Materials Science and Engineering C 58 (2016) 757–767

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Materials Science and Engineering C journal homepage: www.elsevier.com/locate/msec

Poly(hydroxybutyrate)/cellulose acetate blend nanofiber scaffolds: Preparation, characterization and cytocompatibility Cai Zhijiang a,b,⁎, Xu Yi a, Yang Haizheng a, Jianru Jia a, Yuanpei Liu a a b

School of Textiles, Tianjin Polytechnic University, Tianjin 300387, China State Key Laboratory of Hollow Fiber Membrane Material and Processes, No 399 BingShuiXi Street, XiQing District, Tianjin, China, 300387

a r t i c l e

i n f o

Article history: Received 22 April 2015 Received in revised form 11 August 2015 Accepted 10 September 2015 Available online 14 September 2015 Keywords: Poly(hydroxybutyrate) Cellulose acetate Blend nanofiber Scaffold Electrospinning

a b s t r a c t Poly(hydroxybutyrate) (PHB)/cellulose acetate (CA) blend nanofiber scaffolds were fabricated by electrospinning using the blends of chloroform and DMF as solvent. The blend nanofiber scaffolds were characterized by SEM, FTIR, XRD, DSC, contact angle and tensile test. The blend nanofibers exhibited cylindrical, uniform, bead-free and random orientation with the diameter ranged from 80–680 nm. The scaffolds had very well interconnected porous fibrous network structure and large aspect surface areas. It was found that the presence of CA affected the crystallization of PHB due to formation of intermolecular hydrogen bonds, which restricted the preferential orientation of PHB molecules. The DSC result showed that the PHB and CA were miscible in the blend nanofiber. An increase in the glass transition temperature was observed with increasing CA content. Additionally, the mechanical properties of blend nanofiber scaffolds were largely influenced by the weight ratio of PHB/CA. The tensile strength, yield strength and elongation at break of the blend nanofiber scaffolds increased from 3.3 ± 0.35 MPa, 2.8 ± 0.26 MPa, and 8 ± 0.77% to 5.05 ± 0.52 MPa, 4.6 ± 0.82 MPa, and 17.6 ± 1.24% by increasing PHB content from 60% to 90%, respectively. The water contact angle of blend nanofiber scaffolds decreased about 50% from 112 ± 2.1° to 60 ± 0.75°. The biodegradability was evaluated by in vitro degradation test and the results revealed that the blend nanofiber scaffolds showed much higher degradation rates than the neat PHB. The cytocompatibility of the blend nanofiber scaffolds was preliminarily evaluated by cell adhesion studies. The cells incubated with PHB/CA blend nanofiber scaffold for 48 h were capable of forming cell adhesion and proliferation. It showed much better biocompatibility than pure PHB film. Thus, the prepared PHB/CA blend nanofiber scaffolds are bioactive and may be more suitable for cell proliferation suggesting that these scaffolds can be used for wound dressing or tissue-engineering scaffolds. © 2015 Elsevier B.V. All rights reserved.

1. Introduction Fundamental knowledge of cell–substrate interactions is key point for tissue engineering. Topographical cues, independent of biochemistry, generated by an extracellular matrix (ECM) may have significant effects on cellular cell seeding, adhesion, proliferation and differentiation [1–4]. Thus, preparation of matrices with characteristics of natural extracellular matrix (NECM) has attracted much interest in recent years. In general, the tissue development is controlled in three matrix size scales. The gross shape and size of tissue are decided by the macroscopic shape (cm to mm scale) of matrix; cell invasion and growth are controlled by the size and structure of the matrix pore (μm); the adhesion and gene expression of cells are adjusted by the surface chemistry of the matrices (nm scale). A number of processing techniques such as particulate leaching [5,6], template synthesis [7,8], phase separation [9,10], freeze drying [11,12] and self-assembly [13,14] have been used ⁎ Corresponding author at: School of Textiles, Tianjin Polytechnic University, Tianjin 300387, China. E-mail address: [email protected] (C. Zhijiang).

http://dx.doi.org/10.1016/j.msec.2015.09.048 0928-4931/© 2015 Elsevier B.V. All rights reserved.

to prepare biomimetic ECM as tissue engineering scaffolds. However, the diameters of the matrix fibers and pores are often at micron sizes and still far from the NECM which is a three-dimensional network structure composed of natural fibers ranging from 50 to 500 nm [15]. Hence, the design of scaffold composed of nanofibers has become one of the exciting new areas in tissue engineering. Electrospinning technique provides a versatile and effective method to prepare fibers with the diameters in the range of several microns down to a few tens of nanometers. The electrospun fibrous nonwoven mats are characterized by high surface area to volume/mass ratio, high porosity, small pore size with interconnected structure, thus resembling the NECM. These characteristics have made the electrospun fibrous nonwoven mats which can be applied in membrane filtration [16,17], metal ion adsorption [18,19], optical sensors and biosensors [20,21], wound dressings [22,23], and tissue engineering [24,25]. Poly(hydroxybutyrate) (PHB) is a thermoplastic polyester produced by various microorganisms as a reserve energy source. Due to its inherent biocompatible and biodegradable properties, PHB is ideal for various biomedical applications such as controlled release system [26], surgical sutures [27], wound dressing [28], orthopedic uses [29] and dura

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substitute [30]. Preparation of electrospun PHB scaffold has become a topic of current interest. The in vitro biocompatibility studies of PHB scaffolds fabricated by solution-cast solvent and electrospinning techniques have been reported with Schwann cells [31]. The cell adhesion on the surface of PHB film scaffold is good, while those on the surfaces of PHB electrospun scaffold also appear in their characteristic spindle shape, but with the cells being able to penetrate to the inner side of the scaffolds. This result indicates that PHB electrospun scaffold is more suitable for cell proliferation and differentiation. A comparison of PHB electrospun nanofiber scaffolds with poly(e-caprolactone) (PCL), silk, poly-lactic acid (PLA), and polyamide (PA) electrospun nanofiber scaffolds for cardiac repair has been presented by Castellano. PHB electrospun nanofibers modify the inflammatory response to an M2 macrophage phenotype in cardiac tissue, indicating PHB as a superior substrate for cardiac repair [32]. Electrospun nanofiber mats of PHB, PHBV and their blend have been fabricated and used as bone scaffolds [33,34]. In comparison with the corresponding solution-cast film scaffolds, all of the nanofibrous scaffolds exhibit much better support for cell attachment and proliferation, implying a high potential application of these electrospun PHB nanofiber mats as bone scaffolds. PHB/ nanosized hydroxyapatite blend scaffolds manufactured by gas-jet/ electrospinning illustrate that the electrospun scaffolds possess an extracellular matrix-like topography [35]. Biocomposite scaffolds based on electrospun PHB nanofibers and electrosprayed hydroxyapatite nanoparticles have been prepared and used for bone tissue engineering [36]. Electrospun PHB/magnetite nanofibrous nonwoven has been developed using 2,2,2-trifluoroethanol (TFE) as solvent. The degradation rate can be controlled by loading of magnetite nanoparticles [37]. Further studies show that the electrospun scaffolds have positive effects on attachment, proliferation and differentiation of bone marrow stroma cells (BMSCs). PHB/nanotholits scaffolds have also been electrospun for bone tissue regeneration [38]. PHB/chitosan based polymeric scaffolds and PHB/chitosan ultrafine fiber mats as skin regeneration prepared by electrospinning have been reported recently [39,40]. The cytotoxicity assessment with mouse fibroblast cells (L929) is investigated and the cell culture results show that electrospun PHB/chitosan scaffold benefits promoting the cell attachment and proliferation. PHB/ gelatin core–shell structured electrospun fiber mats have been prepared by coaxial electrospinning. The fiber mats support the growth of human dermal fibroblasts and keratinocytes with normal morphology indicating its potential as a scaffold in tissue engineering [41]. Electrospun PHB/poly(L-lactide-co-ε-caprolactone) (PLCL) composite as nanofibrous scaffolds has been reported by Daranarong. Analysis shows that PHB/PLCL nanofibrous scaffolds can promote cell cycle progression and reduce the onset of necrosis compared to their individual PHB and PLCL components suggesting potential in the repair and engineering of nerve tissue [42]. As a well known derivative of cellulose, cellulose acetate (CA) has been used in a variety of applications such as film based photography [43], component in adhesive [44], reverse osmosis [45] and nanofiltration membrane [46]. CA fibrous structures have been produced via the electrospinning technique [47]. By now, CA/carbon nanotube blend nanofibers, CA/metal particle blend nanofibers, CA/metal oxide blend nanofibers, CA/polyacrylonitrile blend nanofibers, CA/polyvinyl alcohol blend nanofibers and CA/chitosan blend nanofibers have been fabricated and shown potential applications in biomedical, tissue engineering, sensor, adsorbent, etc. [48–55]. In this paper, we are reporting the preparation of poly(hydroxybutyrate) (PHB)/cellulose acetate (CA) blend nanofiber scaffolds by electrospinning technique using chloroform/DMF as co-solvent. The blend nanofiber scaffolds were characterized by SEM, FTIR, DSC, XRD, water contact angle and tensile test. The biodegradability and cytocompatibility of the PHB/CA blend nanofiber scaffolds were also preliminarily evaluated by in vitro degradation test and cell attachment studies. The present contribution aims at combining these two biodegradable and biocompatible polymers to fabricate

new blend nanofiber scaffolds with potential application in biomedical field.

2. Materials and methods 2.1. Materials The poly(hydroxybutyrate) (PHB), a white powder sample, was kindly provided by Tianjin TianLu Co. Ltd. (China), Mw = 4.3 × 105 (obtained by G.P.C. in chloroform at 30 °C). CA with an acetyl content of 40% was purchased from Fluka. Chloroform and other chemicals of the highest purity available were used and purchased from Sigma-Aldrich, USA.

2.2. Preparation of PHB film PHB was dissolved in chloroform/DMF (70:30 v/v) to make a 5% solution. Then, the transparent PHB solution was spin-coated on wafer and dried at room temperature for 12 h. To ensure complete elimination of the solvent, the films were then dried at 60 °C for 6 h. The PHB films were obtained by peeling them off from the wafer.

2.3. Preparation of PHB/CA blend nanofiber scaffolds The production parameters for PHB/CA blend nanofiber scaffolds were summarized in Table 1. Electrospinning solutions were prepared at several PHB/CA ratios (100:0, 90:10, 80:20, 70:30, 60:40 and 0:100 w/w), 5% total polymer, in blends of chloroform/DMF (70:30 v/v). Electrospinning of PHB/CA blend solutions was carried out as the following procedures. PHB/CA blend solution was filled into a glass syringe terminated by a stainless steel needle with inner diameter of 0.50 mm. The syringe was placed in an automatic pump and PHB/CA blend solution was extruded out at a constant speed of 0.2 ml/h. High voltage ranging from 24 to 30 kV was applied in the electrospinning process. The tip-to-collector distance was fixed at 25 cm. The PHB/CA blend nanofibers were collected by Al foil. The experiment was done in an environmental chamber with constant temperature at 25 °C and the relative humidity (RH) of 35%.

2.4. Porosity measurements The porosity of scaffold was estimated using Archimedes' principle based on fluid displacement measurement techniques [56]. Briefly, individual scaffolds (disc-shaped: 10 mm diameter, 5 mm thickness) were placed in a graduated cylinder filled with a known volume of ethanol (V1). The total volume following scaffold immersion (V2) was recorded. After 1 h, the scaffolds were removed with the entrapped solvent in the pores, and the volume of ethanol left in the cylinder was denoted by V3. The total volume (VT) of the scaffolds was determined using the equation VT = V2 − V3. The porosity was then determined by calculating

Table 1 Production parameters for PHB/CA blend nanofiber scaffolds. PHB/CA ratios

Solution concentration (%)

Extrusion speed (ml/h)

Applied voltage (kV)

Reception distance (cm)

100/10 90/10 80/20 70/30 60/40 0/100

5 5 5 5 5 5

0.2 0.2 0.2 0.2 0.2 0.2

24 24 26 28 30 24

25 25 25 25 25 25

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the volume of free space within the material with respect to the total volume of the scaffold (n = 5) using the following equation: Porosity% ¼

V 1 −V 3  100%: VT

ð1Þ

2.5. Characterization Scanning electron microscopy (Hitachi S-4200) was used to characterize the morphology of the electrospun nanofiber scaffolds. The samples were coated with silver and examined at an accelerating voltage of 15 kV. The fiber diameter distributions of the scaffolds were obtained by analyzing the SEM micrographs by image-analysis software (Adobe Photoshop 7.0). One hundred fibers were measured for each sample using four SEM images. The Brunauer–Emmett–Teller (BET) surface areas were measured for each sample for three replications by using a surface area analyzer (SAA: Sorptomatic 1990, ThermoFinnigan Co.). FTIR measurements were carried out on a single beam Perkin Elmer Spectra 2000 IR spectrometer under N2 purging. X-ray diffraction (XRD) patterns were recorded on an X-ray diffractometer (D/MAX2500, Rigaku), by using Cu Kα radiation at 40 kV and 30 mA. The diffraction angle was ranged from 5 to 35°. Differential scanning calorimetry (DSC, Model NETZSCH STA 409) tests were performed under a continuously renewed nitrogen atmosphere (flow rate, 20 ml/min). The sample was heated from room temperature to 250 °C at the heating rate of 5 °C/ min. Contact angle measurements were performed at 25 °C in the range of 0.5–20 min by pendant drop method, employing a contact-angle measurement apparatus (type DSA-10, made in KURSS Company, Germany). The contact angle between the scaffold and the 3T3 fibroblast buffer solution and the pure water was measured. For each sample, the mean of five separate points was obtained based on the same contact time for three times. The mechanical properties of single electrospun nanofiber were evaluated using the NANO UTM universal testing systems (Agilent U9820A G200) with a load cell of 0.5 N, a load resolution of 1 μN and a strain rate of 2 × 10−4 s−1. The samples were prepared according to the parameters listed in Table 1. When the electrospinning process became stable, the single nanofiber was collected by Al foil in a second. SEM image was used to determine the diameter of single nanofiber for each test. 10 tests were applied for each sample to calculate the average and standard deviation. The mechanical properties of nanofiber scaffolds were tested on a tensile tester (AGS100A, Shimadzu Co., Japan) with the extension rate of 2 mm/min in ambient condition. The size of the samples was 100 mm length, 20 mm width, and 50 mm distance between two clamps. Young's modulus of samples was found from the tensile test results conducted according to ASTM D-882-97 as a standard test method for tensile elastic properties of thin plastic sheeting. For each sample, three replicates were used. 2.6. In vitro biodegradation The biodegradation studies of the PHB/CA blend nanofiber scaffolds were conducted at 37 °C in phosphate buffer solutions (PBS, Oxoid pH: 7.2–7.4) and lysozyme solution (Sigma, 0.2% solution in PBS). The dried scaffolds were cut into squares and incubated in the reaction solution with shaking, and at various time points the samples were removed, washed in distilled water and allowed to dry in air to constant weight. For each sample, three replicates were used and the weight loss ratio (S%) was determined by the ratio of the weight loss to the initial weight of samples as shown below: S% ¼

W 0 −W t  100% W0

ð2Þ

where Wt and W0 are the weight of samples after being dried and the initial weight respectively.

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2.7. Cell attachment studies The PHB/CA blend nanofiber scaffolds (20 mm diameter × 1 mm height) were used for the cell attachment studies. Prior to cell culture work, the samples were sterilized using ethylene oxide for 18 h. The samples were then pre-treated by immersing in DMEM (Dilbecco's Modified Eagle Medium) for 24 h. After the pre-treatment, the samples were carefully placed in 24 well plates and the cells (3T3 fibroblasts) were seeded at a density of 2.5 × 104 cells/well. And the samples were incubated at 37 °C/5% CO2 for 48 h. For the measurement of cell adhesion, cells were washed twice with PBS to remove non-adherent cells and the attached cells were fixed with 2.5% glutaraldehyde buffer solution (pH 7.4) at 4 °C for 12 h. The samples were subsequently rinsed in distilled water and dehydrated by freeze-dryer at − 40 °C. For each experimental value, three independent experiments were conducted. The cells' morphology was observed by scanning electron microscope (SEM Philips XL-30). Osteoblast cell viability was assessed by MTT method. 100 μl of the reagent solution prepared according to the manufacturer's instructions was added to each well. After incubation for 24 h, 48 h and 72 h at 37 °C in a humidified atmosphere with 5% CO2, all the solutions were removed and the cells were washed gently with PBS for three times. MTT (25 ml) was added into each well at 37 °C for 4 h. Then DMSO (150 ml) was added to dissolve the MTT crystals. Finally, the absorbance of the solution was measured at 490 nm by an enzyme linked immunosorbent assay (ELISA) Reader (MODEL550, Bio-Rad, USA). 2.8. Statistical analysis Data were expressed as mean ± standard deviation. The standard deviation (σ) was calculated with Eq. (3). vffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi u N u1 X  2 σ ¼t x −x ̅ : N i¼1 i

ð3Þ

Results were statistically analyzed using the Stratgraphics Plus Professional 16.0.03 software and compared with Kruskal Wallis test. The differences were considered statistically significant when p b 0.05. 3. Results and discussion 3.1. SEM observation Fig. 1 shows the surface morphology of pure PHB, CA and PHB/CA blend nanofiber scaffolds prepared by electrospinning under different conditions. The porous structure can be observed and nanofibers exhibit cylindrical, uniform, bead-free and random orientation. For pure PHB and CA nanofiber scaffolds, the average diameters of the nanofiber are 670 ± 220 nm and 265 ± 35. The thinner diameter and narrower diameter distribution of CA nanofiber scaffolds might be due to the low molecular weight of materials. As we know, fiber diameter is dependent on many electrospinning variables including applied voltage and polymer concentration. By varying the applied voltage and polymer concentration, it is possible to modify the average fiber diameter of the electrospun nanofiber scaffolds. The diameter distribution of the PHB/ CA blend nanofiber scaffolds plotted as a function of frequency is shown in Fig. 2. The diameter for each fiber was obtained by analyzing the SEM micrographs by image-analysis software (Adobe Photoshop 7.0). Four SEM images were used and 100 fibers were counted totally for each sample. In the present work, the applied voltage is increased from 24 kV to 30 kV. The average diameter as well as porosity and specific surface areas are summarized in Table 2. It is found that the as-electrospun PHB/CA blend nanofiber scaffolds contain nano- and sub-micro sized fibers, diameters ranging from 80 to 680 nm, and have an average

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Fig. 1. Surface morphology of PHB/CA blend nanofiber scaffolds observed by SEM at an accelerating voltage of 15 kV (PHB/CA ratios in the blend nanofiber scaffolds: a 100/0, b 90/10, c 80/ 20, d 70/30, e 60/40 and f 0/100).

porosity of 81 ± 2.1%–85 ± 2.6%. With the increase of applied voltage and CA content, the average diameters of PHB/CA blend nanofiber scaffolds decrease from 450 ± 124 nm to 160 ± 52 nm and the fiber diameter distributions become narrow with the numbers of the peaks decreasing from 9 to 4. The measured BET surface areas of the PHB/CA blend nanofiber scaffolds are in the range of 40.41 ± 4.24–78.37 ± 11.22 m2/g, depending on the fiber diameter. With the diameter of PHB/CA blend nanofiber decreasing, the BET surface areas increase. From the morphological observation, it can be stated that PHB/CA blend nanofiber scaffolds have diameters comparable to collagen fibers (50–500 nm), a major NECM component [15]. The thinnest fiber with an average diameter of 160 nm can be fabricated in this work. By optimizing the electrospinning parameters, thinner PHB/CA blend nanofibers with diameter less than 100 nm can be fabricated. This kind of work is ongoing in our lab and the results will be reported later. 3.2. FT-IR Fig. 3 depicts the FT-IR spectra of PHB, CA and PHB/CA (60/40) blend nanofiber scaffold. For pure PHB (Fig. 3a), the peak at 3433 cm−1 refers to hydroxyl end groups. Peaks at 3063 cm− 1, 29,651 cm− 1 and 2911 cm−1 refer to C–H stretching vibration. A sharp and steep band observed at 1722 cm−1 is assigned to C_O stretching vibration. The peak at 1280 cm−1 refers to C–C–O stretching vibrations. Peaks located at 1130 cm−1 and 1053 cm−1 refer to C–O and C–C stretching vibration [57–60]. For pure CA (Fig. 3c), broad absorbance peak at 3380 cm−1 is

attributed to OH stretching. Peaks at 2910 cm−1 and 2860 cm−1 refer to C–H stretching vibration. The band at 1750 cm− 1 corresponds to the C_O stretching of ester group (carbonyl groups). The peak at 1435 cm− 1 is due to O_C–OR stretching vibration. Peaks located at 1235 cm−1 and 1050 cm−1 refer to C–C–O and C–O stretching vibration. The PHB/CA blend nanofiber scaffold displays both IR features of PHB and CA without new peaks (Fig. 3b). This result confirms that the structural stability of the electrospun fibers is maintained during the electrospinning process and there is no chemical reaction between PHB and CA in the blend nanofiber. However, the frequency is shifted to higher region. As we know, the frequency difference is considered as a measure of the average strength of the intermolecular bond [61]. The frequency difference indicates that intermolecular interactions between PHB and CA occur. One possible cause for this shift is that the intermolecular interactions between the C_O groups in PHB and the OH groups in CA, i.e., CA–O–H⋯O_C–PHB interactions formed in the blend system [62]. Further measurements such as Raman spectroscopy should be necessary to clarify this. 3.3. XRD analysis Fig. 4 shows the X-ray diffraction patterns of pure PHB, CA and PHB/ CA blend nanofiber scaffolds prepared by electrospinning. The crystalline reflection of PHB is an orthorhombic lattice structure (P212121: a = 0.576 nm, b = 1.320 nm and c = 0.596 nm (fiber axis), α-form) with their chains in the left 2/1 helix [63]. X-ray diffraction peaks appear

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Fig. 2. The diameter distribution of the PHB/CA blend nanofiber scaffolds as a function of frequency (PHB/CA ratios in the blend nanofiber scaffolds: a 100/0, b 90/10, c 80/20, d 70/30, e 60/ 40 and f 0/100). The diameter for each fiber was obtained by analyzing the SEM micrographs by image-analysis software (Adobe Photoshop 7.0). Four SEM images were used and 100 fibers were counted totally for each sample.

at 2θ value of 13.2°, 17.1°, 25°, 26.5°, 26.7°, 28.5° and 31.6° assigned to (0 2 0), (1 1 0), (2 0 0), (0 2 1), (1 0 1), (1 2 1) and (0 0 2) of the orthorhombic unit cell, respectively. Pure CA nanofiber scaffold displays amorphous characteristics with broad diffraction peaks at 2θ = 21.4°. For PHB/CA blend nanofiber scaffolds, the characteristic peaks are the same with pure PHB, which indicate that the crystalline structure of PHB is stable during the electrospinning process. However, the height of the (0 0 2) peak decreases and the area ratio of the crystalline peak is also decreased from 0.80 for PHB to 0.61 for PHB/CA (60:40) with

the CA content increasing in the blend nanofiber scaffolds. Obviously, the presence of CA affects the preferential orientation of the (0 0 2) and makes the crystallinity of as-electrospun PHB nanofiber lower than that of bulk materials. The reason should be due to that pure CA is amorphous materials acting as diluents in the blend nanofiber. Moreover, intermolecular interactions between the C_O groups in PHB and the OH groups in CA formed in the blend nanofibers could also restrict the preferential orientation of PHB molecules. 3.4. DSC test

Table 2 Physical properties of PHB/CA blend nanofiber scaffolds. PHB/CA ratios

Average fiber diameter (nm)

BET surface areas (m2/g)

Porosity (%)

100/10 90/10 80/20 70/30 60/40 0/100

670 ± 220 450 ± 124 360 ± 103 234 ± 76 160 ± 52 265 ± 35

27.55 ± 2.66 40.41 ± 4.24 55.17 ± 7.54 67.22 ± 9.74 78.37 ± 11.22 61.82 ± 8.14

86 ± 2.7 85 ± 2.6 83 ± 2.0 83 ± 1.9 81 ± 2.1 83 ± 2.3

Differential scanning calorimetry (DSC) thermograms of the pure PHB, CA and PHB/CA blend nanofiber scaffolds are displayed in Fig. 5. The first heating of the DSC scan is used to represent the aselectrospun PHB/CA blend nanofiber structure. For pure PHB nanofiber scaffold (Fig. 5a), glass transition temperature (Tg) is detected at about 12.6 °C. An endothermic peak with the heat fusion of 72.4 J/g around 152.4 °C is observed which should be due to the melting transition of the crystalline phase. However, only glass transition can be seen around 121.4 °C for pure CA nanofiber scaffold (Fig. 5f). For PHB/CA blend

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Fig. 3. FTIR spectra of PHB/CA blend nanofiber scaffold (PHB/CA ratios in the blend nanofiber scaffolds: a 100/0, b 60/40 and c 0/100).

nanofiber scaffolds, all the samples show an endothermic peak around 150 °C, which should be due to the melting transition of the crystalline phase. With the CA content increasing from 10 to 40%, the melting

Fig. 5. DSC curves of PHB/CA blend nanofiber scaffolds (PHB/CA ratios in the blend nanofiber scaffolds: a 100/0, b 90/10, c 80/20, d 70/30, e 60/40 and f 0/100). The arrow indicates the increasing of glass transition temperature (temperature range: room ~250 °C, heating rate: 5 °C/min).

temperature slowly decreases from 151.3 to 147.6 °C. At the same time, the heat fusion of the crystalline phase (ΔH) also decreases from 54.7 to 25.8 J/g indicating the decrease in crystallinity. Generally, thermal characterization of polymer blends is a wellknown method for determining the miscibility of polymer blends. The miscibility between any two polymers in the amorphous state is detected by the presence of a single Tg intermediate between those of the two component polymers. In this study, only one Tg could be detected for PHB/CA blend nanofiber scaffolds, which indicates that the PHB and CA are miscible in the blend nanofiber and no phase separation occurs during the electrospinning process. The Tg increases from 22.4 °C to 36.5 °C with the CA content increasing from 10 to 40%. The increase in Tg indicates the decrease in chain mobility of polymers, since the segments of polymers have to be mobile at higher temperatures. The reason might be a result of the formation of more intermolecular hydrogen bonds between PHB and CA, which make chain mobility difficult. It is recognized that tissue engineering scaffolds with a Tg lower than 37 °C (physiological temperature) are desirable, since scaffolds having Tg higher than the physiological temperature tend to be brittle and can fracture when subjected to stress in use [64].

3.5. Tensile test

Fig. 4. XRD patterns of PHB/CA blend nanofiber scaffolds (PHB/CA ratios in the blend nanofiber scaffolds: a 100/0, b 90/10, c 80/20, d 70/30, e 60/40 and f 0/100) (diffraction angle range: 5–35°, scanning rate: 2°/min).

The mechanical properties of the electrospun blend nanofiber scaffolds are strongly influenced by the properties of each polymer in the blend, the interaction between each polymer in the nanofibers, each single nanofiber in the blend scaffolds and nanofiber structure in the scaffolds [65]. To investigate the mechanical characteristics of PHB/CA blend nanofiber scaffolds, tensile tests were conducted using NANO UTM universal testing systems for each single PHB/CA blend nanofiber. The mechanical properties found by stress–strain curves are summarized in Table 3.

C. Zhijiang et al. / Materials Science and Engineering C 58 (2016) 757–767 Table 3 Mechanical properties of single electrospun PHB/CA blend nanofiber. PHB/CA ratios

Tensile strength (MPa)

Young's modulus (MPa)

Elongation at break (%)

100/10 90/10 80/20 70/30 60/40 0/100

7.44 ± 0.58 7.86 ± 0.67 6.64 ± 0.55 5.78 ± 0.47 4.52 ± 0.34 1.56 ± 0.19

867.5 ± 201.2 854.2 ± 187.6 835.1 ± 177.2 822.4 ± 174.3 806.9 ± 168.2 41.6 ± 12.3

22.7 ± 1.77 16.7 ± 1.52 14.35 ± 1.21 9.64 ± 0.67 6.53 ± 0.48 2.35 ± 0.24

Pure CA nanofiber shows low tensile strength and Young's modulus with value of 1.56 ± 0.19 MPa and 41.6 ± 12.3 MPa respectively due to its amorphous characteristic. However, PHB nanofiber displays high mechanical properties with tensile strength and Young's modulus of 7.44 ± 0.58 MPa and 867.5 ± 201.2 MPa respectively. For PHB/CA blend nanofiber, its mechanical properties are much higher than pure CA nanofiber. With PHB content increasing from 60% to 90% in the blend nanofiber, the tensile strength, elongation at break and Young's modulus increase from 4.52 ± 0.34 MPa, 6.53 ± 0.48% and 806.9 ± 168.2 MPa to 7.86 ± 0.67 MPa, 16.7 ± 1.52% and 854.2 ± 187.6 MPa, respectively. This behavior can be due to the inherently high modulus and strength of PHB, which allow the mechanical properties of PHB/CA blend nanofiber to improve. It also indicates that the blend nanofiber has good dispersibility of PHB and CA to lead uniform mechanical properties for each fiber. Moreover, the tensile strength of PHB/CA (90:10) blend nanofiber is even higher than that of pure PHB nanofiber. This might be associated with the molecular interaction between PHB and CA in the blend nanofiber. The tensile tests for PHB/CA blend nanofiber scaffolds were performed according to ASTM D-882-97 standard test method. Fig. 6 shows the stress–strain curves of the blend nanofiber scaffolds with pure PHB and CA scaffolds as comparison. For pure CA nanofiber scaffold (Fig. 6f), it shows typical brittle properties. The elongation at break is very small and no yielding point can be observed. After adding with PHB component, the mechanical properties of CA nanofiber scaffold are largely reinforced. The blend nanofiber scaffolds show toughness properties with the tensile strength, yield strength and elongation at break increasing from 3.3 ± 0.35 MPa, 2.8 ± 0.26 MPa, and 8 ± 0.77% to 5.05 ± 0.52 MPa, 4.6 ± 0.82 MPa, and 17.6 ± 1.24% on increasing PHB content from 60% to 90%, respectively. The Young's modulus found from the slope of the curve is 20 ± 1.66 MPa for CA nanofiber

Fig. 6. Stress–strain curves of PHB/CA blend nanofiber scaffolds (PHB/CA ratios in the blend nanofiber scaffolds: a 100/0, b 90/10, c 80/20, d 70/30, e 60/40 and f 0/100) (sample size: 100 mm length, 20 mm width, 50 mm distance between two clamps, extension rate: 2 mm/min, ambient condition).

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scaffold and 500 ± 14.35 MPa for PHB/CA (90/10) blend nanofiber scaffolds. The PHB/CA blend nanofiber scaffolds display 70% and 60% of improvement in tensile strength and yield strength, 165% of increase in elongation at break with PHB content increasing from 60% to 90% in the blend scaffolds. However, the mechanical properties of nanofiber scaffolds are lower compared with single nanofiber. This result should be due to the unconnected joint points of each single nanofiber. Since the mechanical properties of the nanofiber scaffolds depend on both mechanical properties of single nanofiber and join points, posttreatment like heating to make fibers adhered to each other at the joint points might be helpful for mechanical properties' improvement. The kind of work is ongoing at our lab and the results will be reported later. 3.6. Hydrophilicity The contact angle between the electrospun scaffold and the buffer solution containing fibroblast cells (3T3) was measured to determine the hydrophilicity of the nanofiber scaffolds. As a comparison, the contact angle between the scaffolds and the pure water was also measured since the contact angle measurements using the cell solution might be affected by the presence of proteins and their interactions with the electrospun nanofiber surface. The measured contact angle values of pure PHB, CA and PHB/CA blend nanofiber scaffolds are shown in Fig. 7. The difference of contact angle values measured in cell containing solution and pure water is very small. This result suggests that presence of cells has little effect on the hydrophilicity of PHB/CA blend nanofiber scaffolds. As seen from Fig. 7, the contact angles of the pure PHB, PHB/CA (90:10) and PHB/CA (80:20) blend nanofiber scaffold are about 123 ± 3.5°, 112 ± 1.8° and 96.7 ± 2.3°, indicating that the scaffold surface is hydrophobic. It has been reported that a hydrophobic surface is ideal to promote protein bonding. BSA would rather adsorb to the hydrophobic surfaces than the hydrophilic ones [66,67]. However, the cell morphology investigated by immunofluorescent staining shows evenly spread cells on the hydrophilic materials, whereas the cells on the hydrophobic materials group together [68]. Thus, the cell solution cannot spread well on hydrophobic pure PHB, PHB/CA (90:10) and PHB/CA (80:20) blend nanofiber scaffold surface, which may be not favorable for cell spreading and proliferation. With CA content increasing in the blend nanofiber scaffold, the contact angle tends to decrease and the scaffold surface is gradually transformed from hydrophobicity to hydrophilicity. When

Fig. 7. The contact angle of PHB/CA blend nanofiber scaffolds in cell solution and pure water (PHB/CA ratios in the blend nanofiber scaffolds: a 100/0, b 90/10, c 80/20, d 70/ 30, e 60/40 and f 0/100). 3T3 fibroblasts were used in cell solution. The measurement was performed at 25 °C in the range of 0.5–20 min by pendant drop method.

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the CA content is 40%, the PHB/CA blend nanofiber scaffold is quite hydrophilic with contact angle of 60 ± 1.25°. It is cautioned that the values of contact angle measured here are affected by the roughness of the electrospun nanofiber scaffold. With fiber diameter increasing, the roughness increases. Water contact angle increases due to trapped air pockets [69,70]. However, these values can still be used to evaluate the hydrophilicity of the PHB/CA blend nanofiber scaffold. It is clear that with the CA content increasing and fiber diameter decreasing, the electrospun PHB/CA blend nanofiber scaffolds become more hydrophilic. By controlling the fiber diameter, we can adjust the hydrophobicity/ hydrophilicity of the PHB/CA blend nanofiber scaffold. It can be expected that the PHB/CA blend nanofiber scaffolds with high hydrophilicity are more suitable for cell adhesion and proliferation. 3.7. Biodegradation tests It has been reported that the lysozyme can accelerate the degradation rate of PHB/PEG grafting polymer [71]. In this paper, degradation of PHB/CA blend nanofiber scaffolds was performed in lysozyme buffer solution and buffer solution at 37 °C. Fig. 8 illustrates the changes in the weight loss as a function of CA content in blend nanofiber scaffolds over time. The in vitro degradation tests under enzyme condition reveal the low degradation rate of the pure PHB nanofiber scaffold in the scale of the experiment. After degradation for 25 days, the weight loss ratio is less than 15 ± 0.38% of original weight, which is much faster than that of buffer solution without enzyme (3 ± 0.22% weight loss). With the CA introduction, the degradation rate of PHB/CA blend nanofiber scaffolds is greatly improved. For example, the degradation rate of PHB/CA (70:30) blend nanofiber scaffold is triple as high as that of pure PHB nanofiber scaffold. This acceleration of the biodegradation is supposed to be arisen from the lowered crystallinity of PHB in the blend nanofiber scaffolds. Another possible reason is the hydrophilicity of the blend nanofiber scaffold, which makes the enzyme easier to attack. Therefore, the acceleration of the biodegradation is caused by a combined effect of lower crystallinity and better hydrophilicity as well. The corresponding morphological changes of the PHB/CA blend nanofiber scaffold in vitro degradation at 37 °C in lysozyme buffer solution for 25 days are shown in Fig. 9. The electrospun scaffolds cannot sustain its nanofiber structure and most of nanofibers have been broken. In general, the enzymatic biodegradation proceeds from amorphous regions on the surface of the fiber and erosion develops gradually to the inside. The surface erosion may be promoted by the high surface areas of the nanofibers and may be due to the interconnected porosity arising from the nonwoven fibrous nature of the material. With the prolongation of the biodegradation time, the surface becomes rougher and rougher, and many corrosive spots occur, finally fiber breakage

happens. At higher CA content, there are more amorphous regions on the surface of the blend nanofiber and the lost of fiber integrity occurs quickly. It turned out that CA content is an effective way to control the degradation rate of the electrospun PHB/CA blend nanofiber scaffolds. 3.8. Cytocompatibility Cell cytotoxicity is one of the important factors that affect the use of polymers in tissue engineering. Studies have shown that the nanofibers affect cellular behavior by promoting the proliferation and differentiation of cells because cells attach and organize around fibers with diameters smaller than that of cells [72]. In this study, fibroblast cells (3T3) are used to evaluate cytocompatibility of PHB/CA blend nanofiber scaffolds via cell cultivation in vitro. Fig. 10(a) and (b) shows the morphology of fibroblast cells cultured on pure PHB film and PHB/CA (60/40) blend nanofiber scaffold at 37 °C for 48 h. In general, cells do not grow on a solid polymer surface. In fact, cells grow on a layer of protein that interacts with cellular receptors. Blending with CA and PHB/CA blend scaffolds becomes more hydrophilic. In the case of pure PHB film, most of the cells are still in round-shaped (Fig. 10a). However, in the case of PHB/CA blend nanofiber scaffold, cells cultured on the scaffold adhere and completely spread on the surface (Fig. 10b). They have many pseudopodia and form a layer on the surface (Fig. 10c). These results indicate that the cells stretch their morphology and are proliferating. We evaluate the toxicity of PHB/CA fiber scaffolds cultured for different periods of time (24 h, 48 h and 72 h) towards viability of fibroblast cells. The live cells are determined by MTT essay and the results are reported in Fig. 10(d). For relative cell number, the number of viable cells at day 0 after cell attachment for 4 h is taken as one for each sample to compare the proliferation rate of cells in different scaffolds. The cell number increases with culture time but shows clear dependence on the structure of the scaffolds. The PHB/CA blend nanofiber scaffold significantly promotes cell proliferation compared to PHB film after 72 h (Fig. 10c). This preliminary experimental result suggests that PHB/CA blend nanofiber scaffold has better biocompatibility compared with pure PHB film in terms of fibroblast cell culture. The reason might be due to the fact that the PHB/CA blend nanofiber scaffold with fiber diameter in the range of 80–500 nm is very close to the NECM with three-dimensional network structure composed of natural fibers ranging from 50 to 500 nm. The appearance of nano-scale pores in the blend nanofiber scaffold can adjust the surface chemistry to improve the adhesion of cells. This PHB/CA blend nanofiber scaffold should have potentials to be used as tissue regeneration scaffold in vitro. Further investigations such as cellular proliferation and differentiation assays are underway.

Fig. 8. In vitro degradation of PHB/CA blend nanofiber scaffolds (PHB/CA ratios: a 100/0, b 90/10, c 80/20, d 70/30, e 60/40) in buffer solution (A) and enzyme solution (B) (lysozyme concentration: 2%; temperature: 37 °C).

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Fig. 9. The surface morphology of PHB/CA blend nanofiber scaffolds after degradation in enzyme solution for 25 days observed by SEM (PHB/CA ratios: a 100/0, b 80/20, c 70/30, d 60/40).

4. Conclusion In this paper, PHB/CA blend nanofiber scaffolds have been successfully prepared by electrospinning of blend solution of the two polymers using a mixture of chloroform and DMF as the solvent. SEM images showed that the prepared blend nanofiber scaffolds had very well

interconnected porous fibrous network structure and large aspect surface areas. All electrospun blend nanofiber scaffolds exhibited submicron sized fiber diameters, ranging from 80 to 680 nm, and about 40.41 ± 4.24–78.37 ± 11.22 m2/g of specific surface areas with a porosity of 81 ± 2.1%–85 ± 2.6%. The structural stability of the electrospun PHB and CA fibers was maintained during the electrospinning process

Fig. 10(a). 3T3 fibroblast cell attachments on pure PHB film; (b) 3T3 fibroblast cell attachments on PHB/CA 60/40 blend nanofiber scaffold; (c) high magnification for selected region (Incubation at 37 °C/5% CO2 for 48 h) and (d) relative cell number determined by MTT assay.

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and there was no chemical reaction between PHB and CA in the blend nanofiber. However, the presence of CA affected the crystallization of PHB in the blend and resulted in an increase in the glass transition temperature and a decrease in contact angle. In vitro biodegradation test results demonstrated that the biodegradation rates of the electrospun blend scaffolds could be finely tuned with different blend ratios by controlling the crystallinity and fiber diameters as well as specific surface areas. Cell adhesion studies were carried out using 3T3 fibroblast cells. The cells incubated with PHB/CA blend scaffold for 48 h were capable of forming cell adhesion and proliferation. It showed much better biocompatibility compared with pure PHB film. Thus, the electrospun PHB/CA blend scaffold is bioactive and may be more suitable for cell proliferation suggesting that this scaffold can be used for wound dressing or tissue engineering scaffolds. 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