Electrospinning Homogeneous Nanofibrous Poly(propylene

May 13, 2014 - (1, 2) In the tissue engineering field, a highly porous biodegradable ...... S.; Ikada , Y. Enhanced vascularization and tissue granula...
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Electrospinning Homogeneous Nanofibrous Poly(propylene carbonate)/Gelatin Composite Scaffolds for Tissue Engineering Xin Jing,†,‡,⊥ Max R. Salick,‡,§ Travis Cordie,‡,∥ Hao-Yang Mi,†,‡,⊥ Xiang-Fang Peng,*,† and Lih-Sheng Turng*,‡,⊥ †

National Engineering Research Center of Novel Equipment for Polymer Processing, South China University of Technology, Guangzhou, 510640, China ‡ Wisconsin Institute for Discovery, §Department of Engineering Physics, ∥Department of Biomedical Engineering, ⊥Department of Mechanical Engineering, University of Wisconsin−Madison, Madison, Wisconsin 53706, United States ABSTRACT: Nanofibrous poly(propylene carbonate)(PPC)/gelatin composite scaffolds were fabricated by the electrospinning process and characterized for tissue engineering applications. Using trifluoroethanol (TFE) as a solvent and at low mass content of gelatin, nanofibrous scaffold PPC/gelatin membranes with improved wettability and hydrophilicity were produced, despite the fact that PPC is hydrophobic. However, phase separation occurred when the mass content of gelatin was higher than 5%, resulting in a nonuniform fibrous structure and some large splash defects. Inspired by the effect of pH on the behavior of dissolved gelatin, a small amount of acetic acid was added to the PPC/gelatin solution, which enabled the originally turbid solution to become clear immediately. The results showed that the miscibility of PPC and gelatin was enhanced by acetic acid. Using the treated solution, homogeneous PPC/gelatin nanofibers with a smooth surface and a more uniform diameter were obtained, and they performed better in wettability and mechanical tests. Cell culture experiments showed that fibroblast cells had more favorable interactions on the PPC/gelatin composite scaffolds than those of pure PPC membranes. Furthermore, the introduction of acetic acid did not hinder cell growth and proliferation on the modified membrane. The results of this study suggest the potential use of electrospun PPC/gelatin composite scaffolds in the tissue engineering field.

1. INTRODUCTION Tissue engineering is an interdisciplinary field that applies the principles of biology and engineering to the development of functional substitutes as scaffolds with or without living precursor cells as medical therapies for damaged tissues.1,2 In the tissue engineering field, a highly porous biodegradable scaffold is essential to accommodate mammalian cells and guide their growth in three dimensions. The ideal scaffold should have the following characteristics: (1) appropriate levels and sizes of porosity allowing for cell migration and the transport of nutrients and metabolic waste, (2) a suitable surface area and surface chemistry that encourages cell adhesion, growth, migration, and differentiation, and (3) a degradation rate that closely matches the regeneration rate of the desired natural tissue in vivo.2,3 Electrospinning, which allows for the production of polymer fibers with diameters varying from 3 nm to greater than 5 μm1, has been effectively used to generate fibrous scaffolds for tissue engineering purposes.2,4,5 Various synthetic biodegradable and natural polymers have been electrospun into thin fibers to generate fibrous scaffolds, such as polylactide (PLA),6 PLA/chitosan blends,7 poly(epsiloncaprolactone) (PCL),8 poly(lactideco-glycolide) (PLGA),9,10 and polyglycolic acid (PGA).11 Poly(propylene carbonate) (PPC), an aliphatic polycarbonate composed of propylene epoxide and carbon dioxide (CO2), was first synthesized in the late 1960s.12 PPC is amorphous with a glass transition temperature similar in range to human body temperature (35−40 °C), making it highly transparent under these conditions.13 With certain catalysts, it can degrade into propylene and CO2.14 Because of these properties, along © 2014 American Chemical Society

with its good mechanical properties, PPC has been evaluated as an engineering construct for tissue engineering applications.13,15−17 Kim et al. reported that PPC had good biocompatibility and biodegradability in vivo.18 Zhang et al. seeded rat smooth muscle cells (SMCs) on electrospun PPC grafts and the seeded cells integrated into the microstructure of the scaffold to form a three-dimensional cellular network, which indicated that synthetic PPC scaffolds may be good candidates for vascular tissue engineering.16 However, PPC is hydrophobic by nature14 and electrospinning makes scaffolds even more hydrophobic due to the high surface-to-volume ratio of the fibrous porous scaffold produced.19 It is well-known that hydrophilicity of the scaffolds affects protein absorption and hence subsequent cell adhesion on the scaffolds. According to our experiments, the surface water contact angle of electrospun PPC scaffolds was about 110°, which may limit its application as a tissue engineering scaffold because cells may not attach and proliferate well on matrices having a wettability higher than 90°.20−22 Gelatin is a natural biopolymer derived from partial hydrolysis of native collagen. Because of its biological origin, biocompatibility, biodegradability, and commercial availability at relatively low cost, gelatin has been widely used in the pharmaceutical and medical fields.23−26 Furthermore, it has been reported that gelatin contains many integrin binding sites Received: Revised: Accepted: Published: 9391

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room temperature for 2 h, the tensile tests were performed on an Instron 5565 universal testing machine using a 250 N load cell with a crosshead speed of 5 mm/min. At least four samples were tested for each type of electrospun fibrous membrane. The ultimate tensile strength, Young’s modulus, and elongation-at-break were obtained from the stress−strain curves. 2.4. Biocompatibility Characterization. 2.4.1. Cell Culture Prior to Seeding. Swiss mouse NIH 3T3 ECACC (European Collection of Cell Cultures) fibroblasts were used for the biological assays. Cells were cultured in high-glucose DMEM (Invitrogen), supplemented with 20% fetal bovine serum (WiCell), 1 unit/mL penicillin (Invitrogen), 1 μg/mL streptomycin (Invitrogen), and 2 mM L-glutamine (Invitrogen), and maintained on 6-well tissue culture-treated polystyrene (TCPS) plates (BD Falcon) prior to testing. The medium was replaced every 2 days and cells were passaged with ethylene diaminetetraacetic acid (EDTA) at a 1:40 ratio every 6 days during regular maintenance. 2.4.2. Cell Seeding. The fibrous scaffolds were exposed to sterilizing UV light for 30 min on each side and placed into 24well TCPS plates before cell seeding. 3T3 cells were dissociated with ethylene diaminetetraacetic acid (EDTA) for 5 min prior to seeding. The cells were then seeded onto the surface of the sterilized scaffolds at a density of 25 000 cells/well in the highglucose 3T3 medium. The medium was replaced daily for screening samples at 1 mL per day. 2.4.3. Live/Dead Assay. To confirm the viability of cells cultured on the chosen scaffolds, cells were stained with a Live/ Dead Viability/Cytotoxicity kit (Invitrogen). Cell viability was determined 3 days and 10 days after seeding. This kit provided an assay to simultaneously visualize both live and dead cells. The stain utilized green fluorescent Calcein-AM to target esterase activity within the cytoplasm of living cells, and red fluorescent ethidium homodimer-1 (EthD-1) to indicate cell death by penetrating damaged cellular membranes. Blue fluorescent DAPI stain was additionally applied as a nuclear marker. A Nikon A1RsiTi-E confocal microscope was used to image the cells. 2.4.4. Cell Fixing for SEM. The same specimens used in cell viability testing were later imaged using SEM. Samples were rinsed twice with Hanks’ balanced salt solution (HBSS; Thermo Scientific). Paraformaldehyde was diluted with Hy Clone Hy Pure molecular biology grade water (Thermo Scientific) to make a 4% solution. The rinsed samples were then immersed in the solution for 30 min. After that, the samples were dehydrated using a series of ethanol washes (50%, 80%, 90%, and 100% ethanol for 30 min each), and finally the dehydrated samples were dried in a vacuum desiccator for 2 h before gold sputtering for SEM. 2.4.5. Cell Proliferation. The whole process was performed as previously described.27 The Cell Titer 96 Aqueous One Solution Cell Proliferation Assay (Promega) was used to determine the number of cells during culture on the nanofibrous scaffolds. This assay utilized a tetrazolium compound that was bioreduced upon entering a cell. The resulting products predictably altered the absorbance of the media in which the cells resided. Standard curves were established and confirmed by comparison with hemocytometer readings prior to the experiments. Upon testing, cells were treated with an 83% media, 17% MTS solution and allowed to incubate for exactly 1 h. After incubation, 100 μL of spent media were removed and added to a clear 96-well plate. The

for cell adhesion, migration, and differentiation, which are found in natural collagen and other extracellular matrix proteins.11 In this study, gelatin was added to the PPC solution to enhance the hydrophilicity and biocompatibility of the scaffolds. The PPC/gelatin solutions were electrospun with different weight ratios of gelatin to develop electrospun PPC/ gelatin fibrous scaffolds. The properties of PPC/gelatin nanofibrous scaffolds were investigated by Fourier transform infrared spectroscopy (FTIR), SEM, water contact angle, and tensile tests. The viability and proliferation of 3T3 fibroblast cells on the scaffolds were also studied to understand the effects of gelatin incorporation on potential tissue engineering applications.

2. EXPERIMETAL DETAILS 2.1. Materials. PPC (QPAC 40) was purchased from Empower Materials, Inc. Gelatin (type A from porcine skin), acetic acid, and 2,2,2-trifluoroethanol (TFE) were purchased from Sigma-Aldrich. 2.2. Fabrication of Nanofiber Scaffolds. Polymer solutions with a concentration of 10 wt % were prepared by dissolving PPC and gelatin by weight ratios of 100:0, 98:2, 95:5, 90:10, and 85:15 in TFE and stirring for 12 h at room temperature. The solution was loaded into a 6 mL syringe connected to an 18 gauge blunt needle by Teflon tubing. The desired flow rate was 0.5 mL/h using a syringe pump and the applied voltage was kept at 18 kV. A grounded aluminum foil, 15 cm away from the needle tip, was used as collector. In addition, round stainless washers (inner diameter of 8.33 mm) were used to collect the fibers for cytocompatibility evaluation. The collected electrospun scaffolds were put in the vacuum oven for 3 days to help the trapped solvent inside the fibers evaporate completely. 2.3. Characterization of PPC/Gelatin Scaffolds. 2.3.1. Morphological Characterization. The morphologies of the scaffolds were observed by scanning electron microscopy (SEM) (LEO GEMINI 1530) with an accelerating voltage of 3 kV. Prior to SEM imaging, the specimens were sputtered with gold for 40 s. The diameters of the fibers were measured from the SEM images using the Image-Pro Plus analysis software. The average and standard deviation of the fiber diameter were calculated from 50 random measurements using three SEM images. 2.3.2. Fourier Transform Infrared Spectroscopy (FTIR). Chemical analysis of PPC and PPC/gelatin nanofibrous scaffolds were performed using a Bruker Tensor 27 FTIR instrument. The samples were analyzed in absorbance mode in the range of 600 to 4000 cm−1 with a resolution of 4 cm−1. 2.3.3. Determination of Water Contact Angle (WCA). The surface water contact angles (WCA) of various scaffolds were tested using the sessile drop method in air at room temperature by a video contact angle instrument (Dataphysics OCA 15). The droplet size was set as 2 μL. Three samples for each scaffold were tested and the average value was reported with standard deviation (±SD). 2.3.4. Tensile Properties under Dry and Wet Conditions. For electrospun membranes, it is difficult to prepare the standard specimens for tensile tests. Therefore, we cut them into the same dimension, 15 × 10 mm2 (length × width), for better comparison among the specimens. The thickness of the membranes used was about 400 μm. After immersing the specimens into phosphate-buffered saline (PBS, pH = 7.4) at 9392

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Figure 1. SEM micrographs of PPC and PPC-GT2 electrospun fibers: (a and b) PPC; (c and d) PPC-GT2. Scale bars of the images and the insets are 50 and 10 μm, respectively.

fiber diameter decreased from 1134 ± 195 nm for pure PPC to 858 ± 176 nm for PPC-GT5. However, in PPC-GT10, the average fiber diameter increased with a huge variation. As for PPC-GT15, the considerable amount of splash made it difficult to acquire the average fiber diameter. 3.2. Phase Separation Behavior of Electrospinning PPC/Gelatin Solution. Figure 4 shows images of electrospun PPC/gelatin (gelatin content: 15%) after various collection times. The electrospinning time was recorded starting when the solution was taken from the glass bottle where it was undergoing magnetic stirring. After 5 min of electrospinning, the fibers were fairly uniform and smooth (Figure 4a). Bonded and thicker fibers began to appear when the electrospinning time lasted 10 min (Figure 4b). The number of bonded and thicker fibers increased with increased electrospinning time (Figure 4c,d). When the electrospinning continued for 2 h, the fibrous structure became coarser and some large splash defects appeared (Figure 4e). These observations suggest the existence of phase separation and its profound effects on fiber morphology. 3.2.1. Phase Separation Mediated by Acetic Acid. With the use of TFE as a solvent, either PPC or gelatin could be dissolved to form a transparent solution. However, when mixed together, the mixed solution becomes turbid, and gradually separates slightly into different phases after being left to stand for 6 h. To circumvent this problem, a very small amount of acetic acid (HAc) (e.g., 20 μL) was introduced to the PPCGT15 solution (named PPC-GT15 HAc), and visual inspection showed that the turbid solution turned transparent immediately. The HAc content was tried at 5, 10, 20, and 30 μL. It was found that the higher was the volume of HAc added, the higher was the transmittance of the PPC-GT15 solution, which may give rise to better miscibility between PPC and gelatin. When

absorbance of this plate at the 450 nm wavelength was then read with a GloMax-Multi+ Multiplate Reader (Promega), and the subsequent number of cells was determined. 2.4.6. Statistical Analysis. All of the quantitative results were expressed as mean ± standard deviation (SD). Statistical analyses were carried out by means of a one-way analysis of variance (ANOVA). A p-value less than 0.05 was considered statistically significant.

3. RESULTS AND DISCUSSION 3.1. Morphology of Nanofibrous Scaffolds. Figure 1 shows SEM micrographs of fibers electrospun from pure PPC and the PPC/2% gelatin (PPC-GT2) blends. As seen in Figure 1c, when the gelatin content was 2%, the fiber size decreased and the fibers were fairly uniform. In the Figure 2 panels a and b, the average fiber size was further decreased in the PPC/5% gelatin (PPC-GT5) sample, with the fiber diameter mainly ranging from 400 to 1000 nm. In the PPC-GT10 sample (shown in Figure 2 panels c and e), the fibers became nonuniform, with the presence of both large and small fibers. Furthermore, when the gelatin content reached 15% (shown in Figure 2d), instead of the coexistence of large fibers and tiny fibers, bonded fibers and large splash defects appeared. It is believed that the phase separation occurred during the electrospinning process as a result of weak molecular interactions in the PPC/gelatin solution (due to the higher content of gelatin), consequently leading to gradually deteriorated fiber morphologies such as splash, fiber bonding, and varied fiber sizes. This phenomenon was also observed in electrospun collagen/polycaprolactone (PCL) hybrids,28 gelatin/PCL,29 and collagen/PLCL.30 Figure 3 shows the average fiber diameter of PPC/gelatin electrospun nanofibers. As can be seen in Figure 3, the average 9393

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Figure 2. SEM images of PPC/gelatin electrospun fibers: (a) PPC-GT5, (b) histogram of size distribution of panel a, (c) PPC-GT10, (d) PPCGT15, and (e) histogram of size distribution of panel c.

to improved fiber quality in production and uniform mechanical strength. The same electrospinning parameters were employed for electrospinning the HAc-treated PPC-GT15 solution for better comparison among the different groups. Figure 6 shows the SEM images of the fibrous membrane electrospun using the treated PPC-GT15 solution. It can be seen that the resultant fibers were very uniform and smooth, with an average diameter of 668.3 ± 104 nm, which was significantly more uniform than that in the original, untreated solution. Therefore, the introduction of HAc facilitated the formation of a single-phase mixture of PPC/gelatin/TFE. The possible reasons may be follows: the solubility of gelatin was changed by the pH of the PPC/gelatin solution, and it has been pointed out that proteins are least soluble and most percipitable when they are at their isoelectric points (IPs). However, the solubility or precipitability behavior of gelatin can be manipulated in solution by controlling and adjusting the pH of the solution.31 3.3. FTIR Results. FTIR was carried out to characterize PPC and PPC/gelatin nanofibers. Figure 7 shows the FTIR spectra of PPC and PPC/gelatin nanofibrous scaffolds. PPCrelated infrared spectra were observed in the curves of pure

Figure 3. Average fiber size distribution of PPC/gelatin electrospun nanofibers.

the volume of HAc achieved 20 μL, the opaque PPC-GT15 solution had a transparency similar to that of the neat PPC solution (shown in Figure 5). When the treated solution was left without stirring for 12 h, or even longer, there was no sign of phase separation. The whole process is shown in Figure 5. The phase homogeneous solution was hypothesized to give rise 9394

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Figure 4. Fiber morphology of the electrospun PPC-GT15 collected at different time intervals: (a) 5 min, (b) 10 min, (c) 30 min, (d) 1 h, and (e) 2 h.

Figure 5. Photos of PPC-GT15 before and after adding a small amount of HAc. The white object in the solution is the magnetic stirring rods.

PPC and PPC/gelatin scaffolds, which included 2986 cm−1 (CH2 stretching), 1738 cm−1 (carbonyl stretching), 1243 cm−1 (O−C−O stretching), 1165 cm−1 (C−O stretching in −CH−

O−), as well as 1124 and 1065 cm−1 (C−O stretching in −O− CO). 32−34 Common bands of protein appeared at approximately 1650 cm−1 (amide I) and 1540 cm−1 (amide 9395

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Figure 6. (a) SEM image of the nanofibers from electrospinning PPC-GT15 containing a small amount of HAc; scale bar of the inset is 10 μm. (b) Histogram of size distribution of panel a.

Figure 7. FTIR spectra of electrospun PPC and PPC/gelatin nanofiber membranes with and without HAc.

II), which were assigned to the stretching vibrations of CO bonds, and the coupling of the bending of N−H and stretching of C−N bonds, respectively.11,35 It was shown that with increased gelatin, the characteristic peaks of protein at 1650 cm−1 and 1540 cm−1 became sharper, indicating that the gelatin was successfully added into the fibrous scaffolds. After adding a small amount of HAc into the PPC-GT15 solution to enhance the phase separation behavior, the intensity of PPC-GT 15 HAc electrospun membranes became stronger than that of PPC-GT15, which further verified that the addition of HAc was helpful for enhancing poor interactions between PPC and gelatin. 3.4. Surface Wettability of Electrospun PPC and PPC/ Gelatin Scaffolds. Water contact angles reflect the hydrophilicity of the scaffolds, which affect protein absorption and subsequent cell adhesion on the scaffolds. To evaluate the influence of blending gelatin with PPC on the surface wettability of the electrospun scaffolds, the water contact angle test was carried out. As shown in Figure 8, the incorporation of gelatin greatly enhanced the hydrophilicity of the scaffolds; the contact angle of fibrous scaffolds changed from 112.4 ± 0.2° for PPC to 81.1 ± 2.1° for PPC-GT10 and 70.4 ± 0.5° for PPC-GT15. These results indicate that the wettability of hybrid scaffolds evolved from hydrophobic to hydrophilic (contact angle < 90°). For PPC-GT15 HAc membranes, the water droplet was absorbed into the fibrous networks within 5 s, indicating that the hydrophilicity after HAc treatment was improved compared to the untreated counter-

Figure 8. Water contact angle of electrospun PPC/gelatin fibrous membranes.

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used to do the tensile test. Like other samples, before doing tests, the samples were immersed into PBS for 2 h. Figure 9a shows significant inconsistencies among the four curves due to the phase separation in the PPC-GT15 solution during the electrospinning process. However, after adding a small amount of HAc into the PPC-GT15 solution (such as 20 μL of pure HAc added to 10 mL of untreated solution), the tensile behavior of the membrane, shown in Figure 9b, changed significantly. The tensile strength of HAc-modified PPC-GT15 fibrous membranes was twice that of the untreated ones, and the elongation-at-break of the modified membranes was much higher than the original ones. The tensile results indicated that the HAc had a positive effect on the phase miscibility, as shown by the enhancement of the material’s mechanical strength. Moreover, to investigate the effect of the PBS buffer used, the morphological changes of the samples before and after wetting for 2 h were investigated. SEM images of the fibers before and after wetting are shown in Figure 10. There were not obvious morphological changes between the dry samples and the wet samples, indicating that the PBS buffer did not dissolve the gelatin in the samples. 3.6. Cell Viability and Proliferation. As shown in Figure 2d, due to the serious phase separation, it is difficult to get good PPC-GT15 samples due to many splashes on the collectors. Therefore, we chose PPC-GT5 with better uniformity to do biological tests. The viability of cells cultured on the PPC/ gelatin membranes after 3 days (Day 3) and 10 days (Day 10) was investigated by live/dead assay. The results of the Day 3 assessment are shown in Figure 11. In this assay, green fluorescence indicated living cells, which were labeled with Calcein AM. Red fluorescence corresponded to the dead cells labeled with EthD-1. The clear prominence of green in comparison to red in the pictures suggested that the 3T3 cells reacted favorably to all of the fibrous membranes. Both PPC/ gelatin and HAc-treated nanofibrous membranes showed high biocompatibility as the fibroblasts survived and flourished on the specimens. For a more detailed examination of the influence of gelatin and HAc on cell response, SEM images were taken after 3 days of culture (Figure 11). The cells on the pure PPC membrane tended toward a rounded morphology, indicating limited cell spreading. In comparison, on the surface of the PPC-GT5 membrane, the cells spread out, suggesting that the cells had better attachment on the surface. On the surface of modified PPC-GT15 membranes, the cells were more spindle-like and spread out on the surface of the membrane, indicating that the HAc in the solution did not negatively affect cell growth.

part. Because most of the synthetic biodegradable polymers such as PCL, PLLA, and PLGA are hydrophobic, the nanofibrous scaffolds of PPC/gelatin with improved wettability might be beneficial for cell seeding.36 3.5. Mechanical Properties of PPC and PPC/Gelatin Scaffolds. The mechanical properties of electrospun nanofibers were characterized by tensile tests in dry and wet conditions at room temperature. The Young’s modulus, tensile strength, and elongation-at-break of the scaffolds are shown in Table 1. Under wet conditions, the tensile strength of the Table 1. Mechanical Properties of Electrospun PPC/Gelatin Fibers sample PPCa PPC-GT2a PPC-GT5a PPC-GT10a PPC-GT15a PPC-GT15 HAca PPCb PPC-GT2b PPC-GT5b PPC-GT10b PPC-GT15b PPC-GT15 HAcb

tensile strength (MPa)

Young’s modulus (MPa)

elongation at break (%)

± ± ± ± ± ± ± ± ± ± ± ±

429.63 ± 55.39 137.26 ± 8.27 127.96 ± 2.96 93.53 ± 15.74 60.69 ± 14.53 295.72 ± 68.48 432.31 ± 15.4 300.92 ± 8.12 250.91 ± 5.21 245.72 ± 9.33 84.82 ± 62.58 242.95 ± 52.72

154.93 ± 32.27 94.06 ± 49.09 70.15 ± 37.36 49.32 ± 26.16 23.18 ± 19.33 95.26 ± 26.89 167.21 ± 23.7 109.72 ± 18.7 79.24 ± 24.32 59.95 ± 14.13 34.32 ± 48.83 107.95 ± 31.20

7.37 3.82 3.49 3.31 2.08 7.72 7.36 6.25 4.64 4.26 2.88 6.71

1.67 0.09 0.35 0.25 0.44 1.13 0.53 0.08 0.09 0.15 0.82 0.59

a

Samples were tested under dry conditions. bSamples were tested under wet conditions.

scaffolds decreased gradually with gelatin content, from 7.36 ± 0.53 MPa for PPC fibers to 4.26 ± 0.25 MPa for PPC-GT10 fibers. The elongation-at-break and Young’s modulus showed similar trends. With the increasing content of gelatin in the fiber, the elongation-at-break decreased from 167.2 ± 23.7% for pure PPC fibers to 59.9 ± 14.1% for PPC-GT10 fibers. The mechanical properties of the scaffolds tested under dry conditions showed similar trends to those tested under wet conditions. These results indicated that the mechanical strength of the fibrous scaffolds was affected by the presence of gelatin, and that this reduction was likely due to the weak physical properties of gelatin.37 Apart from morphological and compositional homogeneities, the mechanical strength of the electrospun membranes using PPC-GT15 and PPC-GT15 HAc solution was tested, which is shown in Figure 9. For comparison, four samples cut from PPC-GT15 and PPC-GT15 HAc electrospun membrane were

Figure 9. Stress−strain curves of the electrospun membranes: (a) PPC/GT15 and (b) PPC/GT15 HAc. 9397

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Figure 10. SEM results of PPC/gelatin composite nanofibers before and after wetting in PBS for 2 h: (a, f) PPC, (b, g) PPC-GT2, (c, h) PPC-GT5, (d, i) PPC-GT10, and (e, j) PPC-GT15 HAc. (The images on the first line were obtained from the electrospun nanofibers before wetting, while the images on the second line were obtained after wetting the samples for 2 h).

Figure 11. Day 3 fibroblast cell culture results of (a, d) PPC, (b, e) PPC-GT5, and (c,f) PPC-GT15 HAc. Images a−c are fluorescence microscope pictures (scale bar = 100 μm), in which green indicates living cells and red indicates dead cells. Images d−f are SEM images (scale bar = 10 μm).

4. CONCLUSIONS

The cell culture results of the Day 10 assay are shown in Figure 12. There were few dead cells present on the scaffold, indicating that the PPC scaffolds were able to provide a suitable environment for cell growth. The SEM images (Figure 12d through f) showed that a lot of cells grew on the scaffolds, and almost covered the entire surface of the membranes, which indicated that the PPC specimens had good biocompatibility. The MTS assay statistical data of Day 1, Day 3, and Day 5 cell counts are shown in Figure 13. The PPC-GT membranes had a higher cell number than the pure PPC sample. The modified PPC-GT15 had the largest cell population on Day 1, Day 3, and Day 5 among all of the samples, suggesting that the introduction of HAc did not negatively influence cell growth and that the addition of gelatin was good for cell proliferation in the samples. The cell viability and proliferation results indicated that the PPC/gelatin samples were good for fibroblast cell culture, which have the potential to be used as tissue engineering scaffolds in the future.

By blending PPC with gelatin, and using TFE as a dissolving solvent, we produced a viable polymeric scaffold made from electrospun PPC/gelatin fibers. This scaffold has the potential to be useful in various applications in the biomedical field. However, phase separation occurred when the content of gelatin was higher than 5%, with the whole process of phase separation being observed by SEM imaging during electrospinning. Inspired by the different behavior of gelatin in solutions at different pH values, a small amount of HAc was added to the PPC/gelatin. It was found that the addition of HAc increased the miscibility of PPC and gelatin in the solution, changing the solution from hydrophobic to hydrophilic and improving the uniformity and mechanical properties of the electrospun nanofibers. This finding also allowed for a higher loading of gelatin in the PPC solution. 9398

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Figure 12. Day 10 fibroblast cell culture results of (a, d) PPC, (b, e) PPC-GT5, and (c, f) PPC-GT15 HAc. Images a−c are fluorescence microscope pictures (scale bar = 100 μm), in which green indicates living cells and red indicates dead cells. Images d−f are SEM images (scale bar = 10 μm).

ship Council, the financial support of the National Nature Science Foundation of China (No. 51073061, No. 21174044), the Guangdong Nature Science Foundation (No. S2013020013855, No. 9151064101000066), and National Basic Research Development Program 973 (No. 2012CB025902) in China.

■ Figure 13. MTS assay cell count statistical results of electrospun PPC/ gelatin composite scaffolds at Day 1, Day 3, and Day 5 time points (*p < 0.05).

Cell-culture experiments and SEM observations showed that fibroblast cells had favorable interactions with the PPC/gelatin scaffolds compared to pure synthetic PPC scaffolds. Furthermore, it was found that the addition of HAc in the PPC solution did not negatively influence the growth and proliferation of cells. Therefore, the approach of electrospinning PPC with a natural protein such as gelatin may lead to the broad application of PPC in tissue engineering (e.g., vascular scaffolds, nerve tissue engineering, and bone tissue).

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to acknowledge the support of the Wisconsin Institute for Discovery (WID), the China Scholar9399

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dx.doi.org/10.1021/ie500762z | Ind. Eng. Chem. Res. 2014, 53, 9391−9400