BON-10371; No. of pages: 10; 4C: Bone xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Bone journal homepage: www.elsevier.com/locate/bone

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Article history: Received 5 March 2014 Revised 30 April 2014 Accepted 7 May 2014 Available online xxxx

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Keywords: Activin A Fibroblastic differentiation Migration Osteoblastic differentiation Periodontal ligament Proliferation

c

Department of Endodontology and Operative Dentistry, Faculty of Dental Science, Kyushu University, Higashi-ku, Fukuoka 812-8582, Japan Department of Endodontology, Kyushu University Hospital, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan Colgate Australian Clinical Dental Research Centre, School of Dentistry, University of Adelaide, SA 5005, Australia

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a b s t r a c t

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Hideki Sugii a, Hidefumi Maeda b,⁎, Atsushi Tomokiyo c, Naohide Yamamoto a, Naohisa Wada b, Katsuaki Koori b, Daigaku Hasegawa a, Sayuri Hamano a, Asuka Yuda a, Satoshi Monnouchi b, Akifumi Akamine a,b

Periodontal ligament (PDL) tissue plays an important role in tooth preservation by structurally maintaining the connection between the tooth root and the bone. The mechanisms involved in the healing and regeneration of damaged PDL tissue, caused by bacterial infection, caries and trauma, have been explored. Accumulating evidence suggests that Activin A, a member of the transforming growth factor-β (TGF-β) superfamily and a dimer of inhibinβa, contributes to tissue healing through cell proliferation, migration, and differentiation of various target cells. In bone, Activin A has been shown to exert an inhibitory effect on osteoblast maturation and mineralization. However, there have been no reports examining the expression and function of Activin A in human PDL cells (HPDLCs). Thus, we aimed to investigate the biological effects of Activin A on HPDLCs. Activin A was observed to be localized in HPDLCs and rat PDL tissue. When PDL tissue was surgically damaged, Activin A and IL-1β expression increased and the two proteins were shown to be co-localized around the lesion. HPDLCs treated with IL-1β or TNF-α also up-regulated the expression of the gene encoding inhibinβa. Activin A promoted chemotaxis, migration and proliferation of HPDLCs, and caused an increase in fibroblastic differentiation of these cells while down-regulating their osteoblastic differentiation. These osteoblastic inhibitory effects of Activin A, however, were only noted during the early phase of HPDLC osteoblastic differentiation, with later exposures having no effect on differentiation. Collectively, our results suggest that Activin A could be used as a therapeutic agent for healing and regenerating PDL tissue in response to disease, trauma or surgical reconstruction. © 2013 Published by Elsevier Inc.

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Edited by: Hong-Hee Kim

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Effects of Activin A on the phenotypic properties of human periodontal ligament cells

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Original Full Length Article

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Introduction

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Activins, of the TGF-β superfamily, are 25 kDa proteins found in follicular fluid [1,2]. They consist of two gene products, inhibinβa and inhibinβb, which dimerize to form Activin A (βa:βa), Activin B (βb:βb) and Activin AB (βa:βb). Activin A is localized in the bone marrow, central nervous system, placenta, liver, spleen, heart, adrenal glands, adipose tissue and ovaries [3–5], and has been shown to be involved in the regulation of cellular differentiation [6,7], proliferation [8,9] and migration [10,11] in several of these tissue types. In skin fibroblasts, Activin A treatment has been shown to induce cell proliferation and enhance the expression of alpha-smooth muscle actin (α-SMA) and type I collagen (COL1) [12]. Other studies have suggested a role for Activin A in inflammation and skin repair. In normal skin tissue, Activin A is present in low levels but its expression is elevated in wounded skin [13] working at the early stages of inflammation [14] to

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⁎ Corresponding author at: Department of Endodontology, Kyushu University Hospital, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan. Fax: +81 92 642 6366. E-mail address: [email protected] (H. Maeda).

promote the formation of granulation tissue. A similar elevation has also been noted in brain tissue inflammatory responses [15]. Activin A is reported to be induced by pro-inflammatory cytokines, such as interleukin-1β (IL-1β) [16,17] and tumor necrosis factor-α (TNF-α) [18,19], and is down-regulated by matrix metalloproteinase secretion in IL-1-treated chondrosarcoma cells [20]. Additionally, Activin A expression increases in reactive fibroblastic cells and tissues during the healing process of acute burn wounds in human skin [21]. Activin A has also long been linked with bone metabolism [22]. Several studies in the past have shown that Activin A inhibits osteoblast mineralization of rat calvarial cells [23–25] and it is purported to act during the initial stages of bone formation causing a significant shift in the cell cycle toward the G1 phase with a concomitant decrease in the rate of cell death [26]. Despite this knowledge, there is little information concerning the potential expression and role of Activin A in periodontal tissues. The periodontal ligament (PDL) is a soft tissue structure localized between the cementum covering the tooth root and the alveolar bone [27] where it provides support, protection and provision of sensory input to the masticatory system [28]. PDL tissue, however, can become irreversibly damaged by trauma and diseases that cause inflammation,

http://dx.doi.org/10.1016/j.bone.2014.05.021 8756-3282/© 2013 Published by Elsevier Inc.

Please cite this article as: Sugii H, et al, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone (2013), http://dx.doi.org/10.1016/j.bone.2014.05.021

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60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79

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Cell culture

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HPDLCs were isolated from the teeth of three healthy patients who visited Kyushu University for extraction, as described elsewhere [37]: a third molar from a 23-year-old male (3S), a premolar from a 21-year-old female (3Q) and a premolar from 20-year-old female (3O). In this study, HPDLCs were maintained in alpha-minimum essential medium (αMEM; Gibco-BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Biowest, Nuaillé, France), 50 μg/ml streptomycin and 50 U/ml penicillin (10%FBS/αMEM) at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. The cells from passage 4 through 7 were used in this study. All procedures were performed in compliance with the Research Ethics Committee, Faculty of Dentistry, Kyushu University.

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Experimental animal model

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Five-week-old male Sprague–Dawley rats, weighing 140–150 g, were purchased from Kyudo (Saga, Japan). All procedures were performed according to our recent report [38]. Briefly, animals were anesthetized by an intra-peritoneal injection of 2 mg/kg midazolam (Sandoz Inc, Tokyo, Japan), 0.15 mg/kg medetomidine (Kyoritsu Seiyaku Co, Ltd. Tokyo, Japan), and 2.5 mg/kg butorphanol tartrate (Meiji Seika Pharma Co. Ltd., Tokyo, Japan). A periodontal defect, 2 mm in diameter and 2 mm in depth, was created in the left maxilla extending from the mesio-palatal submarginal portion to the palatal root of the first molar. The first molars on the right side were not wounded and used as controls. All procedures were approved by the Animal Ethics Committee and conformed to the regulation of Kyushu University. At 72 h after surgery, animals were sacrificed by transcardial perfusion with 4% paraformaldehyde (PFA) (Merck, Darmstadt, Germany) in phosphate-buffered saline (PBS) using the same anesthetic as described above. Maxillae were removed and decalcified in 10% ethylenediaminetetraacetic acid (Wako Pure Chemical Industries Ltd., Osaka, Japan) at 4 °C for 1 month before dehydration and embedding in paraffin.

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Immunofluorescence and immunochemical analyses

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Five-micrometer sections were prepared, and the tissues blocked with 2% bovine serum albumin (BSA) in PBS for 1 h. The sections were

109 110 111 112 113 114

119 120 121 122 123 124 125 126 127 128 129 130 131 132 133

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IL-1β or TNF-α treatment of HPDLCs

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HPDLCs (6 × 104 cells per well) were cultured in a 6-well plate in 2 ml of culture medium for 24 h before treatment with 10 ng/ml recombinant human IL-1β (PeproTech EC, London, UK) or 10 ng/ml recombinant human TNF-α (PeproTech EC) for 12, 24, and 48 h. The concentration of both cytokines was determined according to a recent study [39], because they examined the expression of inhibinβA mRNA and activin-A protein in human stromal cells treated with these reagents (10 ng/ml). The cells were then processed for quantitative RT-PCR.

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Semi-quantitative RT-PCR

169

Total cellular RNA was isolated with TRIzol Reagent according to the manufacturer's instructions. First-strand cDNA synthesis was performed with an RNA PCR Core kit (Applied Biosystems, Foster City, CA). Total RNA was reverse-transcribed with oligo d(T)16 primer and MuLV reverse transcriptase for 15 min at 42 °C, and the reaction was stopped by incubation for 5 min at 99 °C, followed by 5 min at 5 °C. PCR was performed using Platinum Taq DNA polymerase (Invitrogen) in a PCR Thermal Cycler Dice (Takara Bio, Shiga, Japan) under the following conditions: 94 °C for 2 min, followed by an appropriate number of cycles (specific to different primers) at 94 °C for 30 s, and finally 72 °C for 30 s. Primer sequences, annealing temperatures, cycle number, and product sizes for human Inhibinβa, Activin receptor (ACVR) Ia (ACVRIa), ACVRIb, ACVRIIa, ACVRIIb, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are listed in Table 1. PCR products were separated on a 2% agarose gels (Seakem ME Agarose, Lonza Rockland, Rockland, ME) by electrophoresis and imaged under ultraviolet excitation with ethidium-bromide. Primer sequences were designed using the GenBank database (National Center for Biotechnology Information; NCBI), and a BLAST search of GenBank was performed on the primer sequences to ensure specificity.

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Quantitative RT-PCR

190

First-strand cDNA was synthesized from 1 mg total RNA using an ExScript RT Reagent kit (Takara Bio). Total RNA was reverse-transcribed with random 6-mers and ExScript RTase for 15 min at 42 °C, and the reaction was stopped by incubation for 2 min at 99 °C, followed by 5 min at 5 °C. PCR was performed by using KAPA SYBR FAST qPCR Kit (Kapa Biosystems Inc., Boston, MA) under the following conditions: 95 °C for 10 s (initial denaturation), then 40 cycles of 95 °C for 5 s and 60 °C for 30 s, followed by a dissociation program of 95 °C for 15 s, 60 °C for

191

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107 108

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99 100

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89 90

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86

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Materials and methods

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139 140

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then incubated with a goat polyclonal antibody against human/rat Activin A-βa subunit (1:40; anti-Activin A, R&D Systems, Minneapolis, MN) and a rabbit polyclonal antibody against rat IL-1β (1:100; Santa Cruz Biotechnology, Santa Cruz, CA), followed by Alexa-Fluor568conjugated anti-goat IgG (Invitrogen, Carlsbad, CA) and Alexa-Fluor488conjugated anti-rabbit IgG (Invitrogen) secondary antibodies, respectively, for 1 h. Staining of the nuclei was carried out using VECTASHILD Mounting Medium containing 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA). Sections were imaged and analyzed using a Biozero digital microscope (Keyence Corporation, Osaka, Japan). For immunocytochemical analysis, HPDLCs were fixed with 4% PFA and 0.5% dimethyl sulfoxide (Wako) in PBS for 20 min. After blocking with 2% BSA for 1 h, the cells were incubated for 1 h with anti-Activin A (1:40) and then washed with PBS. The cells were then incubated with a biotinylated rabbit anti-goat secondary antibody, followed by avidin-peroxidase conjugate (Nichirei Biosciences, Inc., Tokyo, Japan). Positive staining was visualized using DAB solution (Nichirei). Nuclei were stained using Mayer's hematoxylin solution (Wako). The expression of Activin A in Rat PDL tissue was also examined by the same method.

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such as severe periodontitis or deep caries. Therefore, much research has been undertaken to understand and explore the potential methods through which tissue engineering can employ cells, signaling molecules and scaffolds to enhance PDL regeneration. PDL tissue is composed of a heterogeneous cell population, various fibers, non-collagenous ECM proteins, nerve fibers, blood vessels, polysaccharides, and others. The principal cells in PDL tissue are fibroblasts [27,29], PDL stem cells (PDLSCs), epithelial cell rests of Malassez, and endothelial cells. PDLSCs have the potential to differentiate into PDL fibroblasts, osteoblasts, and cementoblasts in vivo [30,31], despite their fibroblastic phenotype [32,33]. Thus, it is thought that these endogenous cells will be instrumental sources of multipotent cells for PDL regeneration. Previous studies have shown that Activin A is expressed in the mouse dental follicle—the embryonic precursor of PDL tissue—and in rat tooth-germ mesenchyme [34–36]. However, the biological functions of Activin A on human periodontal ligament cells (HPDLCs) have not been examined. Therefore, the aim of this study was to assess the effect of Activin A on HPDLCs, specifically by investigating (1) the expression of Activin A and activin receptors (ACVRs) in PDL cells, (2) the expression of Activin A in the wounded site of PDL tissue, and (3) the effects of Activin A on the chemotaxis, migration, proliferation, and osteoblastic or fibroblastic differentiation of HPDLCs.

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H. Sugii et al. / Bone xxx (2013) xxx–xxx

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H. Sugii et al. / Bone xxx (2013) xxx–xxx t1:1 t1:2

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Table 1 Primer sequence, product size, annealing temperature, cycle numbers for semi-quantitative RT-PCR. Target gene (abbreviation)

Forward (top) and reverse (bottom) primer sequences

Size of amplified products (bp)

Annealing temperature (°C)

Cycles

t1:4 t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13 t1:14

Inhibinβa

5′-TGCCCTTGCTTTGGCTGAGA-3′ 5′-ACTTTGCCCACATGAAGCTTT-3′

285

55

35

5′-AAGATGAGAAGCCCAAGGTC-3′ 5′-CAGGCAGGCTAAAAGACAT-3′ 5′-TGGCAGAGTTATGAGGCACT-3′ 5′-ATGGGAGTGAGCGAGTCTCT-3′ 5′-ACCAGTGTTGATGTGGATCTT-3′ 5′-TACAGGTCCATCTGCAGCAGT-3′ 5′-ACAGGTAGGCACGAGACGGT-3′ 5′-GTAGTGCCGTTGACCGACCT-3′

340

55

28

266

55

28

455

55

28

357

59

28

Chemotactic assay

213 214 215 216

Chemotaxis was performed using cell culture inserts (pore size, 8 μm; Becton Dickinson Labware, Franklin Lakes, NJ) in 24-well plates (Becton Dickinson Labware). HPDLCs (3.4 × 103 cells in 200 μl culture medium) were placed upon the cell culture inserts. The lower chambers

t2:1 t2:2

Table 2 Primer sequence, product size, annealing temperature, for quantitative RT-PCR.

209 210

C

207 208

217 218

Migration assay

227

A scratch wound healing assay was performed to assess the effect of Activin A on HPDLC migration in response to wounding. Briefly, HPDLCs (3 × 104 cells per well) were seeded on 12-well plates (Becton Dickinson Labware), and allowed to grow to subconfluence (~90%) for 2 days. Then, a scratch-wound (400 μm wide) was made across the diameter of the well using the end of a 200 μl pipette tip, and scraped cells were removed by washing three times with PBS. The cells were

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203 204

were filled with 750 μl culture medium containing 0, 10, or 100 ng/ml recombinant human Activin A (PeproTech EC). After 24 h incubation, the cells on the upper side of the insert were removed with cottontipped swabs. The cells that had passed through the insert membranes were fixed with methanol (Wako) for 2 min and stained with 1% toluidine blue (Sigma-Aldrich Japan K.K., Tokyo, Japan) for 2 min. Stained cells were imaged on an inverted microscope and analyzed with FlvFs software (Flovel Ltd., Tokyo, Japan). Cells were counted in four randomly chosen fields from each chamber. Measurements were made using Scion Image Software (Scion Corporation, Walkersville, MD).

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211

30 s, and 95 °C for 15 s. Human β-actin was used as an internal control. Expression of the target genes was calculated from the ΔΔCt values. Primer sequences, annealing temperatures, and product sizes for human alpha-smooth muscle actin (α-SMA), bone sialoprotein (BSP), collagen (COL) 1 (COL1), COL3, cyclin (CCN) A2 (CCNA2), CCNE1, fibrillin 1 (FBN1), osteocalcin (OCN), periostin (POSTN), periodontal ligamentassociated protein (PLAP1), runt-related transcription factor (RUNX2), scleraxis (SCX), TGF-β1, and β-actin are shown in Table 2. Primer sequences were designed using the GenBank database (NCBI), and a BLAST search of GenBank was performed on the primer sequences to ensure specificity. The expression levels of these genes were normalized against β-actin expression, and the results are shown as the foldincrease of the control.

199 200

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-IIb

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-IIa

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-Ib

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Activin receptor -Ia

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t1:3

Target gene (abbreviation)

Forward (top) and reverse (bottom) primer sequences

t2:4 t2:5 t2:6 t2:7 t2:8 t2:9 t2:10 t2:11 t2:12 t2:13 t2:14 t2:15 t2:16 t2:17 t2:18 t2:19 t2:20 t2:21 t2:22 t2:23 t2:24 t2:25 t2:26 t2:27 t2:28 t2:29 t2:30 t2:31 t2:32 t2:33

β-actin

5′-ATTGCCGACAGGATGCAGA-3′ 5′-GAGTACTTGCGCTCAGGAGGA-3′ 5′-GACAATGGCTCTGGGCTCTGTAA-3′ 5′-CTGTGCTTCGTCACCCACGTA-3′ 5′-CTGGCACAGGGTATACAGGGTTAG-3′ 5′-ACTGGTGCCGTTTATGCCTTG-3′ 5′-CCCGGGTTTCAGAGACAACTTC-3′ 5′-TCCACATGCTTTATTCCAGCAATC-3′ 5′-GCAAATTCACCTACACAGTTCTGGA-3′ 5′-CTTGATCAGGACCACCAATGTCATA-3′ 5′-CCATACCTCAAGTATTTGCCATCA-3′ 5′-AGCTTTGTCCCGTGACTGTGT-3′ 5′-GCCAGCCTTGGGACAATAATG-3′ 5′-CTTGCACGTTGAGTTTGGGT-3 5′-TGCACCTATGGAACCATGTGATAGA-3′ 5′-AAGTGATCCACTGTGTGCCAACTC-3′ 5′-TGTGCCCACCAAGCTGAGAC-3′ 5′-CTGGGCTGGGCAACTCTATGA-3′ 5′-GTGCAGAGTCCAGCAAAGGT-3′ 5′-TCAGCCAACTCGTCACAGTC-3′ 5′-CATTGATGGAGTGCCTGTGGA-3′ 5′-CAATGAATTTGGTGACCTTGGTG-3′ 5′-ATGGGAGTCTTGCTAACATACCAC-3′ 5′-CAGAAGTCATTTACTCCCACTCTTG-3′ 5′-AACCCTTAATTTGCACTGGGTCA-3′ 5′-CAAATTCCAGCAATGTTTGTGCTAC-3′ 5′-AGCCCAAACAGATCTGCACCTT-3′ 5′-TTCTGTCGCGGTCCTTGCT-3′ 5′-AGCGACTCGCCAGAGTGGTTA-3′ 5′-GCAGTGTGTTATCCCTGCTGTCA-3′

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t2:3

COL1 COL3 CCNA2 CCNE1 FBN1 Inhibinβa OCN POSTN PLAP1 RUNX2 SCX TGF-β1

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BSP

N C O

α-SMA

Size of amplified products (bp)

Annealing temperature (°C)

Sequence ID

89

60

NM_001101.3

147

60

NM_001141945.1

182

60

NM_004967.3

148

60

NM_000088.3

147

60

NM_000090.3

79

60

NM_001237.3

104

60

NM_001238.2

190

60

NM_000138.4

124

60

NM_002192.2

110

60

NM_199173.4

167

60

NM_007675.2

154

60

NM_17680.4

145

60

NM_001024630.3

66

60

NM_001080514.2

130

60

NM_000660.5

Please cite this article as: Sugii H, et al, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone (2013), http://dx.doi.org/10.1016/j.bone.2014.05.021

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228 229 230 231 232 233 234

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then cultured in culture medium containing 0, 10, or 100 ng/ml Activin A. For each well, images were taken at 0 and 24 h after wounding, and the number of cells that migrated into the wound space was manually counted from three randomly-selected areas.

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WST-1 proliferation assay

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HPDLCs (5 × 103 cells per well) were cultured in a 48-well plate in 250 μl of culture medium containing 0, 10, or 100 ng/ml Activin A for up to 5 days. On days 0, 3, and 5 of culture, proliferation was measured using a Cell Proliferation Assay kit (Millipore Corp., Billerica, MA). Briefly, at the indicated time-points, 25 μl of kit reagent, WST-1, was added to the culture medium of each well. After 30 min, 110 μl of supernatant was collected from each well, and the density was measured using an ImmunoMini NJ-2300 (System Instruments Co., Ltd., Tokyo, Japan) at absorbance of 450 nm. Experiments were performed in duplicate.

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HPDLCs were cultured with or without 100 ng/ml Activin A for 3 days. The cells were then collected and a cell suspension in PBS was prepared. Ice-cold 70% ethanol was added to the cells and mixed vigorously. Cells were left at 4 °C overnight, washed with PBS and incubated with RNase and propidium iodide. The percentage of cells in the three phases of the growth cycle (G0/G1, G2/M, and S-phase) was analyzed by flow cytometry (EC800 Cell Analyzer, Sony, Tokyo, Japan) and Eclipse software (Sony).

250 251

Osteoblastic differentiation of HPDLCs

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anti-Activin A

control

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%

B

B

PDL

PDL

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anti-Activin A

control

&

HPDLCs 3S 3O 3Q

252 253 254 255 256 257

HPDLCs (1 × 104 cells per well) were cultured in 24-well plates in 259 four different media: 10% FBS/αMEM as control culture medium 260 (Cont), Cont containing 1 mM CaCl2 (Ca), Cont containing 100 ng/ml 261

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C

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N

244 245

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Cell cycle analysis

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H. Sugii et al. / Bone xxx (2013) xxx–xxx

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ACVR Ia

Ib IIa IIb

Inhibinβa GAPDH Fig. 1. Expression of Activin A and ACVR in HPDLCs and rat PDL tissue. Immunolocalization of Activin A in HPDLC-3S (derived from the third molar of a 23-year-old male) (A) and rat PDL tissue (B) was examined. (A) Immunopositive staining was observed in the cytoplasm of HPDLC. (B) Horizontal sections of rat PDL tissue were taken through a root of the first molar in the left maxilla. Positive staining was entirely observed in rat PDL tissue. No staining was observed in control samples (without primary antibody). Nuclei were stained with hematoxylin. Experiments were performed in duplicate (B, bone; R, root; PDL, periodontal ligament; P, dental pulp tissue). Bars = 100 μm. (C) Semi-quantitative RT-PCR analysis revealed an increase in the expression of genes encoding inhibinβa and four ACVRs (Ia, Ib, IIa, IIb) in HPDLC-3S. GAPDH was used as an internal standard.

Please cite this article as: Sugii H, et al, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone (2013), http://dx.doi.org/10.1016/j.bone.2014.05.021

H. Sugii et al. / Bone xxx (2013) xxx–xxx

(A)

5

(B) Non-wounded site

Wounded site Dis

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Dis

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Pa M1

M1

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M2

Wounded site

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M2

N o n- wo u n d ed s it e

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anIL-1β

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PDL

3 2.5 2 1.5 1 0.5 0

**

**

**

PDL

**

R R

PDL

PDL

PDL

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mRNA expression (inhibinβ a/β-actin)

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Merged image

(H)

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anActivinA

Fig. 2. Expression of Activin A around the wounded PDL tissue in rats and IL-1β- or TNF-α-treated HPDLCs. Immunofluorescence analysis of Activin A and IL-1β expression in rat PDL tissue in the surgically wounded side (A; left) and non-wounded side (B; right) was performed using the sections collected at 72 h after surgery. Horizontal sections were prepared through the first and second molars in the rat maxilla from both sides. Immunopositive staining for both anti-Activin A (red) and anti-IL-1β (green) antibodies is shown. Higher magnification views of the squares shown in panels A and B are provided in panels C–G. More intense staining for both antibodies is recognizable around the wounded site (WS) (C), as compared with normal PDL tissue from the second left molar (E) and another root of the first left molar (D) in the wounded side or the first (F) and second (G) molars from the non-wounded side. Co-localization (yellow) is shown in the merged images. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; blue). Data are representative of three independent experiments. Bars = 100 μm. M1, first molar; M2, second molar; Bu, buccal; Dis, distal; Mes, mesial; Pa, palatal; PDL, periodontal ligament; R, root. (H) Gene expression of inhibinβa in HPDLC-3S (derived from the third molar of a 23-year-old male) treated with IL-1β (10 ng/ml) or TNF-α (10 ng/ml) for 12, 24, and 48 h. Untreated cells served as the control. Inhibinβa was examined using quantitative RT-PCR. The expression levels of these genes were normalized against β-actin expression, and the results are shown as the fold-increase of the control. Values are the mean ± SD from three independent experiments. **P b 0.01. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Please cite this article as: Sugii H, et al, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone (2013), http://dx.doi.org/10.1016/j.bone.2014.05.021

(B)

(C)

0.8

120

**

80 40

E

0 ActivinA 0 (ng/ml)

0.6 0.4

ActivinA 0ng/ml

*

10ng/ml

0.2

100ng/ml

0

10 100

0

10

100

S

G2/M

O

G0/G1

R

(D)

N

C

Control G0/G1:79.84% S-phase:6.73% G2/M:12.21%

G0/G1

S

G2/M

ActivinA G0/G1:70.08% S-phase:11.20% G2/M:16.33%

% Cells

R

E

ActivinA (ng/ml)

C

T

12h

**

P

**

OD450nm

160

D

Cell number

0h

F

100

10

0

** 1000 ** 800 600 400 200 0 ActivinA (ng/ml) 0 10 100

O

ActivinA (ng/ml)

Chemotaxisnumber of cells (Cells/field)

(A)

microscope (Keyence Corporation). Using cells grown under the same culture conditions, total RNA was isolated at 3 h, 3 days, and 1 week of culture, and gene expression of bone-related molecules was examined. Additionally, HPDLCs were cultured in 24-well plates in Ca

100 80 60 40 20 0

*

Control Activin *

*

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264 265

Activin A (ACV), and Cont containing both 1 mM CaCl2 and 100 ng/ml Activin A (Ca + ACV). After 3 weeks of culture, the cells were subjected to von Kossa staining and Alizarin red S staining. The area of each Alizarin red-positive region was measured using a Biozero digital

(E) mRNA expression (/β -actin)

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H. Sugii et al. / Bone xxx (2013) xxx–xxx

R O

6

CCNE1

CCNA2 8 6

**

4 3

4

2

2

1

0

0

**

Please cite this article as: Sugii H, et al, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone (2013), http://dx.doi.org/10.1016/j.bone.2014.05.021

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H. Sugii et al. / Bone xxx (2013) xxx–xxx

Statistical analysis

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All values are expressed as mean ± standard deviation. Statistical analysis was performed using a Student's paired t-test. A probability value of P b 0.05 was considered statistically significant.

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Results

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Expression of Activin A and ACVRs in HPDLCs and rat PDL tissue

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Immunocytochemical and immunohistochemical analyses revealed positive staining with an anti-Activin A antibody in cultured HPDLCs (Fig. 1A and Supplemental Fig. 1A) and in rat PDL tissue (Fig. 1B). Control staining (in the absence of the primary antibody) was negative in HPDLCs and PDL tissues (Figs. 1A, B). Semi-quantitative PCR demonstrated that HPDLCs expressed genes encoding inhibinβa and each of the four ACVRs (Fig. 1C and Supplemental Fig. 1B).

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Expression of Activin A and IL-1β in surgically wounded PDL tissue

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We compared the immunolocalization of Activin A and IL-1β between normal and wounded PDL tissue in rats (Figs. 2A, B). The expression of both proteins was increased in the tissue adjacent to the wounded site, with colocalization observed (Fig. 2C). By comparison, the PDL tissue surrounding the second, non-wounded molar on the wounded (left) side and the first and second molars on the control (right) side exhibited no increased expression of Activin A and no expression of IL-1β (Figs. 2E–G). Additionally, even PDL tissue of another root of the first molar, distant from the wounded area showed no upregulation in the expression of either protein, similar to that in the second and control molar sites (Fig. 2D). Overall, little expression of IL-1β was detected anywhere except for around the wounded site. As per these findings, we next sought to investigate the effects of proinflammatory cytokines, IL-1β and TNF-α, on the expression of the gene encoding inhibinβa in HPDLC cultures using quantitative RT-PCR

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The effect of Activin A on HPDLC chemotaxis, migration, and proliferation was examined using a Transwell assay, a scratch wound healing assay, and WST-1 proliferation assay and cell cycle analysis, respectively. Each test was conducted on HPDLCs isolated from the molar teeth of three individual patients (see Materials and methods). In the Transwell assay, Activin A significantly promoted chemotaxis of HPDLCs after 48 h of culture as compared with untreated cells (Fig. 3A and Supplemental Fig. 3A). Similarly, the scratch wound healing assay also revealed a significant increase in the migration of HPDLCs within 12 h of Activin A treatment, as compared with untreated cells (Fig. 3B and Supplemental Fig. 3B). In terms of cell proliferation, the WST-1 assay showed a significant increase in proliferation in the presence of Activin A for 3 and 5 days as compared with untreated cells (Fig. 3C and Supplemental Fig. 3C), and the flow cytometric analysis confirmed an Activin Ainduced proliferation of HPDLCs through the increase in DNA content in the cell-cycle distribution (Fig. 3D). Indeed, cells treated by Activin A exhibited a significant promotion in S-phase (11.74 ± 0.87%) and G2/M phase (16.4 ± 0.35%) as compared with untreated cells (S-phase: 7.68 ± 2.36%; G2/M phase: 13.18 ± 1.19%). To confirm these results, we examined the expression of CCNA2 and CCNE1 that regulate DNA synthesis and mitosis through the cell cycle ([40,41]) in Activin A-treated cells. Compared with untreated cells, treatment with Activin A led to significant up-regulation of both genes (Fig. 3E).

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Chemotaxis, migration, and proliferation in Activin A-treated HPDLCs

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Soluble collagen released into the culture medium from HPDLCs and the collagen in newly formed extracellular matrix (ECM) produced by HPDLCs were quantified by a Sircol collagen assay (Biocolor, Belfast, UK). Cells were cultured with or without Activin A for 1 week. The anionic dye Sirius red (1 ml), which reacts specifically with basic side chain groups of collagens, was added to the supernatant and incubated in a gentle mechanical shaker for 30 min at room temperature. After centrifugation for 10 min at 12,000 × g, the collagen–dye complex at the bottom of the tubes was collected and dissolved in 0.5 M sodium hydroxide. Absorbance was measured at 570 nm by a Model 680 Microplate Reader (Bio-rad, Berkeley, CA). For ECM collagen formed on cell culture plate, the matrix was dissolved using 0.1 mg/ml pepsin in 0.5 M acetic acid overnight at 4 °C and quantified as described above.

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(Fig. 2H and Supplemental Figs. 2A, B). Compared with untreated cells, 315 treatment with IL-1β and TNF-α led to significant up-regulation of 316 inhibinβa in the HPDLCs after 12, 24, and 48 h. 317

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medium under different exposures to Activin A (see Fig. 4C). After 3 weeks of culture, the cells were subjected to aforementioned analyses.

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Effects of Activin A on osteogenic differentiation of HPDLCs

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Our recent study revealed that calcium supplementation can promote osteogenic differentiation and mineralization of HPDLCs [42]. Thus, HPDLCs were cultured with or without Activin A in the presence or absence of 1 mM CaCl2 for 3 weeks to assess the effect of Activin A on osteogenesis. Staining with von Kossa and Alizarin Red S both demonstrated a significant reduction in calcium-induced mineralization in the presence of Activin A (Fig. 4A and Supplemental Fig. 4A). These findings were confirmed by a corresponding decrease in the expression of genes encoding OCN, RUNX2 and BSP in HPDLCs grown in the presence of both Activin A and calcium (Fig. 4B and Supplemental Fig. 4B). We next examined the influence of the timeframe and duration of Activin A exposure in causing these effects on HPDLC mineralization. First, we used four different delivery windows, with Activin A treatment administered on days 0–21, days 0–7, days 7–14, or days 14–21 (Figs. 4Ca and Cb), and stained for mineralization on day 21. We found a significant inhibitory effect of Activin A when it was delivered during days 0–21 and days 0–7, but not during days 7–14 and days 14–21, as compared with calcium treatment alone (Fig. 4Ca and Supplemental Fig. 4C). Next, we changed the length of Activin A exposure within this 21-day time frame: days 0–21, days 3–21, days 7–21, and days 14–21 (Fig. 4Cb and Supplemental Fig. 4C), once again measuring the effects on mineralization at Day 21. We found that Activin A had a less profound impact on

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Fig. 3. Effects of Activin A on chemotaxis, migration, and proliferation of HPDLCs. (A) HPDLC-3S, derived from the third molar of a 23-year-old male, were seeded onto cell inserts with porous membranes. The lower chambers were filled with 10%FBS/αMEM plus Activin A (0, 10, or 100 ng/ml). Cells that passed through the porous membrane are stained with toluidine blue (TB). The graph shows the number of TB-positive cells per cell insert (n = 4). **P b 0.01. Bar = 100 μm. (B) The migratory activity of HPDLC-3S was analyzed using a scratch wound healing assay. After reaching subconfluence, the cell monolayer was scratched, and growth into the denuded area was assessed in 10%FBS/αMEM with or without Activin A (0, 10, or 100 ng/ml). The dashed lines delimit the initially wounded regions. The number of cells that migrated into the wounded space was counted 12 h after wounding. The graph shows the results from three randomly-selected areas on a dish and the results are representative of three separate experiments. **P b 0.01. Bar = 100 μm. (C) Proliferation assay of HPDLC-3S cultured in 10%FBS/αMEM with or without Activin A (0, 10, or 100 ng/ml) for 0, 3 and 5 days in culture was performed using a WST-1 assay kit, with the absorbance measured at 450 nm. (D) The percentage of HPDLC-3S cultured in 10%FBS/αMEM plus Activin A (0 or 100 ng/ml) for 3 days in G0/G1, G2/M, and S phases was examined by flow cytometric analysis. Data are described graphically. Values are the mean ± SD from three independent experiments and experiments were performed in duplicate. *P b 0.05, **P b 0.01. (E) Gene expression of CCNA2 and CCNE1 in HPDLC-3S treated with Activin A (100 ng/ml) for 3 days. Untreated cells served as the control. CCNA2 and CCNE1 were examined using quantitative RT-PCR. The expression levels of these genes were normalized against β-actin expression, and the results are shown as the fold-increase of the control. Values are the mean ± SD from three independent experiments. **P b 0.01.

Please cite this article as: Sugii H, et al, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone (2013), http://dx.doi.org/10.1016/j.bone.2014.05.021

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Fig. 4. Effects of Activin A on osteoblastic differentiation of HPDLCs. (A) HPDLC-3S, derived from the third molar of a 23-year-old male, were cultured in one of four media: 10%FBS/αMEM as a control, 10%FBS/αMEM + 1 mM CaCl2 (Ca), or 10%FBS/αMEM + 100 ng/ml Activin A (ACV), or 10%FBS/αMEM + 1 mM CaCl2 + 100 ng/ml Activin A (Ca + ACV). Mineralization was visualized by Alizarin Red S staining (AR) and von Kossa staining (VK) on day 21, and staining area was quantitated. (B) Gene expression encoding OCN, RUNX2 and BSP in HPDLC-3S cultured in one of the aforementioned conditions. The expression levels of these genes were normalized against β-actin expression, and the results are shown as the fold-increase of the control. (C) HPDLC-3S were cultured for different timeframes and durations in the presence of absence of Activin A treatment with or without 1 mM CaCl2 to assess changes in mineralization after 21 days in culture (a, b). The schedules of Activin A exposure are described. After culturing, mineralization was visualized by AR and VK on day 21, and staining area was quantitated. Data are described graphically. Values are the mean ± SD from three independent experiments. *P b 0.05, **P b 0.01.

inhibiting mineralization the later it was administered, indicating that Activin A has an inhibitory effect only during the early stages (Days 0–7) of osteogenic differentiation of HPDLCs.

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Effects of Activin A on fibroblastic differentiation of HPDLCs

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We examined gene expression related to fibroblastic differentiation (α-SMA, COL1, COL3, and FBN1), and PDL-related gene expression (POSTN, PLAP1, SCX, and TGF-β1) in HPDLCs treated with Activin A for 3 h, 3 days, or 1 week (Fig. 5A and Supplemental Figs. 5A, B). We found a significant up-regulation in all of the genes following Activin A treatment in HPDLCs as compared with untreated cells. In addition, a Sircol collagen assay revealed a significant up-regulation in the production of soluble collagen released into the cell culture medium and insoluble collagen recovered from newly formed ECM that had been deposited onto cell culture plates following Activin A treatment (Fig. 5B).

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Discussion

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This study is the first to examine the effect of Activin A on human PDL cells and to reveal the overlap and increased expression of Activin A and IL-1β within PDL tissue adjacent to wounded tissue regions as compared with normal tissue. Activin A was found to induce proliferation, migration and chemotaxis of HPDLCs, as well as promote fibroblastic differentiation, but block osteogenic differentiation of HPDLCs.

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Healing of damaged PDL tissues occurs through three phases: inflammation, tissue formation and tissue remodeling [43]. During inflammation, the expression levels of pro-inflammatory cytokines, such as IL-1β and TNF-α, are up-regulated. In a past report, IL-1β stimulated gene expression of inhibinβa in human skin fibroblasts during the early phase of wound healing (days 3 to 5) [17]. Our results here also reveal a higher co-expression of Activin A and IL-1β in PDL tissue adjacent to the wounded site on day 3, compared with non-wounded and normal tissues, which showed weak Activin A, but no IL-1β. These findings were confirmed in vitro, with IL-1β- or TNF-α-treated HPDLCs showing an increase in inhibinβa. Uchida et al. (2000) reported that inhibinβa was up-regulated in osteoprogenitor cells within 3 days after injury in a rat femur, later decreasing after 7 days as the osteoblasts matured [44]. Collectively, these reports suggest that Activin A might play a role during the early phase of the healing process in periodontal inflammation. Until now, reports on Activin A up-regulation in wounded tissues have been limited to the brain, bone, intestine, kidney, liver, pancreas, skin and stomach [13,45,46]. Overexpression of Activin A has been shown to promote the formation of granulation tissue in the skin of transgenic mice [47], which is an important step in skin healing and regeneration [48]. Another recent study showed that Activin A stimulated the proliferation and differentiation of pulmonary and renal fibroblasts into myofibroblasts, leading to the wound healing in human lung and kidney [45,49,50]. Together, these studies point to a role of Activin A in tissue healing and regeneration.

Please cite this article as: Sugii H, et al, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone (2013), http://dx.doi.org/10.1016/j.bone.2014.05.021

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**

mRNA expression (/β -actin)

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Periodontal tissue regeneration relies on the proliferation, migration and differentiation of PDL cells at the site of injury. We found that Activin A promoted the proliferation, migration and chemotaxis of HPDLCs, and furthermore induced fibroblastic differentiation and PDL-related gene expression in HPDLCs. Migration of PDL cells into the wounded site and their subsequent proliferation has been shown to regenerate the periodontium, encompassing the production of newly formed PDL tissue and alveolar bone [51]. Thus, considering the findings of the present study, Activin A might drive PDL cells to wounded sites and affect their subsequent expansion and differentiation into PDL-fibroblastic cells to promote tissue repair. Interestingly, we found that Activin A inhibited osteoblastic differentiation of HPDLCs, quite profoundly within the first three days of treatment. Later supplementation after 7 days' growth had little effect on the cellular differentiation. These results suggest that Activin A might play an inhibitory role during the early stages of osteoblastic differentiation of HPDLCs. These findings coincide with previous studies where it was shown that Activin A does not affect further mineralization in highly differentiated osteoblasts [24,52]. Moreover, patients with R206H mutations in the ACVR1 gene exhibit ectopic ossification, known as fibrodysplasia ossificans progressiva (FOP), a rare disorder of the connective tissue [53,54]. From these reports, Activin A may contribute to preventing ectopic ossification in PDL tissue. Another study reported the gradual down-regulation of Activin A in differentiating

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Fig. 5. Fibroblast-related and PDL-related gene expression and collagen expression in Activin A-treated HPDLCs. (A) Expression of α-SMA, COL1, COL3, FBN1, POSTN, PLAP1, SCX, and TGF-β1 in HPDLC-3S treated with 100 ng/ml Activin A was examined by quantitative RT-PCR. HPDLC-3S were derived from the third molar of a 23-year-old male. Untreated cells were used as a control. The expression levels of these genes were normalized against β-actin expression, and the results are shown as the fold-increase of the control. (B) Production of total soluble collagen and ECM collagen were measured using a Sircol collagen assay kit. Results are shown as the fold-increase of the control. Values are the mean ± SD from three independent experiments. *P b 0.05, **P b 0.01.

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We thank Drs. Teramatsu, Yoshida, Serita, Mitarai and Mizumachi for their great support in the preparation of this work. This work was financially supported by Grants-in-Aid for Scientific Research (Project Nos. 23659890, 23689077, 24390426, 24659848, 24792028, 25293388, and 25670811) from Japan Society for the Promotion of Science.

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rat osteoprogenitors during healing in rat injured bone, with a concomitant increase in OCN [44]. Therefore, these results suggest that such inhibitory effects of Activin A may depend on the osteogenic differentiation stage of HPDLCs. In conclusion, this study showed that Activin A is expressed within the entire PDL tissue at low levels and that its expression is upregulated during the early phases of PDL tissue healing, coincident with increased IL-1β expression. Activin A may contribute to the repair of PDL tissue through increasing cell proliferation, migration, chemotaxis and fibroblastic differentiation, as well as by blocking premature osteogenic differentiation of PDL cells, thereby preventing ectopic PDL tissue ossification, but probably not impairing the later maturation of osteoblasts in bone-related sites. Future studies will need to clarify the effects of Activin A on osteoblasts during these tissue healing responses. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.bone.2014.05.021.

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Please cite this article as: Sugii H, et al, Effects of Activin A on the phenotypic properties of human periodontal ligament cells, Bone (2013), http://dx.doi.org/10.1016/j.bone.2014.05.021

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[21] McLean CA, et al. Temporal expression of activin in acute burn wounds—from inflammatory cells to fibroblasts. Burns 2008;34(1):50–5. [22] Sakai R, Eto Y. Involvement of activin in the regulation of bone metabolism. Mol Cell Endocrinol 2001;180(1–2):183–8. [23] Ikenoue T, et al. Inhibitory effects of activin-A on osteoblast differentiation during cultures of fetal rat calvarial cells. J Cell Biochem 1999;75(2):206–14. [24] Eijken M, et al. The activin A-follistatin system: potent regulator of human extracellular matrix mineralization. FASEB J 2007;21(11):2949–60. [25] Alves RD, et al. Activin A suppresses osteoblast mineralization capacity by altering extracellular matrix (ECM) composition and impairing matrix vesicle (MV) production. Mol Cell Proteomics 2013;12(10):2890–900. [26] Rosenberg N, et al. The role of activin A in the human osteoblast cell cycle: a preliminary experimental in vitro study. Exp Clin Endocrinol Diabetes 2010;118(10):708–12. [27] Beertsen W, McCulloch CA, Sodek J. The periodontal ligament: a unique, multifunctional connective tissue. Periodontol 2000 1997;13:20–40. [28] Moxham BJ, Berkovitz BK. Continuous monitoring of the movements of erupting and newly erupted teeth of limited growth (ferret mandibular canines) and their responses to hexamethonium. Arch Oral Biol 1988;33(12):919–23. [29] Berkovitz BK. Periodontal ligament: structural and clinical correlates. Dent Update 2004;31(1):46–50 [52-54]. [30] Seo BM, et al. Investigation of multipotent postnatal stem cells from human periodontal ligament. Lancet 2004;364(9429):149–55. [31] Maeda H, et al. Prospective potency of TGF-beta1 on maintenance and regeneration of periodontal tissue. Int Rev Cell Mol Biol 2013;304:283–367. [32] Kitagawa M, et al. Effect of F-spondin on cementoblastic differentiation of human periodontal ligament cells. Biochem Biophys Res Commun 2006;349(3):1050–6. [33] Nojima N, et al. Fibroblastic cells derived from bovine periodontal ligaments have the phenotypes of osteoblasts. J Periodontal Res 1990;25(3):179–85. [34] Wang XP, et al. Modulation of activin/bone morphogenetic protein signaling by follistatin is required for the morphogenesis of mouse molar teeth. Dev Dyn 2004;231(1):98–108. [35] Ferguson CA, et al. Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition. Genes Dev 1998;12(16):2636–49. [36] Maas R, Bei M. The genetic control of early tooth development. Crit Rev Oral Biol Med 1997;8(1):4–39. [37] Fujii S, et al. Establishing and characterizing human periodontal ligament fibroblasts immortalized by SV40T-antigen and hTERT gene transfer. Cell Tissue Res 2006;324(1):117–25.

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