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Biomaterials 34 (2013) 121e129

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Biomaterials

journal homepage: www.elsevier .com/locate/biomater ia ls

The regulation of epithelial cell proliferation and growth by IL-1 receptorantagonist

Makoto Kondo a,b, Masayuki Yamato b,*, Ryo Takagi b, Hideo Namiki a, Teruo Okano b

aGraduate School of Science and Engineering, Waseda University, 2-2 Wakamatsu-cho, Shinjuku-ku, Tokyo 162-8480, Japanb Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, TWIns, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan

a r t i c l e i n f o

Article history:Received 17 July 2012Accepted 17 September 2012Available online 8 October 2012

Keywords:Cell proliferationEpithelial cellStem cellGrowth factorsInterleukinGene expression

* Corresponding author. Tel.: þ81 3 5367 9945x621E-mail addresses: afujiyama@abmes.twmu.ac.jp,

(M. Yamato).

0142-9612/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.biomaterials.2012.09.036

a b s t r a c t

We have performed clinical translation of epithelial cell sheets fabricated on temperature-responsiveculture surfaces to treat cornea and esophagus. In the preclinical study using animal models, wefound epithelial cell growth potential varied among species. Canine epithelial cell growth was prom-inent, while rat one was poor under 3T3 feeder layer-free condition. The aim of the present study was toidentify growth-promoting factors for epithelial cells. Conditioned medium of canine cell culture har-vested at different time points showed different growth promotive activity for rat epithelial cells. Time-dependent gene expression was quantitatively evaluated for forty growth factors, and compared withconditioned medium results. Statistically significant promotive activity was observed with IL-1RA, andsignificant inhibitory activity was observed with IL-1a. Furthermore, neutralizing anti-IL-1a antibodyalso showed significant promotive activity. Human epidermal keratinocytes were promoted to proliferateby IL-1RA and neutralizing anti-IL-1a antibody, and showed well differentiation to form transplantable,squamous stratified epithelial cell sheets. These findings would be useful to fabricate reproducible,transplantable epithelial cell sheets for regenerative medicine.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

We have investigated tissue regeneration by transplantation ofcell sheets fabricated on temperature-responsive cell culturesurfaces, on which a temperature-responsive polymer, poly(N-iso-propylacrylamide), is covalently immobilized [1]. Various cell typesadhere, spread, and proliferate on the surfaces at 37 �C, and con-fluently cultured cells are harvested as a contiguous single cellsheet by reducing temperature to 20 �C without any need forproteolytic enzyme like trypsin or dispase. Therefore, harvestedcells intactly retain all the membrane proteins including cadherin,growth factor receptors, and ion channels as well as extracellularmatrices (ECM) deposited during culture underneath cell sheets[2]. This non-invasive cell sheet harvest results in quick and idealintegration to transplanted tissue sites. The clinical applications ofcell sheet transplantation have been successfully performed to treatskin [3], cornea [4], heart [5], esophagus [6], and periodontal tissue[7] for human patients.

Patients’ oral mucosal epithelial cells have been used as a cellsource to fabricate transplantable cell sheets in the cases to treat

1; fax: þ81 3 3359 6046.myamato@abmes.twmu.ac.jp

All rights reserved.

cornea [4] and esophagus [6]. Usually, murine fibroblastic 3T3feeder cells derived from mouse embryo and fetal bovine serum(FBS) are used to promote epithelial cell proliferation in vitro [8].But, we have eliminated these xenogeneic materials from theculture by using patient’s own serum and culture inserts havingmicro-porous membrane, which supplies culture medium from thecell bottom [9,10] to avoid possibility of xenogeneic infection andcontamination. Before, the clinical translation to human patients,we performed the preclinical studies [11e13] using several kinds ofexperimental animals, and we interestingly found different prolif-erative capabilities and supplemental requirement of epithelialcells among the species including human (to be submitted). In thepresent study, we compared canine and rat cell proliferation, andinvestigated which cytokine has an essential role to controlepithelial cell growth in an autocrine manner. The obtained resultswould be useful to reproducibly fabricate transplantable stratifiedsquamous epithelial cell sheets in the clinical settings for regen-erative medicine.

2. Materials and methods

2.1. Fabrication of epithelial cell sheets

All the experimental protocols were approved by the Institutional Animal Careand Use Committee of Tokyo Women’s Medical University. Human epidermal

M. Kondo et al. / Biomaterials 34 (2013) 121e129122

keratinocytes were purchased from Kohjin Bio (Saitama, Japan). Oral mucosal tissuewas excised from the buccal cavities of rats (Lewis, male, 8 weeks old) and canines(beagle, male, 12 months old), disinfected with povidone-iodine (Meiji SeikaPharma, Tokyo, Japan), and washed with Dulbecco’s Modified Eagle Medium(DMEM) (SigmaeAldrich, St Louis, MO) containing 100 IU/mL penicillin and 100 mg/mL streptomycin (Life Technologies, Carlsbad, CA). Epithelial tissuewas peeled off byforceps after incubationwith 1000 PU dispase (Godo-shusei, Tokyo, Japan) at 4 �C for15 h. Peeled epithelium was torn with forceps, and dissociated with 1.25% trypsin-0.5% ethylenediaminetetraacetate in Dulbecco’s phosphate buffer saline (PBS)(SigmaeAldrich) at 37 �C for 15 min. Dissociated cell suspension was filtratedthrough a 40-mm cell strainer (BD Biosciences, Franklin Lakes, NJ). Keratinocyteculture medium (KCM) was prepared by mixing of DMEM and Ham’s F-12 (SigmaeAldrich) at the ratio of 3 to 1, supplemented with 5% FBS (Moregate BioTech,Queensland, Australia), 2 nM triiodothyronine (Wako Pure Chemicals, Osaka, Japan),10 ng/mL recombinant human epidermal growth factor (Protein Express, Chiba,Japan), and pharmaceutical drugs of 5 mg/mL insulin (Humalin: Eli Lilly, Indianapolis,IN), 0.4 mg/mL hydrocortisone (Saxizon: Kowa Pharmaceutical, Tokyo, Japan),0.25mg/mL amphotericin B (Fungizone: Bristol-Myers Squibb, Park Avenue, NY) and40 mg/mL gentamicin (Gentacin: Schering-Plough, Kenilworth, NJ). Cholera toxin(List Biological Labs, Campbell, CA) was eliminated from KCM for canine and ratepithelial cell culture, but added at a concentration of 1 nM for human epidermalkeratinocyte culture. Suspension of primary oral mucosal epithelial cells wereseeded on temperature-responsive cell culture inserts (23 mm in diameter, UpCellInsert: CellSeed, Tokyo, Japan) at a density of 4.0e8.0�104 cells/cm2, and cultured inan atmosphere of 5% CO2 at 37 �C for 11e12 days. Proliferating cells were observedwith a phase contrast microscope (ECLIPSE TE2000-U: Nikon, Tokyo, Japan). Strat-ified squamous epithelial cell sheets were harvested by low temperature treatmentat 20 �C for 30 min, and subjected to histological analyses. No 3T3 feeder layer wasused throughout the experiments.

2.2. Gene expression analyses

Expression of mRNA was quantified by realtime quantitative RT-PCR with 7300Real TimePCRSystem(Life Technologies). Culturedcellswere lysedwithQIAshredder(Qiagen, Hilden, Germany). Total RNA was isolated with RNeasy Plus Mini Kit

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Fig. 1. Culture of canine and rat oral mucosal epithelial cells. The cells were seeded at a densin the upper and lower rows represent canine and rat oral mucosal epithelial cells observed acultured oral mucosal epithelial cells of canine and rat (n ¼ 3). Canine oral mucosal epitheliaindicates 10 mm. The section of the cell sheet was fixed and stained with hematoxylin andscale bar indicates 50 mm.

(Qiagen), and subjected to the synthesis of single stranded cDNA with reverse tran-scription reaction with PrimeScript RT reagent Kit (Takara Bio, Shiga, Japan) withiCycler Thermal Cycler (Bio-Rad Laboratories, Hercules, CA). For canine geneexpression analysis, primer pairs and TaqMan MGB probes were designed for b2microglobulin (B2M), brain-derived neurotrophic factor (Bdnf), epidermal growthfactor (Egf), fibroblast growth factor 1 (Fgf1), Fgf2, Fgf4, Fgf5, Fgf6, Fgf7, Fgf8, Fgf9,Fgf10, Fgf12, Fgf13, Fgf14, Fgf16, Fgf18, Fgf19, glial cell line derivedneurotrophic factor(Gdnf), hepatoma-derivedgrowth factor (Hdgf), hepatocyte growth factor (Hgf), c-fosinduced growth factor (Figf), growth factor augmenter of liver regeneration (Gfer),heparin-binding EGF-like growth factor (Hbegf), insulin-like growth factor 1 (Igf1),Igf2, interleukin-1a (Il1a), interleukin-1 receptor antagonist (Il1ra), nerve growthfactor (Ngf), neuregulin 1 (Nrg1), Nrg2, neurotrophin 3 (Ntf3), Ntf4, platelet-derivedgrowth factor alpha polypeptide (Pdgfa), Pdgfb, placental growth factor (Pgf), pleio-trophin (Ptn), transforming growth factor alpha (Tgfa), Tgfb1, Tgfb2, vascular endo-thelial growth factor A (Vegfa), and vascular endothelial growth factor C (Vegfc) forTaqMan Gene Expression Assays� (Life Technologies). Quantitative PCR was per-formed with Premix Ex Taq (Takara Bio) and TaqMan Gene Expression Assays (LifeTechnologies). For human keratinocyte gene expression assay, primer pairs andTaqMan MGB probes were designed for b2 microglobulin (B2M), Ki67, delta N p63(dNp63), cytokeratin 15 (CK15), integrin beta 1 (ITGb1), integrin alpha 6 (ITGa6),laminin beta 3 (LAMb3), zonula occludens-1 (ZO1), E-cadherin (CDH1), CK4, CK13,CK1, CK10, filaggrin (FLG), loricrin (LOR), involucrin (IVL), tumor necrosis factor-a (TNFA), interleukin-1a (IL1A), interleukin-1b (IL1B), interleukin 1 receptor antag-onist (IL1RA), interleukin 1 receptor I (IL1RI), and interleukin 1 receptor II (IL1RII) forTaqMan Gene Expression Assays� (Life Technologies). TaqMan Fast Universal PCRMasterMix and StepOnePlus�Real-Time PCR Systems (Life Technologies)were used.mRNA expression levels were normalized with the expression level of b2 micro-globulin. Human epidermal keratinocytes were seeded at a density of 9.0� 104 cells/cm2, cultured for 6 days, and subjected to RNA extraction. Obtained data from threeindependent cultures were statistically analyzed by Scheffé’s method.

2.3. Conditioned medium

The scheme of conditioned medium experiment is shown in Fig. 2. Primarycanine oral mucosal epithelial cells were cultured with KCM at a density of

Day13

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ity of 4.0 � 104 cells/cm2. Scale bar indicates 200 mm. Phase contrast microscope imagest 5, 7, 9, and 11 days after cell seeding, respectively. The graph shows the number of thel cell sheet was successfully fabricated as shown in the left photograph. White scale bareosin shows a stratified cell sheet structure with approximately 50 mm in thick. Black

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Day 5 Day 7 Day 9 Day 11 Day 13Controlmedium

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Fig. 2. Scheme of rat oral mucosal epithelial cell culture with conditioned medium. Conditioned media were collected from canine oral mucosal epithelial cell culture at 5, 7, 9, 11,and 13 days after 48-h culture. The volumes of collected supernatant samples were adjusted with fresh KCM for obtaining a certain ratio of the number of the cultured cells and thevolume.

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Fig. 3. Culture of rat oral mucosal epithelial cells with conditioned medium. Thenumber of rat oral mucosal epithelial cells cultured with conditioned medium wascounted at 7 days after the cell seeding (n ¼ 3).

M. Kondo et al. / Biomaterials 34 (2013) 121e129 123

4 � 104 cells/cm2. Conditioned media were collected at the time points of 5, 7, 9, 11,and 13 days culture 48 h after medium change. Collected conditioned media werediluted with fresh KCM to normalize corresponding cell numbers in culture. Then,primary rat oral mucosal epithelial cells were seeded at an initial cell density of4 �104 cells/cm2 with conditioned media for seven days, and the cell numbers werecounted (n ¼ 3). KCM pre-incubated at 37 �C for 48 h was used for a control culturecondition.

2.4. Cytokines and neutralizing antibodies

Each of 13 recombinant cytokines was added to KCM, and the proliferation ofprimary rat oral mucosal epithelial cells and human epidermal keratinocyte wereexamined. Culture condition for rat cells was the same as the conditioned mediumexperiment (n ¼ 3), but the initial cell number was 9.0 � 104 and 1.5 � 104 cells/cm2

for human epidermal cell growth and cell sheet formation experiments, respec-tively. Numbers of cultured human epidermal cells were counted at day 6 (n ¼ 3).Obtained data from three independent cultures were statistically analyzed byStudent’s t-test. Recombinant human heparin-binding EGF-like growth factor (HB-EGF), recombinant human interleukin-1a (IL-1a), recombinant human IL-1 receptorantagonist (IL-1RA), recombinant human brain-derived neurotrophic factor (BDNF),recombinant human neuregulin, recombinant human hepatoma-derived growthfactor Isoform 1 (HDGF), recombinant Rat Platelet derived growth factor-BB (PDGF-BB), recombinant human vascular endothelial growth factor-C (VEGF-C), recombi-nant rat b-NGF, recombinant human pleiotrophin were purchased from R&DSystems (Minneapolis, MN). Recombinant human fibroblast growth factor 18(FGF18) and recombinant human transforming growth factor-a (TGF-a) werepurchased from Biovision (Milpitas, CA). Recombinant growth factor, augmenter ofliver regeneration (GFER) and recombinant placental growth factor (PGF) werepurchased from Abnova (Taipei, Taiwan). Each cytokine was added at a finalconcentration of 10 ng/mL. IL-1a and IL-1RA were examined at concentrations of0.001e10 ng/mL and 0.01e100 ng/mL, respectively. Anti-lL-1a neutralizing antibodywas obtained from R&D Systems. Normal goat IgG (R&D Systems) was used asa control. Antibodies were added at a final concentration of 10 mg/mL. KCM withPBS or normal goat IgG was used for a control culture condition.

2.5. Histology

Canine and human epidermal cell sheets were harvested by temperaturereduction to 20 �C after 11 days culture. For histological analyses, harvested cellsheets were fixed with 10% neutral buffered formalin, and routinely processed into3-mm thick paraffin-embedded sections. Hematoxylin and eosin staining was per-formed by conventional methods. For immunohistochemistry, de-paraffinizedsections were washed with PBS, and subjected to proteinase K treatment (Dako-Cytomation, Glostrup, Denmark) or heat treatment with Target Retrieval Solution,Citrate pH6 (DakoCytomation). Sections were then treated with each of thefollowing primary antibodies; mouse monoclonal anti-E-cadherin (1:50 dilution)(NCH-38: DakoCytomation), mouse monoclonal anti-filaggrin (1:100 dilution)(FLG01: Thermo Fisher Scientific, Waltham, MA), mouse monoclonal anti-p63(1:100 dilution) (4A4: Santa Cruz Biotechnology, Santa Cruz, CA), mouse mono-clonal anti-Ki67 (1:100 dilution) (MIB-1: Lab Vision, Fremont, CA), and mousemonoclonal anti-pancytokeratin (1:200 dilution) (AE1/AE3: Progen Biotechnik,Heidelberg, Germany) at 4 �C overnight. All sections were peroxidase-stained usingLSAB2 kit (DakoCytomation), according to the manufacturer’s suggested protocol.

3. Results

Primary canine and rat oral mucosal epithelial cells werecultured with KCM under a 3T3 feeder layer-free condition. Primarycanine oral mucosal epithelial cells exhibited a small, cobblestone-like cell shape, and a higher proliferation than primary rat oralmucosal epithelial cells (n ¼ 3) (Fig. 1). At 11 days of culture, caninecells reached confluency, and kept their confluence more than 13days of culture. Cultured canine oral mucosal epithelial cells weresuccessfully harvested as a contiguous cell sheet from a tempera-ture-responsive cell culture insert at 12 days after seeding (Fig. 1).On the other hand, rat cells were flat and large in cell shape. Theircell proliferation was significantly poor, and failed to reach con-fluency under the culture condition (Fig. 1), while these cellsshowed high proliferative capacity in the presence of cholera toxinand 3T3 feeder layer (data not shown). This observation

Table 1Forty cytokine gene expressions in cultured canine oral mucosal epithelial cells.

Expression patterns Gene symbol

Descending Bdnf, Fgf1, Fgf18, Gfer, Hbegf, Hdgf, Il1a, Il1ra,Ngf, Nrg1, Pdgfb, Ptn, Tgfa, Vegfc

Ascending Egf, Igf2, Ntf3, PgfNon-descript Fgf2, Fgf7, Fgf9, Fgf13, Gdnf, Hgf, Nrg2, Ntf4,

Pdgfa, Tgfb2, VegfaUndetectable Fgf4, Fgf5, Fgf6, Fgf8, Fgf10, Fgf12, Fgf14, Fgf16,

Fgf19, Figf, Igf1, Tgfb1

Relative gene expressions were quantified at 5, 7, 9, 11, and 13 days after cellseeding. The abbreviation of gene of brain-derived neurotrophic factor is Bdnf;hepatoma-derived growth factor, Hdgf; fibroblast growth factor 1, Fgf1; fibroblastgrowth factor 18, Fgf18; growth factor, augmenter of liver regeneration, Gfer;heparin-binding EGF-like growth factor, Hbegf; interleukin 1 alpha, Il1a; interleukin1 receptor antagonist, Il1ra; nerve growth factor, Ngf; neuregulin 1, Nrg1; platelet-derived growth factor beta polypeptide, Pdgfb; pleiotrophin, Ptn; transforminggrowth factor alpha, Tgfa; vascular endothelial growth factor C, Vegfc; epidermalgrowth factor, Egf; insulin-like growth factor 2, insulin-like growth factor 2, Igf2;neurotrophin 3, Ntf3; placental growth factor, Pgf; fibroblast growth factor 2, Fgf2;fibroblast growth factor 7, Fgf7; fibroblast growth factor 9, Fgf9; fibroblast growthfactor 13, Fgf13; glial cell line derived neurotrophic factor, Gdnf: hepatocyte growthfactor, Hgf; neuregulin 2, Nrg2; neurotrophin 4, Ntf4; platelet-derived growth factoralpha polypeptide, Pdgfa; transforming growth factor, beta 2, Tgfb2; vascularendothelial growth factor A, Vegfa; fibroblast growth factor 4, Fgf4; fibroblastgrowth factor 5, Fgf5; fibroblast growth factor 6, Fgf6; fibroblast growth factor 8,Fgf8; fibroblast growth factor 10, Fgf10; fibroblast growth factor 12, Fgf12; fibroblastgrowth factor 14, Fgf14; fibroblast growth factor 16, Fgf16; fibroblast growth factor19, Fgf19; c-fos induced growth factor, Figf; insulin-like growth factor 1, Igf1;transforming growth factor, beta 1, Tgfb1.

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Fig. 4. Gene expression of selected cytokines showing a descending pattern (n ¼ 3). The cy(Fgf1), fibroblast growth factor 18 (Fgf18), growth factor, augmenter of liver regeneration (G(Hdgf), interleukin-1 alpha (Il1a), interleukin-1 receptor antagonist (Il1ra), nerve growthpleiotrophin (Ptn), transforming growth factor, alpha (Tgfa), vascular endothelial growth faccalculated by allowing the expression at day 5 to be 100%.

M. Kondo et al. / Biomaterials 34 (2013) 121e129124

convincingly revealed that supplemental requirement for cellproliferation is different between the two species, and impliescanine epithelial cells would secrete autocrine factors to promotecell proliferation, that lack in rat epithelial cell culture.

Then, we examined whether conditioned media collected fromcanine epithelial cell culture can promote primary rat epithelial cellproliferation (Fig. 2). Interestingly, potent promotive activity of cellproliferation was observed with conditioned media collectedduring 5e9 day of culture (Fig. 3). Therefore, we examined whichcytokine plays a crucial role in the cell proliferation promotiveactivity. First, gene expression of canine epithelial cells was quan-tified for 40 cytokine genes by quantitative RT-PCR with gene-specific TaqMan probes. Referring to the time-course change ofthe cell proliferation promotive activity, cytokine genes werecategorized into four groups; descending, ascending, non-descript,and undetectable (Table 1). Obtained candidate genes to promoteepithelial cell growth were Bdnf, Fgf1, Fgf18, Gfer, Hbegf, Hdgf, IL1a,Il1ra, Ngf, Nrg1, Pdgfb, Ptn, Tgfa, and Vegfc which showeddescending pattern (Fig. 4). Next, thirteen recombinant cytokineproteins among these fourteen genes were added to culture ofprimary rat epithelial cells. As a result, only IL-1RA enhanced theepithelial cell proliferation dose-dependently (Fig. 5A, B). Bycontrast, IL-1a significantly inhibited rat epithelial cell growth(Fig. 5A, B). Other cytokines except GFER didn’t show significant cellproliferation promotive or inhibitory activity. The promotive

me (day)

GferFgf18

9 11 135 7 5 7 9 11 139 11 13

VegfcPtn Tgfa

NgfIl1raIl1a

tokine genes were brain-derived neurotrophic factor (Bdnf), fibroblast growth factor 1fer), heparin-binding EGF-like growth factor (Hbegf), hepatoma-derived growth factorfactor (Ngf), neuregulin 1 (Nrg1), platelet-derived growth factor subunit B (Pdgfb),tor C (Vegfc). Relative gene expressions at 7, 9, 11, and 13 days after cell seeding were

M. Kondo et al. / Biomaterials 34 (2013) 121e129 125

activity of GFER was statistically significant, but much smaller thanthat of IL-1RA (Fig. 5A).

Then, the activities of interleukins and receptor antagonist wereexamined with normal human epidermal keratinocytes undera 3T3 feeder layer-free condition. Certainly, the epithelial cellproliferation was significantly inhibited by IL-1a, but enhanced byIL-1RA. In addition, the cell size was kept smaller, and the cell shapewas kept more cuboidal in the presence of IL-1RA than a controladded with PBS (Fig. 6). IL-1b showed a small but significantnegative effect on the cell growth (Fig. 6), and no difference of cellshape was observed (data not shown). Furthermore, anti-IL-1aneutralizing antibody also significantly promoted human epithelialcell proliferation (Fig. 6).

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Fig. 5. Growth inhibitory and promotive effects of exogenous cytokines on epithelial cerecombinant cytokines for seven days, then cell number was counted (n ¼ 3) (A). **shows pcell proliferation (n ¼ 3) (B).

Gene expressions of normal human epidermal keratinocytecultured for 6 days in the presence of each of PBS, IL-1RA, IL-1a,normal IgG, and anti-IL-1a neutralizing antibody were analyzed interms of epithelial cell differentiation, stem/progenitor cell marker,cell proliferation, ECM, IL-1 and its receptors (Fig. 7). The geneexpression of proliferation marker; Ki-67, was significantly higherin the presence of IL-1RA. A putative epithelial stem/progenitor cellmarker, dNp63 was expressed in a higher degree in the presence ofIL-1RA or anti-IL-1a antibody, although stem progenitor markerCK15 was expressed in a higher degree only in the presence of IL-1RA. To the contrary, IL-1a down-regulated the expressions ofdNp63 and CK15. The gene expression of epithelial extracellularmatrix component; laminin beta 3 was slightly higher in IL-1RA

*

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0.01 0.1 1 10 100 (ng/mL)

IL-1RA

lls. Rat oral mucosal epithelial cells were cultured with supplement of each of 13< 0.01 IL-1a and IL-1RA showed dose-dependent effects on rat oral mucosal epithelial

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Fig. 6. Effect of IL-1 signaling on normal human epidermal keratinocyte growth. Normal human epidermal keratinocytes cultured with KCM supplemented with each of PBS(control), 100 ng/mL IL-1RA, 10 ng/mL IL-1a, 10 ng/mL IL-1b, 10 mg/mL normal goat immunoglobulin G (normal IgG) as the control, or 10 mg/mL neutralizing anti-IL-1a antibody(anti-IL-1a). Phase contrast microscope images show the cells cultured with KCM containing PBS, IL-1RA, normal IgG, and anti-IL1a at 6 days after cell seeding. The relative cellnumber at day 6 after cell seeding was calculated as the ratio to the control (PBS or normal IgG) (n ¼ 3). Scale bar indicates 200 mm. ** and * show p < 0.01 and 0.05, respectively.

M. Kondo et al. / Biomaterials 34 (2013) 121e129126

condition than its control, and slightly lower in IL-1a, whereas anti-IL-1a showed no difference. Gene expressions of epithelial celldifferentiation markers of CK4, CK13, CK1, and CK10 were down-regulated in the presence of IL-1a, but up-regulated in the pres-ence of IL-1RA, whereas anti-IL-1a antibody showed no effect. Thegene expression of filaggrin was significantly lower in the presenceof IL-1a, but higher in IL-1RA and anti-IL-1a. For TNFA, not signif-icant but slight down-regulation in IL-1RA and significant down-regulation in anti-IL-1a condition, and not significant up-regulation was observed in the presence of IL-1a. Loricrin, IL-1a,IL-1b, IL-1 receptor antagonist, IL-1 receptor I, and IL-1 receptor IIshowed no differences among five conditions (Fig. 7). Histologicalevaluation after H-E staining and immunohistochemistry revealedthat the cell sheets cultured in the presence of IL-1RA or anti-IL-1a

antibody showed cell stratification and normal epidermal celldifferentiation (Fig. 8).

4. Discussion

For establishing epithelial regenerative medicine as a wide-spread and standard treatment, a stable epithelial cell culturecondition should be established. The present study aimed tosophisticate epithelial cell culture by investigating applicablematerials having a growth promotional effect on stratified squa-mous epithelial cells. Primary canine oral mucosal epithelial cellsshowed a significantly higher growth potential thanprimary rat oralmucosal epithelial cells under 3T3 feeder-free condition (Fig.1), andthe supernatant of canine cell culture conditioned medium

CK15dNp63 1OZ3bMAL6aGTI1bGTI76iK

CDH1 CK4 CK13 CK1 CK10 FLG LOR

IIR1LIIR1LIAR1LIB1LIA1LILVI TNFA

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Fig. 7. Gene expression of human normal epidermal keratinocyte. The cells were cultured with KCM containing PBS, 100 ng/mL IL-1RA, 10 ng/mL IL-1a, 10 mg/mL normal IgG, or 10 mg/mL neutralizing anti-IL-1a antibody (anti-IL-1a).Gene expressions were determined by quantitative RT-PCR (n ¼ 3). The analyzed genes were Ki67, delta N p63 (dNp63), cytokeratin 15 (CK15), integrin beta 1 (ITGb1), integrin alpha 6 (ITGa6), laminin beta 3 (LAMb3), zonula occludens-1 (ZO1), E-cadherin (CDH1), CK4, CK13, CK1, CK10, filaggrin (FLG), loricrin (LOR), involucrin (IVL), tumor necrosis factor-a (TNFA), interleukin-1a (IL1A), interleukin-1b (IL1B), interleukin 1 receptor antagonist (IL1RA), interleukin 1receptor I (IL1RI), and interleukin 1 receptor II (IL1RII). Relative gene expression against b2 microglobulin was shown.

M.Kondo

etal./

Biomaterials

34(2013)

121e129

127

IL-1RA

HE

p63

Ki67

Filaggrin

E-cadherin

Pancytokeratin

Anti-IL-1α

Fig. 8. Histology of harvested human normal epidermal cell sheets. The photographs on the upper row are cell sheets cultured in keratinocyte culture medium containing 100 ng/mL IL-1RA and 20 mg/mL neutralizing anti-IL-1a antibody (anti-IL-1a), respectively. White scale bars of the upper row photographs indicate 10 mm. HE staining and immuno-histochemistry of p63, Ki67, filaggrin, E-cadherin, and pan-cytokeratin (panCK) were also performed. Black scale bars indicate 50 mm.

M. Kondo et al. / Biomaterials 34 (2013) 121e129128

enhanced the proliferation of rat cells (Fig. 3). These observationsstrongly implied that cultured canine oral mucosal epithelial cellssecreted growth promotive factors in an autocrine manner. Byfocusing on the time-course change of cytokine gene expression(Table 1 and Fig. 4) and growth promotive activity (Fig. 5A), IL-1RAwas identified to promote epithelial cell growth, while IL-1ainhibited the growth (Fig. 5A, B). Previous reports [14e17] show thatIL-1a have a growth promotional effect on epidermal keratinocytesunder a 3T3 feeder co-cultured conditionunder the rationale that IL-1a allows3T3 feeder layer to releaseKGF into culturemedium. In thepresent study, we utilized the culture condition which eliminated3T3 feeder layer [9,10], and discovered direct negative effect of IL-1aon epithelial cell growth (Fig. 5A, B, Fig. 6). Cultured normal kerati-nocyte store a large amount of IL-1a in the cytoplasm [18e21], andsecrete IL-1a and IL-1RA consistently [22]. Although many reportshave been published on IL-1 signaling, and IL-1 pathway is drawn asa huge correlation diagram [23], the mechanism of IL-1a-mediatingepithelial cell growth inhibition is unproven directly. IL-1a agonizeIL-1 receptor, and the signal is transduced through MyD88, IRAK1,IRAK4, and TRAF6 [24]. NFkB activation by IL-1a through TRAF6 andits effector proteins including NEMO, IKKa, and IKKb has been re-ported [25], andoverexpressedNFkBhas a growth inhibitory role forepidermal cells and inhibition of NFkB causes hyperplasia in vivo[26]. Also, NFkB profoundly inhibits cell cycle progression in vitro[27], and activation of NFkB is required for PMA-induced keratino-cyte growth arrest [28]. Presumably, in this study, exogenous IL-1RAinhibited the signal transduction described above. The balance of IL-1a and IL-1RA invitroand invivowouldbeof importance tomaintain

normal epithelial cell growth. Gene expression of p63 was muchhigher in the presence of IL-1RA or anti-IL-1a antibody, while IL-1a(10 ng/mL) down-regulated p63 expression (Fig. 7). p63 is reportedtobinddirectly to the transcription regulating regionof IL-1a, and IL-1a gene expression is lower in p63 knockout mouse [29]. Negativefeedback system of p63 expression by IL-1a signaling mightunderlie. As well as p63 expression, Ki67, a proliferation marker,various types of keratin, cellecell junction, and cell-substrate junc-tionwere up-regulated by hampering IL-1a signaling (Fig. 7). Theseresults indicated that the control of IL-1a signaling should beimportant to fabricate well-differentiated, robust, stratified squa-mous epithelial cell sheets for regenerative medicine.

5. Conclusion

Under 3T3 feeder-free cell culture condition, IL-1a was found toinhibit the proliferation of stratified squamous epithelial cells,whereas IL-1RA to enhance the proliferation. Moreover, significantgrowth promotion was confirmed for human normal epidermalkeratinocytes by adding exogenous IL-1RA or anti-IL-1a antibody toculture medium.

Acknowledgments

The author thanks Mr. H. Sugiyama of Tokyo Women’s MedicalUniversity for the useful comments and technical criticism. Thiswork was supported by JSPS research fellowship, Formation ofInnovation Center for Fusion of Advanced Technologies in the

M. Kondo et al. / Biomaterials 34 (2013) 121e129 129

Special Coordination Funds for Promoting Science and Technology‘Cell Sheet Tissue Engineering Center (CSTEC)’ and the Global COEprogram, the Multidisciplinary Education and Research Center forthe Establishment of Regenerative Medicine (MERCREM), from theMinistry of Education, Culture, Sports, Science and Technology(MEXT), Japan.

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