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In Vitro Deposition of Ca-P Nanoparticles on Air Jet Spinning Nylon 6 Nanofibers Scaffold For Bone Tissue Engineering
Accepted Manuscript Title: Air Jet Spinning of Nylon 6 Nanofiber Scaffolds for Bone Tissue Engineering Author: Abdalla Abdal-hay Yi Seul Oh Ayman Yousef Hem Raj Pant Pablo Vanegas Jae Kyoo Lim PII: DOI: Reference:
S0169-4332(14)00712-0 http://dx.doi.org/doi:10.1016/j.apsusc.2014.03.161 APSUSC 27562
To appear in:
APSUSC
Received date: Revised date: Accepted date:
18-12-2013 21-3-2014 22-3-2014
Please cite this article as: A. Abdal-hay, Y.S. Oh, A. Yousef, H.R. Pant, P. Vanegas, J.K. Lim, Air Jet Spinning of Nylon 6 Nanofiber Scaffolds for Bone Tissue Engineering, Applied Surface Science (2014), http://dx.doi.org/10.1016/j.apsusc.2014.03.161 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 proof before it is published in its final 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.
*Highlights (for review)
Highlights Fabrication of nylon 6 (N6) membrane mat by air jet spinning (AJS) approach
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Solutions at high concentrations were necessary to form well-defined fibers The production rate had the greatest effect on chain structure conformation of N6
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AJS has great potential for the fabrication of hard tissue engineering scaffolds.
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Graphical Abstract (for review)
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*Manuscript
Air Jet Spinning of Nylon 6 Nanofiber Scaffolds for Bone Tissue Engineering
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Abdalla Abdal-hay1,2,3, Yi Seul Oh4, Ayman Yousef2, Hem Raj Pant2, Pablo Vanegas1, Jae Kyoo Lim4,* Dept. of Chemical Engineering, College of Engineering, Universidad de Cuenca, Ecuador
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Dept. of Bionano System Engineering, College of Engineering, Chonbuk National University,
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Jeonju 561-756, Republic of Korea,
Dept. of Engineering Materials and Mechanical Design, Faculty of Engineering, South Valley
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University, Qena, Egypt,
Dept. of Mechanical Design Engineering, Advanced Wind Power System Research Institute,
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Chonbuk National University, Jeonju 561-756, Republic of Korea.
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*Corresponding author: Tel: +82-63-20-2321; Fax: +82-63-270-4439 E-mail: [email protected] (Jae Kyoo Lim)
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Abstract Microporous, non-woven nylon 6 (N6) scaffolds were prepared with an air jet spinning
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(AJS) approach. In this process, polymer fibers with diameters down to the nanometer range
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(nanofibers) were formed by subjecting a fluid jet to high pressure air. The effects of the solution
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conditions on the morphological appearance and average diameter of the as-spun N6 fibers and crystal structure were investigated. The morphology properties of the AJS membrane mats could
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easily be tailored by adjusting the concentration of the polymer solution. Solutions at high
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concentrations were necessary to form well-defined fibers without beads. The production rate (viz. solvent evaporation rate) had the greatest effect on the chain structure conformation of N6.
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The predominant structure phase of the N6 fibers fabricated by AJS was a thermodynamically
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stable α-form while the electrospinning fibers induced the metastable γ-form. AJS significantly
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enhanced the mechanical properties of the N6 mat. The bone formation ability of AJS fibers was evaluated by incubating the fibers in biomimetic simulated body fluid for 5 and 10 days at 37°C. Overall, the new AJS approach developed for membrane structures has great potential for the fabrication of hard and soft tissue engineering scaffolds. Keywords: Air jet spinning; Nylon 6; Bone tissue engineering; Electrospinning
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1. Introduction Tissue engineering scaffolds are an emerging area in contemporary human health care
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administration in which a basic understanding of cellular biology and the application of bioengineering are harnessed to develop feasible substitutes to aid in clinical treatments [1, 2].
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Recent studies have focused on the use of scaffolds with various advanced techniques to create complex guidance channels that precisely mimic a natural repairing process in the human body
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and are quite similar to natural extracellular matrix (ECM). Regarding the scaffold, a highly
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porous microstructure with high mechanical strength and large surface area is conducive to tissue ingrowth that allows for nutrient supply/transport while providing adequate space for cell
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migration and attachment [3].
Electrospinning produces highly porous, non-woven fabrics consisting of ultrafine fibers
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[4]. A wide variety of polymers have been electrospun, and several applications have been
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recently proposed based on small fiber diameters and high porosities. Nevertheless, the
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electrospinning approach has multiple disadvantages that limit its use in several application sectors. Electrospinning requires high-voltage electrical fields that result in imprecise control over fiber orientation, and it is chiefly affected by (i) system parameters, such as the polymer molecular weight, molecular weight distribution and solution properties (e.g. viscosity, surface tension, conductivity); (ii) process parameters, such as flow rate, and electric potential. Furthermore, the process has a very low production rate (very costly and time consuming), and it produces semi-dry fibers that have poor interconnection or bonded fiber properties resulting in lower mechanical properties [1, 2]. Initially, technical difficulties relating to a number of these parameters prevented electrospinning from emerging as a feasible technique to produce smalldiameter polymer fibers [5]. A few strategies have expanded the versatility of electrospinning;
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however, reliable methods are needed to generate well-characterized, porous micro- to nanoscale polymeric 3-D fibrous structures for engineering scaffolds. Recently, our groups have
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developed a novel, simple, effective and low cost strategy for producing 3-D micro-nano scale fibers by air jet spinning (AJS) [6, 7] as shown in Fig. 1A and the video still as supporting
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information. These studies clearly demonstrated the feasibility of fiber spinning without bead
polymer concentration using different polymer types.
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formation within a very short time (5 ml/min) and control of the fiber diameter by varying the
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Nylon 6 (-(CH2)5C(=O)N(H)-) (N6) exhibits several amazing characteristics such as flexibility in surface functionalities and mechanical performance (e.g. stiffness and tensile
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strength) superior to many other forms of synthetic or/and natural polymeric materials [2, 3]. N6 resembles the collagen protein in its backbone structure and active groups, and has excellent
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stability in human body fluids [3, 8-11]. There have been many reports regarding the formation
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of hard tissue scaffolds using N6 [10, 12-14]. N6 is used as a medical polymer in applications
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such as medical threads and artificial skin. Furthermore, N6 can be easily spun within a wide range of process and material parameters, which make it a good candidate for tissue engineering scaffolds [15, 16]. There are no reports on the design of N6 synthetic polymeric scaffolds at the nano- or submicron-scale using an AJS technique. N6 is a semicrystalline polymer and exhibits three crystalline forms (γ, α and β-form) that generally coexist in various amounts, depending on the sample preparation. Stephens et al. reported that the chain conformation (secondary structure) of N6 directly impacts its physical and mechanical properties and biological function [17]. Electrospinning of N6 resulted in the metastable γ-form, whereas film casting resulted in a more stable α-form [9]. This molecular transformation may have been due to the change in evaporation rate of the solvent as
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documented by Giller et al. [18]. These studies indicate that varying the vapor phase concentration and, thus, the rate of solvent evaporation during the fabrication process varies the
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resulting crystal structure of the N6. Furthermore, Hem et.al [19] also reported that the transition of the metastable γ-form into the thermodynamically stable α-form was achieved by
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incorporating lactic acid (LA) in the N6 solution to reduce the solvent evaporation rate (due to
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the high boiling point of LA) during the spinning process. In the present study, due to the high production rate (high solvent evaporation rate) of AJS and thereby the high solvent vapor
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concentration in the surrounding spinning chamber, there was more time for the polymer chains to create the thermodynamically stable α-form. Since the polymorphic behavior stems in part
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from a balance between the rates of crystallization and solvent vapor concentration in the chamber, an evaporation rate faster than the crystallization rate of the thermodynamically stable
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α-form would induce the metastable γ-form, whereas slower evaporation rates would result in the
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α-form. The aim of this study was to fabricate N6 fibrous 3-D membrane mats by an AJS
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approach for tissue scaffold applications and to investigate the effect of the polymer concentration on the fiber morphology and chain conformation of N6 biomimetic polymers. The effects of the electrospun and sol-gel fabrication process on the crystal structure of N6 were also investigated in the present contribution. 2. Experimental
2.1 Air jet spinning of N6 mats
Five N6 solutions with different polymer concentrations (5, 10, 14, 18 and 22 wt%) were dissolved in a solvent mixture of formic and acetic acids with a volume ratio of 80:20 and directly deposited on a polyethylene sheet using a custom designed airbrush spraying device (Pa201, IWATEC, Taiwan; Fig. 1) at room temperature and 45% humidity with a 250-µm nozzle
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diameter with double action/internal mixing and a gravity feed color cup. The process was conducted at an optimized air pressure of 450 kPa. The distance between the collector and AJS
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tip was 25-30 cm. The variations in the spraying distances within this range could create welldefined fibers without bead formation, and a single spraying distance of 25 cm was focused on in
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this study. The airbrush was fixed during the spraying process. The diameter was covered by the
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conical spray jet on the substrate over the duration of the deposition. 2.2. Electrospinning of the N6 mats
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The electrospinning setup used in the present work was described in detail in our previous publication [2]. The electrospinning solution was prepared by dissolving N6 at 22 wt% AJS. The
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solution was loaded into a 5-mL plastic syringe with a 22-gauge needle attached. The solution was dispensed using a syringe pump at an injection rate of 0.5 ml/h. The working distance
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between the tip of the needle and the collector was 25 cm and a 16 kV voltage was applied. The
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mats collected from both processes were placed in a vacuum oven overnight at 40 °C to remove
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any potential residual solvents. 2.3. Characterizations
The surface properties of the collected mats were characterized by field emission scanning electron microscopy (FESEM; Hitachi S-7400, Hitachi Co., Tokyo, Japan) and X-ray diffraction analysis (XRD, (GA-XRD, Philips X’Pert, Holland). The polymer mat layer thickness was assessed by ultrasonic measurement (coating thickness gauge OMEGA instrument, OM179745) to a precision of 1 µm. Fourier transform infrared spectrometry (FT-IR) analysis was used to positively identify the N6 phase structure of the as-obtained mats. FT-IR spectra in the transmission mode were collected using an ABB Bomen MB100 spectrometer (Bomen, Canada) with an FT-IR 4000 in the range of 4000-400 cm-1.
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2.4. Mechanical characterization of the fabricated scaffolds AJS and electrospun N6 mats were subjected to stress-strain analysis using an Instron
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mechanical tester (LLOYD instruments, LR5K plus, UK). For the mechanical tests, samples were trimmed into a “dogbone” (see inset of Fig. 4) with offset ends via die cutting from the as-
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obtained mats to reduce grip effects according to the procedures of ASTM D-638. Testing was
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conducted with the tissue grips moving at a rate of 10 mm/min and the load was applied until the specimen experienced complete failure. The specimen thicknesses were measured using a digital
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micrometer with a precision of 1 µm (the thickness was approximately 150 and 100 µm for the AJS and electrospinning mats, respectively). The tensile modulus was calculated as the slope of
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the initial linear portion of the stress-strain curve. The data acquisition rate was set to 20.0 Hz. Five membrane mat samples of each approach were subjected to tensile testing at room
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temperature. The data presented were expressed as the mean ± standard deviation. Statistical
2.5.
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significant.
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analysis was performed using a Student’s t-test, and a p-value less than 0.05 was considered
Apatite-like formation on the as-obtained scaffolds To produce the bioactive surface and investigate the biomineralization capacity of the
prepared scaffolds, the mats were submitted to a procedure inspired by the so-called biomimetic treatment, a commercially available Hank’s balanced salts (H2387; Sigma Aldrich, Korea), which has a similar composition to a standard simulated body fluid (SBF). Mats with dimensions of 10 mm and 15 mm were immersed in 100 ml solution. The electrolyte was saturated with atmospheric oxygen and the temperature was maintained at 37.5 ± 0.2 ºC during the test. For the standard biomimetic deposition production, the mats were soaked in SBF to form apatite nuclei, and after 5 and 10 days incubation, the samples were removed and gently rinsed in distilled
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water to eliminate non-adherent particles. The morphological characterization was performed with FESEM analysis. FTIR was used to verify the apatite-like formation and potential
3.
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interactions. Results and discussion
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The AJS process is a simple method to produce wet, non-woven webs of micro and nanofibers [6, 7, 20]. AJS depends on a certain arrangement of concentric nozzles (air and
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solution nozzles) in which the prepared polymer solution is ejected through the inner nozzle by
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gravity at a constant feed rate, while high pressure air flows from the outer nozzle. At the tip of the inner nozzle, the forced solution forms a drop that is stretched by the high pressure stream of
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compressed air flowing on the outside of the nozzle. Hence, the compressed air and polymer solution match inside the cone jet (Fig. 1A). The AJS approach makes use of Bernoulli’s
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principle in which changes in pressure are converted into kinetic energy, i.e., as the high pressure
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gas (air) stream exits the outer nozzle, the pressure quickly drops (atmospheric pressure)
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increasing the kinetic energy of the stream and resulting in an increase in the gas velocity. The compressed air exiting the nozzle causes the surface of the drop to shape into a cone similar to the Taylor cone in electrospinning. As the spinning travels through the surrounding environment, the solvent evaporates, leaving behind semi-dry fibers that can be collected on virtually any substrate.
Similar to electrospinning, the AJS polymer concentration (solution viscosity) plays an important role in determining the range of concentrations from which continuous fibers can be obtained [21]. To verify this hypothesis, the processing conditions for fabricating the N6 scaffold were optimized and their properties were strongly influenced by the concentration of the polymeric solution. The as-obtained AJS N6 membrane from 5, 10, 14, 18 and 22 wt% N6 were
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visualized under FE-SEM and different fiber morphologies were captured, as shown in Fig. 2AF, to illustrate the effect of the N6 solution concentration on the morphological appearance of the
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obtained as-spun materials. The results indicate that the formation of well-defined fibers are directly proportional to the polymer concentration and occur with increasing solution
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concentration, which is consistent with our previous reports of PLA membrane fibers fabricated
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using the same way [7]. The 5 wt% N6 concentration produced highly fused morphologies with a random microporous structure on the surface (Fig. 2A). As the N6 concentration was increased
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to 10%, a bead morphology was observed with a few distinct fibers (Fig. 2B), and the fibers became even more apparent at a concentration of 14 and 18 % (Fig. 2C, D). Increasing the
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polymer concentration to 22 wt% produced well-defined fiber morphology without beads leading to smooth fibers morphology (Fig. 2E, F). At this concentration, fibrous scaffolds exhibited
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highly localized alignment (or multiple fiber strands or bundles) (Fig. 2F). The possible reason
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for the localized, aligned nanofibers is that the fluid jet travels in straight paths due to drag air
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forces towards the target. Nevertheless, aligned fibers are very difficult to achieve via electrospinning [21]. The fiber diameter was 93.6-200 nm for the 18 wt% N6 concentration (inset of Fig. 2D) and 180-500 nm for the 22 wt% N6 concentration. The average diameter for 18 wt% N6 was approximately 150 nm (the membrane fibers contain beads) and 220 nm for 22 wt%, which is free of beads as shown in Fig. 2F. Thus, the increased polymer concentration resulted in larger fiber diameters. Oliveira e.al [22] reported that the dependency between the concentration and fiber diameter increase with increasing concentration due to the reduced mobility of the polymer chains in the jet during the spinning process. Alternatively, the trend in the morphology transition from fused/beads to well-defined fibers occurred with increasing difficultly to completely dry the AJS material before it hits the target as the atmosphere becomes
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more concentrated [19]. Attempts to air spin a 22 wt% N6 concentration resulted in a morphology similar to that achieved via electrospinning according to our previous publication
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[23], further validating this hypothesis. Collectively, the polymer concentration drastically affects the formation of the fibers using the standard processing conditions.
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An analysis of these materials on the molecular level shows even more dramatic changes
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as the amount of evaporated solvent is increased with decreasing polymer concentration. Figure 3A shows the X-ray profiles of N6 AJS membranes with different N6 concentrations (14 and 22
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wt% were selected) in the chamber. In addition, the profiles acquired from electrospun membrane fibers at same concentration (22%) for the as-obtained AJS defined fibers are shown
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for comparison. Electrospun N6 fibers consist primarily of the meta-stable γ-form (Curve 1) [19] and the AJS membrane mats consist primarily of the thermodynamically stable α-form structure
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(Fig. 3A, Curves 2, 3). The X-ray profile (Fig. 3A) clearly shows that as the concentration of N6
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increases in the AJS solution, the intensity peaks of the α-form crystal structure of N6 is
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stronger, as observed in the (200) (α) and (002) (α)/(202) (α) reflections at 2θ= 20.3 and 23.5° (Curve C) (monoclinic unit cell: a=0.956, b=1.724 and c=0.801 nm, with the b dimension along the chain axis [24]). The corresponding d-spacings (at 22 wt%) were 0.44 and 0.37 nm, respectively, which represents the projected intermolecular distance within the hydrogen bonded planes and the interplanar distance. The α-form is more stable than the γ-form because of shorter hydrogen bonds [17, 25]. However, electrospun fibers are the exception for forming the γ-phase as a permanent structure, which has hydrogen bonding between parallel chains, resulting in a mismatch of hydrogen-bonding sites [17]. Hence, the diffraction peak at 2θ= 21.3 is characteristic of pseudo-hexagonal γ-phase crystals of the electrospun N6 associated with the (001) crystal planes (monoclinic structure with a=0.933, b=1.688 and c=0.478 nm [24]) with a
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small shoulder on the left side of this peak that represents traces of the α-form N6 crystals. However, several researchers have attempted to induce the chain conformation of a N6
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nanofibers scaffold by the electrospinning process [18, 19]. Hem and collaborators have recently contributed to N6 modification and chain conformation from the meta-stable γ-form to thermally
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stable α-form in recent years. Unfortunately, the investigations of these authors could not induce
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the α-form as a dominant structure, which was easily obtained by the simple AJS technique in the present study. Alternatively, Vasanthan et al. [26] reported that X-ray diffraction does not
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allow a reliable estimation of the N6 crystal structures that only differs by subtle features in the patterns. Conversely, infrared (IR) spectroscopy seems to easily discriminate both the α and γ
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forms, which is more sensitive to the molecular conformation of the polymers [18]. However, IR also showed similar trends to those of the X-ray profiles. The IR of different mats (Fig. 3B)
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showed the effect of both AJS and electrospinning on the crystalline structure. The IR bands of
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N6 have been previously assigned [27]. The formation of the α-form structure on AJS mats and
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formation of the γ-form on the electrospun mat as dominant structures is more clearly observed. The significant decrease or disappearance (note y-axis) in the band intensity at 1120 cm−1 of the electrospun fibers, as compared to the band of the AJS fibers, indicates the formation of N6 from the γ-form for the electrospun and α-form on the AJS fibers. Furthermore, in comparing the IR spectra of the electrospinning and AJS membranes at the same polymer concentration (22 wt%), there were very obvious differences in the intensities, including for the NH-stretching (3293 cm1
), the CC stretch region (900-1150 cm-1), the CNH bending region (1310-1350,1440-1490 cm-1),
amide I (1638cm-1), amide II (1 545cm-1, primarily due to the carbonyl stretching vibration), and amide III (1369 and 1263 cm-1) vibration bands [9, 17]. Further support for the expansion of the formation of the α- and γ-forms can be found elsewhere [27, 28]. The increase in crystallinity of
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the AJS N6 mat compared to the electrospinning mat, which is affected by the amount of N6 in the solution, was also observed. The band at 1120 cm-1 represents the amorphous phase and
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becomes less intense with AJS; indicating an increase in crystallinity [29]. This result was further supported by the XRD pattern shown in Fig. 3A. During the processing of the N6 mats,
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AJS suppresses the formation of γ-form crystals and significantly promotes the formation of αcrystals. The α-form crystal is dominant in the AJS N6 mat at lower polymer concentrations.
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To understand the possible reasons for the formation of the α-form structure on the AJS
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membrane mat and the γ-form on the electrospun mat, the fabrication rates and solvent evaporation rate during both fabrication processes were investigated as the two processes have
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dramatically different solvent evaporation kinetics. A higher fabrication rate induces a faster solvent evaporation rate in the environment of the surrounding chamber. AJS has a very high
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fabrication rate (5 ml/min) compared to electrospinning (0.5 ml/h), which results in greater
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solvent content inside the chamber. The elongation of the polymer solution jet and subsequent
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solvent evaporation of both processes occurs within milliseconds [18, 30]. Based on these experiments, the amount of the α-form was correlated to the solvent vapor concentration in the surrounding chamber. Because all AJS mats were fabricated at different solvent concentrations (solvent to polymer ratio), the α formation was more complete as more solvent evaporated into the AJS atmosphere, strongly indicating that the solvent evaporation kinetics significantly impact the crystal structure of the resultant polymer as clearly shown in the XRD profiles and FTIR bands. Accordingly, the same phase formation and trends were induced on casted films (Fig. 3B). At greater solvent vapor concentrations in the surrounding chamber, the polymer chains likely have more time to pack into the thermodynamically stable α-form, suggesting that the solvent evaporation kinetics inherent to the AJS process drastically slows under these conditions.
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These results are in a very good agreement with previous studies [17, 18]. At lower concentrations of evaporated solvent in the chamber for the electrospinning process, the solvent
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evaporation rate is faster than the crystallization rate of the α-form, and the resultant electrospun N6 is kinetically trapped in the meta-stable γ-form. Giller and co-works [18] investigated the
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solvent concentration effects on the N6 phase structure formation. The γ-form is created at low solvent concentrations in the surrounding chamber during the electrospinning process. Increasing
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the evaporated solvent concentration resulted in the complete disappearance of the γ peak and the
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formation of the α-form. Overall, as noted for the formation of the α-form structure of the AJS N6 mats, the AJS process is associated with a high shear stress and a very low structure
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formation of the polymer material. Furthermore, the rapid solvent evaporation of the consolidating process of the electrospinning may also hinder the formation of perfect crystallites
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such as those of the stable α-phase [17, 31, 32].
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The mechanical property of a scaffold is another important aspect of its design. The
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purpose of a hard tissue scaffold is to provide a surface for new bone formation and maintain mechanical stability at the defect site of the host [23]. Furthermore, to function as tissue engineering scaffolding, a matrix must have sufficient mechanical integrity to withstand manual manipulation. Mechanical properties, such as tensile modulus, ultimate tensile stress, and ultimate strain were evaluated. A representative stress-strain curve of the AJS and electrospinning mats from 22 wt% N6 was constructed from the load-deformation curve illustrated in Fig. 4 and their tensile properties are given in Table. 1. The mechanical strength of the AJS mat was greater than that of the electrospun N6 mat. The ultimate tensile stress of the AJS and electrospun N6 fibers mat was 21.8±1.3 and 12±2.5 MPa, and the ultimate strain was 105.8±3.8 and 106.3±2.7%, respectively. The tensile modulus of the structure was 67.2±12 and
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56±8.96 MPa for the AJS and electrospun N6 fibers, respectively. The increased mechanical strength may be explained considering the locally-oriented N6 fibers using AJS as shown in Fig.
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1F, whereas the electrospun fibers were randomly oriented within the nanofibrous structures (Supporting Information, Fig. S1). Matthews et al. reported that the local orientation of the fibers
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on the AJS scaffolding in the present study directly modulate the material properties of the
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engineered matrix,[33] as noted in Fig. 1F.
The increased mechanical strength was further explained by the polymer chain orientation
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and ability to maximize hydrogen bonding interactions in the α-crystalline phase due to the (200) planes, primarily fixed by hydrogen bonds [28]. The presence of the α-crystalline phase as a
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dominant crystal structure in the AJS mat can provide stronger hydrogen bonding between the N6 backbone chains than the as-obtained AJS mat [18]. The shifting of different IR-bands
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towards a lower frequency in the FT-IR spectra was also observed, which further indicated the
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formation of stronger hydrogen bonds in the AJS mat due to the presence of the α-crystalline
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form. The fundamental relationship between the chemical microstructures of N6 and the mechanical properties of the as-obtained mats was established by the conversion of N6 from the gauche conformation (from electrospinning) to the trans conformation (from AJS). The proposed technique to incorporate a calcium compound on the surface of the polymer fibers is superior to others with blended calcium compounds or nanoparticles (NPs) added prior to spinning process. Most of the spinning composite fibers were prepared by simply mixing the previously obtained HA nanoparticles with viscous spinning solutions of polymer carriers, which typically results in nanocomposites with very limited or no specific interactions between the organic and inorganic phases [34]. There is a common disadvantage in that inorganic particles cannot be distributed within the organic matrices at the nanoscale level. Weak molecular
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interactions and poor dispersion consequently produce problems such as compromised spinnability, reduced nanoparticle loading capacity, and unfavorable cellular responsiveness.
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Alternatively, because N6 dissolves well in single or organic acid mixtures (e.g., acetic acid and formic acid), these acids have a strong negative affinity for calcium groups that dissociate
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calcium phosphates into primary ions, as our group previously reported [2]. These results
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indicate that when pure HA powders were mixed directly with a solution of N6 dissolved in acetic and formic acids, despite being electrospinnable, both the HA-associated haze effect and
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characteristic diffraction rings were absent indicating a loss of the crystalline nature of HA. A better solution for preparing pristine polymer fibers could be using an AJS method and
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subsequently converting them into composite fibers via the well-known in situ biomimetic synthesis approach [2, 35]. Thus, the primary method of utilizing artificial biomaterials as bone
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substitutes in biomimetic-inspired approaches is to produce nanocrystallites of calcium
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compounds that are deposited and well dispersed on the surface of the polymer fibers.
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The in vitro bioactivity formation (mineralized calcium phosphates, noted as apatite-like) on the AJS membrane mat at a polymer concentration of 22 wt% was investigated by immersing the mat in simulated body fluids at 37°C for 5 and 10 days as illustrated in Fig. 5. Previously, our group developed a simulated body fluid (SBF) method to prepare biomimetic N6/apatite composite scaffolds [2, 4]. Deposition of a biomimetic apatite layer throughout the electrospinning membrane structure of 3-D scaffolds is an effective method to control the surface topography and chemistry within large, complex structures. In this study, this method is introduced to fabricate biomimetic AJS N6/apatite composite scaffolds on α-form, N6 fibers crystalline structures. The particle number and size in the scaffold were controlled by the incubation time and ionic concentration of the SBF. The average particle number and size
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increased with incubation time. After five days, a small number of nanoscale and microscale particles were formed on the fiber surfaces, as shown in Fig. 5A. These particles were assembled
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to form larger aggregates with diameters of 0.5-1 µm, Fig. 5B. Their morphology is typical of an apatite-like structure, which has also been observed to form on an electrospun N6 membrane mat
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after incubating in SBF [2, 4]. The surface forms a biologically active apatite layer that provides
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the bonding interface with tissues. The apatite phase that forms on bioactive implants is chemically and structurally equivalent to the mineral phase in bone, providing interfacial
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bonding. To verify the phase properties, the formation of calcium phosphates on the surfaces of as-obtained AJS N6 mats after immersion in SBF was confirmed by FTIR spectra as displayed in
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Fig. 5C. Comparison of the FTIR spectrum of N6/apatite composites and the pristine N6 mat, suggests that characteristic bands of both apatite-like particles and N6 are present in the
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composite. Compared to the pristine mat, the N6/apatite-like scaffold is characterized by
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absorption bands at 1090, 1027-1030, 602, and 963 cm-1 (see marked area) [4, 36] and the
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bending deformation mode of O-H from calcium phosphates, confirming the successful formation of the N6/apatite-like composite membrane scaffold. The N6 network serves as a matrix to the apatite-like particles and provides an anchoring site for apatite particles in the structure, binding them together in the composite fibers. As shown in the FTIR analysis, there were some interactions between the N6 chain molecules and apatite particles on the mat. Additionally, the amide II band (~1540 cm-1) became less intense when the apatite particles were deposited onto the surfaces of the N6 fibers, indicating the apatite-like groups interact with N6 by hydrogen bonding [8]. The broad bands between ~3000 and 3300 cm-1 shifted slightly to a smaller wavelength when the particles were deposited on the surfaces of the N6 fibers due to the hydrogen-bonded –COOH and –NH– groups of the N6. A more detailed study on the structure
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and characteristics of the apatite-like particles and their effect on composite biocompatibility, e.g. using osteoblast cell culture experiments, is the focus of current investigations.
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Although there are numerous reports on the electrospinning of N6, the AJS fabrication process based on high gas pressure or differences in kinetic energies is first reported here. The
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polymer solution concentration during the fabrication process was demonstrated to tailor the
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morphology of the membrane, and the process can also be applied to other polymers to produce micro- to nano-scale fibers for different biomedical applications. Furthermore, AJS differs from
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other techniques (N6 electrospinning) because it generates the α-form crystal structure on N6 fiber mats with the aid of an effective hydrogen bond formation mechanism, improving
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mechanical properties. 4. Conclusion
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In the present contribution, the physicochemical modification of nylon 6 (N6) membrane
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mats was successfully fabricated by a simple, effective and low cost air jet spinning (AJS)
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approach. The effects of the solution concentration on the morphological appearance of the asobtained mats were visually observed from a series of scanning electron micrographs. Solutions with a high polymer concentration were necessary to produce AJS N6 fibers with a nanoscale diameter. The morphology and architecture of the AJS fibers is similar to those from electrospinning. Furthermore, the solvent evaporation kinetics affected the polymer crystallization chain conformation of N6, suggesting that the crystalline state of AJS N6 was largely dependent on the solvent evaporation kinetics during the process that depend on the solvent vapor concentration in the environmental chamber. As a result, the tensile properties of the AJS N6 mat were better than the N6 electrospun mats. The AJS N6 mats are a potential approach for the fabrication of hard and soft tissue engineering scaffolds, and the resulting
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biological behavior for tissue scaffolds is the subject of a forthcoming publication. In particular, the use of AJS instead of electrospinning has potential for soft tissue engineering applications.
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To the authors’ knowledge, this is the first experimental report assessing the fabrication of N6 mats by the AJS approach. Future research should focus on tailoring novel nanocomposite fiber
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scaffolds for bone tissue applications. The AJS technique may be extremely useful in medical
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applications (such as wound dressings), where non-woven mats can be applied directly to tissue cultures or to living tissue for a variety of medical procedures without applying, for example, a
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high electric voltage used in electrospinning. Acknowledgements
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We acknowledge the research funds supported by the Secretary of Higher Education, Science, Technology and Innovation of Ecuador, SENESCYT. Abdalla Abdal-hay acknowledges
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References
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the supporting scholarship-2014 from “SENESCYT” at University of Cuenca.
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[24] T. Jiang, Y.-h. Wang, J.-t. Yeh, Z.-q. Fan, Study on solvent permeation resistance properties of nylon6/clay nanocomposite, European Polymer Journal, 41 (2005) 459-466. [25] J.C. Ho, K.H. Wei, Induced γ→ α crystal transformation in blends of polyamide 6 and liquid crystalline copolyester, Macromolecules, 33 (2000) 5181-5186. [26] N. Vasanthan, D. Salem, FTIR spectroscopic characterization of structural changes in polyamide‐6 fibers during annealing and drawing, Journal of Polymer Science Part B: Polymer Physics, 39 (2001) 536-547. [27] N. Kimura, H.-K. Kim, B.-S. Kim, K.-H. Lee, I.-S. Kim, Molecular Orientation and Crystalline Structure of Aligned Electrospun Nylon-6 Nanofibers: Effect of Gap Size, Macromolecular Materials and Engineering, 295 (2010) 1090-1096. [28] G.-M. Kim, G.H. Michler, F. Ania, F.J.B. Calleja, Temperature dependence of polymorphism in electrospun nanofibres of PA6 and PA6/clay nanocomposite, Polymer, 48 (2007) 4814-4823. [29] H. Arimoto, α–γ Transition of nylon 6, Journal of Polymer Science Part A: General Papers, 2 (1964) 2283-2295. [30] D.H. Reneker, A.L. Yarin, H. Fong, S. Koombhongse, Bending instability of electrically charged liquid jets of polymer solutions in electrospinning, Journal of Applied physics, 87 (2000) 4531. [31] H.R. Pant, M.P. Bajgai, C. Yi, R. Nirmala, K.T. Nam, W.-i. Baek, H.Y. Kim, Effect of successive electrospinning and the strength of hydrogen bond on the morphology of electrospun nylon-6 nanofibers, Colloids and Surfaces A: Physicochemical and Engineering Aspects, 370 (2010) 87-94. [32] H.R. Pant, K.-T. Nam, H.-J. Oh, G. Panthi, H.-D. Kim, B.-i. Kim, H.Y. Kim, Effect of polymer molecular weight on the fiber morphology of electrospun mats, Journal of Colloid and Interface Science, 364 (2011) 107-111. [33] J.A. Matthews, G.E. Wnek, D.G. Simpson, G.L. Bowlin, Electrospinning of Collagen Nanofibers, Biomacromolecules, 3 (2002) 232-238. [34] H.R. Pant, M.P. Bajgai, K.T. Nam, Y.A. Seo, D.R. Pandeya, S.T. Hong, H.Y. Kim, Electrospun nylon-6 spider-net like nanofiber mat containing TiO(2) nanoparticles: a multifunctional nanocomposite textile material, J Hazard Mater, 185 (2011) 124-130. [35] W. Liu, Y.-C. Yeh, J. Lipner, J. Xie, H.-W. Sung, S. Thomopoulos, Y. Xia, Enhancing the stiffness of electrospun nanofiber scaffolds with a controlled surface coating and mineralization, Langmuir, 27 (2011) 9088-9093. [36] W.W. Thein-Han, R.D.K. Misra, Biomimetic chitosan–nanohydroxyapatite composite scaffolds for bone tissue engineering, Acta Biomaterialia, 5 (2009) 1182-1197.
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Figure captions Fig. 1 (A) The AJS mechanism fabrication process and (B) 2-D image of the as-obtained AJS
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mat. Fig. 2 (A-F) FESEM photographs of the fabricated AJS samples at different N6 solution
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concentrations. Inset of (D) illustrates the higher magnification of the dashed circle area.
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Fig. 3 (A) XRD profiles and (B) FTIR spectra of the AJS and electrospun mats. The casted film FTIR spectra was also combined.
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Fig. 4 Stress-strain curves of the AJS and electrospun mats. The inset shows the 2-D image of the tested samples.
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Fig. 5 (A, B) FESEM photographs and (C) FTIR profiles of the AJS mats from a 22 wt% N6
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solution after immersion into simulated body fluids for 5 and 10 days at 37 °C.
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Table 1. Tensile properties of AJS and electrospinning mats prepared from the same polymer concentration (22 wt%) Tensile strength
Young’s modulus
(MPa)
(MPa)
Breaking elongation (%)
67.2± 12
Electrospun fibers
12±2.5
56±8.96
105.8±3.8
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21.8±1.3
106.3±2.7
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AJS fibers
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Sample
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S0169-4332(14)00712-0 http://dx.doi.org/doi:10.1016/j.apsusc.2014.03.161 APSUSC 27562
To appear in:
APSUSC
Received date: Revised date: Accepted date:
18-12-2013 21-3-2014 22-3-2014
Please cite this article as: A. Abdal-hay, Y.S. Oh, A. Yousef, H.R. Pant, P. Vanegas, J.K. Lim, Air Jet Spinning of Nylon 6 Nanofiber Scaffolds for Bone Tissue Engineering, Applied Surface Science (2014), http://dx.doi.org/10.1016/j.apsusc.2014.03.161 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 proof before it is published in its final 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.
*Highlights (for review)
Highlights Fabrication of nylon 6 (N6) membrane mat by air jet spinning (AJS) approach
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Solutions at high concentrations were necessary to form well-defined fibers The production rate had the greatest effect on chain structure conformation of N6
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AJS has great potential for the fabrication of hard tissue engineering scaffolds.
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Graphical Abstract (for review)
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*Manuscript
Air Jet Spinning of Nylon 6 Nanofiber Scaffolds for Bone Tissue Engineering
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Abdalla Abdal-hay1,2,3, Yi Seul Oh4, Ayman Yousef2, Hem Raj Pant2, Pablo Vanegas1, Jae Kyoo Lim4,* Dept. of Chemical Engineering, College of Engineering, Universidad de Cuenca, Ecuador
2
Dept. of Bionano System Engineering, College of Engineering, Chonbuk National University,
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3
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Jeonju 561-756, Republic of Korea,
Dept. of Engineering Materials and Mechanical Design, Faculty of Engineering, South Valley
4
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University, Qena, Egypt,
Dept. of Mechanical Design Engineering, Advanced Wind Power System Research Institute,
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Chonbuk National University, Jeonju 561-756, Republic of Korea.
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*Corresponding author: Tel: +82-63-20-2321; Fax: +82-63-270-4439 E-mail: [email protected] (Jae Kyoo Lim)
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Abstract Microporous, non-woven nylon 6 (N6) scaffolds were prepared with an air jet spinning
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(AJS) approach. In this process, polymer fibers with diameters down to the nanometer range
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(nanofibers) were formed by subjecting a fluid jet to high pressure air. The effects of the solution
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conditions on the morphological appearance and average diameter of the as-spun N6 fibers and crystal structure were investigated. The morphology properties of the AJS membrane mats could
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easily be tailored by adjusting the concentration of the polymer solution. Solutions at high
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concentrations were necessary to form well-defined fibers without beads. The production rate (viz. solvent evaporation rate) had the greatest effect on the chain structure conformation of N6.
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The predominant structure phase of the N6 fibers fabricated by AJS was a thermodynamically
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stable α-form while the electrospinning fibers induced the metastable γ-form. AJS significantly
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enhanced the mechanical properties of the N6 mat. The bone formation ability of AJS fibers was evaluated by incubating the fibers in biomimetic simulated body fluid for 5 and 10 days at 37°C. Overall, the new AJS approach developed for membrane structures has great potential for the fabrication of hard and soft tissue engineering scaffolds. Keywords: Air jet spinning; Nylon 6; Bone tissue engineering; Electrospinning
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1. Introduction Tissue engineering scaffolds are an emerging area in contemporary human health care
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administration in which a basic understanding of cellular biology and the application of bioengineering are harnessed to develop feasible substitutes to aid in clinical treatments [1, 2].
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Recent studies have focused on the use of scaffolds with various advanced techniques to create complex guidance channels that precisely mimic a natural repairing process in the human body
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and are quite similar to natural extracellular matrix (ECM). Regarding the scaffold, a highly
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porous microstructure with high mechanical strength and large surface area is conducive to tissue ingrowth that allows for nutrient supply/transport while providing adequate space for cell
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migration and attachment [3].
Electrospinning produces highly porous, non-woven fabrics consisting of ultrafine fibers
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[4]. A wide variety of polymers have been electrospun, and several applications have been
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recently proposed based on small fiber diameters and high porosities. Nevertheless, the
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electrospinning approach has multiple disadvantages that limit its use in several application sectors. Electrospinning requires high-voltage electrical fields that result in imprecise control over fiber orientation, and it is chiefly affected by (i) system parameters, such as the polymer molecular weight, molecular weight distribution and solution properties (e.g. viscosity, surface tension, conductivity); (ii) process parameters, such as flow rate, and electric potential. Furthermore, the process has a very low production rate (very costly and time consuming), and it produces semi-dry fibers that have poor interconnection or bonded fiber properties resulting in lower mechanical properties [1, 2]. Initially, technical difficulties relating to a number of these parameters prevented electrospinning from emerging as a feasible technique to produce smalldiameter polymer fibers [5]. A few strategies have expanded the versatility of electrospinning;
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however, reliable methods are needed to generate well-characterized, porous micro- to nanoscale polymeric 3-D fibrous structures for engineering scaffolds. Recently, our groups have
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developed a novel, simple, effective and low cost strategy for producing 3-D micro-nano scale fibers by air jet spinning (AJS) [6, 7] as shown in Fig. 1A and the video still as supporting
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information. These studies clearly demonstrated the feasibility of fiber spinning without bead
polymer concentration using different polymer types.
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formation within a very short time (5 ml/min) and control of the fiber diameter by varying the
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Nylon 6 (-(CH2)5C(=O)N(H)-) (N6) exhibits several amazing characteristics such as flexibility in surface functionalities and mechanical performance (e.g. stiffness and tensile
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strength) superior to many other forms of synthetic or/and natural polymeric materials [2, 3]. N6 resembles the collagen protein in its backbone structure and active groups, and has excellent
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stability in human body fluids [3, 8-11]. There have been many reports regarding the formation
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of hard tissue scaffolds using N6 [10, 12-14]. N6 is used as a medical polymer in applications
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such as medical threads and artificial skin. Furthermore, N6 can be easily spun within a wide range of process and material parameters, which make it a good candidate for tissue engineering scaffolds [15, 16]. There are no reports on the design of N6 synthetic polymeric scaffolds at the nano- or submicron-scale using an AJS technique. N6 is a semicrystalline polymer and exhibits three crystalline forms (γ, α and β-form) that generally coexist in various amounts, depending on the sample preparation. Stephens et al. reported that the chain conformation (secondary structure) of N6 directly impacts its physical and mechanical properties and biological function [17]. Electrospinning of N6 resulted in the metastable γ-form, whereas film casting resulted in a more stable α-form [9]. This molecular transformation may have been due to the change in evaporation rate of the solvent as
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documented by Giller et al. [18]. These studies indicate that varying the vapor phase concentration and, thus, the rate of solvent evaporation during the fabrication process varies the
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resulting crystal structure of the N6. Furthermore, Hem et.al [19] also reported that the transition of the metastable γ-form into the thermodynamically stable α-form was achieved by
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incorporating lactic acid (LA) in the N6 solution to reduce the solvent evaporation rate (due to
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the high boiling point of LA) during the spinning process. In the present study, due to the high production rate (high solvent evaporation rate) of AJS and thereby the high solvent vapor
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concentration in the surrounding spinning chamber, there was more time for the polymer chains to create the thermodynamically stable α-form. Since the polymorphic behavior stems in part
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from a balance between the rates of crystallization and solvent vapor concentration in the chamber, an evaporation rate faster than the crystallization rate of the thermodynamically stable
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α-form would induce the metastable γ-form, whereas slower evaporation rates would result in the
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α-form. The aim of this study was to fabricate N6 fibrous 3-D membrane mats by an AJS
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approach for tissue scaffold applications and to investigate the effect of the polymer concentration on the fiber morphology and chain conformation of N6 biomimetic polymers. The effects of the electrospun and sol-gel fabrication process on the crystal structure of N6 were also investigated in the present contribution. 2. Experimental
2.1 Air jet spinning of N6 mats
Five N6 solutions with different polymer concentrations (5, 10, 14, 18 and 22 wt%) were dissolved in a solvent mixture of formic and acetic acids with a volume ratio of 80:20 and directly deposited on a polyethylene sheet using a custom designed airbrush spraying device (Pa201, IWATEC, Taiwan; Fig. 1) at room temperature and 45% humidity with a 250-µm nozzle
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diameter with double action/internal mixing and a gravity feed color cup. The process was conducted at an optimized air pressure of 450 kPa. The distance between the collector and AJS
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tip was 25-30 cm. The variations in the spraying distances within this range could create welldefined fibers without bead formation, and a single spraying distance of 25 cm was focused on in
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this study. The airbrush was fixed during the spraying process. The diameter was covered by the
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conical spray jet on the substrate over the duration of the deposition. 2.2. Electrospinning of the N6 mats
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The electrospinning setup used in the present work was described in detail in our previous publication [2]. The electrospinning solution was prepared by dissolving N6 at 22 wt% AJS. The
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solution was loaded into a 5-mL plastic syringe with a 22-gauge needle attached. The solution was dispensed using a syringe pump at an injection rate of 0.5 ml/h. The working distance
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between the tip of the needle and the collector was 25 cm and a 16 kV voltage was applied. The
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mats collected from both processes were placed in a vacuum oven overnight at 40 °C to remove
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any potential residual solvents. 2.3. Characterizations
The surface properties of the collected mats were characterized by field emission scanning electron microscopy (FESEM; Hitachi S-7400, Hitachi Co., Tokyo, Japan) and X-ray diffraction analysis (XRD, (GA-XRD, Philips X’Pert, Holland). The polymer mat layer thickness was assessed by ultrasonic measurement (coating thickness gauge OMEGA instrument, OM179745) to a precision of 1 µm. Fourier transform infrared spectrometry (FT-IR) analysis was used to positively identify the N6 phase structure of the as-obtained mats. FT-IR spectra in the transmission mode were collected using an ABB Bomen MB100 spectrometer (Bomen, Canada) with an FT-IR 4000 in the range of 4000-400 cm-1.
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2.4. Mechanical characterization of the fabricated scaffolds AJS and electrospun N6 mats were subjected to stress-strain analysis using an Instron
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mechanical tester (LLOYD instruments, LR5K plus, UK). For the mechanical tests, samples were trimmed into a “dogbone” (see inset of Fig. 4) with offset ends via die cutting from the as-
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obtained mats to reduce grip effects according to the procedures of ASTM D-638. Testing was
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conducted with the tissue grips moving at a rate of 10 mm/min and the load was applied until the specimen experienced complete failure. The specimen thicknesses were measured using a digital
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micrometer with a precision of 1 µm (the thickness was approximately 150 and 100 µm for the AJS and electrospinning mats, respectively). The tensile modulus was calculated as the slope of
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the initial linear portion of the stress-strain curve. The data acquisition rate was set to 20.0 Hz. Five membrane mat samples of each approach were subjected to tensile testing at room
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temperature. The data presented were expressed as the mean ± standard deviation. Statistical
2.5.
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significant.
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analysis was performed using a Student’s t-test, and a p-value less than 0.05 was considered
Apatite-like formation on the as-obtained scaffolds To produce the bioactive surface and investigate the biomineralization capacity of the
prepared scaffolds, the mats were submitted to a procedure inspired by the so-called biomimetic treatment, a commercially available Hank’s balanced salts (H2387; Sigma Aldrich, Korea), which has a similar composition to a standard simulated body fluid (SBF). Mats with dimensions of 10 mm and 15 mm were immersed in 100 ml solution. The electrolyte was saturated with atmospheric oxygen and the temperature was maintained at 37.5 ± 0.2 ºC during the test. For the standard biomimetic deposition production, the mats were soaked in SBF to form apatite nuclei, and after 5 and 10 days incubation, the samples were removed and gently rinsed in distilled
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water to eliminate non-adherent particles. The morphological characterization was performed with FESEM analysis. FTIR was used to verify the apatite-like formation and potential
3.
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interactions. Results and discussion
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The AJS process is a simple method to produce wet, non-woven webs of micro and nanofibers [6, 7, 20]. AJS depends on a certain arrangement of concentric nozzles (air and
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solution nozzles) in which the prepared polymer solution is ejected through the inner nozzle by
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gravity at a constant feed rate, while high pressure air flows from the outer nozzle. At the tip of the inner nozzle, the forced solution forms a drop that is stretched by the high pressure stream of
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compressed air flowing on the outside of the nozzle. Hence, the compressed air and polymer solution match inside the cone jet (Fig. 1A). The AJS approach makes use of Bernoulli’s
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principle in which changes in pressure are converted into kinetic energy, i.e., as the high pressure
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gas (air) stream exits the outer nozzle, the pressure quickly drops (atmospheric pressure)
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increasing the kinetic energy of the stream and resulting in an increase in the gas velocity. The compressed air exiting the nozzle causes the surface of the drop to shape into a cone similar to the Taylor cone in electrospinning. As the spinning travels through the surrounding environment, the solvent evaporates, leaving behind semi-dry fibers that can be collected on virtually any substrate.
Similar to electrospinning, the AJS polymer concentration (solution viscosity) plays an important role in determining the range of concentrations from which continuous fibers can be obtained [21]. To verify this hypothesis, the processing conditions for fabricating the N6 scaffold were optimized and their properties were strongly influenced by the concentration of the polymeric solution. The as-obtained AJS N6 membrane from 5, 10, 14, 18 and 22 wt% N6 were
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visualized under FE-SEM and different fiber morphologies were captured, as shown in Fig. 2AF, to illustrate the effect of the N6 solution concentration on the morphological appearance of the
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obtained as-spun materials. The results indicate that the formation of well-defined fibers are directly proportional to the polymer concentration and occur with increasing solution
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concentration, which is consistent with our previous reports of PLA membrane fibers fabricated
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using the same way [7]. The 5 wt% N6 concentration produced highly fused morphologies with a random microporous structure on the surface (Fig. 2A). As the N6 concentration was increased
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to 10%, a bead morphology was observed with a few distinct fibers (Fig. 2B), and the fibers became even more apparent at a concentration of 14 and 18 % (Fig. 2C, D). Increasing the
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polymer concentration to 22 wt% produced well-defined fiber morphology without beads leading to smooth fibers morphology (Fig. 2E, F). At this concentration, fibrous scaffolds exhibited
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highly localized alignment (or multiple fiber strands or bundles) (Fig. 2F). The possible reason
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for the localized, aligned nanofibers is that the fluid jet travels in straight paths due to drag air
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forces towards the target. Nevertheless, aligned fibers are very difficult to achieve via electrospinning [21]. The fiber diameter was 93.6-200 nm for the 18 wt% N6 concentration (inset of Fig. 2D) and 180-500 nm for the 22 wt% N6 concentration. The average diameter for 18 wt% N6 was approximately 150 nm (the membrane fibers contain beads) and 220 nm for 22 wt%, which is free of beads as shown in Fig. 2F. Thus, the increased polymer concentration resulted in larger fiber diameters. Oliveira e.al [22] reported that the dependency between the concentration and fiber diameter increase with increasing concentration due to the reduced mobility of the polymer chains in the jet during the spinning process. Alternatively, the trend in the morphology transition from fused/beads to well-defined fibers occurred with increasing difficultly to completely dry the AJS material before it hits the target as the atmosphere becomes
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more concentrated [19]. Attempts to air spin a 22 wt% N6 concentration resulted in a morphology similar to that achieved via electrospinning according to our previous publication
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[23], further validating this hypothesis. Collectively, the polymer concentration drastically affects the formation of the fibers using the standard processing conditions.
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An analysis of these materials on the molecular level shows even more dramatic changes
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as the amount of evaporated solvent is increased with decreasing polymer concentration. Figure 3A shows the X-ray profiles of N6 AJS membranes with different N6 concentrations (14 and 22
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wt% were selected) in the chamber. In addition, the profiles acquired from electrospun membrane fibers at same concentration (22%) for the as-obtained AJS defined fibers are shown
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for comparison. Electrospun N6 fibers consist primarily of the meta-stable γ-form (Curve 1) [19] and the AJS membrane mats consist primarily of the thermodynamically stable α-form structure
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(Fig. 3A, Curves 2, 3). The X-ray profile (Fig. 3A) clearly shows that as the concentration of N6
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increases in the AJS solution, the intensity peaks of the α-form crystal structure of N6 is
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stronger, as observed in the (200) (α) and (002) (α)/(202) (α) reflections at 2θ= 20.3 and 23.5° (Curve C) (monoclinic unit cell: a=0.956, b=1.724 and c=0.801 nm, with the b dimension along the chain axis [24]). The corresponding d-spacings (at 22 wt%) were 0.44 and 0.37 nm, respectively, which represents the projected intermolecular distance within the hydrogen bonded planes and the interplanar distance. The α-form is more stable than the γ-form because of shorter hydrogen bonds [17, 25]. However, electrospun fibers are the exception for forming the γ-phase as a permanent structure, which has hydrogen bonding between parallel chains, resulting in a mismatch of hydrogen-bonding sites [17]. Hence, the diffraction peak at 2θ= 21.3 is characteristic of pseudo-hexagonal γ-phase crystals of the electrospun N6 associated with the (001) crystal planes (monoclinic structure with a=0.933, b=1.688 and c=0.478 nm [24]) with a
Page 12 of 31
small shoulder on the left side of this peak that represents traces of the α-form N6 crystals. However, several researchers have attempted to induce the chain conformation of a N6
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nanofibers scaffold by the electrospinning process [18, 19]. Hem and collaborators have recently contributed to N6 modification and chain conformation from the meta-stable γ-form to thermally
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stable α-form in recent years. Unfortunately, the investigations of these authors could not induce
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the α-form as a dominant structure, which was easily obtained by the simple AJS technique in the present study. Alternatively, Vasanthan et al. [26] reported that X-ray diffraction does not
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allow a reliable estimation of the N6 crystal structures that only differs by subtle features in the patterns. Conversely, infrared (IR) spectroscopy seems to easily discriminate both the α and γ
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forms, which is more sensitive to the molecular conformation of the polymers [18]. However, IR also showed similar trends to those of the X-ray profiles. The IR of different mats (Fig. 3B)
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showed the effect of both AJS and electrospinning on the crystalline structure. The IR bands of
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N6 have been previously assigned [27]. The formation of the α-form structure on AJS mats and
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formation of the γ-form on the electrospun mat as dominant structures is more clearly observed. The significant decrease or disappearance (note y-axis) in the band intensity at 1120 cm−1 of the electrospun fibers, as compared to the band of the AJS fibers, indicates the formation of N6 from the γ-form for the electrospun and α-form on the AJS fibers. Furthermore, in comparing the IR spectra of the electrospinning and AJS membranes at the same polymer concentration (22 wt%), there were very obvious differences in the intensities, including for the NH-stretching (3293 cm1
), the CC stretch region (900-1150 cm-1), the CNH bending region (1310-1350,1440-1490 cm-1),
amide I (1638cm-1), amide II (1 545cm-1, primarily due to the carbonyl stretching vibration), and amide III (1369 and 1263 cm-1) vibration bands [9, 17]. Further support for the expansion of the formation of the α- and γ-forms can be found elsewhere [27, 28]. The increase in crystallinity of
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the AJS N6 mat compared to the electrospinning mat, which is affected by the amount of N6 in the solution, was also observed. The band at 1120 cm-1 represents the amorphous phase and
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becomes less intense with AJS; indicating an increase in crystallinity [29]. This result was further supported by the XRD pattern shown in Fig. 3A. During the processing of the N6 mats,
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AJS suppresses the formation of γ-form crystals and significantly promotes the formation of αcrystals. The α-form crystal is dominant in the AJS N6 mat at lower polymer concentrations.
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To understand the possible reasons for the formation of the α-form structure on the AJS
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membrane mat and the γ-form on the electrospun mat, the fabrication rates and solvent evaporation rate during both fabrication processes were investigated as the two processes have
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dramatically different solvent evaporation kinetics. A higher fabrication rate induces a faster solvent evaporation rate in the environment of the surrounding chamber. AJS has a very high
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fabrication rate (5 ml/min) compared to electrospinning (0.5 ml/h), which results in greater
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solvent content inside the chamber. The elongation of the polymer solution jet and subsequent
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solvent evaporation of both processes occurs within milliseconds [18, 30]. Based on these experiments, the amount of the α-form was correlated to the solvent vapor concentration in the surrounding chamber. Because all AJS mats were fabricated at different solvent concentrations (solvent to polymer ratio), the α formation was more complete as more solvent evaporated into the AJS atmosphere, strongly indicating that the solvent evaporation kinetics significantly impact the crystal structure of the resultant polymer as clearly shown in the XRD profiles and FTIR bands. Accordingly, the same phase formation and trends were induced on casted films (Fig. 3B). At greater solvent vapor concentrations in the surrounding chamber, the polymer chains likely have more time to pack into the thermodynamically stable α-form, suggesting that the solvent evaporation kinetics inherent to the AJS process drastically slows under these conditions.
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These results are in a very good agreement with previous studies [17, 18]. At lower concentrations of evaporated solvent in the chamber for the electrospinning process, the solvent
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evaporation rate is faster than the crystallization rate of the α-form, and the resultant electrospun N6 is kinetically trapped in the meta-stable γ-form. Giller and co-works [18] investigated the
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solvent concentration effects on the N6 phase structure formation. The γ-form is created at low solvent concentrations in the surrounding chamber during the electrospinning process. Increasing
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the evaporated solvent concentration resulted in the complete disappearance of the γ peak and the
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formation of the α-form. Overall, as noted for the formation of the α-form structure of the AJS N6 mats, the AJS process is associated with a high shear stress and a very low structure
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formation of the polymer material. Furthermore, the rapid solvent evaporation of the consolidating process of the electrospinning may also hinder the formation of perfect crystallites
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such as those of the stable α-phase [17, 31, 32].
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The mechanical property of a scaffold is another important aspect of its design. The
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purpose of a hard tissue scaffold is to provide a surface for new bone formation and maintain mechanical stability at the defect site of the host [23]. Furthermore, to function as tissue engineering scaffolding, a matrix must have sufficient mechanical integrity to withstand manual manipulation. Mechanical properties, such as tensile modulus, ultimate tensile stress, and ultimate strain were evaluated. A representative stress-strain curve of the AJS and electrospinning mats from 22 wt% N6 was constructed from the load-deformation curve illustrated in Fig. 4 and their tensile properties are given in Table. 1. The mechanical strength of the AJS mat was greater than that of the electrospun N6 mat. The ultimate tensile stress of the AJS and electrospun N6 fibers mat was 21.8±1.3 and 12±2.5 MPa, and the ultimate strain was 105.8±3.8 and 106.3±2.7%, respectively. The tensile modulus of the structure was 67.2±12 and
Page 15 of 31
56±8.96 MPa for the AJS and electrospun N6 fibers, respectively. The increased mechanical strength may be explained considering the locally-oriented N6 fibers using AJS as shown in Fig.
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1F, whereas the electrospun fibers were randomly oriented within the nanofibrous structures (Supporting Information, Fig. S1). Matthews et al. reported that the local orientation of the fibers
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on the AJS scaffolding in the present study directly modulate the material properties of the
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engineered matrix,[33] as noted in Fig. 1F.
The increased mechanical strength was further explained by the polymer chain orientation
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and ability to maximize hydrogen bonding interactions in the α-crystalline phase due to the (200) planes, primarily fixed by hydrogen bonds [28]. The presence of the α-crystalline phase as a
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dominant crystal structure in the AJS mat can provide stronger hydrogen bonding between the N6 backbone chains than the as-obtained AJS mat [18]. The shifting of different IR-bands
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towards a lower frequency in the FT-IR spectra was also observed, which further indicated the
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formation of stronger hydrogen bonds in the AJS mat due to the presence of the α-crystalline
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form. The fundamental relationship between the chemical microstructures of N6 and the mechanical properties of the as-obtained mats was established by the conversion of N6 from the gauche conformation (from electrospinning) to the trans conformation (from AJS). The proposed technique to incorporate a calcium compound on the surface of the polymer fibers is superior to others with blended calcium compounds or nanoparticles (NPs) added prior to spinning process. Most of the spinning composite fibers were prepared by simply mixing the previously obtained HA nanoparticles with viscous spinning solutions of polymer carriers, which typically results in nanocomposites with very limited or no specific interactions between the organic and inorganic phases [34]. There is a common disadvantage in that inorganic particles cannot be distributed within the organic matrices at the nanoscale level. Weak molecular
Page 16 of 31
interactions and poor dispersion consequently produce problems such as compromised spinnability, reduced nanoparticle loading capacity, and unfavorable cellular responsiveness.
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Alternatively, because N6 dissolves well in single or organic acid mixtures (e.g., acetic acid and formic acid), these acids have a strong negative affinity for calcium groups that dissociate
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calcium phosphates into primary ions, as our group previously reported [2]. These results
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indicate that when pure HA powders were mixed directly with a solution of N6 dissolved in acetic and formic acids, despite being electrospinnable, both the HA-associated haze effect and
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characteristic diffraction rings were absent indicating a loss of the crystalline nature of HA. A better solution for preparing pristine polymer fibers could be using an AJS method and
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subsequently converting them into composite fibers via the well-known in situ biomimetic synthesis approach [2, 35]. Thus, the primary method of utilizing artificial biomaterials as bone
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substitutes in biomimetic-inspired approaches is to produce nanocrystallites of calcium
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compounds that are deposited and well dispersed on the surface of the polymer fibers.
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The in vitro bioactivity formation (mineralized calcium phosphates, noted as apatite-like) on the AJS membrane mat at a polymer concentration of 22 wt% was investigated by immersing the mat in simulated body fluids at 37°C for 5 and 10 days as illustrated in Fig. 5. Previously, our group developed a simulated body fluid (SBF) method to prepare biomimetic N6/apatite composite scaffolds [2, 4]. Deposition of a biomimetic apatite layer throughout the electrospinning membrane structure of 3-D scaffolds is an effective method to control the surface topography and chemistry within large, complex structures. In this study, this method is introduced to fabricate biomimetic AJS N6/apatite composite scaffolds on α-form, N6 fibers crystalline structures. The particle number and size in the scaffold were controlled by the incubation time and ionic concentration of the SBF. The average particle number and size
Page 17 of 31
increased with incubation time. After five days, a small number of nanoscale and microscale particles were formed on the fiber surfaces, as shown in Fig. 5A. These particles were assembled
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to form larger aggregates with diameters of 0.5-1 µm, Fig. 5B. Their morphology is typical of an apatite-like structure, which has also been observed to form on an electrospun N6 membrane mat
cr
after incubating in SBF [2, 4]. The surface forms a biologically active apatite layer that provides
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the bonding interface with tissues. The apatite phase that forms on bioactive implants is chemically and structurally equivalent to the mineral phase in bone, providing interfacial
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bonding. To verify the phase properties, the formation of calcium phosphates on the surfaces of as-obtained AJS N6 mats after immersion in SBF was confirmed by FTIR spectra as displayed in
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Fig. 5C. Comparison of the FTIR spectrum of N6/apatite composites and the pristine N6 mat, suggests that characteristic bands of both apatite-like particles and N6 are present in the
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composite. Compared to the pristine mat, the N6/apatite-like scaffold is characterized by
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absorption bands at 1090, 1027-1030, 602, and 963 cm-1 (see marked area) [4, 36] and the
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bending deformation mode of O-H from calcium phosphates, confirming the successful formation of the N6/apatite-like composite membrane scaffold. The N6 network serves as a matrix to the apatite-like particles and provides an anchoring site for apatite particles in the structure, binding them together in the composite fibers. As shown in the FTIR analysis, there were some interactions between the N6 chain molecules and apatite particles on the mat. Additionally, the amide II band (~1540 cm-1) became less intense when the apatite particles were deposited onto the surfaces of the N6 fibers, indicating the apatite-like groups interact with N6 by hydrogen bonding [8]. The broad bands between ~3000 and 3300 cm-1 shifted slightly to a smaller wavelength when the particles were deposited on the surfaces of the N6 fibers due to the hydrogen-bonded –COOH and –NH– groups of the N6. A more detailed study on the structure
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and characteristics of the apatite-like particles and their effect on composite biocompatibility, e.g. using osteoblast cell culture experiments, is the focus of current investigations.
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Although there are numerous reports on the electrospinning of N6, the AJS fabrication process based on high gas pressure or differences in kinetic energies is first reported here. The
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polymer solution concentration during the fabrication process was demonstrated to tailor the
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morphology of the membrane, and the process can also be applied to other polymers to produce micro- to nano-scale fibers for different biomedical applications. Furthermore, AJS differs from
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other techniques (N6 electrospinning) because it generates the α-form crystal structure on N6 fiber mats with the aid of an effective hydrogen bond formation mechanism, improving
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mechanical properties. 4. Conclusion
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In the present contribution, the physicochemical modification of nylon 6 (N6) membrane
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mats was successfully fabricated by a simple, effective and low cost air jet spinning (AJS)
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approach. The effects of the solution concentration on the morphological appearance of the asobtained mats were visually observed from a series of scanning electron micrographs. Solutions with a high polymer concentration were necessary to produce AJS N6 fibers with a nanoscale diameter. The morphology and architecture of the AJS fibers is similar to those from electrospinning. Furthermore, the solvent evaporation kinetics affected the polymer crystallization chain conformation of N6, suggesting that the crystalline state of AJS N6 was largely dependent on the solvent evaporation kinetics during the process that depend on the solvent vapor concentration in the environmental chamber. As a result, the tensile properties of the AJS N6 mat were better than the N6 electrospun mats. The AJS N6 mats are a potential approach for the fabrication of hard and soft tissue engineering scaffolds, and the resulting
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biological behavior for tissue scaffolds is the subject of a forthcoming publication. In particular, the use of AJS instead of electrospinning has potential for soft tissue engineering applications.
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To the authors’ knowledge, this is the first experimental report assessing the fabrication of N6 mats by the AJS approach. Future research should focus on tailoring novel nanocomposite fiber
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scaffolds for bone tissue applications. The AJS technique may be extremely useful in medical
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applications (such as wound dressings), where non-woven mats can be applied directly to tissue cultures or to living tissue for a variety of medical procedures without applying, for example, a
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high electric voltage used in electrospinning. Acknowledgements
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We acknowledge the research funds supported by the Secretary of Higher Education, Science, Technology and Innovation of Ecuador, SENESCYT. Abdalla Abdal-hay acknowledges
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References
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the supporting scholarship-2014 from “SENESCYT” at University of Cuenca.
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Figure captions Fig. 1 (A) The AJS mechanism fabrication process and (B) 2-D image of the as-obtained AJS
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mat. Fig. 2 (A-F) FESEM photographs of the fabricated AJS samples at different N6 solution
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concentrations. Inset of (D) illustrates the higher magnification of the dashed circle area.
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Fig. 3 (A) XRD profiles and (B) FTIR spectra of the AJS and electrospun mats. The casted film FTIR spectra was also combined.
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Fig. 4 Stress-strain curves of the AJS and electrospun mats. The inset shows the 2-D image of the tested samples.
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Fig. 5 (A, B) FESEM photographs and (C) FTIR profiles of the AJS mats from a 22 wt% N6
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solution after immersion into simulated body fluids for 5 and 10 days at 37 °C.
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Table 1. Tensile properties of AJS and electrospinning mats prepared from the same polymer concentration (22 wt%) Tensile strength
Young’s modulus
(MPa)
(MPa)
Breaking elongation (%)
67.2± 12
Electrospun fibers
12±2.5
56±8.96
105.8±3.8
cr
21.8±1.3
106.3±2.7
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AJS fibers
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Sample
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