国際シンポジウム
国際シンポジウム＝口腔顎顔面領域の発生と再生＝＝口腔顎顔面領域の発生と再生＝
Dental andandCraniofacial
CraniofacialMorphogenesis
Morphogenesis
DentalandandTissueTissueRegeneration
Regeneration

主催：九州大学大学院 歯学研究院「魅力ある大学院教育イニシアティブ」
九州大学大学院歯学研究院リサーチコア「口腔組織の再生・再建医療研究」
日時：平成１８年３月１６日（木曜日）
１３：００〜１８：００
場所：西鉄グランドホテル「鳳凰の間」

Special Lecture（ 13:00-16:00））

Chair: Kazuaki NONAKA,
Section of Pediatric Dentistry, Faculty of Dental Science, Kyushu University
Hiroshi NAKANISHI,
Laboratory of Oral Aging Science, Faculty of Dental Science, Kyushu University

Making new teeth from stem cells.
Paul Sharpe
Department of Craniofacial Development, Dental Institute, Kings College London
All our organs develop in the early embryo from simple origins, most often from interactions
between just two types of cell, epithelium and mesenchyme. In order to devise methods ofconstructing organs and tissues for replacement or repair, these embryonic epithelialmesenchymal cell interactions must be reproduced in the laboratory. Since the most importantcharacteristic of embryonic cells is their plasticity, stem cells are the most likely source ofcells to be engineered to produce organ primordia that can be used for transplantation. Teeth
are organs that can be easily accessible and non-essential for life. Replacing missing or
damaged teeth using stem cell-based tissue engineering is thus not only clinically andcommercially valuable but can also provide a test case for using stem cells for replacement ofan organ that is not life-threatening to the patient. Teratomas are stem cell tumours of theadult ovary that often include ectopic teeth. Thus in this pathological situation, stem cells, inthis case germ cells, are capable of forming a complete organ. Using this and anunderstanding of the early developmental processes in the embryos, we are developing
methods of producing human tooth primordia for transplantation into the adult mouth. The
procedure involves reproducing the epithelium and mesenchymal cells that form embryonic
tooth primordia from cultured stem cells. In animal studies we have shown that stem cells thatare easily cultured such as embryonic stem cells, embryonic neural stem cells and adult bonemarrow cells can all form tooth primordia that are able to develop into complete teeth in adults.
Similarly studies are now ongoing with human stem cells.

1The molecular regulatory mechanism of palatogenesis.
Yang Chai*, Xun Xu, Jun Han, Yoshihiro Ito, and Pablo Bringas, Jr.
Center for Craniofacial Molecular Biology, University of Southern California
Cleft palate represents one of the major groups of congenital birth defects in the humanpopulation. Despite recent advancements in medical intervention, babies born with cleft palate
often suffer multiple handicaps that significantly compromise the quality of their lives.
Multiple growth and transcription factors have been identified as critical regulators forpalatogenesis that control the interaction and fate determination of the cranial neural crest(CNC) derived palatal mesenchyme and medial edge epithelium (MEE). Specifically,members of the transforming growth factor-β (TGF-β) superfamily mediate a wide range of
biological activities, including cell proliferation, differentiation, extracellular matrix formation,and induction of homeobox genes, suggesting that TGF-β signaling is important in patternformation and organogenesis.
During craniofacial development, TGF-β is expressed in the craniofacial epithelium and the
CNC-derived mesenchyme, suggesting tissue-specific functions in regulating craniofacial
development. To investigate the functional requirement for TGF-β signaling, we generated
animal models with epithelim or CNC-derived mesenchyme specific inactivation of Tgfbr2
gene and investigated the molecular mechanism of TGF-β signaling in regulating the fate of
craniofacial epithelial or the CNC-derived mesenchyme cells, respectively. Using mice withconditional inactivation of Tgfbr2 in the epithelium, we showed that TGF-β signaling was
specifically required for regulating palate fusion. At the cellular level, TGF-β signaling wascritical for mediating apoptosis of MEE cells during palatal fusion. On the other hand,conditional Tgfbr2 gene ablation in the CNC resulted in complete cleft secondary palate,calvaria agenesis, and other skull defects with complete phenotype penetrance. Significantly,
disruption of the TGF-β signaling did not adversely affect CNC migration. Cleft palate in
Tgfbr2 mutant mice resulted from a cell proliferation defect within the CNC-derived palatalmesenchyme. The midline epithelium of the mutant palatal shelf remained functionallycompetent to mediate palatal fusion once the palatal shelves were placed in close contact invitro. Our data suggest that TGF-β IIR plays a critical, cell autonomous role in regulating thefate of MEE and CNC cells during palatogenesis. To explore the molecular mechanism of
TGF-β signaling in regulating craniofacial development, we analyzed downstream target
genes affected by altered TGF-β signaling in either the epithelium or the CNC-derived
mesenchyme. Comprehensive analysis revealed specific TGF-β signaling network inregulating craniofacial development. Collectively, our study demonstrates that TGF-β2

signaling plays an essential role in cell fate determination of the craniofacial epithelium andthe CNC-derived mesenchyme and provides animal models for the study of abnormal
craniofacial development.

3

Roles of Runx2 and BMP in osteoblast differentiation and bone regeneration.
Akira Yamaguchi
Section of Oral Pathology, Department of Oral Restitution, Graduate School of Tokyo
Medical and Dental University
Recent advance in bone cell biology provides us a lot of important information for molecularmechanism underlining bone formation and bone resorption. To develop biology-based
regenerative medicine/dentistry, we conduct BONE REGENERATION PROJECT byinvestigating molecular mechanism underlying osteoblast differentiation and boneregeneration. I will introduce you two topics in this project.
1) Transcription factors regulating osteoblast differentiation: Runx2 is an essential
transcription factor that regulates osteoblast differentiation and bone formation. We
established two clonal cell lines (C2 and C6) from calvarial region of Runx2-deficient mice.
Both cell lines expressed extremely low levels of osteoblast phenotypes, but treatment with
rhBMP-2 induced these cell lines to express substantial levels of osteoblast phenotypes
including alkaline phosphatase and osteocalcin, suggesting that transcription factors otherthan Runx2 play some important roles in regulation of osteoblast differentiation.
Identification of such transcription factors may provide important information to understandmolecular mechanism in osteoblast differentiation and bone formation.
2) Extensive gene expression analyses during bone regeneration: Gene expression profile
during bone regeneration has not been clarified well. We extensively analyzed genes
expressed during bone regeneration using GeneChip (Affimetrix) in a mouse boneregeneration model. We selected 40 genes that were up-regulated at an early stage of boneregeneration. Among these genes, we demonstrated that the genes relating Notch signaling
play important roles in osteoblast differentiation and bone regeneration. Our study indicatedthat Delta1/Jagged1-activated Notch signaling stimulated BMP2-induced osteoblast
differentiation by interacting with BMP signals.
Finally, I would like to discuss roles of Runnx2 and BMP signals in osteoblast differentiation
and bone regeneration.

4

Gap junctional communication regulates ameloblast differentiation.
Satoshi Fukumoto1, Aya Yamada1, Emiko Fukumoto2, Kenji Yuasa1, Kazuaki Nonaka1
1

Division of Pediatric Dentistry, Kyushu University

2

Division of Oral Hearth Services Research, Nagasaki University

Tooth development was regulated by their specific gene expression. To identify the moleculesthat express stage specifically in tooth germ, we performed the digital differential display(DDD) method using pooled molar UniGene library. The molecules that expressed in tooth
germ stronger than other organs were checked their expressions in mouse incisor using theimmunofluorescence. We found that Gja1 mRNA in tooth germ was expressed stronger thanthat of other 9 organs by DDD method. The tissue expression of Gja1 in mouse incisor wasstage specific in amelogenesis, and in odontoblasts. It expressed strongly in secretary stage,and no expression in maturation stage of ameloblasts. Further, Gja1 disrupted mice showed
inhibition of tooth germ, salivary gland, and lung morphogenesis, similar to occulodentdigital
dysplasia (ODDD). These results suggest Gja1 expression may be associated with ODDD
phenotype in teeth, especially anodontia, microdontia and amelogenesis imperfecta.

5Oral Presentation by Graduate School Students ( 16:00-18:00)
Title

Chaired by:

1. Role of neurotrophic factor NT-4 in ameloblast differentiation.
K. Yoshizaki, S. Fukumoto, Y. Kamasaki, K. Hirano, H. Harada, A.
Nakasima, K. Nonaka

S. Fukumoto
T. Kiyoshima

2. Impairment of Interaction between Microglia and Axotomized Facial
Motoneurons and Decreased Neuronal Survival in Cathepsin Sdeficient Mice.
HP Hao, H. Nakanishi

S. Fukumoto
T. Kiyoshima

3. L3/Lhx8, a LIM homeodomain encoding gene, is expressed in thedeveloping maxilla of chick embryo.
M. Inoue, M Kawakami, K Tatsumi, T Manabe, A Wanaka, T Kirita

T. Kiyoshima
I. Kobayashi

4. Possible functions of Pgk1 in the tooth development.
J Honda, I Kobayashi, T Kiyoshima, H Sakai

I. Kobayashi
T. Kiyoshima

5. BMP-2 promotes osteoblast differentiation in Runx2-deficientcalvarial cell lines.
Tingjiao Liu, A Yamaguchi

I. Kobayashi
M. A. Kido

6. Cystatin C Accelerates the Osteogenic Differentiation of Bone
Marrow Mesenchymal Stem Cells and Increases Bone Formation in vivo.
A Danjo, T Yamaza, D Shimohira, MA Kido, T Tanaka

M. A. Kido
I, Kobayashi

7. Effects of enamel matrix derivative on the injured pulp tissues.
H Kaida, H Anan, N Fujishiro, K Maeda

M. A. Kido
H. Maeda

8. Expression and transcriptional regulation of Osterix transcription
factor during odontoblast differentiation.
M Ishikawa, K Iohara, A Akamine, M Nakashima

H. Maeda
M. A. Kido

9. Characterization of clonal human periodontal ligament cell lines.
S Fujii,, H Maeda, N Wada, A Tomokiyo, A Akamine

H. Maeda
S. Matsuya

10. Topical application of statin affects the bone healing around
implants.
Y Moriyama, Y Ayukawa, K Koyano

S. Matsuya
H. Maeda

11. Fabrication of Low-Crystalline Hydroxyapatite from Gypsum Basedon Phosphate Treatment.
Y Suzuki, S Matsuya, M Nakagawa, Y Ayukawa, K Koyano, K Ishikawa

S. Matsuya
H. Maeda

6

Satoshi Fukumoto
Section of Pediatric Dentistry, Faculty of Dental Science, Kyushu University
Tamotsu Kiyoshima
Laboratory of Oral Pathology and Medicine, Faculty of Dental Science, Kyushu University
Ieyoshi Kobayashi
Laboratory of Oral Pathology and Medicine, Faculty of Dental Science, Kyushu University
Mizuho A. Kido
Department of Oral Anatomy and Cell Biology, Faculty of Dental Science, Kyushu University
Hidefumi Maeda
Department of Endodontology and Operative Dentistry, Faculty of Dental Science, Kyushu
University
Sigeki Matsuya
Department of Biomaterials, Faculty of Dental Science, Kyushu University

7Oral Presentation by Graduate School Students ( 16:00-18:00)
1. Role of neurotrophic factor NT-4 in ameloblast differentiation.
K. Yoshizaki1,4, S. Fukumoto1, Y. Kamasaki2, K. Hirano1, H. Harada3, A. Nakasima4, K.
Nonaka11Section of Pediatric Dentistry, Faculty of Dental Science, Kyushu University, 2Division of

Pediatric Dentistry, Nagasaki University, 3Department of Oral Anatomy and Developmental
Biology, Osaka University Graduate School of Dentistry, 4Section of Orthodontics Dentistry,
Faculty of Dental Science, Kyushu University
Objectives: Neurotrophic factors regulate survival, differentiation, and growth in the nervous
system. In addition, they are expressed in tooth germ during their development. However,detail mechanism of tooth development regulated by neurotrophic factors has never beenclearly understood. Here, we focused on the role of neurotrophin-4 (NT-4) in ameloblast
differentiation, which is detected at high level in ameloblasts at early stage of amelogenesis.
Methods: To analyze the expression of enamel matrix in dental epithelial cell line, we
performed RT-PCR after stimulated with or without NT-4. ERK1/2 phosphorylation byexogenous administration of NT-4 was analyzed by western immunoblotting. Further, toexamine the in vivo function of NT-4, NT-4 disrupted mice were used for histologicalanalysis of tooth development.
Results: In vitro assay, NT-4 induced the expression of ameloblastin and enamelin in dental
epithelial cells. Further, enamel matrix thickness of mutant mice was changed at postnatal day
3 (P3) compared with wild type. The mRNA expression of ameloblastin and amelogenin inmutant mice was changed in P3.
Conclusion: NT-4 regulated the expression of enamel matrix proteins, ameloblastin andenamelin both in vitro and in vitro. This result suggests that neurotrophin, especially NT-4,
may be important for regulation of ameloblast differentiation.

8

2. Impairment of interaction between microglia and axotomized facial motoneurons anddecreased neuronal survival in cathepsin S-deficient mice.
Hai Peng Hao, Hiroshi NakanishiLaboratory of Oral Aging Science, Faculty of Dental Science, Kyushu University
Trauma to facial nerve axotomy occurs as a result of traffic accidents or due to damage during
surgical manipulations. It has been reported that expression level of cathepsin S (CS), alysosomal cysteine protease, and its endogenous inhibitor cystatin C (CC) were markedly upregulated in microglia in response to facial nerve axotomy. Besides functions in theendosoma/lysosomal system, there is substantial evidence that CS is involved in extracellular
proteolysis, because CS degrades extracellular matrix molecules even at neutral pH. However,the precise role of CS in activated microglia that is believed to be closely associated withaxonal regeneration is poorly understood. Therefore, we have attempted to elucidate the preciserole of CS in activated microglia after facial nerve axotomy by the use of CS-deficient mice.
In wild-type mice, microglia accumulated in the axotomized side and spread on the surface of
axotomized motoneurons. Both CS and CC were markedly increased at mRNA and protein
levels, whereas cathepsin B (CB) showed no significant change. In response to the axotomy,
microglia were also activated and accumulated in the axotomized side of CS-deficient mice.
However, the mean cell number of microglia accumulated in the axotomized side of CSdeficient mice was significantly smaller than that of wild-type mice. Furthermore, it was also
noted that CS-deficient microglia abutted to axotomized motoneurons, but failed to spread on
the neuronal surface. In contrast to the wild-type mice, both mRNA and protein levels of CB
were significantly increased in the axotomized side of CS-/- mice. The intense
immunoreactivity of CB was observed in both neurons and microglia abutted to the surface offacial motoneurons. Furthermore, primary cultured microglia prepared from CS-/- mice failed
to adhere on the dishes coating with fibronectin, which is known to be increased in the surfaceof axotomized neurons. The mean number of facial motoneurons at 30 days after axotomy inCS-/- mice was significantly smaller than that of wild-type mice. On the basis of theseobservations, it is reasonable to consider that CS-deficiency impaired spreading of microglia on
the surface of axotomized facial motoneurons. This impairment of cell-cell contact may bemainly responsible for the decrease in neuronal survival after axotomy in CS-deficient mice.

93. L3/Lhx8, a LIM homeodomain encoding gene, is expressed in the developing maxillaof chick embryo.
Masahide Inoue1, Masayoshi Kawakami1, Kouko Tatsumi2, Takayuki Manabe2,
Akio Wanaka2, Tadaaki Kirita1
1

Department of Oral and Maxillofacial Surgery and 2Anatomy, Nara Medical University

LIM-homeodomain (Lhx) encoding gene plays critical roles in the control of patternformation and cell type specification. Here we report a chick orthologues of L3/Lhx8 and the
profiles of expression during the upper lip formation. The chick EST clone was highly
homologous to the mouse Lhx8. Whole-mount in situ hybridization showed that dominant
expression of L3/Lhx8 mRNA from stage 19-29 HH in the maxillary process as well as thelateral edge of the frontonasal mass. The signal was restricted to the mesenchyme underlying
the oral ectoderm. To study the regulation of L3/Lhx8 gene expression, maxillary tissues ofstage 23 HH chick embryos were transplanted into limb bud with or without the maxillaryepithelium. The expression of L3/Lhx8 in the presence of the maxillary epithelium was at
significant levels whereas no expression of L3/Lhx8 was detected in the epithelium-freemesenchyme. Beads soaked in recombinant Fgf-8b or Tgf-β3 induced expression of L3/Lhx8
in epithelium-free mesenchymal grafts. Our data suggest that the expression of L3/Lhx8
required epithelial mesenchymal interaction and Fgf-8b and Tgf-β3 may be responsible forLhx gene expression in the maxilla.

104. Possible functions of Pgk1 in the tooth development.
Jun-ya Honda 1, 2, Ieyoshi Kobayashi 1, Tamotsu Kiyoshima 1, Hidetaka Sakai 1
1

Laboratory of Oral Pathology and Medicine, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University
2
Ddepartment of Orthodontics Division of Oral Health, Growth and Development, Faculty of
Dental Science, Kyushu University
We previously performed cDNA subtraction between mouse embryonic day (E) 10.5 andE12.0 mandibles, and found that several genes were differentialy expressed. In this study, we
examined the temporal and spacial expression pattern of Phosphoglycerate kinase (Pgk) 1
which was one of differentially expressed genes, by means of in situ hybridization (ISH)
method in the course of tooth germ formation. Pgk 1 showed specific strong expression
patterns in the mouse tooth germ at E10.5-E18.0. Strong expression was also found in themouse brain, eye, tongue, heart, lung, liver, kidney and intestine at E15.0. We then studied
Pgk1 protein by western blotting (WB) analysis using total protein isolated from tooth germand tissues at E15.0. The results of WB showed distinct two bands, the one is original (49kDa) and the other is high molecular protein. Interestingly, high molecular protein suggested
more positive reaction in embryonic tissues and tooth germ than in adult tissues. We thoughtthat high molecular band is a complex with GAPDH referring past literatures and based onmolecular weight. We had the result of positive bands of Pgk1 and GAPDH with WB
combined with immunoprecipitation (IP) using mouse liver at E15.0. In conclusion, ourpresent results suggested that Pgk1 performed some functional roles in the development of the
tooth germ and other embryonic organs by forming protein complex with GAPDH.

11

5. BMP-2 promotes osteoblast differentiation in Runx2-deficient calvarial cell lines.
Tingjiao Liu, Akira Yamaguchi
Section of Oral Pathology, Department of Oral Restitution, Graduate School of Tokyo
Medical and Dental University
Although Runx2 is an essential transcription factor for osteoblast differentiation, we
previously reported that primary cells isolated from calvariae of Runx2-deficient embryos
expressed osteoblast markers such as alkaline phosphatase (ALP) activity and osteocalcin in
response to rhBMP-2 (Cell 89:755-764,1997). These results suggest that BMP-2 stimulates
osteoblast differentiation by Runx2-independent pathway. To investigate molecularmechanism of osteoblastic differentiation regulated by Runx2-independent pathway, weestablished two clonal cell lines, named C2 and C6, from calvariae of Runx2-deficientembryos. Both cell lines expressed extremely low or undetectable levels of mRNA relating toosteoblast differentiation at basal condition, whereas treatment with rhBMP-2 or transductionof BMP-2 gene by adenovirus vector effectively induced both cells to express mRNA relating
to osteoblast differentiation including ALP, osteocalcin and osterix. Induction of osteoblast
differentiation makers by BMP-2 was more prominent in C6 cells than C2 cells. Transductionof Runx2 gene by adenovirus vector also induced expression of mRNA relating osteoblast
differentiation in both cell lines, but this effect was more prominent in C2 cells than C 6 cells.
These results suggest that osteoblast differentiation in C2 cells is preferentially regulated by
Runx2-dependent pathway, but that in C6 cells is preferentially regulated by Runx2independent pathway using molecules induced by BMP-2. To explore such molecules, we
conducted microarray analysis using C6 cells with or without rhBMP-2 treatment. RNA
microarray analysis revealed that 72 genes were up regulated by rhBMP2 treatment in C6
cells including 14 transcription-relating molecules such as Msx2, Dlx2, Id1, Id2, Id3, Smad6
and Smad7. Genes involved in TGF-beta pathway, Wnt pathway and Notch signaling
pathway were also up regulated in C6 cells by treatment with rhBMP-2. To confirm boneformation ability of C6 cells and calvarial cells isolated from wild type mice, we transplanted
these cells into peritoneal cavity of athymic mice using diffusion chambers coated with
rhBMP-2. Wild type calvarial cells generated mineralized bone and cartilage in the diffusion
chambers, whereas C6 cells generated only cartilage but not mineralized bone. These results
indicate that Runx2-deficient cells are capable of differentiating into certain stages ofosteoblast lineage cells using transcription factors and signaling pathways other than Runx2,
but incapable of generating mineralized bones. These results further confirmed that Runx2 isessential to bone formation.
126. Cystatin C accelerates the osteogenic differentiation of bone marrow mesenchymal
stem cells and increases bone formation in vivo.
Atsushi Danjo, Takayoshi Yamaza*, Daiji Shimohira, Mizuho A. Kido, Teruo Tanaka
Department of Oral Anatomy and Cell Biology, Kyushu University Graduate School of
Dentistry, *National Institute of Dental and Craniofacial Research
Introduction: Cystatin C (CysC) is well known as a natural cysteine proteinase inhibitor andpresent in several body fluids such as milk. Recently, a novel function of CysC was identified
that CysC acts as an antagonist for transforming growth factor (TGF)-β receptor. TGF-β plays
significant roles not only in the cell differentiation including mesenchymal stem cells (MSCs),
but also in the development and regeneration of tissue and organ including bone. However, it
has been not elucidated that CysC affects on the osteogenic differentiation of MSCs, still
more on the bone formation and regeneration. In this study, we examined the effect of CysC
on the osteogenic potentiality of human bone marrow MSCs (hBMMSCs) derived from bonemarrow and in vivo bone formation.
Methods: hBMMSCs were cultured in a growth medium with or without CysC, andexamined for immunofluorescence, cell proliferation and colony forming unit (CFU) assay.
hMSCs were also cultured in a osteogenic medium in the presence of CysC and analyzed
alkaline phosphatese (ALP) activity, mineralized nodule formation and bone sialoprotein(BSP) and Runx2 gene expressions. CysC-stimulated hBMMSCs were transplanted toimmunocompromised mice, and the transplants were analyzed. To evaluate an effect ofsystemic administration for bone formation, CysC was orally administrated in ovariectomized(OVX) mice. The analyses of X-ray, histology and RT-PCR were subjected for the osteogenic
assay both in the transplants and in mice long bones (femur and tibiae).
Results: CysC treatment increased cell proliferation and colony formation of hBMMSCs.
ALP activity and calcified nodule formation were also accelerated in CysC-stimulated group.
CysC induced mRNA expression for BSP and Runx2. In transplant experiment, boneformation was increased in the group transplanted CysC-pretreated hBMMSCs. Gene
expression for BSP and Runx2 was increased in the CysC-pretreated group. In OVX miceadministrated with CysC, bone density was increased by both X-ray and histological analyses.
Osteocalcin (OCN) increasingly expressed in the CysC-treated mice.
Conclusion: Our results indicated that CysC accelerates not only proliferation andosteogenic potential of hBMMSCs, but also bone formation in vivo.

Therefore, it issuggested that CysC is a new candidate for the treatment of osteolytic bone disease such as
osteoporosis and periodontal disease in the field of hBMMSCs therapy.
13

7. Effects of enamel matrix derivative on the injured pulp tissues.
H. Kaida, H. Anan, N. Fujishiro, K. Maeda
Department of Periodontology, Division of Oral Rehabilitation, Faculty of Dental Science,
Kyushu University
Objectives: To investigate the pathobiological mechanisms of wound healing in the injured
pulp tissues after the application of EMDOGAIN® gel containing enamel matrix derivative(EMD).
Methods: Pulpotomy was performed in the mandibular first molars of rats. EMD or
Vitapex® (VIT) containing calcium hydroxide were applied to the exposed pulp tissues. We
especially examined the relationship between the expression of IL-1β, TGF-β1, BMP-2,
BMP-4 and DMP-1, and the behavior of reparative dentine formation showing alkaline
phosphatase activity using immunohistochemical and enzyme-histochemical methods.
Results: In EMD-treated rats, a small number of IL-1β-expressing macrophages wereobserved around the microabscess during the early phase, and then cells expressing BMPs
were rapidly increased in number. In contrast, cells expressing TGF-β1 were hardly seen
throughout the experimental period. Newly formed reparative dentine was observed earlier in
EMD-treated rats than that seen in VIT-treated rats. On the other hand, in VIT-treated rats,
numerous macrophages expressing IL-1β were continuously observed around the abscess,and then cells expressing TGF-β1 were seen in the lesions. Subsequently, cells expressing
BMPs were gradually increased in number with reparative dentine formation.
Conclusion: These results suggest that there is the difference in the time and extent of theexpression of cytokine and growth factor between EMD and VIT-treated rats. It is
speculated that EMD induced BMPs-expressing macrophages in the injured pulp tissues, andthese cells created a favorable environment for promoting wound healing of the injured pulp
tissues.

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8. Expression and transcriptional regulation of Osterix transcription factor duringodontoblast differentiation.
Masaki Ishikawa1*, Koichiro Iohara2, Akifumi Akamine1, Misako Nakashima2
1

Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation,

Faculty of Dental Science, Kyushu University
2

Division of Regeneration of Oral Function, Laboratory of Oral Disease Research, National

Institute for Longevity Sciences, National Center for Geriatrics and Gerontology.
Objectives: Osterix is a zinc finger-containing transcription factor, which is essential for boneformation. Osterix is also expressed in the mesenchyme of tooth germ. The precise function ofOsterix in tooth development, however, is not clear. In this study, transcriptional regulation ofOsterix by Runt-related gene (Runx) family and bone morphogenetic protein 2 (Bmp2) duringodontoblast differentiation was examined.
Methods: Localization of Osterix mRNA was compared with Runx family and Bmp2 during
tooth development by in situ hybridization. The expression of Osterix in tooth germ wascompared in Runx3 homozygotes with wild type. The expression of Osterix was also
examined by real-time RT-PCR after transfection of Bmp2 and co-transfection of Runx2 andRunx3.
Results: Osterix mRNA was primarily expressed in the preodontoblastic layer at the late bell
stage, and strongly expressed in the odontoblastic layer at the terminal differentiation stage.
Runx2, Runx3 were expressed in the dental papilla at the cap and the early bell stage. Runx2
was no more detected at the late bell stage, while Runx3 was restricted to the odontoblastic
layer at the late bell stage, overlapping with Osterix and Bmp2. There was no conspicuous
difference of expression of Osterix, Runx2 and Bmp2 in the tooth germ of Runx3 homozygotes
compared with wild type. Expression of Osterix, Runx2 and Runx3 was much increased 24 hrs
after Bmp2 gene transfection in primary pulp cells. Runx3 gene transfection induced anincrease of Osterix expression, and cotransfection of Runx3 with Runx2 resulted in inhibitionof Runx3-stimulated Osterix expression.
Conclusion: These results suggest that Osterix might be implicated in odontoblast terminal
differentiation, positively regulated by BMP2 and Runx3 and negatively by Runx2. Nodiscernible change in expression of Osterix in Runx3 homozygotes raising the possibility ofoverlapping functions of other signaling factors during odontoblast differentiation.

15

9. Characterization of clonal human periodontal ligament cell lines.
S. Fujii*, H. Maeda, N. Wada, A. Tomokiyo, A. Akamine.
Department of Endodontology and Operative Dentistry, Division of Oral Rehabilitation,
Faculty of Dental Science, Kyushu University
Objectives: The periodontal ligament (PDL) is a highly specialized tissue connecting thecementum with the tooth socket bone, and affects the life span of tooth. However, little is
known about the precise characteristics and regenerative mechanism of human PDL, becauseof the absence of cell lines and specific makers, and the fact that the PDL tissue consists ofheterogeneous cell populations. Therefore, to address these issues, we aimed to establish andcharacterize clonal human PDL cell lines immortalized by the transduction of Simian Virus40
T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT).
Methods: Normal human PDL fibroblasts (HPLF) were obtained from a healthy firstpremolar of one volunteer. HPLF were transfected with SV40T-Ag gene and hTERT gene,
named as STPLF. The expression of SV40T-Ag and hTERT in STPLF was ascertained by RTPCR analysis and Western blotting analysis. Twenty clonal human PDL cell lines wereobtained by limiting dilution of STPLF. Three of them, 1-4, 1-11, and 1-24, which were used
in

this

study,

were

examinedontheexpressionof

STRO-1

andCD146

immunocytochemically, and gene expressions by RT-PCR technique. Moreover, 1-11 was
examined on the potential to differentiate into osteoblastic or adipocytic cells, andtransplanted into SCID mice.
Results: STPLF obviously expressed SV40T-Ag and hTERT, and showed stable proliferation
at more than 120 population doublings (PD), while HPLF stopped at 20 PD. 1-4, 1-11, and 124 expressed RUNX2, Col I, ALP, OPN, OCN, RANKL, OPG, Scx, Periostin, Col XII, and α-SMA
mRNA, comparable to HPLF. Immunocytochemical data showed that CD146 was expressedin 1-4 and 1-11, and STRO-1 was expressed in 1-11 and 1-24. 1-11 produced calcified
matrices in the presence of ascorbic acid and β-glycerophosphate, and showed adipocytic
differentiation in the adipogenic induction medium. When 1-11 was transplanted into SCID
mice with β-TCP for four weeks, transplant produced bone-like structures around β-TCP.
Conclusion: The present results suggest that a clonal human PDL cell line, 1-11, would bederived from the immature cell present in PDL, and that STRO-1 and CD146 would bepossible markers for such immature cells. This clone could be a very helpful model for the
study of characteristics and regeneration of human PDL.

16

10. Topical application of statin affects the bone healing around implants.
Yasuko Moriyama, Yasunori Ayukawa, Kiyoshi Koyano
Department of Removable Prosthodontics, Faculty of Dental Science, Kyushu University
Objectives: HMG-CoA reductase inhibitors (“statin”), which inhibit a key enzyme in themevalonate pathway, are widely used for the treatment of hyperlipidemia. Recent studies
demonstrate that statins stimulate BMP-2 expression in osteoblasts, suggesting an anabolic
effect of statins on the bone enhancement. The aim of the present study was to evaluate if the
topical application of statin enhances the osteogenesis around the titanium implant, using a rat
model.
Methods: Commercially-pure titanium rods were inserted in both tibiae of Wistar rats. Tenweek-old female Wistar rats were divided into 6 groups with 9 rats for each groups: (1)passive control (implant only); (2) active control (implant with 0µg fluvastatin-containing
propylene glycol alginate (PGA)); (3) low-dose fluvastatin (implant with 3µg fluvastatincontaining PGA) treated; (4) medium-dose fluvastatin (implant with 15µg fluvastatincontaining PGA) treated; (5) high-dose fluvastatin (implant with 75µg fluvastatin-containing
PGA) treated; and (6) ultra-high-dose fluvastatin (implant with 300µg fluvastatin-containing
PGA) treated. The animals were sacrificed on first, second and fourth weeks of observation.
Peri-implant bone formation was assessed using histomorphometric procedures by measuringbone-implant contact (BIC) and the volume density of the newly formed peri-implant osteoiddensity (OD) or mineralized bone density (MBD). Statistical differences among groups were
determined by ANOVA and P<0.05 was considered significant.
Results: At week 1, there was no significant difference in BIC values among groups however,OD in 300µg group was significantly higher than other groups. At week 2, in the experimentalgroups, the medullary canal was filled with abundant bone trabeculae with mesh-like structure.
Especially in 75µg group, this bone trabeculae seen in the medullary canal were somewhat
thicker than those of control groups. BIC was significantly higher in both 15 and 75µg groups
than in the passive control group. Peri-implant MBD was also significantly increased in 75
and 300µg groups in comparison with control groups. At 4 weeks, there was no significant
difference in BIC values among the groups, but OD was significantly higher in passive
control group than in the experimental groups.
Conclusion: Our histomorphometrical evaluations revealed the positive effect of topicallyapplied fluvastatin on the bone around the implant.

1711. Fabrication of low-crystalline hydroxyapatite from gypsum based on phosphate
treatment.
Yumiko Suzuki1,2, Shigeki Matsuya1, Masaharu Nakagawa1, Yasunori Ayukawa2, Kiyoshi
Koyano2, Kunio Ishikawa1
1

Department of Biomaterials, Faculty of Dental Science, Kyushu University

2

Department of Removable Prosthodontics, Faculty of Dental Science, Kyushu University

Objectives: Hydroxyapatite (HAP) has been widely used as a bone substitute material. Mostof the HAP products are prepared by sintering at high temperature. However, the original
shape is difficult to retain due to shrinkage brought about by the sintering process.
Furthermore, the sintered HAP cannot be resorbed by the osteoclasts, therefore the sintered
HAP cannot be replaced with the newly-formed bone. In this study, we tried to fabricate a
low-crystalline HAP with various shapes from gypsum block at lower temperature without
sintering. Subsequently, we also investigated the biocompatibility of the fabricated material invivo.
Materials and Methods: Calcium sulfate hemihydrate was mixed with distilled water and set
gypsum was prepared. Gypsum blocks were immersed in phosphate solution at 100ºC for
24hours. The specimens were characterized by powder X-ray diffraction and FT-IR after thereaction. Biocompatibility of the fabricated material was investigated through an in vivo
evaluation using rats. The material was filled into the artificial bone defect created in the rat
tibia. As a control, sintered HAP was also filled in the same manner. After 2 and 4weeks,
1µm-thick sections of the implant sites were observed under light microscopy.
Results: By compositional analysis, we confirmed that low-crystalline HAP was fabricatedwith its original shape retained. Histologically, as early as 2weeks of implantation, thefabricated low-crystalline HAP was almost circumscribed by newly-formed bone. Resorption
by the osteoclasts could also be observed on the fabricated HAP. On the other hand, sintered
HAP was surrounded by connective tissue.
Conclusions: Low-crystalline HAP monolith was successfully prepared from gypsum throughphosphate treatment at lower temperature and it showed much better biocompatibility andosteoconductivity than sintered HAP. Therefore it has a potential to be an ideal bone
substitute material wherein new bone deposition and resorption took place.

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