Salt-Induced Electrospun Patterned Bundled Fibers for Spatially

Subscriber access provided by University of Sussex Library

Article

Salt-Induced Electrospun Patterned (SiEP) Bundled Fibers for Spatially Regulating Cellular Responses Mira Cho, Seung-Hyun Kim, Gyuhyung Jin, Kook In Park, and Jae-Hyung Jang ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b03848 • Publication Date (Web): 11 May 2016 Downloaded from http://pubs.acs.org on May 13, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

ACS Applied Materials & Interfaces is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

ACS Applied Materials & Interfaces

Salt-Induced Electrospun Patterned (SiEP) Bundled Fibers for Spatially Regulating Cellular Responses Mira Cho†, Seung-Hyun Kim†, Gyuhyung Jin†, Kook In Park‡ and Jae-Hyung Jang†* †

Department of Chemical and Biomolecular Engineering, Yonsei University, 50, Yonsei-ro, Seodaemun-gu, Seoul, 120-749, Korea,

‡

Department of Pediatrics, College of Medicine, Yonsei University, 50, Yonsei-ro, Seodaemungu, Seoul, 120-749, Korea

ABSTRACT

Implementing patterned fibrous matrices can offer a highly valuable platform for spatially orchestrating hierarchical cellular constructs, specifically for neural engineering approaches, in which striated alignment or directional growth of axons are key elements for the functional recovery of damaged nervous systems. Thus, understanding the structural parameters of patterned fibrous matrices that can effectively promote neural growth can provide crucial clues for designing state-of-the-art tissue engineering scaffolds. To this end, salt-induced electrospun patterned fiber bundles (i.e., SiEP-bundles) comprising longitudinally stacked multiple fibers were fabricated, and their capabilities of spatially stimulating the responses of neural cells,

ACS Paragon Plus Environment

1


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 2 of 42

including PC12 cells, human neural stem cells (hNSCs), and dorsal root ganglia (DRG), were assessed by comparing them to conventional fibrous matrices having either randomly oriented fibers or individually aligned fibers. The SiEP-bundles possessed remarkably distinctive morphological and topographical characteristics: multi-complexed infra-structures with nanoand micro-scale fibers, rough surfaces, and soft mechanical properties. Importantly, the SiEPbundles resulted in spatial cellular elongations corresponding to the fiber directions and induced highly robust neurite extensions along the patterned fibers. Furthermore, the residence of hNSCs on the topographically rough grooves of the SiEP-bundles boosted neuronal differentiation. These findings can provide crucial insights for designing fibrous platforms that can spatially regulate cellular responses and potentially offer powerful strategies for a neural growth system in which directional cellular responses are critical for the functional recovery of damaged neural tissues.

KEYWORDS Electrospinning, salt-induced bundle, patterned scaffold, neurite outgrowth, neural stem cells, nerve tissue engineering


2

Page 3 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


1. Introduction Biomaterial-based guiding technologies capable of mediating cellular patterns in a spatial manner are essential to many tissue engineering approaches, specifically for nerve regeneration. Manipulating the guidance of neurite extension outgrowth from injured neuronal cell bodies toward distal axons to link them together is highly crucial for the functional recovery of the damaged peripheral or central nervous system.1-4 Importantly, exploiting the characteristic features of the extracellular matrix (ECM), composed of fibrous morphological networks, may offer highly effective strategies for artificially supporting the guidance of cellular patterns. The natural ECM serves as a versatile substrate that can coordinate the networks formed between local cells and numerous receptors, ligands, and signals for determining tissue phenotypes, soluble factor secretion, and other important biochemical processes in a spatial and timely manner. The spatial variations of inductive factors (i.e., receptors, ligands, and signals) along ECM networks can function as key routes for developing unique tissue morphologies and modulating cellular patterns.5-7 Furthermore, the physical aspects of ECM networks, including their stiffness or topographical alterations, have been regarded as crucial factors for tuning intracellular signal cascades, possibly serving as cues to arrange cellular morphologies8-11 or fates.12-16 Therefore, designing smart devices that can potentially mimic the fibrous morphology of the natural ECM and simultaneously manipulating its functional characteristics can be an essential task in designing biomaterial-based systems capable of inducing patterned tissue formation, especially for guiding nerve regenerations in a spatial manner.17-18 The structural similarity of electrospun fibrous matrices compared with ECM exterior morphologies allowed the electrospun fibers to potentially adopt ECM’s key functions,19 such as serving as building blocks for cell growth or facilitating soluble factor reservoir/delivery. Thus,


3


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 4 of 42

these fibers can serve as versatile templates for many biomedical applications, including tissue engineering, drug/gene delivery, and cell therapy.20 Adjusting the surface tension of a polymer solution under a high electric voltage generates collections of numerous fibers that are identical to the natural ECM.21 In addition to their morphological aspects, the fibers’ surfaces or structures can be decorated by immobilizing or integrating various agents, such as drugs,22 gene vectors,23 cells,24 and adhesives,25 which has been performed to adopt the ECM’s major functions, namely manipulating cellular phenotypes and constructing hierarchical cellular structures in local areas.26 Importantly, electrospun fibers fabricated with unique structural features, e.g., uniaxially aligned fibrous mats, tubular conduits, and rolled matrices, have served as efficient physical supports for guiding the spatial arrangement of neuronal cells.4,18,27-31 The majority of these fibrous systems require specialized apparatus18 or modified building-block materials29 to create well-regulated fibrous patterns. To further enhance the technical specificity of fibrous systems, understanding their key design parameters that can efficiently mediate the spatial patterns of neuronal infrastructures must be conducted. In this study, three-dimensional (3D), well-aligned fibrous bundles were fabricated based on a slightly modified version of the salt-inducted electrospinning process.32 The inclusion of ionic salts, such as ethylamine hydrochloride, in a solvent containing polycaprolactone (PCL) prior to electrospinning generated spontaneously elongated fiber bundles on a ground collector.32 Subsequently, solely winding the fiber bundles using a rotor produced highly aligned matrices composed of multiple fiber bundles, referred to as salt-induced electrospun patterned (SiEP)bundles. The SiEP-bundles’ ability to manipulate the spatial responses of neural cells, including PC12 cells, hNSCs, and DRG, was evaluated by comparing it with that of different sets of conventional fibrous systems, namely fibrous matrices comprising i) randomly oriented fibers


4

Page 5 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(i.e., random_fibers) or ii) individually separated, aligned fibers (i.e., aligned_fibers). The resulting fibrous systems possessed highly distinctive characteristic features in terms of their stiffness, fiber morphology, surface topography, and surface roughness, ultimately providing diverse physical cues that can stimulate spatial cellular responses in a variety of ways. Thus, the three fibrous matrices were compared by evaluating their capabilities of extending neurites of neuronal cells such as PC12 cells and dorsal root ganglia (DRG) along the fiber direction. Furthermore, the neuronal differentiation of hNSCs against the exterior deviation of each fibrous system was examined. Finally, the key design parameters of fibrous systems for effectively guiding the spatial responses of neural cells were determined. The parallel examination of two aligned fibrous structures with respect to their abilities to spatially regulate cellular responses (i.e., neurite extension, directions, and neuronal differentiation) has never been conducted. Thus, these findings should provide valuable insights into designing biomaterial systems for orchestrating hierarchical tissue constructs, especially for the peripheral or central nervous system in which directional axonal regeneration or re-myelination along the axons can be crucial for the functional recovery of injured or damaged nervous systems.

2. Experimental Section

Fabrication of electrospun aligned fibrous matrices Three different fibrous structures were fabricated: i) randomly oriented conventional flat PCL mats (i.e., random_fibers); ii) conventionally patterned fibrous matrices comprising numerous individually separated, aligned fibers formed by electrospinning on a high-speed rotating mandrel (i.e., aligned_fibers); and iii) patterned fibrous matrices composed of multiple aligned


5


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 6 of 42

fiber bundles fabricated by a slightly modified salt-induced electrospinning process,32 which were referred to salt-induced electrospun patterned bundles (i.e., SiEP_bundles). All the resulting fibers were electrospun using a conventional apparatus (ESR 100, NanoNC, Seoul, Korea). Two different rotating drums were utilized to generate random and two sets of patterned fibrous systems. The first drum, referred to as a rotating mandrel (DC90, NanoNC, Seoul, Korea), was used to fabricate the individually aligned_fibers. The other drum was manually designed to generate the random_fibers and the SiEP bundles at a low rotating speed (i.e., 25 rpm). Briefly, poly(ε-caprolactone) (PCL; ‫ܯ‬ௐ =80,000, 15% (w/v), Sigma-Aldrich, St. Louis, MO, USA) was dissolved in a mixture comprising dichloromethane (DCM, anhydrous, Duksan pure chemical, Ansan, Korea) and N,N-dimethyl formamide (DMF, anhydrous, Duksan Pure Chemical) (v/v ratio = 1:1) and loaded into a 10 mL syringe connected to a 23G needle (inner diameter = 330.3µm). First, as a control condition, the PCL solution containing no ionic salt was simply ejected from a normal electrospinning nozzle tip and collected on a negatively charged custommade collector rotating at a low speed (i.e., 25 rpm) to produce conventional sheet-like PCL random_fibers. Second, to fabricate the aligned_fibers, the PCL solution was electrospun onto a negatively charged mandrel rotating at a speed of 1200 rpm. Finally, the incorporation of ethylamine hydrochloride (≥ 0.5% (w/v); Sigma-Aldrich) into the PCL solution prior to electrospinning led to the spontaneous generation of fiber bundles. To fabricate the aligned fiber bundles, the PCL solution containing ethylamine hydrochloride was electrospun onto a negatively charged custom-made rotating collector, which ultimately formed the SiEP_bundles. Each electrospinning process was conducted under a high voltage (14~16 kV, working distance: 19 cm) at a constant polymer feed rate (i.e., 1 mL/h). The resulting fibrous matrices were placed


6

Page 7 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


in a desiccator (Dry Active, Korea Ace Scientific, Seoul, Korea) overnight to remove residual solvent and stored until use.

Characterization of fibrous matrices The fibrous morphology of the resulting structures, including the exterior and cross-sectional areas of each fiber, was visualized using a scanning electron microscope (SEM) (JEOL-5410 and JEOL-7001F, JEOL Ltd., Tokyo, Japan). To differentiate the fibrous structures from cells seeded onto the fiber surfaces, coumarin 6 (Sigma-Aldrich) was incorporated into the PCL solutions at a concentration of approximately 0.01% (w/v) prior to electrospinning, and the fibrous structures containing cells were visualized using a confocal laser scanning microscope (CLSM, LSM 700, Carl Zeiss, Thornwood, NY, USA). To readily compare the differences in the fiber distribution of each system, the fluorescence images of fibrous matrices were reconstructed threedimensionally using Imaris 7.4.2 (Bitplane, UK). The distribution analysis of fiber orientation was further performed using the Orientation J plugin in ImageJ software, as previously described.24 The void volume in each fibrous structure was calculated as previously reported in a study, based upon the volume/mass of each matrix and the density of the polymeric material used as a building block.33 The crystalline portions of polymer chains were examined by X-ray diffraction (XRD; Rigaku Miniflex, Rigaku, Tokyo, Japan). The mechanical properties of the fibrous matrices, captured by their stress-strain behaviors, were examined using a universal testing machine (Multi Test 1-i, Mecnnesin, Slinfold, UK) with a 50 N load. The surface morphology and roughness of each fibrous substrate were investigated using atomic force measurement (AFM; XE-BIO AFM, Park Systems, Suwon, Korea).


7


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 8 of 42

Cell culture To characterize the responses of cells on the fibrous structures, which varied in their fibrous morphologies and scales, two different types of cells, PC12 cells and human fatal neural stem cells (hNSCs), were cultured on each fibrous mat. These neural cells were employed as model cells to determine the design parameters of the resulting fibrous systems and thereby effectively mediate the directional elongation of neurites or neuronal cells along the patterned fibers. The PC12 cells, which are derived from a pheochromocytoma of the rat adrenal medulla, were cultured in Roswell Park Memorial Institute medium (RPMI) 1640 (Life technology, Grand Island, NY, USA) with 7.5% fetal bovine serum (FBS, Corning, Corning, NY, USA), 7.5% horse serum (Life technology) and 1% penicillin and streptomycin (Life technology) at 37°C and 5% CO2. The hNSCs, which were directly derived from telencephalon (HFT13) as previously described,34 were cultured in Dulbecco’s modified Eagle’s medium/F12 (DMEM/F12; Life technology, Carlsbad, CA, USA) containing 1% N-2 supplement, 8 mg/ml heparin (SigmaAldrich), 20 ng/ml fibroblast growth factor-2 (FGF-2; R&D Systems, Minneapolis, MN, USA), 10 ng/ml leukemia inhibitory factor (LIF; Chemicon, Temecula, CA, USA) and 2% penicillin and streptomycin (Life technology) at 37°C and 6% CO2.

Cell seeding on fibrous structures A highly dense suspension of PC12 cells (1 × 105 in 50 µL) or hNSCs (1 × 105 in 20 µL) was prepared to seed cells into the porous fibrous structures. Prior to placing the cells, each structure was incubated in 70% ethanol for 30 minutes for sterilization and rinsed vigorously with PBS five times to fully hydrate each fibrous matrix. During this rinsing process, the additive,


8

Page 9 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


ethylamine hydrochloride, could be substantially leached out from the bundled fibrous mats to leave an amount that was not detrimental to the viability of cells. The residual PBS was blotted on a filter paper to fully provide room for cell infiltration across the matrix interior. Subsequently, the cell suspension was directly dropped onto the fibrous matrices and incubated for 30 minutes to allow cells to adhere onto the fiber surfaces. Finally, the resulting constructs were transferred to fresh 24-well plates containing 600 µL medium to further grow cells within the structures prior to analysis.

Cell viability studies The viability of the cells (i.e., PC12) growing on each fibrous structure was determined using a WST1 cell cytotoxicity assay kit (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s protocol. Briefly, at 2 days post-culture, the cell-fibrous matrix composites were placed in fresh wells to exclude non-bound cells on the fibrous substrates, then a 10% (v/v) WST-1 solution suspended in cell-culture medium was directly dropped into each well and incubated for 3 hours at 37°C. Subsequently, the colorimetric change of the supernatant was measured at 440 nm using a spectrophotometer (NanoDrop 2000, Thermo Scientific, West Palm Beach, FL, USA), and the absolute absorbance values (i.e., Absorbance (440 nm)) were plotted to evaluate the cell viability for each fibrous matrix.

Analysis of PC12 cell responses on the patterned fibrous matrices The spatial responses of PC12 cells against each fibrous system were analyzed by investigating the alterations in cellular morphology. To characterize the cytoskeletal morphologies of PC12 cells on each fibrous matrix, both CLSM imaging and SEM analysis were used. For CLSM


9


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 10 of 42

imaging, actin filaments of PC12 cells were stained with a rhodamine-labeled phalloidin (1:50 dilution; Life technology), and the nuclei of cells were stained with a 4',6-diamidino-2phenylindole (DAPI; 3 µg/mL in PBS, Vector Laboratories, Burlingame, CA, USA). Briefly, 1 × 105 PC12 cells were grown within each fibrous matrix for two days. The cell-fiber composites were rinsed three times with PBS supplemented with 0.01% Triton X-100 (PBS-T; SigmaAldrich), and cells were then fixed by 4% paraformaldehyde (PFA; Sigma-Aldrich) for 20 minutes at 4°C. Subsequently, the cells were rinsed three times with PBS-T and incubated with the rhodamine-labeled phalloidin/PBS-T for 30 minutes in the dark, washed three times with PBS-T, and counterstained with DAPI for 30 minutes. The trajectories of actin filaments were determined under a confocal microscope (CLSM, LSM 700), and the projected areas of PC12 cells were quantified using the ImageJ software program (National Institutes of Health, Bethesda, MD, USA). To determine the position of PC12 cells along the fiber constructs, a coumarin dye (0.01% (w/v); Sigma-Aldrich) was incorporated into each PCL fiber prior to electrospinning, and the nuclei of PC12 cells were stained with DAPI according to the manufacturer’s manual. For SEM analysis, cells were pre-fixed with a Karnovsky fixative, which was a mixture of 2% glutaraldehyde (Millipore, Billerica, MA, USA), 2% PFA, and 0.5% calcium chloride (Sigma-Aldrich), and adjusted with 0.1 M phosphate buffer (pH 7.4) for at least 6 hours. The pre-fixed cells were then post-fixed with 1% osmium tetroxide (OsO4; Polysciences, Warrington, PA, USA) in 0.1 M phosphate buffer for 2 hours. The post-fixed cells were washed with the 0.1 M phosphate buffer solution for 10 minutes and dehydrated sequentially in 50, 60, 70, 80, 90, 95, and 100% alcohol for 10 minutes each. The dehydrated cells were freeze-dried using t-butyl alcohol (Sigma-Aldrich), coated with a gold layer (thickness: 30 nm; coater: 108 Manual sputter coater, TED PELLA INC., Redding, CA, USA),


10

Page 11 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


and finally examined using a field emission-scanning electronic microscope (FE-SEM; S-800 FE-SEM, Hitachi, Tokyo, Japan) at an accelerating voltage of 20 kV.

Neurite extension from PC12 cells along the patterned fibrous matrices Both the direction and the length of neurites extending from PC12 cells along the patterned fibrous matrices were examined to further analyze the spatial cellular responses. The PC12 cellfiber composites were exposed for 4 days to normal culture medium supplemented with 100 ng/ml mouse-β-nerve growth factor (NGF; ProSpec-TanyTechnogene, Ltd., East Brunswick, NJ, USA), which was added to the culture medium at 12 hours post-culture. At 4 days post-culture with NGF, cells were fixed with 4% PFA for 20 minutes and blocked with 5% goat serum in PBS-T (0.3%) for 40 minutes at room temperature. Cells were then incubated overnight at 4°C with mouse anti-β-III-tubulin (1:500 dilution; Sigma-Aldrich), washed with PBS-T three times, and stained with goat anti-mouse Alexa 633 (1:250 dilution; Life Technologies) for 3 hours at room temperature. The nuclei of PC12 cells were counterstained with DAPI. Finally, the fluorescently labeled neurites and PC12 cells were imaged using a confocal microscope (CLSM, LSM 700).

Dorsal root ganglion (DRG) culture on each fibrous matrix To examine the spatial patterns of the fibrous structures on the patterned cellular responses, both the directions and the lengths of neurites extended from the dorsal root ganglia (DRG) were assessed. The DRG, whose dimensions ranged from approximately 440 µm to 1000 µm, were isolated from 9-day-old chick embryos. After DRG isolation, approximately 3 DRG / matrix were immediately placed on the sterilized fibrous matrices without surface coating and cultured


11


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 12 of 42

in DMEM/F12 supplemented with 1% N-2 (Life Technologies) and 50 ng/ml NGF. At 3 days post-exposure to NGF, to examine neurite extension from the DRG bodies, the DRG-fiber composites were incubated in 4% PFA for 20 minutes at 4°C and blocked with 5% goat serum in PBS-T for 2 hours at 25°C. The neurites extending from DRG on each fibrous matrix were incubated with mouse anti-β-III-tubulin (1:500 dilution in PBS-T; Sigma-Aldrich) overnight at 4°C, washed three times with PBS-T, and stained with goat anti-mouse Alexa 633 (1:250 dilution in PBS-T, Life Technology). Fluorescence images of neurite extension along the fibers were acquired under a CLSM (LSM880). To count the number of cells that stretched neurites from DRG, the nuclei of cells comprising DRG explants were stained with DAPI, and the number of cells with branching neurites was manually counted and normalized to the number of DRG explants, which was used for counting the cell nuclei. The stained neurites were marked manually, and both the directions and the length of neurites extending from the DRG bodies were quantified using the ImageJ software program (NIH).

Analysis of spatial hNSC responses on each fibrous matrix To examine the structural effect of the aligned fibrous structures on hNSC differentiation and the distribution of differentiated neurons along the fibers, hNSCs (1 × 105) were grown on each fibrous matrix and exposed to the plain culture medium. To examine neuronal differentiation on each fibrous matrix, immunostaining for β-III-tubulin and glial fibrillary acidic protein (GFAP) was performed to identify neurons and astrocytes among the total cell population, respectively. At 2 weeks post-culture, the fibrous systems containing hNSCs were rinsed twice with PBS-T (0.3% Triton-X), and hNSCs were fixed with 4% PFA for 20 minutes at 4°C and blocked with 5% goat serum in PBS-T for 2 hours at room temperature. Subsequently, hNSCs were incubated


12

Page 13 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


overnight at 4°C with primary antibodies: mouse anti-β-III-tubulin (1:500 dilution in PBS-T; Sigma-Aldrich) and guinea-pig anti-GFAP (1:500 dilution in PBS-T; Synaptic systems, Goettigen, Germany). Cells were rinsed three times with PBS-T and incubated with secondary fluorescent-conjugated antibodies, including goat anti-mouse Alexa 633 (1:250 dilution in PBST; Life Technologies, Carlsbad, CA, USA) and goat anti-guinea pig Alexa 488 (1:250 dilution in PBS-T; Life Technologies). Finally, fluorescence images of stained cells were acquired using CLSM (LSM 700).

Statistics All experiments were performed at least in triplicate, and all data were presented as the mean ± the standard deviation (SD). A one-way analysis of variance (ANOVA) with a post-hoc Dunnett’s test using the SPSS 21.0 software package (IBM Corporation, Somers, NY, U.S.A.) was performed to test statistical significance.

3. Result and discussion


13


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 14 of 42

Figure 1. Salt-induced electrospinning process for fabricating self-bundled fibers. (A) Schematic description of the process using ethylamine hydrochloride to from the salt-induced fiber bundles. The inset image is a digital image of the bundled fibers, which were directly elongated from the collector up to the nozzle tip. (B) SEM images illustrating the morphological transitions of electrospun fibers, transforming from randomly oriented conventional fibrous mats via multilayered electrospun fibers (i.e., SpONGE) into bundled fibers by varying the concentration of ethylamine hydrochloride in the polymer solution (i.e., 0 – 0.5 % (w/v)). The scale bar indicates 50 µm. (C) Conductivity of the solvent used for dissolving PCL polymers prior to electrospinning.

The effects of structural deviations of fibrous matrices on the spatial responses of neural cells were investigated. Initially, the fibrous bundled structures were spontaneously formed by the salt-induced electrospinning of a PCL solution containing ethylamine hydrochloride (Figure 1A). The incorporation of ionic salts (i.e., ethylamine hydrochloride) into the PCL solution prior to electrospinning resulted in unique fibrous morphological transitions (Figure 1B), as consistently


14

Page 15 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


shown in previous studies.26,32 The concentration of ionic salts served as a crucial parameter for varying the structure of the fibrous matrices.26,32,35 As the concentration of ethylamine hydrochloride in the polymer solution was increased to 0.4%, the exterior morphology of the randomly oriented two-dimensional (2D) thin fibrous mats (top panel in Figure 1B) was spontaneously transformed into a three-dimensional (3D) fibrous structure composed of multiple fibrous layers (middle panel in Figure 1B), as shown in our previous study.26 Importantly, when more ethylamine hydrochloride (≥ 0.5%) was added to the polymer solution, the multi-layered fibrous matrices further transformed into a unique fibrous architecture: a fibrous matrix containing multiple individual fibers stacked together (bottom panel in Figure 1B), which were continuously connected directly from the collector to the nozzle (the inset digital image of Figure 1A). Two different ionic salts, FeCl3 and NaIO4, were used to confirm the role of ionic salts in the fibrous morphological transition from the randomly oriented fibers via multiple layered fibers to self-bundled fibers. Regardless of the type of ionic salt used, the fibrous morphological transitions were observed (Figure S1). The proportions of the ionic salts within the polymer solution for mediating the self-bundled fibrous structures varied slightly depending on the salt type (i.e., FeCl3 and NaIO4: 0.4%). The accumulation of electrons within fibers, caused by the presence of ionic salts in the solvent, was key for diversifying the exterior morphologies of the fibrous structures and transforming the structures from multi-layered constructs into striated fiber bundles. As the amount of salt in the solvent increases, more electrons can be collected within fibers that are newly formed on the ground collector, as confirmed by the increase in the electrical conductivity of the salt-containing solvent (Figure 1C). Increasing the concentration of ionic salts markedly enhanced electron transfer throughout the ejected fibers, possibly resulting in attractive


15


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 16 of 42

interactions between ejected fibers and the positively charged needle tip.26,36 As the amount of ionic salt exceeded a certain threshold, for example, transitioning from 0.4% to 0.5% of ethylamine hydrochloride, sufficiently strong attractive forces between the fibers and the positively charged tip formed striated-fiber bundles that were elongated from the needle tip to the collector (inset image of Figure 1A). The enhanced mobility of electrons, which might result from the moisture or solvent residues residing in the surroundings of the fibers or within the fibers, was a key factor that could readily manipulate the fibrous morphologies. The remnant moisture adjacent to the ejected fibers might facilitate electron mobility, thereby readily spreading the net charges throughout the fibrous networks.32 Accordingly, electrospinning under humid surroundings or with surrounding residual solvent likely triggered the morphological transition toward the bundled structures (not shown).

Figure 2. Fabrication of uniaxially aligned fibrous matrices. (A) Schematic illustration of fabrication methods: randomly oriented fibrous mat (left; random_fibers), fibrous matrices


16

Page 17 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


comprising individually aligned fibers (middle; aligned fibers), and salt-induced electrospun patterned (SiEP)-bundles (right). (B) Digital images of each resulting fiber matrix, which was cut into 1 × 1 cm pieces for analysis. (C) Representative SEM images showing exterior surfaces of the resulting fibrous matrices. The images on the upper and lower rows indicate the ones captured at low and high magnification, respectively. The white arrows in panel C indicate the direction of rotating spin. The scale bars on the upper and lower rows are 100 µm and 10 µm, respectively.

Figure 3. Fiber alignment by varying rotating speed. (A) SEM images exhibiting the extent of fiber alignment with or without ethylamine hydrochloride prior to electrospinning. The scale bars on the upper and lower rows indicate 100 µm and 10 µm, respectively. (B) Distribution analysis


17


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 42

of fiber orientation depending on rotating speed (i.e., 25, 200, and 600 rpm), performed using ImageJ software.

The resulting fiber bundles were further processed to assign uniaxially aligned patterns to the fiber structures by solely rotating the bundles. Thus, the final fibrous structures were referred to as salt-induced electrospun patterned (SiEP)-bundles. To examine the effects of the structural variations of the fibrous structures on neural cell responses, such as cell position, elongation, and differentiation, additional fibrous matrices with different morphological and topographical features compared with those of the SiEP-bundles were fabricated (Figure 2A): i) randomly oriented, conventional flat mats and ii) uniaxially aligned fibrous mats comprising individually separated fibers. The fibrous systems were referred to as random_fibers and aligned_fibers, respectively (left and middle panels of Figure 2A, respectively). The random_fibers containing no ionic salt were simply formed using a conventional electrospinning methodology on a mandrel rotating at a low speed (i.e., 25 rpm), producing randomly oriented flat fibrous mats (top panel in Figure 2B and 2C). As illustrated in the middle panel of Figure 2A, to fabricate the individually aligned_fibers, the ejected polymers were collected on a mandrel rotating at a high speed (i.e., 1200 rpm); digital images of the fibers are shown in the middle panel of Figure 2B. Consequently, fibrous structures comprising closely distributed aligned fibers, whose directions mostly corresponded to that of the rotating spin, were obtained (Figure 2C). Winding the fiber bundles on a rotator at a low rotating speed was the sole step required to create the SiEP-bundles containing directionally patterned fibrous bundles (right panel of Figure 2A). The SiEP-bundles were composed of multi-complexed infrastructures heterogeneously stacked with multiple nano- and micro-scale fibers, thereby comprising much thicker fibrous


18

Page 19 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


networks than the aligned_fibers (Figure 2C). Thus, the dimensions of the fiber bundles roughly ranged from 10 to 100 µm, and spacious voids were observed across the entire matrices. Rotating a mandrel at a speed of at least 600 rpm was mandatory to forming well-aligned fibrous matrices containing no salts (i.e., aligned_fibers) (Figure 3A). However, a remarkably reduced rotating speed (i.e., 25 rpm) was required to create the uniaxially aligned fiber bundles (i.e., SiEPbundles). Regardless of the type of ionic salt used, well-patterned fibrous bundles were readily formed at a slowly rotating speed (Figure S2A). To quantitatively analyze the fiber alignment depending on the rotating speed, the angles of individual fibers with respect to a fixed reference line were enumerated, and the frequencies of the detected fiber angles were quantified as shown in Figure 3B. Thus, the narrow peak around a specific angle can be related to the high rates of fiber alignment. Regardless of salt type, when ionic salts were included, highly sharp peaks were observed even at 25 rpm, whereas a sharp peak appeared at 600 rpm when the aligned_fibers were formed without salts (Figure 3B and Figure S2B). Ethylamine hydrochloride was selected as a representative ionic salt to form the SiEP-bundles for further studies.


19


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 20 of 42

Figure 4. Characterization of the resulting fibrous matrices. (A) Diameters of individual fibers. At least 100 fibers captured by SEM were randomly selected, and their dimensions were measured by ImageJ software. (B) The cross-sectional SEM images of individual fibers or fiber bundles. The scale bar indicates 10 µm. (C) Void volumes not occupied by fibers within a designated space. (D) CLSM images of each fibrous matrix containing a coumarin dye. Subsequently, each fluorescence dye-encapsulating fibers were three-dimensionally re-


20

Page 21 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


constructed using IMARIS software. The scale bar indicates 30 µm. (E) The tensile stress-strain curves of random_fibers, aligned_fibers, and SiEP-bundles. (F) The crystallite size analysis for each fibrous matrix, which was confirmed by XRD analysis. The symbols *, and ** shown in this figure indicate significant differences (P

Salt-Induced Electrospun Patterned Bundled Fibers for Spatially

Recommend Documents