Author’s Accepted Manuscript Fabrication, characterization and biomedical application of two-nozzle electrospun polycaprolactone/ zein- calcium lactate composite nonwoven mat Nina Liao, Mahesh Kumar Joshi, Arjun Prasad Tiwari, Chan-Hee Park, Cheol Sang Kim www.elsevier.com/locate/jmbbm

PII: DOI: Reference:

S1751-6161(16)00044-8 http://dx.doi.org/10.1016/j.jmbbm.2016.02.006 JMBBM1802

To appear in: Journal of the Mechanical Behavior of Biomedical Materials Received date: 11 November 2015 Revised date: 20 January 2016 Accepted date: 2 February 2016 Cite this article as: Nina Liao, Mahesh Kumar Joshi, Arjun Prasad Tiwari, ChanHee Park and Cheol Sang Kim, Fabrication, characterization and biomedical application of two-nozzle electrospun polycaprolactone/ zein- calcium lactate composite nonwoven mat, Journal of the Mechanical Behavior of Biomedical Materials, http://dx.doi.org/10.1016/j.jmbbm.2016.02.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Fabrication, characterization and biomedical application of two-nozzle electrospun polycaprolactone/ zein- calcium lactate composite nonwoven mat

Nina Liao1, 2, Mahesh Kumar Joshi1, 3, Arjun Prasad Tiwari1, Chan-Hee Park1, 4*, Cheol Sang Kim1, 4*

1

Department of Bionanosystem Engineering, Chonbuk National University, Jeonju

561-756, Republic of Korea 2

Center for Translational Medicine Research and Development, Shenzhen Institute of

Advanced Technology, Chinese Academy of Science, China 3

Department of Chemistry, Tri-Chandra Multiple Campus, Tribhuvan University,

Kathmandu, Nepal 4

Division of Mechanical Design Engineering, Chonbuk National University, Jeonju

561-756, Republic of Korea

*Corresponding authors: Cheol Sang Kim ([email protected]) Chan Hee Park ([email protected]) Tel.: +82-63-270-4284; fax: +82-63-270-2460. E-mail addresses: [email protected] , [email protected]

1

Abstract The objective of the current work is to incorporate calcium lactate (CL) into polycaprolactone

(PCL)/zein

composite

micro/nanofibrous

scaffolds

via

electrospinning to engineer bone tissue. In this study, a composite micro/nano fibrous scaffold was fabricated using a single two-nozzle electrospinning system to combine indicative nanofibers from a blended solution of zein-CL and micro-sized fibers from a PCL solution. Incorporation of the CL into the PCL/zein fibers were shown to improve the wettability, tensile strength and biological activity of the composite mats. Moreover, the composite mats have a high efficiency to nucleate calcium phosphate from simulated body fluid (SBF) solution. An in vitro cell culture with osteoblast cells demonstrated that the electrospun composite mats possessed improved biological properties, including a better cell adhesion, spread and proliferation. This study has demonstrated that the PCL/zein-CL composite provides a simple platform to fabricate a new biomimetic scaffold for bone tissue engineering, which can recapitulate both the morphology of extracellular matrix and composition of the bone. Keywords: Two-nozzle electrospinning; Micro-nano fibers; Calcium lactate; Biomedical application; Bone tissue;

2

1. Introduction Micro/nano fibers have been extensively studied for wide range of biomedical applications such as tissue engineering, wound dressing, and drug delivery due to their highly porous structures and large surface area (Kasoju et al., 2009; Santos et al., 2008). The micro fibers provides the mechanical support to the scaffolds, and the nano fibers enhance the cellular activity (Zhang et al., 2011a). As a result, micro/nano fibers are extensively used in biomedical applications, including bone tissue engineering (Zhang et al., 2008), drug delivery (Cui et al., 2010) and wound dressing (Unnithan et al., 2014). Among the various techniques, electrospinning is a facile and efficient technique to produce a variety of fibrous structures (Pham et al., 2006; Ramakrishna et al., 2006).

This technique consists of three major components – a

high voltage power supply, a spinneret and a collector plate (grounded) – and it can be used to generate fibers (diameter range from tens of nanometers to a few micrometers) from different polymers. There are two types of polymers synthetic polymers and natural polymers. Electrospun fibers of synthetic polymers possesses good mechanical properties and the degradation time for some polymers can be controlled (Rosso et al., 2005). However, synthetic polymers may induce a pro-inflammatory response due to the lack of cell recognition sites (Kim et al., 2011). On the other hand, naturally derived polymers exhibit excellent biological properties, including improved cell adhesion, biodegradability, and biocompatibility (Jux et al., 2003). However, the weak mechanical properties of natural polymers limit their application as tissue scaffolds (Lu et al., 2013). Composite fibers generated from both synthetic and natural 3

polymers possess the mechanical strength of the synthetic polymer and the good biological properties of the natural polymer (Bhattarai et al., 2009). However, different solvent systems and electrospinning parameters are needed for different polymers, which prevent mixing and blending their solutions to produce a micro/nano fibrous morphology using a traditional electrospinning system. To overcome this obstacle, two-nozzle electrospinning is used to produce composite fibers from two or more different polymers (Frey and Li, 2007). Several studies have shown that the mechanical strength of the electrospun mats can be improved by using a two-nozzle at an angle of 80° between the tips of two nozzles instead of a traditional electrospinning system (Tijing et al., 2013; Zhang et al., 2011b). In addition, the two-nozzle electrospinning system can be used to fabricate a composite membrane of the micro and nanofibers (Zhang et al., 2011b), and such an architecture can improve cell growth, adhesion and proliferation (Tuzlakoglu et al., 2005). Bone tissue engineering is a biomedical field where scaffolds play an important role for repair and replacement. An ideal scaffold possesses the mechanical strength necessary to support the affected area and a highly porous structure to promote cell adhesion and cell proliferation (Porter et al., 2009). In recent years, several studies have reported that electrospun scaffolds mimic the extra-cellular matrix and improve the cellular activity (Kim et al., 2005) and bone forming ability (Zhang et al., 2008). Moreover, composite micro/nano electrospun scaffolds made from synthetic and natural polymers enriched with a calcium compound could be strategic for hydroxyapatite nucleation. 4

Various synthetic and natural polymers, such as polycaprolactone (PCL), poly(vinyl alcohol) (PVA), polyvinyl acetate (PVAc), polyethylene oxide (PEO), polyethylene glycol (PEG), poly(lactic-co-glycolic acid) (PLGA), poly-L-lactic acid (PLLA), chitosan, alginates and gelatin, have been extensively studied to fabricate tissue scaffolds (Pant et al., 2013b). Of the various synthetic polymers that are available, PCL possesses many desirable properties, such as a proper biodegradability, good biocompatibility, excellent processibility and the proper mechanical strength (Kim et al., 2013). Therefore, PCL has been extensively studied for tissue engineering applications, including bone, skin and nerve regeneration (Pan et al., 2012). However, its poor hydrophilicity leads to a low cell adhesion, proliferation, and differentiation, which limit its use for tissue regeneration (Ghasemi-Mobarakeh et al., 2008; Kim et al., 2006). The hydrophilicity and biological activity of electrospun PCL fibers could be improved by incorporating calcium L-lactate (CL) in the nanofibers. Calcium L-lactate hydrate is a salt of lactate acid, which is a common source of calcium in food, and it has also been reportedly used for firming agents (Main et al., 1986) and food additives (B.M. et al., 1982). Furthermore, the tensile strength and water content capacity of biocomposite films can be improved by incorporating CL (Sedlarik et al., 2009). In another study, it has been reported that calcium lactate-coated nylon 6 mats improved cell adhesion, growth and proliferation (Pant et al., 2013c). However, this process is time-consuming and tedious since the lactic acid-coated nylon 6 nanofibers have to be prepared and then neutralized with calcium hydroxide to generate calcium lactate in the nanofibers. Therefore, a more reliable and easy process to form 5

composite nanofibers could involve the direct incorporation of CL into the polymer solution. As we observed in this study, a result of the lack of a suitable solvent and miscibility with the polymers, CL cannot be blended directly with the polymer solution for electrospinning. To overcome this obstacle, a two-nozzle electrospinning process can be used where CL can be electrospun with zein (amphiphilic biopolymer) from one nozzle and another desired polymer can be electrospun through another nozzle. Zein, a protein from corn, is an amphiphilic biopolymer that has polar and non-polar amino acids groups (Paliwal and Palakurthi, 2014), and it is adequate to dissolve CL in order to form a blended solution for electrospinning. Furthermore, Zein has proline and glutamine in abundance (Shewry and Halford, 2002). Polar groups in proline and glutamine may have ionic interaction with calcium lactate salt. Zein has been extensively studied for use in biomedical applications, such as for drug delivery (Liu et al., 2005; Yang et al., 2013), bone tissue engineering (Salerno et al., 2010; Wu et al., 2012), and wound dressing applications (Lin et al., 2012; Unnithan et al., 2014). In this study, micro/nano fibrous PCL/zein-CL scaffold structures for use in bone tissue engineering applications were fabricated with different CL contents by using a two-nozzle electrospinning process. The relevant properties of the scaffolds, such as their hydrophilicity, mechanical strength, and cell adhesive ability were investigated. Furthermore, the bone-forming ability of the scaffolds was evaluated by incubating them in biomimetic simulated body fluid (SBF). 2. Materials and methods 6

2.1 Materials Polycaprolactone (PCL, Mw=70,000~90,000, Sigma Aldrich, USA), zein (a protein from corn, Sigma Aldrich, Korea), and calcium L-lactate hydrate (CL, Sigma Aldrich, Netherlands) were used. Dichloromethane (DCM) and dimethylformamide (DMF) were supplied by Samchun in Korea. Cell counting kit-8 (CCK-8, Dojindo, Japan) was used to measure the cell proliferation. 4’6-diamidino-2-phenylindole (DAPI) was purchased from Life Technologies. 2.2 Preparation of the electrospinning solution Firstly, the 35 wt% zein solution was prepared by dissolving zein powder into the DMF. The blend solutions of zein and calcium lactate were prepared by mixing the varying amounts of calcium L-lactate hydrate (1, 3, 5 and 10 %) to the zein solution. Each solution was stirred for 4h prior to electrospinning for homogeneous mixing. A 10 wt% PCL solution was prepared by dissolving PCL in DCM/DMF (4/1, w/w) and then stirred for 12 h. 2.3. Electrospinning set-up A two-nozzle electrospinning set-up was used for this experiment (Figure 1A). The setup consisted of a high-voltage power supply, a rotating aluminum collector covered with a Teflon sheet, two syringe pumps with 12-ml plastic syringes, 2 metal nozzles with diameters 0.51 mm (21G), and a copper sheet to connect the two nozzles to the power supply. The angle between two nozzles was set to 80°, and the PCL solution (5 ml) was loaded in one syringe and the zein-CL solution (5 ml) in the other syringe. The electrospinning parameters included a voltage of 17 kV with a flow rate 7

of 1 ml/h for each solution. An 18-cm distance was maintained between the tip of the nozzle and the collector. The electrospun mat that was obtained was then vacuum dried for 24 h to remove residual solvents. The electrospun mats were denoted as M1, M2, M3 and M4 to indicate the zein/PCL mats with 1, 3, 5, and 10 % CL content, respectively. 2.4 Characterization The fiber morphology of the electrospun nanofibrous mats was determined via scanning electron microscopy (SEM, JSM-5900, Japan), and the diameter and size distribution of the fibers in the SEM images was analyzed by using the ImageJ software (NIH, USA). The diameter was measured for 70 random fibers to calculate the average and standard deviation of the fiber diameters. The viscosity and conductivity of the different solutions were respectively measured using a programmable rheometer (Brookfield) and an EC meter (DKKTOA). Fourier transform infrared (FT-IR) spectroscopy was conducted for the different mats using an ABB Bomen MB100 spectrometer (Bomen, Canada). The mechanical properties of each mat were tested using a universal testing machine (AG-5000G, Shimadzu, Japan) at room temperature, under a crosshead speed of 5 mm/min. According to ASTM Standard D 638, the samples were prepared in the form of standard dumbbell shapes (Figure 5A) by die cutting and tested in the machine. The modulus values were determined from the linear region of the stress-strain curve at 12.5% strain. Tensile strength, yield point and deformation were calculated by stress-strain curves. 8

The surface wettability of the electrospun mats was analyzed by using a contact angle meter (GBX, Digidrop, France) with deionized water. The deionized water was automatically dropped (with a drop diameter of 6 μm) onto the surface of the mats. The micrographs were taken after 5 s and the contact angles were measured. For each sample, contact angle was measured at three different positions and the mean value is displayed. 2.5 Biomimetic mineralization of the scaffolds For biomimetic mineralization, different mats were incubated into the simulated body fluid (SBF) solution up to 7 days. SBF solution was prepared using Hank’s balanced salt (Sigma Aldrich, USA) according to our previous report (Joshi et al., 2016) . Briefly, a bottle of Hank’s balanced salt, magnesium sulfate (0.097 g), sodium hydrogen carbonate (0.350 g), and calcium chloride (0.185 g) were dissolved in distilled water in a 1000 ml volumetric flask. The volume of the solution was filled up to the mark, and the pH of the solution was maintained at 7.4 using (CH2OH)3CNH2 (0.5 g/L). The solution was filtered and stored at 5 °C. Electrospun mats were cut into discs with diameters of 8 mm. Samples were then incubated in 5 mL of SBF solution at 37 °C with a 5% CO2 atmosphere for different intervals of time. The SBF solution was replaced every 24 h, and after incubation, the scaffolds were rinsed with distilled water and dried in an oven at 37 °C for further analysis. The mineralization of pure PCL, PCL/zein and PCL/zein-CL mats was observed via field-emission scanning electron microscopy (FESEM, S-7400, Hitachi, Japan). 7-days SBF treated scaffolds were analyzed in order to evaluate the ability of different 9

scaffolds for the nucleation of the calcium compound. 2.6 Quantification of the mineralization using Alizarin Red S The calcium phosphate that was deposited in the fiber was quantified according to our previous report (Pant et al., 2013d). A solution consisting of 40 mM of Alizarin Red S (ARS) dissolved in distilled water (pH adjusted to 4.1 by using 0.1 mM NaOH) was used to determine the mineralization of the scaffolds by storing them in the dark. The scaffolds that had been incubated in SBF for 7 days were rinsed three times in distilled water, fixed in 3.7% buffered formaldehyde for 30 min, and stained with ARS solution for 20 min on an orbital shaker. The scaffolds were rinsed with distilled water to remove the excess dye, and the samples were then transferred into a 2 ml tube containing 50% acetic acid for 30 min. The dissolved dye was pipetted out, and the pH was adjusted to 4.1. The absorbance was measured at 550 nm in a 96-well plate using a SpectraMax spectrophotometer (Molecular Devices). For each mat, three samples were analysed and the average value was displayed. 2.7 In-vitro biocompatibility A CCK assay was performed to measure the proliferation of the MC3T3-E1 (ATCC) mouse osteoblast cells at 1, 3, and 6 days of the cell culture. The electrospun scaffolds were cut to the same size, sterilized under UV light for 1 h, and thoroughly rinsed with phosphate buffer saline (pH 7.4). Later, transferred to a 48-well plate and rinsed with medium prior to cell seeding. 200 µl MC3T3-E1 cell suspension (20th passage) at a density of 1×104 cells/well were seeded on the surface of scaffolds and were incubated at 37°C in a 5% CO2 atmosphere. After 2h, 800 µl fresh medium were 10

added to each well and further incubated in 1, 3 and 6 days. The culture medium was changed every 2 days, and the cell viability was measured according to the manufacturer’s instruction. 100 µl of cultured medium was transferred to a 96-well plate, and 20 µl of a CCK-8 solution was added to each well and incubated. After 3h incubation, absorbance was measured at a wavelength of 450 nm using an iMark™ Microplate reader. A standard curve was established by measuring known number of cells prior to the experiment and cell viability was determined from the standard curves. Among the groups, the cell viability of polystyrene well plate was set as control and viability for other mats was expressed with its reference. The relative cell viability (%) was calculated for each extracts according to equation as following: Cell viability (%) =

OD450(sample) OD450(cell control)

× 100%

Where OD represents the optic density. To examine cell attachment and spreading, samples were chemically fixed. The three- and six- day cell cultured samples were rinsed with phosphate buffer saline (pH 7.4), and then fixed with 2.5% glutaraldehyde for 1 h followed by washing with 25%, 50%, 75% and 100% ethanol for 10 min. The samples were dried overnight in a laminar flow hood, and the cell morphology was determined via SEM. In addition to SEM images, a fluorescent microscope (IX71 Inverted microscope, ONLYMPUS) was employed to further confirm the cell biocompatibility and spreading behavior toward the scaffolds. Accordingly, cells were cultured on the scaffolds for a designated time (3 or 6 days) and stained with DAPI. Then, cells were fixed with 4% paraformaldehyde, and stained according to the manufacturer’s 11

protocol. Finally, the stained mats were examined using fluorescent microscope. 2.8 Statistical analysis The quantitative data were expressed as mean ± standard deviation, analyzed data using Student’s t-test and repeated measures for the analysis of variance (ANOVA) test. A probability of less than 0.05 was considered to be statistically significant. 3. Results and discussion 3.1 Physiochemical properties of composite mats The

PCL/zein-CL composite

mat

was

prepared

using

a

two-nozzle

electrospinning system as previously described. Figure 1A shows a diagrammatic sketch of the electrospinning system that was used in this study. As shown in the diagram, both nozzles were configured side by side at an angle of 80 ° between the tips of two nozzles, as reported in our previous studies (Tijing et al., 2013). Here, we found that the CL is immiscible in the PCL solution and miscible in the zein solution. Therefore, the PCL solution was electrospun from one nozzle, and the CL-zein blend solution from another nozzle. Interestingly, CL was observed to be insoluble in pure DMF and in the PCL solution but soluble in the zein solution prepared in DMF. The digital image (Figure 1B) clearly shows that CL is insoluble in DMF but soluble in the zein solution at ambient conditions (Figure 1B (b)). The solubility of CL in the zein solution might be the result of the interaction between the functional groups that are present in zein with CL (Figure 1B (c)). At first, the CL-zein solutions were prepared with different amounts of CL, and several physiochemical properties of the CL-zein solutions and the corresponding composite mats were studied before 12

co-electrospinning with PCL. The concentration of the CL in the zein solution showed a vivid effect on the physicochemical properties of the zein-CL blend solution and the corresponding composite mats. Table 1 shows the viscosity and conductivity of the various CL-zein blended solutions and the fiber diameter of the corresponding composite mats. The viscosity of the blended solution gradually increased while the conductivity gradually decreased as the CL content increased in the zein solution (Table 1). The viscosity of the solution obviously decreases as the concentration of the solute increases. The decrease in the conductivity of the blend solution with an increasing amount of CL might be due to the interaction of the CL with polar functional groups that are present in zein. Figure 2 shows the surface morphologies of the pristine zein and the CL-zein composite mats with varying amounts of CL in the zein solution. Smooth and continuous fibers were observed when the concentration of CL was within 0 to 5 wt % of the zein solution. However, the fibers became uneven and were more extensively fused when the concentration of the CL increased to 10 wt% of the zein solution. The average diameter of the fiber gradually increased from 156 nm to 240 nm with an increase in the CL concentration from 0% to 10% in the zein solution (Table 1). The discrepancy in the fiber diameter of the composite mats might be a result of the physiochemical properties, particularly the viscosity and conductivity of the electrospinning solution (Deitzel et al., 2001). The increase in viscosity for the solution gives rise to a larger fiber diameter (Deitzel et al., 2001), and the increase in conductivity improved the spinnability of the solution, resulting in a smaller diameter for the fibers (Fong et al., 1999). These results suggest that CL is 13

miscible with the zein solution, and CL concentration in the zein solution affected both the electrospinning process and the fiber morphologies. Figure 3 shows the SEM images and the corresponding fiber diameters of the pristine PCL, PCL/zein, and PCL/ CL-zein composite mats with varying amounts of CL. The electrospun fibers have an ultrafine matrix of interlocking micron to sub-micron sized fibers as well as a high porosity with non-woven mats that are randomly oriented. The single-nozzle pristine PCL (Figure 3a) fibers show uniform non-woven micron-sized (>1µm) fibers with while the zein-CL composite nanofibers (Figure 2) showed ultra-fine true nanofibers (less than 250 nm) with a uniform fiber diameter. Furthermore, the electrospun micro-nano fibers produced by the two-nozzle composite mat had curly, random micro- and nano-sized fibers with inter-fiber bonds in two different fiber diameter ranges (with point bond structures). Moreover, the composite mats (Fig. 3(b-e)) exhibited a micro/nanofibrous morphology with two distinct types of fibers (microfibers and nanofibers). This morphology is attributed to the different bending and axisymmetrical instabilities of the electrospun jets that are dependent on the composition of the solution (Tijing et al., 2013). The single-nozzle electrospun mats made from PCL and zein-CL nanofibers were compared to the two-nozzle PCL/ zein-CL nanofibers. It can be deduced that the microfibers were produced from the PCL while the nanofibers were produced from the CL-zein blended solution. The micro fibers in the composite mats obtained from the two-nozzle system were straight and continuous with point bond structures, in contrast to the pristine PCL mats. 14

The fabrication of composite nanofiber is further confirmed from FTIR spectra. The FTIR spectra of the zein, PCL, PCL/zein and PCL/zein-CL fibrous mats are shown in Figure 4A. The FTIR bands for zein (Figure 4A (a)) at 3700 to 3100 cm−1, 1700 to 1600 cm−1, and 1500 to 1400 cm−1 are attributed to amide A, amide I and amide II since these are the typical absorption for protein (Forato et al., 1998). Calcium lactate (Figure 4A (b)) showed a broad hydroxyl peak at 3750-3500 cm-1, and the absorption bands at 1211, 1127, 1097, and 1046 cm-1 correspond to the vibrations of various C-O bonds in the molecules (Petibois et al., 2000). A typical band at 1571 cm-1 in calcium lactate is assigned to the carboxylate (COO-) groups. The FTIR spectra of PCL (Figure 4A (c)) showed typical absorption bands at 2946 and 2867 cm-1 (CH2 stretch), 1722 cm-1 (C=O stretch), 1241 and 1160 cm-1 (C-O-C stretch) (Bolgen et al., 2005). The zein-CL composite mat showed a broadening in the IR band at 3400-3600 cm-1, and the peak intensity increased relative to that of other peaks. The composite fibers (Figure 4A (e)) showed a broad spectrum at 3000-3600 cm-1 (wide band for OH), 1571 cm-1 (carboxylate, COO-, groups) and 1122 cm-1 (C-O stretching) in addition to the peaks of the pristine material. These results confirm the formation of a composite fiber. An SEM-EDS analysis further confirms the presence of calcium lactate in the composite fiber (Figure 4B). The CL-fabricated composite mat clearly showed the presence of calcium, which indicates that calcium lactate was incorporated in the fibrous matrix. The mechanical properties of the scaffolds are important parameters for hard tissue engineering. Figure 5A shows the tensile stress-strain curves of the different 15

micro/nano fibrous mats. Young’s modulus, yield point, tensile stress and % strain are presented in Table 2. Yield point is defined as the stress at which a material begins to deform plastically. The amount of CL in the zein-CL solution affected the mechanical properties of the electrospun mats. Pristine PCL mats showed the highest deformation (1442.01 ± 5.76 %) while the composite mats have lower sustenance to deformation. M3 showed the highest tensile strength (11.33 ± 0.14 MPa) compared to the other mats. As the CL content increased from 1 to 5 wt% in the zein-CL blend, the mechanical strength, yield point and Young’s modulus of the mats gradually increased (Table 2). The increase in the mechanical strength might be attributed to the hydrogen bonds that were created between the hydroxyl group of the calcium lactate and the functional groups present in zein and PCL. Sedlarik and coworkers also reported that the incorporation of CL into Poly(vinyl alcohol) improved the tensile strength of the composite films (Sedlarik et al., 2009). Furthermore, the point-bond structure (Figure 3, red circle) between the nanofibers and microfibers may have also increased the mechanical strength of the PCL/zein-CL composite nanofibers. Several studies reported that the attachment of fibers by means of point bonding can enhance the tensile properties of the nonwoven membranes nonwovens (Pant et al., 2011; Gajanan S. Bhat and Nanjundappa, 2013). However, a CL content greater than 5 wt % resulted in a decrease in the mechanical strength of the composite mat. A higher concentration of CL may hinder the proper orientation of the polymer molecules and may cause a decrease in the tensile strength of the mat (Kim et al., 2014). This result revealed that a CL of 5 wt% 16

was suitable to fabricate PCL/zein-CL composite fibers. The hydrophilicity of the micro/nano fiber membranes affects the cell adhesion and proliferation. The measurement for the water contact angle is suitable to assess the hydrophilicity of the biomaterials. Figure 5B shows the contact angle of the different composite mats. The pure PCL mats exhibited hydrophobic properties with a contact angle of around 124.9 ± 0.9° (Figure 5B (a)). The contact angle was slightly lower for the PCL/zein composite mat at 112.7 ± 0.3° (Figure 5B (b)). Interestingly, incorporation of the CL has a great influence on the hydrophilicity of the composite mats. The water contact angle of the composite mats decreased from 102.6° to 55.7° as the CL content increased from 1 to 5 wt% of zein. This result demonstrates that the incorporation of CL into the nanofibers can enhance the surface wettability of electrospun mat. The increase in the hydrophilicity of the composite mats is attributed to the polar carboxylic and hydroxyl groups of calcium lactate in the composite mat. Here, M3 exhibited more hydrophilic surfaces and is expected to have a better affinity for the cells. 3.2 Biomimetic mineralization in-vitro In vitro incubation in an SBF solution is the proper technique to assess the hydroxyapatite nucleation ability of the composite mats. Figure 6(A) shows FE-SEM images of different mats incubated in SBF solution for 7 days. The pristine PCL scaffold exhibited the deposition of the calcium phosphate compound (CPC) on the surface fibers (red circle, Figure 6A (a)) while the nanofibers beneath the surface layer remained smooth without CPC crystals (Figure 6A (a)). The hydrophobic nature of 17

the PCL nanofibers makes the capillary force ineffective. Therefore, the SBF solution could not penetrate to the inner fibers, limiting mineralization to the surface fibers of the electrospun mat (Joshi et al., 2015). On the other hand, composite scaffolds (PCL/Zein-CL, Figures 6A (b-d)) showed a homogeneous mineralization throughout the fibrous matrix. The superior performance of the composite mat (Figures 6A (b-d)) is attributed to its improved wettability, which allowed the SBF solution to enter into the pores of the mat via capillary action, and the calcium compound was deposited throughout the matrix. Furthermore, the calcium ions in the PCL/zein-CL composite fibers might have provided nucleation sites for the nucleation of the calcium compounds from the SBF solution. Deposition of calcium compounds on the seven days SBF-treated mats was further studied using ARS assay. Figure 6B shows the absorbance values of ARS extracted from the mineralized scaffolds immersed in the SBF solution after 7 days. As expected, the increase in CL concentration resulted in an increase in the absorbance values. The control value for a pure PCL scaffold was of 0.088. 7-days SBF –treated M3 mat showed the absorbance of 0.166 that was significantly higher than the untreated M3 mat. The ARS data suggests that 7 days of incubation in SBF solution were sufficient to induce mineralization in the scaffolds. 3.3 Biocompatibility test It is important to investigate the cell viability in order to evaluate the biocompatibility of the different biomaterials in vitro (Pant et al., 2013a). Therefore, osteoblast cells were cultured for 1, 3 and 6 days, and the viability was determined by 18

using a CCK assay. As shown in Figure 7A, the proliferation in the CL-fabricated mat was higher than that for pure PCL and PCL/zein composites, indicating that the incorporation of CL and zein has accelerated the proliferation and differentiation of the osteoblast cells. Figure 7B shows the attachment of the cells onto the different scaffolds. The pure PCL mat showed an aggregation of cellular mass in a small area while the PCL-zein composite mat showed cells that had spread through the surface of the mat with extended pseudopodia. Moreover, the CL-synthesized composite mat (PCL/zein-CL) had a well spread cellular mass throughout the fiber matrix and some of the cells were infiltrated in the nanofibrous mesh (as shown with the red circle in Figure 7B). Further, cell morphologies were confirmed using Fluorescence microscopy. Fluorescent microscopic images of cells on the scaffolds after 3 and 6 days’ culture are shown in Figure 7C. PCL/zein-CL (5%) mat showed profound cell growth throughout the mat compared to pristine PCL and PCL/zein mat. CCK results, SEM images and Fluorescent microscopic images showed that the osteoblasts could attach and grow in pristine and composite mats. However, the PCL/zein-CL composite mats exhibited improved cell adhesion and proliferation relative to pristine and PCL/zein composite mats. The improved biochemical interaction of the cells and the CL, as well as hydrophilicity of composite scaffolds, might result in an improvement in the interaction between the cells and the PCL/zein-CL (5%) composite fibers, as compared to the PCL/zein and pristine PCL fibers. 4. Conclusion Micro/nano fibrous PCL/zein-CL scaffolds were successfully fabricated by using 19

an advanced two-nozzle electrospinning method. The composite scaffolds with 5 wt% CL exhibited excellent hydrophilicity and mechanical properties. In addition, the mineralization in SBF showed that the composite scaffolds with 5 wt% CL had a good ability to accelerate CPC deposition. The in vitro cell culture indicated that the composite scaffolds were supposed to have good bioactivity, a high cell affinity, and adequate growth. These results indicated that the PCL/zein-CL (5%) fibrous scaffolds have improved properties with potential for biomedical applications, especially those related to bone tissue engineering. Acknowledgements This research was supported with a grant from the Basic Science Research Program through the National Research Foundation of Korea (NRF) from the Ministry of Education, Science and Technology (Project no: 2013R1A2A2A04015484). We would also like to thank the Centre for University Research Facility (CURF) of Chonbuk National University for the spectroscopic measurements. 5. References B.M. et al., 1982 B.M., S., S.A., M., B.G., B., 1982. Preliminary Studies on Calcium Lactate as an Anticaries Food Additive. Caries Research 16, 12-17.doi:DOI:10.1159/000260570 Bhattarai et al., 2009 Bhattarai, N., Li, Z.S., Gunn, J., Leung, M., Cooper, A., Edmondson, D., Veiseh, O., Chen, M.H., Zhang, Y., Ellenbogen, R.G., Zhang, M.Q., 2009. Natural-Synthetic Polyblend Nanofibers for Biomedical Applications. Adv Mater 21, 2792-+.doi:DOI 10.1002/adma.200802513 Bolgen et al., 2005 Bolgen, N., Menceloglu, Y.Z., Acatay, K., Vargel, I., Piskin, E., 2005. In vitro and in vivo degradation of non-woven materials made of poly(epsilon-caprolactone) nanofibers prepared by electrospinning under different conditions. J Biomat Sci-Polym E 16, 1537-1555.doi:Doi 10.1163/156856205774576655 Cui et al., 2010 Cui, W.G., Zhou, Y., Chang, J., 2010. Electrospun nanofibrous materials for tissue engineering and drug delivery. Sci Technol Adv Mat 11.doi:Artn 014108 Doi 10.1088/1468-6996/11/1/014108 Deitzel et al., 2001 Deitzel, J.M., Kleinmeyer, J., Harris, D., Tan, N.C.B., 2001. The effect of processing variables on the morphology of electrospun nanofibers and textiles. Polymer 42, 261-272.doi:Doi 20

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23

Table 1 Viscosity, conductivity and fiber diameter of the various solutions/mats Conductivity Sample

Viscosity (cP)

Zein Zein-CL(1%) Zein-CL(3%) Zein-CL(5%) Zein-CL(10%)

791.56 1231.60 1375.40 1800.50 2674

± ± ± ± ±

3.20 41.01 18.08 3.42 25.51

(mS m−1) 14.80 14.63 13.30 12.75 11.42

± ± ± ± ±

0.25 0.35 0.18 0.53 0.30

Fiber diameter (nm) Distribution 62 78 60 139 142

– – – – –

239 255 232 296 365

Mean 156 172 183 201 240

± ± ± ± ±

39 49 29 37 52

Note: Conductivity and viscosity were measured three times for each solution and Mean±SD values are displayed in the table. The fiber diameter was measured for different mats (70 fibers from different images for each mat) using the ImageJ program and Mean±SD values is displayed.

Table 2 Mechanical properties of different fibrous scaffolds. Sample PCL PCL/zein PCL/zein-CL(1%) PCL/zein-CL(3%) PCL/zein-CL(5%) PCL/zein-CL(10%)

Tensile strength (MPa) 3.59 ± 0.28 2.19 ± 0.25 2.80 ± 0.31 4.58 ± 0.59 6.00 ± 0.32 4.08 ± 0.48

Young's modulus (MPa) 2.38 ± 0.40 4.12 ± 1.40 5.09 ± 0.99 10.04 ± 0.65 11.33± 0.14 6.61 ± 2.97

Yield point (MPa) 1.04 ± 0.61 1.79 ± 0.09 2.61 ± 0.26 4.53 ± 0.67 5.77 ± 0.32 3.91 ± 0.17

Deformation (strain at Maximum %) 1442.01 ± 5.76 179.26 ± 28.41 124.09 ± 24.85 91.81 ± 20.84 95.80 ± 14.03 125.66 ± 21.10

Note: Each sample was evaluated for five times. The mean values and corresponding standard deviation is displayed.

24

Figure caption Figure. 1.

(A) Schematic images of the two-nozzle electrospinning set-up and the modified nozzle configuration with two nozzles placed side-by-side with an 80° angle between them. (B) Digital images of the different solutions: (a) water, (b) CL in the DMF solution, (c) CL in the zein solution (zein dissolved in DMF).

Figure. 2.

SEM images of fibrous membranes electrospun from different CL concentrations: (a) pure zein, (b) zein-CL (1%), (c) zein-CL (3%), (d) zein-CL (5%), (e) zein-CL (10%).

Figure. 3.

SEM images of fibrous membrane electrospun mats: (a) pure PCL, (b) PCL/zein, (c) M1, (d) M2, (e) M3 and (f) M4.

Figure. 4.

(A) FTIR spectra of different samples: (a) Zein nanofibers, (b) CL powders, (c) pure PCL mat, (d) Zein-CL (10wt%) nanofibers, (e) M4 mat. (B) EDX spectra of different electrospun mats.

Figure. 5.

(A) Stress-strain curves of the electrospun fibrous mats and (B) Water contact angle on the surface of the corresponding fibrous mats for 5 seconds: (a) pure PCL, (b) PCL/zein, (c) M1, (d) M2, (e) M3 and (f) M4. The data is reported as mean ± SD (n = 3 and p