BASIC SCIENCE Nanomedicine: Nanotechnology, Biology, and Medicine 11 (2015) 693 – 704

Regenerative Nanomedicine (Ed. A. Seifalian)

nanomedjournal.com

Highly aligned nanocomposite scaffolds by electrospinning and electrospraying for neural tissue regeneration Wei Zhu, MS a , Fahed Masood b , Joseph O'Brien, MD c, d , Lijie Grace Zhang, PhD a, e,⁎ a

Department of Mechanical and Aerospace Engineering, The George Washington University, Washington, DC, USA b University of Maryland, Collage Park, MD, USA c Department of Orthopaedic Surgery, The George Washington University, Washington, DC, USA d Department of Neurosurgery, The George Washington University, Washington, DC, USA e Department of Medicine, The George Washington University, Washington, DC, USA Received 4 May 2014; revised 19 October 2014; accepted 4 December 2014

Abstract Neural tissue engineering offers a promising avenue for repairing neural injuries. Advancement in nanotechnology and neural scaffold manufacturing strategies has shed light on this field into a new era. In this study, a novel tissue engineered scaffold, which possesses highly aligned poly-ε-caprolactone microfibrous framework and adjustable bioactive factor embedded poly (D, L-lactide-co-glycolide) core-shell nanospheres, was fabricated by combining electrospinning and electrospraying techniques. The fabricated nanocomposite scaffold has cell favorable nanostructured feature and improved hydrophilic surface property. More importantly, by incorporating core-shell nanospheres into microfibrous scaffold, a sustained bioactive factor release was achieved. Results show rat pheochromocytoma (PC-12) cell proliferation was significantly promoted on the nanocomposite scaffold. In addition, confocal microscope images illustrated that the highly aligned scaffold increased length of neurites and directed neurites extension along the fibers in both PC-12 and astrocyte cell lines, which indicates that the scaffold is promising for guiding neural tissue growth and regeneration. From the Clinical Editor: In an attempt to direct neural cell growth, biomimetic neural scaffold was produced by electrospinning integrated with co-axial electrospraying techniques. In-vitro data provided a framework for future designs for neuronal regeneration. © 2015 Elsevier Inc. All rights reserved. Key words: Nanocomposite; Electrospinning; Electrospraying; Axonal extension; Neural tissue engineering

Neural degeneration resulted from various traumas and diseases, represents a prevalent burden on healthcare systems around the world. Although various cell therapies and implants have been investigated, repairing damaged nerves and achieving full functional recovery are still challenging [1]. The gold standard, autografts, are always limited by insufficient donor nerves from patients and possible functional impairment of the donor site [2]. Other traditional neuroprosthetic devices and nerve guidance conduits used for neural tissue repair have been limited by the presence of increased scarring and fibrosis, as well

Conflict of interest and disclosure: N/A All sources of support for research: This work was supported by award number UL1TR000075 from the NIH National Center for Advancing Translational Sciences and by the George Washington Institute for Nanotechnology. ⁎Corresponding author at: Washington DC, 20052. E-mail address: [email protected] (L.G. Zhang).

as insufficient cytocompatibility properties for sustained nerve regrowth [3]. Given the poor self-regeneration capacity of the nervous system and inadequate clinical therapeutic treatments, the development of novel strategies to improve and guide neural regeneration is imperative. Neural tissue engineering provides an attractive option in improving therapeutic effects when compared with traditional approaches. The fundamental principle of neural tissue engineering involves combination of three-dimensional biological scaffolds and various nerve cells to create a biomimetic implantable substitute [4,5]. Figure 1, A shows several important parameters for an ideal tissue engineered neural construct. An ideal neural scaffold should be biocompatible to maximally improve cell adhesion, migration, proliferation and axonal extension without any cytotoxicity [6]. In addition, the scaffold should be biodegradable, and provide sufficient mechanical support in the early period of implantation, then degrade in vivo with the new neural tissue formation to avoid complications caused by a surgical removal procedure. Moreover, since neural

http://dx.doi.org/10.1016/j.nano.2014.12.001 1549-9634/© 2015 Elsevier Inc. All rights reserved. Please cite this article as: Zhu W, et al, Highly aligned nanocomposite scaffolds by electrospinning and electrospraying for neural tissue regeneration. Nanomedicine: NBM 2015;11:693-704, http://dx.doi.org/10.1016/j.nano.2014.12.001

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Figure 1. Schematic illustration of (A) the key components of an ideal neural scaffold, and our electrospinning set-up for (B) aligned and (C) random microfiber fabrication. (D) Schematic diagram of the coaxial electrospraying technique for producing PLGA nanospheres into PCL eletrospun scaffolds.

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cells directly interact with nanostructured extra-cellular matrices (ECM) which have various bioactive factors such as proteins and growth factors [5,7], the design of a neural scaffold with biomimetic nano features and bioactive growth factor environments are highly desirable [8–10]. For in vivo applications, current available bioactive proteins and growth factors face ongoing issues related to short-term retention, quick half-life in circulation, and quick loss of biological activity in vivo even when administered at higher doses [11]. These deficiencies and others limit their potential to promote prolonged neural tissue formation within a targeted site. Thus, in this study, we fabricated a sustained bioactive factor delivery system based on biodegradable core-shell nanospheres for improved neural regeneration in the long term. Specifically, poly lactic-co-glycolic acid (PLGA) nanospheres with encapsulated model bovine serum albumin (BSA) were fabricated via a coaxial electrospraying technique. The coaxial electrospraying technique allows easy fabrication of a controllable core-shell nanosphere with intact biologically active factors within the core and polymeric outer shell [12,13]. In addition, different from traditional solvent emulsion methods, it enables the separation of organic and aqueous phases, and incorporation of biologically active components into the aqueous phase without exposing them to harmful organic solvents. BSA is a large globular protein with numerous biochemical applications and can be used as a nutrient to improve neural cell behavior [14]. It can be added with target bioactive factors to preserve their bioactivity and is a popular component as a release model drug for neural tissue engineering application [15]. PLGA was selected to serve as the carrier material housing the bioactive factors in our study due to its excellent biocompatibility, mechanical properties as well as biodegradability, render it ideal for many controlled therapeutic delivery and tissue engineering applications [16,17]. For 3D tissue scaffold fabrication, currently a variety of nano/ microfabrication techniques including phase separation, 3D bioplotting, electron beam lithography, photolithography, self-assembly, and electrospinning have been developed [5,8,18–21]. Among them, electrospinning has presented great promise in fabrication of neural scaffolds because of the ease with one can modulate desired physical and mechanical dimensions, and incorporate nanomaterials [22,23]. It can create aligned nano/microfibrous scaffolds that mimic natural ECM from a wide array of synthetic and natural biomaterials [24]. Studies have shown that aligned electrospun neural scaffolds can promote neural cell growth and guide neurites to propagate from the proximal stump along the direction of the fibers to the distal segment of the injured nerve to eventually restore function [25–27]. From the viewpoint of biomaterials for electrospun neural scaffolds, a variety of natural and synthetic biomaterials such as collagen, poly-ε-caprolactone (PCL), poly-L-lactic acid (PLLA), polyglycolic acid (PGA), polyurethane (PU), and poly(organo)phosphazenes, have been explored as potential biomaterial candidates for neural regeneration [9]. In particular, synthetic biomaterials have excellent cytocompatibility and offer a higher degree of customizability due to their flexible structure and properties, but generally lack the similar biomimetic properties to natural biomaterials.

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In this work, we uniquely integrated electrospinning with the co-axial electrospraying techniques to fabricate novel highly aligned biomimetic neural scaffold which has both nano to micro features and a sustained bioactive factor release environment. For this purpose, we electrospun aligned PCL microfibers, and then electrosprayed core-shell PLGA nanospheres to achieve nanocomposite scaffolds to direct neural cell growth. The aligned microfibers are expected to play a critical role in guiding axon propagation to bridge the injured area. Besides the sustained bioactive factor delivery effects, we also expect the nanospheres can influence the surface properties of our scaffolds and contribute to improving cell behavior.

Methods Fabrication of nanocomposite scaffolds Random and aligned PCL microfibrous scaffolds were fabricated via our lab's electrospinning set-up as shown in Figure 1, B and C. Specifically, PCL (Mw = 70,000-90,000, Sigma-Aldrich, USA) was dissolved in chloroform under sonication to form 12% (w/v) clear solution. Then the PCL solution in a 5 mL standard syringe attached to a 26 G blunt needle was extruded at a flow rate of 4.5 mL/hour and electrospun under a 5 kV voltage. Electrospun microfibers were collected at a distance of 12 cm from the needle tip to a static aluminum foil collector for random scaffolds and to a rotating mandrel for aligned scaffolds. Core-shell PLGA nanospheres with encapsulated BSA aqueous solution were produced by co-axial electrospraying process using a core-shell needle with 20 G outer and 26 G inner and sprayed onto PCL microfibrous scaffolds as shown in Figure 1, D. Briefly, 1 mg/mL core BSA aqueous solution was pumped through the core feed inlet. PLGA (50:50 with inherent viscosity range from 0.55 to 0.75 dL/g in Hexafluoroisopropanol, Sigma-Aldrich) was dissolved in acetone in a concentration of 2.5% w/v and fed through the shell feed inlet encapsulating the aqueous mixture via hydrophobic interaction. 7 kV voltage was applied to form BSA embedded core-shell nanospheres. Then, nanospheres were collected on an electrically grounded PCL mat placed at 15 cm vertical distance to the needle tip. As control, equivalent volume of BSA in solution was directly sprayed onto PCL scaffolds in the absence of PLGA nanospheres. Characterization of nanocomposite scaffolds Surface topography of electrospun nanocomposite scaffolds was examined via scanning electron microscope (SEM, Zeiss NVision 40 FIB) under an accelerating voltage of 1-2 kV. All samples for SEM were sputter-coated with gold. Distribution and orientation of various electrospun fibers were analyzed using the OrientationJ plug-in for imageJ (National Institutes of Health, USA). The contact angles of the nanocomposite scaffolds were measured by a drop shape analyzer (DSA4, Krüss) equipped with a camera to determine surface hydrophilicity and energy of the scaffolds. In brief, circular samples with 8 mm diameters were placed on glass slides. 0.9 μL of ultra pure water was pumped automatically on the samples' surface using a syringe.

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Temporal images 30 s after the droplet fell down were selected from videos for contact angle measurement. Surface energy (Esurface) was calculated using the equation Esurface = Elv × cosθ, where Elv = 72.8 mJ/m 2 at 20 °C for pure water and θ is the measured contact angle [28]. All experiments were conducted in ambient conditions and were performed at least 5 times per sample. Tensile mechanical properties were evaluated via a tabletop axial tester under a load cell capacity of 50 N. The scaffolds were cut into rectangular strips of 1 cm × 6 cm dimensions and mounted vertically on the gripping unit to keep a 2 cm effective length. Young's modulus was calculated from obtained tensile stress–strain curve of each scaffold in the linear region. Raman spectrometer (Horiba Scientific) was used to analyze the nanocomposite scaffold at a 532.06 nm line of Ar + laser for excitation over a range of 800-4000 cm − 1. Experiments were conducted at 25 °C and 45% humidity. In vitro drug release profile study and nano/microsphere characterization BSA embedded PLGA core-shell nanospheres were collected in ultra pure water followed by centrifuging, freezing and lyophilizing for drug release study. BSA solution was also sprayed on the PCL scaffolds directly as control group. In addition, PLGA microspheres with encapsulated BSA were prepared using water/oil/water double emulsion solvent extraction technique to compare the release behavior with core-shell nanospheres in vitro. The method of microspheres fabrication followed an established protocol [29]. Briefly, the aqueous phase was prepared by dissolving 10 mg/mL BSA in water. 50 mg PLGA was dissolved in 1 mL chloroform, and used as the organic phase. Afterward, 500 μL BSA solution was emulsified for 30 s with the organic phase using a sonicator (VWR Ultrasonicator) followed by injecting the resulting emulsion (w/o) into the second water phase. The obtained emulsion (w/o/w) was stirred for 1 hour at 1000 rpm at room temperature to remove the remaining organic solvent and then lyophilized. The morphology of synthesized microspheres and core-shell nanospheres was imaged and compared using SEM. The encapsulation efficacy of nano and microshperes was calculated by dividing the mass of BSA encapsulated in the spheres by the total mass of BSA used in the spheres fabrication, as shown in the equation below. Encapsulation efficacy ðw=w%Þ BSA encapsulated in spheres  100% ¼ Total weight of BSA The loading efficacy was determined by the following equation: Loading efficacy ðw=w%Þ ¼

BSA encapusulated in spheres  100% Total weight of spheres

For the in vitro BSA release study, 10 mg nanospheres, microspheres and control scaffold with aqueous sprayed BSA were separately placed in microcentrifuge tubes containing 1 mL of phosphate buffered solution (PBS) at 37 °C in an incubator.

At the end of each immersion period (4 hours, 10 hours, 1 day, 3 day, 6 day, 8 day, and 10 day), the samples were centrifuged at 10,000 rpm for 6 minutes. All supernatants were collected and identical fresh buffer was added. Supernatants were analyzed by BCA™ Protein Assay Reagent kit (Pierce Biotechnology) and the BSA content was quantified. Each sample was prepared in quintuple. PC-12 cell culture and proliferation studies Rat pheochromocytoma cells (PC-12, ATCC, USA) were used to evaluate cell behaviors on the nanocomposite scaffolds. PC-12 cells were cultured in RPMI 1640 (ATCC, USA), a high glucose media, supplemented with 10% horse serum (ATCC, USA), 5% fetal bovine serum (FBS, ATCC, USA) and 1% L-glutamine (Sigma-Aldrich, USA) and penicillin-streptomycin (10,000 U/mL penicillin and 10,000 μg/mL streptomycin, Gibco, USA). Cells were cultured on collagen coated flasks (Becton, Dickinson and Company, USA) for cell expansion under a humidified atmosphere with 5% CO2 at 37 °C. Medium was changed every two days. Electrospun scaffolds were punched into small circle samples with 5 mm in diameter and taped on glass coverslips. The samples were then sterilized by UV for 15 min, rinse twice using PBS, and pre-wetted in medium at 37 °C before cells seeding. PC-12 cells at a density of 30,000 per scaffold were seeded onto the circle samples for cell proliferation study. Cell number was quantified via a CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay (MTS assay) (Promega) at days 2, 4, and 6. Specifically, at each prescribed time, scaffolds were transferred to a new well plate, and rinsed three times with PBS. Then cells were lifted using Trypsin-EDTA (trypsin-ethylenediaminetetraacetic acid), reacted with MTS solution and analyzed using a Thermo Scientific Multiskan GO Spectrophotometer at 490 nm wavelength light after incubating 1 hour at 37 °C. The proliferation study was repeated three times with three replicates per group, totaling 9 samples per group. Immunocytochemistry of PC-12 cells and astrocytes in scaffolds PC-12 cells were cultured in standard medium with 50 ng/mL nerve growth factor (NGF) on random and aligned scaffolds with and without nanospheres for 7 and 10 days. Then, samples were rinsed with PBS, fixed with 4% formaldehyde for 20 minutes at room temperature. The cells were further fixed with 0.2% Triton X-100 in PBS for 6 min followed by blocking with PBS containing 5% BSA. Diluted primary antibodies, rabbit antiMAP2 antibody (1:500; Abcam), mouse anti-TuJ1 (1:1,000, Covance), were gently added into scaffolds and incubated at room temperature in a moist environment to prevent drying. This was followed by second antibodies incubation with Alexa Fluor 488 (Abcam) goat anti-rabbit, and Alexa Fluor 594 goat anti-mouse (Life technologies) at room temperature. The cell nuclei were stained by 10 μg/mL 4′-6-diamidino-2-phenylindole (DAPI) (Life technologies). Furthermore, immortalized human astrocytes obtained from Dr. Michael Keidar's lab at the George Washington University were selected as another model cell line to evaluate the guidance function of the nanocomposite scaffold. After culturing in scaffolds for 3 days, astrocytes were double

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stained by rhodamine phalloidin (Life technologies) and DAPI. Laser scanning confocal microscopy (LSCM 710, Zeiss) was employed to visualize and monitor the 3D cell growth and neurites extension of PC-12 and astrocyte cells. Statistical analysis All quantitative data are expressed as average ± standard error of the mean. Numerical data were analyzed via student's t-test to determine differences amongst the groups. Statistical significance was considered at P b 0.05.

Results Nanocomposite scaffold characterization The orientation of the electrospun fibers is an important consideration for neural tissue engineering applications. It is possible to control the aligned or random fiber orientation during fabrication via altering the collectors and optimizing solution concentration and electrospinning parameters. Studies have revealed that the alignment degree of electrospun fibers is influenced by the rotating speed of the collecting surface. The increase of rotating rate resulted in more evenly distributed orientation in deposited fibers [30]. In the present study, the optimal rotating rate was determined to be 1000 rpm. Figure 2 displays the SEM morphology of random and highly aligned PCL fibrous scaffolds which were collected in static foil (Figure 2, A and E) and rotating cylindrical mandrel (Figure 2, B and F), respectively. It reveals that 12% PCL can produce highly aligned and interconnected porous architecture under controlled conditions. The aligned fibers (Figure 2, D) collected on a rotating mandrel presented high uniform orientation with a narrow angular distribution compared with the random fibers fabricated on static foil (Figure 2, C). High magnification of SEM images showed PLGA core-shell nanospheres were homogenously distributed in the scaffolds (Figure 2, G and H). In addition, porous structure was observed on aligned fibers, which can be attributed to quick evaporation of solvent when collector rotated at a high speed. Porosity correlates to the adsorption/immobilization of proteins from cell culture media on the fiber surface which is pivotal to cell adhesion and spreading [31]. Figure 3, A shows that contact angle was decreased from 148 ± 4° to 127 ± 4° after incorporation of PLGA nanospheres onto PCL scaffold. Meanwhile, the surface energy was altered to − 43 from − 62 mJ/m 2. These indicate that the presence of PLGA nanospheres on the scaffolds can improve hydrophilicity, which may contribute to better cell adhesion. Aligned fibrous scaffolds presented anisotropic behavior, the circumferential and axial directions exhibit different mechanical properties [32]. The tensile properties testing of aligned fibrous scaffold are applied in the circumferential direction. Electrospun aligned fibrous scaffolds are utilized in this direction to regenerate the nerve gaps in most cases. The Young's modulus of the electrospun scaffolds were 1.06 ± 0.07 MPa, and 2.33 ± 0.03 MPa, respectively for pure PCL, and PLGA nanospheres loaded scaffolds

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(Figure 3, B). The obvious increase in Young's modulus is mainly because of the incorporation of PLGA nanospheres. Surface Raman spectroscopy characterization of the electrospun scaffolds with and without PLGA nanospheres was carried out to characterize the functional groups present on the scaffolds (Figure 3, C). Representative Raman spectra of PCL were observed on both scaffolds. These include typical double-bond carbon and oxygen stretching vibration (νC = O) that appeared at 1727 cm −1, δCH2 at 1421-1469 cm−1, ωCH2 at 1287-1308 cm −1; and skeletal stretching at 1112 cm −1 [33]. The peak between 2870 cm −1 and 2913 cm −1 assigned to CH2 symmetric and asymmetric stretching in PCL [34]. At the same time, new peak between 3100 and 3650 cm − 1 in nanospheres coated scaffolds, which correspond to overlapping absorption of hydroxyl group component of PLGA and NH of BSA, appeared in addition to the characteristic peaks of PCL [35]. Nano/microsphere characterization and in vitro sustained BSA release Figure 4, A and B showed the SEM images of PLGA core-shell nanospheres and microspheres and their size distribution. The nanospheres have a nanostructured spherical shape and homogeneously diameter distribution around 81 to 327 nm. The same well-defined spherical shape was observed in microspheres but with much larger diameter (mean size 2 μm). Table 1 summarized the encapsulation efficacy, the drug loading efficacy and mean particle size of nano/microspheres. The results showed that the electrosprayed core-shell PLGA nanospheres have higher encapsulation rate (80.45%) and loading efficacy (41.25%) when compared to PLGA microspheres (75.3 and 37.65%, respectively) fabricated via the traditional w/o/w double emulsion solvent extraction technique. The results of in vitro cumulative release study demonstrated that PLGA nanospheres presented much lower initial burst release than others (Figure 4, C). In the initial 10 hours, almost full release was observed for the samples directly sprayed with BSA, and BSA released more than 90% of the total amount from the microspheres. In contrast, BSA only released less than 60% from the core-shell nanospheres at the same time point. The cumulative drug release of nanospheres was observed to be significantly lower than the other groups in 24 hours (P b 0.05). Cell growth and differentiation studies The in vitro cell-scaffold interaction studies were performed using PC-12 cells. Figure 5 revealed that the PC-12 cell grew well on all scaffolds. The cell proliferation on PCL scaffolds with BSA embedded PLGA nanospheres is significantly higher than PCL controls and PCL scaffolds with directly sprayed BSA after 4 and 6 days. According to the release result shown in Figure 4, C, directly sprayed bare BSA on scaffold can make BSA to be fast released in a short time. Because of their rapid release profiles, majority of BSA was removed when changing the medium at the early time point and only a small amount of BSA was further released thereafter, which cannot provide sustained support for cell proliferation after 4 and 6 days. In contrast, the PLGA core-shell nanospheres can decrease initial burst release

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Figure 2. SEM images of low and high magnification of (A, E) random PCL scaffold; and (B, F) highly aligned PCL scaffold; (C) and (D) are orientation analysis of random and aligned electrospun fibers with OrientationJ plug-in for imageJ; and SEM images of PCL nanocomposite scaffold with core-shell PLGA nanospheres at (G) low magnification and (H) at high magnification.

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Figure 3. (A) Contact angles, surface energy and (B) tensile testing analysis of scaffolds with and without PLGA nanospheres. Data are mean ± standard error of the mean, n = 5; *P b 0.05. (C) Raman spectra of pure PCL and PLGA nanospheres embedded PCL scaffolds.

of BSA and keep a slow and steady release over the entire proliferation study period to promote PC-12 cells proliferation. Since PC-12 cells will stop dividing and start to differentiate in the presence of NGF. Medium containing 50 ng/mL NGF was employed for the studies of PC-12 cell neuronal differentiation.

Figure 6 shows the confocal micrographs of PC-12 cells cultured in completed medium with NGF for 7 and 10 days on aligned and random scaffolds with and without nanospheres. It can be observed the PC-12 cells attached and grew well on various scaffolds. The outgrowth and extension of neurites were seen on

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Figure 4. SEM images and particle size distributions of (A) PLGA core-shell nanospheres and (B) PLGA microspheres. (C) Cumulative BSA release profiles of nanosphere, microsphere and bare BSA in scaffolds. Data are mean ± standard error of the mean, n = 5, *P b 0.05 when compared other groups at 4, 10, and 24 hours.

both aligned and random fibers. Two neuronal markers TuJ1 and MAP2 that reveal the early and late stages of neuronal differentiation [36] were used to image the neural differentiation of PC-12 cells. Figure 6, A shows that the PC-12 cells have differentiated after 7 days of culture on all scaffolds. More importantly, the orientation of differentiated neurites preferentially extended exactly along the long axis of aligned fibers in parallel with neighboring cells, while axons extended radially

without any specific directionality when cultured on fibrous scaffolds with random fiber orientation (Figure 6, A and B). Meanwhile, the bared aligned scaffold and the nanospheres incorporated scaffold have comparable neurites extension (Figure 6, A), and the length of neurites in the aligned scaffold is higher than the random scaffold (Figure 6, B). In addition, we evaluated astrocytes growth on various scaffolds (aligned scaffolds with or without BSA incorporated nanospheres, and

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Table 1 Encapsulation efficacy, loading efficacy, and mean size of nano and microspheres. Samples

Encapsulation efficacy (w.t. %)

Loading efficacy (w.t. %)

Mean particle size (nm)

Nanospheres Microspheres

80.45 75.3

41.25 37.65

160.7 2000.4

random scaffold) after 3 days of culture. Figure 6, C shows that our aligned scaffolds can well guide the cell growth and spreading morphology.

Discussion

Figure 5. Greatly enhanced PC-12 cell proliferation in nanocomposite scaffold after 4 and 6 days. Data are mean ± standard error of the mean, n = 9; *P b 0.05.

Aligned nanocomposite scaffold for neural regeneration Despite many clinical approaches for neural injury repair, successful regeneration is limited due to the complexity and limitations of nervous system anatomy. Recently, electrospun fibrous scaffolds have been presented as a potential avenue for neural tissue repair [26]. Interest in employing electrospun scaffolds for neural application is primarily due to the versatility in mechanical properties, and biodegradation through selecting polymers and adjusting process parameters. Particularly, aligned fibrous scaffolds with topographical cues have shown a superior capacity in directing neurite outgrowth and extension, and successfully connecting target injured tissues. It is known that surface topography of scaffolds plays a critical role in regulating many cell behaviors, such as adhesion, migration, and differentiation, both in vitro and in vivo [37]. Aligned but not random fibrous neural conduits influence endogenous neural repair mechanisms, and are much more conducive to neurite growth even without requiring additional exogenous growth factors [25]. Meanwhile, aligned fibers can not only offer a better substrate for neural cells, such as Schwann cell, migration than random fibers, but also benefit formation of longitudinally oriented Büngner bonds in a long distance [38,39]. The bonds of Büngner include aligned strands of Schwann cells and laminin, which exert an essential key element in nerve repair. They can selectively guide axon regrowth to target organs and provide neurotrophic factors, and cell adhesion bioactive molecules [39]. Studies documented that biodegradable PCL conduits are suitable for the survival and differentiation of various neural cells with a low risk of being rejected [40]. For instance, in a study conducted by Frattini et al., PCL tubes were implanted into sciatic nerve gaps, sciatic functional index was great improved after 6 weeks without any inflammation reported [40]. PCL electrospun nanocomposite scaffolds in this study exhibits involvement of the important aligned structure cues as illustrated by SEM micrographs. It is believed that the 3D configuration of aligned fibrous scaffold can provide topographical directional cues for axonal regeneration across long nerve gaps. Sørensen et al. reported grooved topography in the micrometer range can support neural cell alignment [41]. In our study, enhanced neurite outgrowth and oriented extension were observed on aligned fibrous scaffolds compared to random

fibers (Figure 6). The results further confirmed the phenotypes of astrocytes and differentiated PC-12 cells influenced by the topography of scaffolds. The fibers in micro-scale probably provided a groove-like geography for directing cell body and neurite extension. Unlike the neurites on aligned scaffolds, the neurites on randomly oriented fibers presented random distribution and became entangled. Random fibers provide multiple random adhesion sites while aligned surface topography enhanced contact direction for cellular extension along the orientation of fibers. It is necessary to reduce branching of neurites and increase their length so as to prevent formation of neurolemoma in the process of neural repair [42]. Successful nerve regeneration depends on the extension of axons and formation of synaptic junctions. Therefore, it was suggested the aligned nanocomposite scaffolds promote the outgrowth and extension of neurites and provide potential for reforming synaptic junctions. Sustained nano drug release system for enhanced cell response Neurotrophy is pivotal in maintaining surviving neural cells. Administration of sustained release exogenous neurotrophic factors into neural scaffolds is an important consideration during the design of tissue engineered scaffolds in order to efficiently link with a drug delivery system. The combination of neural tissue engineering and drug delivery might mimic the in vivo release profiles of growth factors and neurotrophies during natural tissue self-repair [43]. Microspheres as drug delivery systems have been well developed and employed in tissue engineering [44]. However, problems such as inactivation of the drug during fabrication, and poor control of drug release rate in relation to the initial burst, impeded applications of microspheres in medicine [45]. In our study, the novel PLGA nanospheres fabricated by electrospraying present a desirable drug release behavior. The developed nanocomposite scaffold can incorporate a variety of contents inside of the nanospheres, such as bioactive proteins, NGF, or other neurtrophies. The controlled release of neurotrophies is paramount in neural regeneration through coordinating cell responses. The BSA release profile of our designed core-shell nanospheres presented a lower initial burst and longer release

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Figure 6. Confocal microscopy images of PC-12 cell and astrocyte spreading morphology. (A) Double staining of MAP2 and TuJ1 to detect PC-12 differentiation on various scaffolds after 7 days of culture. (B) PC-12 cell neurite extension along highly aligned fibers and unidirectional cell growth on random scaffolds, and quantification of neurite length after 10 days culture. Neurite length was analyzed using imageJ software. Data are mean ± standard error of the mean, n = 4, *P b 0.05. (C) Low and high magnification images of astrocytes growth on scaffolds, red color represents cell cytoskeleton and blue color represents cell nuclei.

compared to microspheres and bare BSA. The high encapsulation and loading efficacies as well as sustained drug release profile over the period of 10 days make these core-shell PLGA

nanospheres a promising delivery system for the controlled release of various neurotrophic factors in neural regeneration. Moreover, we, for the first time, integrated the sprayed PLGA

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Figure 6 (continued).

core-shell nanospheres with the highly aligned PCL scaffold, which altered the surface and physical properties of scaffold and provided the favorable microenvironment for improved PC-12 cell proliferation. Since natural neural tissue ECM has many nanostructured features, the creation of nanostructured surface topography and sustained bioactive factors can help mimic the nature of neural tissue, and lead to a more cell-favorable environment [46]. In addition, the enhanced hydrophilicity and mechanical properties that originated from the involvement of nanospheres also distinguished our designed neural scaffold from more traditional designs. Surface wettability and surface energy of biomaterial scaffolds are believed to be related to mediating cell adhesion, proliferation and differentiation on substrates [47,48]. A more hydrophilic surface can contribute to more specific proteins adsorption and further higher cell adhesion in the initial period of cell culture [49]. Our results (Figure 3, A) showed that the involvement of PLGA nanospheres can decrease the contact angle which may partly contribute to better cytocompatibility properties of nanocomposite scaffolds than PCL controls. Furthermore, the PLGA core-shell nanospheres can provide an altered surface chemistry (Figure 3, C) and unique nano-topography (Figure 2) on aligned PCL microfibrous scaffolds that may further improve neural function. It has been found that the nanoroughness and correspondingly increased surface areas of electrospun scaffolds can increase neural cell growth rate to 50 % in comparison with smooth electrospun scaffolds [50]. In summary, in this study, a novel highly aligned PCL nanocomposite scaffold was fabricated by integrating electrospinning and electrospraying techniques. Our results demonstrated core-shell nanospheres can create a cell-favorable nano environment and serve as a model system for a sustained release

of bioactive factors. Greatly enhanced proliferation and aligned neurite extension in PC-12 cells have been observed on our nanocomposite scaffold which combined both physical guidance and neurotrophy delivery system. The nanocomposite scaffolds meet several key criteria of an ideal neural scaffold (Figure 1, A), thus warranting further exploration for neural tissue regeneration.

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Highly aligned nanocomposite scaffolds by electrospinning and electrospraying for neural tissue regeneration.

Neural tissue engineering offers a promising avenue for repairing neural injuries. Advancement in nanotechnology and neural scaffold manufacturing str...
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