Kyudai Oral Bioscience 2013
—7th International Symposium —March 8-9th, 2013
Fukuoka Recent Hotel, Fukuoka, JapanPROGRAM & ABSTRACTS

Kyushu University Faculty of Dental Science

〇会期：平成 24 年 3 月 8 日〜9 日
3 月 8 日（金）13:00~18:30、懇親会 18:30~20:00
3 月 9 日（土）9:00~17:20〇会場：福岡リーセントホテル 舞鶴の間 A（２階）：下図参照
住所：福岡市東区箱崎 2-52-1
電話：092‐641‐7741〇主催：九州大学大学院 歯学研究院
歯学研究院研究プロジェクト「口腔組織の再生・再建医療」、「口腔健康科学」

文部科学省

特別教育研究経費「歯学連携ネットワークによる口腔からＱＯＬ向上を目指す研究」

連絡先：

中西 博
九州大学大学院 歯学研究院
口腔機能分子科学分野〒812‐8582 福岡市東区馬出 3-1-1
電話：092‐642‐6413
ファックス：092‐642‐6415
Ｅ-mail：nakan@dent.kyushu-u.ac.jp1

Information for Speakers■ Presentation Instrument

・Presentations are restricted to computer presentations using your own
personal computer.
・Please use a computer running Windous XP, Windows Vista, Windows 7
or Machintosh OS X or later, and fitted with an external monitor output
terminal.
・All speakers are also required to bring the data of your presentation on a
USB or a CD-ROM. Please make do not forget to mark your name andthe session number (e.g. S1A-1) on the file. Please make sure that virus
check is excuted beforehand.
■ Making Presentations

・For the speakers in Sessions 1A, 2A and 3, please bring your own personal
computers to the Computer Operating Desk (on the left-hand side facing
toward inside the presentation venue) at least 20 min prior to yourpresentation.
・For the speakers in Sessions 1B, 1C, 2B, please bring your own personal
computers to the Computer Operating Desk during Coffee Break just before
your Session.
・All speakers are requested to operate the computer by themselves.
・Your cooperation in finishing your presentation within the allotted time isappreciated.
・After your presentation, please reclaim your computer from the Computer
Operator Desk.

2

PROGRAM3

March 8th (Fri)MAIZURU Room A（2nd Floor）

Opening remarks
13:00 - 13:05
13:05 - 13:10

Hiroshi Nakanishi (vice Dean)
Akifumi Akamine (Dean, Faculty of Dental Science, Kyushu University)

Session 1: Dental and Craniofacial Morphogenesis and Tissue Regeneration
Session 1A■Chair person: Kazuaki Nonaka (Department of Pediatric Dentistry)
13:10-13:40

13:40-14:10

14:10-14:40

S1A-1 Yuji Mishina
Department of Biologic and Material Sciences, University of Michigan,
Ann Arbor, MI, USA
S1A-2 Han-Sung Jung
Department of Oral Biology, Yonsei University College of Dentistry,Seoul, Korea
S1A-3 Jonathan Schoenecker
Vanderbilt University Medical Center, Nashville, TN, USA

14:40-15:00 Coffee BreakSession 1B■Chair person: Seiji Nakamura (Section of Oral and Maxillofacial Oncology)
15:00-15:30

15:30-15:50

S1B-1 Takayoshi Sakai
Department of Oral-Facial Disorders,
Osaka University, Graduate School of Dentistry, Osaka, Japan
S1B-2 Haruyoshi Yamaza
Faculty of Dental Science, Kyusyu University, Fukuoka, Japan

15:50-16:10 Coffee BreakSession 1C■Chair person: Toshio Kukita (Dept of Molecular Cell Biology and Oral Anatomy)
16:10-16:50

16:50-17:20

17:20-17:50

S1C-1 Agnès Vignery
Orthopedic and Rehabilitation and Cell Biology
Yale University School of Medicine, New Haven, CT, USA
S1C-2 Kenichi Tezuka
Department of Oral Maxillofacial Science, Gifu University
Graduate School of Medicine, Gifu, Japan
S1C-3 Akihito Yamamoto
Department of Oral and Maxillofacial Surgery,
Nagoya University Graduate School of Medecine, Nagoya, Japan

18:00-20:00 Getting Together Party, MC: Ichiro Takahashi (vice Dean)

CRYSTAL Room（2nd Floor）

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March 9th (Sat)

MAIZURU Room A（2nd Floor）

Session 2: Regulation of Chronic Inflammation and Diseases
Session 2A■Chair person: Katsumasa Maeda (Department of Periodontology)
9:00- 9:25

S2A-1
Terukazu Sanui
Department of Periodontology, Faculty of Dental Science, Kyusyu University,
Fukuoka, Japan
9:25-10:00 S2A-2 Thomas E. van Dyke
Department of Periodontology, The Forsyth Institute Cambridge, MA, USA
10:00-10:25

S2A-3 Koichi Tabeta
Center for Transdiciplinary Research, Niigata University, Niigata, Japan

10:25-10:50 Coffee BreakSession 2B■Chair person: Hiroshi Nakanishi (Department of Aging Science and Pharmacology)
10:50-11:15

S2B-1 Zhou Wu
Department of Aging Science and Pharmacology. Faculty of
Dental Science, Kyusyu University, Fukuoka, Japan
11:15-11:45 S2B-2 Jessica L TeelingCentre for Biological Science, University of Southampton, Southampton, UK
11:45-12:10 S2B-3 Sung Joong Lee
Department of Neuroscience and Oral Physiology, and Dental ResearchInstitute, School of Dentistry, Seoul National University, Seoul, Korea
12:30-13:30

Lunch
For the Invited Speakers: Restaurant SARAFURU (1st Floor)

Session 3: Oral Health Science■Chair person: Yoshihisa Yamashita (Section of Preventive and Public Health Dentistry)
13:30-13:50

S3-1
Toru Takeshita
Section of Preventive and Public Health Dentistry, Faculty of Dental Science,
Kyusyu University, Fukuoka, Japan
13:50-14:20 S3-2
Todd D. Taylor
Laboratory for MetaSystems Research, Computational Biology Research Core,
RIKEN Quantitative Biology Center, Yokohama, Japan
14:20-14:50 S3-3
Liping Zhao
Shanghai Center for System Biomedicine, Shanghai, China
14:50-15:10 Coffee Break5

Session 4: PhD Student Session■Chair person: Masato Hirata (Laboratory of Molecular and Cellular Biochemistry)
15:10-15:20
15:20-15:30
15:30-15:40
15:40-15:50
15:50-16:00

S4-1
S4-2
S4-3
S4-4
S4-5

Lan Ma (Department of Pediatric Dentistry)
Kyosuke Toyoda (Department of Periodontology)
Urara Tanaka (Department of Periodontology)
Tomohiro Onimura (Section of Oral and Maxillofacial Oncology)
Xin-Wen Zhang (Department Aging Science and Pharmacology)

16:00-16:20 Coffee Break■Chair person: Takeshi Yokoyama (Department of Dental Anesthesiology)
16:20-16:30 S4-6 Yu Yasutake (Laboratory of Molecular and Cellular Biochemistry)
16:30-16:40
16:40-16:50
16:50-17:00
17:00-17:10

S4-7
S4-8
S4-9
S4-10

Daichi Muramatsu (Section of Oral and Maxillofacial Oncology)
Saori Sako (Department of Dental Anesthesiology)
Jane Harland (Department of Biomaterials)
XNT Tram (Department of Biomaterials)

Closing remarks
17:10-17:20 Seiji Nakamura (vice Director, Kyushu University Hospital)

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ABSTRACTS

7

Session 1
Dental and Craniofacial Morphogenesis
and Tissue Regeneration

8

S1A-1
Smad-dependent BMP Signaling through Type IA Receptor
in Cranial Neural Crest Cells Directs Their Cell Fate
towards Chondrocytes to Cause Craniosynostosis
Yuji Mishina
Department of Biologic and Materials Sciences,
University of Michigan, Ann Arbor, MI, USA
Dr. Mishina’s laboratory is interested in functions of bone morphogenetic protein (BMP) signaling
during bone development/remodeling and craniofacial development. We recently developed several
mouse lines to conditionally decrease or increase levels of BMP signaling using a Cre-loxP system.
Using these systems, we have found that BMP signaling in osteoblasts is critical for maintenance ofbone mass and biomechanical properties, BMP signaling in early embryos is critical for ciliogenesisthat is essential to establish a left-right asymmetry, and BMP signaling in cardiac neural crest cells is
important for valve functions during heart development. In this seminar, I would like to talk about
our recent findings how BMP signaling is critically involved in skull morphogenesis through a tightregulation of its signaling activity.
Craniosynostosis describes conditions in which one or more sutures of the infant skull areprematurely fused, resulting in facial deformity and delayed brain development. Approximately 20%
of human craniosynostoses are thought to result from gene mutations altering growth factor
signaling; however, the molecular mechanisms by which these mutations cause craniosynostosis areincompletely characterized, and the causative genes for diverse types of syndromic craniosynostosis
have yet to be identified.
We recently found that enhanced BMP signaling through the BMP type IA receptor (BMPR1A)
in cranial neural crest cells, but not in osteoblasts, causes premature suture fusion in mice. In support
of a requirement for precisely regulated BMP signaling, this defect was rescued on a Bmpr1a
haploinsufficient background, with corresponding normalization of Smad phosphorylation. Moreover,in vivo treatment with LDN-193189, a selective chemical inhibitor of BMP type I receptor kinases
resulted in rescue of craniosynostosis. The finding that relatively modest augmentation of
Smad-dependent BMP signaling leads to premature cranial suture fusion suggests an important
contribution of dysregulated BMP signaling to syndromic craniosynostoses, and potential strategies
for early intervention.

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S1A-2A Wnt/Shh/Sostdc1 Negative Feedback Loop Governs
the Spatial Patterning of Teeth
Han-Sung Jung
Department of Oral Biology, College of Dentistry, Yonsei University, Seoul, Korea

Each vertebrate species displays specific tooth patterns in each quadrant of the jaw: the mouse has
one incisor and three molars, which develop at precise locations and at different times. Sonic
hedgehog (Shh), Wnt and Sostdc1 are key signalling molecules involved in the spatial patterning ofteeth and other ectodermal organs such as hairs, vibrissae and feathers. For example, conditional
Shh- and Smo-deficient mice such as K14-Cre;Shhflox/flox and K14-Cre;Smoflox/flox exhibit samemorphological aberrations in tooth development: the dental lamina is absent, and the first (M1) andsecond molars (M2) are fused. Interestingly, M1/M2 fusion is also observed in Sostdc1–/– and Lrp4–/–
mice, as have supernumerary molars and incisors. While it has been reported that a Wnt/Shh
negative feedback loop exists in tongue papillae and that Sostdc1 (also known as USAG-1, Ectodin
and Wise) is a secreted inhibitor of the Wnt pathway, the relationship between Shh and Sostdc1 is
yet to be clarified. Here, by utilizing maternal transfer of 5E1 (an IgG1 monoclonal antibody against
Shh protein) through the placenta, we show that blocking Shh signalling by 5E1 results in molar fusionand supernumerary tooth formation and that Sostdc1 is a downstream target of Shh, which suggests a
Wnt/Shh/Sostdc1 negative feedback loop governing the spatial patterning in teeth. Furthermore, we
propose a new reaction–diffusion model, in which Wnt, Shh and Sostdc1 act as the activator,
mediator and inhibitor, respectively, confirming that such interactions do indeed lead to patterning
consistent with tooth patterning of mouse.

10S1A-3
Plasmin but not Fibrin is Essential for Fracture Healing1

Masato Yuasa, 1Nicholas Mignemi, 1Heather A Cole,1
Lynda O’ Rear 1,1Jeffry S Nyman, 2Justin M Cates,
1,2
Herbert S Schwartz, 1,2,3Jonathan G Schoenecker

1

Department of Orthopaedic and Rehabilitation, 2Pathology, 3Pediatrics
Anderbilt University Medical Center, Nashville, TN, USA

INTRODUCTION: For a century the formation of a fibrin clot has been considered to be the initial,and essential, phase of fracture repair. In addition to providing hemostasis, the clot is thought torecruit stem cells and localize growth factors that are required for fracture healing. Hence, many
principles of fracture care and pharmaceuticals have been developed to enhance a fibrin matrix in thefracture bed (1).
Despite its beneficial and essential role in the initial phases of healing, the persistentaccumulation of fibrin has been implicated as a factor of delayed skin wound (2). In addition, recent
evidence in chronic diseases such as Alzheimer’s (3), multiple sclerosis (4) and muscular dystrophy(5) have also implicated the accumulation of fibrin as a key molecular component of thepathophysiology of disease. From these studies we formulated the hypothesis that accumulation offibrin would impair fracture healing.
Materials and Methods: All mice were used for this study at 8 weeks of age. To avoid sex-related
confounding effects on developmental bone growth and fracture, only male mice were used in thisstudy (12-16g). Mice were placed under anesthesia using 60 mg/kg ketamine and 4 mg/kg xylazine.
We created a mid-shaft femur fracture by open method on mice (Wild type (WT)); n= 15,
plasminogen deficient (Plg-/-) which cannot remove fibrin; n= 14, and fibrinogen deficient (Fbg-/-)which cannot make a fibrin clot; n=6). 23 gauge needles (0.6414mm) were used for fixation withretrograde fashion. Fracture healing was followed radiographically using a Faxitron X-ray system(Lincolnshire, IL) weekly from 1 week to 6 weeks post-fracture (PF). We sacrificed the mice at 2 and6 weeks PF. Fracture healing was analyzed by X-ray, micro Computed Tomography (uCT; uCT40
Scanco medical AG, Switzerland), Microfil (Flow Tech Inc., Carver, MA) injection and histology.
We dissected the femur and fixed them in 4 % paraformaldehyde and embedded into paraffin. After
sectioned, we stained the sections by H&E, Safranin-O, CD31 immunohistochemical (IHC) (antimouse CD31, BD Pharmigen) and Fibrin IHC staining (given by Dr. J. Degen, Division of
Developmental Biology, Children’s Hospital Research Foundation, Cincinnati, Ohio). The extent of
fracture healing in the mice was quantified by both X-ray and micro CT. We quantified the healing11

by a grading scale, which accounts for development of ossified callus and mineral remodeling by
x-rays and by measuring their structural and tissue level properties of bone by uCT. We injected
antisense oligonucleotide of fibrinogen to Plg (-/-) mice (Plg-/- ASO treated; n=10) weekly to restrict
fibrinogen level in blood 2 weeks prior to fracture. Fibrinogen levels in plasma were measured 6
weeks PF by fibrinogen ELISA (Immunology Consultants Laboratory, Inc, OR). STATISTICS:
Comparisons among the groups were performed using the one-way analysis of variance with theleast significant difference procedure was used for analyzing 2 or multiple groups, respectively. A
level of p< 0.05 was considered significant for the differences between mean values (*).
RESULTS: Weekly X-rays revealed that WT and Fbg (-/-) mice formed ossified bridging callus by 3
weeks PF which was almost completely remodeled by 6 weeks PF. uCT and histological results
revealed that callus formation in WT and Fbg (-/-) mice has completely bridged by 6 weeks PF.
However, Plg (-/-) mice failed to develop an organized mineralized callus and failed to unite or
remodel the disorganized matrix (Figure.1). uCT at 6 weeks PF in Plg (-/-) mice revealed that they
still retained poor remodeled callus formation and disorganized mineralization at the fracture site andmoreover 71% (ten out of fourteen mice) displayed non-bridging callus. Microfil injection
demonstrated that, at 2 weeks PF, vascularity in the callus was significantly reduced in Plg (-/-) micecompared to WT and Fbg (-/-) mice (Figure2-a). Consistent with these findings, there remained
abundant avascular cartilage in Plg (-/-) mice at 2 weeks PF in histological sections compared to WT
mice. We confirmed the migrating neovessels into avascular cartilage by CD31 IHC staining. Fibrin
IHC staining showed abundant fibrin interposed between the avascular cartilage and vascularizedbone (Figure2-b). At 6 weeks, whereas in WT, vessels crossed over the fracture site, those in Plg(-/-) mice did not cross over the non bridging callus. Fibrinogen level in blood of plg (-/-) micetreated with ASO was significantly reduced although that of Plg (-/-) mice was 2-2.5 folds higher
than WT mice. Fracture healing was impaired in Plg (-/-) mice and was partially rescued by ASO
injection when we measure the fracture healing by grading scale. uCT revealed that structuralproperties of the fracture site in Plg (-/-) mice at 6 weeks PF were statistically lower than those of
WT, Fbg (-/-) and Plg (-/-) ASO treated mice.
DISCUSSION: This is the first study to demonstrate that, as opposed to what has been accepted,
fibrin is not essential for fracture healing and that accumulation of fibrin can result in delayed,disorganized non-unions. From our data, it is speculated that with insufficient fibrinolysis, fibrin canbecome a structural barrier to cell invasion, in particular endothelial cells and osteoblasts into
avascular fracture callus. These results may provide valuable insight into novel means of improving
fracture healing in these populations by targeting fibrin degradation.
SIGNIFICANCE: These results demonstrate that dysfunction of fibrinolysis leads disruption ofneovascularization in the fracture healing. These findings have potential clinical implications in

12

disease conditions which are known to have alteration of fibrin clots rendering it more resistant tofibrinolysis as well as primary dysfunction of the fibrinolytic system such as diabetes, smoking (6)and aging.
1: Richard M Berg, Calcification in callus formation and fracture repair. ANNALS of Surgery Vol
93(5); 1931
2: Thomas H. Bugge, Loss of Fibrinogen Rescues Mice from the Pleiotropic Effects of Plasminogen
Deficiency. Cell Vol. 87, 709–719, November 15, 1996
3: Marieke van Oijen, Fibrinogen Is Associated With an Increased Risk of Alzheimer Disease and
Vascular Dementia. Stroke 2005, 36:2637-2641
4: Akassoglou K., Fibrin depletion decreases inflammation and delays the onset of demyelination ina tumor necrosis factor transgenic mouse model for multiple sclerosis. Proc Natl Acad Sci U S A.
2004,101(17): 6698–6703
5: Berta Vidal, Fibrinogen drives dystrophic muscle fibrosis via a TGFB /alternative macrophageactivation pathway. GENES & DEVELOPMENT 2008, 22:1747–1752
6: Rajat S. Barua, Effects of Cigarette Smoke Exposure on Clot Dynamics and Fibrin Structure : An
Ex Vivo. Arterioscler Thromb Vasc Biol. 2010;30:75-79

13

S1B-1
Exploration of Functional Regulators for
Branching Morphogenesis of
Salivary Gland by Microanalysis of Gene Expression
Takayoshi Sakai
Department of Oral-Facial Disorders, Graduate School of Dentistry,
Osaka University, Osaka, Japan
Many organs such as salivary glands, kidneys and lungs form multiple epithelial clefts during theprocess of branching morphogenesis in embryonic development.
The developing submandibular salivary gland provides an excellent model system for clarifying
the mechanisms comprising this phenomenon. In cleft formation, buds are delineated by theconversion of epithelial cell-cell adhesions to cell-matrix adhesions, but the mechanisms of cleft
formation are not clear. Here we show a set of approaches being used to identify and characterizemolecules necessary for branching morphogenesis. A combination of laser microdissection with
T7-SAGE has been established as a method for gene discovery of candidate molecules that may beessential for early organ morphogenesis (Current Protocols in Cell Biology 19.3.1-19.3.30, 2002).
We have identified Fibronectin and Btbd7 at cleft-forming sites and provided insights into themechanisms of dynamic cleft propagation in branching morphogenesis (GSE22374: T7-SAGE
transcriptome of cleft versus end bud epithelial cells of embryonic mouse salivary glands). The
matrix protein fibronectin is required for salivary, kidney, and lung branching. Wedges of fibronectintranslocate inward as clefts form between randomly motile epithelial cells, accompanied by loss ofthe cell-cell adhesion molecule E-cadherin in cells adjacent to the fibronectin (Nature 423, 876-881,
2003). How a matrix molecule can drive cleft formation and branching is unknown. We haveidentified Btbd7 as a dynamic regulator of branching morphogenesis. Btbd7 provides a mechanistic
link between the extracellular matrix and cleft propagation through its highly focal expression
leading to local regulation of Snail2 (Slug), E-cadherin, and epithelial cell motility (Science 329,
562-565, 2010). Inhibition experiments show that Btbd7 is required for branching of embryonicmammalian salivary glands and lungs. Hence, Btbd7 is a regulatory gene that promotes epithelial
tissue remodeling and formation of branched organs. Progress in understanding the mechanisms ofsalivary branching morphogenesis should provide novel approaches to future tissue engineering or
regeneration of damaged salivary glands.

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S1B-2
Caloric Restriction in HealthHaruyoshi Yamaza
Department of Pediatric Dentistry, Graduate School of Dental Science,
Kyushu University, Fukuoka, Japan
Caloric restriction (CR) retards aging processes and extends mean and maximum lifespan, and theeffects of CR are demonstrated in a wide range of animals including C. elegans, Drosophila androdents. Although the findings have been confirmed repeatedly in many laboratories, the mechanismof CR has not been elucidated. Recent discoveries of longevity assurance genes emphasize theimportance of reduced growth hormone (GH)–insulin-like growth factor (IGF)-1 signaling with adecrease in serum insulin levels.
We previously reported a new long-lived transgenic (Tg) dwarf rat model, in which the
GH–IGF-1 axis is selectively suppressed by overexpression of the antisense GH gene, thatsignificantly lives 7%–10% longer than wild-type (WT) rats. Subsequent studies revealed that theseTg rats, which fed the foods ad libitum (AL), have similar phenotypes to WT-CR rats. The plasma
IGF-1 levels are moderately reduced in both groups of rats as compared to WT-AL rats. In WT-CR
and Tg-AL rats, glucose tolerance was normal or slightly improved, whereas the glucose-induced
insulin response was lower. To investigate the potential role of the GH–IGF-1 axis in the CR effect,
recombinant human (rh)IGF-1 was administered continuously to WT-CR, WT-AL, and Tg-AL rats
for 14 days and their responses were analyzed. Infusion of a physiologic dose of rhIGF-1 elicitednegative feedback regulation of rat IGF-1 production, thus the total IGF-1 concentrations did notincrease compared with the infusion of the normal saline in all three rat groups. In addition, rhIGF-1
infusion in WT-CR and Tg-AL reduced the plasma concentration of adiponectin, which wasincreased in long-lived animals by CR and the reduction of GH-IGF-1 axis, on the level of control
group.
The homolog of FoxO, which is the downstream of insulin/IGF-1 signal and transcription factor,
is involved in the lifespan extension in C. elegans and Drosophila. We focused on FoxO1. Therefore,
we investigated the roles of FoxO1 in the effects of CR using FoxO1 knockout (KO) mice. Although
the lifespan did not differ significantly between the WT and KO mice in AL or CR conditions, theanti-neoplastic effect of CR, as indicated by reduced incidence of tumors at death in the WT-CR
mice, was mostly abrogated in the KO-CR mice
In conclusion, adiponectin and FoxO1 could be the important factors through insulin/IGF-1
signaling pathway for CR effects such as lifespan extension and anti-neoplasm.

15

S1C-1The Making of Multinucleate Macrophages:
Osteoclasts and Giant cells
Agnès Vignery
Yale School of Medicine, Orthopaedics and Cell Biology, New Haven, CT, USA

Macrophages are innate immune cells that are omnipresent in tissues and play key roles in immunity,
development, remodeling, and tissue repair. In addition, in rare instances, macrophages can fuse toform a multinucleate osteoclast, in bone, or a giant cell, in chronic inflammatory reactions. While ourunderstanding of the fusion mechanism of viruses with host cells, to inject their DNA or RNA, hasmade major progress, the fusion mechanism of macrophages remains poorly understood. Indeed, thefusion of cells in general, remains obscure. Such cell-cell developmental fusion events include
fertilization, which results from the fusion of a sperm cell with an oocyte; the formation ofmultinucleate skeletal muscle cells, which originate from the fusion of myoblasts; and the formationof the placenta, in which cytotrophoblasts fuse into syncytiotrophoblasts. The molecular mechanismsthat mediate cell-cell fusion remain to be discovered.
Our hypothesis has been that viruses have stolen from macrophages their fusion machinery.
Because macrophages are ubiquitously present in tissues and organs as mononucleate cells, wehypothesize that the fusion machinery must be transiently induced at the onset of fusion so as to limit
fusion in time, hence in cell size. As a result of cDNA microarray and monoclonal antibody screens,
we have identified SIRPa, its receptor CD47, CD200 and its receptor CD200R, CD44 andKCa3.1/SK4 as molecules transiently induced at the onset of fusion. Because SIRPa-CD47 andCD200-CD200R belong to the superfamily of immunoglobulins, we propose that they allow
macrophages to recognize each other as self, hence adhere and fuse with one another to form a new
multinucleate cell rather than to kill one another. Such a recognition mechanism is therefore essential
and might have played a central role in the evolution of the innate immune system.
We therefore regard the fusion machinery of macrophages as a complex molecular assembly thathas evolved to ensure the recognition of non-self by macrophages to protect organisms against
pathogens and foreign bodies, which are bigger than pathogens, such as implants. In the case of
osteoclasts, they can be regarded as a task force that shaped up in response to the formation of
calcified bone, which appeared evolutionary as a new challenge.

16

S1C-2
Dental Pulp Cells as a Source for iPS Cell Banking
Ken-ichi Tezuka
Department of Tissue and Organ Development,
Gifu University Graduate School of Medicine, Gifu, Japan
Human dental pulp cells (DPCs) are present in the cell population isolated from dental pulp tissues(Gronthos et al., PNAS 2000, JDR 2002; Takeda et al., JDR 2008; Iida el al. Arch. Oral Biol. 2010).
We reported that viral introduction of four transcription factors (OCT3/4, SOX2, KLF4, and c-MYC)
can reprogram DPCs into induced pluripotent stem (iPS) cells, which closely resemble embryonicstem cells (Tamaoki et al., JDR 2010, Okita et al. Nature Methods 2011). However, we also foundthat establishing quality-controlled iPS cell lines from a large number of individual patients is noteasy and validation of them requires considerable time and cost.
Human leukocyte antigen (HLA) plays an important role in rejection of tissues and cells
transplanted from allogenic donors. In recent days, tissue transplantation is conducted withoutcomplete matching of HLA because of shortage of the donors. Instead of matching HLA types,
immnosuppressants are frequently used to prevent immune reaction both host to graft and graft tohost directions; however, when the immune system function is suppressed, there is an increased
susceptibility to infectious diseases and cancers.
To solve this problem, usage of HLA haplotype-homo donors has been considered in iPS celltherapy. HLA haplotype-homo donors have a couple of identical HLA gene sets, resulting inpresentation of HLA molecules half in the variation. Therefore, iPS cells derived from HLA
haplotype-homo donors are expected to be successfully transplanted to many patients with lesspossibility of rejection. Potential risk of GVHD will be omitted by making iPS cells to differentiate
strictly into desired cell types without contamination of hematopoietic cells. We screened
approximately 200 DPC lines to find three patients having only one genotype in each of three HLAloci, A, B, DRB1. If iPS cells will be established from these three patients, they are expected to showcomplete match with approximately 20% of the Japanese population.
We will discuss about the advantages and disadvantages of DPCs as one of the somatic cellsources for future iPS cell banking and regenerative medicine.

17

S1C-3
Multifaceted Neuro-Regenerative Activities of Human
Dental Pulp Stem Cells Promote Locomotor Recovery
after Spinal Cord Injury in Rats
Akihito Yamamoto
Department of Oral and Maxillofacial Surgery,
Nagoya University Graduate School of Medicine, Nagoya, Japan
Spinal cord injury (SCI) often leads to persistent functional deficits due to loss of neurons and glia
and to limited axonal regeneration after injury. We have reported that transplantation of adult dental
pulp stem cells (DPSCs) and stem cells from human exfoliated deciduous teeth (SHEDs) into thecompletely transected adult rat spinal cord resulted in marked recovery of hindlimb locomotor
function. Our study revealed that engrafted DPSC and SHED exhibited three major therapeutic
benefits for recovery after SCI, including (a) inhibition of SCI-induced multicellular apoptosis,
which promoted the preservation of neural fibers and myelin sheaths; (b) regeneration of thetransected axon through the direct inhibition of multiple axonal growth inhibitor signals derived from
SCI-circumstance; (c) replacement of lost or damaged oligodendrocytes after SCI through specificdifferentiation into mature oligodendrocytes under the extreme conditions of SCI. Thus, the data
indicate that SHED/DPSCs promote substantial functional recovery after SCI through bothcell-autonomous/cell-replacement and paracrine/trophic effects.
In current study, we have examined the level of benefits of paracrine effects alone for thetreatment of SCI. We found that the intrathecal administration of serum-free conditioned medium(CM) derived from SHED, but not bone marrow stromal cells or skin fibroblasts, promoted
remarkable functional recovery. This recovery was supported by a strong immunoregulatory functionof SHED-CM, which dramatically converted the pro-inflammatory SCI-circumstance into a tissue
repair/regenerating platform by modulating the microglia/macrophage phenotype. SHED-CM
induced robust differentiation of the anti-inflammatory M2 microglia/macrophages in the injured SC.
Furthermore; we found that in vitro SHED-CM directly induced IL-10-producing M2 microglia in amanner synergistic with a major extracellular matrix component of glial scar. In addition, I will
introduce you remarkable benefits of SHED-CM for the treatment of hypoxic-ischemic (HI) brain
injury in neonatal infants. HI is a major cause of acute mortality and chronic neurologic morbidity
for which there currently is no promising therapy. Taken altogether, our study demonstrates that
SHED-CM may enable the establishment of new stem-cell-based neuroregenerative therapies that do
not involve cell transplantation.
References
Sakai, K. and Yamamoto, A., et al. (2012). Human dental pulp-derived stem cells promote locomotor
recovery after complete transection of the rat spinal cord by multiple neuro-regenerative mechanisms.
J Clin Invest, 122 (1), 80–90.
Yamagata, M and Yamamoto, A., et al. (2012). Human dental pulp-derived stem cells protect against
hypoxic-ischemic brain injury in neonatal mice. Stroke, in press.
18

Session 2
Regulation of Chronic Inflammation
and Diseases

19

S2A-1
Inhibition of Sprouty2 Polarizes Macrophages
toward M2 Phenotype in Periodontitis
Terukazu Sanui, Urara Tanaka, Kyosuke Toyoda,
Takao Fukuda, Ryo Atomura, Takafumi Hamachi, and Katsumasa Maeda
Department of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science,
Kyushu University, Fukuoka, Japan
Periodontal disease is a chronic inflammatory response to bacterial pathogens involving the
supporting tissues of the teeth. The anerobic bacterium, Porphyromonas gingivalis, has been implicatedas a major etiologic agent in the development and progression of periodontitis. Lipopolysaccharide(LPS) from P. gingivalis induces pro-inflammatory cytokines in host macrophages. Macrophages canacquire distinct functional phenotypes, referred to as classically activated, pro-inflammatory
macrophages (M1) and alternatively activated, anti-inflammatory macrophages (M2). M2
macrophages are associated with homeostatic functions linked to wound healing and tissue repair.
Therefore, the inhibition of pro-inflammatory cytokine production in macrophages is the importantbiological reaction for the homeostasis after elimination of the etiologic agent.
Sprouty proteins are identified as inhibitors of fibroblast growth factor (FGF) receptor.
Specifically Sprouty2 (Spry2) functions as a negative regulator of receptor tyrosine kinases (RTKs)
signaling, and is expressed in several developing organs including kidney, lung, brain, heart, skeletal
muscle and craniofacial area. Furthermore, Spry2 has conserved function to modulate morphogenesis
in several tissues. Recently, we demonstrated that inhibition of Spry2 induced cell proliferation anddifferentiation of MC3T3-E1osteoblastic cells, while it diminished cell proliferation of GE1gingival
epithelial cells in vitro.
We report here that suppression of Spry2 polarizes J774 mouse macrophages toward alternative
macrophage activation (M2) phenotype when J774 cells are stimulated with LPS from P. gingivalis.
This polarization is defined by surface protein expression, cytokine production and arginase activity.
These data provide evidence that Spry2 inhibitors affect the inflammatory process at the level of themacrophage and shifts macrophage polarization from pro-inflammatory properties towardanti-inflammatory ones in periodontitis.
Additionally, we are confirming how this response affects the periodontal tissue regeneration or
the para-inflammation of periodontitis.

20

S2A-2
Resolution of Inflammation in
Periodontal Regeneration
Thomas E. van Dyke
Department of Periodontology, The Forsyth Institute Cambridge, MA, USA

Resolution of inflammation is an active; agonist mediated well-orchestrated return of tissue
homeostasis. Lipoxins and Resolvins are endogenous lipid mediators (eicosanoids) that actively
regulate the resolution of acute inflammation. Lipoxin A4 and Resolvin E1, derived from arachidonic
acid and eicosapentaenoic acid, respectively, are endogenous anti-inflammatory and pro-resolving
mediators that regulate leukocyte migration and enhance macrophage phagocytosis of apoptotic
neutrophils. These small lipid molecules act through specific G-protein linked receptors on
inflammatory cells. Resolution receptor agonists have been shown to have significant impact ininflammatory bone diseases, such as periodontitis and arthritis. Periodontitis is an inflammatory
disease that is initiated by oral microbial biofilm. It is the host response to the biofilm that destroys
bone (osteoclastic resorption) in the pathogenesis of the disease. Resolution agonists have been
demonstrated to promote regeneration of bone lost to disease and to prevent inflammatory bone loss
in animals. Mechanistic studies have revealed that bone cells, particularly osteoclasts and osteoblasts,
express functional resolution receptors on their surface that regulate RANKL:OPG ratios and theconsequent osteogenic and osteolytic responses. The potential for therapeutic benefit of resolution
agonists in clinical periodontal regeneration is being examined in large animal models.

21

S2A-3Mechanisms Linking Periodontitis toAtherosclerotic Diseases- Oral Infection and Lipid Metabolism Koichi Tabeta
Department of Oral Biological Science,
Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan

Coronary heart disease (CHD) is the leading cause of death in Japan and other developed countries.
The major pathway underlying CHD pathology is atherosclerosis. Several epidemiological studies
have suggested a link between atherosclerosis and infection/inflammation such as Chlamydia
pneumoniae infection, Helicobacter pylori infection, and periodontitis.
Our project adopted as Periodontal Medicine project in Niigata University Transdisciplinary
Research aimed to reveal underlying mechanisms linking periodontitis to atherosclerotic diseases,
which we believe gives us opportunity to develop novel clinical markers reflecting periodontal status
in terms of increasing progression of atherosclerosis or considering risk of atherothrombotic vascular
event. Using animal model has an advantage to address dissecting mechanisms of the pathogenesis
linking two diseases. Several studies have suggested that certain periodontopathic bacteria accelerate
atherogenesis in mouse model. However, the results varied except for accelerating atherogenesis dueto the study design such as infection method, infection period and genetic background. In fact, themechanisms linking cholesterol accumulation to periodontal infection-induced inflammation arelargely unknown.
To address these issues, we orally infected both C57BL/6 and hyperlipidemic
C57BL/6.KOR-Apoeshl (B6.Apoeshl) mice with Porphyromonas gingivalis and evaluatedatherogenesis, gene expression in the aorta and liver and systemic inflammatory and lipid profiles inthe blood. The changes in the expression of key genes involved in cholesterol turnover, such as liver
X receptor and ATP-binding cassette A1 were observed by P. gingivalis infection. P. gingivalis
infection had a potential role of shifting genes targeting the expression of LDL receptor, which
resulted in the disturbance of regulatory mechanisms of the cholesterol level in vitro study.
Our animal study indicated periodontal infection itself does not cause atherosclerosis, but it
accelerates it by inducing deteriorating lipid metabolism in addition to systemic inflammation and,particularly when underlying hyperlidemia or susceptibility to hyperlipidemia exists, it may
contribute to the development of coronary heart disease.

22

S2B-1Cathepsin B-dependent
Novel IL-1β Production Pathway in Microglia
Zhou Wu and Hiroshi Nakanishi
Department of Aging Science and Pharmacology,
Faculty of Dental Sciences, Kyushu University, Fukuoka, Japan
Interleukin-1β (IL-1β) promotes various chronic neurological disorders such as dementia and pain.
Activated microglia are the main cellular source of IL-1β in the brain. However, it remains unclear
why the IL-1β production during acute inflammatory reactions becomes chronic. Production of
IL-1β is tightly regulated. Two signals mediated by Toll-like receptors (TLRs) and Nod-likereceptors (NLRs) are generally required for the production and secretion of IL-1β. TLRs lead to thetranscriptional induction of pro-IL-1β though activation of nuclear factor (NF)-κB. The NLRP3
inflammasome contributes to the conversion of pro-caspase-1 to mature caspase-1.
Recently, we have found that chromogranin A (CGA), a neuroendocrine secretory proteins, canpotently induce the production and secretion of IL-1β by microglia in a cathepsin B (CatB), alysosomal cysteine protease, dependent manner. CGA activated NF-κB to drive pro-IL-1βtranscription through the activation of TLR4 and its co-receptor CD14. CGA also bound on the
scavenger receptor class A to form phagolysosomes and then increasingly induced expression of
CatB. CatB in turn induced proteolytic maturation of pro-caspase-1 in phagolysosomes of microglia
without activation of the NLRP3 inflammasome (Terada et al., 2010; Sun et al., 2012).
We further explored the roles of CatB in age-dependent impairment of hippocampal long-term
potentiation (LTP), which is the cellular basis for learning and memory. Chronic systemic
inflammation drives microglia to produce IL-1β (Wu et al., 2005, 2007, 2008) and impaired theformation of hippocampal LTP (Liu et al., 2012) even in the middle-aged animals. Minocycline, aknown microglial activation inhibitor, significantly suppressed microglial production of IL-1β andrestored the formation of LTP in the middle-aged animals subjected to systemic inflammation. The
age-dependent impairment of LTP was not observed in the aged CatB-/- mice. Furthermore, CatB-/mice was resistant to the chronic inflammatory pain. Interestingly, CGA could cause chronic pain
through induction of CatB-dependent IL-1β production by spinal microglia (Sun et al., 2012).
We conclude that CGA is a CatB-dependent endogenous potent IL-1β inducer of microglia.
Therefore, the CGA-CatB pathway can induce chronic inflammation, which is associated withage-dependent cognitive deficits and inflammatory pain.
23

S2B-2
Immune Adaptation in the Central Nervous System in
Response to Systemic Infections
U. Püntener, S.G. Booth, V.H. Perry, and J.L. Teeling
CNS Inflammation Group, Centre for Biological Sciences,
University of Southampton, Southampton, UK
Systemic bacterial infections are a common cause for morbidity and mortality in the elderly, and inparticular those with a neurodegenerative disease, but the mechanism underlying these observations
remain unclear. Experimental models of neurodegeneration have shown that LPS-induced systemic
inflammation increases neuronal damage, a process that is believed to be mediated by activation ofprimed microglia. The effects of a real systemic bacterial infection on the innate immune cells of
healthy, aged and diseased brain are less well described, and therefore, we have investigated theacute and long term effects on microglia and brain vascular endothelial cells in response to an
experimentally induced bacterial infection. Young (3m), aged (18m) and mice with a pre-existingneurodegenerative disease, were given a single systemic injection of live Salmonella typhimurium.
Inflammatory cytokines were measured in serum, spleen and brain, and microglia and endothelial
cell phenotypes studied by immunohistochemistry. Serum cytokine levels (i.e. IFNγ, IL-1β, IL-6)
peaked at day 7 after infection with S. typhimurium and were significantly reduced three weeks postinfection. In contrast, brain cytokine levels (IL-1β, IL-12) in S. typhimurium infected mice increased
over three weeks, following high circulating IFN-γ levels. Furthermore, systemic bacterial infection
was associated with a heightened response to a secondary challenge in the brain, suggesting primingof innate immune cells in the CNS in response to a systemic bacterial infection. Infection of aged
mice and mice with a pre-existing neurodegenerative disorder showed increased phenotype changesof microglial cells, increased sickness behaviours, increased metabolic changes and a significant
increase of infiltrating CD3+ T cells into the brain parenchyma.
These studies reveal that the innate immune cells in the brain are activated by systemic
infections, which may lead to prolonged and damaging cytokine production. This exacerbation of theinflammation and the priming of the brain innate immune cells may have a profound effect on the
progression of pre-existing neurodegenerative disease.

24

S2B-3Preceding Role of Satellite Glia in Nerve Injury-Induced
Microglia Activation and Pain Hypersensitivity
Sung Joong Lee
Department of Neuroscience and Oral Physiology, and Dental Research Institute,
Seoul National University School of Dentistry, Seoul, Korea

Increasing evidence supports the notion that spinal cord microglia activation plays a causal role in thedevelopment of neuropathic pain after peripheral nerve injury; yet the mechanisms for microglia
activation remain elusive. Previously, we have reported that nerve injury-induced microglia
activation and subsequent pain hypersensitivity are attenuated in toll-like receptor 2 (TLR2) andNox2 knockout mice, suggesting an important role of TLR2-Nox2-ROS signaling pathways in thenerve injury-induced spinal cord microglia activation and neuropathic pain. More recently, we foundthat proinflammatory gene expression and macrophages infiltration in the dorsal root ganglion(DRG) after nerve injury are also reduced in the TLR2 knockout mice. In these mice, the nerve
injury-induced spontaneous pain behavior was also compromised. However, macrophages
infiltration in the DRG per se has minimal effects on the spinal cord microglia activation in thenerve-injured mice suggesting putative role of satellite glial cells (SGC) but not macrophages in thedevelopment of neuropathic pain. To address the function SGC in the nerve injury-induced spinal
cord microglia activation and pain hypersensitivity, we generated IKKf/f/CNPase-Cre mice, in which
IKK/NF-kB-dependent SGC activation is abrogated. In these mice, we found that nerve
injury-induced spinal cord microglia activation and pain hypersensitivity was significantly attenuatedcompared to control mice. These data demonstrate that SGC activation is preceding to the spinal cord
microglia activation and pain hypersensitivity due to peripheral nerve injury.

25

Session 3
Oral Health Science

26

S3-1
Dental Plaque Development in Adults withNo Previous Experience of Dental Caries
Toru Takeshita
Section of Preventive and Public Health Dentistry, Division of Oral Health, Growth andDevelopment, Kyushu University Faculty of Dental Science, Fukuoka, Japan

Dental caries is now recognized to be caused by dental plaque microbiota developed into ahighly acidogenic community, rather than an emergence of a single or multiple pathogens. To
explore a ‘healthy’ development pattern of plaque bacterial community, we compared an
assembly of plaque bacterial community in young adults with and without caries experience.
Plaque samples accumulated for 1, 2, 3, 4, 5 and 7 days were collected from 19 subjects(mean age, 23 ± 6 years) using a retrievable hydroxyapatite disk model. In both caries-freeand -active subjects, total bacterial amount and microbial diversity steadily increased overtime. On the other hand, the bacterial amount in caries-free subjects increased more slowly
than caries-active subjects. The early plaque of caries-free subjects exhibited a more diverse
community with higher proportions of Neisseria, Gemella and TM7 genera incertae sedis
compared with caries-active subjects, and the relative abundances of Granulicatella,Actinomyces and Veillonella less markedly increased in the later stage. This study identified a
succession pattern of plaque bacterial community in caries-free adults. Our results suggestthat inter-individual difference of species arrangement within plaque is associated withsusceptibility to dental caries.

27

S3-2
Metagenomics:
An Introduction and Applications to Dentistry
Todd D. Taylor
Laboratory for MetaSystems Research, Computational Biology Research Core,
RIKEN Quantitative Biology Center, Yokohama, Japan
Metagenomics (sometimes referred to as environmental genomics, ecogenomics orcommunity genomics) is the study of metagenomes, genetic material recovered directly fromenvironmental samples as opposed to that grown from clonal cultures. Living organisms
generally do not reside in isolation, but in most cases live in harmony with many otherspecies. Human beings depend on several diverse microbial-rich communities, for example
in the intestinal tract and oral cavity, and on the skin, for normal health maintenance. Just as
importantly, in the environment, complex microbial communities in soil, water, air, andelsewhere are responsible for maintaining the health of our planet. Only a small fraction ofthese microbes can be cultivated in the laboratory, so currently very little is known about
them. Without understanding this microbial compliment, we cannot truly understand the vast
majority of life on earth, or the human genome itself. Identification of the microbial proteins,both known and novel, and hence their functions in these sometimes extreme environments
will provide clues for understanding how they work, how the microbes cooperate andcompete with one another, how the microbes interact with the host or other organisms, andhow we may manipulate them to our advantage in order to improve human health and better
manage our environment. The ultimate goal of metagenomic research is to be able to modeland predict the behavior and interactions of whole environments of organisms. Eventually wewould like to understand how the entire 'meta-system' (host genome, microbes, viruses, othereukaryotes, metabolites, environment, etc.) works and to be able to predict how perturbationsin the system impact its various parts. By combining new high-throughput sequencing
technologies and next-generation supercomputing capabilities we will finally able to study
these complex environments on an enormous scale. I will present an introduction tometagenomics and give some examples of how it is being applied to the field of dentistry andoral health.

28

S3-3
Microbiome-wide Association Studies (MiWAS)
for Hunting down the Obesity Bugs
Liping Zhao
School of Life Sciences and Biotechnology, Shanghai Jiao Tong University,
Shanghai, China

Accumulating evidence indicates that gut microbiota may play a pivotal role in onset andprogression of obesity and insulin resistance, the core conditions of metabolic syndrome (MetS) via
two different but complementary pathways, i.e. regulation of energy metabolism and provocation ofchronic inflammation. However, these mechanistic findings are obtained almost exclusively withrodent models. Their relevance to humans still remains a question. It is also controversial whether
the obesity-associated changes of gut microbiota happen at broad taxonomic-level or are more
relevant with specific phylotypes. Microbiome-wide association studies, MiWAS, try to answer thisquestion by correlating structural variations of gut microbiota with variations of host metabolic
phenotypes. The structural variations can be characterized with next-generation sequencing of either
the phylogenetic marker gene 16S rRNA or the whole microbiome. Host metabolic phenotypes canbe captured as bodyweight, body fat content, insulin sensitivity, pro- or anti-inflammatory markers,
and lipo-polysaccharide-binding protein (LBP), etc. Multi-variate statistics are used to identify
associations between phylotypes/functional genes and host metabolic parameters. Due to thestructural elasticity of gut microbiota, nutritional or drug interventions can be implemented tomodulate gut microbiota of a diseased population to achieve improvement of host metabolic
phenotypes. Samples collected from the same host before and after the intervention can yield muchstronger associations than cross-sectional strategies such as those in classical genome-wide
association studies (GWAS). We employed this MiWAS strategy in dietary therapy of morbid
obesity/diabetes in humans to show that specific phylotypes which are more relevant with MetS canbe identified, isolated and demonstrated to be causatively contributing to MetS development in
humans.

29

Session 4
PhD Student Session

30

S4-1
Long Term Cryopreserved Dental Pulp Tissues of Exfoliated Deciduous
Teeth is a Feasible Stem Cell Resource for Regenerative Medicine
Lan Ma1,2, Yusuke Makino1,3, Haruyoshi Yamaza2, Kentaro Akiyama4, Guangtai
Song5, Toshio Kukita1, Songtao Shi4, Kazuaki Nonaka2, Takayoshi Yamaza1
Departments of 1Molecular Cell Biology and Oral Anatomy, 2Pediatric Dentistry and 3Fixed
Prothodontics, Graduate School of Dental Science, Fukuoka, Japan.
5
Department of Pedodontics, School of Stomatology, Wuhan University, Wuhan, China.
4Center for Craniofacial Molecular Biology, Herman Ostrow School of Dentistry of USC,
University of Southern California, CA, USA.
Human exfoliated deciduous teeth have been considered to be a promising source for stem cell-based
regenerative therapy because they contain unique postnatal mesenchymal stem cells (MSCs) capableof multipotency and immunomodulatory function. However preservation technique of the teeth has
not been developed for future therapy although it is impractical to isolate stem cells from human
exfoliated deciduous teeth (SHED) immediately after exfoliated. In this study, we tested whether thatcryopreserved dental pulp tissues of human exfoliated deciduous teeth contain retrievable SHED andcompared the therapeutic efficacy of SHED isolated from the cryopreserved tissues (SHED-Cryo)with SHED isolated from the fresh tissues (SHED-Fresh). SHED-Cryo isolated from thecryopreserved dental pulp tissues for over 2 years (25-30 months) owned MSC properties including
clonogenicity, self-renew, MSC marker expression, multipotency and in vivo tissue regenerative
capacity and expressed similar MSC properties of SHED-Fresh. To examine the immunomodulatory
functions of SHED-Cryo on human systemic lupus erythematosus (SLE), SHED-Cryo wereintravenouslytransplanted
into
human
SLE
model
MRL/lprmice.
Systemic
SHED-Cryo-transplantation improved SLE-like syndrome including short lifespan, elevatedautoantibody levels and nephritis-like renal dysfunction. SHED-Cryo amended elevated levels of
IL-17-secreting helper T (Th17) cells in MRL/lpr mice and directly inhibited the differentiation of
Th17 cells under in vitro co-culture system. SHED-Cryo expressed similar immunomodulatory
efficacy to SHED-Fresh. Furthermore, we examined SHED-Cryo-mediated bone regeneration incalvarial bone-defect model mice. SHED-Cryo implantation was able to repair the defect with
substantial bone formation resembled to SHED-Fresh implantation. These data suggest thatcryopreservation of deciduous teeth provides a desirable clinical approach for potential therapeutic
applications.

31

S4-2
Proteomic Approach to Understanding Endocytosis of Amelogenin
Kyosuke Toyoda, Urara Tanaka, Takao Fukuda, Terukazu Sanui, Ryo Atomura,
Takafumi Hamachi and Katsumasa Maeda
Department of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science,
Kyushu University

Objectives: Emdogain (enamel matrix derivative, EMD) is used to stimulate regeneration of
periodontal tissues. However, the exact molecular mechanisms of periodontal regeneration remain
largely unknown. In this study, we investigated the proteins bound to amelogenin, the major (>95%)
component of EMD, to elucidate the molecules involved in the periodontal tissue regeneration.
Methods: We firstly examined the pattern of cellular internalization of amelogenin by addingrAMEL exogenously to SaOS-2 osteoblastic cells and human PDL cells. Secondary, we screened theproteins bound to amelogenin by GST pull down assay followed by proteomic analysis. Purified
GST fusion amelogenin immobilized on glutathione was incubated with the cell lysates. Afterextensive washing, bound proteins ware resolved by using two-dimensional polyacrylamide gelelectrophoresis (2D-PAGE), and protein spots were visualized by CBB staining and silver staining.
To identify proteins, the major spots were excised from gel, and subjected to matrix-assisted laser
desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis. The identified
protein was examined by Western blot methods in the SaOS-2 osteoblastic cells.
Results: Confocal microscopy indicated that rAMEL attached to the plasma membrane andinternalized in both cell lines. Our proteomic approach showed number of proteins bound toamelogenin. We identified 16 proteins in lysate of SaOS-2 osteoblastic cells, and 13 proteins in themembrane fraction of human PDL cells.
Conclusions: We identified new amelogenin binding proteins. Several membrane-associatedproteins were identified as amelogenin binding partners. They include endoplasmic reticulum
proteins, mitochondrial membrane proteins, and nuclear proteins. We identified Grp78/Bip protein
both in cytosolic and membrane-enriched fractions.

32

S4-3
The Role of Sprouty2 Protein in Osteoblasts and Periodontal Ligament Cells
Urara Tanaka, Kyosuke Toyoda, Terukazu Sanui, Takao Fukuda, Ryo Atomura,
Takafumi Hamachi, and Katsumasa Maeda
Department of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science,
Kyushu University

Objectives: Sprouty proteins are identified as inhibitors of fibroblast growth factor (FGF) receptor.
Specifically Sprouty2 functions as a negative regulator of receptor tyrosine kinases (RTKs) signaling.
On the other hand, osteoblasts and PDL (periodontal ligament) cells are critical participants during
the periodontal tissue regeneration process. However, the regenerative mechanisms induced by
Sprouty2 in osteoblasts and PDL cells remain largely elusive. The purpose of this study was toinvestigate whether Sprouty2 could be new therapeutic targets for periodontal tissue regeneration.
Methods: MC3T3-E1 osteoblastic cells and human PDL cells were transfected with a control
plasmid, or a dominant negative mutant plasmid of Sprouty2. For immunoblot analysis, cells wereextracted after growth factor stimulation and proteins were detected by various antibodies. Using
MTT assay, growth factor-induced cell proliferation was measured in transfected osteoblasts and
PDL cells. A scratch wound healing assay was conducted with transfected PDL cells stimulated by
growth factors. In order to evaluate osteoblast differentiation, ALP assay was performed using
Sprouty2 dominant negative mutant MC3T3-E1 cells.
Results: Transduction of Sprouty2 mutation enhanced bFGF+EGF-induced ERK activation in
MC3T3-E1 cells and PDL cells. In addition, when osteoblastic cells and PDL cells were stimulatedwith bFGF+EGF, cell proliferation assay revealed that Sprouty2 mutant cells proliferated faster thancontrol cells. Migration activity of bFGF+EGF-stimulated Sprouty2 mutant PDL cells was slightly
higher than control cells. ALP activity was enhanced in Sprouty2 mutant MC3T3-E1 cells.
Conclusions: We demonstrated that Sprouty2 dominant negative mutants activate cell proliferationof MC3T3-E1 osteoblastic cells and PDL cells stimulated by bFGF+EGF, and that suppression of
Sprouty2 induces cell migration of PDL cells and ALP activity of osteoblasts. In other words, the useof Sprouty2 inhibitors and bFGF+EGF to bony defects may effectively allow alveolar bone to grow.

33

S4-4
Sprouty2 Controls Osteoblast Proliferation and Differentiation
via MAPK and Smad Pathways
Tomohiro Onimura1, Takaharu Taketomi1, Daigo Yoshiga2, Daichi Muratsu1,
and Seiji Nakamura1
1

Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University
2
Division of Oral and Maxillofacial Reconstructive Surgery, Kyushu Dental College

Objectives: Fibroblast growth factor (FGF) and bone morphogenetic protein (BMP) play essential
roles for bone formation and osteoblast activity via mitogen-activated-protein-kinase (MAPK) andSmad pathways. Sprouty family is known as intracellular inhibitor of the FGF signaling, and four
orthologs of Sprouty have been identified in mammals. Among them, Sprouty2 and Sprouty4 double
knockout mice show the abnormal teeth, mandible, and limb. Furthermore, MAPK pathway has been
also reported to participate in BMP signaling. In this study, we clarified the role of Sproutys for the
proliferation and differentiation of osteoblast.
Materials and Methods: The expression of Sproutys was examined by RT-PCR, in humanosteoblast like cells (SaOS-2). After Myc-tagged Sprouty2 plasmid was transfected into SaOS-2, thecell proliferation was determined by WST-8 assay. In SaOS-2, each phosphorylation of MAPK and
Smad1/5/8 by FGF or BMP-2 stimulation was investigated by western blotting, and the expression of
RUNX2 and ALP known as transcription factor for osteoblast differentiation was analyzed byreal-time PCR.
Results: mRNA of Sprouty2 and Sprouty4 was induced by FGF stimulation in SaOS-2. In terms ofthe degree of cell proliferation, the number of cells overexpressed with Sprouty2 decreased
compared to that of controls. The phosphorylation of MAPK and Smad1/5/8 were downregulated in
SaOS-2 transfected with Sprouty2. Furthermore, Sprouty2 suppressed the mRNA expression of
RUNX2 and ALP.
Conclusion: These results suggest that Sprouty2 can negatively control osteoblast proliferation anddifferentiation by downregulating MAPK and Smad pathways, and also suppress the induction oftranscription factors for osteoblast differentiation.

34

S4-5
Cathepsin S Contributes to the Development of Chronic Pain
Xin-Wen Zhang, Yoshinori Hayashi, Ryo Okada, Zhou Wu, andHiroshi Nakanishi
Department of Aging Science and Pharmacology, Faculty of Dental Sciences,
Kyushu University
Neuropathic pain is a significant clinical problem because two well-established classes of analgesics,
opiates and the nonsteroidal anti-inflammatory drugs (NSAIDs), have limited efficacy. The lack ofsuitable therapy has motivated us to identify novel pharmacological targets for neuropathic pain. It is
well known that spinal microglia play essential roles in the induction of neuropathic pain. Cathepsin
S (CatS) is a member of cysteine protease family, which is preferentially expressed in cells ofmononuclear-phagocytic origin. We may therefore speculate that CatS plays specific roles in thereaction of microglia and/or monocytic cells during the development of neuropathic pain. In thepresent study, we have thus examined the effects of CatS-deficiency and a specific CatS inhibitor on
the development of neuropathic pain.
CatS-deficiency significantly attenuated the development of neuropathic pain and the mean
number of Iba1-positive cells accumulated in the dorsal spinal cord after nerve injury. CatS
significantly increased in spinal microglia from 1 day after nerve injury. It is generally believed thatmicroglia are involved especially in the induction of neuropathic pain (up to 3 days after nerve
injury). However, we found that increase in the cell number of Iba1-positive cells peaked at 14 days.
Furthermore, Z-FL-COCHO, a specific inhibitor of CatS, significantly reversed already establishedneuropathic pain. Therefore, some populations of Iba1-positive cells in the dorsal spinal cord may beoriginated by infiltrated inflammatory monocytes because CatS is essential for the recruitment ofinflammatory monocytes through proteolytic degradation of the basal membrane. To examine this,we conducted immunoblot analyses of cathepsin F (CatF), which is expressed inmonocytes/macrophages, but not in microglia. We found the expression of CatF in the spinal cord ofwild-type, but not CatS-deficient mice, after nerve injury. Finally, we examined the effect of
splenectomy on the development of neuropathic pain, because spleen is a major source of monocyte
development after injury. The splenectomy significantly inhibited the development of neuropathic
pain.
Our results strongly suggest the prevention of monocyte recruitment by CatS inhibitors can be auseful therapeutic strategy for the pain relief.

35

S4-6
Zoledronic Acids Enhance Lipopolysaccharide-Stimulated
Inflammatory Reaction in Macrophages
Daichi Muratsu1, Daigo Yoshiga2, Takaharu Taketomi1, Tomohiro Onimura1,
and Seiji Nakamura1
1

Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University
2
Division of Oral and Maxillofacial Reconstructive Surgery, Kyushu Dental College

Purpose: Critical side effects of Bisphosphonates (BP) are reported to occur bisphosphonate-related
osteonecrosis of the jaw (BRONJ). Recent study reported that the initiation of BRONJ was involvedin the innate immunity. The activation of immune cells is induced by inflammation, and excessive
inflammation causes tissue destruction. In this study, to examine BRONJ correlate innate immunity,
we used the macrophage which is one of the most important cells in the innate immunity, andanalysis the effects of BPs on the macrophage.
Material, and Methods: A murine macrophage cell line, RAW264.7 was pretreated with Zoledronic
acid (ZOL) which is a nitrogen-containing bisphosphonate, and analyzed the expression of Toll-likereceptor 4 (TLR4). To determine the effects of inflammatory mediators, inflammatory cytokines
expression was investigated by real-time PCR and nitric oxide (NO) production by Griess methods.
The activation of IκB-α as an intracellular signaling molecule was examined by Western blot
analysis. LPS-induced apoptosis was measured by flow cytometry.
Result: The TLR4 expression showed no significant difference by pretreated with ZOL comparedwith the control. On the other hand, cytokines and NO production were increased by pretreated with
ZOL. Furthermore, ZOL induced the activation of NF-κB through the enhancement of IκB-αdegradation. LPS-induced apoptosis was increased by pretreated with ZOL.
Conclusion: These results suggest that ZOL induce the activation of innate immunity, and the bone
destruction found in BRONJ may be caused by the excessive production of inflammatory cytokines.

36

S4-7
Roles of the Bone-Derived Osteocalcin as a GLP-1 Secretagogue
Yu Yasutake, Akiko Mizokami, and Masato Hirata
Laboratory of Molecular and Cellular Biochemistry,
Faculty of Dental Science, Kyushu University

It has recently been reported that bone is an endocrine organ, since osteocalcin (OC) produced byosteoblasts is revealed to trigger the secretion of insulin; OC in uncarboxylated form (ucOC) but notcarboxylated GlaOC acts as a potent insulin secretagogue, regulating energy metabolism byincreasing energy expenditure and insulin sensitivity in the target organs. Incretin hormone
glucagon-like peptide 1 (GLP-1) is a gastrointestinal hormone secreted by enteroendocrine L cells inthe small intestine, and has a numerous physiological actions including potentiation of glucose
stimulated insulin secretion, enhancement of β-cell growth and survival.
We here found that mouse small intestine and mouse enteroendocrine cell line STC-1 cells
expressed Gprc6a, a putative ucOC receptor. Serum GLP-1 was increased by ucOC at both
intraperitoneal and oral applications, but such an effect was not observed with GlaOC. STC-1 cells
also responded to ucOC to secrete GLP-1 into the culture medium. GlaOC was effective only in thecase of oral administration. Moreover, serum insulin level was increased in response to ucOC
application, which was inhibited by GLP-1 receptor antagonist exendin(9-39), indicating that theinsulin-releasing effect of ucOC is in part attributed to the action of GLP-1 released from the gut.
These findings indicate that ucOC acts through its receptor in the small intestine, triggering
secretions of GLP-1 and insulin.

37

S4-8
Swallowing Action May Attenuate Fentanyl-Induced CoughSaori Sako, Shoko Itoda, Naho Zen, Noriko Karube, Eiji Sakamoto,Takeshi Yokoyama
Department of Dental Anesthesiology, Faculty of Dental Science,
Kyushu University

Aim: Intravenous fentanyl often induces cough during induction of anesthesia. This fentanyl-induced
cough is not always benign, and sometimes spasmodic and/or explosive. But the mechanism is still
not clear. We found swallowing action, when it was asked at induction of anesthesia to reduce
pharyngeal discharge for facilitating laryngoscopy, attenuated that fentanyl-induced cough. In thecurrent study, we investigated the relation between occurrence of fentanyl-induced cough andswallowing action retrospectively.
Method: We checked the anesthesia records of patients, 21-60 year-old, ASA-PS I or II, who
underwent head and neck surgery from October 2010 to May 2012. Subjects were selected on
condition that their anesthesia was induced with intravenous fentanyl first. Additionally, they weredivided into two groups, swallowing action (S) when fentanyl was administered and no swallowing
action (non-S). Patient’s background, dose of fentanyl and occurrence of cough were investigatedfrom their records. The data were compared between the groups using Mann-Whitney U-test or Chi
square test. A p-value less than 0.05 were considered as the level of statistical significance.
Result: Forty-eight patients were suitable for this study. There were no significant differences intheir background and dose/weight of fentanyl between S group (n=23) and non-S group (n=25). In S
group, 4 of 23 patients (17.4%) had cough, while 13 of 25 patients (52.0%) had cough in non-Sgroup. The occurrence of fentanyl-induced cough was significantly less in S group (p=0.027). Thisswallowing action was a simple and effective method to reduce fentanyl induced cough.
Conclusion: Swallowing action, when fentanyl is administered intravenously, may be useful to avoid
cough during induction of anesthesia.
Key Words: fentanyl-induced cough, swallowing action

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S4-9
Increasing Mechanical Strength of Carbonate Apatite Blockby Ca Salt Introduction

Jane Harland1, Kanji Tsuru1, Michito Maruta2, Giichiro Kawachi1,
Kunio Ishikawa1
1
2

Department of Biomaterials, Faculty of Dental Science, Kyushu University

Section of Bioengineering, Department of Dental Engineering, Fukuoka Dental College

Carbonate apatite (CO3Ap) is an ideal bone substitute as it has excellent osteoconductivity andbioresorbability. Research has shown that CO3Ap block can be fabricated using calcium
carbonate (calcite: CaCO3) block as a precursor, based on the dissolution-precipitation reaction,and the mechanical strength of CO3Ap block was increased by introduction of calcium lactate.
However, the resultant CO3Ap block still needs improvement, as higher mechanical strength isdesirable for clinical use in load-bearing sites. The objectives of this study were to investigate
the feasibility of using calcium acetate in the Ca salt introduction method and examine theeffect of reducing porosity, by the addition of extra CO3Ap crystals, on the mechanical strengthof CO3Ap block. Calcium acetate (1 M) was introduced into the micropores of CO3Ap block
under vacuum at room temperature. Next, the CO3Ap block was exposed to carbon dioxide(CO2) gas under atmospheric pressure for 14 days to carbonate the calcium salt to calcite.
Finally, the CO3Ap block was immersed in 1 M disodium hydrogen phosphate (Na2HPO4)
aqueous solution at 60°C for 14 days to transform the calcite into CO3Ap. Results suggest that
the Ca salt introduction method enables fabrication of reinforced CO3Ap block with greater
mechanical strength than the original CO3Ap block and sufficient strength to be a promisingbone substitute in load-bearing areas.

39S4-10Hydrothermal Conversion of Calcite Foam to Carbonate Apatite
Tram N. X. T.1, Maruta, M.2, Tsuru, K.1, Matsuya, S.2 and Ishikawa, K1.
1

2

Department of Biomaterials, Faculty of Dental Science, Kyushu University
Section of Bioengineering, Department of Dental Engineering, Fukuoka Dental College

We have previously reported an inverse ceramic foam method aiming to improve mechanicalstrength of calcite foam that has interconnected porous structure. The modified foam was obtainedby multiple polyurethane coating. In previous study, calcium hydroxide (Ca(OH)2) slurry was poured
into the foam. After drying, the foam was burnt out to remove polyurethane and to carbonate
Ca(OH)2 under mixed O2-CO2 atmosphere. Ca(OH)2 was completely converted to calcite. Those
calcite foams are the useful precursors for the preparation of the three-dimensional (3D)interconnected porous carbonate apatite (CO3Ap) foam. Therefore, we investigated the hydrothermal
treatment of calcite foam to CO3Ap foam in phosphate solution. The results indicated that theconversion ratio depends on the degree of porosity of calcite precursor. The CO3Ap foam showed theincrease in the compressive strength compared to conventional method. So, it is concluded that
CO3Ap foam fabricated in this study could be a good candidate for the bone replacement material inclinical application.

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