Properties and fibroblast cellular response of soft and hard thermoplastic polyurethane electrospun nanofibrous scaffolds Hao-Yang Mi,1,2,3 Xin Jing,1,2,3 Max R. Salick,2,4 Travis M. Cordie,2 Xiang-Fang Peng,1 Lih-Sheng Turng2,3 1
The Key Laboratory for Polymer Processing Engineering of Ministry of Education, South China University of Technology, Guangzhou, 510640, China 2 Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, Wisconsin 53715 3 Department of Mechanical Engineering, University of Wisconsin–Madison, Madison, Wisconsin 53706 4 Department of Engineering Physics, University of Wisconsin–Madison, Wisconsin 53706 Received 23 April 2014; revised 18 July 2014; accepted 8 August 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33271 Abstract: Soft and hard thermoplastic polyurethane (TPU) and their blends were electrospun to fabricate nanofibrous scaffolds with various properties in order to investigate the substrate property effects on cellular response. The scaffolds were characterized with Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, water contact angle tests, and protein absorption tests. It was found that the hard segment content in the scaffold increased with the hard TPU ratio, which resulted in improved hydrophobicity and decreased over all protein absorption. 3T3 fibroblasts were cultured on those scaffolds to investigate the cellular response. On soft TPU scaffolds,
the cells formed were round in shape and aggregated into clusters. However, on hard TPU scaffolds, the cells exhibited a spindle shape and spread out on the scaffolds, indicating preferred cell–substrate interaction. The cell viability and proliferation of cells on hard scaffolds were higher than on soft C 2014 Wiley scaffolds and on 50% hard/50% soft scaffolds. V Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000– 000, 2014.
Key Words: polyurethane, electrospinning, wettability, protein absorption, fibroblast
How to cite this article: Mi H-Y, Jing X, Salick MR, Cordie TM, Peng X-F, Turng L-S. 2014. Properties and fibroblast cellular response of soft and hard thermoplastic polyurethane electrospun nanofibrous scaffolds. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Tissue engineering is an interdisciplinary field that applies the principles of biology and engineering to the development of functional substitutes for damaged tissue.1 Tissue scaffolds act as extracellular matrices (ECMs) for living cells to grow on and provide support to damaged tissues, then subsequently degrade in vivo.2 Recently, fabricating viable scaffolds for tissue engineering is attracting more and more attention from both academia and industry. Many methods have been developed to produce biodegradable scaffolds including solvent casting/particulate leaching,3 thermally induced phase separation,4 electrospinning,5 rapid prototyping,6 batch foaming,7 and microcellular injection molding.8 Among these methods, electrospinning is widely used to produce nanofibers for tissue engineering applications due to its simple setup and its ability to generate fibers with
diameters that range from 50 nm to a few micrometers.9 This fibrous structure also leads to high scaffold interconnectivity and high surface areas. In electrospinning, a stream of polymer solution continuously flows out from a blunt needle which is connected to a high electrical charge. The polymer solution starts to form fibers when the electrostatic repulsion forces from the surface charges overcome the surface tension.10 To mimic the ECM, a scaffold has to possess the following characteristics: (1) a three-dimensional (3D), highly porous structure with an interconnected network to facilitate cell attachment, migration, proliferation, and differentiation as well as free transportation of nutrients, metabolic waste, and paracrine factors11; (2) be biocompatible and biodegradable with controllable degradation rates12; (3) have a suitable surface chemistry to support the
Additional Supporting Information may be found in the online version of this article. Correspondence to: L.-S. Turng; e-mail: [email protected] or X.-F. Peng; e-mail: [email protected] Contract grant sponsor: Wisconsin Institute for Discovery (WID) and China Scholarship Council; contract grant number: 51073061 Contract grant sponsor: National Nature Science Foundation of China; contract grant numbers: 51073061; 21174044 Contract grant sponsor: The Guangdong Nature Science Foundation; contract grant number: S2013020013855 Contract grant sponsor: The Fundamental Research Funds for the Central Universities; contract grant number: 2011ZZ0011 Contract grant sponsor: 973 Program; contract grant number: 2012CB025902
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abovementioned cell functions13; and (4) have ideal mechanical properties that match the surrounding tissue of the implantation site and provide support.11 Among these, scaffold surface chemistry has the most important impact on cellular behavior since it is the initial site that interacts with the cells. Wettability and protein absorption of the scaffolds are the most commonly used methods to characterize scaffold surface properties. Protein is an important factor in mediating cell and biomaterial interactions since the biological recognition of cells happens between the cells and the proteins adsorbed to the biomaterials.14 The wettability of the biomaterials can affect protein absorption significantly. However, contradictory opinions exist in understanding this relationship. It is widely accepted that hydrophilic surfaces tend to absorb water molecules and form a water layer rather than adsorb proteins,15 while on a hydrophobic surface, proteins may unfold and their hydrophobic side chains may come in contact with the surface16 and cause protein immobilization. However, many publications have reported that for tissue scaffold applications, hydrophilic materials display a higher affinity for cells.17,18 The hydrophilic scaffolds show higher protein absorption and cell reproduction, and can overcome the 3D scaffold flotation problem in culture media.19,20 Moreover, scaffolds with hydrophilic properties improved by the addition of chitosan,21 gelatin,22 or collagen23 showed enhanced biocompatibility. Therefore, it is worth putting more effort into further verifying the relationship between scaffold wettability and biocompatibility. Thermoplastic polyurethane (TPU) is a linear polymer that consists of soft and hard segments. TPU, as a class of PU, has been widely used in medical applications, mainly because of its biocompatibility, high fracture strain, moderate tensile strength, and excellent abrasion and tear resistances. In most TPUs, soft segments such as aliphatic polyether polyols or aliphatic polyester polyols are coupled with hard segments of diisocyanates, such as toluene diisocyanate, diphenylmethane diisocyanate, and 1,6-hexamethylene diisocyanate, followed by a chain extension with a diol in the presence of catalysts. Thus, the molecular weight and molecular structure of a TPU can be adjusted via the chemical synthesis formula and ratio of soft and hard segments. Through adjusting the combination of soft and hard segments, TPU can be synthesized into both hydrophilic and hydrophobic TPU. It is worth investigating the cellular response to electrospun TPU scaffolds with different wettability characteristics since, to the best of our knowledge, no studies have been done on this to date. Yuan and coworkers24 reported on a PU/polyethylene glycol vascular scaffold with improved hydrophilicity that proved suitable for endothelia cells attachment and proliferation. Kim et al.25 showed that hydrophilicity can be improved by adding gelatin into TPU, with the resulting scaffolds displaying better cell proliferation. However, this improvement of cell response could be attributed to either enhanced hydrophilicity or the bioactivity provided by the natural materials. In this study, hydrophilic and hydrophobic TPUs and their blends were electrospun to produce scaffolds with dif-
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TABLE I. Solution Formula Solution Formula Soft TPU (S) 25%H 50%H 75%H Hard TPU (H)
Concentration (wt./vol.) 20% 15% 15% 15% 10%
ferent properties. These scaffolds were fully characterized via Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), water contact angle (WAC) tests, and protein absorption tests. Furthermore, the fibroblast cell response to these scaffolds was investigated by live/dead assay and 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. EXPERIMENTAL
Materials Two kinds of medical grade TPUs—Tecophilic SP-80A-150 (soft TPU, Mw 5 179464 Da; Mn 5 63,492 Da) and Carbothane PC-3572D (hard TPU, Mw 5 201,873 Da; Mn 5 94,212 Da)—were supplied by Lubrizol. The molecular weight of TPU was measured using gel permeation chromatography. The solvent used to dissolve TPU was N, N-dimethylformamide (DMF) purchased from Sigma-Aldrich. All materials were used as received. Electrospinning process. The TPU pellets were dried at 80 C for 2 h prior to solution preparation. Pure TPU and the soft/hard TPU mixtures were prepared at various ratios with different concentrations by dissolving TPU pellets in DMF for 8 h with magnetic stirring at 60 C. The solutions prepared are listed in Table I. The solution concentrations were determined based on a previous study to produce uniform, bead-less electrospun fibers.26 The prepared solution was loaded in a plastic syringe connected to an 18 gauge blunt-end needle mounted on a digital syringe pump. The electrospinning procedure was carried out using a voltage of 15 kV, a working distance of 150 mm, and a flow rate of 0.5 mL/h. The electrospun fibers were collected using a thin aluminum foil. The same conditions were used for all of the solutions in order to eliminate the effect of processing parameters. All of the solutions were used immediately after they were prepared to avoid possible phase separation of soft and hard TPUs. NIH 3T3 fibroblast cell culture. Prior to cell culture testing, 3T3 cells were maintained on 6-well tissue culture-treated polystyrene plates (BD Falcon). Cells were fed every other day with a high-glucose 20% serum medium consisting of high-glucose DMEM (Gibco), 20% fetal bovine serum (WiCell), 2 mM L-glutamine (Invitrogen), and penicillin– streptomycin (Invitrogen). Cells were passaged at a 1:40 ratio every 6 days via a 5-minute EDTA treatment (Invitrogen). The samples used for cell culture were directly
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FIGURE 1. X-ray photoelectron spectroscopy test results of soft (S) and hard (H) TPUs: (a) the survey scan of S and H, (b) the proportion of carbon peaks within S and H from C1 core-level signals for (c) S and (d) H. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
electrospun on sterilized stainless steel washers and then cut off from the electrospun membrane. The scaffolds were cultured with an inert metal fixture (washer) to prevent scaffold flotation so that the area of the cells could be determined. The casted pure soft (S) and hard (H) TPU films and tissue culture plates (TCPs) were tested as control groups. For the cell culture testing, samples were placed in 24-well tissue culture-treated polystyrene plates after UV light sterilization on each side (30 min per side). 3T3 cells were treated with EDTA for 5 min and washed with phosphatebuffered saline (PBS) prior to seeding. Cells were then seeded at a density of 1.25 3 105 cells/cm2. Spent medium was aspirated and replaced with 1 mL of fresh medium daily for screening samples. Characterization X-ray photoelectron spectroscopy. X-ray Photoelectron Spectroscopy (XPS) survey scans and C1 core-level signals were analyzed to identify the elements and the proportion of carbon bonds on the surface of soft and hard scaffolds in detail to get a clear understanding of the difference between these two materials. XPS measurements were performed on an X-ray photoelectron spectrometer with a focused, monochromatic K-alpha X-ray source and a monoatomic/cluster ion gun (Thermo Scientific). The spectra were Gaussian fit-
ted and the proportion of each bond was determined from the peak area ratios. Fourier transform infrared spectroscopy FTIR was used to identify the functional groups of the scaffolds. Each scaffold was folded twice to increase its thickness and measured with a Bruker Tensor 27 FTIR instrument. Samples were analyzed in the absorbance mode in the range of 600 to 4000 cm21. Thermogravimetric analysis. TGA was used to determine the portion of hard and soft segments in the scaffolds and their decomposition behavior. A sample of approximately 10 mg was used for the test. The test was initiated at room temperature and then equilibrated to 100 C to eliminate any water in the sample. The temperature was then increased to 600 C using a heating rate of 10 C/min on a TA Q50 instrument. The scaffold weight was measured against the temperature. Scanning electron microscopy The microstructure of electrospun nanofibers was characterized using a fully digital LEO GEMINI 1530 SEM with an accelerating voltage of 3 kV. The electrospun membranes were cut into small pieces with a scalpel and sputtered with
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FIGURE 2. FTIR test results of S, 25%H, 50%H, 75%H, and H electrospun scaffolds. Four areas were enlarged to show the intensity variation clearly. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
gold for 40 s before imaging. The fiber diameter of each scaffold, which was measured by Image-Pro Plus software, was the average value of 50 individual fibers. Differential scanning calorimetry. Differential scanning calorimetry (DSC) measurements were performed with a DSC Q20 (TA Instruments). Samples weighing 5 to 8 mg were measured and encapsulated in standard aluminum pans. Samples were then heated to 200 C and kept isothermal for 5 min, then cooled to 280 C and reheated to 200 C. The scanning rate of the test was 5 C/min and all tests were carried out under the protection of a nitrogen atmosphere. Wettability. Wettability of the electrospun fibers was characterized with WAC test which was performed at room temperature in a Dataphysics OCA 15 optical contact angle measuring system using the sessile drop method. Two microliters of deionized water were dropped on the scaffolds and images at 0 and 2 s were taken and measured. The WCA of each scaffold was the average value of three different spots. Protein absorption. The scaffolds were first sterilized by UV treatment, then specimen dimensions were determined. The specimens were washed three times with Dulbecco’s phosphate buffer saline (DPBS, Life Technologies), at 1 h intervals to remove any impurities. Samples were then placed into the 3T3 fibroblast culture medium for a predetermined amount of time (e.g., 15, 50, and 150 h) inside the cell culture incubator at 37 C. Following incubation, the samples were removed from the medium and placed into a fresh plate and washed once with DPBS. The amount of adsorbed protein on each specimen was measured using a Thermo ScientificTM PierceTM BCATM Protein Assay with the
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aid of a GloMax Multi1 Detection System (Promega). The tests were repeated three times for each sample. 3T3 fibroblast cell viability. Fibroblast cell viability was determined 3 days and 10 days after cell seeding. Viability was assessed via a Live/Dead Viability/Cytotoxicity Kit (Invitrogen). The stain utilized green fluorescent Calcein-AM to target living cells, red fluorescent ethidium homodimer-1 (EthD-1) to indicate cell death, and blue fluorescent DAPI to identify the cell nucleus. Stained cells were imaged with a Nikon Ti-E Confocal microscope. Advanced Research v.3.22 software was used for image analysis. To assess the percent of live cells, the stained cells were detached using TrypLE (Life Technologies) for 5 min at 37 C and then collected and centrifuged at 200 rpm for 5 min. The supernatant was aspirated and the cells resuspended in 600 lL of PBS and filtered prior to analysis. The percentage of green fluorescent cells was acquired with an Accuri C6 flow cytometer (BD Biosciences). Cell fixation for SEM. The same samples used for cell viability testing were rinsed twice with PBS. Samples were submerged in a 4% paraformaldehyde solution (electron microscopy solutions) for 30 min. The samples were then dehydrated using a series of ethanol washes (50, 90, and 100% for 30 min each), and finally dried in a vacuum desiccator for 2 to 3 h before gold sputtering for SEM imaging. MTS assay. The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was used to determine the number of cells on the scaffolds. Standard curves were established by performing the tests on cells seeded on the cell culture wells and confirmed by comparison to hemocytometer readings prior to testing. Upon testing, cells were treated with an 83% media, 17% MTS solution and allowed
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FIGURE 4. TGA results of S, 25%H, 50%H, 75%H, and H electrospun scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
RESULTS
FIGURE 3. SEM images of electrospun scaffolds: (a) S, (b) 25%H, (c) 50%H, (d) 75%H, and (e) H. Left images are at low magnification with a scale bar of 20 lm; right images are at high magnification with a scale bar of 2 lm. The insets show the fiber diameter distribution. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
to incubate for exactly one hour. One hundred microliters of spent media were removed and added to a clear 96-well plate. The absorbance of this plate at the 450 nm wavelength was read using a GloMax-Multi 1 Multiplate Reader (Promega) and the subsequent number of cells was determined relative to the negative control. Statistical analysis. All biological experiments contain at least three samples. Statistical analysis was performed using one-way analysis of variance (ANOVA) and the difference significance between groups was compared as well, with the level of significance set at p < 0.05 and p < 0.01.
Scaffold component characterization The XPS results on S and H TPUs are shown in Figure 1, which characterized the basic differences between S and H. Figure 1(a) shows that both TPUs contain C, O, and N elements, while, for S TPU, the ratio of O was higher and the ratio of N was lower than that of H TPU according to the intensity of C1, O1, and N1 peaks. Furthermore, the fitted C1 peak results [Figure 1(c,d)] and the proportion of carbon peak results [Figure 1(b)] also showed that the carbon–oxygen bonds that belonged to the polyol soft segments had higher ratio in S TPU, while the carbon–nitrogen bonds that belonged to the isocyanate hard segments were dominant in H TPU.27,28 The FTIR results, which are shown in Figure 2, showed that soft and hard TPUs have the same peaks, although the peak intensities were different, thus indicating different component ratios. As one can see, the peak intensity of amide I (1692 cm21), amide II (1530 cm21), and amide III (1245 cm21) groups29 that belong to the hard segment of TPU increased with increasing hard TPU content in the sample. On the contrary, the ether group (CAOAC) at 1245 cm21, which existed in the soft segments of the TPU molecular chain,30 increased with increasing soft TPU content in the scaffold. These results suggest that the proportion of soft and hard segments in the scaffolds can be adjusted effectively by varying the soft and hard TPU blending ratio. In addition, the peak of absorbed water located around 3550 cm21 became higher as the soft segment content increased, hence suggesting that the moisture absorbed by the scaffold increased. Morphology The morphologies of the electrospun scaffolds are shown in Figure 3. The distribution of the fiber diameter was uniform, with most fiber diameters ranging from 200 to 800 nm. At the same solution concentration [Figure 2(b–d)], the fiber diameter increased as the hard segment content increased in the solution. It was found in a previous study26
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FIGURE 5. Differential scanning calorimetry results of S, 25%H, 50%H, 75%H, and H electrospun scaffolds: (a) second heating period and (b) first cooling period. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
that increasing both the solution concentration and the hard segment proportion of the solution could improve the TPU molecular chain entanglement. This is important because once the chain entanglement becomes larger than a critical value, continuous uniform fiber structures can be obtained. Furthermore, it was noticed that the fiber surface of the S (soft TPU) scaffold [Figure 3(a)] was smooth, while the fiber surface became rougher as the hard segments content increased, especially in the H (hard TPU) scaffold [Figure 3(e)]. This was because of the increase in the rigid hard segment portion in the fiber. Thermal properties The thermal properties of the scaffolds were evaluated via TGA and DSC tests. As shown in Figure 4, all of the scaffolds started to decompose at about the same temperature (280 C). The H scaffold lost weight dramatically as the temperature increased because the amide bonds in the hard segments were easier to cleave than the ether bonds in the soft segments as the temperature increased.31 In addition, the weight loss of the scaffolds had two obvious steps that belonged to the decomposition of the hard and soft segments. The transition points of the two steps indicated the weight portion of the soft and hard segments in the scaffolds. It was noticed again that the proportion of hard segments in the scaffolds increased with increasing hard TPU content, and that the scaffolds had better thermal stability with more soft segments.
Figure 5 shows the DSC test results of the electrospun scaffolds. It was found that both soft and hard TPU were amorphous without obvious melting peaks and crystallization peaks. The H scaffold showed a wide glass transition region from 258 to 85 C, while it was 254 to 218 C for the S scaffold. All of the blended scaffolds showed two glass transition slopes, which indicating the low miscibility between these two kinds of TPUs. The peaks initiated at 0 C in both first cooling and the second heating periods were due to the existence of moisture in the scaffolds. The water content increased as the soft segment proportion increased, corresponding to the FTIR results. Wettability The wettability of scaffolds is an important characteristic that determines cell adhesion. As shown in Figure 6, the H scaffold was hydrophobic with a WCA constantly over 120 , while the S scaffold was highly hydrophilic with a WAC reduced to 0 in 2 s after the water bead was dropped on the scaffold. The scaffolds blended by S and H showed higher WCAs with more hard segments, and that angle reduced over time. Protein absorption The S, 50%H, and H scaffolds were chosen to evaluate the protein absorption as proteins always mediate the interactions between the cell and the biomaterial. As shown in Figure 7, all of the scaffolds absorbed more proteins as time passed. The S scaffold absorbed more protein than the
FIGURE 6. WAC test results of S, 25%H, 50%H, 75%H, and H electrospun scaffolds at 0 and 2 s after the water bead was dropped on the scaffold.
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FIGURE 7. Protein absorption test results of S, 50%H, and H electrospun scaffolds at 15, 50, and 150 h time points. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
50%H and H scaffolds over all three time periods. Furthermore, the protein concentration for the S scaffold increased more from 50 to 150 h, while for the H scaffold, it increased more from 12 to 50 h. This may indicate that the S scaffold can absorb protein continuously into the scaffold, while the H scaffold adsorbed protein only on the scaffold surface. 3T3 fibroblast cell culture To further investigate the cellular response to the TPU scaffolds prepared, 3T3 fibroblast cells were cultured on S,
50%H, and H scaffolds for 10 days and imaged at day 3 and day 10 time points. The number of cells was measured by MTS assay at days 3, 6, and 10. Day 3 results are shown in Figure 8, from which it was found that the cells formed clusters on the S [Figure 8(a)] and 50%H [Figure 8 (b)] scaffolds, while they spread out very well on the H [Figure 8(c)] scaffolds. In addition, most cells on the scaffolds were alive at day 3, as indicated by large areas of green fluorescence and few red spots. Individual cell morphologies can be seen from the SEM images. As one can see, the cells spread only on the H scaffold [Figure 8(f)], while cells on the S and 50%H scaffolds presented round shapes and aggregated into clusters. A similar cell response was observed at day 10 as shown in Figure 9. It can be seen from Figure 9(a–c) that the cells were denser and covered a larger area. The 50%H scaffold showed a larger cell area than the S scaffold. More dead cells were observed in all scaffolds compared to the day 3 results, especially the S scaffold. The increase in cell death may have been due to an increase in cell competition as the cells proliferated. From the SEM images, it was found that cells still agglomerated into clusters and presented round shapes on the S and 50%H scaffolds, while spreading into elongated shapes on the H scaffolds. In addition, the absence of fibrous structures in the S scaffolds may have been caused by the ethanol dehydration procedure when preparing the SEM sample since soft TPU swells in ethanol. The control groups showed a similar cell response to the materials. The cells formed clusters on the casted S film and
FIGURE 8. Day 3 cell culture fluorescence images (a–c) and SEM images (d–f) of S [(a) and (d)], 50%H [(b) and (d)], and H [(c) and (f)] electrospun scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 9. Day 10 cell culture fluorescence images (a–c) and SEM images (d–f) of S [(a) and (d)], 50% H [(b) and (d)], and H [(c) and (f)] electrospun scaffolds. Separated fluorescence of image (a) indicates (g) cell nucleus, (h) live cells, and (i) dead cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
spread out on the casted H film and the TCPs, as shown in the Supporting Information Figure S1. The cell viability results from the live/dead assays and the number of cells from the MTS assay are shown in Figure 10(a,b), respectively. The cell viability showed the same trend at day 3 and day 10, which was that the H scaffolds had higher cell viability than the S and 50% H scaffolds, as well as that of the S and H film controls. The S scaffold seemed to perform better than the S film, although the difference was not statistically significant. The cell proliferation results showed that the number of cells increased as time passed. It was also noticed that at each time period, the H scaffold had significantly more cells than the 50% H scaffold, which had more
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cells than the S scaffold. At day 10, the H scaffold had significantly more cells than the H film, although the difference was not significant at day 3. It was found that the TCP control group presented the highest cell viability and cell proliferation at all time points due to its specially treated surface. Therefore, the statistical comparisons to the TCP groups in the ANOVA test were not shown in the figure so as to emphasize the important comparison groups. The results from these tests suggested that the cells could reproduce better on hydrophobic H scaffolds with a higher viability than on the S scaffolds and the casted films. For the same material, a fibrous structure was superior for cells than a flat film due to the higher aspect ratio of the fibers.
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FIGURE 10. (a) Cell viability and (b) number of cells at day 3 and day 10 of S, 50% H, and H scaffolds and control groups of S films, H films, and TCPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
DISCUSSION
The scaffold is a crucial part in the tissue engineering process. Preparation of scaffolds with proper attributes is important to in vitro and in vivo cell culture. Different properties of scaffolds can determine the cellular response in terms of cell adhesion, proliferation, differentiation, and migration.13,32 The substrate properties that affect the cellular response include the mechanical properties33; surface chemistry and topography (wettability, softness, stiffness, and roughness)34; and microstructure (porosity, pore size, pore shape, and interconnectivity).35,36 In this study, the TPU electrospun scaffolds were characterized with SEM, WCA tests, and protein absorption tests. The ability of the scaffolds to absorb proteins is important as the amount of serum proteins—such as fibronectin and vitronectin— absorbed by the scaffold affects cell adhesion significantly.37 The scaffold with more soft segments showed higher hydrophilicity (Figure 6) and higher protein absorption (Figure 7) than those with more hard segments. References 18, 19, and 38 suggested that elevated hydrophilicity and protein absorption would result in improved cell proliferation. The cell number from the MTS assay (Figure 10), however, showed lower cell numbers on scaffolds with more soft segments. Similar decrease of goat bone marrow stromal cell proliferation as scaffold hydrophilicity increase has been reported.39 To investigate the cause of cell death on scaffold S at day 10, the colors in Figure 9(a) were separated to indicate cell nuclei (blue), live cells (green), and dead cells (red), as shown in Figure 9(g–i). It was noticed from the figure that most dead cells were located in the center of the cluster and were surrounded by live cells. Therefore, the cells in the center of the cluster died because they could not obtain enough nutrients from the cell culture media. In fact, it has been found that poor interactivity between the substrate and the cells, as well as the low amount of surfaceimmobilized, cell-attachment proteins, would lead to a balled up cell morphology and the formation of cell clusters.40 The substrate hydrophilicity is one of the main concerns that affect the interactivity between cells and
substrate. The favorable hydrophilicity of the substrate varied with different cell types; neither highly hydrophilic nor highly hydrophobic surfaces were favorable for cells. For example, the best WCA for endothelial cell attachment and proliferation was about 50 , while it was 76 for chondrocytes.34,41 Cui et al.38 reported that the poly(lactic acid)/chitosan scaffolds with WCA of 0 improved human dermal fibroblasts proliferation dramatically. However, Cheung et al.42 recently found that a polar hydrophobic polyurethane scaffold enhanced human gingival fibroblast proliferation. In this study, hydrophobic hard TPU performed better than hydrophilic soft TPU in terms of cell attachment and proliferation for both electrospun fibers and casted films, while the electrospun fibers were superior to the casted films for the same material. Since the cells formed similar aggregates on the S scaffold and the casted S film, and formed similar spread shapes on both the H scaffold and the H film, the effect of structural differences can be disregarded. Therefore, the main reason might be the proteins had been absorbed inside of the hydrophilic soft TPU, while a water layer formed on the scaffold surface.15 The formed water layer prevented cell attachment and spreading, hence leading to the formation of ball-like shapes and cell clusters. On the contrary, the scaffolds that had more hard segments possessed hydrophobic surfaces that enabled the proteins to be adsorbed on the scaffold surface, even though the proteins could not penetrate into the scaffold. The adsorbed proteins facilitated cell attachment and migration. During long culture times, the cells on the H scaffold divided and migrated, covering the whole surface. On the S scaffold, however, cells were more likely to stick to each other when they divided, which led to cell agglomeration and cell death in the center of the agglomerate.
CONCLUSIONS
Soft and hard TPU and their blends were electrospun to fabricate scaffolds for tissue engineering applications. The properties of these scaffolds were characterized by FTIR,
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SEM, TGA, DSC, WCA tests, and protein absorption tests. The 3T3 fibroblast cell response was investigated via live/ dead assay and MTS assay. It was found that the hard segment content in the scaffolds increased as the hard TPU proportion increased in the blends, while scaffold decomposition was delayed as the soft segment content increased. Scaffolds with more hard segments showed higher hydrophobicity and lower protein absorption than those with more soft segments. Cell culture tests proved that cells exhibited round shapes and aggregated into clusters on the scaffolds with more soft segments, even though those scaffolds were highly hydrophilic and absorbed more proteins. On the contrary, scaffolds with more hard segments had more cells, and the cells spread into spindle shapes. This was because the hydrophilic soft TPU absorbed proteins inside of the scaffold, meanwhile, a water layer was form on the scaffold surface which prevented cell attachment. The hard TPU, on the other hand, adsorbed proteins on the scaffold surface and showed better cell interactions and higher cell viability and cell proliferation.
REFERENCES 1. Langer R, Vacanti JP. Tissue Engineering. Science 1993;260:920– 926. 2. Lee J, Cuddihy MJ, Kotov NA. Three-dimensional cell culture matrices: State of the art. Tissue Eng Part B-Rev 2008;14:61–86. 3. Mikos AG, Thorsen AJ, Czerwonka LA, Bao Y, Langer R, Winslow DN, Vacanti JP. Preparation and characterization of poly(L-lactic acid) foams. Polymer 1994;35:1068–1077. 4. Nakamatsu J, Torres FG, Troncoso OP, Yuan ML, Boccaccini AR. Processing and characterization of porous structures from chitosan and starch for tissue engineering scaffolds. Biomacromolecules 2006;7:3345–3355. 5. Wang L, Shi J, Liu L, Secret E, Chen Y. Fabrication of polymer fiber scaffolds by centrifugal spinning for cell culture studies. Microelectron Eng 2011;88:1718–1721. 6. Park SA, Kim HJ, Lee SH, Lee JH, Kim HK, Yoon TR, Kim W. Fabrication of nano/microfiber scaffolds using a combination of rapid prototyping and electrospinning systems. Polym Eng Sci 2011;51: 1883–1890. 7. Corre YM, Maazouz A, Duchet J, Reignier J. Batch foaming of chain extended PLA with supercritical CO2: Influence of the rheological properties and the process parameters on the cellular structure. J Supercrit Fluids 2011;58:177–188. 8. Kramschuster A, Turng LS. An injection molding process for manufacturing highly porous and interconnected biodegradable polymer matrices for use as tissue engineering scaffolds. J Biomed Mater Res Part B Appl Biomater 2010;92B:366–376. 9. Zhang XH, Cai Q, Liu HY, Zhang S, Wei Y, Yang XP, Lin YH, Yang ZG, Deng XL. Calcium ion release and osteoblastic behavior of gelatin/beta-tricalcium phosphate composite nanofibers fabricated by electrospinning. Mater Lett 2012;73:172–175. 10. Geng XY, Kwon OH, Jang JH. Electrospinning of chitosan dissolved in concentrated acetic acid solution. Biomaterials 2005;26: 5427–5432. 11. Hutmacher DW. Scaffold design and fabrication technologies for engineering tissues—State of the art and future perspectives. J Biomater Sci-Polym Ed 2001;12:107–124. 12. Kweon H, Yoo MK, Park IK, Kim TH, Lee HC, Lee HS, Oh JS, Akaike T, Cho CS. A novel degradable polycaprolactone networks for tissue engineering. Biomaterials 2003;24:801–808. 13. Mandal BB, Kundu SC. Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials 2009;30:2956–2965. 14. Hynes RO. Integrins—Versatility, modulation, and signaling in cell-adhesion. Cell 1992;69:11–25.
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15. Elbert DL, Hubbell JA. Surface treatments of polymers for biocompatibility. Annu Rev Mater Sci 1996;26:365–394. 16. Horbett TA, Lew KR. Residence Time effects on monoclonalantibody binding to adsorbed fibrinogen. J Biomater Sci-Polym Ed 1994;6:15–33. 17. Lampin M, WarocquierClerout R, Legris C, Degrange M, SigotLuizard MF. Correlation between substratum roughness and wettability, cell adhesion, and cell migration. J Biomed Mater Res Part A 1997;36:99–108. 18. Mohan N, Nair PD. Polyvinyl alcohol-poly(caprolactone) semi IPN scaffold with implication for cartilage tissue engineering. J Biomed Mater Res Part B-Appl Biomater 2008;84B:584–594. 19. Oh SH, Kang SG, Kim ES, Cho SH, Lee JH. Fabrication and characterization of hydrophilic poly(lactic-co-glycolic acid)/poly(vinyl alcohol) blend cell scaffolds by melt-molding particulate-leaching method. Biomaterials 2003;24:4011–4021. 20. Niu XF, Li XM, Liu HF, Zhou G, Feng QL, Cui FZ, Fan YB. Homogeneous chitosan/poly(L-lactide) composite scaffolds prepared by emulsion freeze-drying. J Biomater Sci-Polym Ed 2012;23:391–404. 21. Lin HY, Chen HH, Chang SH, Ni TS. Pectin-chitosan-PVA nanofibrous scaffold made by electrospinning and its potential use as a skin tissue scaffold. J Biomater Sci-Polym Ed 2013;24:470–484. 22. Binulal NS, Natarajan A, Menon D, Bhaskaran VK, Mony U, Nair SV. Gelatin nanoparticles loaded poly(epsilon-caprolactone) nanofibrous semi-synthetic scaffolds for bone tissue engineering. Biomed Mater 2012;7. 23. Lee H, Kim G. Three-dimensional plotted PCL/beta-TCP scaffolds coated with a collagen layer: preparation, physical properties and in vitro evaluation for bone tissue regeneration. J Mater Chem 2011;21:6305–6312. 24. Wang HY, Feng YK, Fang ZC, Yuan WJ, Khan M. Co-electrospun blends of PU and PEG as potential biocompatible scaffolds for small-diameter vascular tissue engineering. Mater Sci Eng CMater Biol Appl 2012;32:2306–2315. 25. Kim SE, Heo DN, Lee JB, Kim JR, Park SH, Jeon S, Kwon IK. Electrospun gelatin/polyurethane blended nanofibers for wound healing. Biomed Mater 2009;4. 26. Mi HY, Jing X, Jacques BR, Turng LS, Peng XF. Characterization and properties of electrospun thermoplastic polyurethane blend fibers: Effect of solution rheological properties on fiber formation. J Mater Res 2013;28:2339–2350. 27. Mishra AK, Chattopadhyay DK, Sreedhar B, Raju KVSN. FT-IR and XPS studies of polyurethane-urea-imide coatings. Prog Org Coat 2006;55:231–243. 28. Wang TL, Yu CC, Yang CH, Shieh YT, Tsai YZ, Wang NF. Preparation, characterization, and properties of polyurethane-grafted multiwalled carbon nanotubes and derived polyurethane nanocomposites. J Nanomater 2011;2011: 814903. 29. Pinto UA, Visconte LLY, Nunes RCR. Mechanical properties of thermoplastic polyurethane elastomers with mica and aluminum trihydrate. Eur Polym J 2001;37:1935–1937. 30. Dong ZH, Li YB, Zou Q. Degradation and biocompatibility of porous nano-hydroxyapatite/polyurethane composite scaffold for bone tissue engineering. Appl Surf Sci 2009;255:6087–6091. 31. Buczek O, Krowarsch D, Otlewski J. Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI). Protein Sci 2002;11:924–932. 32. Mandal BB, Kundu SC. Osteogenic and adipogenic differentiation of rat bone marrow cells on non-mulberry and mulberry silk gland fibroin 3D scaffolds. Biomaterials 2009;30:5019–5030. 33. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–689. 34. Arima Y, Iwata H. Effect of wettability and surface functional groups on protein adsorption and cell adhesion using welldefined mixed self-assembled monolayers. Biomaterials 2007;28: 3074–3082. 35. Chang H-I, Wang Y. Cell responses to surface and architecture of tissue engineering scaffolds. In: Eberli D, editor. Regenerative Medicine and Tissue Engineering - Cells and Biomaterials. Rijeka, Croatia: In Tech; 2011. pp 569–588 36. O’Brien FJ, Harley BA, Yannas IV, Gibson LJ. The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 2005;26:433–441.
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37. Steele JG, Johnson G, Underwood PA. Role of serum vitronectin and fibronectin in adhesion of fibroblasts following seeding onto tissue-culture polystyrene. J Biomed Mater Res 1992;26:861–884. 38. Cui WG, Cheng LY, Li HY, Zhou Y, Zhang YG, Chang J. Preparation of hydrophilic poly(L-lactide) electrospun fibrous scaffolds modified with chitosan for enhanced cell biocompatibility. Polymer 2012;53:2298–2305. 39. Ivirico JLE, Salmeron-Sanchez M, Ribelles JLG, Pradas MM, Soria JM, Gomes ME, Reis RL, Mano JF. Proliferation and differentiation of goat bone marrow stromal cells in 3D scaffolds with tunable hydrophilicity. J Biomed Mater Res Part B-Appl Biomater 2009;91B:277–286.
40. Ishihara K, Ishikawa E, Iwasaki Y, Nakabayashi N. Inhibition of fibroblast cell adhesion on substrate by coating with 2methacryloyloxyethyl phosphorylcholine polymers. J Biomater Scie-Polym Ed 1999;10:1047–1061. 41. Zhu HG, Ji J, Lin RY, Gao CY, Feng LX, Shen JC. Surface engineering of poly(DL-lactic acid) by entrapment of alginate-amino acid derivatives for promotion of chondrogenesis. Biomaterials 2002;23:3141–3148. 42. Cheung JWC, Rose EE, Santerre JP. Perfused culture of gingival fibroblasts in a degradable/polar/hydrophobic/ionic polyurethane (D-PHI) scaffold leads to enhanced proliferation and metabolic activity. Acta Biomater 2013;9:6867–6875.
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The Key Laboratory for Polymer Processing Engineering of Ministry of Education, South China University of Technology, Guangzhou, 510640, China 2 Wisconsin Institute for Discovery, University of Wisconsin–Madison, Madison, Wisconsin 53715 3 Department of Mechanical Engineering, University of Wisconsin–Madison, Madison, Wisconsin 53706 4 Department of Engineering Physics, University of Wisconsin–Madison, Wisconsin 53706 Received 23 April 2014; revised 18 July 2014; accepted 8 August 2014 Published online 00 Month 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: 10.1002/jbm.b.33271 Abstract: Soft and hard thermoplastic polyurethane (TPU) and their blends were electrospun to fabricate nanofibrous scaffolds with various properties in order to investigate the substrate property effects on cellular response. The scaffolds were characterized with Fourier transform infrared spectroscopy, thermogravimetric analysis, scanning electron microscopy, water contact angle tests, and protein absorption tests. It was found that the hard segment content in the scaffold increased with the hard TPU ratio, which resulted in improved hydrophobicity and decreased over all protein absorption. 3T3 fibroblasts were cultured on those scaffolds to investigate the cellular response. On soft TPU scaffolds,
the cells formed were round in shape and aggregated into clusters. However, on hard TPU scaffolds, the cells exhibited a spindle shape and spread out on the scaffolds, indicating preferred cell–substrate interaction. The cell viability and proliferation of cells on hard scaffolds were higher than on soft C 2014 Wiley scaffolds and on 50% hard/50% soft scaffolds. V Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 00B: 000– 000, 2014.
Key Words: polyurethane, electrospinning, wettability, protein absorption, fibroblast
How to cite this article: Mi H-Y, Jing X, Salick MR, Cordie TM, Peng X-F, Turng L-S. 2014. Properties and fibroblast cellular response of soft and hard thermoplastic polyurethane electrospun nanofibrous scaffolds. J Biomed Mater Res Part B 2014:00B:000–000.
INTRODUCTION
Tissue engineering is an interdisciplinary field that applies the principles of biology and engineering to the development of functional substitutes for damaged tissue.1 Tissue scaffolds act as extracellular matrices (ECMs) for living cells to grow on and provide support to damaged tissues, then subsequently degrade in vivo.2 Recently, fabricating viable scaffolds for tissue engineering is attracting more and more attention from both academia and industry. Many methods have been developed to produce biodegradable scaffolds including solvent casting/particulate leaching,3 thermally induced phase separation,4 electrospinning,5 rapid prototyping,6 batch foaming,7 and microcellular injection molding.8 Among these methods, electrospinning is widely used to produce nanofibers for tissue engineering applications due to its simple setup and its ability to generate fibers with
diameters that range from 50 nm to a few micrometers.9 This fibrous structure also leads to high scaffold interconnectivity and high surface areas. In electrospinning, a stream of polymer solution continuously flows out from a blunt needle which is connected to a high electrical charge. The polymer solution starts to form fibers when the electrostatic repulsion forces from the surface charges overcome the surface tension.10 To mimic the ECM, a scaffold has to possess the following characteristics: (1) a three-dimensional (3D), highly porous structure with an interconnected network to facilitate cell attachment, migration, proliferation, and differentiation as well as free transportation of nutrients, metabolic waste, and paracrine factors11; (2) be biocompatible and biodegradable with controllable degradation rates12; (3) have a suitable surface chemistry to support the
Additional Supporting Information may be found in the online version of this article. Correspondence to: L.-S. Turng; e-mail: [email protected] or X.-F. Peng; e-mail: [email protected] Contract grant sponsor: Wisconsin Institute for Discovery (WID) and China Scholarship Council; contract grant number: 51073061 Contract grant sponsor: National Nature Science Foundation of China; contract grant numbers: 51073061; 21174044 Contract grant sponsor: The Guangdong Nature Science Foundation; contract grant number: S2013020013855 Contract grant sponsor: The Fundamental Research Funds for the Central Universities; contract grant number: 2011ZZ0011 Contract grant sponsor: 973 Program; contract grant number: 2012CB025902
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abovementioned cell functions13; and (4) have ideal mechanical properties that match the surrounding tissue of the implantation site and provide support.11 Among these, scaffold surface chemistry has the most important impact on cellular behavior since it is the initial site that interacts with the cells. Wettability and protein absorption of the scaffolds are the most commonly used methods to characterize scaffold surface properties. Protein is an important factor in mediating cell and biomaterial interactions since the biological recognition of cells happens between the cells and the proteins adsorbed to the biomaterials.14 The wettability of the biomaterials can affect protein absorption significantly. However, contradictory opinions exist in understanding this relationship. It is widely accepted that hydrophilic surfaces tend to absorb water molecules and form a water layer rather than adsorb proteins,15 while on a hydrophobic surface, proteins may unfold and their hydrophobic side chains may come in contact with the surface16 and cause protein immobilization. However, many publications have reported that for tissue scaffold applications, hydrophilic materials display a higher affinity for cells.17,18 The hydrophilic scaffolds show higher protein absorption and cell reproduction, and can overcome the 3D scaffold flotation problem in culture media.19,20 Moreover, scaffolds with hydrophilic properties improved by the addition of chitosan,21 gelatin,22 or collagen23 showed enhanced biocompatibility. Therefore, it is worth putting more effort into further verifying the relationship between scaffold wettability and biocompatibility. Thermoplastic polyurethane (TPU) is a linear polymer that consists of soft and hard segments. TPU, as a class of PU, has been widely used in medical applications, mainly because of its biocompatibility, high fracture strain, moderate tensile strength, and excellent abrasion and tear resistances. In most TPUs, soft segments such as aliphatic polyether polyols or aliphatic polyester polyols are coupled with hard segments of diisocyanates, such as toluene diisocyanate, diphenylmethane diisocyanate, and 1,6-hexamethylene diisocyanate, followed by a chain extension with a diol in the presence of catalysts. Thus, the molecular weight and molecular structure of a TPU can be adjusted via the chemical synthesis formula and ratio of soft and hard segments. Through adjusting the combination of soft and hard segments, TPU can be synthesized into both hydrophilic and hydrophobic TPU. It is worth investigating the cellular response to electrospun TPU scaffolds with different wettability characteristics since, to the best of our knowledge, no studies have been done on this to date. Yuan and coworkers24 reported on a PU/polyethylene glycol vascular scaffold with improved hydrophilicity that proved suitable for endothelia cells attachment and proliferation. Kim et al.25 showed that hydrophilicity can be improved by adding gelatin into TPU, with the resulting scaffolds displaying better cell proliferation. However, this improvement of cell response could be attributed to either enhanced hydrophilicity or the bioactivity provided by the natural materials. In this study, hydrophilic and hydrophobic TPUs and their blends were electrospun to produce scaffolds with dif-
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TABLE I. Solution Formula Solution Formula Soft TPU (S) 25%H 50%H 75%H Hard TPU (H)
Concentration (wt./vol.) 20% 15% 15% 15% 10%
ferent properties. These scaffolds were fully characterized via Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM), water contact angle (WAC) tests, and protein absorption tests. Furthermore, the fibroblast cell response to these scaffolds was investigated by live/dead assay and 3-(4,5dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) assay. EXPERIMENTAL
Materials Two kinds of medical grade TPUs—Tecophilic SP-80A-150 (soft TPU, Mw 5 179464 Da; Mn 5 63,492 Da) and Carbothane PC-3572D (hard TPU, Mw 5 201,873 Da; Mn 5 94,212 Da)—were supplied by Lubrizol. The molecular weight of TPU was measured using gel permeation chromatography. The solvent used to dissolve TPU was N, N-dimethylformamide (DMF) purchased from Sigma-Aldrich. All materials were used as received. Electrospinning process. The TPU pellets were dried at 80 C for 2 h prior to solution preparation. Pure TPU and the soft/hard TPU mixtures were prepared at various ratios with different concentrations by dissolving TPU pellets in DMF for 8 h with magnetic stirring at 60 C. The solutions prepared are listed in Table I. The solution concentrations were determined based on a previous study to produce uniform, bead-less electrospun fibers.26 The prepared solution was loaded in a plastic syringe connected to an 18 gauge blunt-end needle mounted on a digital syringe pump. The electrospinning procedure was carried out using a voltage of 15 kV, a working distance of 150 mm, and a flow rate of 0.5 mL/h. The electrospun fibers were collected using a thin aluminum foil. The same conditions were used for all of the solutions in order to eliminate the effect of processing parameters. All of the solutions were used immediately after they were prepared to avoid possible phase separation of soft and hard TPUs. NIH 3T3 fibroblast cell culture. Prior to cell culture testing, 3T3 cells were maintained on 6-well tissue culture-treated polystyrene plates (BD Falcon). Cells were fed every other day with a high-glucose 20% serum medium consisting of high-glucose DMEM (Gibco), 20% fetal bovine serum (WiCell), 2 mM L-glutamine (Invitrogen), and penicillin– streptomycin (Invitrogen). Cells were passaged at a 1:40 ratio every 6 days via a 5-minute EDTA treatment (Invitrogen). The samples used for cell culture were directly
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FIGURE 1. X-ray photoelectron spectroscopy test results of soft (S) and hard (H) TPUs: (a) the survey scan of S and H, (b) the proportion of carbon peaks within S and H from C1 core-level signals for (c) S and (d) H. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
electrospun on sterilized stainless steel washers and then cut off from the electrospun membrane. The scaffolds were cultured with an inert metal fixture (washer) to prevent scaffold flotation so that the area of the cells could be determined. The casted pure soft (S) and hard (H) TPU films and tissue culture plates (TCPs) were tested as control groups. For the cell culture testing, samples were placed in 24-well tissue culture-treated polystyrene plates after UV light sterilization on each side (30 min per side). 3T3 cells were treated with EDTA for 5 min and washed with phosphatebuffered saline (PBS) prior to seeding. Cells were then seeded at a density of 1.25 3 105 cells/cm2. Spent medium was aspirated and replaced with 1 mL of fresh medium daily for screening samples. Characterization X-ray photoelectron spectroscopy. X-ray Photoelectron Spectroscopy (XPS) survey scans and C1 core-level signals were analyzed to identify the elements and the proportion of carbon bonds on the surface of soft and hard scaffolds in detail to get a clear understanding of the difference between these two materials. XPS measurements were performed on an X-ray photoelectron spectrometer with a focused, monochromatic K-alpha X-ray source and a monoatomic/cluster ion gun (Thermo Scientific). The spectra were Gaussian fit-
ted and the proportion of each bond was determined from the peak area ratios. Fourier transform infrared spectroscopy FTIR was used to identify the functional groups of the scaffolds. Each scaffold was folded twice to increase its thickness and measured with a Bruker Tensor 27 FTIR instrument. Samples were analyzed in the absorbance mode in the range of 600 to 4000 cm21. Thermogravimetric analysis. TGA was used to determine the portion of hard and soft segments in the scaffolds and their decomposition behavior. A sample of approximately 10 mg was used for the test. The test was initiated at room temperature and then equilibrated to 100 C to eliminate any water in the sample. The temperature was then increased to 600 C using a heating rate of 10 C/min on a TA Q50 instrument. The scaffold weight was measured against the temperature. Scanning electron microscopy The microstructure of electrospun nanofibers was characterized using a fully digital LEO GEMINI 1530 SEM with an accelerating voltage of 3 kV. The electrospun membranes were cut into small pieces with a scalpel and sputtered with
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FIGURE 2. FTIR test results of S, 25%H, 50%H, 75%H, and H electrospun scaffolds. Four areas were enlarged to show the intensity variation clearly. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
gold for 40 s before imaging. The fiber diameter of each scaffold, which was measured by Image-Pro Plus software, was the average value of 50 individual fibers. Differential scanning calorimetry. Differential scanning calorimetry (DSC) measurements were performed with a DSC Q20 (TA Instruments). Samples weighing 5 to 8 mg were measured and encapsulated in standard aluminum pans. Samples were then heated to 200 C and kept isothermal for 5 min, then cooled to 280 C and reheated to 200 C. The scanning rate of the test was 5 C/min and all tests were carried out under the protection of a nitrogen atmosphere. Wettability. Wettability of the electrospun fibers was characterized with WAC test which was performed at room temperature in a Dataphysics OCA 15 optical contact angle measuring system using the sessile drop method. Two microliters of deionized water were dropped on the scaffolds and images at 0 and 2 s were taken and measured. The WCA of each scaffold was the average value of three different spots. Protein absorption. The scaffolds were first sterilized by UV treatment, then specimen dimensions were determined. The specimens were washed three times with Dulbecco’s phosphate buffer saline (DPBS, Life Technologies), at 1 h intervals to remove any impurities. Samples were then placed into the 3T3 fibroblast culture medium for a predetermined amount of time (e.g., 15, 50, and 150 h) inside the cell culture incubator at 37 C. Following incubation, the samples were removed from the medium and placed into a fresh plate and washed once with DPBS. The amount of adsorbed protein on each specimen was measured using a Thermo ScientificTM PierceTM BCATM Protein Assay with the
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aid of a GloMax Multi1 Detection System (Promega). The tests were repeated three times for each sample. 3T3 fibroblast cell viability. Fibroblast cell viability was determined 3 days and 10 days after cell seeding. Viability was assessed via a Live/Dead Viability/Cytotoxicity Kit (Invitrogen). The stain utilized green fluorescent Calcein-AM to target living cells, red fluorescent ethidium homodimer-1 (EthD-1) to indicate cell death, and blue fluorescent DAPI to identify the cell nucleus. Stained cells were imaged with a Nikon Ti-E Confocal microscope. Advanced Research v.3.22 software was used for image analysis. To assess the percent of live cells, the stained cells were detached using TrypLE (Life Technologies) for 5 min at 37 C and then collected and centrifuged at 200 rpm for 5 min. The supernatant was aspirated and the cells resuspended in 600 lL of PBS and filtered prior to analysis. The percentage of green fluorescent cells was acquired with an Accuri C6 flow cytometer (BD Biosciences). Cell fixation for SEM. The same samples used for cell viability testing were rinsed twice with PBS. Samples were submerged in a 4% paraformaldehyde solution (electron microscopy solutions) for 30 min. The samples were then dehydrated using a series of ethanol washes (50, 90, and 100% for 30 min each), and finally dried in a vacuum desiccator for 2 to 3 h before gold sputtering for SEM imaging. MTS assay. The CellTiter 96 Aqueous One Solution Cell Proliferation Assay (Promega) was used to determine the number of cells on the scaffolds. Standard curves were established by performing the tests on cells seeded on the cell culture wells and confirmed by comparison to hemocytometer readings prior to testing. Upon testing, cells were treated with an 83% media, 17% MTS solution and allowed
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FIGURE 4. TGA results of S, 25%H, 50%H, 75%H, and H electrospun scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
RESULTS
FIGURE 3. SEM images of electrospun scaffolds: (a) S, (b) 25%H, (c) 50%H, (d) 75%H, and (e) H. Left images are at low magnification with a scale bar of 20 lm; right images are at high magnification with a scale bar of 2 lm. The insets show the fiber diameter distribution. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
to incubate for exactly one hour. One hundred microliters of spent media were removed and added to a clear 96-well plate. The absorbance of this plate at the 450 nm wavelength was read using a GloMax-Multi 1 Multiplate Reader (Promega) and the subsequent number of cells was determined relative to the negative control. Statistical analysis. All biological experiments contain at least three samples. Statistical analysis was performed using one-way analysis of variance (ANOVA) and the difference significance between groups was compared as well, with the level of significance set at p < 0.05 and p < 0.01.
Scaffold component characterization The XPS results on S and H TPUs are shown in Figure 1, which characterized the basic differences between S and H. Figure 1(a) shows that both TPUs contain C, O, and N elements, while, for S TPU, the ratio of O was higher and the ratio of N was lower than that of H TPU according to the intensity of C1, O1, and N1 peaks. Furthermore, the fitted C1 peak results [Figure 1(c,d)] and the proportion of carbon peak results [Figure 1(b)] also showed that the carbon–oxygen bonds that belonged to the polyol soft segments had higher ratio in S TPU, while the carbon–nitrogen bonds that belonged to the isocyanate hard segments were dominant in H TPU.27,28 The FTIR results, which are shown in Figure 2, showed that soft and hard TPUs have the same peaks, although the peak intensities were different, thus indicating different component ratios. As one can see, the peak intensity of amide I (1692 cm21), amide II (1530 cm21), and amide III (1245 cm21) groups29 that belong to the hard segment of TPU increased with increasing hard TPU content in the sample. On the contrary, the ether group (CAOAC) at 1245 cm21, which existed in the soft segments of the TPU molecular chain,30 increased with increasing soft TPU content in the scaffold. These results suggest that the proportion of soft and hard segments in the scaffolds can be adjusted effectively by varying the soft and hard TPU blending ratio. In addition, the peak of absorbed water located around 3550 cm21 became higher as the soft segment content increased, hence suggesting that the moisture absorbed by the scaffold increased. Morphology The morphologies of the electrospun scaffolds are shown in Figure 3. The distribution of the fiber diameter was uniform, with most fiber diameters ranging from 200 to 800 nm. At the same solution concentration [Figure 2(b–d)], the fiber diameter increased as the hard segment content increased in the solution. It was found in a previous study26
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FIGURE 5. Differential scanning calorimetry results of S, 25%H, 50%H, 75%H, and H electrospun scaffolds: (a) second heating period and (b) first cooling period. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
that increasing both the solution concentration and the hard segment proportion of the solution could improve the TPU molecular chain entanglement. This is important because once the chain entanglement becomes larger than a critical value, continuous uniform fiber structures can be obtained. Furthermore, it was noticed that the fiber surface of the S (soft TPU) scaffold [Figure 3(a)] was smooth, while the fiber surface became rougher as the hard segments content increased, especially in the H (hard TPU) scaffold [Figure 3(e)]. This was because of the increase in the rigid hard segment portion in the fiber. Thermal properties The thermal properties of the scaffolds were evaluated via TGA and DSC tests. As shown in Figure 4, all of the scaffolds started to decompose at about the same temperature (280 C). The H scaffold lost weight dramatically as the temperature increased because the amide bonds in the hard segments were easier to cleave than the ether bonds in the soft segments as the temperature increased.31 In addition, the weight loss of the scaffolds had two obvious steps that belonged to the decomposition of the hard and soft segments. The transition points of the two steps indicated the weight portion of the soft and hard segments in the scaffolds. It was noticed again that the proportion of hard segments in the scaffolds increased with increasing hard TPU content, and that the scaffolds had better thermal stability with more soft segments.
Figure 5 shows the DSC test results of the electrospun scaffolds. It was found that both soft and hard TPU were amorphous without obvious melting peaks and crystallization peaks. The H scaffold showed a wide glass transition region from 258 to 85 C, while it was 254 to 218 C for the S scaffold. All of the blended scaffolds showed two glass transition slopes, which indicating the low miscibility between these two kinds of TPUs. The peaks initiated at 0 C in both first cooling and the second heating periods were due to the existence of moisture in the scaffolds. The water content increased as the soft segment proportion increased, corresponding to the FTIR results. Wettability The wettability of scaffolds is an important characteristic that determines cell adhesion. As shown in Figure 6, the H scaffold was hydrophobic with a WCA constantly over 120 , while the S scaffold was highly hydrophilic with a WAC reduced to 0 in 2 s after the water bead was dropped on the scaffold. The scaffolds blended by S and H showed higher WCAs with more hard segments, and that angle reduced over time. Protein absorption The S, 50%H, and H scaffolds were chosen to evaluate the protein absorption as proteins always mediate the interactions between the cell and the biomaterial. As shown in Figure 7, all of the scaffolds absorbed more proteins as time passed. The S scaffold absorbed more protein than the
FIGURE 6. WAC test results of S, 25%H, 50%H, 75%H, and H electrospun scaffolds at 0 and 2 s after the water bead was dropped on the scaffold.
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FIGURE 7. Protein absorption test results of S, 50%H, and H electrospun scaffolds at 15, 50, and 150 h time points. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
50%H and H scaffolds over all three time periods. Furthermore, the protein concentration for the S scaffold increased more from 50 to 150 h, while for the H scaffold, it increased more from 12 to 50 h. This may indicate that the S scaffold can absorb protein continuously into the scaffold, while the H scaffold adsorbed protein only on the scaffold surface. 3T3 fibroblast cell culture To further investigate the cellular response to the TPU scaffolds prepared, 3T3 fibroblast cells were cultured on S,
50%H, and H scaffolds for 10 days and imaged at day 3 and day 10 time points. The number of cells was measured by MTS assay at days 3, 6, and 10. Day 3 results are shown in Figure 8, from which it was found that the cells formed clusters on the S [Figure 8(a)] and 50%H [Figure 8 (b)] scaffolds, while they spread out very well on the H [Figure 8(c)] scaffolds. In addition, most cells on the scaffolds were alive at day 3, as indicated by large areas of green fluorescence and few red spots. Individual cell morphologies can be seen from the SEM images. As one can see, the cells spread only on the H scaffold [Figure 8(f)], while cells on the S and 50%H scaffolds presented round shapes and aggregated into clusters. A similar cell response was observed at day 10 as shown in Figure 9. It can be seen from Figure 9(a–c) that the cells were denser and covered a larger area. The 50%H scaffold showed a larger cell area than the S scaffold. More dead cells were observed in all scaffolds compared to the day 3 results, especially the S scaffold. The increase in cell death may have been due to an increase in cell competition as the cells proliferated. From the SEM images, it was found that cells still agglomerated into clusters and presented round shapes on the S and 50%H scaffolds, while spreading into elongated shapes on the H scaffolds. In addition, the absence of fibrous structures in the S scaffolds may have been caused by the ethanol dehydration procedure when preparing the SEM sample since soft TPU swells in ethanol. The control groups showed a similar cell response to the materials. The cells formed clusters on the casted S film and
FIGURE 8. Day 3 cell culture fluorescence images (a–c) and SEM images (d–f) of S [(a) and (d)], 50%H [(b) and (d)], and H [(c) and (f)] electrospun scaffolds. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
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FIGURE 9. Day 10 cell culture fluorescence images (a–c) and SEM images (d–f) of S [(a) and (d)], 50% H [(b) and (d)], and H [(c) and (f)] electrospun scaffolds. Separated fluorescence of image (a) indicates (g) cell nucleus, (h) live cells, and (i) dead cells. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
spread out on the casted H film and the TCPs, as shown in the Supporting Information Figure S1. The cell viability results from the live/dead assays and the number of cells from the MTS assay are shown in Figure 10(a,b), respectively. The cell viability showed the same trend at day 3 and day 10, which was that the H scaffolds had higher cell viability than the S and 50% H scaffolds, as well as that of the S and H film controls. The S scaffold seemed to perform better than the S film, although the difference was not statistically significant. The cell proliferation results showed that the number of cells increased as time passed. It was also noticed that at each time period, the H scaffold had significantly more cells than the 50% H scaffold, which had more
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cells than the S scaffold. At day 10, the H scaffold had significantly more cells than the H film, although the difference was not significant at day 3. It was found that the TCP control group presented the highest cell viability and cell proliferation at all time points due to its specially treated surface. Therefore, the statistical comparisons to the TCP groups in the ANOVA test were not shown in the figure so as to emphasize the important comparison groups. The results from these tests suggested that the cells could reproduce better on hydrophobic H scaffolds with a higher viability than on the S scaffolds and the casted films. For the same material, a fibrous structure was superior for cells than a flat film due to the higher aspect ratio of the fibers.
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FIGURE 10. (a) Cell viability and (b) number of cells at day 3 and day 10 of S, 50% H, and H scaffolds and control groups of S films, H films, and TCPs. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]
DISCUSSION
The scaffold is a crucial part in the tissue engineering process. Preparation of scaffolds with proper attributes is important to in vitro and in vivo cell culture. Different properties of scaffolds can determine the cellular response in terms of cell adhesion, proliferation, differentiation, and migration.13,32 The substrate properties that affect the cellular response include the mechanical properties33; surface chemistry and topography (wettability, softness, stiffness, and roughness)34; and microstructure (porosity, pore size, pore shape, and interconnectivity).35,36 In this study, the TPU electrospun scaffolds were characterized with SEM, WCA tests, and protein absorption tests. The ability of the scaffolds to absorb proteins is important as the amount of serum proteins—such as fibronectin and vitronectin— absorbed by the scaffold affects cell adhesion significantly.37 The scaffold with more soft segments showed higher hydrophilicity (Figure 6) and higher protein absorption (Figure 7) than those with more hard segments. References 18, 19, and 38 suggested that elevated hydrophilicity and protein absorption would result in improved cell proliferation. The cell number from the MTS assay (Figure 10), however, showed lower cell numbers on scaffolds with more soft segments. Similar decrease of goat bone marrow stromal cell proliferation as scaffold hydrophilicity increase has been reported.39 To investigate the cause of cell death on scaffold S at day 10, the colors in Figure 9(a) were separated to indicate cell nuclei (blue), live cells (green), and dead cells (red), as shown in Figure 9(g–i). It was noticed from the figure that most dead cells were located in the center of the cluster and were surrounded by live cells. Therefore, the cells in the center of the cluster died because they could not obtain enough nutrients from the cell culture media. In fact, it has been found that poor interactivity between the substrate and the cells, as well as the low amount of surfaceimmobilized, cell-attachment proteins, would lead to a balled up cell morphology and the formation of cell clusters.40 The substrate hydrophilicity is one of the main concerns that affect the interactivity between cells and
substrate. The favorable hydrophilicity of the substrate varied with different cell types; neither highly hydrophilic nor highly hydrophobic surfaces were favorable for cells. For example, the best WCA for endothelial cell attachment and proliferation was about 50 , while it was 76 for chondrocytes.34,41 Cui et al.38 reported that the poly(lactic acid)/chitosan scaffolds with WCA of 0 improved human dermal fibroblasts proliferation dramatically. However, Cheung et al.42 recently found that a polar hydrophobic polyurethane scaffold enhanced human gingival fibroblast proliferation. In this study, hydrophobic hard TPU performed better than hydrophilic soft TPU in terms of cell attachment and proliferation for both electrospun fibers and casted films, while the electrospun fibers were superior to the casted films for the same material. Since the cells formed similar aggregates on the S scaffold and the casted S film, and formed similar spread shapes on both the H scaffold and the H film, the effect of structural differences can be disregarded. Therefore, the main reason might be the proteins had been absorbed inside of the hydrophilic soft TPU, while a water layer formed on the scaffold surface.15 The formed water layer prevented cell attachment and spreading, hence leading to the formation of ball-like shapes and cell clusters. On the contrary, the scaffolds that had more hard segments possessed hydrophobic surfaces that enabled the proteins to be adsorbed on the scaffold surface, even though the proteins could not penetrate into the scaffold. The adsorbed proteins facilitated cell attachment and migration. During long culture times, the cells on the H scaffold divided and migrated, covering the whole surface. On the S scaffold, however, cells were more likely to stick to each other when they divided, which led to cell agglomeration and cell death in the center of the agglomerate.
CONCLUSIONS
Soft and hard TPU and their blends were electrospun to fabricate scaffolds for tissue engineering applications. The properties of these scaffolds were characterized by FTIR,
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SEM, TGA, DSC, WCA tests, and protein absorption tests. The 3T3 fibroblast cell response was investigated via live/ dead assay and MTS assay. It was found that the hard segment content in the scaffolds increased as the hard TPU proportion increased in the blends, while scaffold decomposition was delayed as the soft segment content increased. Scaffolds with more hard segments showed higher hydrophobicity and lower protein absorption than those with more soft segments. Cell culture tests proved that cells exhibited round shapes and aggregated into clusters on the scaffolds with more soft segments, even though those scaffolds were highly hydrophilic and absorbed more proteins. On the contrary, scaffolds with more hard segments had more cells, and the cells spread into spindle shapes. This was because the hydrophilic soft TPU absorbed proteins inside of the scaffold, meanwhile, a water layer was form on the scaffold surface which prevented cell attachment. The hard TPU, on the other hand, adsorbed proteins on the scaffold surface and showed better cell interactions and higher cell viability and cell proliferation.
REFERENCES 1. Langer R, Vacanti JP. Tissue Engineering. Science 1993;260:920– 926. 2. Lee J, Cuddihy MJ, Kotov NA. Three-dimensional cell culture matrices: State of the art. Tissue Eng Part B-Rev 2008;14:61–86. 3. Mikos AG, Thorsen AJ, Czerwonka LA, Bao Y, Langer R, Winslow DN, Vacanti JP. Preparation and characterization of poly(L-lactic acid) foams. Polymer 1994;35:1068–1077. 4. Nakamatsu J, Torres FG, Troncoso OP, Yuan ML, Boccaccini AR. Processing and characterization of porous structures from chitosan and starch for tissue engineering scaffolds. Biomacromolecules 2006;7:3345–3355. 5. Wang L, Shi J, Liu L, Secret E, Chen Y. Fabrication of polymer fiber scaffolds by centrifugal spinning for cell culture studies. Microelectron Eng 2011;88:1718–1721. 6. Park SA, Kim HJ, Lee SH, Lee JH, Kim HK, Yoon TR, Kim W. Fabrication of nano/microfiber scaffolds using a combination of rapid prototyping and electrospinning systems. Polym Eng Sci 2011;51: 1883–1890. 7. Corre YM, Maazouz A, Duchet J, Reignier J. Batch foaming of chain extended PLA with supercritical CO2: Influence of the rheological properties and the process parameters on the cellular structure. J Supercrit Fluids 2011;58:177–188. 8. Kramschuster A, Turng LS. An injection molding process for manufacturing highly porous and interconnected biodegradable polymer matrices for use as tissue engineering scaffolds. J Biomed Mater Res Part B Appl Biomater 2010;92B:366–376. 9. Zhang XH, Cai Q, Liu HY, Zhang S, Wei Y, Yang XP, Lin YH, Yang ZG, Deng XL. Calcium ion release and osteoblastic behavior of gelatin/beta-tricalcium phosphate composite nanofibers fabricated by electrospinning. Mater Lett 2012;73:172–175. 10. Geng XY, Kwon OH, Jang JH. Electrospinning of chitosan dissolved in concentrated acetic acid solution. Biomaterials 2005;26: 5427–5432. 11. Hutmacher DW. Scaffold design and fabrication technologies for engineering tissues—State of the art and future perspectives. J Biomater Sci-Polym Ed 2001;12:107–124. 12. Kweon H, Yoo MK, Park IK, Kim TH, Lee HC, Lee HS, Oh JS, Akaike T, Cho CS. A novel degradable polycaprolactone networks for tissue engineering. Biomaterials 2003;24:801–808. 13. Mandal BB, Kundu SC. Cell proliferation and migration in silk fibroin 3D scaffolds. Biomaterials 2009;30:2956–2965. 14. Hynes RO. Integrins—Versatility, modulation, and signaling in cell-adhesion. Cell 1992;69:11–25.
10
MI ET AL.
15. Elbert DL, Hubbell JA. Surface treatments of polymers for biocompatibility. Annu Rev Mater Sci 1996;26:365–394. 16. Horbett TA, Lew KR. Residence Time effects on monoclonalantibody binding to adsorbed fibrinogen. J Biomater Sci-Polym Ed 1994;6:15–33. 17. Lampin M, WarocquierClerout R, Legris C, Degrange M, SigotLuizard MF. Correlation between substratum roughness and wettability, cell adhesion, and cell migration. J Biomed Mater Res Part A 1997;36:99–108. 18. Mohan N, Nair PD. Polyvinyl alcohol-poly(caprolactone) semi IPN scaffold with implication for cartilage tissue engineering. J Biomed Mater Res Part B-Appl Biomater 2008;84B:584–594. 19. Oh SH, Kang SG, Kim ES, Cho SH, Lee JH. Fabrication and characterization of hydrophilic poly(lactic-co-glycolic acid)/poly(vinyl alcohol) blend cell scaffolds by melt-molding particulate-leaching method. Biomaterials 2003;24:4011–4021. 20. Niu XF, Li XM, Liu HF, Zhou G, Feng QL, Cui FZ, Fan YB. Homogeneous chitosan/poly(L-lactide) composite scaffolds prepared by emulsion freeze-drying. J Biomater Sci-Polym Ed 2012;23:391–404. 21. Lin HY, Chen HH, Chang SH, Ni TS. Pectin-chitosan-PVA nanofibrous scaffold made by electrospinning and its potential use as a skin tissue scaffold. J Biomater Sci-Polym Ed 2013;24:470–484. 22. Binulal NS, Natarajan A, Menon D, Bhaskaran VK, Mony U, Nair SV. Gelatin nanoparticles loaded poly(epsilon-caprolactone) nanofibrous semi-synthetic scaffolds for bone tissue engineering. Biomed Mater 2012;7. 23. Lee H, Kim G. Three-dimensional plotted PCL/beta-TCP scaffolds coated with a collagen layer: preparation, physical properties and in vitro evaluation for bone tissue regeneration. J Mater Chem 2011;21:6305–6312. 24. Wang HY, Feng YK, Fang ZC, Yuan WJ, Khan M. Co-electrospun blends of PU and PEG as potential biocompatible scaffolds for small-diameter vascular tissue engineering. Mater Sci Eng CMater Biol Appl 2012;32:2306–2315. 25. Kim SE, Heo DN, Lee JB, Kim JR, Park SH, Jeon S, Kwon IK. Electrospun gelatin/polyurethane blended nanofibers for wound healing. Biomed Mater 2009;4. 26. Mi HY, Jing X, Jacques BR, Turng LS, Peng XF. Characterization and properties of electrospun thermoplastic polyurethane blend fibers: Effect of solution rheological properties on fiber formation. J Mater Res 2013;28:2339–2350. 27. Mishra AK, Chattopadhyay DK, Sreedhar B, Raju KVSN. FT-IR and XPS studies of polyurethane-urea-imide coatings. Prog Org Coat 2006;55:231–243. 28. Wang TL, Yu CC, Yang CH, Shieh YT, Tsai YZ, Wang NF. Preparation, characterization, and properties of polyurethane-grafted multiwalled carbon nanotubes and derived polyurethane nanocomposites. J Nanomater 2011;2011: 814903. 29. Pinto UA, Visconte LLY, Nunes RCR. Mechanical properties of thermoplastic polyurethane elastomers with mica and aluminum trihydrate. Eur Polym J 2001;37:1935–1937. 30. Dong ZH, Li YB, Zou Q. Degradation and biocompatibility of porous nano-hydroxyapatite/polyurethane composite scaffold for bone tissue engineering. Appl Surf Sci 2009;255:6087–6091. 31. Buczek O, Krowarsch D, Otlewski J. Thermodynamics of single peptide bond cleavage in bovine pancreatic trypsin inhibitor (BPTI). Protein Sci 2002;11:924–932. 32. Mandal BB, Kundu SC. Osteogenic and adipogenic differentiation of rat bone marrow cells on non-mulberry and mulberry silk gland fibroin 3D scaffolds. Biomaterials 2009;30:5019–5030. 33. Engler AJ, Sen S, Sweeney HL, Discher DE. Matrix elasticity directs stem cell lineage specification. Cell 2006;126:677–689. 34. Arima Y, Iwata H. Effect of wettability and surface functional groups on protein adsorption and cell adhesion using welldefined mixed self-assembled monolayers. Biomaterials 2007;28: 3074–3082. 35. Chang H-I, Wang Y. Cell responses to surface and architecture of tissue engineering scaffolds. In: Eberli D, editor. Regenerative Medicine and Tissue Engineering - Cells and Biomaterials. Rijeka, Croatia: In Tech; 2011. pp 569–588 36. O’Brien FJ, Harley BA, Yannas IV, Gibson LJ. The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 2005;26:433–441.
PROPERTIES AND CELL RESPONSE STUDY OF ELECTROSPUN TPU SCAFFOLDS
ORIGINAL RESEARCH REPORT
37. Steele JG, Johnson G, Underwood PA. Role of serum vitronectin and fibronectin in adhesion of fibroblasts following seeding onto tissue-culture polystyrene. J Biomed Mater Res 1992;26:861–884. 38. Cui WG, Cheng LY, Li HY, Zhou Y, Zhang YG, Chang J. Preparation of hydrophilic poly(L-lactide) electrospun fibrous scaffolds modified with chitosan for enhanced cell biocompatibility. Polymer 2012;53:2298–2305. 39. Ivirico JLE, Salmeron-Sanchez M, Ribelles JLG, Pradas MM, Soria JM, Gomes ME, Reis RL, Mano JF. Proliferation and differentiation of goat bone marrow stromal cells in 3D scaffolds with tunable hydrophilicity. J Biomed Mater Res Part B-Appl Biomater 2009;91B:277–286.
40. Ishihara K, Ishikawa E, Iwasaki Y, Nakabayashi N. Inhibition of fibroblast cell adhesion on substrate by coating with 2methacryloyloxyethyl phosphorylcholine polymers. J Biomater Scie-Polym Ed 1999;10:1047–1061. 41. Zhu HG, Ji J, Lin RY, Gao CY, Feng LX, Shen JC. Surface engineering of poly(DL-lactic acid) by entrapment of alginate-amino acid derivatives for promotion of chondrogenesis. Biomaterials 2002;23:3141–3148. 42. Cheung JWC, Rose EE, Santerre JP. Perfused culture of gingival fibroblasts in a degradable/polar/hydrophobic/ionic polyurethane (D-PHI) scaffold leads to enhanced proliferation and metabolic activity. Acta Biomater 2013;9:6867–6875.
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