Colloids and Surfaces B: Biointerfaces 132 (2015) 177–184
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Preparation and properties of PLGA nanofiber membranes reinforced with cellulose nanocrystals Yunfei Mo a,b , Rui Guo a,b,∗ , Jianghui Liu c , Yong Lan a,b , Yi Zhang a,b , Wei Xue a,b , Yuanming Zhang d,∗∗ a
Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China c Department of Emergency, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China d Department of Chemistry, Jinan University, Guangzhou 510632, China b
a r t i c l e
i n f o
Article history: Received 12 February 2015 Received in revised form 13 May 2015 Accepted 15 May 2015 Available online 23 May 2015 Keywords: Cellulose nanocrystals (CNCs) Polylactide–polyglycolide (PLGA) Nanofibers Mechanical properties Biocompatibility Tissue engineering
a b s t r a c t Although extensively used in the fields of drug-carrier and tissue engineering, the biocompatibility and mechanical properties of polylactide–polyglycolide (PLGA) nanofiber membranes still limit their applications. The objective of this study was to improve their utility by introducing cellulose nanocrystals (CNCs) into PLGA nanofiber membranes. PLGA and PLGA/CNC composite nanofiber membranes were prepared via electrospinning, and the morphology and thermodynamic and mechanical properties of these nanofiber membranes were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). The cytocompatibility and cellular responses of the nanofiber membranes were also studied by WST-1 assay, SEM, and confocal laser scanning microscopy (CLSM). Incorporation of CNCs (1, 3, 5, and 7 wt.%) increased the average fiber diameter of the prepared nanofiber membranes from 100 nm (neat PLGA) to ∼400 nm (PLGA/7 wt.% CNC) and improved the thermal stability of the nanofiber membranes. Among the PLGA/CNC composite nanofiber membranes, those loaded with 7 wt.% CNC nanofiber membranes had the best mechanical properties, which were similar to those of human skin. Cell culture results showed that the PLGA/CNC composite nanofiber membranes had better cytocompatibility and facilitated fibroblast adhesion, spreading, and proliferation compared with neat PLGA nanofiber membranes. These preliminary results suggest that PLGA/CNC composite nanofiber membranes are promising new materials for the field of skin tissue engineering. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering, an important emerging topic in biomedical engineering, has shown tremendous promise in creating biological alternatives for harvested tissues, implants, and prostheses [1]. Three elements, namely cells, scaffolds, and biomolecules, play determinant roles in tissue regeneration [2]. Using nanofibers as scaffolds or films in tissue engineering is of particular interest because these materials mimic the structure of native extracellular matrix (ECM) proteins, which provide a platform for cell adhesion, differentiation, and proliferation [3]. The nanofibers can be
∗ Corresponding author at: Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China. Tel.: +86 20 85224338; fax: +86 20 85224338. ∗∗ Corresponding author. Tel.: +86 20 85224338; fax: +86 20 85224338. E-mail addresses: [email protected] (R. Guo), [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.colsurfb.2015.05.029 0927-7765/© 2015 Elsevier B.V. All rights reserved.
produced in several ways, including self-assembly [4], phase separation [5], template-assisted synthesis [6], and electrospinning [7]. Electrospinning is an especially versatile technique because it can be used to fabricate a variety of polymeric nanofiber membranes and is able to generate continuous fibers having diameters from a few nanometers to several micrometers [8,9]. Ideal nanofiber membranes prepared via electrospinning should simulate the ECM in structure and function to promote tissue regeneration and facilitate cell differentiation and proliferation [10,11]. To achieve these goals, several synthetic and natural polymers have been successfully electrospun into nanofibrous scaffolds or films to act as base matrices. These polymers include poly(l-lactide) (PLA) [12], poly(εcaprolactone) (PCL) [13], cellulose acetate (CA) [14], polyvinyl acetate (PVA) [15], poly(ethylene oxide) (PEO) [16], chitosan [17], and collagen [18,19]. The synthetic polymer poly(lactic-co-glycolic acid) (PLGA) has excellent biocompatibility and a controllable degradation rate that
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can be adjusted by modifying its crystal structure [20,21]. Scaffolds or films fabricated with PLGA have found widespread application in tissue engineering such as for the regeneration and repair of skin, bone, and nerves. However, PLGA is still not suitable for use in many tissue engineering situations because of its limited mechanical strength and hydrophobicity, which depresses cell adhesion and proliferation [22]. Extensive research has been conducted to improve its hydrophilicity and mechanical properties. A nanofibrous matrix of PLGA/type I collagen produced via electrospinning showed improved hydrophilicity and has been used in biomimetic scaffolds [10]. Chitosan-graft-PLGA (CS-graft-PLGA) produced by electrospinning had improved hydrophilicity and protein absorption, with acceptable mechanical properties [10]. Electrospun PLGA and nano-hydroxyapatite (HA) had reduced chain mobility, which improved the mechanical properties [23]. Cellulose nanocrystals (CNCs) obtained by sulfuric acid hydrolysis of native cellulose have excellent mechanical properties such as high strength (∼7 GPa), low extension to break, high aspect ratio, high surface area, and biocompatibility [24]. CNCs have recently attracted the interest of researchers in the fields of tissue engineering and regenerative medicine for use as scaffolds or drug-delivery systems. CNCs dispersed within a cellulose acetate propionate (CAP) matrix formed a three-dimensional rigid percolating network, which was contemplated for use as a scaffold in small-diameter vascular grafts [25]. Huang et al. [26] prepared electrospun silk fibroin nanofiber mats reinforced with CNCs; the tensile strength and Young’s modulus of these mats were almost twice those of unreinforced silk fibroin mats when the CNC content was 2 wt.%. Enhanced mechanical properties were also reported for bacterial cellulose nanocrystals, which acted as a nanofiller, in silk fibroin nanofiber membranes [27]. Collagen/CNC multilayered films have been proposed for layer-by-layer (LBL) assembly as a way to maximize the interaction between the two structural materials [28]. Collagen-based composite films reinforced with CNCs designed by our group had mechanical properties that were significantly better than those of pure collagen film. In vitro 3T3 fibroblasts culture results revealed that the composite films were not cytotoxic and facilitated cell adhesion [29]. CNC-reinforced collagen scaffolds containing gelatin microspheres (GMs) capable of releasing basic fibroblast growth factors (bFGF) have also been fabricated. Those collagen/CNCs/bFGF–GMs scaffolds contained a significantly higher number of newly formed and mature blood vessels, which suggested great potential in skin tissue engineering [30]. In this work, electrospinning was used to fabricate PLGA nanofiber membranes reinforced with biodegradable CNCs derived from microcrystalline cellulose (MCC). The morphology, hydrophilicity, thermodynamic, and mechanical properties of the composite nanofiber membranes were investigated. Cell–nanofiber interactions were also studied using 3T3 fibroblasts cultured on these nanofiber membranes.
2.2. Preparation of cellulose nanocrystals (CNCs) CNCs were prepared by acid hydrolysis of MCC according to a previously described procedure [31]. In brief, 10 g of MCC powder was added to 95 mL of 65 wt.% sulfuric acid under vigorous mechanical stirring. The hydrolysis was performed at 55 ◦ C for 5 h, and the mixture was diluted fivefold to quench the hydrolysis reaction. The resulting suspension was centrifuged at 5000 rpm for 10 min to separate the crystals, which were then washed and then treated ultrasonically for 15 min. The precipitate was further dialyzed against distilled water until the water pH reached 7.0. Finally, dialysis for 1 week against deionized water with a Viskase 3500 MWCO dialysis membrane was performed to remove residual sulfuric acid. 2.3. Transmission electron microscopy (TEM) Drops of aqueous dispersions of CNCs (0.01%, w/v) were deposited on carbon-coated electron microscope grids (Protochips Inc.), negatively stained with uranyl acetate, and allowed to dry. The grids were observed with a Hitachi HF-2000 transmission electron microscope operated at an accelerating voltage of 20 kV. 2.4. Electrospinning For the electrospinning of PLGA reinforced with CNCs, the CNCs were dispersed in a 15% (w/v) PLGA solution (HFIP solvent) at loads corresponding to 1, 3, 5, and 7 wt.% (based on the PLGA weight). The needle was connected to the high voltage supply, which could generate positive DC voltages and current up to 15 kV. Freshly prepared dispersions were electrospun at a varying flow rate (1.0–2.0 mL/h), and the distance between the needle tip and the ground electrode was 10 cm. The produced nanofiber membranes were collected over 2–3 h. All electrospinning experiments were carried out at room temperature. 2.5. Fourier transform infrared spectroscopy (FT-IR) The infrared spectra of the neat PLGA nanofiber membranes and composite nanofiber membranes were obtained using an FTIR spectrometer (Vertex 70; Bruker). The spectra were recorded in the absorbance mode using a diamond crystal plate and obtained in the spectral region of 500–4000 cm−1 at a resolution of 4 cm−1 and 20 scans per sample. 2.6. Scanning electron microscopy (SEM) The morphologies of the neat PLGA nanofiber membranes and composite nanofiber membranes were characterized by SEM (LEO1530 VP; Philips) after gold coating. The average diameter and diameter distribution were obtained by analyzing SEM images at least one hundred nanofibres using ImageJ software (NIH).
2. Materials and methods 2.1. Materials
2.7. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)
Microcrystalline cellulose (50 m) was obtained from Aladdin Reagents (Shanghai) Co. Ltd. PLGA having a lactic acid:glycolic acid ratio of 75:25 and a molecular weight of 97,000 Da was obtained from Polysciences, Inc. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was purchased from J&K Chemical Ltd. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphatebuffered saline (DPBS), penicillin, and streptomycin were acquired from Life Technologies. All other reagents were of analytical grade and used as received. Triple-distilled water was used throughout the study.
Thermal analysis has been extensively used to study the thermostability of polymeric blends. The thermal degradation behavior of films with and without incorporated CNCs was investigated using TGA (Tarsus 209F3; Netzsch) under a N2 atmosphere. Data were collected after placing ∼5 mg of sample in a clean platinum pan and heating from ambient temperature to 600 ◦ C (at a heating rate of 20 ◦ C/min). DSC measurements were performed using a TA Instruments Q100. In a typical experiment, ∼10 mg of sample was placed in the DSC cell and heated from −60 to 100 ◦ C (at a heating rate of 10 ◦ C/min). The melting temperature Tm was taken as the onset
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temperature of the melting endotherm. For crystallization studies, the sample was first heated to 100 ◦ C, kept at this temperature for 5 min to ensure complete melting of the PLGA, and then cooled to 0 ◦ C at a cooling rate of 5 ◦ C/min. The crystallization temperature Tc was taken as the peak of the crystallization exotherm.
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were rinsed with 1% bovine serum albumin (BSA, Sigma–Aldrich) solution to remove unbound dye. Cell nuclei were stained by further incubation with 200 L of propidium iodide (Sigma–Aldrich). The samples were then thoroughly rinsed with 1% BSA solution. 2.13. Statistical analysis
2.8. Dynamic mechanical analysis (DMA) DMA was performed in tensile mode using a TA Instruments Q242. The measurements were carried out at a constant frequency of 1 Hz and a strain amplitude of 0.03% over 25–40 ◦ C using a heating rate of 1 ◦ C/min and a gap between jaws of ∼10 mm. Nonlinear deformations (tensile tests) were obtained in the controlled force mode at 25 ◦ C and with ramping at 2 N/min. The DMA samples consisted of 5-mm-wide strips cut from the respective nanofiber webs. Three samples were used to characterize each material. 2.9. Water contact angle (WCA) Static WCA measurements were recorded with a CAM 200 Optical Contact Angle Meter (KSV Instruments Ltd.). Immediately after deposition of an ultrapure water droplet onto the surface, 20 images were recorded at 1-s intervals. The Young–Laplace equation was used to calculate the average contact angle for each image except the first. The WCA at t = 0 was determined via linear regression through the remaining points.
Data are expressed as the means of at least three replicates ± standard deviation (SD). Statistical comparisons were performed using ANOVA (t-test). All statistical computations were performed using the SPSS software (v. 16.0; IBM), and differences with a value of P < 0.05 were considered statistically significant. 3. Results and discussion 3.1. Characterization of CNCs CNCs were produced by sulfuric acid hydrolysis of MCC. The morphology of these nanocrystals was characterized by TEM (Fig. S1). The mean values of the length and diameter of the prepared rodlike CNCs were 185 ± 20 and 7 ± 1.3 nm, respectively, giving an aspect ratio (L/D) of ∼26, which agreed with values reported for ramie CNCs produced under the same conditions [32,33]. The prepared CNCs had a stiff, rodlike structure, and their tight agglomeration demonstrated the presence of intermolecular hydrogen bonding within the cellulose chains and strong hydrophilic interactions between cellulose sheets [29].
2.10. Cell viability assay 3.2. Characterization of composite nanofiber membranes The cell viability and proliferation on nanofiber membranes were determined using the WST-1 reagent (Beyotime Institute of Biotechnology). For the test, 3T3 fibroblasts were grown on Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic/antimycotic solution, and when enough cells were produced, 1.0 × 103 3T3 fibroblasts per well were seeded in 96-well plates with 100 L of growth medium and cultured at 37 ◦ C under a 5% CO2 atmosphere. After 24 h of incubation, the supernatants were evaluated at 450 nm. The results are reported in terms of the optical density (OD). 2.11. Cell morphology The organization and morphology of the 3T3 fibroblasts on neat PLGA and PLGA reinforced with CNC nanofiber membranes were observed by SEM. First, the nanofiber membranes were washed with DPBS and the cells were fixed with 2.5% (w/v) glutaraldehyde (Sigma–Aldrich) in 0.01 M DPBS for 30 min at room temperature. Then, they were dehydrated in ethanol series (i.e., 50%, 70%, 90%, and 100%) and rinsed with DPBS for 5 min. After complete drying, the nanofiber membranes were mounted on aluminum stubs and coated with gold–palladium prior to morphological observation by SEM (JSM-TE300, JEOL). 2.12. Cytoskeletal observation by confocal laser scanning microscopy (CLSM) Examination of the actin cytoskeleton was performed using a CLSM 510 confocal microscope (Carl Zeiss MicroImaging GmbH) with fluorescently stained cells. After 24 h of incubation, the cell culture medium was removed and the cells were fixed with 2.5% glutaraldehyde in 0.01 M DPBS for 30 min. The fixed cells were permeabilized with 0.1% Triton-X100 (Sigma–Aldrich) in DPBS for 5 min. After washing with DPBS, 200 L of Alexa Fluor 488 phalloidin (Life Technologies) was placed on each sample; the samples were incubated in the dark for 20 min to label F-actin. The samples
Neat PLGA nanofiber membranes and composite nanofiber membranes were made via electrospinning. Fig. 1 shows SEM images of the composite nanofiber membranes. The nanofiber membranes were homogeneous and their diameters were all in the nanoscale range. Some anomalies such as beads (Fig. 1b) and diameter enlargement (Fig. 1e) within single fibers were revealed, which are typical and common in electrospinning [10]. All composite nanofiber membranes were nanometer-sized, and their surfaces appeared to be smooth except those filled with 1% CNCs. The incorporation of CNCs led to nanofiber membranes that were thicker and stronger than neat PLGA nanofiber membranes. Fig. S2a shows that the neat PLGA nanofiber membranes had a distribution with the majority of fibers in the 100–200 nm range. With the addition of 1% CNCs (Fig. S2b), the fiber diameter distribution shifted to the lower diameters of the 100–300 nm range, but this was accompanied by a large number of beadings, which may be caused by the applied electric voltage, the humidity of electrospinning chamber and the charge density of polymer fluids. It is worth pointing out that the overall effect is negligible [34]. At higher concentrations (3%, 5% and 7%) (Fig. S2c–e), a broad distribution of fibers ranging from 300 to 600 nm was observed, with the majority toward the upper nanometer limit. A similar trend of increasing nanofiber diameter with increasing CNC content has been reported [10]. All CNC-reinforced PLGA nanofiber membranes were nanometer-sized, and the surfaces became rough as CNCs increased. Fig. 2 shows the FT-IR spectra of CNCs, neat PLGA and PLGA composite nanofiber membranes. CNCs (Fig. 2a) exhibit characteristic absorption peaks at 3340, 2900, 1650, 1453 and 1310 cm−1 , which arise from the aliphatic acid chain ascribed to C O and O C O stretching vibrations. The neat PLGA nanofiber membranes (Fig. 2b) displayed strong characteristic absorption peaks at ∼1785, 1450, 1260, and 1150 cm−1 corresponding to C O bonds, C O bonds, C O C ether groups, and C H methyl groups, respectively. As expected, the characteristic CNC peaks at 2994, 3103, and 3615 cm−1 were observed in all of the other spectra (Fig. 2c–f); these bands were attributable to C H stretching vibrations of the
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Fig. 1. SEM images of neat PLGA nanofiber membranes (a) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (b), 3% (c), 5% (d) and 7% (e). The bar indicates 1 m.
Fig. 2. FTIR spectra of CNCs (a), neat PLGA nanofiber membranes (b) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (c), 3% (d), 5% (e) and 7% (f).
aliphatic acid chain segments of the C O and O C O groups. The electrospun PLGA/CNC composite nanofiber membranes revealed peaks that were unique to the CNCs.
3.3. Hydrophilicity of the composite nanofiber membranes Hydrophilicity is one of the most important surface characteristics of biomedical materials because it influences cell adhesion and proliferation [35,36]. The wettability of the composite nanofiber membranes was determined by static WCA analysis (Table 1). The value for the neat PLGA nanofiber membranes was 135.08 ± 3.50◦ , indicating hydrophobic surfaces. However, the WCAs of the PLGA/CNC composite nanofiber membranes were lower than that of the neat PLGA nanofiber membranes
Table 1 Static contact angles of neat PLGA nanofiber membranes, and PLGA nanofiber membranes reinforced with CNCs at loadings of 1, 3, 5, and 7 wt.% (n = 3 for each group). Nanofiber membranes
Contact angle (◦ )
PLGA PLGA/1% CNCs PLGA/3% CNCs PLGA/5% CNCs PLGA/7% CNCs
135.08 113.72 107.1 96.22 94.46
± ± ± ± ±
3.5 2.37 2.56 2.15 2.11
because the CNCs introduced hydrophilic hydroxyl groups [10]. The addition of the CNC-reinforcements reduced the contact angle from 113.72 ± 2.37◦ to 94.46 ± 2.11◦ , indicating a steady improvement in the hydrophilicity of the PLGA nanofiber membranes
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Fig. 3. TGA (A), DTG (B) and DSC (C) curves of CNCs (a), neat PLGA nanofiber membranes (b) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (c), 3% (d), 5% (e) and 7% (f).
[37,38]. As noted above, the presence of intermolecular hydrogen bonding within the cellulose chains also helped to improve the hydrophilicity of the composite nanofiber membranes. Similar contact angle results were reported for electrospun CS-graft-PLGA
nanofiber membranes [10,39]. The improved hydrophilicity of biomaterials can be engineered by tailoring their chemical and mechanical properties, each of which directly influences cell fate [40].
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3.4. Thermal properties of the composite nanofiber membranes TGA and DSC were conducted to investigate the effect of CNCs on the thermal stability of the nanofiber membranes and also to obtain deeper insight into the interactions between the PLGA matrix and the CNC reinforcements. Fig. 3A shows the TG curves of CNCs, neat PLGA and PLGA composite nanofiber membranes respectively. CNCs started to degrade at ∼40 ◦ C and finished at ∼450 ◦ C while the neat PLGA nanofiber membranes started to degrade at ∼250 ◦ C and finished at ∼400 ◦ C with nearly 100% weight loss. Little difference was observed in the starting degradation temperatures (∼250 ◦ C) for the PLGA/CNC composite nanofiber membranes. This was related to the increased amount of CNC adhered to the PLGA surface; the weight losses decreased when the PLGA/CNC composite nanofiber membranes were heated up to 400 ◦ C at which temperature only the PLGA completely degraded, leaving the CNCs. The TGA and FTIR results were in excellent agreement for the composite nanofiber membranes. Differential thermograms of CNCs, neat PLGA and the PLGA/CNC composite nanofiber membranes are shown in Fig. 3B. The neat PLGA matrix had only one decomposition process with the main peak at 355 ◦ C. The initial thermal degradation of CNCs, neat PLGA and the PLGA/CNC composite nanofiber membranes occurred at low temperatures (between 0 and 250 ◦ C) as indicated by a weight loss. This initial weight loss was attributable to water loss, and the difference noted in the first-order derivative curve before and after electrospinning was negligible. The thermograms of the neat PLGA and the composite nanofiber membranes showed two regions; the temperature corresponding to maximum mass loss at ∼250 related to chain-stripping produced by chain scission and decomposition. The last thermal event at ∼400 ◦ C is related to the elimination of carbonaceous material. For the composite nanofiber membranes, no clear shift in thermal decomposition was observed even when the CNC loading was 7 wt.%. Fig. 3C shows the DSC curves of CNCs, neat PLGA and the composite nanofiber membranes. Neat PLGA showed an endothermic melting peak at 51 ◦ C. This characteristic peak of the neat PLGA nanofiber membranes appeared in all of the PLGA/CNC composite nanofiber samples. It shows that the cold crystallization temperature of PLGA/CNC nanocomposite mats initially decreased, and then increased with increased CNC contents. It is hypothesized that CNCs promoted the cold crystallization at lower temperatures, while the aggregates of CNCs occurred at high loading levels were less effective in promoting crystallization due to decreased contact area between CNCs and PLGA matrix. The results of this study demonstrated that CNCs enhanced the thermal stability of PLGA nanofiber membranes by improving the interfacial adhesion between the
Table 2 Tensile test results for neat PLGA nanofiber membranes and PLGA nanofiber membranes reinforced with CNCs at loadings of 1, 3, 5, and 7 wt.% (n = 3 for each group). Nanofiber membranes
Young’s modulus (MPa)
Neat PLGA PLGA/1% CNCs PLGA/3% CNCs PLGA/5% CNCs PLGA/7% CNCs
4.52 7.24 9.68 15.83 21.28
± ± ± ± ±
0.31 0.15 0.63 0.74 0.37
Strength (MPa) 0.72 0.75 0.80 1.43 1.68
± ± ± ± ±
0.17 0.08 0.25 0.22 0.13
Strain at break (%) 47.7 72.1 79.7 85.5 89.2
± ± ± ± ±
7.8 8.4 8.6 6.2 5.3
PLGA chains and chemically bonded CNCs on the surface of the PLGA nanofiber membranes. 3.5. Mechanical properties of composite electrospun nanofiber membranes Several post-processing treatments have been advanced to improve mechanical properties [20–22,29,41]. Nano-structured networks of nanofiber membranes are considered to influence mechanical properties such as the tensile strength and elongation [39]. Herein, DMA measurements were performed on the PLGA/CNC composite nanofiber membranes; the storage modulus as a function of temperature is plotted in Fig. 4A. The modulus was slightly lower at temperatures between 25 and 32 ◦ C. This phenomenon is explained by progressive melting of the PLGA matrix with coexistence of amorphous, rubbery, and crystalline domains. The storage modulus sharply decreased as the temperature increased from 32 to 45 ◦ C; this softening was irreversible because the crystalline zones of the PLGA matrix completely melted. The storage moduli of the composite electrospun nanofiber membranes were significantly higher than that of the neat PLGA nanofiber membranes. This behavior is partially attributable to the larger diameter of the composite nanofiber membranes and is in agreement with published data for PCL reinforced with CNCs [22]. Hence, the improved mechanical behavior is attributable to reinforcement by the CNCs within the fibers. Thus, the mechanical properties of the composite nanofiber membranes clearly are mostly associated with the effect of the CNCs and especially the fiber diameter. The behavior of the nanofiber membranes under nonlinear tensile deformations was also measured; Table 2 summarizes the results. Neat PLGA nanofiber membranes showed an ultimate strength of 0.72 MPa, a Young’s modulus of 4.52 MPa, and a maximum strain of 47.7%. A definite improvement in these properties was observed with the incorporation of CNCs. The nanofiber membranes reinforced with 7% CNCs showed the greatest improvement over neat PLGA, i.e.,
Fig. 4. DMA (A) and fibroblast cell viability (B) of neat PLGA nanofiber membranes (a) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (b), 3% (c), 5% (d) and 7% (e), n = 3 for each group.
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Fig. 5. SEM micrographs of cultured on the nanofiber membranes of neat PLGA nanofiber membranes (a) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (b), 3% (c), 5% (d) and 7% (e).
a ∼4.7-fold increase in Young’s modulus, ∼2.3-fold increase in ultimate strength, and ∼1.9-fold increase in strain at break. The tensile-tension results indicate that reinforcing PLGA nanofiber membranes with CNCs via electrospinning improves the mechanical properties of the nanofiber membranes. Generally, the results imply desirable mechanical properties of the electrospun nanofiber membranes compared to natural skin which possess tensile modulus of 15–150 MPa and ultimate strain of 35–115% in order to be utilized as suitable skin graft [42]. 3.6. Cell viability and spreading on composite nanofiber membranes As a potential scaffold for tissue engineering, composite nanofiber membranes should promote cell proliferation and maintain their physiological function. In this study, comparing Neat PLGA nanofiber membranes as control, the viability and proliferation of 3T3 fibroblasts on the composite nanofiber membranes were
evaluated by WST-1 assay. Fig. 4B shows cell viability in the neat PLGA and PLGA/CNC composite nanofiber membranes. Neat PLGA nanofiber membranes had a cell viability of 65%, and cell viability improved with increasing addition of CNCs to the PLGA nanofiber membranes. Fig. 5 shows field-emission SEM (FE-SEM) images of fibroblasts on neat PLGA and PLGA/CNC composite nanofiber membranes. After 24 h of cell seeding, the fibroblasts on the neat PLGA nanofiber membranes had a rounded morphology (Fig. 5a), but on the composite nanofiber membranes, the fibroblasts started stretching with increasing loading of CNCs. Cell spreading was more prominent and the cells had a spindle-shaped morphology on the PLGA nanofiber membranes reinforced with 7 wt.% CNCs. CLSM imaging of DAPI- and phalloidin-stained cells showed adherence and the desirable spread of cells on the surface of neat PLGA nanofiber membranes and PLGA/CNC composite nanofiber membranes after 24 h (Fig. 6). All of the incubated cells expressed red fluorescence, indicating better cell–matrix interactions,
Fig. 6. Confocal laser scanning microscopy of 3T3 fibroblasts cultured for 24 h on neat PLGA nanofiber membranes (a) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (b), 3% (c), 5% (d) and 7% (e). The bar indicates 10 m.
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but cells incubated on the PLGA/CNC composite nanofiber membranes were more widely spread out than those incubated on the neat PLGA. Incorporation of CNCs increased the surface hydrophilicity, which may have led to the improved cell-adhesion strength [29,34,43,44]. Cell adhesion and proliferation are affected by mechanical properties and the hydrophilicity of a material [23,45,46]. The DMA, WCA, and SEM data for the PLGA/CNC composite nanofiber membranes together indicated improved 3T3 fibroblasts adhesion, but with a change in the cell morphology to an elongated, flat, and uniform shape. 4. Conclusions In this study, neat PLGA and PLGA/CNC composite nanofiber membranes based on biodegradable PLGA and CNCs were successfully prepared via an electrospinning technique. FE-SEM images showed uniform morphology of the electrospun nanofiber membranes with an average diameter of 100 nm for the neat PLGA nanofiber membranes and 200–500 nm for the PLGA/CNC composite nanofiber membranes. These homogeneous composite nanofiber membranes with diameters in the nanoscale region had improved thermomechanical properties and mechanical strengths compared with neat PLGA nanofiber membranes. The PLGA/7 wt.% CNC composite nanofiber membranes had a tensile modulus of 21.28 MPa and an ultimate strain of 89.2 ± 5.3%, which are similar to the values of human skin. Additionally, the composite nanofiber membranes were more hydrophilic than the neat PLGA nanofiber membranes. Moreover, PLGA nanofiber membranes reinforced with CNCs supported a sustained 3T3 fibroblasts proliferation, indicating excellent biocompatibility; the fibroblasts were well adhered and widely dispersed on the composite nanofiber membranes. In light of these results, we believe that the novel composite nanofiber membranes, with their good biocompatibility, degradability, mechanical properties, stable functionality, and lower cytotoxicity, constitute potential candidates for tissue engineering. Acknowledgments This study was supported financially by the Natural Science Foundation of China (51303064), Natural Science Foundation of Guangdong (S2012040008003), the Ph.D. Programs Foundation of the Ministry of Education of China (20124401120015), and the Fundamental Research Funds of the Central Universities (21612327). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.05. 029 References [1] C.P. Barnes, S.A. Sell, E.D. Boland, D.G. Simpson, G.L. Bowlin, Adv. Drug. Deliv. Rev. 59 (14) (2007) 1413–1433. [2] M. He, A. Callanan, Tissue Eng. B 19 (3) (2013) 194–208.
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Colloids and Surfaces B: Biointerfaces journal homepage: www.elsevier.com/locate/colsurfb
Preparation and properties of PLGA nanofiber membranes reinforced with cellulose nanocrystals Yunfei Mo a,b , Rui Guo a,b,∗ , Jianghui Liu c , Yong Lan a,b , Yi Zhang a,b , Wei Xue a,b , Yuanming Zhang d,∗∗ a
Key Laboratory of Biomaterials of Guangdong Higher Education Institutes, Jinan University, Guangzhou 510632, China Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China c Department of Emergency, The First Affiliated Hospital of Sun Yat-sen University, Guangzhou 510080, China d Department of Chemistry, Jinan University, Guangzhou 510632, China b
a r t i c l e
i n f o
Article history: Received 12 February 2015 Received in revised form 13 May 2015 Accepted 15 May 2015 Available online 23 May 2015 Keywords: Cellulose nanocrystals (CNCs) Polylactide–polyglycolide (PLGA) Nanofibers Mechanical properties Biocompatibility Tissue engineering
a b s t r a c t Although extensively used in the fields of drug-carrier and tissue engineering, the biocompatibility and mechanical properties of polylactide–polyglycolide (PLGA) nanofiber membranes still limit their applications. The objective of this study was to improve their utility by introducing cellulose nanocrystals (CNCs) into PLGA nanofiber membranes. PLGA and PLGA/CNC composite nanofiber membranes were prepared via electrospinning, and the morphology and thermodynamic and mechanical properties of these nanofiber membranes were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA), differential scanning calorimetry (DSC), and dynamic mechanical analysis (DMA). The cytocompatibility and cellular responses of the nanofiber membranes were also studied by WST-1 assay, SEM, and confocal laser scanning microscopy (CLSM). Incorporation of CNCs (1, 3, 5, and 7 wt.%) increased the average fiber diameter of the prepared nanofiber membranes from 100 nm (neat PLGA) to ∼400 nm (PLGA/7 wt.% CNC) and improved the thermal stability of the nanofiber membranes. Among the PLGA/CNC composite nanofiber membranes, those loaded with 7 wt.% CNC nanofiber membranes had the best mechanical properties, which were similar to those of human skin. Cell culture results showed that the PLGA/CNC composite nanofiber membranes had better cytocompatibility and facilitated fibroblast adhesion, spreading, and proliferation compared with neat PLGA nanofiber membranes. These preliminary results suggest that PLGA/CNC composite nanofiber membranes are promising new materials for the field of skin tissue engineering. © 2015 Elsevier B.V. All rights reserved.
1. Introduction Tissue engineering, an important emerging topic in biomedical engineering, has shown tremendous promise in creating biological alternatives for harvested tissues, implants, and prostheses [1]. Three elements, namely cells, scaffolds, and biomolecules, play determinant roles in tissue regeneration [2]. Using nanofibers as scaffolds or films in tissue engineering is of particular interest because these materials mimic the structure of native extracellular matrix (ECM) proteins, which provide a platform for cell adhesion, differentiation, and proliferation [3]. The nanofibers can be
∗ Corresponding author at: Department of Biomedical Engineering, Jinan University, Guangzhou 510632, China. Tel.: +86 20 85224338; fax: +86 20 85224338. ∗∗ Corresponding author. Tel.: +86 20 85224338; fax: +86 20 85224338. E-mail addresses: [email protected] (R. Guo), [email protected] (Y. Zhang). http://dx.doi.org/10.1016/j.colsurfb.2015.05.029 0927-7765/© 2015 Elsevier B.V. All rights reserved.
produced in several ways, including self-assembly [4], phase separation [5], template-assisted synthesis [6], and electrospinning [7]. Electrospinning is an especially versatile technique because it can be used to fabricate a variety of polymeric nanofiber membranes and is able to generate continuous fibers having diameters from a few nanometers to several micrometers [8,9]. Ideal nanofiber membranes prepared via electrospinning should simulate the ECM in structure and function to promote tissue regeneration and facilitate cell differentiation and proliferation [10,11]. To achieve these goals, several synthetic and natural polymers have been successfully electrospun into nanofibrous scaffolds or films to act as base matrices. These polymers include poly(l-lactide) (PLA) [12], poly(εcaprolactone) (PCL) [13], cellulose acetate (CA) [14], polyvinyl acetate (PVA) [15], poly(ethylene oxide) (PEO) [16], chitosan [17], and collagen [18,19]. The synthetic polymer poly(lactic-co-glycolic acid) (PLGA) has excellent biocompatibility and a controllable degradation rate that
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can be adjusted by modifying its crystal structure [20,21]. Scaffolds or films fabricated with PLGA have found widespread application in tissue engineering such as for the regeneration and repair of skin, bone, and nerves. However, PLGA is still not suitable for use in many tissue engineering situations because of its limited mechanical strength and hydrophobicity, which depresses cell adhesion and proliferation [22]. Extensive research has been conducted to improve its hydrophilicity and mechanical properties. A nanofibrous matrix of PLGA/type I collagen produced via electrospinning showed improved hydrophilicity and has been used in biomimetic scaffolds [10]. Chitosan-graft-PLGA (CS-graft-PLGA) produced by electrospinning had improved hydrophilicity and protein absorption, with acceptable mechanical properties [10]. Electrospun PLGA and nano-hydroxyapatite (HA) had reduced chain mobility, which improved the mechanical properties [23]. Cellulose nanocrystals (CNCs) obtained by sulfuric acid hydrolysis of native cellulose have excellent mechanical properties such as high strength (∼7 GPa), low extension to break, high aspect ratio, high surface area, and biocompatibility [24]. CNCs have recently attracted the interest of researchers in the fields of tissue engineering and regenerative medicine for use as scaffolds or drug-delivery systems. CNCs dispersed within a cellulose acetate propionate (CAP) matrix formed a three-dimensional rigid percolating network, which was contemplated for use as a scaffold in small-diameter vascular grafts [25]. Huang et al. [26] prepared electrospun silk fibroin nanofiber mats reinforced with CNCs; the tensile strength and Young’s modulus of these mats were almost twice those of unreinforced silk fibroin mats when the CNC content was 2 wt.%. Enhanced mechanical properties were also reported for bacterial cellulose nanocrystals, which acted as a nanofiller, in silk fibroin nanofiber membranes [27]. Collagen/CNC multilayered films have been proposed for layer-by-layer (LBL) assembly as a way to maximize the interaction between the two structural materials [28]. Collagen-based composite films reinforced with CNCs designed by our group had mechanical properties that were significantly better than those of pure collagen film. In vitro 3T3 fibroblasts culture results revealed that the composite films were not cytotoxic and facilitated cell adhesion [29]. CNC-reinforced collagen scaffolds containing gelatin microspheres (GMs) capable of releasing basic fibroblast growth factors (bFGF) have also been fabricated. Those collagen/CNCs/bFGF–GMs scaffolds contained a significantly higher number of newly formed and mature blood vessels, which suggested great potential in skin tissue engineering [30]. In this work, electrospinning was used to fabricate PLGA nanofiber membranes reinforced with biodegradable CNCs derived from microcrystalline cellulose (MCC). The morphology, hydrophilicity, thermodynamic, and mechanical properties of the composite nanofiber membranes were investigated. Cell–nanofiber interactions were also studied using 3T3 fibroblasts cultured on these nanofiber membranes.
2.2. Preparation of cellulose nanocrystals (CNCs) CNCs were prepared by acid hydrolysis of MCC according to a previously described procedure [31]. In brief, 10 g of MCC powder was added to 95 mL of 65 wt.% sulfuric acid under vigorous mechanical stirring. The hydrolysis was performed at 55 ◦ C for 5 h, and the mixture was diluted fivefold to quench the hydrolysis reaction. The resulting suspension was centrifuged at 5000 rpm for 10 min to separate the crystals, which were then washed and then treated ultrasonically for 15 min. The precipitate was further dialyzed against distilled water until the water pH reached 7.0. Finally, dialysis for 1 week against deionized water with a Viskase 3500 MWCO dialysis membrane was performed to remove residual sulfuric acid. 2.3. Transmission electron microscopy (TEM) Drops of aqueous dispersions of CNCs (0.01%, w/v) were deposited on carbon-coated electron microscope grids (Protochips Inc.), negatively stained with uranyl acetate, and allowed to dry. The grids were observed with a Hitachi HF-2000 transmission electron microscope operated at an accelerating voltage of 20 kV. 2.4. Electrospinning For the electrospinning of PLGA reinforced with CNCs, the CNCs were dispersed in a 15% (w/v) PLGA solution (HFIP solvent) at loads corresponding to 1, 3, 5, and 7 wt.% (based on the PLGA weight). The needle was connected to the high voltage supply, which could generate positive DC voltages and current up to 15 kV. Freshly prepared dispersions were electrospun at a varying flow rate (1.0–2.0 mL/h), and the distance between the needle tip and the ground electrode was 10 cm. The produced nanofiber membranes were collected over 2–3 h. All electrospinning experiments were carried out at room temperature. 2.5. Fourier transform infrared spectroscopy (FT-IR) The infrared spectra of the neat PLGA nanofiber membranes and composite nanofiber membranes were obtained using an FTIR spectrometer (Vertex 70; Bruker). The spectra were recorded in the absorbance mode using a diamond crystal plate and obtained in the spectral region of 500–4000 cm−1 at a resolution of 4 cm−1 and 20 scans per sample. 2.6. Scanning electron microscopy (SEM) The morphologies of the neat PLGA nanofiber membranes and composite nanofiber membranes were characterized by SEM (LEO1530 VP; Philips) after gold coating. The average diameter and diameter distribution were obtained by analyzing SEM images at least one hundred nanofibres using ImageJ software (NIH).
2. Materials and methods 2.1. Materials
2.7. Thermogravimetric analysis (TGA) and differential scanning calorimetry (DSC)
Microcrystalline cellulose (50 m) was obtained from Aladdin Reagents (Shanghai) Co. Ltd. PLGA having a lactic acid:glycolic acid ratio of 75:25 and a molecular weight of 97,000 Da was obtained from Polysciences, Inc. 1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) was purchased from J&K Chemical Ltd. Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphatebuffered saline (DPBS), penicillin, and streptomycin were acquired from Life Technologies. All other reagents were of analytical grade and used as received. Triple-distilled water was used throughout the study.
Thermal analysis has been extensively used to study the thermostability of polymeric blends. The thermal degradation behavior of films with and without incorporated CNCs was investigated using TGA (Tarsus 209F3; Netzsch) under a N2 atmosphere. Data were collected after placing ∼5 mg of sample in a clean platinum pan and heating from ambient temperature to 600 ◦ C (at a heating rate of 20 ◦ C/min). DSC measurements were performed using a TA Instruments Q100. In a typical experiment, ∼10 mg of sample was placed in the DSC cell and heated from −60 to 100 ◦ C (at a heating rate of 10 ◦ C/min). The melting temperature Tm was taken as the onset
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temperature of the melting endotherm. For crystallization studies, the sample was first heated to 100 ◦ C, kept at this temperature for 5 min to ensure complete melting of the PLGA, and then cooled to 0 ◦ C at a cooling rate of 5 ◦ C/min. The crystallization temperature Tc was taken as the peak of the crystallization exotherm.
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were rinsed with 1% bovine serum albumin (BSA, Sigma–Aldrich) solution to remove unbound dye. Cell nuclei were stained by further incubation with 200 L of propidium iodide (Sigma–Aldrich). The samples were then thoroughly rinsed with 1% BSA solution. 2.13. Statistical analysis
2.8. Dynamic mechanical analysis (DMA) DMA was performed in tensile mode using a TA Instruments Q242. The measurements were carried out at a constant frequency of 1 Hz and a strain amplitude of 0.03% over 25–40 ◦ C using a heating rate of 1 ◦ C/min and a gap between jaws of ∼10 mm. Nonlinear deformations (tensile tests) were obtained in the controlled force mode at 25 ◦ C and with ramping at 2 N/min. The DMA samples consisted of 5-mm-wide strips cut from the respective nanofiber webs. Three samples were used to characterize each material. 2.9. Water contact angle (WCA) Static WCA measurements were recorded with a CAM 200 Optical Contact Angle Meter (KSV Instruments Ltd.). Immediately after deposition of an ultrapure water droplet onto the surface, 20 images were recorded at 1-s intervals. The Young–Laplace equation was used to calculate the average contact angle for each image except the first. The WCA at t = 0 was determined via linear regression through the remaining points.
Data are expressed as the means of at least three replicates ± standard deviation (SD). Statistical comparisons were performed using ANOVA (t-test). All statistical computations were performed using the SPSS software (v. 16.0; IBM), and differences with a value of P < 0.05 were considered statistically significant. 3. Results and discussion 3.1. Characterization of CNCs CNCs were produced by sulfuric acid hydrolysis of MCC. The morphology of these nanocrystals was characterized by TEM (Fig. S1). The mean values of the length and diameter of the prepared rodlike CNCs were 185 ± 20 and 7 ± 1.3 nm, respectively, giving an aspect ratio (L/D) of ∼26, which agreed with values reported for ramie CNCs produced under the same conditions [32,33]. The prepared CNCs had a stiff, rodlike structure, and their tight agglomeration demonstrated the presence of intermolecular hydrogen bonding within the cellulose chains and strong hydrophilic interactions between cellulose sheets [29].
2.10. Cell viability assay 3.2. Characterization of composite nanofiber membranes The cell viability and proliferation on nanofiber membranes were determined using the WST-1 reagent (Beyotime Institute of Biotechnology). For the test, 3T3 fibroblasts were grown on Dulbecco’s Modified Eagle’s medium (DMEM) supplemented with 10% (v/v) fetal bovine serum and 1% (v/v) antibiotic/antimycotic solution, and when enough cells were produced, 1.0 × 103 3T3 fibroblasts per well were seeded in 96-well plates with 100 L of growth medium and cultured at 37 ◦ C under a 5% CO2 atmosphere. After 24 h of incubation, the supernatants were evaluated at 450 nm. The results are reported in terms of the optical density (OD). 2.11. Cell morphology The organization and morphology of the 3T3 fibroblasts on neat PLGA and PLGA reinforced with CNC nanofiber membranes were observed by SEM. First, the nanofiber membranes were washed with DPBS and the cells were fixed with 2.5% (w/v) glutaraldehyde (Sigma–Aldrich) in 0.01 M DPBS for 30 min at room temperature. Then, they were dehydrated in ethanol series (i.e., 50%, 70%, 90%, and 100%) and rinsed with DPBS for 5 min. After complete drying, the nanofiber membranes were mounted on aluminum stubs and coated with gold–palladium prior to morphological observation by SEM (JSM-TE300, JEOL). 2.12. Cytoskeletal observation by confocal laser scanning microscopy (CLSM) Examination of the actin cytoskeleton was performed using a CLSM 510 confocal microscope (Carl Zeiss MicroImaging GmbH) with fluorescently stained cells. After 24 h of incubation, the cell culture medium was removed and the cells were fixed with 2.5% glutaraldehyde in 0.01 M DPBS for 30 min. The fixed cells were permeabilized with 0.1% Triton-X100 (Sigma–Aldrich) in DPBS for 5 min. After washing with DPBS, 200 L of Alexa Fluor 488 phalloidin (Life Technologies) was placed on each sample; the samples were incubated in the dark for 20 min to label F-actin. The samples
Neat PLGA nanofiber membranes and composite nanofiber membranes were made via electrospinning. Fig. 1 shows SEM images of the composite nanofiber membranes. The nanofiber membranes were homogeneous and their diameters were all in the nanoscale range. Some anomalies such as beads (Fig. 1b) and diameter enlargement (Fig. 1e) within single fibers were revealed, which are typical and common in electrospinning [10]. All composite nanofiber membranes were nanometer-sized, and their surfaces appeared to be smooth except those filled with 1% CNCs. The incorporation of CNCs led to nanofiber membranes that were thicker and stronger than neat PLGA nanofiber membranes. Fig. S2a shows that the neat PLGA nanofiber membranes had a distribution with the majority of fibers in the 100–200 nm range. With the addition of 1% CNCs (Fig. S2b), the fiber diameter distribution shifted to the lower diameters of the 100–300 nm range, but this was accompanied by a large number of beadings, which may be caused by the applied electric voltage, the humidity of electrospinning chamber and the charge density of polymer fluids. It is worth pointing out that the overall effect is negligible [34]. At higher concentrations (3%, 5% and 7%) (Fig. S2c–e), a broad distribution of fibers ranging from 300 to 600 nm was observed, with the majority toward the upper nanometer limit. A similar trend of increasing nanofiber diameter with increasing CNC content has been reported [10]. All CNC-reinforced PLGA nanofiber membranes were nanometer-sized, and the surfaces became rough as CNCs increased. Fig. 2 shows the FT-IR spectra of CNCs, neat PLGA and PLGA composite nanofiber membranes. CNCs (Fig. 2a) exhibit characteristic absorption peaks at 3340, 2900, 1650, 1453 and 1310 cm−1 , which arise from the aliphatic acid chain ascribed to C O and O C O stretching vibrations. The neat PLGA nanofiber membranes (Fig. 2b) displayed strong characteristic absorption peaks at ∼1785, 1450, 1260, and 1150 cm−1 corresponding to C O bonds, C O bonds, C O C ether groups, and C H methyl groups, respectively. As expected, the characteristic CNC peaks at 2994, 3103, and 3615 cm−1 were observed in all of the other spectra (Fig. 2c–f); these bands were attributable to C H stretching vibrations of the
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Fig. 1. SEM images of neat PLGA nanofiber membranes (a) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (b), 3% (c), 5% (d) and 7% (e). The bar indicates 1 m.
Fig. 2. FTIR spectra of CNCs (a), neat PLGA nanofiber membranes (b) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (c), 3% (d), 5% (e) and 7% (f).
aliphatic acid chain segments of the C O and O C O groups. The electrospun PLGA/CNC composite nanofiber membranes revealed peaks that were unique to the CNCs.
3.3. Hydrophilicity of the composite nanofiber membranes Hydrophilicity is one of the most important surface characteristics of biomedical materials because it influences cell adhesion and proliferation [35,36]. The wettability of the composite nanofiber membranes was determined by static WCA analysis (Table 1). The value for the neat PLGA nanofiber membranes was 135.08 ± 3.50◦ , indicating hydrophobic surfaces. However, the WCAs of the PLGA/CNC composite nanofiber membranes were lower than that of the neat PLGA nanofiber membranes
Table 1 Static contact angles of neat PLGA nanofiber membranes, and PLGA nanofiber membranes reinforced with CNCs at loadings of 1, 3, 5, and 7 wt.% (n = 3 for each group). Nanofiber membranes
Contact angle (◦ )
PLGA PLGA/1% CNCs PLGA/3% CNCs PLGA/5% CNCs PLGA/7% CNCs
135.08 113.72 107.1 96.22 94.46
± ± ± ± ±
3.5 2.37 2.56 2.15 2.11
because the CNCs introduced hydrophilic hydroxyl groups [10]. The addition of the CNC-reinforcements reduced the contact angle from 113.72 ± 2.37◦ to 94.46 ± 2.11◦ , indicating a steady improvement in the hydrophilicity of the PLGA nanofiber membranes
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Fig. 3. TGA (A), DTG (B) and DSC (C) curves of CNCs (a), neat PLGA nanofiber membranes (b) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (c), 3% (d), 5% (e) and 7% (f).
[37,38]. As noted above, the presence of intermolecular hydrogen bonding within the cellulose chains also helped to improve the hydrophilicity of the composite nanofiber membranes. Similar contact angle results were reported for electrospun CS-graft-PLGA
nanofiber membranes [10,39]. The improved hydrophilicity of biomaterials can be engineered by tailoring their chemical and mechanical properties, each of which directly influences cell fate [40].
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3.4. Thermal properties of the composite nanofiber membranes TGA and DSC were conducted to investigate the effect of CNCs on the thermal stability of the nanofiber membranes and also to obtain deeper insight into the interactions between the PLGA matrix and the CNC reinforcements. Fig. 3A shows the TG curves of CNCs, neat PLGA and PLGA composite nanofiber membranes respectively. CNCs started to degrade at ∼40 ◦ C and finished at ∼450 ◦ C while the neat PLGA nanofiber membranes started to degrade at ∼250 ◦ C and finished at ∼400 ◦ C with nearly 100% weight loss. Little difference was observed in the starting degradation temperatures (∼250 ◦ C) for the PLGA/CNC composite nanofiber membranes. This was related to the increased amount of CNC adhered to the PLGA surface; the weight losses decreased when the PLGA/CNC composite nanofiber membranes were heated up to 400 ◦ C at which temperature only the PLGA completely degraded, leaving the CNCs. The TGA and FTIR results were in excellent agreement for the composite nanofiber membranes. Differential thermograms of CNCs, neat PLGA and the PLGA/CNC composite nanofiber membranes are shown in Fig. 3B. The neat PLGA matrix had only one decomposition process with the main peak at 355 ◦ C. The initial thermal degradation of CNCs, neat PLGA and the PLGA/CNC composite nanofiber membranes occurred at low temperatures (between 0 and 250 ◦ C) as indicated by a weight loss. This initial weight loss was attributable to water loss, and the difference noted in the first-order derivative curve before and after electrospinning was negligible. The thermograms of the neat PLGA and the composite nanofiber membranes showed two regions; the temperature corresponding to maximum mass loss at ∼250 related to chain-stripping produced by chain scission and decomposition. The last thermal event at ∼400 ◦ C is related to the elimination of carbonaceous material. For the composite nanofiber membranes, no clear shift in thermal decomposition was observed even when the CNC loading was 7 wt.%. Fig. 3C shows the DSC curves of CNCs, neat PLGA and the composite nanofiber membranes. Neat PLGA showed an endothermic melting peak at 51 ◦ C. This characteristic peak of the neat PLGA nanofiber membranes appeared in all of the PLGA/CNC composite nanofiber samples. It shows that the cold crystallization temperature of PLGA/CNC nanocomposite mats initially decreased, and then increased with increased CNC contents. It is hypothesized that CNCs promoted the cold crystallization at lower temperatures, while the aggregates of CNCs occurred at high loading levels were less effective in promoting crystallization due to decreased contact area between CNCs and PLGA matrix. The results of this study demonstrated that CNCs enhanced the thermal stability of PLGA nanofiber membranes by improving the interfacial adhesion between the
Table 2 Tensile test results for neat PLGA nanofiber membranes and PLGA nanofiber membranes reinforced with CNCs at loadings of 1, 3, 5, and 7 wt.% (n = 3 for each group). Nanofiber membranes
Young’s modulus (MPa)
Neat PLGA PLGA/1% CNCs PLGA/3% CNCs PLGA/5% CNCs PLGA/7% CNCs
4.52 7.24 9.68 15.83 21.28
± ± ± ± ±
0.31 0.15 0.63 0.74 0.37
Strength (MPa) 0.72 0.75 0.80 1.43 1.68
± ± ± ± ±
0.17 0.08 0.25 0.22 0.13
Strain at break (%) 47.7 72.1 79.7 85.5 89.2
± ± ± ± ±
7.8 8.4 8.6 6.2 5.3
PLGA chains and chemically bonded CNCs on the surface of the PLGA nanofiber membranes. 3.5. Mechanical properties of composite electrospun nanofiber membranes Several post-processing treatments have been advanced to improve mechanical properties [20–22,29,41]. Nano-structured networks of nanofiber membranes are considered to influence mechanical properties such as the tensile strength and elongation [39]. Herein, DMA measurements were performed on the PLGA/CNC composite nanofiber membranes; the storage modulus as a function of temperature is plotted in Fig. 4A. The modulus was slightly lower at temperatures between 25 and 32 ◦ C. This phenomenon is explained by progressive melting of the PLGA matrix with coexistence of amorphous, rubbery, and crystalline domains. The storage modulus sharply decreased as the temperature increased from 32 to 45 ◦ C; this softening was irreversible because the crystalline zones of the PLGA matrix completely melted. The storage moduli of the composite electrospun nanofiber membranes were significantly higher than that of the neat PLGA nanofiber membranes. This behavior is partially attributable to the larger diameter of the composite nanofiber membranes and is in agreement with published data for PCL reinforced with CNCs [22]. Hence, the improved mechanical behavior is attributable to reinforcement by the CNCs within the fibers. Thus, the mechanical properties of the composite nanofiber membranes clearly are mostly associated with the effect of the CNCs and especially the fiber diameter. The behavior of the nanofiber membranes under nonlinear tensile deformations was also measured; Table 2 summarizes the results. Neat PLGA nanofiber membranes showed an ultimate strength of 0.72 MPa, a Young’s modulus of 4.52 MPa, and a maximum strain of 47.7%. A definite improvement in these properties was observed with the incorporation of CNCs. The nanofiber membranes reinforced with 7% CNCs showed the greatest improvement over neat PLGA, i.e.,
Fig. 4. DMA (A) and fibroblast cell viability (B) of neat PLGA nanofiber membranes (a) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (b), 3% (c), 5% (d) and 7% (e), n = 3 for each group.
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Fig. 5. SEM micrographs of cultured on the nanofiber membranes of neat PLGA nanofiber membranes (a) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (b), 3% (c), 5% (d) and 7% (e).
a ∼4.7-fold increase in Young’s modulus, ∼2.3-fold increase in ultimate strength, and ∼1.9-fold increase in strain at break. The tensile-tension results indicate that reinforcing PLGA nanofiber membranes with CNCs via electrospinning improves the mechanical properties of the nanofiber membranes. Generally, the results imply desirable mechanical properties of the electrospun nanofiber membranes compared to natural skin which possess tensile modulus of 15–150 MPa and ultimate strain of 35–115% in order to be utilized as suitable skin graft [42]. 3.6. Cell viability and spreading on composite nanofiber membranes As a potential scaffold for tissue engineering, composite nanofiber membranes should promote cell proliferation and maintain their physiological function. In this study, comparing Neat PLGA nanofiber membranes as control, the viability and proliferation of 3T3 fibroblasts on the composite nanofiber membranes were
evaluated by WST-1 assay. Fig. 4B shows cell viability in the neat PLGA and PLGA/CNC composite nanofiber membranes. Neat PLGA nanofiber membranes had a cell viability of 65%, and cell viability improved with increasing addition of CNCs to the PLGA nanofiber membranes. Fig. 5 shows field-emission SEM (FE-SEM) images of fibroblasts on neat PLGA and PLGA/CNC composite nanofiber membranes. After 24 h of cell seeding, the fibroblasts on the neat PLGA nanofiber membranes had a rounded morphology (Fig. 5a), but on the composite nanofiber membranes, the fibroblasts started stretching with increasing loading of CNCs. Cell spreading was more prominent and the cells had a spindle-shaped morphology on the PLGA nanofiber membranes reinforced with 7 wt.% CNCs. CLSM imaging of DAPI- and phalloidin-stained cells showed adherence and the desirable spread of cells on the surface of neat PLGA nanofiber membranes and PLGA/CNC composite nanofiber membranes after 24 h (Fig. 6). All of the incubated cells expressed red fluorescence, indicating better cell–matrix interactions,
Fig. 6. Confocal laser scanning microscopy of 3T3 fibroblasts cultured for 24 h on neat PLGA nanofiber membranes (a) and PLGA nanofiber membranes reinforced with CNCs at weight loads of 1% (b), 3% (c), 5% (d) and 7% (e). The bar indicates 10 m.
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but cells incubated on the PLGA/CNC composite nanofiber membranes were more widely spread out than those incubated on the neat PLGA. Incorporation of CNCs increased the surface hydrophilicity, which may have led to the improved cell-adhesion strength [29,34,43,44]. Cell adhesion and proliferation are affected by mechanical properties and the hydrophilicity of a material [23,45,46]. The DMA, WCA, and SEM data for the PLGA/CNC composite nanofiber membranes together indicated improved 3T3 fibroblasts adhesion, but with a change in the cell morphology to an elongated, flat, and uniform shape. 4. Conclusions In this study, neat PLGA and PLGA/CNC composite nanofiber membranes based on biodegradable PLGA and CNCs were successfully prepared via an electrospinning technique. FE-SEM images showed uniform morphology of the electrospun nanofiber membranes with an average diameter of 100 nm for the neat PLGA nanofiber membranes and 200–500 nm for the PLGA/CNC composite nanofiber membranes. These homogeneous composite nanofiber membranes with diameters in the nanoscale region had improved thermomechanical properties and mechanical strengths compared with neat PLGA nanofiber membranes. The PLGA/7 wt.% CNC composite nanofiber membranes had a tensile modulus of 21.28 MPa and an ultimate strain of 89.2 ± 5.3%, which are similar to the values of human skin. Additionally, the composite nanofiber membranes were more hydrophilic than the neat PLGA nanofiber membranes. Moreover, PLGA nanofiber membranes reinforced with CNCs supported a sustained 3T3 fibroblasts proliferation, indicating excellent biocompatibility; the fibroblasts were well adhered and widely dispersed on the composite nanofiber membranes. In light of these results, we believe that the novel composite nanofiber membranes, with their good biocompatibility, degradability, mechanical properties, stable functionality, and lower cytotoxicity, constitute potential candidates for tissue engineering. Acknowledgments This study was supported financially by the Natural Science Foundation of China (51303064), Natural Science Foundation of Guangdong (S2012040008003), the Ph.D. Programs Foundation of the Ministry of Education of China (20124401120015), and the Fundamental Research Funds of the Central Universities (21612327). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.colsurfb.2015.05. 029 References [1] C.P. Barnes, S.A. Sell, E.D. Boland, D.G. Simpson, G.L. Bowlin, Adv. Drug. Deliv. Rev. 59 (14) (2007) 1413–1433. [2] M. He, A. Callanan, Tissue Eng. B 19 (3) (2013) 194–208.
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