Fabrication of Porous Poly (ε-caprolactone) Scaffolds Containing

Article pubs.acs.org/IECR

Fabrication of Porous Poly(ε-caprolactone) Scaffolds Containing Chitosan Nanofibers by Combining Extrusion Foaming, Leaching, and Freeze-Drying Methods Xin Jing,†,‡ Hao-Yang Mi,†,‡ Travis Cordie,‡,§ Max Salick,‡,⊥ Xiang-Fang Peng,*,† and Lih-Sheng Turng*,‡,∥ †

The Key Laboratory of Polymer Processing Engineering of Ministry of Education, National Engineering Research Center of Novel Equipment for Polymer Processing, South China University of Technology, Guangzhou, China, 510641 ‡ Wisconsin Institutes for Discovery, §Department of Biomedical Engineering, ∥Department of Mechanical Engineering, and ⊥ Department of Engineering Physics, University of WisconsinMadison, Madison, Wisconsin 53706, United States ABSTRACT: Biomimetic porous poly(ε-caprolactone) (PCL) scaffolds containing chitosan nanofibers (CSNF) were produced by using a hybrid process that combines extrusion foaming, water-soluble phase leaching, and freeze-drying. The high porosity and interconnectivity of the scaffolds were achieved by supercritical fluid foaming and the addition of water-soluble poly(ethylene oxide) (PEO) and sodium chloride (NaCl) as porogens. The porosity of the scaffolds after leaching was over 78%. The structure of the chitosan nanofibers introduced into the micropores of the scaffolds was similar to the collagen bundles in the extracellular matrix (ECM). The chitosan nanofibers enhanced the compressive modulus of the scaffolds slightly and improved the water uptake ability by as much as 35% compared to the scaffolds without chitosan. A 3T3 fibroblast cell culture was performed to investigate the biocompatibility of the scaffolds, which revealed that the cells proliferated better on the PCL/chitosan scaffolds than on the PCL scaffolds. Regarding cell morphology, cells on the PCL scaffolds were rounded in the form of tumoroids, suggesting poor cell adhesion and poor cell−scaffold interactions. In contrast, cells on the PCL/chitosan scaffolds were elongated and spindle-shaped, indicating favorable cell−scaffold interactions. Therefore, the highly porous PCL scaffolds containing chitosan nanofibers fabricated in this study have the potential to be used in tissue engineering applications.

1. INTRODUCTION Tissue engineering, which aims to develop biological substitutes that restore, maintain, or improve tissue function, has been attracting more and more attention in recent years.1 This technique has been successfully used to improve the recovery of many kinds of tissues such as skin, bones, blood vessels, and nerve conduits.2 Three-dimensional polymeric scaffolds are essential in tissue engineering as they are the foundation for cell attachment and subsequent tissue formation.3−5 Ideally, the scaffold should have pores that are open, interconnected, and hierarchical in size, and have a uniform distribution throughout the scaffold, all while maintaining the required mechanical properties.6 Open interconnected pores allow cells to infiltrate the scaffold and also allow for the mass transport of cell nutrients and waste. Hierarchical in size refers to variable pore sizes on different scales: macrosized pores (>50 μm) influence tissue shape, microsized pores (1−50 μm) influence cell function, and nanosized pores (1−1000 nm) influence nutrient diffusion.7 A large variety of approaches have been developed to produce porous scaffoldssuch as electrospinning, phase separation, particulate (particle) leaching, three-dimensional (3D) printing, and gas foamingand extensive reviews are available detailing these methods.8−11 Polymer leaching is an attractive and cost-effective technique to produce porous scaffolds that can yield an interconnected porous structure. This method mainly focuses on blending a water-soluble polymer with the scaffold matrix materials; a porous structure can then be created after leaching the water© 2014 American Chemical Society

soluble phase such as poly(vinyl alcohol) (PVA) and poly(ethylene oxide) (PEO).12−14 However, this technique is limited to a maximum porosity of 50−60%. Combining polymer leaching and particulate leaching can yield better pore size control, which enables the fabrication of scaffolds with multimodal pore size distributions. Recently, Reignier et al. combined cocontinuous extrusion blending using two biodegradable polymers, poly(ε-caprolactone) (PCL) and poly(ethylene oxide) (PEO), with salt particulate leaching to prepare porous scaffolds. After extraction of the continuous PEO and mineral salts, highly porous PCL scaffolds with fully interconnected pores were created. Human dura mater stem cells were cultured on the prepared PCL scaffolds for 3 weeks and the results showed that the scaffolds could support proper proliferation and differentiation for cell growth, demonstrating the potential application of PCL porous scaffolds in bone tissue engineering. However, in this technique, during extrusion blending, the salts might be broken by the shear forces during blending, thus causing smaller and unpredictable pore sizes in the scaffolds.15 The Botchwey group16 also did a similar study. Instead of using salt as porogens, they employed the batch foaming technique combined with extruded PCL and watersoluble PEO blends to fabricate porous PCL scaffolds. Received: Revised: Accepted: Published: 17909

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PEO was chosen as a sacrificial polymer due to its water solubility and similarity in melting temperature to that of PCL. PEO, with an average molecular weight of 100 000, was purchased in powder form from Sigma-Aldrich. It has a specific gravity of 1.13 and a melting temperature of 65 °C. 2.1.3. Sodium Chloride (NaCl). On the basis of previous reports,29,30 NaCl was used as the particulates. NaCl (reagent grade) was purchased from Sigma-Aldrich with a specific gravity of 2.17. Its high melting temperature (801 °C) means that it will not melt at normal processing conditions used in this study (i.e., under 150 °C). 2.1.4. Supercritical N2. Nitrogen (N2) was chosen as the blowing agent for the extrusion foaming process. It has a lower solubility in most polymers and yielded a finer porous structure in the foaming process.31 2.1.5. Chitosan (CS). Chitosan with medium molecular weight (degree of deacetylation, 75−85%) was provided by Sigma-Aldrich. CS was used in this study to create a nanofibrous structure in the porous PCL scaffolds. 2.2. Methods. Before compounding, PCL and PEO were dried in a vacuum oven at 40 °C for 12 h. NaCl particles were sieved to a size range of 150−425 μm using a sieve shaker (H4328, Humboldt Mfg). The sieved NaCl was then dried at 150 °C for 2 h before using. According to the viscosity of PCL and PEO, and the rheological viscosity−volume fraction equation proposed by Jordhamo et al.,32 a cocontinuous structure should be obtained with a composition containing approximately equal amounts of PCL and PEO (i.e., 50/50% PCL/PEO). On the other hand, the porous scaffolds should maintain their mechanical strength. Therefore, three kinds of blends with different PCL/PEO mass ratios were investigated in this study to prepare the cocontinuous scaffolds. After choosing suitable compositions for the blends, 10 wt % of NaCl pellets was added to the prepared PCL/PEO blends by extrusion compounding and foaming to create an interconnected pore network. The extrusion compounding and foaming were performed on a twin-screw extruder (Leistritz ZSE 18 HPe) equipped with a supercritical gas supply system. The optimized processing parameters, which are listed in Table 1, were used in the

Although toxic solvents were absent in their technique, the batch foaming in the second step was time-consuming and hard to scale up. Therefore, in our study, a new method was developed where the polymer blend extrusion method was followed by continuous gas foaming and particulate leaching to create highly porous scaffolds with an optimal pore size distribution. Since the blending and molding of the scaffolds (prior to leaching) can be made using mass-production polymer processing equipment, this process provides promise for the large scale production of porous scaffolds of many sizes in a cost-effective way. Recently, it was found that nanofibrous structures mimic collagen fibers in natural extracellular matrix (ECM) and can promote cell attachment, proliferation, migration, and differentiation. A series of nanofibrous matrixes using natural or synthetic polymers have been investigated, such as poly(Llactide) (PLLA), PCL, gelatin, collagen, etc.17−21 However, nanoscale matrixes usually show much lower mechanical properties than scaffolds with a solid wall structure due to their lack of integrity, which is a big drawback for nanofibrous scaffolds in clinical applications. Moreover, cells may struggle to attach and proliferate on the scaffolds prepared with synthetic polymers due to the lacking of integrin binding on the surface of the scaffolds.22 Therefore, a natural biocompatible polymer that is rich in integrin for cell attachment needs to be introduced into the micropores in traditional porous scaffolds to mimic the three-dimensional collagen nanofibrous network structure. Natural polymer chitosan has been widely used as an important tissue engineering scaffold material in recent years.23−25 As a unique cationic polysaccharide, chitosan has characteristics of outstanding biocompatibility, biodegradability, bioadhesion, and nontoxicity. In addition, chitosan has a structure similar to that of glucosaminoglycan, which is an important constituent of ECM and plays a key role in modulating cell morphology and cell functions.26,27 Furthermore, it has been reported that chitosan nanofibers with diameters of 50−500 nm have been prepared by thermally induced phase separation.28 The purpose of this study is to evaluate the feasibility of a preparation method combining extrusion foaming and particulate leaching for the preparation of highly interconnected three-dimensional polymeric scaffolds with controlled pore sizes. The porosity, pore size, and morphology of the scaffolds will be addressed. Moreover, to further improve the biocompatibility of the PCL scaffolds prepared using this approach, chitosan nanofibers (CSNF) were introduced into the micropores of the scaffolds through freeze-drying (also known as thermally induced phase separation). The mechanical properties and water uptake abilities of the scaffolds with and without chitosan nanofibers were investigated, and the biocompatibility was evaluated in vitro by 3T3 fibroblast cell culture.

Table 1. Processing Parameters Used in the Extrusion Compounding and Foaming Process

a

key param

value

composition of blend (PCL/PEO) temperature screw speed gas content

70/30, 60/40, 50/50 (w/w) 90/95/95/95/95/95/100a 100 rad/min 0.5%

Sequence from hopper to die.

extrusion foaming process to achieve the proper pore structure for tissue engineering scaffold applications. The effects of the extrusion foaming conditions on the porous structure have been extensively investigated elsewhere.33−37 The foamed samples with different compositions were then transferred to individual containers filled with deionized water with magnetic rotation at 300 rad/min to leach out the watersoluble PEO and NaCl. The deionized water was changed every 4 h. Three days later, the washed samples were dried in a vacuum oven for 24 h and porous PCL scaffolds were obtained. Finally, in order to introduce chitosan nanofibers (CSNF) into the porous PCL scaffolds, the prepared scaffolds were soaked in a chitosan solution (0.01% w/v), which was prepared

2. EXPERIMENTAL SECTION 2.1. Materials. 2.1.1. Poly(ε-caprolactone) (PCL). PCL (Capa 6500 from Perstorp U.K. Ltd.), a biocompatible and biodegradable polymer, was used as the matrix material in this study. It has a melt flow index of around 7 g/10 min (160 °C/ 2.16 kg). Its glass transition temperature is −60 °C, with a melting temperature of 58−60 °C and a specific gravity of 1.10. 2.1.2. Poly(ethylene oxide) (PEO). In order to create the cocontinuous network to connect the pores in the PCL matrix, 17910

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Figure 1. Preparation of PCL/CSNF scaffolds by gas foaming/particulate leaching and the thermally induced phase separation technique.

700 °C at a heating rate of 10 °C/min. The flow rate of the protection gas (nitrogen) was 60 mL/min. 2.3.5. Porosity. In accordance with ref 38, the porosities of the leached samples were determined by weighing them and measuring the dimensions of the weighed samples to obtain the volume. Equation 2 was then applied to determine the porosity. The reported values are the average of five tests for each group.

by dissolving 10 mg of chitosan in 100 mL (0. 5% v/v) of acetic acid aqueous solution at room temperature overnight. Then the PCL/chitosan composite was removed from the chitosan solution and immersed in liquid nitrogen immediately for 1 h. The PCL/CSNF scaffolds were obtained after 24 h of lyophilization. The whole process of preparing foamed PCL/ CSNF scaffolds is shown in Figure 1. The prepared scaffolds measured 8 mm × 5 mm (diameter × thickness). 2.3. Characterization. 2.3.1. Rheology. The viscosities of the two materials were tested via a rheometer (AR 2000ex) to determine the compositions of the cocontinuous blends composed of PCL and PEO. A 25 mm parallel-plate geometry was used, and the tests were carried out at 120 °C. The complex viscosity was investigated with an increase of angular frequency from 0.1 to 200 rad/s. 2.3.2. Morphological Analysis. The morphology of the samples was evaluated using a scanning electron microscope (SEM, LEO 1530) operated at an accelerating voltage of 3 kV. Prior to observation by SEM, the samples were cryogenically fractured using liquid nitrogen and then sputtered with gold for 40 s. ImageJ software was used to determine the average pore size and help with the calculation of the pore density. Three SEM images of each sample group were used to obtain the average diameter of the pores. Equation 1 was used to calculate the volumetric pore density: pore density = (N /A)3/2

porosity = (Vthρ − Wm)/Vthρ · 100%

(2)

where Wm was the measured weight, ρ was the known density of the PCL matrix, and Vth was the volume of the measured samples. 2.3.6. Water Uptake. Water uptake tests were carried out as previously reported.39 Briefly, the prepared porous PCL and PCL/CSNF scaffolds were placed into phosphate buffered saline (PBS) for 2, 4, 6, 12, and 24 h at 37 °C until equilibrium. The swollen samples were weighed after removal of excess surface water with filter paper. Water uptake of each scaffold was calculated using eq 3. water uptake = (Ws − Wd)/Wd·100%

(3)

where Ws was the weight of the swollen scaffolds and Wd was the weight of dry scaffolds. 2.3.7. Mechanical Properties. The mechanical properties of the scaffolds were characterized via compression tests. These tests were performed on five cylinder specimens of each type using a universal testing machine (Instron 5967, USA) with a 250 N load cell. Each specimen was compressed to a strain of −50% at a rate of 5 mm/min. The reported values are the average of five tests for each sample group. All of these tests were carried out at ambient temperature (25 °C). The compressive modulus was evaluated from the entire linear elastic region of the stress−strain curve. 2.4. Biocompatibility Characterizations. 2.4.1. Cell Culture. In order to investigate their biocompatibility and explore their potential to be used as tissue engineering scaffolds, the prepared scaffolds were subjected to preliminary cytotoxicity screening. Swiss mouse NIH 3T3 ECACC (European Collection of Cell Cultures) fibroblasts were used for the biological assays. Cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen),

(1)

where N was the number of pores and A was the area of the SEM image. 2.3.3. Differential Scanning Calorimetry (DSC). Thermal property measurements were performed with a DSC Q20 (TA Instruments). Samples were heated to 150 °C at a heating rate of 5 °C/min and held isothermally for 5 min to eliminate any prior thermal history. Samples were then cooled to −100 °C at 5 °C/min and then reheated to 150 °C at the same rate. All tests were carried out under the protection of nitrogen atmosphere. 2.3.4. Thermogravimetric Analysis (TGA). The residual NaCl content in the scaffolds was analyzed using a thermogravimetric analyzer (TGA; TA Instruments Q500). After drying in an oven, the samples were heated from 30 to 17911

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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 six-well tissue culture-treated polystyrene (TCPS) plates (BD Falcon) prior to testing. The medium was replaced every other day, 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 composite scaffolds were exposed to sterilizing UV light for 30 min on each side and placed into 96well TCPS plates before cell seeding. 3T3 cells were dissociated with trypsin−EDTA for 5 min prior to seeding. The cell suspension was then seeded onto the surface of the sterilized scaffolds at a density of 20 000/well in the high-glucose 3T3 medium. The medium was replaced daily for screening samples 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 2 and 7 days after seeding. This kit allows simultaneous visualization of both live and dead cells. The stain utilizes 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 A1Rsi Ti-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 (Electron Microscopy Sciences) 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, 70, 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. Flow Cytometry. The proliferation of cells on the scaffolds was investigated after 2 and 7 days of seeding. The seeded samples were transferred to new well plates to ensure that the cells collected were not growing on the surface of the well plates. Then the transferred samples were washed twice with phosphate buffered saline (PBS), and 250 μL of Trypsin (Life Technologies) per well was used for 5 min at 37 °C to detach the cells. After incubation, the cells were collected and centrifuged at 1000 rpm for 5 min. The supernatant was then aspirated and the cells were resuspended in 600 μL of PBS and filtered prior to analysis. Data were acquired with an Accuri C6 (BD Biosciences). 2.5. Statistical Analysis. All data presented are expressed as mean ± standard deviation (SD). Statistical analysis was carried out using single-factor analysis of variance (ANOVA). A value of p < 0.05 and p < 0.01 were considered statistically significant.

Figure 2. Complex viscosity of pure PCL and PEO at 120 °C as a function of frequency.

about 50 s−1, the two viscosity curves crossed each other and the PCL/PEO viscosity ratio was around 1.2 at 100 s−1. According to the Cox−Merz rule40 and a vast majority of theoretical models for the prediction of phase inversion points, the cocontinuous blend morphology can be found in a concentration range that depends on the viscosity ratio between the polymeric blend components. In our study, according to the rheology test results, the complex viscosity differences between the blend components were relatively modest and a phase inversion point close to the 50/50 (by weight ratio) may be expected. Therefore, it is expected that the blend with 50:50 PCL:PEO would show a cocontinuous structure. 3.2. Morphology of the Foamed PCL/PEO Blends. To verify the above expectation, three different compositions including PCL/PEO = 70/30, PCL/PEO = 60/40, and PCL/ PEO = 50/50 were chosen for this study. Figure 3 shows the morphology of extrusion foamed PCL/PEO blends with different compositions after leaching for 3 days. No salt particles were used in these samples. The number of larger pores increased as PEO increased in the blends. Porous channels could be seen throughout all of the blends; however, the channels became longer with an increase of PEO in the blends. In the PCL/PEO 70/30 blends (Figure 3a,d), the porous channels were not uniformly distributed throughout the samples, which might indicate that some of the PEO phase was encapsulated by the PCL phase and was not leached out. Compared with PCL/PEO 60/40 (Figure 3b,e) blends, interconnected channels were formed in the PCL/PEO 50/ 50 (Figure 3c,f) blends after the removal of PEO. Channels on the pore walls were visible indicating the formation of a cocontinuous structure, which corresponded to the expectation in the rheology test. 3.3. Detect Residual PEO in the Blends after Leaching. To further verify the removal of PEO in the blends with different compositions, DSC tests were carried out to detect the residual PEO phase; the results are shown in Figure 4. As shown in Figure 4a, the crystallinity peak of neat PCL was around 35 °C while the second crystallinity peak belonged to PEO, which indicates that the two polymers in the study are immiscible. After 3 days of leaching, in the PCL/PEO 50/50 blend, the second peak of crystallinity disappeared, demonstrating that the PEO in the blend was completely leached out. However, for the PCL/PEO 60/40 and PCL/PEO 70/30 blends, tiny peaks in the curves can still be seen, indicating that some PEO remained in the leached samples. Therefore, by SEM observation and DSC tests, we chose PCL/PEO 50/50 as

3. RESULTS AND DISCUSSION 3.1. Determination of the Composition of Cocontinuous Blends. The complex viscosity of the neat materials at 120 °C is shown in Figure 2. PCL showed a well-defined Newtonian plateau at low frequency, while PEO demonstrated shear-thinning behavior in the probed frequency range. At 17912

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Figure 3. Porous PCL/PEO blends with different compositions after leaching: (a, d) PCL/PEO 70/30, (b, e) PCL/PEO 60/40, and (c, f) PCL/ PEO 50/50.

Figure 4. DSC results of the blends before and after leaching. (a) Cooling segments of DSC results before leaching and (b) cooling segments of DSC results after leaching.

the master batch to do the following experiments and further tests. 3.4. Morphology of the Prepared PCL/PEO/NaCl Samples. For the PCL/PEO 50/50 foamed sample after leaching, even though interconnected channels were formed, the porosity and the interconnections among pores remained limited (recall Figure 3c). Therefore, 10 wt % of NaCl particles was added into the PCL/PEO 50/50 blends as additional porogens to further enhance pore interconnectivity, and then the PCL/PEO/NaCl blends were foamed via extrusion foaming followed by leaching for 3 days. Figure 5 shows the morphology of the foamed PCL/PEO/NaCl blends before and after leaching. Before leaching, it can be seen from Figure 5a,b that the pores created by gas foaming were uniform with an average pore diameter of 230 μm, and the NaCl particles were inlaid in the pores and on the interfaces among the pores. After leaching, as shown in Figure 5c,d, the pore size became larger (265.9 μm) and many of the micropores became connected due to the removal of NaCl particles at the pore interfaces (as shown in the green circles) as well as the PEO phase. From Table 2, it was found that the addition of NaCl caused smaller pore diameters and much larger pore densities and larger porosities in the scaffolds after leaching. This was because, during the extrusion process, the strong shear forces of the extruder broke some of the salt particles and those small particles acted as nucleating agents during the foaming process, resulting in smaller pores in the scaffolds.

Figure 5. (a, b) SEM images at two different magnifications of the surface of PCL/PEO/NaCl blends before dissolution of PEO and NaCl. (c, d) SEM results at two different magnifications of the surface of porous PCL scaffolds after extraction of PEO and NaCl. In all blends, the PCL/PEO composition ratio was kept constant at 50/50 by mass ratio.

3.5. Leaching Effect on PCL/PEO/NaCl Scaffolds. To characterize the residual content of NaCl in the blends after leaching, TGA tests were performed on the neat PCL, PEO, 17913

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polymers such as collagen, gelatin, and chitosan, PCL often suffers from poor cell adhesion due to its hydrophobicity and its lack of cell recognition sites.41 In order to take advantage of both natural and synthetic polymers, chitosan nanofibers (CSNF) were introduced into the prepared porous PCL scaffolds via the freeze-drying method. Different chitosan solutions (0.05% w/v, 0.02% w/v, and 0.01% w/v) were tried to optimize the chitosan concentration for preparing PCL/ CSNF scaffolds. The results are shown in Figure 7. It was found that the morphology of chitosan changed from an aligned macroporous structure to a fibrous structure when the chitosan concentration decreased from 0.05 to 0.01%. Therefore, it is believed that the concentration of the chitosan solution plays a major role in the formation of the different structures. In Figure 7c, a three-dimensional nanofibrous chitosan network with a fiber diameter ranging from 10 to 1000 nm was formed within the micropores of the PCL scaffold, indicating that the chitosan nanofibers dispersed well over the whole scaffold, showing a weblike structure present both in the pores and on the pore interfaces as shown in Figure 7d. This kind of fibrous structure mimics the structure of collagen nanofibers in natural ECM and is believed to be favorable for cell attachment, proliferation, and differentiation.42 Based on the aim of this study, 0.01% w/v chitosan solution was chosen for the following experiments. 3.7. Compressive Properties of the Scaffolds with or without Chitosan Nanofibers. Compression tests were employed to evaluate the mechanical strength of the tissue engineering scaffolds, and the results are shown in Figure 8. It can be seen that the scaffolds with and without chitosan nanofibers showed similar stress−strain curve patterns and the scaffolds with chitosan nanofibers had higher compressive moduli than the scaffolds without chitosan nanofibers due to the presence of the additional fibrous structures. These results indicate that the introduction of chitosan nanofibers to the micropores of PCL could slightly improve the compressive properties of the porous PCL scaffolds. 3.8. Water Uptake Ability of the Scaffolds. The water uptake ability of a scaffold is an important factor for cell seeding, which affects the cell distribution throughout the whole scaffold and the transfer efficiency of nutrition and wastes in the scaffold. Water uptake test results are shown in

Table 2. Pore Size, Pore Density, and Porosity of the Scaffoldsa sample

composition

pore diam (μm)

pore density

porosity (%)

1 2

PCL/PEO PCL/PEO/NaCl

334.9 265.9

1.3 × 104 3.1 × 104

77.9 ± 0.2 86.4 ± 1.1

a

All samples used were already leached in deionized water.

and PCL/PEO/NaCl scaffolds before and after leaching as shown in Figure 6. The curves indicated that the onset

Figure 6. TGA curves of neat PCL, PEO, PCL/PEO, and PCL/PEO/ NaCl before and after leaching.

decomposition temperature of neat PEO was lower than that of neat PCL. After extrusion foaming, the onset decomposition temperature of the blends was between the two cocontinuous phases of PCL and PEO, indicating that NaCl had a minimal effect on the thermal properties of the blends. Because the decomposition temperature of NaCl was higher than 800 °C, the residual of the burnt samples was caused by NaCl content, which was about 10 wt %. After leaching, the TGA curve of the leached samples almost overlapped with the neat PCL curve, indicating that there was almost no NaCl residual in the porous PCL scaffolds after leaching. 3.6. Morphology of the Scaffolds after Chitosan Nanofibers Introduction. Even though PCL is one of the promising biodegradable polymers, compared with natural

Figure 7. SEM images of PCL/CSNF scaffolds from freeze-drying chitosan solutions with different concentrations: (a) 0.05% (w/v), (b) 0.02% (w/ v), and (c) 0.01% (w/v) chitosan in 0.5% (v/v) acetic acid; (d) and (e) are higher magnifications of image (c). 17914

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Figure 8. Mechanical tests of prepared scaffolds: (a) typical stress−strain curves of the scaffolds and (b) compressive modulus of the tested scaffolds.

Figure 9, from which it can be seen that the incorporation of chitosan into the PCL scaffolds (PCL/CSNF) improved the

Figure 9. Water uptake ability of porous PCL scaffolds with and without chitosan nanofibers (CSNF).

water uptake ability significantly compared with the PCL scaffolds without chitosan. Initially, after 1 h of incubation, the water absorbed by PCL/CSNF scaffold was 35% higher than the PCL scaffold, which was attributed to the introduction of the chitosan and the enhanced porosity. The hydrophilicity of chitosan would cause the chitosan to swell in the water, which would increase the water uptake of the scaffolds. Furthermore, the enhanced porosity of the PCL/CSNF scaffolds would be more favorable for water penetration than the PCL scaffolds. Therefore, the PCL/CSNF scaffolds were able to absorb more water than the PCL scaffolds. As the incubation time became longer, the water uptake rate increased for both scaffolds. After 5 h of incubation, both scaffolds showed a similar pattern of slow water uptake due to the slow water absorption of PCL. 3.9. Cell Viability and Proliferation. After 2 and 7 days of in vitro cell culture, cell viability assays were performed on the PCL and PCL/CSNF scaffolds; the results are shown in Figure 10. After 2 days of culture, most cells on both PCL and PCL/ CSNF scaffolds were alive, as indicated by the green fluorescence spots, and only a few dead cells were observed, which were presented as red fluorescence spots. Some chitosan nanofibers on the PCL/CSNF scaffolds appeared as purple and light red strips according to Figure 10b. After being cultured for 7 days, there were more and denser cells on both scaffolds. Most cells were still alive, indicating that both scaffolds showed good biocompatibility. Furthermore, the cells on the PCL/ CSNF scaffolds were larger and more elongated than those on the PCL scaffolds. These observations demonstrated that the

Figure 10. 3T3 fibroblast cell culture results: (a) PCL and (b) PCL/ CSNF after 2 days culture, and (c) PCL and (d) PCL/CSNF after 7 days culture. Green indicates living cells and red indicates dead cells.

introduction of chitosan was helpful for cell attachment and growth. To further investigate the influence of chitosan on the cell proliferation, a flow cytometer was employed to count the number of live cells on the scaffolds. Figure 11 shows the 3T3

Figure 11. Comparison of 3T3 cell proliferation on porous PCL and PCL/CSNF scaffolds at day 2 and day 7. Asterisk (∗): p < 0.05 and (**): p < 0.01. 17915

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cell proliferation on the PCL and PCL/CSNF scaffolds at day 2 and day 7 culture time points. The results demonstrated that the cells proliferated faster on the PCL/CSNF scaffolds than on the PCL scaffolds, and the differences were statistically significant at day 7. These results revealed the positive effect of chitosan nanofibers within the PCL scaffolds on cell survival and cell growth. This could be attributed to the resemblance of the chitosan nanofiber structure to native ECM structure, as well as the hydrophilicity provided by the chitosan.43 3.10. Cell Morphology on the Scaffolds. Scaffold properties play a pivotal role in controlling cell growth and morphology, and have a direct influence on intracellular responses. From the SEM results shown in Figure 12, after 2

Figure 13. SEM results of 3T3 cells cultured on scaffolds after 7 days: (a, b) PCL at different magnifications and (c, d) PCL/CSNF at different magnifications.

ratio to achieve two continuous phases was obtained by rheological study, and the high pore interconnection was verified by SEM observation. NaCl particles were added into the PCL/PEO blends to further improve the porosity and pore interconnectivity. DSC and TGA results proved that the PEO and NaCl could be leached out effectively. Chitosan nanofibers were introduced into the fabricated PCL scaffolds using the freeze-drying method to mimic the structure of native ECM fibrillar collagen and improve the biocompatibility of the scaffolds. The addition of chitosan nanofibers enhanced the compressive modulus slightly and increased the water uptake rate of the scaffolds. The in vitro cell culture results demonstrated that the scaffolds containing chitosan nanofibers had better biocompatibility compared to those without chitosan, which might be ascribed to the biomimetic chitosan nanofibers that promoted cell adhesion and proliferation. The proposed method in this study provided a new green tissue engineering scaffold (e.g., bone tissue) fabrication method and combined the advantages of natural and synthetic materials to create structures similar to those in an in vivo environment.

Figure 12. SEM results of 3T3 cells cultured on scaffolds after 2 days: (a, b) PCL at different magnifications and (c, d) PCL/CSNF at different magnifications.

days of culture, cells proliferated and migrated through the pores and covered a large area of the scaffolds, especially on the PCL/CSNF scaffold. From the high magnification images, it was observed that the cells on the PCL/CSNF scaffold presented a predominantly fusiform shape and bridged the micropores with their pseudopodia (Figure 12b), while the cells on the PCL scaffold presented a round shape without clear cell pseudopodia (Figure 12d). After 7 days of culture, the cell coverage area became larger for both scaffolds according to the low magnification images, thus indicating cell reproduction. The cells cultured on the porous PCL scaffolds (Figure 13b) showed a rounded morphology and aggregated into cell clusters. Instead of spreading out on the substrate, the cells grew on top of each other, showing poor interactions with the substrate material. On the other hand, the cells on the PCL/ CSNF scaffolds (Figure 13d) connected with each other to form stratified cell layers, where each cell was fully stretched out and spread into a flat shape with their pseudopodia attached to the substrate or other cells. These results suggest that the introduction of chitosan nanofibers into the PCL scaffolds greatly improved cellular interactions with the substrate scaffolds, as confirmed by the favorable cell morphology.

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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 Scholarship 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 the National Basic Research Development Program 973 (No. 2012CB025902) in China.

4. CONCLUSIONS In this study, an environmentally friendly scaffold fabrication method was developed by combining extrusion foaming, leaching, and freeze-drying methods. The optimal PCL/PEO 17916

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caprolactone) and polysaccharides in tissue engineering applications. Biomacromolecules 2005, 6, 1961. (23) Thein-Han, W. W.; Misra, R. D. K. Biomimetic chitosannanohydroxyapatite composite scaffolds for bone tissue engineering. Acta Biomater. 2009, 5, 1182. (24) Huang, Y.; Onyeri, S.; Siewe, M.; Moshfeghian, A.; Madihally, S. V. In vitro characterization of chitosan-gelatin scaffolds for tissue engineering. Biomaterials 2005, 26, 7616. (25) Jana, S.; Florczyk, S. J.; Leung, M.; Zhang, M. Q. High-strength pristine porous chitosan scaffolds for tissue engineering. J. Mater. Chem. 2012, 22, 6291. (26) Nishikawa, H.; Ueno, A.; Nishikawa, S.; Kido, J.; Ohishi, M.; Inoue, H.; Nagata, T. Sulfated glycosaminoglycan synthesis and its regulation by transforming growth factor-beta in rat clonal dental pulp cells. J. Endod. 2000, 26, 169. (27) Yayon, A.; Klagsbrun, M.; Esko, J. D.; Leder, P.; Ornitz, D. M. Cell-Surface, Heparin-Like Molecules Are Required for Binding of Basic Fibroblast Growth-Factor to Its High-Affinity Receptor. Cell 1991, 64, 841. (28) Zhao, J. H.; Han, W. Q.; Chen, H. D.; Tu, M.; Zeng, R.; Shi, Y. F.; Cha, Z. G.; Zhou, C. R. Preparation, structure and crystallinity of chitosan nano-fibers by a solid-liquid phase separation technique. Carbohydr. Polym. 2011, 83, 1541. (29) Mikos, A. G.; Thorsen, A. J.; Czerwonka, L. A.; Bao, Y.; Langer, R.; Winslow, D. N.; Vacanti, J. P. Preparation and Characterization of Poly(L-Lactic Acid) Foams. Polymer 1994, 35, 1068. (30) Ma, P. X.; Choi, J. W. Biodegradable polymer scaffolds with well-defined interconnected spherical pore network. Tissue Eng. 2001, 7, 23. (31) Di Maio, E.; Mensitieri, G.; Iannace, S.; Nicolais, L.; Li, W.; Flumerfelt, R. W. Structure optimization of polycaprolactone foams by using mixtures of CO2 and N2 as blowing agents. Polym. Eng. Sci. 2005, 45, 432. (32) Jordhamo, G. M.; Manson, J. A.; Sperling, L. H. Phase Continuity and Inversion in Polymer Blends and Simultaneous Interpenetrating Networks. Polym. Eng. Sci. 1986, 26, 517. (33) Tai, H. Y.; Mather, M. L.; Howard, D.; Wang, W. X.; White, L. J.; Crowe, J. A.; Morgan, S. P.; Chandra, A.; Williams, D. J.; Howdle, S. M.; Shakesheff, K. M. Control of pore size and structure of tissue engineering scaffolds produced by supercritical fluid processing. Eur. Cells Mater. 2007, 14, 64. (34) Salerno, A.; Guarnieri, D.; Iannone, M.; Zeppetelli, S.; Netti, P. A. Effect of Micro- and Macroporosity of Bone Tissue ThreeDimensional-Poly(epsilon-Caprolactone) Scaffold on Human Mesenchymal Stem Cells Invasion, Proliferation, and Differentiation In Vitro. Tissue Eng., Part A 2010, 16, 2661. (35) Salerno, A.; Zeppetelli, S.; Di Maio, E.; Iannace, S.; Netti, P. A. Novel 3D porous multi-phase composite scaffolds based on PCL, thermoplastic zein and ha prepared via supercritical CO2 foaming for bone regeneration. Compos. Sci. Technol. 2010, 70, 1838. (36) Xu, Z. M.; Jiang, X. L.; Liu, T.; Hu, G. H.; Zhao, L.; Zhu, Z. N.; Yuan, W. K. Foaming of polypropylene with supercritical carbon dioxide. J. Supercrit. Fluids 2007, 41, 299. (37) Arora, K. A.; Lesser, A. J.; McCarthy, T. J. Preparation and characterization of microcellular polystyrene foams processed in supercritical carbon dioxide. Macromolecules 1998, 31, 4614. (38) Mi, H. Y.; Salick, M. R.; Jing, X.; Jacques, B. R.; Crone, W. C.; Peng, X. F.; Turng, L. S. Characterization of thermoplastic polyurethane/polylactic acid (TPU/PLA) tissue engineering scaffolds fabricated by microcellular injection molding. Mater. Sci. Eng., C 2013, 33, 4767. (39) Jiao, Y. P.; Liu, Z. H.; Zhou, C. R. Fabrication and characterization of PLLA-chitosan hybrid scaffolds with improved cell compatibility. J. Biomed. Mater. Res., Part A 2007, 80A, 820. (40) Kitano, T.; Nishimura, T.; Kataoka, T.; Sakai, T. Correlation of Dynamic and Steady Flow Viscosities of Filled Polymer Systems. Rheol. Acta 1980, 19, 671. (41) He, W.; Ma, Z. W.; Yong, T.; Teo, W. E.; Ramakrishna, S. Fabrication of collagen-coated biodegradable polymer nanofiber mesh

REFERENCES

(1) Langer, R.; Vacanti, J. P. Tissue Engineering. Science 1993, 260, 920. (2) Mi, H. Y.; Salick, R. M.; Jing, X.; Jacques, R. B.; Wendy, C. C.; Peng, X. F.; Turng, L.-S. Characterization of thermoplastic polyurethane/polylactic acid (TPU/PLA) tissue engineering scaffolds fabricated by microcellular injection molding. Mater. Sci. Eng., C 2013, 33, 4767. (3) O’Brien, F. J. Biomaterials & scaffolds for tissue engineering. Mater. Today 2011, 14, 88. (4) Hutmacher, D. W. Scaffold design and fabrication technologies for engineering tissuesstate of the art and future perspectives. J. Biomater. Sci., Polym. Ed. 2001, 12, 107. (5) Ma, P. X. Biomimetic materials for tissue engineering. Adv. Drug Delivery Rev. 2008, 60, 184. (6) Hollister, S. J. Porous scaffold design for tissue engineering. Nat. Mater. 2005, 4, 518. (7) Bonfield, W. Designing porous scaffolds for tissue engineering. Philos. Trans. R. Soc., A 2006, 364, 227. (8) Hutmacher, D. W. Scaffolds in tissue engineering bone and cartilage. Biomaterials 2000, 21, 2529. (9) Karageorgiou, V.; Kaplan, D. Porosity of 3D biomaterial scaffolds and osteogenesis. Biomaterials 2005, 26, 5474. (10) Cheung, H. Y.; Lau, K. T.; Lu, T. P.; Hui, D. A critical review on polymer-based bio-engineered materials for scaffold development. Composites, Part B 2007, 38, 291. (11) Agrawal, C. M.; Ray, R. B. Biodegradable polymeric scaffolds for musculoskeletal tissue engineering. J. Biomed Mater. Res. 2001, 55, 141. (12) Mi, H. Y.; Jing, X.; Salick, M. R.; Turng, L. S.; Peng, X. F. Fabrication of thermoplastic polyurethane tissue engineering scaffold by combining microcellular injection molding and particle leaching. J. Mater. Res. 2014, 29, 911. (13) Cui, Z. X.; Nelson, B.; Peng, Y. Y.; Li, K.; Pilla, S.; Li, W. J.; Turng, L. S.; Shen, C. Y. Fabrication and characterization of injection molded poly (epsilon-caprolactone) and poly (epsilon-caprolactone)/ hydroxyapatite scaffolds for tissue engineering. Mater. Sci. Eng., C 2012, 32, 1674. (14) Kramschuster, A.; Turng, L. S. 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 2010, 92B, 366. (15) Reignier, J.; Huneault, M. A. Preparation of interconnected poly(epsilon-caprolactone) porous scaffolds by a combination of polymer and salt particulate leaching. Polymer 2006, 47, 4703. (16) Aronin, C. E. P.; Cooper, J. A.; Sefcik, L. S.; Tholpady, S. S.; Ogle, R. C.; Botchwey, E. A. Osteogenic differentiation of dura mater stem cells cultured in vitro on three-dimensional porous scaffolds of poly(epsilon-caprolactone) fabricated via co-extrusion and gas foaming. Acta Biomater. 2008, 4, 1187. (17) McManus, M. C.; Boland, E. D.; Simpson, D. G.; Barnes, C. P.; Bowlin, G. L. Electrospun fibrinogen: Feasibility as a tissue engineering scaffold in a rat cell culture model. J. Biomed Mater. Res., Part A 2007, 81A, 299. (18) Cipitria, A.; Skelton, A.; Dargaville, T. R.; Dalton, P. D.; Hutmacher, D. W. Design, fabrication and characterization of PCL electrospun scaffoldsa review. J. Mater. Chem. 2011, 21, 9419. (19) Chong, E. J.; Phan, T. T.; Lim, I. J.; Zhang, Y. Z.; Bay, B. H.; Ramakrishna, S.; Lim, C. T. Evaluation of electrospun PCL/gelatin nanofibrous scaffold for wound healing and layered dermal reconstitution. Acta Biomater. 2007, 3, 321. (20) Matthews, J. A.; Wnek, G. E.; Simpson, D. G.; Bowlin, G. L. Electrospinning of collagen nanofibers. Biomacromolecules 2002, 3, 232. (21) Prabhakaran, M. P.; Venugopal, J.; Ramakrishna, S. Electrospun nanostructured scaffolds for bone tissue engineering. Acta Biomater. 2009, 5, 2884. (22) Ciardelli, G.; Chiono, V.; Vozzi, G.; Pracella, M.; Ahluwalia, A.; Barbani, N.; Cristallini, C.; Giusti, P. Blends of poly-(epsilon17917

dx.doi.org/10.1021/ie5034073 | Ind. Eng. Chem. Res. 2014, 53, 17909−17918

Industrial & Engineering Chemistry Research

Article

and its potential for endothelial cells growth. Biomaterials 2005, 26, 7606. (42) Chu, X. H.; Shi, X. L.; Feng, Z. Q.; Gu, Z. Z.; Ding, Y. T. Chitosan nanofiber scaffold enhances hepatocyte adhesion and function. Biotechnol. Lett. 2009, 31, 347. (43) Zhao, J. H.; Han, W. Q.; Chen, H. D.; Tu, M.; Huan, S. W.; Miao, G. Q.; Zeng, R.; Wu, H.; Cha, Z. G.; Zhou, C. R. Fabrication and in vivo osteogenesis of biomimetic poly(propylene carbonate) scaffold with nanofibrous chitosan network in macropores for bone tissue engineering. J. Mater. Sci.: Mater. Med. 2012, 23, 517.

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