Accepted Manuscript Title: Bioactive fish collagen/polycaprolactone composite nanofibrous scaffolds fabricated by electrospinning for 3D cell culture Author: Da Jeong Choi Seung Mi Choi Hae Yeong Kang Hye-Jin Min Rira Lee Muhammad Ikram Fazli Subhan Song Wan Jin Young Hun Jeong Jong-Young Kwak Sik Yoon PII: DOI: Reference:

S0168-1656(15)00026-7 http://dx.doi.org/doi:10.1016/j.jbiotec.2015.01.017 BIOTEC 6994

To appear in:

Journal of Biotechnology

Received date: Revised date: Accepted date:

22-8-2014 12-1-2015 16-1-2015

Please cite this article as: Choi, D.J., Choi, S.M., Kang, H.Y., Min, H.J., Lee, R., Ikram, M., Subhan, F., Jin, S.W., Jeong, Y.H., Kwak, J.-Y., Yoon, S.,Bioactive fish collagen/polycaprolactone composite nanofibrous scaffolds fabricated by electrospinning for 3D cell culture, Journal of Biotechnology (2015), http://dx.doi.org/10.1016/j.jbiotec.2015.01.017 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.

Bioactive fish collagen/polycaprolactone composite nanofibrous scaffolds

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fabricated by electrospinning for 3D cell culture

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Da Jeong Choia,e, Seung Mi Choia,e, Hae Yeong Kanga,e, Hye-Jin Mina,e, Rira Leea,e, Muhammad Ikrama,e, Fazli Subhana,e, Song Wan Jinb,e, Young Hun Jeongc,e, JongYoung Kwakd,e, Sik Yoona,e,*

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Department of Anatomy, Pusan National University School of Medicine, Yangsan, Gyeongsangnam-do, 626-870, b Department of Mechanical Engineering, Korea Polytechnic University, Siheung, 429-793, c Department of Mechanical Engineering, Kyungpook National University, Daegu, 702-701, d Department of Biochemistry, School of Medicine, Dong-A University, Busan, 602-714, e Pioneer Research Center, Republic of Korea Running title: Fabrication of collagen/PCL nanofibrous scaffolds for 3D cell culture

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* Corresponding author. Tel.: +82 51 510 8044; fax: +82 51 510 8049

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* E-mail addresses: [email protected]

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Highlights

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- We fabricated a novel electrospun fish collagen/PCL composite nanofiber

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scaffold.

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- The diameter of the nanofibers decreased as fish collagen content was increase

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proliferation.

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- The scaffold promoted the cell adhesion, spreading, protrusions, and

- The scaffold stimulated the expression of cell adhesion and thymopoietic molecules.

- The scaffold offers a potential platform for a wide range of 3D cell culture models.

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ABSTRACT:

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One of the most challenging objectives of 3D cell culture is the development of scaffolding

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materials with outstanding biocompatibility and favorable mechanical strength. In this study, we fabricated a novel nanofibrous scaffold composed of fish collagen (FC) and

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polycaprolactone (PCL) blends by using the electrospinning method. Nanofibrous scaffolds were characterized using a scanning electron microscope (SEM), and it was revealed that the

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diameter of nanofibers decreased as FC content was increased in the FC/PCL composite nanofibers. The cytocompatibility of the FC/PCL scaffolds was evaluated by SEM, WST-1

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assay, confocal microscopy, western blot, and RT-PCR. It was found that the scaffolds not

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only facilitated the adhesion, spreading, protrusions, and proliferation of thymic epithelial

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cells (TECs) but also stimulated the expression of genes and proteins involved in cell adhesion and T cell development. Thus, these results suggest that the FC/PCL composite nanofibrous scaffolds will be a useful model of 3D cell culture for TECs, and may have wide applicability in the future for engineering tissues or organs.

Keywords:

Electrospun nanofiber, Composite scaffold, Fish collagen, Polycaprolactone (PCL), Thymic epithelial cells, 3D cell culture

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1. Introduction

Cell culture systems are fundamental and essential tools for a wide range of basic and

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clinical in vitro studies in the research fields of life science, biotechnology, and biomedical science. However, conventional two-dimensional (2D) cell cultures poorly recapitulate the

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conditions in living tissues where cells grow in three-dimensional (3D) environments. Hence,

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a variety of critical biological phenomena such as gene expression, survival, proliferation, adhesion, migration, development, and differentiation of cells have been shown to more

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closely resemble in vivo circumstances in 3D cell culture than in 2D culture (Bonaventure et al., 1994; Li et al., 2008; Chang et al., 2009; Qutub and Popel, 2009; Ayala et al., 2010; Bott

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et al., 2010; Lei et al., 2013). Consequently, data obtained from 2D cell culture systems may be less reliable and significant compared with those from 3D systems. In this regard, 3D cell

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culture is gaining considerable attention recently. Scaffolding plays a pivotal role in

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supporting cells for 3D culture as well as in tissue engineering. Therefore, fabrication of a

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suitable scaffold is one of the major challenges in these research areas. Among the available scaffolds for 3D cell culture, nanofibrous scaffolds were shown to be a prototypical and favorable matrix for 3D cell culture compared with other scaffolds (Carletti et al., 2011; Liu et al., 2012). Electrospinning is a simple, effective, versatile, inexpensive, and scalable technique capable of producing nanofibrous scaffolds from a variety of synthetic or natural polymers with diameters down to the nanoscale (Agarwal et al., 2009; Liu et al., 2012). An ideal scaffold would be one that could provide the cells with an environment that closely resembles their native extracellular matrix (ECM). Thus, electrospun nanofibers are believed to be an efficient scaffold for 3D cell culture and for tissue engineering applications since 4

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they create a connected and porous scaffold mimicking the ECM structurally and mechanically (Ku and Park, 2010). Various kinds of synthetic polymers, such as polycaprolactone (PCL), and natural

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polymers, such as collagen, have widely been used as sources of biomaterials for the fabrication of electrospun nanofibers. Recently, composite electrospun nanofibers of natural

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and synthetic materials have attracted increasing worldwide attention because of their

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advantages over pure synthetic or pure natural polymer nanofibers (Gloria et al., 2010; Ingavle and Leach, 2014). In particular, a key advantage of synthetic polymer scaffolds is

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their superior mechanical properties, while naturally occurring polymers can provide cells with a high degree of biocompatibility and biodegradability. As a result, synthetic polymers

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can be used to reinforce the mechanical properties of matrix materials while natural

(Zhang et al., 2007).

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biopolymers can be used to impart bioactivity to the biologically passive synthetic polymers

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Collagen, the main constituent of the ECM of most tissue types, plays a crucial role in

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maintaining the biologic and structural integrity of the ECM architecture. The interaction between collagen and cells is critically involved in cell adhesion and spreading and consequently plays an important role in determining the pathways of cellular differentiation, growth, and survival (Abraham et al., 2008). On this account, collagens have been used as an attractive biomaterial in a variety of biomedical studies. Fish collagen (FC) is the major form of marine collagen derived from various marine

organisms such as fish, seaweed, or jellyfish. FC is usually extracted from the scale, skin, and bone of fish, and has been widely used in the food and cosmetic industries. It has been demonstrated that the amino acid composition of FC is similar to that of mammalian collagen 5

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(Kimura, 1983; Bae et al., 2008). Numerous attempts have recently been made to use FC as a biomaterial (Yamada et al., 2014). The primary reasons for using FC include its excellent biocompatibility and low immunogenicity (Sugiura et al., 2009; Pati et al., 2012; Yamada et

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al., 2014); no risk in the transmission of bovine spongiform encephalopathy (BSE) and transmissible spongiform encephalopathies (TSEs), which are serious health issues to be

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considered when using animal collagen (Song et al., 2006); low cytotoxic effects; high cell

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viability (Song et al., 2006; Sugiura et al., 2009; Yamada et al., 2014); and high degree of biodegradability (Sugiura et al., 2009). In particular, porous collagen scaffolds were produced

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by freeze-drying and carbodiimide hydrochloride (EDC)/N-hydroxysuccinimide (NHS) cross-linking using marine collagen extracted from jellyfish, and were found to have high

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porosity and an interconnected pore structure (Song et al., 2006). In addition, it was also shown that an effective vascular graft was developed by using a FC hydrogel scaffold (Nagai

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et al., 2008). Although collagen used for biomedical purposes has predominantly been

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obtained from mammalian sources, these results suggest that FC scaffolds have potential as a

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suitable biomaterial, acting as an alternative to conventional mammalian collagen for 3D cell culture and biomedical applications. The thymus serves a vital role in the development of T cells which are critical to the

adaptive immune system. The thymus may undergo involution due to various stimuli including aging, severe stress, ionizing radiation, and cytotoxic agents such as anti-neoplastic agents. Following thymic involution, depletion of T cells in peripheral tissues can occur and thereby host immunity may be suppressed. Thus, it is critical to develop therapeutic strategies for restoring the damaged thymic tissue. Thymus tissue engineering has been highlighted as one of the most promising ways for repairing the involuted thymus or synthesizing a new 6

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thymus. In particular, development of an effective 3D culture technique of thymic epithelial cells (TECs) which play a critical role in T-cell development through their intercellular interactions is important because it is an essential step in the successful generation of tissue-

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engineered thymus.

In the current study, we fabricated novel electrospun FC/PCL nanofibrous composite

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scaffolds with different ratios of FC/PCL in the electrospinning solution. These scaffolds

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were then characterized by analyzing the morphological structure and fiber diameter distribution. We further examined the biocompatibility of these scaffolds using mouse TECs

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by evaluating their morphology, adhesion, spreading, protrusions, proliferation, and

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gene/protein expression.

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2. Materials and Methods

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2.1. Preparation of electrospinning solution

Polycaprolactone (PCL, 80000 g/mol MW, Sigma-Aldrich, St. Louis, MO, USA) was

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dissolved in chloroform (Sigma-Aldrich) at a concentration of 8.8 wt% polymer in solvent.

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Fish collagen (FC) extracted from fish scale (Geltech, Busan, South Korea) was dissolved in deionized water (DW) to prepare a stock solution at a concentration of 50% (w/v).

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Considering the successful generation of a FC-blended PCL-based nanofibrous mat with water content in the electrospinning solution, the emulsions were prepared by mixing well a

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50% (w/v) FC stock solution with an 8.8 wt% PCL solution via stirring at room temperature for 6 h in ratios of 0.4:9.6, 1:9, and 2:8 (v/v). Viscosity measurements of the electrospinning

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solutions were determined using a Brookfield digital viscometer (Model DV-II+ Pro,

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Middleboro, MA). Polymer solutions were taken in the inbuilt stainless steel container of the

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viscometer and the measurements were carried out using S04 spindle at an RPM of 10.

2.2. Electrospinning

The polymer solution for electrospinning was placed in a 1 mL syringe equipped with a

27-gauge needle. The solution was dispersed by a syringe pump at a feeding rate of 0.4 mL/h, a humidity of 45–55%, and a temperature of 21–22°C. An electrospinning voltage of 9 kV DC was applied to the needle using a high voltage power supply (HV Generator, HV60, Nano NC, Seoul, Korea). The distance between the tip of the syringe and the collector was 8 cm. The resultant nanofibrous scaffolds were collected on a stainless steel wire mesh 8

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collector (wire diameter 0.5 mm; inner gap dimensions 1 mm × 1 mm). The nanofiber-coated wire meshes were then placed in a vacuum dryer overnight to remove any remaining solvent. The electrospun nanofibrous scaffolds were cut into 5 mm by 5 mm squares in all samples.

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After the scaffolds were disinfected by soaking in 70% ethanol for 1 h, they were rinsed by sterile water 3 times (each for 5 min) followed by rinsing with sterile PBS 3 times (each for 5

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min).

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2.3. Characterization

The thickness of the scaffolds was measured at three different points using a micrometer

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(Model 103-137, Mitutoyo, Tokyo, Japan) and averaged. For determination of the electrospun nanofiber porosity, the apparent density of nanofiber mats was firstly calculated

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by the measurement of volume and mass of samples as the following equation.

The morphological studies of the nanofibrous scaffolds were examined using a scanning electron microscope (S-4200, Hitachi, Tokyo, Japan) after samples were mounted onto stubs and coated with gold using a sputter-coater for 100 s. 9

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Fourier transform infrared spectroscopy (FT-IR) spectra of the electrospun scaffolds were obtained with an attenuated total reflection (ATR) technique using a Spectrum GX FTIR spectrometer (Perkin–Elmer, Waltham, MA, USA) at a resolution of 4 cm-1 in the range of

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600–4,000 cm-1.

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2.4. Cell line and culture maintenance

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Mouse thymic cortical epithelial reticular cells (CREC, 1308.1), a type of TECs, were kindly provided by Dr. Barbara B. Knowles (The Jackson Laboratory, Bar Harbor, ME,

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USA). The cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM), containing 10% (v/v) fetal bovine serum (FBS), 100 U/mL penicillin, and 100 μg/mL streptomycin (all

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from Gibco/Invitrogen Life Technologies, Grand Island, NY, USA) at 37°C in a 5% CO2

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incubator. Subconfluent cells were harvested with trypsin-EDTA and used for further

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experiments. Fresh medium was replenished every second day.

2.5. Scanning electron microscopy

The morphology of entrapped CRECs was examined by SEM. In brief, the porous

scaffolds with cells were washed with Dulbecco’s phosphate buffered saline (DPBS, pH 7.4). The cells were then fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4). Cellseeded scaffolds were washed with 0.1 M phosphate buffer (pH 7.4). Subsequently, the cells 10

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were fixed with 1% osmic acid, and washed with 0.1 M phosphate buffer (pH 7.4). For freeze-drying, the scaffolds were rinsed twice with PBS and then with DW. They were then quickly frozen in liquid nitrogen and transferred to a freeze-dryer (FDS series, ilShin BioBase,

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Gyeonggi-do, Korea) for drying. All samples were kept in a dry environment before subsequent preparation procedures. Finally, samples were coated with gold using a sputter-

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coater for 100 s before examination using an SEM (S-4200, Hitachi).

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2.6. Cell proliferation assay

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CRECs were seeded on PCL or FC/PCL (ratios of 0.4:9.6, 1:9 and 2:8) nanofibrous scaffolds at 2 × 104 cells per scaffold and incubated for 1, 3, or 5 days. Then, cell viability

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was determined using the colorimetric WST-1-based cell viability/cytotoxicity assay (EZ-

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Cytox assay, Daeil Lab Service, Seoul, Korea). In brief, 10 µL of the EZ-Cytox reagent were

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added to each well and incubated at 37°C under 5% CO2 in a humidified incubator for 1 h. Absorbance of the formazan dye, generated by the reaction of dehydrogenase with WST in viable metabolically active cells, was measured using a microplate reader (Tecan Group Ltd., Männedorf, Switzerland) at 450 nm according to the manufacturer’s instructions.

2.7. Confocal laser scanning microscopy

For confocal microscopic analysis, CRECs were seeded on PCL or FC/PCL (ratios of 11

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0.4:9.6 and 2:8) nanofibrous scaffolds at 1 × 104 cells per scaffold and incubated for 1, 2, 3, or 5 days. The cultured cells were washed with PBS solution and fixed with 4% paraformaldehyde in 0.1 M phosphate buffer for 10 min at 4°C. Subsequently, the fixative

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was removed by washing the coverslips three times for 5 min each with cold PBS followed by permeabilization with 0.1% Triton X-100 in PBS for 5 min. The cells were washed again

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with cold PBS and then incubated with 1% bovine serum albumin (BSA, Sigma-Aldrich) for

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60 min at room temperature. Excess solution was shaken off and the cells were incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)-phalloidin (Promega,

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Madison, WI, USA, diluted 1:150), anti-E-cadherin (BD Biosciences, San Jose, CA, USA, diluted 1:50), anti-vinculin (Sigma-Aldrich, diluted 1:400), anti-ICAM-1 (BioLegend, San

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Diego, CA, USA, diluted 1:50), and anti-cytokeratin-8 (Troma-1, Developmental Studies Hybridoma Bank from The University of Iowa, Ames, IA, USA). Following incubation with

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the primary antibodies, the cells were washed three times for 5 min each with cold PBS.

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Afterwards, the sections were treated with an affinity-purified F(ab′)2-fragment donkey anti-

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rabbit Texas Red-conjugated antibody (Jackson ImmunoResearch Laboratories, diluted 1:100) or goat anti-mouse IgG antibody for 1 h (Santa Cruz Biotechnology, Santa Cruz, CA, USA; diluted 1:100). The cells were then rinsed in cold PBS and mounted on glass slides using Vectashield® containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, USA). Fluorescence of cells was observed using a confocal laser scanning microscope (Olympus, FV1000-IX81, Tokyo, Japan). To comparatively examine the extent of cell infiltration on different scaffold types, fluorescence-stained cell-seeded constructs were mounted and monitored on Olympus FV1000-IX81 laser confocal microscope.

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2.8. RNA extraction and RT-PCR analysis

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Total RNA was isolated from cultured cells on nanofibrous scaffolds with TRIzol reagent (Invitrogen). Poly(A) mRNA was purified from total RNA using the Poly(A)-Tract

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mRNA isolation system (Promega). First-strand cDNA was obtained via reverse transcription

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using 1 µg of total RNA. The reaction was conducted in 20 μL of buffer containing 0.5 µg of oligo(dT)12–18 primer (Promega), 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 40

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mM dithiothreitol, 0.5 mM deoxynucleotide triphosphate mixture (Promega), 10 U RNase inhibitor (Promega), and 200 U Moloney murine leukemia virus (MMLV) reverse

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transcriptase (Promega). After incubation at 37°C for 60 min, the reaction was stopped via heating at 70°C for 5 min. To remove the remaining RNA, we added 1 μL of Escherichia coli

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RNase H (4 mg/mL) to the reaction mixture and incubated the mixture at 37°C for 30 min.

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The cDNA was used as a template for PCR amplification with gene-specific primers. As a

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standard control, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was amplified using the primer pair GAPDH-S (5′-CAA CTC CCT CAA GAT TGT CAG C-3′) and GAPDH-AS (5′-GGG AGT TGC TGT TGA AGT CAC A-3′) derived from mouse GAPDH (GenBank accession no. NM_017008), IL-7-S (5′-GCC TGT CAC ATC ATC TGA GTG CC-3′) and IL7-AS (5′-CAG GAG GCA TCC AGG AAC TTC TG-3′) derived from mouse IL-7 (GenBank accession no. NM_008371), GM-CSF-S (5′-GTC ACC CGG CCT TGG AAG CAT-3′) and GM-CSF-AS (5′-ACA GTC CGT TTC CGG AGT TGG-3′) derived from mouse GM-CSF (GenBank accession no. NM_009969), ICAM-1-S (5′-TGC GTT TTG GAG CTA GCG GA3′) and ICAM-1-AS (5′-CGA GGA CCA TAC AGC ACG TG-3′) derived from mouse 13

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ICAM-1 (GenBank accession no. NM_010493). PCR amplification of the cDNA was performed in an automated thermal cycler (PC 320, Astec, Osaka, Japan) in a final volume of 25 μL containing 4 μL of cDNA solution, 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 1.5 mM

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MgCl2, 0.1% Triton X-100, 0.2 mM deoxynucleotide triphosphate mixture (Promega), 0.5 pmol of each primer, and 5 U Taq DNA polymerase (Promega). After PCR, the amplified

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products were analyzed with electrophoresis in a 2% agarose gel and visualized using

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ethidium bromide staining under ultraviolet light. Band intensities of the PCR products were measured using an image analysis program (MetaMorph, Universal Imaging Corporation,

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Downingtown, PA, USA). Data were expressed as ratios of each mRNA normalized to GAPDH mRNA amplified from the same cDNA sample to correct any error in

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2.9. Western blot analysis

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spectrophotometric RNA quantification or pipetting.

PCL and FC/PCL nanofibrous scaffolds were placed into wells of a 24-well plate after

sterilization of the scaffolds with 70% ethanol. CRECs were seeded onto each scaffold at a density of 3.3 × 105 cells/well and cultured at 37°C under 5% CO2 in a humidified incubator for 48 h. Cells were harvested using lysis buffer in PBS with 1% Triton X-100 and cocktails of protease and phosphatase inhibitors (Roche, Basel, Switzerland) at 4°C. The lysates were centrifuged at 13,000 rpm for 20 min at 4°C. Protein concentrations were measured using a BCA protein assay kit (Sigma-Aldrich). Equal amounts of protein samples were heated for 5 min at 100°C in sample buffer and separated by 10% sodium dodecyl sulfate polyacrylamide 14

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gel electrophoresis, using a Mini-Protean III system (Bio-Rad, Hercules, CA, USA). The proteins were transferred onto a polyvinylidene fluoride membrane (Bio-Rad) via semi-dry transfer (Bio-Rad), and the membrane was incubated overnight at 4°C with anti-IL-7 (sc-

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1268, Santa Cruz Biotechnology), anti-pFAK (sc-166378, Cell Signaling Technology, Danvers, MA, USA), anti-cytokeratin-8 (Troma-1, Developmental Studies Hybridoma Bank

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from The University of Iowa), and anti-β-actin (Abcam, Cambridge, UK) antibodies in Tris-

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buffered saline (TBS, 20 mM Tris-HCl, 150 mM NaCl, pH 7.4) containing 2% BSA. After three washes with TBS-T (TBS containing 0.1% Tween 20), the membrane was incubated for

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1 h at room temperature with secondary antibodies, rabbit anti-goat IgG-HRP (sc-2768, Santa Cruz Biotechnology), donkey anti-rat IgG-HRP (7077, Cell Signaling Technology), and goat

immunoreactivity

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detected

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anti-rabbit IgG-HRP (sc-2004, Santa Cruz Biotechnology). After three washes with TBS-T, with

enhanced

chemiluminescence

(West-Q

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Chemiluminescent Substrate kit, GenDEPOT, Barker, TX, USA) according to the

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manufacturer’s instructions. Images were captured and quantified with a LAS-3000 imaging

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system (Fujifilm, Tokyo, Japan).

2.10. Statistical analysis

The results of the present study are expressed as the mean ± SD under all conditions and

statistically analyzed using a two-tailed Student’s t-test. A value of p < 0.05 was considered to indicate a statistically significant difference.

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Results and Discussion

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3.1. Characterization of electrospinning solution and nanofibers

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In this study, a novel porous nanofibrous composite scaffold composed of FC and PCL as well as a porous nanofibrous PCL scaffold were successfully constructed under optimized

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electrospinning conditions (Fig. 1). Since solution viscosity plays a key element in electrospinning, rheometry was applied to measure the viscosities of different polymer

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solutions. The viscosity of fiber diameters were determined to be 1837 ± 15 cP, 3680 ± 40 cP, 3727 ± 50 cP, and 11123 ± 86 cP for pure PCL, FC/PCL 0.4:9.6, FC/PCL 1:9, and FC/PCL

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2:8, respectively. The viscosity was higher in the FC/PCL solutions compared to pure PCL solutions.

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The amount of spinning solution per scaffold was calculated to be 1.98 ± 0.1 μL for all

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types of nanofibrous scaffolds. The weight, thickness, apparent density, and porosity of the

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same-sized scaffolds are presented in Table 1. All types of the electrospun scaffolds displayed a high degree of porosity between 85 and 89%. Porosity is a critical parameter to be characterized when selecting nanofibrous scaffolds for cell culture or tissue engineering. The porosity of nanofiber scaffolds in the range of 60-90% is generally considered to be suitable for cellular penetration (Chong et al., 2007). The respective loadings of FC and PCL were directly related to the morphology of the

electrospun fiber. As shown in the SEM micrographs in Fig. 1A, all types of nanofibrous scaffolds generated in this study appeared as non-woven fibers with interconnected pores, presenting a highly uniform and smooth morphology without bead. To measure the average 16

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diameter of the nanofibers, a minimum of 100 fibers in the SEM micrograph were randomly selected, and the average value of their measured diameter was considered as the average fiber diameter. The fiber diameters were determined to be 1.57 ± 1.0 μm, 1.06 ± 0.5 μm, 0.99

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± 0.5 μm, and 0.60 ± 0.4 μm for pure PCL, FC/PCL 0.4:9.6, FC/PCL 1:9, and FC/PCL 2:8, respectively (Fig. 1C). The average fiber diameter decreased with the increase in FC

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concentration in FC/PCL blends (Fig. 1C).

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Fig. 1B shows the histogram of fiber diameters for different samples. The fiber diameter distribution of pure PCL and FC/PCL (0.4:9.6, 1:9, and 2:8) scaffolds showed that the

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majority (82%, 89%, 91%, and 86%, respectively) of the electrospun nanofibers in the scaffolds had a diameter ranging from 0.69 μm to 2.59 μm, 0.6 μm to 1.38 μm, 0.52 μm to

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1.21 μm, and 0.26 μm to 0.86 μm, respectively. Our results on fiber diameter and distribution are similar to those reported in previous studies on PCL/collagen, PCL/gelatin or

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PCL/chitosan composite nanofibrous scaffolds (Prabhakaran et al., 2008; Lee et al., 2009;

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Chakrapani et al., 2012; Gautam et al., 2013).

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In the present study, the diameter of the electrospun nanofibers was found to have an inverse relationship with viscosity (i.e., the fiber diameter decreases with increasing solution viscosity) in contrast to a general concept on the relationship of fiber diameter with viscosity that solution viscosity strongly affects fiber diameter, which increases with increasing solution viscosity according to a power law relationship (He et al., 2011). These facts suggest that the fiber diameter in this study might be attributed to some factors which are unknown, rather than the viscosity of the solution. Therefore, further studies are needed to provide new insights into the underlying mechanisms of this phenomenon. To confirm the interaction of PCL with collagen, the electrospun PCL and FC/PCL 17

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nanofibers were subjected to FT-IR analysis. Fig. 2 exhibits the FT-IR spectra (600–4000 cm1

) of PCL and FC/PCL nanofibers. An intense sharp peak at 1721.54 cm-1 due to C=O bonds,

a characteristic structure of the PCL (Hong et al., 2005) was observed in the spectra of both

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PCL nanofibers (Fig. 2a) and FC/PCL nanofibers (Fig. 2b). FT-IR image of FC/PCL nanofibers showed three characteristic absorption bands at 3314.02, 1651.74, and 1555.09

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cm-1, which are the amide A, I and II bands (Jackson et al., 1995). The amide A, I and II

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represent N-H stretch, C=O stretch, and 60% NH stretch combined with 40% C-N stretch in collagen, respectively.

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There was a broad peak appeared at 3314.02 cm-1. The broadening of this peak could be due to the hydrogen bonding interaction of collagen with PCL (Chakrapani et al., 2012).

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Taken these results into consideration, it is clear that the combined electrospun scaffolds contains both FC and PCL.

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Collectively, these results indicate not only that the blended FC/PCL fibers were

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successfully engineered without beads, but also that nanofiber diameters and their

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distributions in electrospun FC/PCL scaffolds could easily be modified by blending of FC with PCL where a greater loading of FC favored smaller diameters and a more homogeneous distribution.

3.2. Cell attachment and spreading

As shown in Fig. 3, the morphology of CRECs attached on PCL and FC/PCL (0.4:9.6 and 2:8) nanofibrous scaffolds and the contacts between CRECs and scaffolds were subjected to 18

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SEM investigation after culture for three days. The CRECs adhered strongly and showed far more spreading on both types of FC/PCL scaffolds compared with those on pure PCL scaffolds. Consequently, each cell covered a relatively larger area on the FC/PCL scaffolds in

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comparison with those on pure PCL scaffolds. CRECs on the FC/PCL scaffolds also displayed a multipolar polygonal shape with multiple long, tapering cytoplasmic processes,

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indicating a functionally active phenotype. In contrast, the cellular protrusions of CRECs on

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the pure PCL scaffolds were poorly developed, revealing that FC promoted the outgrowth of protrusions in CRECs. In particular, CRECs on the FC/PCL scaffolds exhibited an extensive

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connection with each other by their elongated cytoplasmic processes, suggesting enhanced cell-cell interactions. Therefore, the good cell protrusion activity of CRECs could constitute a

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crucial property of the FC/PCL scaffolds required for adequate physiological function of TECs, such as TEC-TEC interaction, TEC-thymocyte interaction, and thymopoietic activity

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since well-developed cytoplasmic processes play key roles in 3D thymic architecture,

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complex organizations of TECs, and T cell development and differentiation in the in vivo

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environments of the thymus. These results demonstrate that FC/PCL composite nanofibrous scaffolds promoted the adhesiveness of CRECs and the expression of their characteristic morphology.

3.3. Cell proliferation

To quantify the proliferation of adherent CRECs on the PCL and FC/PCL (0.4:9.6, 1:9, and 2:8) nanofibrous scaffolds, a WST-based cell viability assay was performed (Fig. 4). 19

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With respect to the first cell proliferation experiment, After one day of incubation postseeding, the cells continuously grew in number, and greater numbers of cells were observed on the FC/PCL (1:9 and 2:8) scaffolds compared with the PCL scaffolds, consistent with

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better cell adhesion and spreading on these substrates. At day 5, the cell number on the FC/PCL (1:9 and 2:8) scaffolds was significantly higher than that on the PCL scaffolds,

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which suggested that the FC/PCL (1:9 and 2:8) surfaces supported the highest increase in the

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number of cells for all proliferation periods, indicating beneficial effects of FC on cell proliferation. However, the FC/PCL 0.4:9.6 scaffold exhibited only a slight increase in cell

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proliferative activity compared with the PCL scaffolds. Taken together, these results suggest that the amounts of FC in the FC/PCL scaffolds likely contributed to the differences seen in

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cell proliferation.

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3.4. Morphological and immunocytological observation by confocal microscopy

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As presented in Fig. 5, confocal micrographs of the stained nuclei and actin cytoskeletons of CRECs cultured on PCL and FC/PCL (0.4:9.6 and 2:8) nanofibrous scaffolds for 1, 3, and 5 days show the cell adhesion morphology and growth pattern of CRECs on each scaffold. After one day of incubation, the cells were well attached to the FC/PCL nanofibrous scaffolds and their actin cytoskeletons were well developed compared with those on the PCL nanofibrous scaffolds (Fig. 5). A trend in increasing cell attachment to scaffolds in the order of PCL < FC/PCL 0.4:9.6 < FC/PCL 2:8 nanofibrous scaffolds was observed (Fig. 5). With increasing time, a gradual increase in the number of cells attached to the scaffolds was found in all types of scaffolds (Fig. 5). Fig. 4 shows a dense cellular mass of CRECs on FC/PCL 20

Page 20 of 50

(0.4:9.6 and 2:8) nanofibrous scaffolds on day 5. Generally, the FC/PCL scaffolds exhibited higher cell proliferation than the PCL nanofibrous scaffolds did. The immunofluorescent images shown in Fig. 5 are in excellent agreement with the WST-1 assay results.

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Confocal z-stack microscopy allowed us to section the samples in the z-direction to observe the penetration of cells into the interiors of scaffolds by optical slicing. As shown in

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the confocal z-stack images of different planes stacked from top to bottom with 2 ㎛ plane

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thickness, and the reconstructed 3D projection image in each scaffold in Fig. 6, cells

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infiltrated well into the all types of scaffolds at 3 days of cell culture. Cells in the PCL and FC/PCL 0.4:9.6 scaffolds were able to penetrate to the interior deeper than the other types of

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the nanofibrous scaffolds but the number of cells attached to the PCL nanofiber in each plane was very less compared with FC/PCL 1:9 and FC/PCL 2:8 scaffolds (Fig. 6), indicating that

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cells in the FC/PCL 1:9 and FC/PCL 2:8 scaffolds proliferate much better than those in the

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PCL nanofibrous scaffolds. Taken together, these results suggest that FC/PCL 1:9 and

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FC/PCL 2:8 scaffolds are more suitable for 3D culture of TECs than the PCL nanofiber.

3.5. Cell adhesion molecule expression

To evaluate the expression of key cell adhesion molecules, including vinculin and E-

cadherin, we seeded CRECs at a density of 2 × 104 cells per scaffold in a 96-well plate. These cells were cultured on PCL and FC/PCL nanofibrous scaffolds for 48 h, and were subjected to confocal laser microscopic analysis. Vinculin, a scaffolding protein in focal adhesion plaques recruited at integrin-associated complexes that contributes to mechanotransduction at cell21

Page 21 of 50

matrix adhesions, is required for strong cell adhesion via the linkage of integrin adhesion molecules to the actin cytoskeleton (Dufour et al., 2013). The expression of vinculin was increased in CRECs cultured on the FC/PCL nanofibrous scaffolds compared with those on

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the PCL scaffolds (Fig. 7A). Since quantifying the expression of vinculin provides evidence related to the effect of biomaterials on the extent of integrin recruitment for focal adhesion

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formation (Hunter et al., 1995), our results suggest that FC/PCL scaffolds facilitated the

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adhesion of CRECs to the scaffold. The expression of E-cadherin, a calcium-dependent cellcell adhesion molecule with pivotal roles in cell adhesion between epithelial cells and

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epithelial cell behavior, was enhanced in CRECs cultured on the FC/PCL nanofibrous scaffolds compared with those on the PCL scaffolds, pointing out that the FC/PCL scaffolds

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aided the adhesion of CRECs to the scaffold (Fig. 7B). The results of immunofluorescent staining proved that the expressions of vinculin and E-cadherin were more predominant on

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the FC/PCL nanofibrous scaffolds compared with the PCL nanofibrous scaffold, indicating

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that the FC/PCL nanofibrous scaffolds provided favorable chemical and biological cues for

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the growth of CRECs. The observed results show that the expression of cell adhesion proteins was higher in the FC/PCL nanofibrous scaffolds than in the PCL nanofibrous scaffolds. Western blot analysis demonstrated that CRECs cultured on the FC/PCL nanofibrous

scaffolds promoted the phosphorylation and activation of focal adhesion kinase (FAK), a protein tyrosine kinase (Fig. 7C). We observed a significant increase in the activation of pFAK (Tyr576/577) in CRECs cultured on the FC/PCL 0.4:9.6 and FC/PCL 2:8 scaffolds than in cells cultured on the PCL scaffolds (Fig. 7C). Since activation of FAK initiates numerous signal transduction pathways that ultimately lead to increased survival and proliferation, our results support and validate the current concept that FC promotes diverse 22

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crucial biological processes, including cell spreading, migration, and survival.

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3.6. Thymopoietic protein expression

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To evaluate whether FC/PCL scaffolds can stimulate the expression of IL-7, a

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thymopoietic protein, and cytokeratin-8, a cytoskeletal protein, in CRECs, western blot analysis was performed to quantify the amounts of IL-7 and cytokeratin-8 present in the

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whole population of cells grown on the PCL and FC/PCL scaffolds (Fig. 8). The expressions of IL-7 and cytokeratin-8 are key indicators of the physiological function of CRECs. CRECs

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cultured for 48 h on both FC/PCL 0.4:9.6 and FC/PCL 2:8 nanofibrous scaffolds induced more enhanced expressions of IL-7 and cytokeratin-8 than those on the PCL scaffolds did,

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with higher IL-7 and cytokeratin-8 expressions on FC/PCL 2:8 compared with FC/PCL

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0.4:9.6 (Fig. 8). Thus, these results indicate that the bioactivity of the FC/PCL scaffolds for

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CRECs was superior to that of scaffolds consisting of only synthetic biomaterials.

3.7. Thymopoietic gene expression

To determine whether FC/PCL nanofibrous scaffolds could modulate the gene expression

of essential thymopoietic molecules, including IL-7, GM-CSF, and ICAM-1, in CRECs after 3D culture, we seeded CRECs at a density of 2 × 104 cells per scaffold in a 96-well plate. These cells were cultured on PCL and FC/PCL nanofibrous scaffolds for 48 h, and were 23

Page 23 of 50

subjected to either RT-PCR or confocal laser microscopic analysis. Thymocytes, which are hematopoietic stem cells, progenitor cells, and precursor cells possessing T cell lineage potential, are present in the thymus. Thymopoiesis, a process in the thymus by which

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thymocytes differentiate into mature T cells, is crucial for the development and maintenance of an adequate and healthy immune system. TECs constitute a major component of the

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thymic microenvironment in which immunocompetent T cells are selected and produced

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principally by TEC-thymocyte interactions. These important processes are regulated by multiple factors including cytokines, hormones, and chemokines. IL-7 is a vital thymopoietic

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cytokine predominantly expressed by TECs. GM-CSF facilitates T cell development by enhancing the expression of IL-6 involved in thymocyte activation (Papiernik et al., 1992).

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ICAM-1 is one of the pivotal molecules involved in thymocyte-TEC adhesion (Marlin and Springer., 1987). As shown in Fig. 9A, the gene expressions of IL-7, GM-CSF, and ICAM-1

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were significantly potentiated in CRECs cultured on the FC/PCL nanofibrous scaffolds

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compared with those on the PCL scaffolds. Consistent with this finding, upregulation of

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ICAM-1 on the surface of CRECs was confirmed by confocal microscopic analysis (Fig. 9B). These results suggest that FC/PCL nanofibrous scaffolds enhanced the adhesion of thymocytes to TECs by upregulating ICAM-1 expression. Taken together, these data suggest that FC/PCL nanofibrous scaffolds possess a robust bioactivity for CRECs. The most favorable advantage of natural polymers is their inherent biocompatibility as

they promote cell properties and behaviors, such as cell attachment, spreading, viability, proliferation, and function. This biocompatibility is induced via polymer binding with cells through specific cell recognition sites (Haslauer et al., 2011). Among natural polymers, such as collagen, silk, and chitosan, which are common biomaterials for electrospinning, collagen 24

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is one of the most frequently used polymers for the fabrication of electrospun nanofibrous scaffolds since it is the major ECM components in most tissue types in the body. The results of the current study are consistent with those of other studies, which have shown that

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scaffolds made of collagen-blended synthetic polymers have biomimetic and bioactive properties. It was demonstrated that composite nanofibrous scaffolds containing collagen

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promoted cell attachment, spreading, viability, proliferation, differentiation, migration, and

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guidance (Venugopal et al., 2006; Schnell et al., 2007; Zhang et al., 2007; Haslauer et al., 2011; Solouk et al., 2011; Gerdon et al., 2012; Laco et al., 2013). Furthermore, in agreement

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with our results, it has been documented that mixed solutions of synthetic polymers and collagen produced finer electrospun fibers such that the diameter of co-electrospun synthetic

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polymer/collagen composite nanofibers gradually decreased with increasing collagen content under fixed electrospinning conditions (Kwon and Matsuda, 2005; Zhang et al., 2007). Taken

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together, our results indicate that the FC/PCL scaffolds have good biocompatibility with

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CRECs and offer a physiologically relevant 3D cellular microenvironment, further suggesting

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their promising potential for 3D culture of TECs and synthesis of artificial thymic tissues and other tissues.

In conclusion, a novel FC/PCL composite nanofibrous scaffold was successfully

fabricated by electrospinning techniques in the present study. Our data on the adhesion and spreading of CRECs to the composite scaffold, the expression of the molecular machineries for cell adhesion, spreading, migration and cytoskeleton, and the cell viability, proliferation and characteristic cell morphologies, indicate the suitability of FC/PCL scaffolds for 3D cell culture and tissue engineering applications. Moreover, the results of western blot and RT-PCR assays for the evaluation of the expression of thymopoietic genes and proteins proved that the 25

Page 25 of 50

properties of the FC/PCL scaffolds were favorable for TECs, with a potential for 3D culture and tissue engineering in the study of T cell differentiation, regeneration, and reconstitution. Thus, these results confirm the biocompatibility and bioactivity of the FC/PCL composite

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nanofibrous scaffolds and provide new insights into their use for various biomedical applications including regenerative medicine.

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In particular, combined with the results of the present study, further studies of related

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technologies underlying the creation of a bioengineered thymus, such as the generation, expansion and differentiation of thymic epithelial stem/progenitor cells, the in vitro culture,

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expansion and differentiation of thymocytes, the coculture of multiple thymic cell types, the modification of thymic epithelial cells to be optimized for supporting T cell development, the

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modelling of 3D thymic architecture, the identification of the characteristic molecular signature of thymic microenvironment, the design and fabrication of scaffolds mimicking the

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in vivo thymic tissue, the induction of angiogenesis in tissue-engineered scaffolds, the

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implantation of 3D-cultured thymic cells, and the development of therapeutic strategies to

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promote thymus regeneration, will contribute to synthesis of bioartificial thymus.

Acknowledgements

This work was supported by the Pioneer Research Center Program through the National Research Foundation of Korea funded by the Ministry of Science, ICT & Future Planning (201210130003).

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biological responses and bioresorption of tilapia scale collagen as a potential biomaterial. J. Biomater. Sci. Polym. Ed. 20, 1353-1368.

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Venugopal, J.R., Zhang, Y., Ramakrishna, S., 2006. In vitro culture of human dermal fibroblasts on electrospun polycaprolactone collagen nanofibrous membrane. Artif.

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Yamada, S., Yamamoto, K., Ikeda, T., Yanagiguchi, K., Hayashi, Y., 2014. Potency of fish collagen as a scaffold for regenerative medicine. Biomed. Res. Int. 2014, 302932.

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Zhang, Y.Z., Su, B., Venugopal, J., Ramakrishna, S., Lim, C.T., 2007. Biomimetic and bioactive nanofibrous scaffolds from electrospun composite nanofibers. Int. J.

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Nanomedicine. 2, 623-638.

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Legends for figures

Fig. 1. Characterization of electrospun FC/PCL nanofibers. (A) Scanning electron

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microscopic characterization of PCL (a), FC/PCL 0.4:9.6 (b), FC/PCL 1:9 (c), and FC/PCL 2:8 (d) nanofibrous scaffolds. (B) Histograms of fiber diameters, obtained from a minimum

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of 100 measurements, are shown for fibers prepared from each material. (C) Bar graph

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represents average fiber diameter of each scaffold type. 3000×. Data are expressed as mean ±

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SD. Statistical difference between groups is indicated (***p < 0.001). Scale bar = 10 μm.

Fig. 2. FT-IR spectra of PCL and FC/PCL nanofibrous scaffold. (a) PCL; (b) FC/PCL

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(0.4:9.6).

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Fig. 3. Scanning electron microscopic images of CRECs grown on PCL (a, b), FC/PCL

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Scale bar = 10 μm.

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0.4:9.6 (c, d), and FC/PCL 1:9 (e, f) electrospun nanofibrous scaffolds for three days. 1500×.

Fig. 4. Cell proliferation assay for CRECs cultured on electrospun PCL and FC/PCL (0.4:9.6, 1:9, and 2:8) scaffolds after 1, 3, and 5 days of growth as assessed by a WST-1-based cell viability assay. Data are expressed as mean ± SD (n = 5). Statistical difference between groups is indicated (**p < 0.01, ***p < 0.001).

Fig. 5. Confocal laser scanning micrographs of CRECs seeded onto the electrospun PCL and FC/PCL (0.4:9.6 and 2:8) nanofibrous scaffolds over varying time points. Cells were stained 33

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with FITC-phalloidin (green) for F-actin cytoskeleton and DAPI for nucleus (blue). Scale bar = 50 μm.

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Fig. 6. Confocal microscope z-stack images of CRECs seeded onto the PCL (A), FC/PCL 0.4:9.6 (B), FC/PCL 1:9 (C), and FC/PCL 2:8 (D) nanofibrous scaffolds after 3 days of

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growth. In each group, a reconstructed 3D projection image (a), a stacked (b) and unstacked

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(c) z-stack images of different planes showing from surface to bottom with 2 ㎛ plane

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thickness are shown. Cells were stained with FITC-phalloidin (green) for F-actin

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cytoskeleton and DAPI for nucleus (blue). Scale bar = 50 μm

Fig. 7. CRECs cultured for 48 h on the FC/PCL (0.4:9.6, 1:9, and 2;8) nanofibrous scaffolds

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display higher expressions of vinculin, E-cadherin, and pFAK. Confocal microscopy for

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staining of (A) vinculin (green), (B) E-cadherin (red), and DAPI for nucleus (blue) in the PCL and FC/PCL 1:9 nanofibrous scaffolds. (C) Western blot and fold graph of densitometric

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quantification of pFAK normalized to β-actin. Scale bar = 30 μm

Fig. 8. CRECs cultured for 48 h on the FC/PCL (0.4:9.6 and 2:8) nanofibrous scaffolds induced enhanced expressions of IL-7 and cytokeratin-8. (A) Western blots and fold graph of densitometric quantification of IL-7 and cytokeratin-8 normalized to β-actin, and (B) Confocal microscopy for staining of cytokeratin-8 (green) and DAPI for nucleus (blue) in the PCL and FC/PCL 0.4:9.6 nanofibrous scaffolds. Total protein isolated from CRECs was used as a positive control (Control). Scale bar = 30 um. . 34

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Fig. 9. CRECs cultured for 48 h on the FC/PCL nanofibrous scaffolds induced the upregulated expression of major thymopoietic molecules (IL-7, GM-CSF, and ICAM-1). (A) RT-PCR analysis for the measurement of mRNA levels of IL-7, GM-CSF, and ICAM-1

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expressed in CRECs grown on the PCL and FC/PCL (0.4:9.6 and 1:9) nanofibrous scaffolds, and fold graph of densitometric quantification of IL-7, GM-CSF, and ICAM-1 normalized to

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GAPDH. (B) Confocal microscopy for staining of ICAM-1 (green) and DAPI (blue) in the

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PCL and FC/PCL 0.4:9.6 nanofibrous scaffolds. Scale bar = 30 μm.

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Table 1 Weight (㎎)

Thickness (㎛)

Apparent density (g/cc)

Porosity (%)

PCL

0.65 ± 0.03

165 ± 26

0.161 ± 0.025

88.9 ± 1.5

FC/PCL 0.4:9.6

0.42 ± 0.08

92 ± 16

0.186 ± 0.029

86.8 ± 1.8

FC/PCL 1 : 9

0.29 ± 0.02

61 ± 6

0.190 ± 0.018

FC/PCL 2 : 8

0.24 ± 0.01

48 ± 6

0.204 ± 0.027

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Nanofibers

86.7 ± 1.1

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d

M

an

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85.1 ± 1.7

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Figure 2

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Figure 3

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Figure 4

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Figure 6A

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Figure 6B

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Figure 9A

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Figure 9B

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