the 6th international joint symposium on“dental and craniofacial morphogenesis
and tissue regeneration”
and“oral health science”
4­5th March, 2011
at Fukuoka Recent Hotel
Fukuoka, Japan（BMC Dev Biol 10:115, 2010 より引用）

Program & Abstracts

○ 会期：平成２３年３月４日〜５日○ 会場：福岡リーセントホテル（下図参照）○ 主催：九州大学 大学院 歯学研究院「口腔組織の再生・再建医療」プロジェクト「口腔健康科学」プロジェクト
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：坂井 英隆
九州大学 大学院 歯学研究院
口腔顎顔面病態学講座 口腔病理学研究分野〒812-8582 福岡市東区馬出３丁目１番１号
電話：092-642-6325〜6328
ファックス：092-642-6329
E-mail: hsakaiop@dent.kyushu-u.ac.jp

Program=4th March, 2011=
Opening remarks
12:50 - 12:55 Masato Hirata ( Council member of Kyushu University)
12:55 - 13:00 Hiroshi Nakanishi ( Dean, F aculty of Dental Science, Kyushu University)
Special Lectures (Dental and Craniofacial Morphogenesis and Tissue
Regeneration)
Chairpersons: Katsumasa Maeda (Section of Periodontology, Kyushu University)
Ichiro Takahasi (Section of Orthodontics and Dentofacial Orthopedics,
Kyushu University)SL­1 13:00 ­ 13:50
FGF­2 stimulates Periodontal Regeneration
Shinya Murakami
Professor and Chairman, Department of Periodontology, Graduate School of Dentistry,
Osaka UniversitySL­2 14:00 ­ 14:50
Periodontal Disease and Rheumatoid Arthritis
P. Mark Bartold
Professor and Director, Colgate Australian Clinical Dental Research Centre, Universityof AdelaideSL­3 15:00 ­ 15:30
The new approaches for bone regeneration through the activation of
Wnt/β­catenin signaling pathway
Etsuko Matsuzaki1, Fumi Takahashi­Yanaga2, Katsumasa Maeda11Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science,2
Department of Clinical Pharmacology, Faculty of Medical Sciences, Kyushu
University

Break

1

Oral Session (Part I)
Chairperson: Hidefumi Maeda (1­1~1­3)
1­1 16:00 – 16:10
The roles of stretch loading in human periodontal ligament cells
Satoshi Monnouchi1, Hidefumi Maeda2, Shinsuke Fujii2, Atsushi Tomokiyo1,
Naohisa Wada2, Kiyomi Kono1, and Akifumi Akamine1, 2
1
Department of Endodontology and Operative Dentistry, Division of Oral
Rehabilitation, Faculty of Dental Science, Kyushu University
2
Department of Endodontology, Kyushu University Hospital1­2 16:15 – 16:25
Anti­Dkk1 antibody induces osteoblast differentiation through the Wnt/β­cateninsignaling pathway
M. Kobayashi, E. Matsuzaki, T. Hamachi, S. Hiratsuka, Y. Aida, and K. Maeda
Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Sciences,
Kyushu University
1­3 16:30 – 16:40
Sphingosine­1­phosphate activates the Wnt/β­catenin signaling pathway andincreases osteoprotegerin gene expression
S. Hiratsuka, E. Matsuzaki, M. Kobayashi, T. Hamachi, Y. Aida, and K. Maeda
Section of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Sciences,
Kyushu University
Chairperson: Haruyoshi Yamaza (1­4~1­5)1­4 16:45 – 16:55
Functional implication of thymosin beta­4 in the morphogenesis of the tooth germ
via the regulation of odontogenic gene expressions
Yukiko Ookuma1, 2, Ieyoshi Kobayashi1, Tamotsu Kiyoshima1, Kengo Nagata1,
Hiroko Wada1, Kazuaki Nonaka2 and Hidetaka Sakai1
１
Laboratory of Oral Pathology, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University
2
Section of Pediatric Dentistry, Division of Oral Health, Growth and Development,
Faculty of Dental Science, Kyushu University2

1­5 17:00 – 17:10
Expression pattern and possible function of thymosin beta­10 in developing tooth
germ compared with thymosin beta­4
Maho Shiotsuka1, 2, Hiroko Wada1, Tamotsu Kiyoshima1, Ieyoshi Kobayashi1,
Kengo Nagata1, Hiroaki Fujiwara1, Ichiro Takahashi2 and Hidetaka Sakai1
１
Laboratory of Oral Pathology, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University
2
Section of Orthodontics and Dentofacial Orthopedics, Division of Oral Health,Growth and Development, Faculty of Dental Science, Kyushu University
Chairperson: Hiroshi Takeuchi (1­6~1­7)
1­6 17:15 – 17:25
Involvement of PRIP in the bone formation
Koshiro Tsutsumi1, 2, Miho Kotani1, Ayako Murakami1, Miho Matsuda1
and Masato Hirata1
1
Laboratory of Molecular and Cellular Biochemistry, Faculty of Dental Science,
Kyushu University
2
Division of Fixed Prosthodontics, Faculty of Dental Science, Kyushu University
1­7 17:30 – 17:40
Effects of allergic inflammation on orthodontically induced tooth root resorption
Naohisa Murata1, 2 Hideki Ioi2, Masahiro Ohuchi1,2, Tomoka Takao1, Hanako Oida1,
Takayoshi Yamaza1, Ichiro Takahashi2, Mizuho A. Kido1
1
Department of Molecular Cell Biology and Oral Anatomy, Graduate school of Dental
Science, Kyushu University
2
Department of Orthodontics, Graduate school of Dental Science, Kyushu University
Chairperson: Yasunori Ayukawa (1­8~1­10)1­8 17:45 – 17:55
Stress distribution of maxillary implant supported overdentures in vitro
Asuka Haruta, Yasuyuki Matsushita, Yoshihiro Tsukiyama and Kiyoshi Koyano
Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation,
Faculty of Dental Science, Kyushu University
1­9 18:00 – 18:10
Resistant factors of the conservative therapy to BRONJ
Goto Y1, Kawano S 1, Hayashida J 1, Ikari T 2, Kurahara S 2, Toyoshima T 1,
Minamizato T1, Kiyosue T1, Matsubara R1, Oobu K1, Takenoshita Y2, Mori Y2,
Shiratsuchi Y1, Nakamura S1
1
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University
2
Section of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University3

1­10 18:15 – 18:25
Ca2+ stimulates the expression of bone morphogenetic protein­2 throughcalcium­sensing receptor in fibroblasts
Yoshie Yahara
Section of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University
19:00 – 21:00
Getting Together Party(MC: Masato Hirata, Council member of Kyushu University)4

=5th March, 2011=
Opening remarks
9:50 - 9:55 Seiji Nakamura (Section of Oral and Maxillofacial Oncology, Kyushu
University)
Special Lectures (Oral Health Science)

Chairperson: Yoshihisa Yamashita (Section of Preventive and Dental Public Health,
Kyushu University)SL­4 10:00 – 10:50
Re­Engineering of Gut Microbiota with Traditional Chinese Medicinal Foods forPreventive Management of Metabolic Diseases
Liping Zhao
Laboratory of Molecular Microbial Ecology and Ecogenomics, School of Life Sciences
and Biotechnology, Shanghai Jiao Tong University, Shanghai Center for Systems
Biomedicine, 800 Dongchuan Road, Shanghai, 200240, CHINA
SL­5 11:00 – 11:50
Effects of butyric acid, the bacterial metabolite, on the periodontal tissue andreactivation of latent HIV­1
Kuniyasu Ochiai
Department of Microbiology, Nihon University School of Dentistry

Lunch

Oral Session (Part II)
Chairperson: Zhou Wu (2­1~2­3)
2­1 13:00 – 13:10
Phosphatidylserine containing liposomes facilitate osteogenic maturation and boneformation of rats
Hong Mei Ma1, Zhou Wu1, Yin Ji Li2, Toshio Kukita2 and Hiroshi Nakanishi1
1
Department of Oral Aging Science and Pharmacology, Faculty of Dental Science,
Kyushu University
2
Department of Molecular Cell Biology and Oral Anatomy, Faculty of Dental Science,
Kyushu University5

2­2 13:15 – 13:25
A possible involvement of cathepsin B in nociceptive pain through the processing
and secretion of Interleukin­1 family from microglia
Li Sun, Zhou Wu, Yoshinori Hayashi, Hiroshi Nakanishi
Department of Oral Aging Science and Pharmacology, Faculty of Dental Science,
Kyushu University
2­3 13:30 – 13:40
Effect of PRIP deficiency in autophagy
Hisanori Umebayashi1, Miho Matsuda1, Takashi Kanematsu2, and Masato Hirata1
1
Laboratory of Molecular and Cellular Biochemistry, Graduate School of Dental
Science, Kyushu University
2
Department of Dental Pharmacolgy, Graduate School Hiroshima University
Chairperson: Masaharu Nakagawa (2­4~2­5)
2­4 13:45 – 13:55
Fabrication of interconnected porous calcium phosphate cement
Trung Kien Pham1, Kanji Tsuru1, Michito Maruta1, Masaharu Nakagawa1,
Shigeki Matsuya2 and Kunio Ishikawa1
1
Department of Biomaterials, Faculty of Dental Science, Kyushu University
2
Section of Bioengineering, Department of Dental Engineering, Fukuoka Dental
College2­5 14:00 – 14:10
Fabrication of bioactive polyethylen terephthalate (PET) substrates byozone­calcium treatment
Ahmed Nafis Rashid, Masaharu Nakagawa, Kanji Tsuru, Giichiro Kawachi
and Kunio Ishikawa
Department of Biomaterials, Faculty of Dental Science, Kyushu University
Break

Chairperson: Kanji Tsuru (2­6~2­8)
2­6 14:30 – 14:40
Mechanical strength improvement of carbonate apatite foam bone substitute by
PLGA reinforcement
Girlie M. Munar1, Melvin L. Munar1, Kanji Tsuru1, Shigeki Matsuya2
and Kunio Ishikawa1
1
Department of Biomaterials, Faculty of Dental Science, Kyushu University
2
Section of Bioengineering, Department of Dental Engineering, Fukuoka Dental
College62­7 14:45 – 14:55
Effect of O3 treatment on the basic properties of apatite cement
Ira Artilia, Michito Maruta, Kanji Tsuru and Kunio Ishikawa
Department of Biomaterials, Faculty of Dental Science, Kyushu University2­8 15:00 – 15:10
Effect of surface modification on initial setting time of α­tricalcium phosphatebased apatite cement
Ruslin1, Michito Maruta1, Kanji Tsuru1, Giichiro Kawachi1, Shigeki Matsuya2
and Kunio Ishikawa1
1
Department of Biomaterials, Faculty of Dental Science, Kyushu University
2
Section of Bioengineering, Department of Dental Engineering, Section of
Bioengineering, Fukuoka Dental College
Chairperson: Tsuyoshi Sugiura (2­9~2­10)
2­9 15:15 – 15:25
Association of cytokeratin 17 expression with differentiation of tumor cells in oral
squamous cell carcinoma
Ryoji Kitamura, Takeshi Toyoshima, Shintaro Kawano, Kazunari Oobu,
Takahiro Kiyosue, Ryota Matsubara, Yuichi Goto, and Seiji Nakamura
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University
2­10 15:30 – 15:40
Localization of Th subsets in salivary gland of Sjögren syndrome
Takashi Maehara, Masafumi Moriyama, Jun−nosuke Hayashida, Akihiko Tanaka,
Shouichi Shinozaki, Kaori Matsumura, and Seiji Nakamura
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University

Closing remarks

15:45 ­ 15:50 Seiji Nakamura
15:50 ­ 15:55 P. Mark Bartold
15:55 ­ 16:00 Liping Zhao

7

Abstracts

8

SL­1
FGF­2 stimulates Periodontal Regeneration
Shinya Murakami, D.D.S., Ph.D.
Professor and Chairman,
Department of Periodontology,
Graduate School of Dentistry, Osaka University
Periodontitis progressively destroys the periodontal tissue and no conventional
periodontal treatments can ever regenerate lost periodontal tissue or normal structureand functionality.

However, improving the biological potential of endogenous

mesenchymal stem cells and/or progenitor cells within periodontal ligaments andstimulating the regeneration of periodontal tissue are now recognized as clinicallypossible. One of the most physiologically efficient methods for stimulating the cells isthe use of recombinant cytokines.

Basic fibroblast growth factor (FGF­2) displays

strong angiogenic activity and inductivity to proliferate mesenchymal cells.

We

revealed that FGF­2 is efficacious in regenerating periodontal tissue in models ofartificial loss of periodontal tissue in beagles and non­human primates.

Furthermore,

we recently conducted a double­blinded clinical trial (phase II) with patients frommultidental facilities.

The purpose was to both clarify the activity of FGF­2 toregenerate periodontal tissue in periodontitis patients and to confirm drug safety.
Subjects underwent periodontal surgical treatment during which we also administered
200 ml of the appropriate investigational drug (FGF­2 plus 3% hydroxypropylcellulose:
HPC) to sites displaying periodontal tissue defect. As a result, standardised radiographs
revealed significantly increased alveolar bone mass in 0.3%­FGF­2 Group compared toPlacebo Group (HPC alone). Throughout the investigation period, no emergence of any
serious adverse effects was identified.

This result suggests that topical application of

FGF­2 can induce regeneration of periodontal tissue in periodontitis patients.
In this symposium, I would like to discuss the present status and future outlook of
FGF­2 therapy in the field of periodontal therapy.

9

SL­2
Periodontal Disease and Rheumatoid Arthritis
P. Mark Bartold, D.D.Sc., Ph.D.
Professor and Director, Colgate Australian Clinical Dental Research Centre,
University of Adelaide
Periodontitis and rheumatoid arthritis (RA) appear to share many pathologic features.
In this presentation the common pathologic mechanisms of these two common
chronic conditions are explored. Emerging evidence now suggests a strong
relationship between the extent and severity of periodontal disease and RA. While thisrelationship is unlikely to be causal, it is clear that individuals suffering fromadvanced RA are more likely to experience more significant periodontal problems
compared to their non­RA counterparts, and vice versa. A case is made that thesetwo diseases could be very closely related through common underlying dysfunction of
fundamental inflammatory mechanisms.

The nature of such dysfunction is still

unknown. Nonetheless, there is accruing evidence to support the notion that bothconditions manifest as a result of an imbalance between pro­inflammatory andanti­inflammatory cytokines. As a result, new treatment strategies are expected toemerge for both diseases that may target the inhibition of pro­inflammatory cytokinesand destructive proteases. The clinical implications of the current data dictate thatpatients with RA should be carefully screened for their periodontal status.

10

SL­3
The new approaches for bone regeneration through the activation of
Wnt/β­catenin signaling pathway
Etsuko Matsuzaki1, Fumi Takahashi­Yanaga2, Katsumasa Maeda1
1Periodontology Section, Division of Oral Rehabilitation,
Faculty of Dental Sciences,2

Department of Clinical Pharmacology, Faculty of Medical Sciences,
Kyushu University

Cell signaling cascades activated by Wnt proteins (collectively the Wnt signaling
pathways) have been well conserved throughout evolution. As well as regulating
cellularprocesses

includingproliferation,differentiation,

motility

andsurvival/apoptosis, the Wnt signaling pathways play key roles in embryonicdevelopment and maintenance of homeostasis in mature tissues. Among the described
Wnt signaling pathways, the Wnt/β­catenin signaling pathway (canonical pathway) is
best characterized and it has been shown that this signaling pathway is involved in bone
biology. We previously reported that differentiation­inducing factor­1 (DIF­1), amorphogen of Dictyostelium, altered osteoblast differentiation, including the alkaline
phosphatase (ALP) expression, by suppressing the Wnt/β­catenin signaling pathway.
For the development of new drugs targeting to the bone regeneration, we investigatedthe possible activators of Wnt/β­catenin signaling pathway on osteoblast differentiation.
Dkk1 (Dickkopf­1) is a soluble inhibitor of Wnt signaling pathway by binding to theWnt co­receptor low­density lipoprotein receptor­related protein (LRP) 5/6 and inhibits
the bone formation.

Sphingosine­1­phosphate (S1P) is well known for the signaling

sphingolipid and it has been reported that S1P activated osteoblast and suppressed
osteoclast, resulting in the inhibition of bone resorption.

Therefore, we examined theeffect of anti­Dkk1 antibody and S1P on osteoblast differentiation. We found that bothanti­Dkk1 antibody and S1P activated the Wnt/β­catenin signaling pathway and induced
ALP activation and mineralization. In this symposium, summarizing our recent findingson the possible activators of the Wnt/β­catenin signaling pathway on the osteoblast
differentiation, we will show the possibility for development of bone regenerator by
activating this signaling pathway.

11

SL­4
Re­Engineering of Gut Microbiota with Traditional Chinese Medicinal Foods forPreventive Management of Metabolic Diseases
Liping Zhao
Laboratory of Molecular Microbial Ecology and Ecogenomics, School of Life
Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai Center for
Systems Biomedicine, 800 Dongchuan Road, Shanghai, 200240, CHINA
Gut microbiota, as the second genome for humans, must work in harmonious
integration with the first genome to sustain a healthy immunity and metabolism for thehost. In animal models, diet has been shown to play a dominating role in shaping gut
microbiota. Long term intake of high­fat diet can override host genetics that wildtype
animals with no known relevant genetic defect can develop severe obesity and insulin
resistance. It has been shown that high­fat diet can disrupt gut microbiota to increase
population levels of opportunistic pathogens such as sulfate­reducing bacteria anddecrease the levels of gut barrier­protecting bacteria, such as bifidobacteria. This longterm dysbiosis would lead to increased antigen load in host bloodstream to provoke anunresolved chronic inflammation impairing insulin receptor, vessel walls etc fordevelopment of metabolic diseases. This new understanding of the key mediating roleof gut microbiota between high­fat diet and deranged host metabolism opens vastpossibilities for modulating gut microbiota with designed foods in preventive
management of the devastating epidemic of metabolic diseases.
Traditional Chinese medicine has a legacy of using foods as drugs to prevent chronic
diseases. These “medicinal foods” are regulated as common foods but have been
repeatedly demonstrated to have medicinal effects over thousands of years of “humantrials”. The scientific foundation has yet to be elucidated with modern methodologies.
We designed a diet pattern with ingredients from TCM foods. 123 volunteers (BMI﹥30) were recruited in a self­controlled clinical trial to test the efficacy of thisTCM­based diet for management of weight and metabolic syndrome. Clinical data andblood, urine and fecal samples were collected before, during and after the intervention.
Preliminary data showed a significant effect of TCM foods for modulating gut
microbiota, attenuating inflammation and alleviating the metabolic syndrome.
Metagenomics, metabonomics and multivariate statistics are being used as whole­body12

systems biology approaches to understand the dynamic associations between rebalanced
architects of gut microbiota, attenuated inflammation, improved insulin sensitivity,
weight reduction and global metabolic homeostasis.

13

SL­5
Effects of butyric acid, the bacterial metabolite, on the periodontal tissue andreactivation of latent HIV­1
Kuniyasu Ochiai
Department of Microbiology,
Nihon University School of Dentistry
Butyric acid, an extracelluar metabolite from periodontopathic bacteria, induces
apoptosis in T­cells and macrophages, and exerts immuno­ modulatory properties. We
demonstrated that human gingival fibroblasts (HGFs) rescue butyric acid­induced T­cell
apoptosis via the proinflammatory cytokines, which were produced in HGFs stimulatedwith butyric acid. Moreover, T­cells adhered to HGFs is enhanced by butyric acid andbutyric acid­induced T­cell apoptosis is down­regulated by T­cell adhesion to HGFs.
These data suggested that the cell­cell communication among T­cells and HGFs isessential for maintaining a normal gingival tissue.
Latently infected cells harbor the HIV­1 proviral DNA integrated in heterochromatins
allowing the persistence of transcriptionally­silent proviruses. Hypoacetylation ofhistone proteins by histone deacetylases (HDACs) is involved in the maintenance ofHIV­1 latency by repressing transcription from HIV­1 proviral DNA. Progression of
AIDS is associated with development of severe periodontitis. Although it is known thatbutyric acid is involved in reactivation of the repressed chromatin, it is not known
whether periodontitis is involved in progression of HIV­infection. Thus, we examined
whether infection of periodontopathic bacteria could facilitate progression of AIDS byreactivating the latent HIV provirus. Our results indicated that periodontopathic bacteria
produce high concentrations of butyric acid that act as a potent inhibitor of HDACs andappear to induce acetylation of histone, thus eventually leading to reactivation of HIV­1in latently infected cells. These results suggested that periodontal diseases could act as arisk­factor for HIV reactivation in the latently infected individuals.

14

1-1
The roles of stretch loading in human periodontal ligament cells
Satoshi Monnouchi1, Hidefumi Maeda2, Shinsuke Fujii2, Atsushi Tomokiyo1,
Naohisa Wada2, Kiyomi Kono1, and Akifumi Akamine1, 2
1
Department of Endodontology and Operative Dentistry, Division of Oral
Rehabilitation, Faculty of Dental Science, Kyushu University, and 2Department ofEndodontology, Kyushu University Hospital
The mechanical loading caused by occlusion and mastication is known to play anessential role in maintaining periodontal ligament (PDL) tissues. Our recent study
demonstrated that stretch loading up­regulated transforming growth factor­β1, andalkaline phosphatase in human PDL cells (HPDLCs) in vitro, that was mediated byincreased angiotensin II. Therefore, the present study purposed to detect other biologicalfactors yielded in HPDLCs subjected to stretch loading.
HPDLCs obtained from healthy third molars were pre­cultured in flexible­bottomed
culture chambers coated with type I collagen until reaching sub­confluence. HPDLCs
were subjected to stretch loading (0.5 sec stretch and 0.5 sec relaxation per a cycle) for
1 hr. Gene expressions were analyzed by quantitative RT­PCR, and protein expressions
were analyzed by enzyme­linked immunosorbent assay.
Here, we identified up­regulated gene and protein expressions of interleukin­11(IL­11) in HPDLCs that were exposed to stretch loading. HPDLCs stimulated byangiotensin II also increased gene expressions of IL­11. Furthermore,immuno­localization of IL­11 was detected in rat PDL tissues, but not in pulp tissues,dentin and bone matrices. Additionally, the intense reaction was detected at theextracellular matrices in the bone side of PDL tissues.
It has been reported that IL­11 is a multi­functional cytokine involved actively in bonemetabolism, and recent reports have discussed that IL­11 induced by mechanical stressenhanced osteoblastogenesis. Moreover, in PDL cells, it has been shown that IL­11
expression stimulates osteoblastic differentiation.
These results suggested that IL­11 was up­regulated via angiotensin II induced bystretch loading in HPDLCs, and that it might contribute to bone metabolism under theenvironment of mechanical load in PDL tissues.

15

1-2
Anti­Dkk1 antibody induces osteoblast differentiation
through the Wnt/β­catenin signaling pathway
M. Kobayashi, E. Matsuzaki, T. Hamachi, S. Hiratsuka, Y. Aida, and K. Maeda
Section of Periodontology, Division of Oral Rehabilitation,
Faculty of Dental Science, Kyushu University
The Wnt/β­catenin signaling pathway is central to bone development and homeostasis.
Dkk1 is a soluble inhibitor of this signaling pathway by binding to the Wnt co­receptor
LRP5/6, and inhibits bone formation. It has been reported that anti­Dkk1 antibody waseffective in preventing the development of osteolytic bone disease in multiple myeloma.
However, the role of anti­Dkk1 antibody on osteoblast differentiation is still unknown.
In this study, we investigated the effect of anti­Dkk1 antibody on osteoblast
differentiation and the Wnt/β­catenin signaling pathway.
MC3T3­E1 and SaOS­2 cells, osteoblast­like cells, were used for this study. First, we
examined the effect of anti­Dkk1 antibody on cell proliferation and alkaline phosphatate(ALP) activity, a crucial marker of osteoblast differentiation. We found that anti­Dkk1
antibody significantly increased ALP activity in both MC3T3­E1 and SaOS­2 cells
without affecting cell proliferation. We also investigated the expression levels of mRNAand protein of osteoblast differentiation makers, ALP and osteocalcin (OCN). BothmRNA and protein expression levels of ALP and OCN were increased after treatment ofanti­Dkk1 antibody in MC3T3­E1 cells. Next, we studied the effect of anti­Dkk1
antibody on osteoblast­mediated mineralization. Anti­Dkk1 antibody increased
osteoblast­mediated mineralization.
Subsequently, we examined the effect of anti­Dkk1 antibody on the Wnt/β­cateninsignaling pathway. In this signaling pathway, β­catenin is a key player. Activation ofthis signaling induces the stabilization of cytoplasmic β­catenin, and accumulatedβ­catenin enters the nucleus to regulate target gene expression with T­cell
factor/lymphoid enhancer­binding factor (TCF/LEF) transcriptional factors. The
expression level of β­catenin protein was increased by treatment of anti­Dkk1 antibody
and TCF transcriptional activities were also increased. Furthermore, we investigated theeffect of anti­Dkk1 antibody on human ALP promoter activity mediated by the putative
TCF binding site (­1023/­1017bp) and found that anti­Dkk1 antibody significantly
increased ALP promoter activity.
These results suggest that anti­Dkk1 antibody induces Wnt/β­catenin signaling
pathway, resulting in osteoblast differentiation. Therefore, anti­Dkk1 antibody might
have a therapeutic value to induce bone regeneration.
16

1-3
Sphingosine­1­phosphate activates the Wnt/β­catenin signaling pathway andincreases osteoprotegerin gene expression
S. Hiratsuka, E. Matsuzaki, M. Kobayashi, T. Hamachi, Y. Aida, and K. Maeda
Section of Periodontology, Division of Oral Rehabilitation,
Faculty of Dental Science, Kyushu University[Objective]
Sphingosine­1­phosphate (S1P) is well known for the signaling sphingolipid and thebioactive lipid mediator that impacts migration, proliferation, and survival in diverse
cell types. Most of the biological effects of S1P are mediated by signaling through thecell surface G­protein­coupled receptors S1P1­S1P5. Recently, it has been reported that
S1P stimulates osteoblast migration and survival. Furthermore, S1P has been shown to
inhibit osteoclast differentiation and bone resorption. However, the effects of S1P on
osteoblast differentiation and bone formation are still unknown.
In this study, we investigated the effect of S1P on osteoblast differentiation. We
particularly paid attention to the possible involvement of the Wnt/β­catenin signaling
pathway because this signaling pathway plays an important role in osteoblast
differentiation and bone metabolism.
[Materials and Methods]
Osteoblast­like cell lines MC3T3­E1 and SaOS­2 cells were used in this study. The
effect of S1P on osteoblast differentiation was monitored by alkaline phosphatase (ALP)
activity. mRNA and protein expressions were analyzed by northern blotting/RT­PCR
and western blotting. Reporter gene assays were also carried out to examine the effectof S1P on the Wnt/β­catenin signaling pathway. Von Kossa staining and alizarin red
staining were also performed to detect osteoblast­mediated mineralization.
[Results]
S1P significantly increased ALP activity compared with control. Next, we examinedthe effects of S1P on osteoprotegerin (OPG) and receptor activator of nuclear
factor­kappa B ligand (RANKL) expressions, the crucial markers forosteoblast­osteoclast coupling. S1P increased the mRNA expression levels of both OPG
and RANKL.
Wnt proteins control the expression of many genes including ALP, OPG and RANKL
through the inhibition of GSK­3β and the subsequent activation of β­catenin/TCF/LEF
transcriptional activity. Therefore, we investigated the effect of S1P on the
Wnt/β­catenin signaling pathway. Not only phosphorylation level of Ser9 on GSK­3β,
but S1P increased TCF transcriptional activity. Furthermore, S1P elevated the OPG
17

promoter activity. These results indicated that S1P activated the Wnt/β­catenin signaling
pathway. In addition, S1P induced the osteoblast­mediated mineralization.

18

1-4
Functional implication of thymosin beta­4 in the morphogenesis of the tooth germ
via the regulation of odontogenic gene expressions
Yukiko Ookuma1, 2, Ieyoshi Kobayashi1, Tamotsu Kiyoshima1, Kengo Nagata1,
Hiroko Wada1, Kazuaki Nonaka2 and Hidetaka Sakai1
１
Laboratory of Oral Pathology, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University
2
Section of Pediatric Dentistry, Division of Oral Health, Growth and Development,
Faculty of Dental Science, Kyushu University
We previously detected thymosin beta­4 (Tβ4) as a differentially expressed gene inembryonic day 12.0 (E12.0) mouse mandible. We reported that the temporal expression
pattern of Tβ4 mRNA in the developing tooth germ of the mouse embryo and neonate,
and demonstrated the possible association of Tβ4 with the morphogenesis anddevelopment of the tooth germ in the 3rd symposium. However, the precise functionalroles of Tβ4 have not yet been clarified in the initiation and development of the tooth
germ. In this study, we focused on functional implications of Tβ4 in the mouse firstmolar development. First, an inhibition assay using Tβ4 antisense sulfur­substituted
oligodeoxynucleotide (AS S­ODN) was performed in cultured E11.0 mouse mandibles
or E15.0 tooth germs. Enamel organ formation in the cultured E11.0 mandibles at 8th
day was significantly suppressed by the treatment with AS S­ODN. However, the ASS­ODN treatment was negligibly affected on morphological development in thecultured E15.0 tooth germ at 8th day. The quantitative analysis using real­time PCR
revealed that the expression of type II/III Runx2 and dentin matrix protein 1 (Dmp1)
mRNA was significantly suppressed in both the cultured E11.0 mandible and E15.0
tooth germ by the AS S­ODN treatment. Next, in using dental epithelial or pulpal cellline treated with AS S­ODN, we analyzed the mRNA expressions by real­time PCR,
enzymatic activities of secretory matrix metalloproteinase­2/­9 (MMP­2/­9) by gelatin
zymography, and cell motility. The expression of type II/III Runx2, nucleolin (Ncl),
Mmp2/9 and Dmp1 was decreased by the AS S­ODN treatment in both the cells,suggesting that Tβ4 could be involved in those transcriptions. Enzyme activity of
MMP­2 was inhibited by the AS S­ODN treatment in both the cells. The dental
epithelial cells treated with AS S­ODN significantly migrated into the acellular gap.
Tβ4 may be critical for expression of Runx2, Ncl, MMP­2 and Dmp1 in both the cells,
and for cell motility in the dental epithelial cell.
In conclusion, we have proven multi­functional implications of Tβ4 in thedevelopment of the mouse lower first molar, via its effects on various­gene expressions.
19

1-5
Expression pattern and possible function of thymosin beta­10
in developing tooth germ compared with thymosin beta­4
Maho Shiotsuka1, 2, Hiroko Wada1, Tamotsu Kiyoshima1, Ieyoshi Kobayashi1,
Kengo Nagata1, Hiroaki Fujiwara1, Ichiro Takahashi2 and Hidetaka Sakai1
１
Laboratory of Oral Pathology, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University
2
Section of Orthodontics and Dentofacial Orthopedics, Division of Oral Health,Growth and Development, Faculty of Dental Science, Kyushu University[Objectives] Our previous study has shown the expression pattern and function ofthymosin beta­4 (Tβ4) in developing tooth germ. Thymosin beta­10 (Tβ10), of which
sequence is highly homologous to T4, has been reported to play an important role incell shaping and motility by forming a complex with G­actin similarly to T4, whileT10 acts as a potent inhibitor of angiogenesis unlike T4. So far, there is no report on
the role of T10 in tooth development. In this study, we investigated the expression
pattern and possible function of T10 in developing tooth germ compared with those ofT4.
[Materials and Methods] We performed in situ hybridization of these two genes tostudy their detailed expression pattern in the mouse tooth germs at embryonic day 10.5
(E10.5) to postnatal day 7.0 (P7.0), and used real­time PCR to examine the quantity ofT4 and T10 mRNA expression levels in mandibles at E10.5 and E12.0 and in tooth
germs at E15.0, E18.0, P1.0, and P5.0.
[Results] The in situ expression pattern for T10 in tooth development was apparently
different from that of T4 at the same days. T10 mRNA was intensely expressed in themesenchymal cells and odontoblasts. On the other hand, the in situ signal for T4 wasdetected mainly in dental epithelial cells and ameloblasts. Real­time PCR demonstrated
that both genes were highly expressed in the early stage of odontogenesis and toothmatrix formation after birth.
[Conclusion] These results suggest that T10 participates in differentiation of theodontogenic mesenchymal cells, differentiation of odontoblasts, and dentinogenesis in
the developing tooth germ. Meanwhile, T4 is associated with differentiation of theodontogenic epithelial cells, differentiation of ameloblasts, and amelogenesis.

20

1-6
Involvement of PRIP in the bone formation
Koshiro Tsutsumi1, 2, Miho Kotani1, Ayako Murakami1,
Miho Matsuda1 and Masato Hirata1
1
Laboratory of Molecular and Cellular Biochemistry,
Faculty of Dental Science, Kyushu University
2
Division of Fixed Prosthodontics, Faculty of Dental Science, Kyushu University
PRIP (phospholipase C­related, but catalytically inactive protein) is a signaling protein
isolated as an inositol 1,4,5­trisphoshpate binding protein. PRIP has a domainorganization similar to phospholipase C­d but lacking the enzyme activity.
PRIP­deficient mice (PRIP­KO) mice showed an increased gonadotropin, but adecreased sex steroid hormone. The difference was similar to that for the cause of bonedisease, such as osteoporosis. In the present study, therefore, we analyzed PRIP­KO
mice with a special reference to the bone condition. We first performed three
dimensional analysis of the femur of female mice. The bone mineral density andtrabecular bone volume was higher in the mutant mice. We further performed
histomorphometrical assay of bone formation parameters: bone formation rate, mineralapposition rate, osteoid thickness and osteoblast number were increased in the mutant,indicating that the increased bone mass is caused by the enhancement of bone formation
ability. We then performed the primary culture of calvaria prepared from both genotypes.
In cultures from the mutant mice, osteoblast differentiation was accelerated as assessedby the differentiation marker gene expression including alkaline phosphatase, runx2,
osteonectin etc. Phosphorylation of Smad1/5/8 in response to BMP4 lasted longer,
probably causing the increased expression of osteoblast differentiation markers. These
results indicate that PRIP is involved in the negative regulation of the bone formation.

21

1-7
Effects of allergic inflammation on orthodontically induced tooth root resorption
Naohisa Murata1, 2 , Hideki Ioi2, Masahiro Ohuchi1,2, Tomoka Takao1, Hanako Oida1,
Takayoshi Yamaza1, Ichiro Takahashi2, Mizuho A. Kido1
1
Department of Molecular Cell Biology and Oral Anatomy,
Graduate school of Dental Science, Kyushu University
2
Department of Orthodontics, Graduate school of Dental Science, Kyushu University
The success of orthodontic treatment depends on dynamic alveolar bone metabolism,
which leads to tooth movement. There are variations in the time required and theamount of movement achieved, and some patients show tooth root resorption at the endof the treatment. Epidemiologic study reported that patients who suffer from allergic
conditions have higher rates of tooth root resorption. In this study, we investigated theeffects of allergies on tooth movement and bone/tooth root resorption. Tooth movement
and bone and tooth root resorption were increased in ovalbumin (OVA)­sensitized
Brown­Norway rats subjected to orthodontic force (OF). The expression levels of
RANKL and pro­inflammatory cytokines such as tumor necrosis factor­α andinterleukin­1β were increased in periodontal tissues of OVA­sensitized animals
subjected to OF, compared with non sensitized rats. Furthermore, mRNA expression
levels of enzymes involved in the lipid mediator biosynthetic pathway, and unique
receptors for 5­lipoxygenase­ derived lipid mediators were also low­dose aspirin toOVA­sensitized OF rats. Aspirin reversed the increase in tooth movement andsuppressed tooth root resorption. The results of this study suggest that systemic allergies
can affect bone metabolism, and that aspirin or 5­lipoxygenase­mediated lipid mediators
could be potential therapeutic agents to combat tooth root resorption.

22

1-8
Stress distribution of maxillary implant supported overdentures in vitro
Asuka Haruta, Yasuyuki Matsushita, Yoshihiro Tsukiyama and Kiyoshi Koyano
Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation,
Faculty of Dental Science, Kyushu University
Objectives
Maxillary implant­supported overdentures have been documented with a high implantloss relative to other implant treatment options. Some studies reported that at least four
implants were necessary to support maxillary implant­supported overdentures, but there
is still a lack of studies on the influence of anchorage design on force distribution. The
purpose of this in vitro study is to evaluate the influence of splinted and unsplinted
implant of maxillary implant­supported overdentures by measuring strain of implants.
Materials and Methods
In this study, edentulous maxilla model was used, which were simulated anatomical
structure and bone quality. Four implants were inserted in right and left, canine andpremolar position. The surface of the model was covered with 4mm thickness layer ofimpression material to simulate oral mucosa, and a palateless overdenture wasfabricated. Two different types of attachment were considered; splinted Dolder bar
attachment with clip and unsplinted ball attachment. The maxilla model was fixed and
50N concentrated load was applied at anterior (loading point A), first premolar (loading
point B) and first molar region (loading point C) of the overdenture. Strains inabutments were measured and axial force and bending moment were calculated.
Additionally, denture base movement was measured by a position sensor attached in theleft first molar region.
Results
With the loading point A and B, axial forces on bar attachment were larger than that onball attachment, while with loading point C, axial force on ball attachment were larger.
With the loading point A and C, bending moment on ball attachment was smaller thanthat on bar attachment. With the loading point A, denture movement was the largest on
both attachments. With the loading point B and C, denture movements were larger onball attachment.
Conclusion
Within the limits of the study, unsplinted design could be advantageous for maxillary
implant­supported overdentures based on the lower stress and better denture stability.

23

1-9
Resistant factors of the conservative therapy to BRONJ
Goto Y1, Kawano S 1, Hayashida J 1, Ikari T 2, Kurahara S 2, Toyoshima T 1,
Minamizato T 1, Kiyosue T 1, Matsubara R 1, Oobu K 1, Takenoshita Y 2, Mori Y 2,
Shiratsuchi Y 1, Nakamura S 1
1
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University
2
Section of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University
Objectives: Treatment strategy for bisphosphonate­related osteonecrosis of the jaws
(BRONJ) is not established definitely. We have mainly provided the conservative
therapy including sequestrectomy and debridement to the patients with BRONJ. The
purpose of this study was to assess the resistant factors of conservative therapy.
Patients and methods: Thirty­two patients with BRONJ (2 males and 30 females;
mean age, 72.0years; range 46­88years), who clinically diagnosed from 2004 to 2010 atthe Department of Oral and Maxillofacial Surgery in Kyushu University Hospital, were
evaluated clinical parameters (sex, age, route of administration, duration ofbisphosphonates therapy, systemic factors, local risk factors, treatment methods, drug
holidays, outcome of the treatment, and duration to cure completely).
Results: Oral bisphosphonates (OB) were used in 15 patients. OB combined withcorticosteroids (OC) were used in 5 patients. Intravenous bisphosphonates (IB) wereused in 12 patients. The treatment methods for BRONJ were local irrigation in all cases,
antibiotic therapy in 29, sequestrectomy in 13, debridement in 11, and hyperbaric
oxygen therapy in 6. As for outcomes of management, 15 cases (46.9%) werecompletely cured and 17 (53.1%) were under treatment at this time. The rates ofcomplete cure were 10 of 15 patients (66.7%) in OB, 3 of 5 (60.0%) in OC and 2 of 12(16.7%) in IB. The average duration of therapy to cure completely was 446.9days. The
duration in the patients with corticosteroids therapy (1167.3days) was significantly
longer than that in the patients without (269.0days).
Conclusion: It is difficult to cure in the cases of BRONJ caused by IB or OC, though
the conservative therapy for BRONJ is useful.

24

1-10
Ca2+ stimulates the expression of bone morphogenetic protein­2 throughcalcium­sensing receptor in fibroblasts
Yoshie Yahara
Department of Oral and Maxillofacial Surgery,
Graduate School of Dental Science, Kyushu University
Calcium­sensing receptor (CaSR), which is a seven­transmembrane GTP­binding
protein­coupled receptor, regulates various pathophysiological functions. We haveshown that CaSR is expressed in the fibroblasts isolated from keratocystic odontogenic
tumors (KCOTs) and gingiva, and high extracellular Ca2+ concentration ([Ca2+]o)
stimulates the expression of cyclooxygenase­2 and the secretion of prostaglandin E2(PGE2) in the fibroblasts through CaSR. Since PGE2 regulates bone metabolism byregulating osteoclastogenesis and osteoblast differentiation, Ca2+ might affect bonemetabolism through CaSR in fibroblasts. Bone morphogenetic protein (BMP)­2affects bone metabolism via stimulation of osteoblastic cell differentiation andmaturation and also direct and/or indirect regulation of osteoclastogenesis. Recently, it
was reported that the activation of CaSR stimulated the expression of BMP­2 in colonic
myofibroblasts. This finding leads us to speculate the released Ca2+ from bone might
stimulate the fibroblasts around the bone and affect the bone metabolism by inducing
not only PGE2 secretion but also BMP­2 expression through CaSR. In this study, weinvestigated whether Ca2+ activates BMP­2 expression through CaSR in KCOT andgingival fibroblasts.
Five mM [Ca2+]o and CaSR activator neomycin enhanced the expression of BMP­2mRNA and protein in KCOT and gingival fibroblasts. In the KCOT fibroblasts, both
phospholipase C inhibitor U­73122 and protein kinase C inhibitor staurosporin
attenuated the Ca2+­induced expression of BMP­2 mRNA. Furthermore, specificinhibitors for p38 mitogen­activated protein kinase (MAPK) SB203580 and nuclear
factor κB (NF­κB) PDTC attenuated the Ca2+­induced expression of BMP­2 mRNA.
Neither PGE2 nor indometacin affected the elevated [Ca2+]o­induced expression of
BMP­2 mRNA in the KCOT fibroblasts.
Thus, in KCOT fibroblasts high concentration of extracellular Ca2+ may enhance
BMP­2 expression via p38 signal transduction pathway through CaSR without PGE2.

25

2-1
Phosphatdiylserine containing liposomes facilitate osteogenic maturation
and bone formation of rats
Hong Mei Ma1, Zhou Wu1, Yin Ji Li2, Toshio Kukita2 and Hiroshi Nakanishi1
1
Department of Oral Aging Science and Pharmacology, 2Department of Molecular Cell
Biology & Oral Anatomy, Faculty of Dental Science, Kyushu University
Posphatidylserine­containing liposomes (PS liposomes) can be engulfed bymononuclear phagocytes to promote their production of TGF­b1 and PGE2. We havedemonstrated that PS liposomes inhibit osteoclastogenesis and arthritis induced boneloss (Wu et al., J Immunol. 2010). In present study, we investigated the effects of PS
liposomes on osteogenesis. The cultured rat calvarial osteoblast­like cells (OBs) wereused to determine the effects of PS­liposomes on cell proliferation, alkaline phosphatase(ALP) activity, osteogenesis­related gene expression, and mineralized nodule formationin OBs. We found that PS­liposomes did not affect the proliferative activity of OBs at
day 7 of culture. On the other hand, PS­liposomes significantly increased the ALP
activity at day 7, and PS­liposomes significantly increased the expression of osteocalcin,
a marker for osteogenic maturation after day 14 by real time RT­PCR. Furthermore,PS­liposomes markedly increased mineralized nodule formation after day 21 of culture.
Moreover, local treatment with PS­liposomes significantly improved the bone formationin rat tooth extraction model. Dependent on about results, PS liposomes strongly
promote osteogenesis. Together with the inhibitory effects on osteoclastogenes, PS
liposomes are involved in regulation of bone remodeling.

26

2-2
A possible involvement of cathepsin B in nociceptive pain through the processing
and secretion of Interleukin­1 family from microglia
Li Sun, Zhou Wu, Yoshinori Hayashi, Hiroshi Nakanishi
Department of Oral Aging Science and Pharmacology,
Faculty of Dental Science, Kyushu University
It is now evident that caspase­1, an essential enzyme for maturation of interleukin­1(IL­1) family, can be activated through the assembly of a cytosolic protein complex thatis known as the inflammasome. However, there is still evidence suggesting theexistence of lysosomal­proinflammatory caspase pathways. We have recently reported
that cathepsin B­deficiency abrogated the pro­IL­1β maturation and its release frommicroglia following stimulation with chromogranin A (Terada et al., Glia, 2010).
Furthermore, the maturation of pro­caspase­1 in chromogranin­treated microglia wasalso significantly inhibited by CA­074Me, a specific inhibitor of cathepsin B. In thepresent study, we further found that the maturation and secretion of both IL­18 andIL­33 from microglia was significantly suppressed by cathepsin B­deficiency orCA­074Me treatment. These results strongly suggest that cathepsin B plays a key role inthe maturation of pro­forms of IL­1 family, including IL­1β, IL­18 and IL­33, throughthe caspase­1 activation in chromogranin A­treated microglia. Next, we examined theeffect of cathepsin B­deficiency on the nociceptive processing, because IL­1 family is
known as “painful cytokine”. As expected, cathepsin B­deficient mice showed asignificant resistance against inflammation­ and nerve injury­induced pain. Therefore,
either pharmacological or genetic inhibition of cathepsin B may provide an effective
therapeutic intervention in inflammatory and naturopathic pain.

27

2-3
Effect of PRIP deficiency in autophagy
Hisanori Umebayashi1, Miho Matsuda1, Takashi Kanematsu2, and Masato Hirata1
1
Laboratory of Molecular and Cellular Biochemistry,
Graduate School of Dental Science, Kyushu University
2
Department of Dental Pharmacolgy, Graduate School of Biomedical Science,
Hiroshima University
We isolated a novel protein as an inositol 1,4,5­triphosphate binding protein molecule
similar to phospholipase C, but catalytically inactive, then we termed it PRIP
(PLC­related catalytically inactive protein). We found that PRIP binds GABARAP(GABA receptor associated protein)、regulating the membrane trafficking of GABAA
receptor. Recently it was reported that GABARAP family are involved in autophagy.
Therefore, we studied the possible involvement of PRIP in autophagy. Autophagy is amechanism for the degradation of cellular material either as a way to provide nutrientsduring times of starvation or as a quality control mechanism that eliminates unneeded
proteins and/or organelles during normal growth and development. Of 34
autophagy­related proteins (ATG) identified so far, ATG8 (LC3 in mammal) is used as a
specific marker to monitor the process of autophagy. ATG8/LC3 is a member of
GABARAP family. We first confirmed the interaction of PRIP with GABARAP family.
PRIP interacted with GABARAP, and also with LC3 and other GABARAP family
molecules, albeit to a lesser extent. Immunoprecipitation assay showed that PRIP
interacted with both LC3­I (cytosolic form) and LC3­II (membrane bound form).
Secondly, autophagy activities were monitored by Western blotting analysis using
lysates obtained from wild­type (WT) and PRIP­deficient (PRIP­KO) mouse embryonic
fibroblast (MEF) starved by amino acid­deprivation along with lysosomal inhibitors.
The ratio of LC3­II/LC3­I was higher, and degradation of LC3­II (Autophagic flux) was
more increased in PRIP­KO MEF. Thirdly, we generated WT and PRIP­KO mice,expressing transgene of GFP­LC3 by mating and prepared MEF from the mice.
Enhanced autophagic activity, as assessed by more GFP dots observed under a confocal
microscopy, was observed in the PRIP­KO MEF. Finally, PRIP gene was transfected to
PRIP­KO MEF. The number of GFP dots was decreased in an inverse proportion to theexpression level of PRIP. These results suggest that PRIP deficiency induces more
autophagy, thus indicating that PRIP plays a negative role in autophagy, probably byinteracting with LC3.

28

2-4
Fabrication of interconnected porous calcium phosphate cement
Trung Kien Pham1, Kanji Tsuru1, Michito Maruta1, Masaharu Nakagawa1,
Shigeki Matsuya2 and Kunio Ishikawa1
1
Department of Biomaterials, Faculty of Dental Science, Kyushu University
2
Section of Bioengineering, Department of Dental Engineering,
Fukuoka Dental College
For bone tissue regeneration, the interconnected porous structure plays an importantrole since this porous structure allows cell penetration and cell revascularization. In thisstudy, we fabricate calcium phosphate cement with fully­interconnected porous
structure based on setting reaction of ­TCP microsphere with 0.2 mol∙L­1 monocalcium
phosphate monohydrate ­ 0.1 mol∙L­1 acid phosphoric (MCPM­H3PO4) solution at 37oC.
For the basic study, set two­­TCP microsphere specimens were evaluated by scanningelectron microscope (SEM) and X­ray diffraction (XRD) to understand the setting
mechanism. SEM results revealed that new crystals were formed at the interface
between microspheres after the reaction for a minute and then grown and interlocked
together after 5 minutes. XRD results also indicated that the surface of ­TCP
microsphere was transformed to brushite when treated with MCPM­H3PO4 solution.
Based on the basic study, calcium phosphate cement with fully­interconnected porous
structure was fabricated by using many ­TCP microspheres and MCPM­H3PO4
solution at 37oC. The interconnectivity of set porous cement was checked by ­CT
analysis. The interconnected porous calcium phosphate cement, which has in­situ
setting property at 37oC, is expected to become an ideal bone substitute for bone tissue
regeneration.

29

2-5
Fabrication of bioactive polyethylen terephthalate (PET) substrates
by ozone­calcium treatment
Ahmed Nafis Rashid, Masaharu Nakagawa, Kanji Tsuru,
Giichiro Kawachi and Kunio Ishikawa
Department of Biomaterials, Faculty of Dental Science, Kyushu University
At present, polyethylene terephthalate (PET) has been widely used as several
biomedical applications especially for ligaments and vascular grafts. Considering
artificial ligaments, PET prosthesis prevents full integration with the body because it issynthetic, thus prosthesis failure is induced. PET with bioactive surface might help toovercome this problem. We previously reported that ozone­calcium chloride treatment
could induce osteoconductivity to titanium and concluded that Ca ions might beimmobilized with hydroxyl groups on the surface. This method might be applicable forpolymers which can form hydroxyl and carboxylic groups during ozone (O3) treatment.
The present study focused on PET as a target substrate. PET films were subjected toozone treatment in the presence of 1 mol/L CaCl2 solution for 6 hours in atmosphericpressure. For comparison, PET films were treated by O3 in distilled water (DW). Peakfitting of C1s spectra by X­ray photoelectron spectroscopy (XPS) showed increased
amount of carboxyl groups and Ca2p spectra by XPS showed immobilization of Ca2+
onto the substrate after O3­CaCl2 treatment. Scanning electron microscope (SEM)
images revealed no morphological changes based on O3­CaCl2 treatment. Water contact
angle was found to be decreased after O3­CaCl2 treatment. Initial cell attachment of
NIH3T3 cell­line (fibroblast cells) for 3 hours showed higher cell attachment rate incase of O3­CaCl2 treated samples than non treated and O3­DW treated ones. Based on
the results obtained, O3­CaCl2 treatment method might be applicable for surface
modification of PET and to improve interactions between the polymeric implant andsurrounding tissue.

30

2-6
Mechanical strength improvement of carbonate apatite foam bone substituteby PLGA reinforcement
Girlie M. Munar1, Melvin L. Munar1, Kanji Tsuru1,
Shigeki Matsuya2 and Kunio Ishikawa1
1
Department of Biomaterials, Faculty of Dental Science, Kyushu University
2
Department of Dental Engineering, Section of Bioengineering, Fukuoka Dental
College
Carbonate apatite foam bioceramics is a potential bone defect filler or scaffold for bone
tissue engineering due to its close similarities to the biological apatite of bone,morphology which is characterized by interconnecting open pore structure andosteoclast­induced bioresorption. In spite of these merits, the primary drawback
carbonate apatite foam is low resistance to fracture. This makes handling difficult likecutting and shaping to fit the bone defect. As the organic phase of bone provides thestrength and resiliency, reinforcing the carbonate apatite foam with organic material may
also improve its mechanical strength for sufficient handling. In this study, an attempt toimprove the mechanical strength of carbonate apatite foam by the addition of an organic
phase was performed. The carbonate apatite foam was first fabricated and then reinforced
with biodegradable copolymer of poly(DL­lactide­co­glycolide) (PLGA) using
freeze­vacuum infiltration technique. After PLGA reinforcement, the compressive
strength increased from 0.04 MPa up to 1.5 MPa while porosity remained as high as 82%.
SEM observation showed the preservation of the three­dimensional interconnecting pore
structure and presence of PLGA both in the internal hollow structures and externalsurface of the foam. X­ray diffraction and FT­IR analysis revealed no compositionaltransformation of the carbonate apatite phase. In conclusion, addition of organic polymer
such as PLGA is a useful method to increase the mechanical strength of carbonate apatite
foam without changing its morphology and composition.

31

2-7
Effect of O3 treatment on the basic properties of apatite cement
Ira Artilia, Michito Maruta, Kanji Tsuru and Kunio Ishikawa
Department of Biomaterials, Faculty of Dental Science, Kyushu University
Ozone (O3) treatment of powder phase of apatite cement (AC) was found to improve
the wettability of AC’s powder phase and thus result in improved handling properties
without changing setting time, compositional transformation to apatitic mineral as wellas mechanical strength of the set mass. At the same handling property of its paste phase,the mechanical strength of O3 treated AC was approximately twice higher whencompared to non­treated AC. Smaller amount of the liquid to perform higher handlingproperty index and resulting reduced porosity of the set mass was a key for the
improved mechanical strength of the O3 treated AC.

32

2-8
Effect of surface modification on initial setting time of α­tricalcium phosphatebased apatite cement
Ruslin1, Michito Maruta1, Kanji Tsuru1, Giichiro Kawachi1,
Shigeki Matsuya2 and Kunio Ishikawa1
1
Department of Biomaterials, Faculty of Dental Science, Kyushu University
2
Department of Dental Engineering, Section of Bioengineering,
Fukuoka Dental College
The key advantage of apatite cement (AC) is its self­setting ability and appropriate
setting time is required for its clinical application. AC should have an initial setting timethat enables the clinician to fill the bone defect with the apatite having exactly the same
shape with the bone defect. Otherwise, if AC fails to set and powder is released into soft
tissue, it will cause an inflammatory response called crystalline inflammatory response.
Typically the initial setting time is close to 10 minutes. Unfortunately, setting time ofα­tricalcium phosphate (α­TCP) based AC is too long if free from additive. Therefore,chelating agent such as succinic acid or citric acid is employed for the initial setting
reaction of AC. Even though, it prevents compositional transformation to apatite. In thisstudy, effect of surface modification of αTCP powder with distilled water (DW) on basic
setting reaction of AC has been investigated. Briefly, αTCP powder was soaked in DW
with different temperature and periods. After the treatment, the treated powder was
immersed in acetone for 3 min to stop the reaction followed by drying in an oven at 60°C.
The αTCP powder thus obtained was used as powder phase of AC whereas DW was used
as its liquid phase. AC was mixed with liquid to powder of 0.5 and packed in Teflon mold.
The packed cements were allowed to set at 37°C and 100% relative humidity. Setting
time of this cement was confirmed by a Vicat needle method. When αTCP powder was
pre­treated at 50°C for 30 minutes, the setting time became much shorter, whileapparently, a slight morphological changes and crystal transformation could be observedby Scanning Electron Microscope (SEM) and X­Ray Diffraction (XRD). Furthermore,when α­TCP powder was pretreated at 50°C for 90 minutes, the setting time becamemuch shorter than that of 30 minutes pre­treatment. In this pre­treatment condition, newfine precipitations could be observed by SEM. Moreover, XRD measurement indicatedthat 17 % α­TCP transformed to calcium deficient apatite. Finally, after immersion at
50°C for 180 minutes of α­TCP powder, the setting time becomes 9 minutes. It was
concluded that the immersion of αTCP powder in DW may be useful to reduce the initial
setting time of α­TCP which clinically acceptable.

33

2­9
Association of cytokeratin 17 expression with differentiation of tumor cells
in oral squamous cell carcinoma
Ryoji Kitamura, Takeshi Toyoshima, Shintaro Kawano, Kazunari Oobu,
Takahiro Kiyosue, Ryota Matsubara, Yuichi Goto, and Seiji Nakamura
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University
Objectives: Cytokeratin (CK) is expressed in basal cells of normal oral mucosa andsquamous cell carcinoma cells, however, little is known about the CK expression
pattern in oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate
the relevance of various CKs to clinicopathological parameters of OSCC.
Methods: In 62 samples from patients with primary OSCC prior to either chemotherapy
or radiotherapy, the expression of CKs in tumor cells was immunohistochemicallyexamined by anti­CK14, anti­CK16, anti­CK17, anti­CK18, anti­CK19, and anti­CK20
monoclonal antibodies. A comparison of CK expression patterns withclinicopathological parameters (tumor region and size, lymph node states, mode ofinvasion by Yamamoto­Kohama’s criteria, differentiation grade, and clinical stage) was
carried out, to clarify the clinically pronounced CKs from 6 kinds of CKs. The stained
sections with more than 10% reactive cells in tumor cells were defined as positive.
Results: Of 62 samples, CK14 was detected in 62 samples (96.8%), CK17 in 44(71.0%), CK16 in 36 (58.1%), CK19 in 22 (37.1%), CK18 in 15 (24.2%), and CK20 in
14 (22.6%). CK17 was significantly expressed in N0 cases (p<0.05) more thanN­positive cases. Moreover, CK17 was significantly expressed in YK­1, YK­2, andYK­3 cases (p<0.01) more than YK­4C and YK­4D cases. CK20 was significantly
expressed in T3 and T4 cases (p<0.01) more than T1 and T2 cases. There were no
significant differences between other CKs (CK14, CK16, CK18, and CK19) andclinicopathological parameters.
Conclusion: The expression of CK17 was associated with metastasis of neck lymph
nodes and characteristic of tumor invasion. The expression of CK20 was associatedwith clinical progression of OSCC.

34

2-10
Localization of Th subsets in salivary gland of Sjögren syndrome
Takashi Maehara, Masafumi Moriyama, Jun−nosuke Hayashida, Akihiko Tanaka,
Shouichi Shinozaki, Kaori Matsumura, and Seiji Nakamura
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University
Objectives: Sjögren’s syndrome (SS) is an autoimmune disease characterized bylymphocytic infiltration into the salivary and lacrimal glands with concomitant
destruction of the glandular tissue. We had reported that T helper (Th) 1 / Th2 balance
was associated with the initiation and progression of SS. Recently, Th17 and regulatory
T cells (Treg) had been shown to have critical function in a variety of autoimmune
disease. Moreover, the localization of Th subsets such as Th1, Th2, Th17, and Treg at
each part of lesion has not been still poorly understood. Therefore, the aim of this study
was to investigate the localization of these Th subsets in the labial salivary glands(LSGs) from SS patients.
Methods: Thirty­two patients with SS and 16 healthy controls were included in thisstudy. Th subsets of infiltrating lymphocytes in the LSGs were examined byimmunohistochemical staining. The mRNA expression of these Th type molecules
including cytokines, chemokines, and transcription factors was determined by real­time
polymerase chain reaction. In addition, lymphocytes without germinal center (GC) :(GC (−) ) and lymphocytes with GC : (GC (+)) were extracted by using laser capture
microdissection, and then, mRNA expression of these molecules in each part of lesion
compared between two groups.
Results: In SS patients, the expression of Th17 type molecules was increased more thanthat in healthy controls. Whereas the expression of Th17 and Treg type molecules had
no association with the degree of lymphocytic infiltration. In addition, Th1 and Th17
type molecules were detected strongly in the GC (−), while Th2 type molecules were in
the GC (+).
Conclusion: These results suggest that SS has definitive localization of Th subsets inthe lesions of LSGs. Furthermore, SS might be initiated by Th1 and Th17 cells andprogress in association with Th2 cells.

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