Kyudai Oral Bioscience&
OBT Research Center・DDR Research Center

7th Joint International Symposium 2023

PROGRAM & ABSTRACTS

February 18, 2024
Lecture Room AB / Zoom meeting
Faculty of Dental Science,
Kyushu University

u Date:February 18, 2024

u Hybrid:
Zoom meeting / Lecture room AB
u Organization
Kyudai Oral Bioscience
Dento-Craniofacial Development and Regeneration
Research Center
Oral Health・Brain・Health Total Health Research Centeru Contact
Satoshi Fukumoto
Dento-Craniofacial Development and Regeneration Research Center
Section of Pediatric Dentistry, Faculty of Dental Science,
Kyushu University, Fukuoka 812-8582, Japan
Tel: +81 92-092-642-6402
E-mail: fukumoto@dent.kyushu-u.ac.jp

7th KOB & OBT & DDRJoint International Symposium 2023

PROGRAM

February 18 (Sunday)

Time

Title

Presenter

9:00­9:10

Opening Remark

Prof. Fusanori Nishimura

Session 1

Yonsei University Session

Chair:
Dr. Shingo Takai

9:10­9:25

Construction of 3D anatomical data using ultrasound and motion capture technology.

Hyungkyu Bae

9:25­9:40

Salt diet affect salt preference in Drosophila melanogaster.

LINNI JIN

9:40­9:55

Tuning circadian rhythm of cancer-associated fibroblasts against cancer progression.

Shujin Li

9:55­10:10

Break

Session 2

DDR Session

Chair:
Prof. Satoshi Fukumoto

10:10­10:50

Dual nature of K8 expressing differentiated cells drives epithelial regeneration.

Han-Sung Jung

10:50­11:10

Human fibrotic autoimmune disease: latest immunological approaches into the pathogenesis Takashi Maehara

11:10­11:30

Temperature-controlled ex vivo culture for organ development and drug-induced side
effect mitigation: insights from mouse tooth germ model

11:30­11:45

Break

Session 3

Undergaduate Student̀s Session

11:45­12:00

Short term summer study abroad program at Universitas Airlangga (Indonesia)

12:00­12:15

Study Abroad at The University of Waikato, Waikato College (New Zealand)

12:15­13:15

Lunch Break

13:15­13:25

Award Presentation (IF and FWCI Award)

Prof. Fusanori Nishimura

Session 4

Award Lectures (IF Award winners)

Chair:
Prof. Takashi Kanematsu

13:25­13:45

p130Cas is required for androgen-dependent postnatal development regulation ofsubmandibular glands.

Jing Gao

13:45­14:05

Distinct disease-specific Tfh cell populations in two different fibrotic diseases: IgG4-relateddisease and Kimuraʼs disease.

Ryusuke Munemura

14:05­14:25

GATA3 mediates nonclassical β-catenin signaling in skeletal cell fate determination andectopic chondrogenesis.

Daigaku Hasegawa

14:25­14:45

Long-term administration of bisphosphonates enhances aversive behavioral responses to
HCl.

Asami Oike

14:45­15:00

Break

Session 5

Graduate Student̀s Session

Chair: Dr. Takao Fukuda
Dr. Takashi Maehara

15:00­15:15

The function of membrane-permeable itaconate in cancer cell growth.

Ayaka Saeki

15:15­15:30

Arl4c is involved in tooth germ development through osteoblastic differentiation.

Truong Thi Kim Thinh

15:30­15:45

Involvement of B cells in tumor immunity of oral squamous cell carcinoma.

Junsei Sameshima

15:45­16:00

Theme of presentation: Fabrication and histological evaluation of ant-nest type porous
carbonate apatite artificial bone.

Janice Tan Lay Tin

16:00­16:15

Cytoplasmic signaling molecule CySM2 in LTi cells plays critical role on Payerʼs patch
formation.

Naoko Kiya

16:15­16:30

Fabrication of granular apatite cement using partially set α-tricalcium phosphate granules.

Abdulrahman Diabi

16:30­16:45

Establishment of a new peripheral nerve regeneration therapy using artificial nerves withextracellular vesicles derived from dental pulp stem cells.

Aiko Yano

16:45­16:55

Closing Remarks

Prof. Satoshi Fukumoto

Tian Tian

Chair:
Dr. Kenichi Ogata

Kanto Nakamura
Miku Tokuhisa

7th KOB & OBT & DDRJoint International Symposium 2023

Session1

Yonsei University Session

Chaird by Dr. Shingo Takai

Construction of 3D anatomical data using ultrasound andmotion capture technology.

Hyungkyu Bae1, Kang-Woo Lee1, Soo-Bin Kim1, Kyung-Seok Hu1, Hee-Jin Kim1,2
1Division in Anatomy and Developmental Biology, Department of Oral Biology, Human Identification

Research Institute, BK21 Four Project, Yonsei University College of Dentistry, Seoul, Republic of
Korea
2Department of Materials Science & Engineering, College of Engineering, Yonsei University, Seoul,

Republic of Korea

Ultrasound (US) imaging is widely used in both treatment and diagnosis because it
can visualize the internal structure in real time, is simple to utilize, and less harmful
to patients. Recently, various three-dimensional (3D) methods including 3D
scanning, computed tomography (CT), magnetic resonance imaging (MRI) havebeen used in the field, but US has been able to be analyzed 3D only in a limitedarea due to its characteristics. The aim of the study was to construct and verify 3D
anatomic data by specifying the location of the US transducer using motion capture
technology.
A total of 57 healthy Korean volunteers (average age 43.2 years, 57 females) were
included in this study. Real-time two-dimensional B-mode ultrasound images of theintraoral structures were acquired using a high-frequency (11.5 MHz) intraoraltransducer. Image reconstruction was additionally performed to identify structures
in intraoral US images. A motion capture camera was used to track using an
intraoral transducer attached with a marker, and the structure was scanned at thereference points. Simultaneously, the marker attached to the subject's forehead wastracked and used as the Reference Coordinate System. The face of the volunteer
was scanned with the mouth open 2.7cm using an Artec Space Spider scanner,which is a high-resolution 3D scanner based on blue light technology. The US
image position of the tracked intraoral transducer and the scanned face werevisualized with software that is part of the SlicerIGT extension. For the verification,
MRI images were taken from one volunteer using a 3.0T scanner. The visualized
image and MRI of the volunteer were superimposed on the nose and ear and used
for image analysis.
The superimposed model of the MRI and 3D scanned face with tracked intraoral US
image was obtained. The location of the Internal anatomic structures of intraoral
area were confirmed in the model and verified through comparison with MRI image.
The association with motion capture technology can extend the ultrasonographic
visualization from two dimensions to the three dimensions. This method canprovide 3D model for the internal structure in real time, and it is expected to be
utilized as a powerful diagnostic tool for clinicians.

Salt diet affect salt preference in Drosophila melanogaster.

LINNI JIN1 and Seok Jun Moon1
1Department of Oral Biology, BK 21 FOUR Project, Yonsei University College of Dentistry, Seoul

03722, Korea

Taste plays a pivotal role in evaluating the nutritional value and potential toxicity
of food. Typically, sweet and umami are perceived as appealing, while bitter andsour are considered aversive, and salt elicits a concentration-dependent preference.
Salt taste is essential for maintaining electrolyte homeostasis and facilitating
nutrient absorption; however, prolonged high salt intake can be detrimental. In
mammals, elevated salt consumption is linked to an increased risk of cardiovascularand chronic kidney diseases. Despite considerable knowledge about the molecularand cellular underpinnings of salt taste in mammals and insects, the impact of daily
dietary salt on salt perception remains unclear. In this study, utilizing the
Drosophila model system, we investigated the effects of daily salt intake on salt
perception. Our findings reveal that daily salt intake suppresses low salt sensitivity
so that reduces the intake of salt-containing food in flies. Exposure to a sodium-richdiet leads to the abolition of low salt electrophysiological responses in low saltsensing gustatory neurons through receptor-mediated endocytosis, reshaping both
low salt perception and feeding behavior. While the desensitization mechanism
exhibits sexual dimorphism, both male and female flies counteract salt
desensitization by inhibiting clathrin-mediated endocytosis following the salt diet.
Notably, the macropinocytosis pathway also modulates low salt perception
exclusively in female flies after the salt diet. Collectively, our study provides
insights into how salt diet-induced alterations in salt perception are regulated in
Drosophila, shedding light on the intricate interplay between dietary factors andsensory mechanisms.

Tuning circadian rhythm of cancer-associated fibroblasts
against cancer progression
Shujin Li1, Yan Cui1, Senthil Kumar Baskaran1, Ji-Sun Kim1, Anish Ashok
Adpaikar1, Hyun-Yi Kim1, Han-Sung Jung*
1

Division in Anatomy and Developmental Biology, Department of Oral Biology, Taste Research Center,
Oral Science Research Center, BK21 FOUR Project, Yonsei University College of Dentistry, Seoul,
Korea
2NGeneS Inc., Ansan-si, Gyeonggi-do, South Korea

Cancer-associated fibroblasts (CAFs) are essential stromal cell populations of thetumor microenvironment (TME) with diverse functions, including matrix deposition
and remodeling, extensive reciprocal signaling interactions with cancer cells andcrosstalk with infiltrating leukocytes. However, owing to the limitation ofunderstanding the origin and biological function of CAFs, many challenges arepresent in ongoing attempts to modulate CAFs for therapeutic benefit.
The circadian clock coordinates the daily rhythmicity of metabolic, physiologic,
and behavioral processes with a 24-hour periodicity in humans. Intriguingly,
dysfunction of the circadian clock is intimately related to tumorigenesis andfacilitates the circadian rhythm disruption of the adjacent tissues.
In the present study, we are utilizing ameloblastoma, an odontogenic tumor
arising in the jawbone, as a model system, to investigate the circadian rhythm of
CAFs and the subsequential biological effect on tumor progression. We establishedthe stromal-rich ameloblastoma tumoroid that recapitulated the histological andmolecule characteristics of ameloblastoma. The CAFs that isolated fromameloblastoma patients, showed accelerated growth and metabolic rhythm anddisrupted circadian clock expression pattern compared to the control. Time-series
RNA sequencing results confirmed that circadian clock genes were upregulated, therhythmically expressed genes were decreased, and gene ontology terms related to
extracellular matrix remodeling, secretory process, and metabolism process wereenriched in ameloblastoma tumoroid co-cultured with the CAFs group. Interestingly,
CRISPR/Cas9 based-BMAL1 knockout in CAFs results in the reduces of the growth
factors and inflammatory cytokines secretion, matrix contraction in transcription
levels. Furthermore, administration of the BMAL1 inhibitor (GSK4112) in CAFs
increase the anti-tumor efficacy of PLX4023 on ameloblastoma tumoroid. In
conclusion, we demonstrated that targeting CAFs by tuning circadian rhythm
regulate the secretion and ECM remodeling properties that beneficial for the anticancer treatment on ameloblastoma.
This work was supported by the National Research Foundation of Korea (NRF)
Grant funded by the Korea Government (MSIT) (NRF-2022R1A2B5B03001627).

7th KOB & OBT & DDRJoint International Symposium 2023

Session2

DDR Session

Chaird by Prof. Satoshi FukumotoDual nature of K8 expressing differentiated cells drives
epithelial regeneration

Han-Sung Jung, Anish Adpaikar, Jong-Min Lee
Division in Anatomy and Developmental Biology, Department of Oral Biology,
Taste Research Center, Oral Science Research Center, BK21 FOUR Project,
Yonsei University College of Dentistry, Seoul 03722, Korea

Taste receptor cells are epithelial cells dependent upon the innervating nerve forcontinuous renewal and maintained by resident tissue stem/progenitor cells.
Transection of the innervating nerve causes degeneration of taste buds and taste
receptor cells. Genetic lineage tracing has identified that Lgr5+ cells present at the
trench of the circumvallate papilla give rise to all taste and non-taste epithelial cells
during homeostasis and regeneration. However, whether Lgr5 expressing cells are
the only source of stem cells in circumvallate papillae (CVP) remains to beinvestigated. This study revealed that a subset of the taste receptor cells ismaintained without nerve contact after glossopharyngeal nerve transection in the
CVP of adult mice.
Furthermore, the injury caused by nerve transection triggers these surviving
differentiated K8-positive taste receptor cells to dedifferentiate and acquire atransient progenitor cell-like state. Dedifferentiated taste receptor cells proliferate,
express progenitor cell markers (K14, Sox2, PCNA), and form organoids in vitro. In
another model, using Lgr5-DTR mice, ablation of Lgr5+ expressing cells led to thedepletion of half the CVP organ by disrupting the basal cells and basement
membrane after 24 hours. Interestingly, the entire CVP with taste buds was
regenerated within 2 weeks. This data indicates that another population of cells acts
as reserve stem cells in the CVP that activate upon Lgr5 stem/progenitor cell
depletion.
This work was supported by the National Research Foundation of Korea (NRF)
Grant funded by the Korea Government (MSIP) (2022R1A2B5B03001627 and NRF2021R1A2C1005506)

Human fibrotic autoimmune disease: latest immunological approaches
into the pathogenesis
Takashi Maehara1,2

1 Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical

Sciences, Faculty of Dental Science, Kyushu University, Fukuoka, Japan
2 Dento-craniofacial Development and Regeneration Research Center, Faculty of Dental Science,

Kyushu University, Fukuoka, Japan

We examined tissue infiltrating T cells in human fibrotic atutoimmune disease.
IgG4-related disease (IgG4-RD) is an immune-mediated fibrotic disordercharacterized by the dysregulated resolution of severe inflammation and woundhealing and represents an example of a Type I atutoimmune disease. We performed
single-cell RNA-sequencing and T-cell receptor (TCR) sequencing to obtain acomprehensive, unbiased view of tissue infiltrating T-cells. Almost all clonallyexpanded T-cells in these lesions were either GZMK expressing CD4+ cytotoxic Tcells (CTLs) or GZMK+ CD8+ CTLs. These Granzyme K+ cytotoxic T cells also
express amphiregulin and TGFb, but did not express immune-checkpoints, and thetissue infiltrating CD8+ T cells are phenotypically heterogeneous. Other abundant
infiltrating T-cells in IgG4-RD lesions included two T helper cell populations,
IL10+LAG3+Foxp3- Tfh cells and CXCL13+TIGIT+PD1+Tfh cells. Infiltrating
regulatory T cells with an altered dysregulated transcriptome similar to that seen intumors and severe COVID-19 were also observed in this inflammatory disease. We
also conducted quantitative analyses of the T cell subsets in 68 IgG4-RD and 30
Sjögrenʼs syndrome (SjS) patients. Our findings, validated using orthogonal
approaches, support a central role for cytotoxic CD4+ and CD8+T-cells expressing
Granzyme K, amphiregulin and TGFb in the pathogenesis of inflammatory fibrotic
disorders. Therapies should be focused on targeting these cells and studies on
pathogenesis should examine the antigenic sources and mechanisms of theirgeneration.

Temperature-controlled ex vivo culture for organ development and druginduced side effect mitigation: insights from mouse tooth germ model

Tian Tian1,2, Satoshi Fukumoto1,2,3
1Dento-Craniofacial Development and Regeneration Research Center, Kyushu University Faculty of

Dental Science, Fukuoka, Japan
2Section of Pediatric Dentistry, Division of Oral Health, Growth and Development, Kyushu University

Faculty of Dental Science, Fukuoka, Japan
3 Division of Pediatric Dentistry, Department of Community Social Dentistry, Tohoku University

Graduate School of Dentistry, Sendai, Japan

In recent years, with the advancement of regenerative technologies, the prospectof organ regeneration using pluripotent stem cells has become increasingly realistic.
However, for the clinical application of these achievements in regenerative
medicine, the timely provision of regenerated organs to patients is crucial. This
study aimed to establish a temperature screening method for controlling thedevelopment of organs by applying an ex vivo organ culture model using mouse
tooth germs. The development of cultured tooth germs was delayed by lowtemperature stimulation and could be resumed by subsequent cultivation at 37 C.
Furthermore, the optimal temperature for the long-term preservation of organs wasfound to be 25 C, maintaining the expression of stem cell markers under these
conditions. Comprehensive analysis revealed that low-temperature stimulationinduces the expression of cold shock proteins (CSP). These results suggest thatmaintaining stem cell viability and CSP expression may play an important role in
the long-term preservation of organs under the low-temperature culture conditions
at 25 C.
Additionally, with a focus on clinical applications, we investigated the potential of
avoiding the side effects of anticancer drugs that affect developing organs. By
applying low-temperature stimulation, it was possible to circumvent the inhibitionof tooth formation caused by cyclophosphamide (CPA), an anticancer drug used in
the treatment of childhood leukemia. Furthermore, low-temperature stimulation
inhibited Rb phosphorylation and induced cell cycle arrest at the G1 phase during
CPA treatment. These findings suggest the potential for preventing cell damage
associated with DNA replication caused by CPA through low-temperature
stimulation.
This study indicates the potential utility of a screening method for organ cultureusing tooth germs in screening for organ regeneration technology and medical
techniques.

7th KOB & OBT & DDRJoint International Symposium 2023

Session3

Undergraduate Student Session

Chaird by Dr. Kenichi Ogata

7th KOB & OBT & DDRJoint International Symposium 2023

Session4

Award Lectures

Chaird by Prof. Takashi Kanematsu

p130Cas is required for androgen-dependent postnatal
development regulation of submandibular glands

Jing Gao1, Aonan Li1, Shinsuke Fujii2,3, Fei Huang1, Tamotsu Kiyoshima2,
Eijiro Jimi1,4
1Laboratory of Molecular and Cellular Biochemistry, 2Laboratory of Oral Pathology, 3Dento-craniofacial Development and Regeneration Research Center, 4OBT Research Center, Faculty of
Dental Science, Kyushu University, Japan

Salivary glands develop through epithelial-mesenchymal interactions and areformed through repeated branching. The Crk-associated substrate protein (p130Cas)
serves as an adapter that forms a complex with various proteins via integrin andgrowth factor signaling, with important regulatory roles in several essential cellular
processes. We found that p130Cas is expressed in ductal epithelial cells of thesubmandibular gland (SMG). We generated epithelial tissue-specific p130Casdeficient (p130CasΔepi‒) mice and aimed to investigate the physiological role of
p130Cas in the postnatal development of salivary glands.
Submandibular gland were extracted from p130Casflox/flox (control) andp130CasΔepi‒ mice and measured for weight. Mice were anesthetized and stimulatedwith pilocarpine to collect saliva for secretion measurement and components
analysis. For histological analysis, submandibular gland were processed for H/E
staining, immunohistochemistry and transmission electron microscopy (TEM),
respectively. Quantitative RT-PCR was performed to examine mRNA expression
levels in submandibular gland.
Histological analysis showed immature development of granular convoluted
tubules (GCT) of the SMG in male p130CasΔepi‒ mice. Immunofluorescence staining
showed that nuclear-localized androgen receptors (AR) were specifically decreasedin GCT cells in p130CasΔepi‒ mice. Furthermore, epidermal growth factor (EGF)positive secretory granules contained in GCT cells were significantly reduced in
p130CasΔepi‒ mice with downregulated AR signaling. GCTs lacking p130Cas showed
reduced numbers and size of secretory granules, disrupted subcellular localizationof the cis-Golgi matrix protein GM130, and sparse endoplasmic reticulum
membranes in GCT cells. These results suggest that p130Cas plays a crucial role inandrogen-dependent GCT development accompanied with ER-Golgi network
formation in SMG by regulating the AR signaling.

Distinct disease-specific Tfh cell populations in two different
fibrotic diseases: IgG4-related disease and Kimuraʼs disease
Ryusuke Munemura1, Takashi Maehara1,10, Yuka Murakami1,
Risako Koga1, Ryuichi Aoyagi1, Naoki Kaneko1, Atsushi Doi2,
Cory A. Perugino3,4, Emanuel Della-Torre5,Takako Saeki6, Yasuharu Sato7,
Hidetaka Yamamoto8, Tamotsu Kiyoshima9, John H. Stone3, Shiv Pillai4,
and Seiji Nakamura1.
1the Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical

Sciences, Faculty of Dental Science, Kyushu University, Japan; 2Cell Innovator Inc, Japan; 3theDivision of Rheumatology, Allergy, and Immunology, Massachusetts General Hospital, Harvard
Medical School, USA; 4the Ragon Institute of MGH, MIT, and Harvard, Massachusetts General
Hospital, Harvard Medical School, USA; 5the Unit of Immunology, Rheumatology, Allergy, and Rare
Diseases, IRCCS San Raffaele Scientific Institute, Italy; 6the Department of Internal Medicine, Nagaoka
Red Cross Hospital, Japan; 7the Okayama University Graduate School of Medicine, Dentistry, andPharmaceutical Sciences, Japan; 8the Division of Diagnostic Pathology, Kyushu University Hospital,
Japan; 9the Laboratory of Oral Pathology, Division of Maxillofacial Diagnostic and Surgical Sciences,
Faculty of Dental Science, Kyushu University, Japan; and 10the Dento-craniofacial Development andRegeneration Research Center, Faculty of Dental Science, Kyushu University, Japan.

How T follicular (Tfh) cells contribute to many different B-cell class-switching
events during T-cell‒dependent immune responses has been unclear. Diseases withpolarized isotype switching offer a unique opportunity for the exploration of Tfh
subsets. Secondary and tertiary lymphoid organs in patients with elevated tissue
expression levels of IgE (Kimura disease, KD) and those of IgG4 (IgG4-relateddisease, IgG4-RD) can provide important insights regarding cytokine expression by
Tfh cells. We sought to identify disease-specific Tfh cell subsets in secondary andtertiary lymphoid organs expressing IL-10 or IL-13 and thus identify different
cellular drivers of class switching in 2 distinct types of fibrotic disorders: allergic
fibrosis (driven by type 2 immune cells) and inflammatory fibrosis (driven bycytotoxic T lymphocytes).
Peripheral blood and affected organs of 11 patients with KD and 25 patients with
IgG4-RD were used. Single-cell RNA sequencing, in situ sequencing, and multicolor
immunofluorescence analysis were used to investigate B cells, Tfh cells, andinfiltrating type 2 cells in lesion tissues from patients with KD or IgG4-RD.
Infiltrating Tfh cells in tertiary lymphoid organs from IgG4-RD were divided into 6
main clusters. We encountered abundant infiltrating IL-10‒expressing LAG3+Tfh
cells in patients with IgG4-RD. Furthermore, we found that infiltrating
AICDA+CD19+ B cells expressing IL-4, IL-10, and IL-21 receptors correlated with
IgG4 expression. In contrast, we found that infiltrating IL-13‒expressing Tfh cells
were abundant in affected tissues from patients with KD. Moreover, we observed
few infiltrating IL-13 expressing Tfh cells in tissues from patients with IgG4-RD,
despite high serum levels of IgE (but low IgE in the disease lesions). Cytotoxic T
cells were abundant in IgG4-RD;in contrast, type 2 immune cells were abundant in KD.
Our analysis revealed a novel subset of IL-10+LAG3+Tfh cells infiltrating theaffected organs of IgG4-RD patients. In contrast, IL-13+Tfh cells and type 2 immune
cells infiltrated those of KD patients. IL-10+LAG3+Tfh cells might contribute to IgG4
isotype switching in SLOs and TLOs of patients with IgG4-RD. In contrast, IL13+Tfh
cells might contribute to high-affinity IgE secretion by B cells in KD.

GATA3 mediates nonclassical β-catenin signaling in skeletal
cell fate determination and ectopic chondrogenesis

Takamitsu Maruyama1,2†, Daigaku Hasegawa1,2†, Tomas Valenta3, Jody
Haigh4, Maxime Bouchard5, Konrad Basler3, Wei Hsu1,2,6,7,8*
1Forsyth Institute, Cambridge, MA, 2University of Rochester Medical Center, Rochester, NY,
3Department of Molecular Life Sciences, University of Zürich, Zürich, Switzerland, 4CancerCare

Manitoba Research Institute, Department of Pharmacology and Therapeutics, University of Manitoba,
Winnipeg, Canada, 5Goodman Cancer Institute and Department of Biochemistry, McGill University,
Montreal, Canada, 6Faculty of Medicine, Harvard University, Boston, MA, 7Harvard School of Dental
Medicine, Boston, MA, 8Harvard Stem Cell Institute, Cambridge, MA.
†These authors contributed equally to this work, *Corresponding author.

Skeletal precursors are mesenchymal in origin and can give rise to distinct
sublineages. Their lineage commitment is modulated by various signaling pathways.
The importance of Wnt signaling in skeletal lineage commitment has been
implicated by the study of β-catenin‒deficient mouse models. Ectopic
chondrogenesis caused by the loss of β-catenin leads to a long-standing belief incanonical Wnt signaling that determines skeletal cell fate. As β-catenin has otherfunctions, it remains unclear whether skeletogenic lineage commitment is solely
orchestrated by canonical Wnt signaling. The study of the Wnt secretion regulator
Gpr177/Wntless also raises concerns about current knowledge.
Therefore, we have created several mouse models to examine details of theskeletal cell fate decision mediated by β-catenin. Our findings provide evidence
supporting that an alternative mechanism mediated by β-catenin independent of
the transcriptional output of canonical Wnt signaling is necessary for inhibiting
ectopic chondrogenesis. The whole genomics study further examines downstream
effectors, leading to the identification of GATA3 as the key modulator associatedwith this alternative signaling effects of β-catenin. Functional analyses further
demonstrate that the GATA3 transcription factor is sufficient to promote thecommitment of skeletogenic mesenchyme to chondrocyte lineage. In this study, thesingle pioneer factor GATA3 can program skeletal cells to become chondrocytesthat are responsible for cartilage formation, demonstrating its essential role inmediating nonclassical β-catenin signaling in skeletogenic lineage specification.

Long-term administration of bisphosphonates enhances
aversive behavioral responses to HCl.

Asami Oike1, Ayaka Hirayama2, Yuki Nagasato2, Yuko Kawabata2, Shingo
Takai2, Keisuke Sanematsu2,3,4, Naohisa Wada1, Noriatsu Shigemura2,3.
1Section of Interdisciplinary Dentistry, Graduate School of Dental Science, Kyushu University,

Fukuoka, Japan; 2Section of Oral Neuroscience, Graduate School of Dental Science, Kyushu University,
Fukuoka, Japan; 3Research and Development Center for Five-Sense Devices, Kyushu University,
Fukuoka, Japan;4Oral Health/Brain Health/Total Health Research Center, Faculty of Dental Science,
Kyushu University, Fukuoka, Japan

Drug-induced taste disorders cause malnutrition and reduce the quality of life,
but little is known about the molecular mechanism. One of taste disorders is known
adverse effects of bisphosphonates, which are administered as anti-osteoporotic
drugs or anti-bone-resorptive and temporary. Therefore, the present study evaluatedthe effects of risedronate (a bisphosphonate) on taste bud cells. Bisphosphonates
inhibit farnesyl diphosphate synthase (FDPS), which is a key enzyme in themevalonate pathway that mediates cholesterol synthesis. Expression analyses
revealed that FDPS was present in a subset of mouse taste bud cells, especially typeIII sour-sensitive taste cells. Behavioral analyses (short-term lick tests) revealed thatmice administered risedronate for 28 days exhibited a significant reduction in theirpreference for sour tastant (HCl) but not for other taste solutions (salty; NaCl, KCl,sweet; sucrose, bitter; quinine HCl, and umami; monopotassium glutamate)compared to controls. In contrast, taste nerve (the chorda tympani nerve and theglossopharyngeal nerve) responses showed no significant differences in responses toall taste substances. On the other hand, the taste buds of mice administered
risedronate for 28 days exhibited significantly higher mRNA expression of FDPS andsignificantly lower mRNA expression of desmoglein-2, an integral component ofdesmosomes. Taken together, these findings suggest that risedronate may interact
directly with FDPS to inhibit the mevalonate pathway in type III taste cells, thereby
affecting the structure of desmosomes in the plasma membrane related withparacellular permeability to H+ and/or Cl- in the taste buds. These findings provide
new insights into the mechanisms of FDPS in taste bud cells by whichbisphosphonates may cause taste-like disorders.

7th KOB & OBT & DDRJoint International Symposium 2023

Session5
Graduate Student̀s Session

Chaird by

Dr. Takao Fukuda
Dr. Takashi Maehara

The function of membrane-permeable itaconate in cancer cell
growthAyaka Saeki1, Yoshikazu Hayashi1,2,3, Shohei Yoshimoto3,4, Yuji
Hatakeyama2, Masato Hirata3, Eijiro Jimi1, Tomoyo Kawakubo-Yasukochi1.
1 OBT Research Center, Faculty of Dental Science, Kyushu University
2 Division of Functional Structure, Department of Morphological Biology, Fukuoka Dental College
3 Oral Medicine Research Center, Fukuoka Dental College
4 Section of Pathology, Department of Morphological Biology, Division of Biomedical Sciences,

Fukuoka Dental College

Introduction: Itaconate (IA) is synthesized in the citric acid cycle via cis-aconitate
decarboxylase (ACOD1). Previous reports demonstrated that IA is an antiinflammatory metabolite in macrophage-mediated acute inflammation, making it
possible to link with cellular and molecular events involved in various
pathophysiological conditions such as cancer. In this study, we thus focused on theeffect of IA on cancer progression by using 4-occtyl itaconate (OI) which is amembrane-permeable IA.

Materials and Methods: OI, IA (membrane-impermeable IA), and 2-ethylhexanoic
acid (EHA) were applied to in vitro (murine (B16) and human (COLO679 and G361)
melanoma cell lines) and in vivo (C57BL/6N mice) assays. Cell proliferation
potential was evaluated by using the WST-8 and the BrdU incorporation assays.
RNA sequencing was performed with the MGI DNBSEQ G400 FAST. LCMS-8050
was used for HPLC-MS/MS assays. Glutathione, ROS, and γH2AX quantifications
were measured by GSSG/GSH quantification kit, ROS assay kit, and DNA damage
detection kit-γH2AX, from Dojindo, respectively. Lipid hydroperoxide (LPO) and
ROS were quantified by FACSAria Fusion and FlowJo software from BD Bioscienceswith Liperfluo (Dojindo). Succinate dehydrogenase (SDH) activity was measured byComplex II assay kit from Cayman Chemical. Mitochondrial respiratory capacity
was assessed using a Seahorse XF Extracellular Flux analyzer.

Results: Cell proliferation of melanoma cells treated with OI, but not IA and EHA,
weresignificantly
suppressedin
its
concentration-dependent
manner.
Comprehensive transcriptome analysis in B16 cells revealed that OI treatment
induced a marked change in the expression of genes related to glutathione
metabolism. Subsequent analysis showed OI treatment induced a decreased
glutathione levels followed by increased levels of ROS, LPO, and γH2AX.
Furthermore, OI treatment suppressed the activity of mitochondrial complex II andinduced a marked decrease in mitochondrial respiratory capacity. In addition, OI
markedly suppressed tumor growth in vivo, partly caused by abnormal glutathione
metabolism, with the minimum side effect.

Conclusion: This study demonstrated the direct antiproliferative effects of OI on
melanoma cells, mediated by increased ROS accumulation, resulted from
impaired glutathione metabolism and dysfunction of mitochondrial respiratory
capacity. These intracellular events consequently led the melanoma cells tosenescence.

Arl4c is involved in tooth germ development throughosteoblastic differentiation.

Truong Thi Kim Thinh, Shinsuke Fujii, Tamotsu Kiyoshima

Laboratory of Oral Pathology

Murine tooth germ development proceeds in continuous and sequential steps
with reciprocal interactions between the odontogenic epithelium and the adjacentmesenchyme, and several growth factor signaling pathways and their activation are
required for tooth germ development. The expression of ADP-ribosylation factor(Arf)-like 4c (Arl4c) has been shown to induce cell proliferation, and is thereby
involved in epithelial morphogenesis and tumorigenesis. In contrast, the otherfunctions of Arl4c (in addition to cellular growth) are largely unknown. Although
our laboratory members recently demonstrated the involvement of the upregulated
expression of Arl4c in the proliferation of ameloblastoma (J Pathol., 2022), which
has the same origin as odontogenic epithelium, its effect on tooth germdevelopment remains unclear.
In this study, single-cell RNA sequencing (scRNA-seq) analysis revealed thatthe expression of Arl4c, among 17 members of the Arf-family, was specificallydetected in odontogenic epithelial cells, such as those of the stratum intermedium,stellate reticulum and outer enamel epithelium, of postnatal day 1 (P1) mouse
molars. scRNA-seq analysis also demonstrated the higher expression of Arl4c in
non-ameloblast and inner enamel epithelium, which include immature cells, of P7mouse incisors. In the mouse tooth germ rudiments culture, treatment with SecinH3(an inhibitor of the ARNO/Arf6 pathway; a possible downstream of Arl4c signaling)
reduced the size, width and cusp height of the tooth germ and thickness of theeosinophilic layer, which would involve the synthesis of dentin and enamel matrixorganization. In addition, loss-of-function experiments using siRNAs and shRNA
revealed that Arl4c expression was involved in cell proliferation and osteoblastic
cytodifferentiation in odontogenic epithelial cells. Finally, RNA-seq analysis withgene set enrichment analysis (GSEA) and Gene Ontology (GO) analysis showed that
osteoblastic differentiation-related gene sets and/or GO terms were downregulated
in shArl4c-expressing odontogenic epithelial cells.
These results suggest that the Arl4c-ARNO/Arf6 pathway axis contributes totooth germ development through osteoblastic differentiation.

Involvement of B cells in tumor immunity of oral squamous cell
carcinoma
Junsei Sameshima1), Naoki Kaneko1), Hu Chen1), Shiho Yokomizo1), Tomoki
Sueyoshi1), Haruki Nagano1), Yuka Miyahara1), Yan Lijin1), Mizuki
Sakamoto1) , Masafumi Moriyama2,3), Shintaro Kawano1)
1.
2.
3.

Section of Oral and Maxillofacial Oncology Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University, Fukuoka, Japan
Section of Oral and Maxillofacial Surgery Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University, Fukuoka, JapanOBT Research Center, Faculty of Dental Science, Kyushu University, Fukuoka, Japan(Background)
Understanding the significance of lymphocyte infiltration in the tumor lesion ispivotal in cancer immunotherapy. Traditionally, cytotoxic T cells have been shown
to play a significant role in eliminating tumor cells. Recent research has also
exhibited that tumor-infiltrating B cells strikingly influence the prognosis andefficacy of cancer immunotherapy. While B cells primarily produce humoral
immunity through antibody production, their role in tumor immunity remains
unclear. This study thus aimed to clarify the role of B cells infiltrating locally andantibodies in oral squamous cell carcinoma (OSCC).
(Materials and Methods)
Quantitative analysis of infiltrating antibody-secreting cells (ASCs) and the bindingof IgG antibodies to tumor cells were conducted using multiplex
immunofluorescence. Additionally, we performed gene expression analysis and B
cell receptor (BCR) repertoire analysis through single-cell RNA-sequencing (scRNAseq) on CD45-positive cells extracted from three OSCC resected tissues. Moreover,
we generated recombinant antibodies with binding sites identical to the BCRs thatshowed substantial expansion among B cells infiltrating OSCC. Subsequently, weinvestigated the binding of these antibodies to tongue SCC cell lines using multiplex
immunofluorescence.
(Results)
Multiplex immunofluorescence revealed significant infiltration of ASCs in the lesionof OSCC and more frequent IgG binding to tumor cells compared with that toadjacent oral mucosal epithelial cells. The scRNA-seq with BCR repertoire analysis
demonstrated the infiltration of B cells at various differentiation stages in OSCC,
particularly showing clonal expansion of the ASCs cluster with high IgG expression.
The binding of recombinant antibodies referring to BCR sequences of clonallyexpanded ASCs to the tongue SCC cell lines was also observed. The investigation
into the impact of IgG antibody binding to tumor cells on prognosis revealed nosignificant correlation with clinical findings.
(Conclusion)
This study suggests that B cells, especially ASCs, may play an essential role intumor immunity by producing tumor-specific IgG antibodies in the lesion of OSCC.
However, no significant correlation was found between IgG antibody binding totumor cells and clinical outcomes, including prognosis. Further investigation isrequired to elucidate the antibodies produced locally and their functions, withidentifying these antigens being particularly imperative.

Fabrication and histological evaluation of ant-nest type porous
carbonate apatite artificial bone

Janice Tan Lay Tin, Akira Tsuchiya, Masaya Shimabukuro,

Ryo Kishida and Kunio Ishikawa
Biomaterials

Background
Carbonate apatite (CO3Ap) artificial bone has been used as bone substitute due toits superior osteoconductivity. Besides composition, morphology of the artificial
bone is an crucial factor for bone regeneration. Generally, the porosity andmechanical strength of artificial bone are trade-offs. Previously, the polyurethane
foam have been used as replica template to fabricate interconnected porous CO3Ap
with a low compressive strength (25kPa) which is not suitable to be used in load
bearing area. In contrast, polyurethane foam used as porogen able to provide
CO3Ap an ant-nest type porous (ANP) structure without creating an excessive
amount of pores. This study aimed to evaluate the effectiveness of using
polyurethane foam as a porogen in fabricating CO3Ap artificial bone and its
potential in bone regeneration.
Materials and methods
The porogen employed in this investigation was polyurethane foam with strut
thickness of approximately 150 µm. Calcium carbonate (CaCO3) slurry wasincorporated into the polyurethane foam's pores, followed by the burned out of thefoam to produce an interconnected porous CaCO3 precursor block. CaCO3 block
were then transformed into CO3Ap block through a dissolution-precipitation
reaction. The resulting CO3Ap blocks were characterized in terms of composition
and morphology. Additionally, the ANP-structured CO3Ap artificial bone's
mechanical and biological performances were investigated.
Results & Discussion
The polyurethane foam was completely burned out after the heat treatment. As aresult, the samples were converted to interconnected porous CO3Ap blocks which
revealed an ANP structure with a compressive strength comparable to that ofhuman trabecular bone (around 15MPa). Moreover, in vivo result showed that the
migration of cells and tissues towards the interior of the artificial bone through the
ANP structure. The results indicated that the the usage of polyurethane foam as aporogen gave rise to an ANP-structured CO3Ap block which possessed adequate
mechanical strength and promoted bone regeneration.
Conclusion
The fabrication of an ANP-structured CO3Ap artificial bone with adequate
mechanical strength was successful. The interconnected porous architecture aided
cell and tissue migration into the CO3Ap artificial bone, thereby replacing CO3Ap
with new bone. This approach were found to be beneficial in creating artificial bone
for bone regeneration.

Cytoplasmic signaling molecule CySM2 in LTi cells plays
critical role on Payerʼs patch formation.
Naoko Kiya1,２, Miho Matsuda3）and Shinichiro Sawa1）
Division of Mucosal Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences, Faculty of
Dental Science, Kyushu University, Fukuoka, Japan
3 Laboratory of Molecular and Cellular Biochemistry, Division of Oral Biological Sciences, Kyushu University ,Fukuoka, Japan
1
2＜Background＞
The intestinal tract is the largest immune organ continuously exposed to antigens
from ingested food, symbiotic bacteria and pathogens. Intestinal mucosal immunity
develops gut-associated lymphoid tissue (GALT) which achieves both tolerance toharmless symbionts and defense against pathobionts. Failure of such fine-tuned
intestinal immune system is supposed to cause inflammatory bowel diseases. Peyer's
patches (PPs) and ILFs are major components of GALT formed via such common
cellular mechanism as mutual activation of Lymphoid Tissue inducer (LTi) cells andmesenchymal fibroblastic cell called Lymphoid Tissue organizer (LTo) cells.
Recently, we coincidentally found that ubiquitously expressed cytoplasmic signaling
molecule family, hereafter CySM, was involved in the development of PPs and ILFs
in mouse. From these results, we hypothesized that CySM molecules contribute to
PPs and ILFs via previously unappreciated mechanisms.
＜Methods＞
In mouse, CySM is composed of two subfamily molecules CySM 1 and CySM 2.
First, to determine which CySM molecule plays predominant roles on GALT
development, we generated Cysm 1/2-DKO, Cysm 1-KO, and Cysm 2-KO mice.
Second, to determine cell population which plays critical role on CySM-dependent
GALT formation, we generated lymphocyte-specific Cysm2-deficient Cd127Cre;
Cysm2 flox/flox mice and two mesenchymal-specific Cysm2-deficient mouse lines suchas Cxcl13Cre; Cysm2 flox/flox and Twist2Cre; Cysm2 flox/flox. In these Cysm deficientand the control mice, formations of PPs and ILFs were assessed with microscopy.
＜Results and Discussion＞
In Cysm 1/2-DKO mice, PP formation was totally impaired. As the size andnumber of PP in Cysm2 KO mice were significantly reduced compared to WT or
Cysm1 KO mice, we concluded that CySM 2 plays predominant role on PP
formation. While mesenchymal-specific Cysm2-deficient mice showed no significant
loss of PP in size and number, lymphocyte-specific CySM 2-deficient mice showed
severely impaired PP formation. From these results, we speculate that CySM 2 inLTi cells plays critical role on PP formation. However, precise role of CySM2 in LTi
cells during PP formation remains to be elucidated.

Fabrication of granular apatite cement using partially set αtricalcium phosphate granules

Abdulrahman Diabi
Department of Biomaterials, Faculty of Dental Science, Kyushu University, 3-1-1 Maidashi

Background
Apatite cements are synthetic bone grafts with the ability to set and harden
in bone defects. These are prepared by mixing a powder, typically α-tricalcium
phosphate (α-TCP), with a liquid to form a moldable paste. This paste sets bytransforming into apatite products such as hydroxyapatite (HAp). However,traditional apatite cements form dense blocks with limited bone regeneration inside.
To address this, introducing pores into the cement is crucial. Using larger particles,
or granules, instead of fine powder, helps create porous structures by leaving gaps
between the granules. The challenge, however, lies in maintaining a high surface
area with larger particles, as this is crucial for the setting reaction. Our study
introduces a novel method for producing α-TCP granules with a high specificsurface area, aiming to develop a granular apatite cement that is both porous andsets effectively.
Materials and methods
We created α-TCP granules by freeze-drying partially set apatite cement.
The process involved mixing α-TCP powder with a sodium phosphate solution,
incubating it at 37 C for partial setting, followed by freeze-drying and crushing into
granules of 50 ­ 100 μm diameter. For comparison, we also produced α-TCP
granules using a traditional sintering method, where β-tricalcium phosphate wascompacted and sintered at 1500 C.
Results and discussion
The partial setting of α-TCP created a unique microporous, raspberry-likestructure in the granules, maintaining gaps between particles and resulting in alarge specific surface area (5.9 m²/g), significantly higher than the sintered granules(0.2 m²/g). Importantly, these freeze-dried granules preserved the original α-TCP
composition. When mixed with a weakly acidic sodium phosphate solution, thegranules set within 25 minutes, which is clinically acceptable, unlike the slower
setting sintered granules (91 minutes). The freeze-dried granules rapidlytransformed into HAp, while the sintered ones predominantly remained as α-TCP
even after a week. The final set blocks featured macropores around 20­40 μm.
These findings indicate that our method is promising for creating apatite cementwith enhanced properties suitable for clinical applications.

Establishment of a new peripheral nerve regeneration therapy
using artificial nerves with extracellular vesicles derived fromdental pulp stem cells.
Aiko Yano1, Kenichi Ogata 1,2, Masafumi Moriyama1,3, Tatsuya
Kawado1, Mayu Matsumura1, and Shintaro Kawano 1
1Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic and Surgical Sciences,
Faculty of Dental Science, Kyushu University, Fukuoka, Japan2
Department of Dentistry and Oral Surgery, Karatsu Red Cross Hospital, Saga, Japan
3 OBT Research Center, Faculty of Dental Science, Kyushu University, Fukuoka, Japan.

Backgrounds;
Recently, extracellular vesicles (EVs) released by stem cells have also attracted
attention, as they are transported to target cells and provide various biological
functions such as regulating immune functions, suppressing inflammatory reactions,and even repairing tissues. Previous reports have shown that EVs from dental pulp
stem cells (DPSCs) have higher anti-inflammatory and tissue regeneration properties
than those from other stem cells.
In this study, we compared EVs from DPSCs (DPSC-EVs) with known artificial
nerves in vivo to confirm whether the addition of DPSCs-EVs to artificial nerves,which are already commercially available, improves nerve regeneration.
Methods;
EVs were collected from the conditioned media of human DPSCs using
ultracentrifugation; EVs were evaluated by Western blotting using CD9 and CD81,
and their presence was confirmed by cryo-EM. In vivo study, a 15-mm transectedsciatic nerve model of 8-week-old male SD rats was created, and five groups were
established. At 1 and 2 weeks after the nerve defect, the footprints were measured
and the sciatic functional index (SFI) was calculated. Two weeks later, the artificial
nerve was harvested, and electrophysiological evaluation was performed using anoscilloscope. Finally, the gastrocnemius muscle innervated by the sciatic nerve washarvested and its weight measurement and Massonʼs trichrome staining were used
to compare the degree of fibrosis of the muscle.
Results;
DPSC-EVs were CD9-positive and CD81-positive by Western blot, and were
spherical in size, around 200 nm, and surrounded by a bilayer membrane. In vivo
study, electrophysiological evaluation showed that the EVs group recovered tovalues closer to the amplitude of normal nerves than the PBS group. In functional
evaluation, SFI values in the EVs group were improved over those in the PBS group;
histological findings showed infantile nerve regeneration with angiogenesis in the
EVs group compared to the PBS group. In addition, gastrocnemius muscle wascollected and weighed, which was heavier in the EVs group than in the PBS group.
Conclusion;
In the EVs group, it was found that nerve regeneration was triggered earlier bystimulating angiogenesis from the surrounding area.