cells were cultured in DMEM, High Glucose, Pyruvate (GIBCO, Gaithersburg, MD, USA) supplemented with 10% Super Neonatal Bovine Serum (NBS, Sijiqing, Hangzhou, China), 100 U/ml Penicillin G (Sangon, Shanghai, China) and 100 ␮g/ml streptomycin (Sangon, Shanghai, China). The cells were maintained at 37 ◦ C, 5% CO2 in 25 cm2 -cell culture flasks until confluence at the time of inoculation. The culture medium was refreshed every 2 days. 2.5. Cell morphology and proliferation on the scaffolds

The scaffold morphology was observed by scanning electron microscopy (SEM, Hitachi, S-3400N II, Japan). The scaffolds were sputter-coated with gold and viewed using an accelerating voltage of 15 kV and a working distance of 18–20 mm. The mean fibre diameter was measured from SEM images using Adobe Photoshop CS3 Software. A total of 100 randomly selected fibres were measured per sample. The mean pore diameter of the scaffold was determined by capillary flow analysis (Porous Materials, Inc., 1100 AEX

Cell morphologies on the PU/PVDF scaffolds were observed by laser scanning confocal microscopy (LSCM, Leica, TCS SP5, Germany) and SEM. Briefly, 5 × 104 cells were seeded on the scaffolds in 24-well culture plates. The adherent cells were fixed in 4% paraformaldehyde/PBS after culturing for 1, 3 and 5 days. For LSCM, fixed cells were stained with Actin-Tracker Green (Beyotime, Shanghai, China) and DAPI (Sigma, St. Louis, MO, USA). For SEM,

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the scaffolds were sputter-coated with gold and viewed using an accelerating voltage of 15 kV and a working distance of 18–20 mm. Cell proliferation on the PU/PVDF scaffolds was determined using cell counting kit-8 (CCK-8, Dojindo, Kumamoto, Japan). Briefly, 5 × 104 cells were seeded on the scaffolds in 24-well culture plates. After 1, 3, 5, and 7 days, the test was performed according to the manufacturer’s instructions. On a microplate reader (Thermo Scientific, Multiskan Spectrum, USA), absorbances at wavelengths of 450 and 600 nm were measured for each sample. Cells seeded directly on the bottom of the 24-well culture plates were used as controls. 2.6. Wound-healing assay The piezoelectric effect of the PU/PVDF scaffolds on fibroblast migration was investigated using wound-healing assay. To excite piezoelectricity, flexible bottomed culture plates (Flexcell, BF-3001U, USA) were used with the Flexercell tension plus system (Flexcell, FX-4000T, USA) that provided equibiaxial strain to cells in monolayer cultures. The tension system consisted of a computercontrolled vacuum unit and base plates that held the culture plates. The computer system controlled the frequency of deformation and the negative pressure applied to the culture plates. Before cell inoculation, the scaffolds were stuck to the silicone bottom of the 6-well culture plates with a 5% PLLA/Dioxane glue. Pressure applied to the silicone membrane was then transmitted to the scaffolds, thereby exciting their piezoelectricity. After that, 5 × 105 DiI (Beyotime, Shanghai, China)-stained cells were seeded on the scaffolds and cultured to confluence. The scaffolds were subjected to intermittent deformation of 4%, 8% and 12% at a frequency of 0.5 Hz or 1 Hz for 24 h. Scratch wounds were made using a 20 ␮l pipette tip. The cells were cultured in low serum medium (1% NBS) at 37 ◦ C, 5% CO2 . The scratched areas were observed at 0, 6, 12 and 24 h, respectively. Control cells were cultured on the nonpiezoelectric-excited scaffolds (0% deformation) and piezoelectric-excited PU scaffolds (same deformation, frequency and time). The results were analysed by measuring the confluence of the cells that migrated to the scratched area using Image-Pro Plus 6.0 Software. Ten randomly selected fields of the scratched area were measured per sample. 2.7. Cell-adhesion assay The piezoelectric effect of the PU/PVDF scaffolds on fibroblast adhesion was analysed by cell-adhesion assay. The preparations for piezoelectric excitation were performed as described in Section 2.6. After that, 5 × 105 cells were seeded on the PU/PVDF scaffolds and cultured to confluence. The scaffolds were then subjected to an intermittent deformation of 8% at a frequency of 0.5 Hz for 24 h. Then, 5 × 105 Hoechst 33342 (Beyotime, Shanghai, China) stained cells were seeded on the scaffolds and incubated at 37 ◦ C, 5% CO2 for 1 h. The 6-well culture plates were vortexed on an orbital shaker (Kylin-Bell, TS-1, China) at a speed of 120 rpm for 5 min. The stained cells that adhered to cell layers on the scaffolds were observed by LSCM. The results were analysed by counting the nuclei of the adherent cells using Image-Pro Plus 6.0 Software. Ten randomly selected fields were measured per sample. 2.8. Quantitative RT-PCR and Western blot analyses The preparations for piezoelectric excitation were performed as described in Section 2.6. After that, 5 × 105 cells were seeded on the PU/PVDF scaffolds and cultured to confluence. The scaffolds were then subjected to an intermittent deformation of 8% at a frequency of 0.5 Hz for 24 h. The mRNA expression levels of collagen type I (Colla1), elastin (Eln) and fibronectin 1 (Fn1) were analysed by quantitative RT-PCR using GoTaq 2-Step RT-qPCR

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System (Promega, Madison, WI, USA) on ABI PRISM 7000 Sequence Detection System (Applied Biosystems, USA) according to the manufacturers’ instructions. Briefly, the total RNA was isolated using TRIZOL Reagent (Invitrogen, Carlsbad, CA, USA). Reverse transcription of the total RNA was performed with Oligo (dT)15 Primer, Random Primer and GoScript Reverse Transcriptase at 42 ◦ C for 1 h. After denaturing at 70 ◦ C for 15 min, a PCR amplification reaction was conducted for 40 cycles at 95 ◦ C for 15 s and 60 ◦ C for 1 min. The mRNA levels of the target genes were normalised to those of the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The forward and reverse primers used were available in supplement Table 1. All primer sets were designed using Primer Premier 5.0 Software. The protein expression levels of the target genes were analysed by Western blotting. Briefly, cells were lysed and the proteins were separated by electrophoresis and transferred onto PVDF membranes. The membranes were incubated with Rabbit Anti-Collagen I (Bioss, Beijing, China), Rabbit Anti-Elastin/ELN (Bioss, Beijing, China), Rabbit Anti-FN (Bioss, Beijing, China) and Anti-Beta Actin (control) (Bioss, Beijing, China), followed by incubation with Goat Anti-rabbit IgG/HRP (Bioss, Beijing, China). The target proteins were detected with SuperSignal West Femto Maximum Sensitivity Substrate (Pierce, Rockford, IL, USA). The luminescence bands were scanned using Gel Doc 2000 and Chemi Doc Systems (Bio-Rad, USA). The protein levels of the target genes were normalised to that of the housekeeping gene ␤ actin. The relative intensities of the bands were quantified by Quantity One 4.4.0 Software. 2.9. Subcutaneous implantation of the scaffolds Seventeen male Sprague-Dawley (SD) rats weighing 250–300 g were obtained from Third Military Medical University animal facility. The rats were anaesthetised with a combination of 5% chloral hydrate and 5% sodium bromide at 0.7 ml/100 g intraperitoneally and were placed in prone and supine positions. The operative regions (vertex, abdomen and back) were shaved, scrubbed with a povidone-iodine solution and wiped clean with 70% ethanol. Two 1-cm subcutaneous incisions were made on both sides of the vertex median line, followed by implantation of the PU/PVDF scaffolds and the PU scaffolds (0.5 cm × 1.0 cm). Two 3-cm subcutaneous incisions were made on both sides of the ventral median line and dorsal median line, followed by implantation of the PU/PVDF scaffolds and the PU scaffolds (1.0 cm × 3.0 cm). The experimental protocol adhered to the rules of the Animal Welfare Act (CFR 9) and was approved by the Institutional Animal Care and Use Committee (IACUC). 2.10. Characterisation of the explanted scaffolds Explantations were performed 14 days after implantation. The scaffolds were carefully peeled off without any visible subcutaneous tissue. The scaffolds were fixed, dehydrated, embedded in paraffin, sliced with a microtome (Leica, RM2235, Germany) and stained with haematoxylin and eosin (H&E). The cells on the scaffolds were characterised by immunofluorescence staining and flow cytometry. For immunofluorescence staining, the fixed scaffolds were incubated with Rabbit Anti-FSP (fibroblast surface protein, Bioss, Beijing, China) followed by incubation with Cy3-labeled Goat Anti-Rabbit IgG (H+L) (Beyotime, Shanghai, China). The scaffolds were then observed by LSCM. For flow cytometry (FCM), the explanted scaffolds were cut into 0.25 cm × 0.25 cm pieces. The pieces were digested with collagenase type I (GIBCO, Gaithersburg, MD, USA) and the detached cells were incubated with Rabbit Anti-FSP/Spastin/Cy3 (Bioss, Beijing, China) and Rabbit IgG/Cy3 (control) (Beyotime, Shanghai, China),

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Fig. 2. In vitro cytocompatibility of the PU/PVDF scaffolds. (A) SEM and LSCM images of the NIH3T3 cells on the scaffolds after culturing for 1, 3 and 5 days (scale bar 100 ␮m). (B) Proliferation of the NIH3T3 cells cultured on the scaffolds and TCPS. Data are expressed as means ± SD. *p < 0.05, significantly different from control group.

Fig. 3. In vitro piezoelectric effect of the PU/PVDF scaffolds on fibroblast migration. (A) Scratched areas of the NIH3T3 cell layers on the piezoelectric-excited PU/PVDF scaffolds and the control scaffolds at different times (scale bar 200 ␮m). (B) Average wound-healing speeds of the NIH3T3 cells on the piezoelectric-excited PU/PVDF scaffolds and the control scaffolds. Data are expressed as means ± SD. *p < 0.05, significantly different from control group.

respectively. The cells were analysed using a flow cytometer (BD, FACScalibur, USA).

blot analyses was performed using one-way analysis of variance (ANOVA). Probability values (p) of <0.05 are considered to indicate statistically significant differences. The results are expressed as means ± standard deviation (SD).

2.11. Statistics

3. Results and discussion

Each experiment was repeated at least three times. Statistical analysis of the data for the cell proliferation assay was performed using independent samples t-test, while that of the data for the mechanical testing, piezoelectric testing, wound-healing assay, cell adhesion assay, quantitative RT-PCR and Western

3.1. Scaffolds morphology, mechanical property and piezoelectric property Increased voltage and other parameters that lead to smallerdiameter fibres are keys for obtaining greater ␤-phase fraction

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Table 1 Mechanical property of PU/PVDF scaffolds with different composition ratios. Data are expressed as means ± SD. Data for each group is significantly different from that for the adjacent groups.

Tensile strength (MPa) Elongation at break (%) Piezoelectric coefficient (d33 ) (pC/N)

PU

PU/PVDF (3/1)

PU/PVDF (2/1)

PU/PVDF (1/1)

PU/PVDF (1/2)

PU/PVDF (1/3)

PVDF

9.632 ± 0.927 188.71 ± 22.40 0.16 ± 0.06

7.433 ± 1.106 156.09 ± 31.72 8.26 ± 1.44

6.860 ± 0.976 123.78 ± 46.56 11.24 ± 1.21

5.984 ± 1.249 107.94 ± 25.80 13.96 ± 1.05

5.562 ± 0.884 88.40 ± 26.41 18.06 ± 2.02

4.107 ± 1.364 94.75 ± 20.00 20.82 ± 2.70

4.016 ± 0.732 76.47 ± 36.46 24.90 ± 2.88

[27]. However, inappropriate parameters result in beads that dramatically influence the morphology and mechanical property of the fibres [28]. To obtain fibres with greater ␤-phase fractions while avoiding bead formation, we used relatively high voltage and deliberately adjusted the flow rate as well as the collecting distance. The parameters for bead-free scaffolds produced in the PU/PVDF solutions were as follows: flow rate 0.8 ml/h, applied voltage 15 kV, collecting distance 20 cm, ambient temperature 20–25 ◦ C and humidity 40–50%. PU exhibited improved mechanical property compared with PVDF. Increasing the ratio of PVDF in the blended scaffolds increased the piezoelectric coefficient (d33 ) but decreased the tensile strength and elongation at break (Table 1). To keep the balance between mechanical property and piezoelectric property, the PU/PVDF (1/1) scaffolds were used for the following experiments. The mean fibre diameter and pore size of the scaffolds were 1.41 ± 0.32 ␮m and 11.47 ± 1.14 ␮m, respectively (supplement Fig. S1). 3.2. Crystalline phase characterisation XRD revealed that the main reflection peak of the PVDF powder was at 2 = 19.9◦ (␣1 1 0), and three other peaks occurred at 17.7◦ (␣1 0 0), 18.4◦ (␣0 2 0) and 26.4◦ (␣0 2 1). The main peak

shifted slightly to 20.6◦ (␤1 1 0 and ␤2 0 0) for the PU/PVDF scaffolds whereas the other three peaks became invisible. Moreover, there was a distinct peak at 36.5◦ (␤0 2 0) [29]. The PU scaffolds showed a single reflection peak at 19.7◦ (supplement Fig. S2). Infrared spectra of the PVDF powder had characteristic absorption bands at 535 cm−1 (CF2 bending), 618 cm−1 (CF2 bending and skeletal bending), 764 cm−1 (CF2 bending and skeletal bending) and 796 cm−1 (CH2 rocking), which indicated ␣ phase formation [30,31]. These ␣-phase characteristic bands were much less intense for the PU/PVDF scaffolds, whereas the characteristic absorption bands at 510 cm−1 (CF2 bending) and 841 cm−1 (CH2 rocking) were observed, which indicated ␤ phase formation [30,31]. The PU scaffolds showed no obvious absorption bands in the range of 500–900 cm−1 (supplement Fig. S3). Thermograms of the PVDF powder and the PU/PVDF scaffolds showed different endothermic peaks. The lower (171.2 ◦ C) and higher (175.2 ◦ C) temperature peaks were attributed to the ␣ and ␤ phases, respectively. The PU scaffolds showed no endothermic peaks in the scanning temperature range (supplement Fig. S4). The crystalline phase of PVDF in the PU/PVDF scaffolds was different from that in the PVDF powder, indicating that the electrospinning process had altered the PVDF crystalline phase. The XRD and FTIR analyses confirmed the crystalline phase transformation of PVDF from the ␣ phase to the ␤ phase. However, this

Fig. 4. In vitro piezoelectric effect of the PU/PVDF scaffolds on fibroblast adhesion. (A) Hoechst 33342-stained NIH3T3 cells adhered to the NIH3T3 cell layers on the piezoelectric-excited PU/PVDF scaffolds and the control scaffolds (scale bar 100 ␮m). (B) Amounts of the cells that adhered to the cell layers on the piezoelectric-excited PU/PVDF scaffolds and the control scaffolds. Data are expressed as means ± SD. *p < 0.05, significantly different from control group.

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conclusion was controversial according to the DSC results. Some previous studies showed that the ␤ phase had a higher temperature endothermic peak compared with the ␣ phase [27,29,30,32], whereas other reports held the opposite view [11,33]. In the current study, we attributed the higher temperature peak to the ␤ phase, because its cell density is the highest amongst other phases. The all-trans planar zigzag conformation of the ␤ phase provides more packing and higher melting-temperature crystals [34]. 3.3. In vitro cytocompatibility Cytocompatibility studies demonstrated that NIH3T3 cells grew and proliferated normally on the PU/PVDF scaffolds. SEM and LSCM revealed the morphology of the cells on the scaffolds. Cells exhibited an elongated and spread-out morphology after culturing for 1 day. Further, the extracellular matrix was secreted, and the cells proliferated and intermingled with each other to form a continuous cell layer. After culturing for 5 days, the cell layers had almost covered the scaffolds (Fig. 2A). The amount of the cells on the scaffolds was comparable to that of the cells on the tissue culture polystyrene (TCPS) after culturing for 3 days (Fig. 2B). 3.4. In vitro piezoelectric effect on fibroblast activities The wound-healing assay indicated that piezoelectricity enhanced migration of the NIH3T3 cells (Fig. 3A). The intermittent deformation of 8% at 0.5 Hz was the most effective piezoelectric excitation parameter of those tested. The cells on the piezoelectricexcited PU/PVDF scaffolds exhibited more rapid wound healing than those on the control scaffolds. At 24 h after scratching, confluence of the cells that had migrated to the scratched area reached 100% for the piezoelectric-excited PU/PVDF scaffolds whereas those for the control scaffolds were approximately 50%. In a 24-h period, the average wound-healing speed of the cells on piezoelectricexcited PU/PVDF scaffolds was approximately twice as those of the cells on the control scaffolds (Fig. 3B). The cell-adhesion assay revealed that piezoelectricity enhanced adhesion of the NIH3T3 cells (Fig. 4A). The number of cells that adhered to the cell layers on the piezoelectric-excited PU/PVDF scaffolds was approximately 1.6-fold above those of the cells adhered to the cell layers on the control scaffolds (Fig. 4B). Quantitative RT-PCR and Western blot analyses indicated that piezoelectricity enhanced secretion of the NIH3T3 cells. Three extracellular matrix proteins were detected. The cells on the piezoelectric-excited PU/PVDF scaffolds showed higher mRNA and protein expression levels of the target genes than those on the control scaffolds (Fig. 5A and B). In order to confirm the piezoelectric effect of the PU/PVDF scaffolds on fibroblast activities, nonpiezoelectric-excited PU/PVDF scaffolds and piezoelectric-excited PU scaffolds were used as controls, respectively. The results indicated that the enhanced migration, adhesion and secretion were due to the piezoelectric effect rather than the scaffolds composition or the piezoelectric excitation process. The enhanced cell activities were the expressions of fibroblast response to the piezoelectrical stimulation, which was caused by piezoelectric excitation of the PU/PVDF scaffolds. However, the mechanisms by which the piezoelectricity influenced the fibroblast gene expression were unknown. We speculated that the piezoelectricity may alter the permeability of the cell membranes to Na+ or Ca2+ , and thus generate action potentials. The internal cellular environment was then influenced by variations in the intracellular sodium and potassium channels, which was followed by changes in cell status. Identifying the exact underlying mechanisms deserves further consideration. It is notable that

Fig. 5. In vitro piezoelectric effect of the PU/PVDF scaffolds on fibroblast secretion. (A) Colla1, Eln and Fn1 mRNA levels of the NIH3T3 cells on the piezoelectric-excited PU/PVDF scaffolds and the control scaffolds. (B) Colla1, Eln and Fn1 protein levels of the NIH3T3 cells on the piezoelectric-excited PU/PVDF scaffolds and the control scaffolds. Data are expressed as means ± SD. *p < 0.05, significantly different from control group.

the NIH3T3 cells cultured on the nonpiezoelectric-excited PU/PVDF scaffolds and the piezoelectric-excited PU scaffolds showed no significant differences in fibroblast activities (statistical data not shown), which seems to be contradictory to the knowledge that the tensile mechanical strain could also increase the fibroblast activities. The possible reason for this phenomenon is that the strain was attenuated by the PLLA glue layer and the stuck scaffold, rather than directly loaded on the cell layer. 3.5. In vivo biocompatibility Both PU and PVDF are nondegradable polymers. During the in vivo tests, no behaviour changes or visible signs of physical impairment that indicated systemic or neurological toxicity were observed. No gross physical damage, shrinking or stretching was observed in any of the implanted scaffolds. H&E staining indicated that there were a number of cells on the scaffolds (Fig. 6). Immunofluorescence staining revealed that the cells on the PU/PVDF scaffolds explanted from the back of the SD rats were

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Fig. 6. H&E staining of the PU/PVDF scaffolds and the PU scaffolds explanted from the vertex, abdomen and back of the SD rats (scale bar 50 ␮m).

mainly fibroblasts (supplement Fig. S5), and flow cytometry indicated that the ratio was 80.25 ± 4.59%. Characterisation of the cells on the PU/PVDF scaffolds explanted from the vertex and abdomen of the SD rats and those on the PU scaffolds explanted from these regions showed similar results (data not shown). Foreign-body inflammation with minimal chronic inflammation and no apparent neutrophilic or lymphoplasmacytic response was found in the scaffolds.

3.6. In vivo piezoelectric effect on fibroblast activities To evaluate the piezoelectric effect of the PU/PVDF scaffolds on fibroblasts in vivo, the scaffolds were subcutaneously implanted in different regions of the SD rats together with the control PU scaffolds. The scaffolds implanted in these regions were subjected to different degrees of bending deformation. The vertex is regarded as a region with rich blood supply that helps to accelerate wound

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healing (fibrosis). This role was confirmed by the current study, in which fibrosis levels of PU scaffolds explanted from the vertex were higher than those of the PU scaffolds explanted from the abdomen and back. However, fibrosis levels of the PU/PVDF scaffolds explanted from the abdomen and back were comparable to those of the PU/PVDF scaffolds explanted from the vertex (Fig. 6). Because of the support of cranium, deformation of the scaffolds implanted in the vertex was much less frequent than that of the scaffolds implanted in the abdomen and back. Thus, we believed that fibrosis levels of the PU/PVDF scaffolds implanted in the abdomen and back were increased as a result of piezoelectrical stimulation, which was caused by random animal movements followed by mechanical deformation of the scaffolds. Besides, fibrosis levels of the PU/PVDF scaffolds explanted from the vertex, abdomen and back of the SD rats were higher than those of the PU scaffolds explanted from the same regions (Fig. 6). Since the two kinds of scaffolds were implanted symmetrically and should have the same degree of bending deformation, we confirmed our argument that the piezoelectricity played an important role in the fibrosis of PU/PVDF scaffolds. 4. Conclusion PU/PVDF blended scaffolds of different composition ratios were prepared by electrospinning. The PU/PVDF (1/1) scaffolds were chosen to keep the balance between mechanical property and piezoelectric property. Crystalline phase characterisation indicated that the PVDF in the scaffolds was mainly in the piezoelectric ␤ phase. The scaffolds were biocompatible, and the piezoelectricity caused by mechanical deformation of the scaffolds could enhance fibroblast activities in vitro and in vivo. The scaffolds are potential candidates for wound healing applications. Acknowledgement This work was supported by the National Science Foundation of China (No: 30672535 and 81000670). Appendix A. Supplementary data Supplementary data associated with cle can be found, in the online http://dx.doi.org/10.1016/j.colsurfb.2012.03.014.

this artiversion, at

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