Functionalized silk fibroin film scaffold using β-Carotene for cornea endothelial cell regeneration

Accepted Manuscript Title: Functionalized silk fibroin film scaffold using ␤-Carotene for cornea endothelial cell regeneration Authors: Do Kyung Kim, Bo Ra Sim, Jeong In Kim, Gilson Khang PII: DOI: Reference:

S0927-7765(17)30793-2 https://doi.org/10.1016/j.colsurfb.2017.11.052 COLSUB 9003

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date: Revised date: Accepted date:

15-5-2017 13-11-2017 21-11-2017

Please cite this article as: Do Kyung Kim, Bo Ra Sim, Jeong In Kim, Gilson Khang, Functionalized silk fibroin film scaffold using ␤Carotene for cornea endothelial cell regeneration, Colloids and Surfaces B: Biointerfaces https://doi.org/10.1016/j.colsurfb.2017.11.052 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.

Article

Functionalized silk fibroin film scaffold using β-Carotene for cornea endothelial cell regeneration

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Do Kyung Kim1, †, Bo Ra Sim1, †, Jeong In Kim 2,*,Gilson Khang1,* Department of BIN Fusion Technology, Department of Polymer Nano Science &

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Technology and Polymer BIN Research Center, Chonbuk National University, Deokjin-gu, Jeonju 561-756, Republic of Korea

Department of Bionanosystem Engineering, Graduate School, Chonbuk National

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University, Jeonju 561-756, Republic of Korea

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† Equally-Contributed Authors

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Graphical Abstract

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Corresponding author. Phone: 82-63-270-2848; E-mail: [email protected]

Highlights  

β-C/SF scaffold provided favorable environment on CEnCs. β-C/SF scaffold showed enhanced cell adhesion, proliferation and gene expression. 1

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β-C/SF film scaffold may possess several advantages over previous scaffolds.

Abstract: Design of corneal endothelium substitute is important for replacement of cadaveric cornea tissue. Our previous study has shown the suitability of silk fibroin (SF) as a biomaterial for cornea scaffold. In this study, we used β-Carotene (β-C) to enhance the

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regeneration of corneal endothelial cells (CEnCs) and maintain CEnC specific function. The fabricated film scaffolds showed desired transparency and hydrophilic properties which are crucial factors for vision recovery. The cell viability, phenotype and gene expression was

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examined using MTT assay, immunofluorescence and reverse transcription polymerase chain reactions. Compared with pristine SF scaffold, proper amount of β-C incorporated with SF

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scaffolds showed higher initial cell attachment, cell viability and mRNA expression. The

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results indicate that the fabricated SF film scaffold incorporated with small amount of β-C

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might be the suitable alternative corneal endothelium substitute for transplantation.

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1. Introduction

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Keywords: cornea endothelial cells; beta carotene; silk fibroin; scaffold; tissue engineering Corneal endothelium is inner layer of cornea located under corneal stroma which regulates

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corneal hydration and water transportation in the eyes. CEnCs are monolayer cells maintain

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corneal transparency by its metabolic activity and ATPase pump function [1,2]. The ideal CEC density for human is about 2500 - 3000 cells/mm2. When human CEnC density reaches under minimum level (500 cells/mm2) by damage or injury, CEnC losses its function and

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cornea edema starts to occur due to its limited self-regeneration capacity and inability of cell division in vivo. The physical damage, surgical intervention, trauma and severe injury cause permanent cell loss by arresting of G1 phase cycle [1,3,4]. In this case, penetrating keratoplasty (PK) is the most common surgical procedure with whole cornea incision [1]. 2

However, PK takes long recovery time and unstabilizes eyes after surgery. PK needs fresh cadaveric tissue meets standard without graft rejection. To meet a desired cornea tissue for patients, it takes long waiting time until it reaches patient’s location [2]. In this reasons, Descemet’s stripping and endothelial keratoplasty (DESK) is replacing PK with its rapid

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recovery time, fast rehabilitation and reduced inflammatory response with small incision around cornea limbus area [5]. Descemet’s membrane attached on corneal stroma is

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physically stripped off with damaged CEnCs and replace with donated cornea underlying confluent healthy CEnCs [1,2]. While the advantage of DSEK is proved, a shortage of donated cadaveric with high quality CEnCs for transplantation is a challenge. As an

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alternative, bioengineered scaffold is required for transplantation in anterior chamber of the

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eyes. The bioengineered cornea should be transparent and clear as normal human cornea.

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Moreover, artificial cornea should possess proper biodegradability, good biocompatibility

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and permeability of water and essential nutrients for cornea endothelium. The suitable mechanical properties for ease handling is important as human DM underlying cornea

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endothelium makes difficult for direct transplantation during DSEK surgery.

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The selection of proper biomaterials for CEnC scaffold construction is crucial for artificial cornea to support cell adhesion, proliferation and migration. We selected silk SF isolated silk

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worm cocoons from bombyx mori which has been used as a suture for decades due to its great mechanical strength and biocompatibility [2,4,6]. The SF based film scaffolds offer

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controllable biodegradability, transparency and non-cytotoxicity [7–9]. SF is a natural organic polymer consisting of two main proteins. The outer layers covering SF are sericin and the inner part of SF is fibroin which had been reported to prevent immune rejection [2,10]. The fibroin based scaffolds promotes cell attachment and proliferation by mimicking the extracellular matrix (ECM) [10,11]. Since SF-based scaffolds exhibited huge potential in 3

tissue engineering applications, and β-C is an acknowledged bioactive component for cornea. We were interested in investigating SF and β-C together for CEnC regeneration. β-C is one of nature derived chemicals also known as carotenes or carotenoids. β-C is a precursor of vitamin A found in orange color vegetables such as pumpkins, carrots and sweet potatoes

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[12]. β-C plays crucial role in human health maintenance and increase metabolism. The antioxidant ability and immune system regulation of β-C protects patients suffering from

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cancer, eye disease and heart disease [13]. β-C has been reported to enhance the cell

proliferation and differentiation [14]. As β-C can be easily found in non-toxic nature such as vegetables and fruits, its abundance will support mass production which could not only

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reduce the cost but also the toxic waste. The biocompatible β-C could enhance CEnC

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proliferation in the anterior chamber of the eyes and beneficial for maintaining CEnC

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function.

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The ultimate goal of this research is to fabricate transplantable SF based film scaffold

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functionalized using β-C to enhance CEnC proliferation and regulate cell phenotype. The β-C film scaffolds were analyzed for various mechanical properties such as FTIR, contact angle,

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transparency and FESEM. For biological evaluation, initial attachment, MTT assay, RT-PCR, FESEM and immunofluorescence were conducted to examine biocompatibility and suitability

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of β-C scaffolds for CEnC carrier. 2. Materials and Methods

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2.1.Preparation of SF solution To prepare aqueous SF solution, we used silkworm cocoons from bombyx mori cut into small pieces. The small pieces of silkworm cocoons were boiled in 0.02 M Na2CO3 (Showa Chemical, Japan) dissolved in distilled water for 30 min to remove sericin. After boiling, 4

degummed silkworm cocoons were washed 3 times with distilled water and dried under the fume hood at room temperature. Fully dried cocoons were dissolved in 9.3 M LiBr (Kanto chemical, Japan) in the 60℃ oven for 4 h. Dissolved silk solution was dialyzed using dialysis tube (Snake SkinR○ Dialysis Tubing 3,500 MWCO (molecular weight cut-off), Thermo

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SCIENCE, USA) in the distilled water for 72 h. Silk aqueous solution was kept at 4℃ before use.

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2.2.Fabrication of β-C/SF film scaffold

Fresh SF aqueous solution (2 ml) and various concentration of β-C (1, 10, 100, 500 mM)

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prepared in 2 μl DMSO were incorporated successfully and poured into a glass dish. The

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final concentration of β-C/SF film scaffolds were 1, 10, 100 and 500 μM including pristine

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SF scaffold. The solution was fully dried under the clean bench to avoid contamination. Dried

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times with distilled water.

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film scaffolds were crosslinked with Methanol for 30 mins at room temperature and washed 3

2.3.Field Emission Scanning Electron microscopy (FESEM)

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The surface characterization of β-C/SF scaffolds were evaluated using field emission scanning electron microscopy (SN-SUPRA 40VP, Carl Zeiss, Germany). On the each β-C/SF

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scaffold, rabbit cornea endothelial cells (rCEnCs) were seeded on the substrate (1.9 x104 cells/scaffold) for cell morphology analysis. After 5 days of cell culture when rCEnCs

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reached confluent on the scaffold, cells were washed with phosphate buffered saline (PBS) and fixed with 2.5% glutaraldehyde (Sigma-Aldrich, USA) at 4℃ overnight. The cell cultured β-C/SF scaffolds were dehydrated with graded series of EtOH in distilled water (60, 70, 80, 90, and 100 %) for 20 min each and dried under the fume good at room temperature. Fully dried samples were observed by FESEM. 5

2.4.Transparency Transparency of fabricated β-C/SF scaffolds were analyzed by SYNERGY Mx spectrophotometer (BioTek, USA) at the wavelength range of 380 nm to 780 nm after immersed in phosphate buffer saline (PBS). Transparency of cell cultured scaffolds were

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measured after 5 days of cell culture when CEnCs reach confluency on the substrate. Cultured medium was replaced with fresh PBS and examined.

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2.5.Fourier Transform Infrared (FTIR) spectroscopy

The structural investigation of β-C/SF scaffolds and bare β-C powder were carried out using

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FTIR (Perkin Elmer, USA) at the spectra wavelength range of 4000 cm-1 to 400 cm-1.

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2.6.Contact angle

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The hydrophilicity and hydrophobicity of different concentration of β-C/SF scaffolds were

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evaluated by measuring the contact angle of a water droplet on the film scaffolds. Contact

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angle was obtained using water contact goniometer (Phoenix10, S.E.O., South Korea) from 0 min (initial time) to 5 min.

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2.7.Isolation of rCEC and culture

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To isolate rabbit corneal endothelial cells (rCEnCs) from rabbit cornea, whole eyes were extracted from 3 weeks old New Zealand white rabbits. All experimental procedure for cell culture was performed under the clean bench. The eye balls were moved to PBS stratightly

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and washed 3 times with PBS. The surrounding tissues around the eye ball were removed and cornea was cut from the eye ball. The DM carrying rCEnCs were stripped off from the corneal stroma under dissecting microscope. The rCEnCs underlying DM were digested using 0.2 % collagenase A (Roche, Germany) for 40 min in the 37 ℃ humidified 5% CO2 6

incubator. Digest solution was centrifuged at 1500 rpm for 15 min. The CEnC pellet was suspended on cell culture dish (corning, USA) with endothelial growth medium-2 (EGM-2, Lonza, USA) containing epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), insulin-like growth factor (IGF), hydrocortisone,

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gentamicin, amphotericin-B, 10 % fetal bovine serum (FBS, Biotechnics, USA) and 1% penicillin (Gibco, USA). The cultured medium was changed every 2 days. The passage 1 of

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primary rCEnCs was used for experiment. 2.8.Cell viability

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The rCEnC viability on the β-C/SF scaffolds was evaluated using MTT (3-[4,-

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dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide;thiazolyl blue) assay at 1day, 3days and 5 days. The 1.9 x104 rCEnCs were seeded on each scaffold for MTT assay. The cultured

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medium was replaced with 1 ml of fresh medium and 100 μl MTT solution (5 mg/ml in PBS) was added to each sample. For formazan crystal formation, cell cultured scaffolds were

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incubated for 4 h at 37 ℃, humidified 5% CO2 incubator. After reaction, solution was removed and 1 ml of dimethyl sulfoxide (DMSO) was added for dissolution of formazan

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(n=3)

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crystal. The dissolved solution was read at 570 nm using microplate reader (Biotek, USA).

2.9.Initial attachment

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The rCEnCs (1000 cells/mm2) were cultured on each scaffold and Tissue culture polystyrene (TCP) to evaluate initial attachment in endothelial basal medium (EBM, Lonza, USA) without serum for 30 min. After 30 min, medium was replaced with new fresh medium and fixed with Methanol at 4 ℃ for overnight. After fixation, samples were washed with PBS and

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stained with DAPI (Santa Cruz, USA). Images were observed under fluorescence microscopy (Nikon, Japan) and counted using ImageJ program. (n=4) 2.10.

mRNA expression

The expression of total RNA extracted from cultured rCEnCs (1.9 x104 cells/scaffold) on β-

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C/SF scaffolds was determined by reverse transcription polymerase chain (RT-PCR). The

total RNA was extracted using TRIzol (Takara, Japan) following manufacturer’s instruction.

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The extracted samples were quantified using BioSpectrometer (Eppendorf, Germany) and amplifed. The CEnC related genes, Na+/K+-ATPase (NaK), Aquaporin-1 (AQ1), chloride channel protein 3 (CL3), sodium/bicarbonate co-transporter (N1) and voltage-dependent

Immunohistological Analysis

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2.11.

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anion channel 2 (V2) were evaluated and normalized using housekeeping gene, β-actin.

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The Na+/K+-ATPase expression of rCEnCs cultured on β-C/SF scaffolds was evaluated after

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5 days of cell culture. The samples were fixed with 4 % paraformaldehyde (Sigma-Aldrich, USA) at 4 ℃ for overnight and washed with PBS. Briefly, protein blocking solution (DAKO,

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USA) was added for 15 min in the dark to prevent non-specific binding. As primary antibody, anti -Na+/K+-ATPase (1:200, Santa Cruz, USA) was treated at 4 ℃ for overnight. As

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secondary antibody, Alexa FluorR○ 594-conjugated AffiniPure Donkey Anti-Rabbit IgG (1:300, Jackson Immuno Research Laboratories, Inc., USA) was added for 30 min at room

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temperature in the dark. After detection, immunohistological images were captured using confocal laser scanning microscope (LSM 510 META, Carl Zeiss, Germany). 2.12.

Statistical analysis

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All measurements were presented as mean ± standard deviation and analyzed with one-way analysis of variance (one-way ANOVA test). The differences were considered significant at P<0.05(*). 3. Results and Discussion

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3.1.Transparency

The optical intensity of clear human acellular corneal stroma has 0.1 – 0.13 at the wavelength

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of 380 nm to 780 nm and higher than 0.1 only at “far red” (>700 nm) [1,2]. The transparency of scaffold for cornea substitute is important for accurate clear vision and cell monitoring

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after surgery [4,15]. Transparency of bare β-C/SF scaffolds and scaffolds with confluent

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monolayer rCEnCs were measured by spectrophotometer at the wavelength range of 380 nm

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to 780 nm. Overall, pristine SF film scaffold showed highest transparency among the

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experimental groups. The transparency of both bare scaffolds and cell cultured scaffolds were in order of SF < 1 μM β-C/SF < 10 μM β-C/SF < 100 μM β-C/SF < 500 μM β-C/SF. (Fig. 1C

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and 1D) However, there was no significant difference between SF, 1 μM β-C/SF and 10 μM β-C/SF. Compared with transparency of human cornea, transparency of β-C/SF film scaffolds

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showed proper value for cornea endothelium scaffold.

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3.2.Fourier Transform Infrared (FTIR) spectroscopy To analyze the components in β-C/SF scaffolds and structure confirmation of SF and β-C,

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FTIR spectroscopy was conducted. Figure 1B shows the infrared spectrum of β-C powder showing specific peaks at 2915 cm-1, 1363 cm-1, 965 cm-1 and broad peak around 3406 cm-1. The crystallization of SF was shown in Figure 1A at 1450 - 1750 cm-1. The SF based film scaffolds presented absorption band at 1620 cm-1 (amide I, C=O bond), 1514 cm-1 (amide II, N=H stretching) and 1229 cm-1 (amide III). As the concentration of β-C present in SF was 9

increased, the absorption band showed deeper depth due to the hydrogen bond between β-C and SF which refers to successful incorporation during scaffold fabrication process. It has been reported that hydrogen bond of macromolecules is stronger than polymer hydrogen bond of polymers.

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3.3.Hydrophilicity Hydrophilicity of scaffolds for cornea regeneration not only influences cell proliferation and

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migration but also initial attachment of cells on the substrate [4]. The hydrophilic scaffolds preserve moist which is related to maintaining body fluid and preventing loss of essential

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nutrients in vivo [16]. Moreover, hydrophilic scaffolds which maintain the moisture, not only

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stimulate cell migration but also protect from bacterial infections [17].

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The contact angle of a water droplet on β-C/SF scaffold was measured for 5 mins. The contact angle of a droplet on the β-C/SF scaffold showed in order of 500 μM β-C/SF < 100

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μM β-C/SF < 10 μM β-C/SF < 1 μM β-C/SF < SF at all-time points (Figure 2A and 2B). As

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the concentration of β-C incorporated in SF increased. This may due to the interaction between SF and β-C and hydrophilic carotenoid of β-C [18]. The difference between the

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contact angle of water droplet on the SF and β-C incorporated with SF scaffolds was not significant at the initial time-point. (0 min) However, the difference between the contact angle of SF and β-C/SF scaffold showed bigger difference after 5 mins. The contact angle

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result of β-C/SF film scaffolds showed the suitability of the fabricated scaffolds for cornea substitute. Thus, hydrophilic β-C/SF scaffolds can be suggested as a CEnC carrier for transplantation by supporting tissue integration in the anterior chamber of the eyes. 3.4.Surface roughness and rCEnC morphology 10

Surface properties of film scaffolds fabricated for CEnC regeneration influences cell to cell interaction and proliferation [19]. The surface roughness of β-C/SF scaffolds and morphology of rCEnCs cultured on the scaffolds were evaluated by FESEM. As shown in Figure 3, the roughness of the scaffolds showed no significant difference. However, the morphology of

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rCEnCs cultured on different concentration of β-C incorporated in SF scaffolds showed different appearance. The rCEnCs were monitored after 5 days of cell culture when the

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monolayer rCEnCs reached confluence on the scaffolds. Compared with rCEnC morphology cultured on pristine SF and other experimental groups, rCEnC morphology on 100 μM βC/SF scaffold showed polygonal shape and higher ECM secretion. The rCEnC morphology

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on 500 μM β-C/SF scaffold showed long directional fibroblastic morphology. The specific

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polygonal shape of CEnC morphology interacting with ECM controls the hydration of

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corneal stroma and maintains the cornea transparency in the anterior chamber of eyeball [15].

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The ECM cell binding components support and maintain rCEnC to form the monolayer [20]. FESEM result demonstrates that 500 μM β-C/SF scaffold facilitates rCEnC proliferation and

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migration with polygonal morphology secreted by uniform ECM.

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3.5.Cell viability and initial attachment

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The initial attachment of rCEnCs was analyzed after cell culture (1000 cells/mm2) for 30 mins. High Initial attachment rate of CEnCs on the film scaffold for cornea regeneration influences the cell proliferation and differentiation. Moreover, high initial attachment rate

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affects the time span for the cells to reach confluence rapidly and affinity of specific substrate towards the cultured cells [21–23]. The rapid time span of the cells to be confluent on the substrate is beneficial for preparation of donor cornea for transplantation and influence adaptability after surgery. The initial cell attachment of β-C/SF scaffolds (1 μM β-C/SF: 786 ± 20 cells/mm2, 10 μM β-C/SF: 820 ± 39 cells/mm2, 100 μM β-C/SF: 857 ± 53 cells/mm2, 11

500 μM β-C/SF: 827 ± 49 cells/mm2) was higher than pure SF scaffold (690 ± 41 cells/mm2) (Figure 4B). To evaluate rCEnC viability and proliferation on β-C/SF scaffolds, MTT assay was conducted at 1 day, 3 days and 5 days of cell culture (Figure 4A). CEnC viability on the scaffold for transplantation is important for vision recovery. The desired minimum density of

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CEnC to avoid cornea edema and maintain function is about 500 cells/mm2 [3]. The damaged CEnC and decreased CEnC number only can be recovered by existing cells. The CEnCs of

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patients in adulthood are lack of self-repair ability and proliferation [2]. Until 3 days of cell

culture, there was no significant difference between the experimental groups. However, after 5 days of cell culture, β-C/SF scaffold showed higher cell proliferation rate than SF scaffold.

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Especially, proliferation rate of 500μM β-C/SF showed highest rate among the experimental

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groups. These results imply that β-C/SF scaffolds with proper amount of β-C provide desired

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3.6.mRNA expression of rCEnCs

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environment for rCEnC initial attachment and support cell proliferation.

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The gene expression of rCEnCs cultured on β-C/SF scaffolds was studied with following genes: NaK, AQ1, CL3, N1 and V3 (Figure 5). The expression of NaK is related to the

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maintenance of cornea transparency and hydration by regulation of NaK pump function and

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cornea edema [24]. AQ1 expresses fluid transportation in cornea which enhances penetrability of water molecule [25]. CL3 plays important role in physiological phenomenon such as pH controlling, organic molecule transportation, differentiation, migration and

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proliferation of cell. Moreover, CL3 maintains specific morphology shape and size of cell. N1 has been reported to increase permeability of basolateral or apical HCO(3)(-) flux in bovine CEnC [26]. V3 regulates proteins and molecules in cell-cell interaction [27]. All genes normalized by β-actin were expressed well. Interestingly, 100μM β-C/SF showed the highest rate in all gene expressions among the other experimental groups. However, the 12

expression of 500μM β-C/SF showed the lowest level on NaK, AQ1, CL3 and N1. This may due to the hypersensitivity of β-C affected the expression of rCEnC specific genes. Compared with pristine SF, gene expression of rCEnCs cultured on SF film scaffolds incorporated with proper amount of β-C (100μM) showed the most desired scaffold environment for rCEnCs

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among the experimental groups. 3.7.Histological evaluation

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The immunofluorescence staining of Na+K+-ATPase shows the cell morphology and specific gene expression. The expression of Na+K+-ATPase is related to cornea transparency by

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purifying water in the cornea. The CEnCs actively transport water molecules from the corneal

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stroma by sodium potassium pump. The transportation and balance of water molecule and essential nutrient in cornea is important for clear vision [24,28,29]. Compared with pristine

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SF scaffold, β-C/SF scaffolds showed more tight cell junctions and uniform cell size. As

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shown in Figure 6, morphology of rCEnCs cultured on SF and 500μM β-C/SF scaffold

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showed fibrous morphology having directions. Notably, morphology of rCEnC cultured on 100μM β-C/SF scaffold showed polygonal shape, high staining intensity, uniform cell

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distribution and tight cell junction. The polygonal shape of rCEnC, which is the characteristic shape of CEnC has been reported to regulate the cornea hydration thorugh Na+K+-ATPase

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pump [2,15]. In this regard, the proper amount of β-C enhances the proliferation and function maintenance of rCEnC. Especially, immunofluorescence evaluation demonstrates 100μM β-

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C/SF scaffold as a promising CEnC carrier for implantation. 4. Conclusion In conclusion, β-C/SF scaffold was fabricated well as an obtainable cell carrier with CEnC favorable environment. Overall, mechanical properties of β-C/SF scaffold like hydrophilicity 13

and transparency results were acceptable for implantable scaffold into the cornea anterior chamber of the eyes. Compared with pristine SF scaffold, film scaffold incorporated with proper amount of β-C showed enhanced initial cell adhesion, proliferation, desired cell morphology and gene expression. Exclusively, β-C existed in SF film scaffold enhanced the

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ATPase pump function of rCEnCs which is the most important function to maintain corneal hydration. The physical evaluation and biological evaluation of β-C/SF scaffold indicate that

scaffolds for delivery of CEnCs to replace diseased CEnCs. Acknowledgements

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bioengineered β-C/SF film scaffold may possess several advantages over other previous

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This research was supported by Basic Science Research Program through the National

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Planning (NRF-2017R1A2B3010270).

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Research Foundation of Korea(NRF) funded by the Ministry of Science, ICT & Future

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Figures

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Figure 1. FTIR result of β-C/SF scaffolds. (A) FTIR of bare β-C powder. (B) Transparency

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of bare SF scaffold and different concentration of β-C/SF scaffolds at wavelength 380 nm –

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780 nm without cell culture (C) with confluent monolayer rCEnC on scaffolds. (D)

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Figure 2. Contact angle of β-C/SF scaffolds analyzed for 5 mins. (A) Contact angle images

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of a water droplet on β-C/SF scaffolds (n=5). (B)

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Figure 3. FESEM images of β-C/SF scaffold surface and rCEC morphology cultured on β-

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C/SF scaffolds.

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Figure 4. Cell viability and proliferation using MTT assay. rCEnCs were cultured for 1, 3

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and 5 days in EGM-2 β-C/SF scaffolds. (A) Initial attachment of rCEnCs on β-C/SF

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scaffolds. (B) Images of DAPI staining for initial attachment evaluation. (C) (n=3, P<0.05(*))

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Figure 5. Specific gene expressions of rCEnC cultured on different β-C/SF scaffolds by RT-

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PCR, normalized by β-actin. (n=3, P<0.05(*))

Figure 6. Immunofluorescence staining images of Na+/K+-ATPase expression on rCEnCs

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cultured on β-C/SF scaffolds.

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