Kyudai Oral Bioscience&
OBT Research Center
Joint International Symposium 2021
PROGRAM & ABSTRACTS

February 6, 2021
Lecture Room A/B, Faculty of Dental Science,
Kyushu University

K OB

2021

■ Date:February 6, 2021
■ Zoom Meeting■ Organization
Kyudai Oral Bioscience
Oral Health・Brain Health・Total Health Research Center■ Contact
Eijiro Jimi
Oral Health・Brain Health・Total Health Research Center
Laboratory of Molecular and Cellular Biochemistry,
Faculty of Dental Science, Kyushu University,
Fukuoka 812-8582, Japan
Tel & Fax: +81 92-642-6332
E-mail: ejimi@dent.kyushu-u.ac.jp

KOB & OBT Joint International Symposium 2021

PROGRAM

February 6 (Saturday)

Time

Title

Presenter

9:00-9:10

Opening Remark

Prof. Seiji Nakamura

Session 1

Special Lecture (1)Chair:
Dr. Keisuke Sanematsu

9:10-10:10

An integrative structure of the human sweet taste receptor and protein engineering using deep mutagenesis

Dr. Erik Procko

10:10-10:25 BreakSession 2

Graduate Student's Session (1)Chair:
Kozue Yamashita

10:25-10:40

Secreted frizzled-related protein 1 promotes odontoblastic differentiation and reparative dentin formation by
regulating Notch signaling in dental pulp cells

Keita Ipposhi

10:40-10:55 Effect of isometric tongue lifting exercise on oral function, physical function, and body composition in the elderly

Asuka Tani

10:55-11:10 Identification of microRNA regulating NF-κB pathway in macrophage

Rongzhi Li

11:10-11:25 The role for non-coding RNAs on pathogenesis of IgG4-related diseases

Soi Kimura

11:25-11:40 BreakSession 3

Undergraduate Student's Session

Chair:
Dr. Tomoyo Yasukochi

11:40-11:55 Effect of prosthodontic treatment on standing movement function

Yuriko Takamoto

11:55-12:10 Comprehensive analysis of non-essential ribosomal subunits

Ronghao Tang

12:10-12:25

Functional analysis of Semaphorin3A expression, which is an axonal guidance factor, in salivary glanddevelopment and adenoid cystic carcinoma cell proliferation

Tatsufumi Fujimoto

12:25-13:15 Lunch Break
13:15-13:25 Award Presentation (IF and FWCI Award)
Session 4

Award Lectures (IF Award winners)

Chair:
Prof. Hidefumi Maeda

13:25-13:45 Impacts of Pore Architecture on Bone Regeneration in Honeycomb Scaffold

Dr. Koichiro Hayashi

13:45-14:05 Epithelial and Connective Tissue Sealing around Titanium Implants with Various Typical Surface Finishes

Dr. Ikue Narimatsu

14:05-14:25

Disrupted tongue microbiota and detection of nonindigenous bacteria on the day of allogeneic hematopoietic stem
Dr. Saori Oku
cell transplantation

14:25-14:45

Activated M2 Macrophages Contribute to the Pathogenesis of IgG4-Related Disease via Toll-like Receptor
7/Interleukin-33 Signaling

Dr. Noriko Ishiguro-Kakizoe

14:45-15:00 BreakSession 5

Special Lecture (2)15:00-15:40 Glial activation and neurodegeneration in neuroinflammation

Chair:
Dr. Zhou Wu
Dr. Sadayuki Hashioka

15:40-15:55 BreakSession 6

Graduate Student's Session (2)Assessment of periodontitis by next-generation sequencing method focusing on subgingival plaque-specificbacteria in saliva
P. gingivalis LPS Induces Inflammatory Bone destruction and Alzheimer’s Disease-Like Brain Pathologies in
16:10-16:25
middle aged mice
15:55-16:10

16:25-16:40 Porphyromonas gingivalis infection induces leptomeningeal cells-mediated synaptic failure in neurons
Cathepsin B inhibition blocks neurite outgrowth in cultured neurons by regulating lysosomal trafficking andremodeling
16:55-17:10 Break
16:40-16:55

Session 7

Special Lecture (3)Chair:
Muzhou Jiang
Jiale Ma
Yebo Gu
Wanyi Huang
Muzhou Jiang
Chair:
Prof. Fusanori Nishimura

17:10-18:10 FORENSIC ODONTOLOGY IN INDONESIA from A to Z

Prof. Mieke Sylvia

18:10

Prof. Eijiro Jimi

Closing Remarks

KOB & OBT Joint International Symposium 2021

ABSTRACTS

KOB & OBT Joint International Symposium 2020

OBT Special Lecture 1
By Dr. Erik Procko
Chaired by Dr. Keisuke SanematsuAn integrative structure of the human sweet taste receptor and protein
engineering using deep mutagenesis
Erik Procko1
1

Department of Biochemistry, University of Illinois, Urbana, USA.

In a deep mutational scan, next generation sequencing is used to follow the in vitro
selection of a diverse collection of protein variants. By observing which variants
increase or decrease in frequency, it becomes possible to determine the relative effects ofthousands of protein mutations in a single experiment, provided a suitable selection
strategy is available.
In the human heterodimeric sweet taste receptor, surface trafficking of the T1R3
subunit is dependent on co-expression with the T1R2 subunit. A selection and deep
mutational scan based on co-trafficking of T1R2-T1R3 to the plasma membrane therefore
informs how mutations influence the subunits’ physical association. Analysis of nearly
20,000 mutations in T1R2 revealed key contacts to T1R3 within the ligand-binding
domain, cysteine-rich domain, and on specific helices of the transmembrane domain.
Guided by the mutagenesis data, an integrative structural model of the complete sweet
taste receptor in an active conformation was solved. There is a twisted arrangement of
domains around a central axis and a continuous folded structure between transmembrane
domain loops and extracellular domains, providing an elegant mechanism for how sugar
binding to an extracellular site causes conformational changes to propagate to thereceptor’s cytoplasmic surface for intracellular signaling.
Recently, the same technology developed for deep mutagenesis of the human sweet taste
receptor was rapidly repurposed for studying the interaction between the spike of SARS
coronavirus 2 and its host entry receptor, ACE2. SARS-CoV-2 is responsible for thecomplex vasculature disease called COVID-19, which includes unusual neurologicalsymptoms such as loss of taste and smell. A deep mutational scan of ACE2 identified
mutations that increase affinity for the viral spike, informing the design of soluble decoy
receptors that rival monoclonal antibodies for tight binding and potent neutralization of
SARS-CoV-2.

KOB & OBT Joint International Symposium 2021

Graduate Student’s Session (1)Chaired by Kozue Yamashita

Secreted frizzled-related protein 1 promotes odontoblastic
differentiation and reparative dentin formation by regulating Notch
signaling in dental pulp cells
Keita Ipposhi¹, Atsushi Tomokiyo², Taiga Ono¹, Kozue Yamashita¹, M.
Anas Alhasan¹, Daigaku Hasegawa², Sayuri Hamano1,3, Shinichiro
Yoshida², Hideki Sugii², Tomohiro Itoyama¹, Marina Ogawa¹, Hidefumi
Maeda1,2
1

Department of Endodontology and Operative Dentistry, Division of Oral
Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka, Japan;
2
Department of Endodontology, Kyushu University Hospital, Fukuoka, Japan;
3
OBT center, Faculty of Dental Science, Kyushu University, Fukuoka, Japan.

Direct pulp capping is an effective treatment for preserving dental pulp against carious ortraumatic pulp exposure via the formation of protective reparative dentin by odontoblastlike cells. Reparative dentin formation can be stimulated by several signaling molecules;
therefore, we investigated the effects of a secreted frizzled-related protein (sFRP) 1 thatwas reported to be strongly expressed in odontoblasts of newborn molar tooth germs onodontoblastic differentiation and reparative dentin formation. In developing rat incisors,
cells in the dental pulp, cervical loop and inner enamel epithelium, as well as ameloblasts
and preodontoblasts weakly expressed sFRP1; however, sFRP1 was strongly expressedin mature odontoblasts. Human dental pulp cells (HDPCs) showed stronger expression ofsFRP1 compared with periodontal ligament cells and gingival cells. sFRP1 knockdown
in HDPCs abolished calcium chloride-induced mineralized nodule formation andodontoblast-related gene expression and decreased Notch-related gene expression.
Conversely, sFRP1 stimulation enhanced nodule formation and expression of Notchrelated genes. Direct pulp capping treatment with sFRP1 induced the formation of aconsiderable amount of reparative dentin in a dose-dependent manner. Our results
indicate that sFRP1 is crucial for dentinogenesis and is important in promoting reparative
dentin formation in response to injury by regulating Notch signaling in dental pulp cells.

Effect of isometric tongue lifting exercise on oral function, physicalfunction, and body composition in the older people
Asuka Tani1, Shinsuke Mizutani1,2, Kiyomi Iyota1, Harukaze Yatsugi3, Saori Oku1,
Tianshu Chu3, Xin Liu3, Hiro Kishimoto3,4, Haruhiko Kashiwazaki1
1

Section of Geriatric Dentistry and Perioperative Medicine in Dentistry, Division of
Maxillofacial Diagnostic and Surgical Science, Faculty of Dental Science Kyushu
University, Japan
2
OBT Research Center, Faculty of Dental Science Kyushu University, Japan3
Department of Behavior and Health Sciences, Graduate School of HumanEnvironment Studies, Kyushu University, Japan
4
Faculty of Arts and Science, Kyushu University, Japan

Background: On average, the older people in Japan need long-term care using nursing
care insurance for 10 years. Frailty prevention is essential to avoid this substantial needfor long-term care. Oral frailty is considered a symptom of pre-frailty; however, a few
studies have examined whether an intervention for oral frailty may prevent frailty.
Methods: This interventional study included 49 participants (25 males; aged 65–79 years;
mean ± SD, 73.1 ± 3.1 years) from a larger group of participants of the “Itoshima Frail
Study,” in Itoshima City, Japan. Strength training of the tongue muscle with an isometrictongue lifting tool (Peko-pandaⓇ) was conducted for 3 months. Tongue pressure wasmeasured on a monthly basis with a tongue-pressure measuring device. We also measured
the number of remaining teeth; tongue and lip motor functions (oral diadochokinesis;
ODK); body composition; and physical function, including open-eyed one-leg standing,
sit-to-stand motion, and 3m timed up & go (TUG), at baseline and follow up. Data after
3 months of training were compared with data at baseline using the Wilcoxon signed-rank
test. The correlation coefficients between the amount of change for each item and change
in tongue pressure were assessed using bivariate analysis calculated with Spearman’srank correlation coefficient.
Results: Tongue pressure and ODK significantly improved after the intervention (p <0.001). In terms of physical function, the time for open-eyed one-leg standing, sit-to-standmotion, and 3m TUG significantly improved (p = 0.004, p < 0.001, p = 0.019,
respectively). However, the amount of change in tongue pressure was not related to theamount of change in any of the other measured items. In addition, a decrease insubcutaneous fat percentage and an increase in skeletal muscle ratio were observed
significantly in the non-frailty group (p<0.001, p=0.036, respectively), but no significant
change was observed in the frailty / pre-frailty group.
Conclusion: Our results showed that isometric tongue lifting exercise is effective in
improving oral functions in the older people. This training may also have positive
indirect effects on physical function and body composition.

Identification of microRNA regulating NF-κB pathway in macrophage
Rongzhi Li1, Tomomi Sano2, Takanori Shinjo1,
Misaki Iwashita1, Akiko Yamashita1, Takao Fukuda1, Fusanori Nishimura1
1

Section of Periodontology, Kyushu University Faculty of Dental Science, Kyushu
University, Japan2
Departmant of Cell Biology and Pharmacology, Faculty of Dental Science, Kyushu
University, Japan.

We previously detected abnormally dysregulated micro (mi) RNAs in the adipose tissueof mice fed a high-fat-diet (HFD) by microarray analysis. Here, we aimed to elucidate therole of dysregulated miRNAs that were not completely characterized.
First, qPCR was performed to verify the results of the microarray analysis. LPSstimulated RAW264.7 cells were used to confirm the changes of these miRNAs under
inflammatory conditions. Then, miRNA mimic was transfected into RAW264.7 cells to
identify the potential targets, and mRNA and protein levels of these target molecules were
measured. In addition, expression of inflammatory markers was evaluated in RAW264.7
cells treated with or without miRNA mimic followed by LPS stimulation.
Selected miRNA was significantly down-regulated in adipose tissue of HFD-fed micecompared with normal diet fed mice, as well as in gingival tissue of ligature-induced
periodontitis mice compared with their control littermates. Moreover, we noticed asignificant reduction of the selected miRNA in RAW264.7 cells stimulated with LPS
compared with those without LPS stimulation. mRNA levels of several putative targets
were lowered after the transfection of miRNA mimic, but only one putative target
molecule exhibited reduction at protein level. Moreover, this selected miRNA appeared
to regulate inflammation.
The predicted gene was found to play an important role on NF-κB signaling pathway bysubsequent analysis. miRNA mimic transfection into macrophages resulted in reduced
expression of target genes as well as altered TNF-α expression. Thus, it is suggested that
this miRNA might play a key role in regulating inflammation in adipose and, also, in
periodontal tissues.

The role for non-coding RNAs on pathogenesis of
IgG4-related diseases
Soi Kimura1,2, Tomoyo Kawakubo-Yasukochi1, Masafumi Moriyama2, Seiji
Nakamura2, Eijiro Jimi1
1

OBT Research Center, Faculty of Dental Science, 2Section of Oral and Maxillofacial
Oncology, Kyushu University, Fukuoka, Japan

IgG4-related disease is a disease in which infiltration of IgG4-positive plasma cells into
various organs and hyper-IgG4eima are observed. However, its pathogenic mechanism isstill unknown. Almost all previous studies on this disease have been performed byimmunological approach, and no analysis has yet been performed to the pathogenesis
process from normal tissue.
In this study, we focused on microRNA (miRNA) in patient serum and analyzed thepathogenesis mechanism using normal salivary gland cells, in order to identify miRNA(s)involved in the pathogenesis of IgG4-related diseases.
miRNA is a short non-coding RNA (ncRNA) of about 18 to 25 bases, and they are
involved in various biological phenomena and pathogenesis by controlling the expressionof genes that have a sequence highly complementary to their own base sequence. In
particular, circulatory miRNAs exist in body fluids are transported being wrapped in
vesicles such as exosomes, and are stable in body fluids such as blood and saliva.
Therefore, miRNA is expected as a non-invasive biomarker to grasp the pathogenesis andprogression of various diseases.
We perfomed a comprehensive miRNA-mRNA pairing analysis, focusing on patientserum miRNA and intracellular mRNA in rat normal salivary epithelial cells. As a result,the miRNA, highly expressed in patients with IgG4-related diseases, and its target mRNA,
localized in salivary gland epithelial cells, were identified. Furthermore, histologicalanalysis revealed that expression pattern of the target factor related with differentiationin pathological region was significantly different from that in human normal salivary
gland tissue.
From these results, it was suggested that the ncRNA in serum has an important role in
the pathogenesis of IgG4-related diseases.

KOB & OBT Joint International Symposium 2021

Undergraduate Student’s Session

Chaired by Dr. Tomoyo Yasukochi

Effect of prosthodontic treatment on standing movement function.
Yuriko Takamoto1, Kyosuke Oki2, Mikio Imai3, Yoko Takemura3,
Yoichiro Ogino2, Kiyoshi Koyano2,3
1

Undergraduate Student, Faculty of Dental Science, Kyushu University ,Fukuoka,
Section of Fixed Proshodontics, Division of Oral Rehabilitation, Faculty of Dental
Science, Kyushu University, Fukuoka, 3 Section of Implant and Rehabilitative Dentistry,
Division of Oral Rehabilitation, Faculty of Dental Science, Kyushu University, Fukuoka
2

Problem/hypothesis; Recently, Japan is experiencing “super-aging” society that isunprecedented in the world. It was reported elderly people with frailty increased risk offalls. However, the appropriate intervention can improve “frail”. The objective of thisstudy was to reveal the effect of prosthodontic treatment (restoration of occlusal support)
on standing movement. Moreover, it also leads to show the role and the meaning ofdentistry in super-aging society.
Methods; The subjects were 48 people and more than 65 years-old who received denture
treatment. in University Hospital (Section of Fixed Prosthodontics and Removable
Prosthodontics). The subjects were grouped two. One is included 24 subjects who haveocclusal support in molar region (Eichner B1〜B3), and the other is included 24 subjects
who don’t have it (Eichner B4〜C3). The oral function (the occlusal number of teeth,
occlusal area of contact, occlusal force and masticatory performance) and the ability ofstanding movement(①power, ②speed, ③muscle strength, ④balance) before and after
a prosthodontic treatment were evaluated. 1) The occlusal force and the masticatory
performance were compared with and without dentures. 2) All subjects were categorizedby 2 groups which are with and without occlusal support in molar region and evaluated.
Statistical analyses were performed using a Wilcoxon signed rank test.
Results; 1) The occlusal force and the masticatory performance were compared with andwithout dentures, and the results with dentures were significantly improved comparedwithout dentures (p<0.05). Among the evaluation items of standing movement, “balance”,“speed” and “muscle strength” were significantly improved with dentures, compared withthe condition without dentures (p<0.05). 2)The occlusal force and the masticatory
performance with dentures were significantly improved compared without dentures inboth groups. In the groups of without occlusal support (Eichner B4~C group), a part ofmasticatory performance was improved with dentures compared without dentures.
Conclusion; It was suggested that the prosthodontic treatments affect not only systemic
functions but also patients without occlusal support in the molars.

Comprehensive analysis of non-essential ribosomal subunits
Ronghao Tang1,2, Akinobu Matsumoto1, Keiichi I. Nakayama1,
1

Division of Cell Biology, Medical Institute of Bioregulation, Kyushu University, Japan;
2
School of Dentistry, Kyushu University, Japan.

The eukaryotic ribosome, which mainly consists of 79 ribosomal proteins (RPs) as
well as 4 ribosomal RNAs (rRNAs), is the cellular translational machinery responsible
for protein synthesis from messenger RNAs (mRNA). However, emerging evidences
reveals that not all ribosomal subunits are essential for ribosome function, and that theribosome is composed of essential subunits and non-essential subunits that modulatetranslation efficiency (TE). For instance, the mice lacking RPS19 are embryonic lethal
prior to implantation, whereas RPL22-null mice are viable and only show impaired αβ Tcell development.
We thus applied the CRISPR-Cas9 knockout screening to comprehensively identify
the non-essential subunits based on their viability. As a result, we identified eight nonessential subunits and established their knockout clones, and double-checked theirdeletion by Western blotting and mass spectrometry. Since the loss of these subunits may
affect the TE of specific mRNAs, we are performing ribosome profiling (Ribo-seq) in theknockout cell lines to measure the TE of each mRNAs.
We also focused on tissue-specific non-essential subunits and found that RPL3L, agene with 74% sequence identity to the RPL3, is specifically expressed in striated muscle
tissues such as heart and skeletal muscle. Notably, mutations in RPL3L have been
identified in patients with dilated cardiomyopathy (DCM) [Ganapathi M et al, Hum.
Genet. (2020)]. Therefore, we generated RPL3L-deficient mice and are currently
performing phenotypic analysis by echocardiography and electrocardiography, andanalysis of TE changes by Ribo-seq.
In summary, we identified non-essential RPs of constitutive and tissue-specificribosomes and generated cells and mice lacking these RPs. We are now analyzing thesecells and mice to elucidate the relationship of non-essential subunits to biologicalprocesses and diseases.

Functional analysis of Semaphorin3A expression, which is an axonal
guidance factor, in salivary gland development and adenoid cystic
carcinoma cell proliferation.
Tatsufumi Fujimoto1,2, Shinsuke Fujii2, Tamotsu Kiyoshima2
1

School of Dentistry, 2Laboratory of Oral Pathology, Division of Maxillofacial
Diagnostic and Surgical Sciences, Faculty of Dental Science,
Kyushu University, Japan

Recently, we demonstrated that Wnt/β-catenin signaling negatively regulated cellular
growth through reduced Semaphorin3A (Sema3A) expression in odontogenic epithelial
cells and its involvement in tooth germ development (Sci Rep, 2019). Although thedevelopmental process in salivary gland may share the same mechanisms with tooth germ,the effect of Sema3A signaling on salivary gland development remains unclear. Here, we
conducted to investigate the function of Sema3A signaling in salivary gland development
and adenoid cystic carcinoma (ACC) cell proliferation.
We developed the salivary gland organ culture system with murine submandibular
salivary gland (SMG) rudiments at embryonic day 13. In the organ culture system, normal
temporospatial development of the SMG rudiment or salivary gland organ, which issimilar to in vivo situations, was confirmed. The treatment with CHIR99021, a GSK3βinhibitor, activated Wnt/β-catenin signaling but decreased Sema3A expression in the
SMG rudiment culture and ACC cell lines. Immunohistochemical analysis demonstrated
that Sema3A was highly expressed in the salivary gland epithelial cells. Loss-of-function
experiments with a Sema3A inhibitor showed the decrease of total epithelial area, thenumbers of buds and proliferating cells, and AKT activation. Consistently, loss-offunction experiment reduced cell proliferation and AKT activation in the ACC cell lines.
These results suggest that Wnt/β-catenin signaling reduces Sema3A expression and theSema3A-AKT axis promotes epithelial cell proliferation to regulate salivary glanddevelopment and ACC cell proliferation.

Mr. Tatsufumi Fujimoto is the best presenter of the Joseph Lister Award 2020 ofJapanese Association for Dental Research (JADR).

KOB & OBT Joint International Symposium 2021

Award Lectures

Chaired by Prof. Hidefumi Maeda

Impacts of Pore Architecture on Bone Regeneration in Honeycomb
Scaffolds
Koichiro Hayashi1
1Faculty of Dental Science, Department of Biomaterials, Kyushu University, Japan

Autografts, allografts, and xenografts are used for treatment for critical-size bone
defects. In terms of invasiveness, infection, supply, and cost, however, synthetic
materials are preferred as scaffolds for bone regeneration. Synthetic scaffolds shouldhave biocompatibility, high osteoconductivity, bioresorbability at a rate commensuratewith bone remodeling, and high mechanical strength. To satisfy these requirements, thedesign of pore architecture, as well as chemical composition, is critical. Honeycomb
construction is considered an ideal pore architecture. In this study, we fabricatedhoneycomb scaffolds consisting of carbonate apatite, which is analogous to thecomposition of natural bone, and evaluated the effects of macropores or channels (> 100μm), micropores (submicron-10 μm), and nanopores (<submicron) on boneregeneration. Carbonate apatite honeycomb scaffolds were fabricated by the following
procedures: 1) mixing calcium compounds (CaSO4, Ca(OH)2, or CaCO3) and a waxbased binder; 2) the fabrication of honeycomb green bodies by extruding the mixture; 3)removal of organics from the honeycomb green bodies by heating; 4) the composition
conversion of the honeycombs subjected to heat treatment into carbonate apatite via thedissolution-precipitation reactions. For in vivo evaluation, a cylindrical hole (diameter =6 mm, depth = 5 mm), which represented a critical-sized bone defect, was drilled into
the femur condyle of the rabbit. The carbonate apatite honeycomb scaffolds were
implanted into the holes. At 4 and/or 12 weeks post-implantation, the condyle of eachfemur was harvested to perform μ-CT imaging and histological analyses. For evaluating
the effects of channel size, honeycomb scaffolds with ~100-, 200-, and 300-μm
channels were used. The ~300-μm channels were extensively occupied by new bone,compared to the ~100- and 200-μm channels. The effects of micropores were evaluatedusing honeycomb scaffolds with ~210-, 200-, and 190-mm3/g micropore volumes. The
replacement of scaffolds by bone was accelerated with increasing micropore volume.
Notably, bone marrow-like tissues were formed in the honeycomb scaffolds with ~210mm3/g micropore volume. The effects of nanopores were evaluated using honeycomb
scaffolds with ~180-, 150-, and 70-mm3/g nanopore volumes. When the nanopore
volume was 150 mm3/g, osteoclastogenesis and osteogenesis progressed harmonically,
resulting in scaffold replacement with new bone.

Epithelial and Connective Tissue Sealing around Titanium Implants
with Various Typical Surface Finishes
Ikue Narimatsu1, Ikiru Atsuta2, Yasunori Ayukawa1, Wakana Oshiro1,
Noriyuki Yasunami1, Akihiro Furuhashi1, and Kiyoshi Koyano1
1Section of Implant and Rehabilitative Dentistry, Division of Oral Rehabilitation,
Faculty of Dental Science, Kyushu University, Fukuoka, Japan
2
Division of Advanced Dental Devices and Therapeutics, Faculty of Dental Science,
Kyushu University, Fukuoka, Japan

Background: The success of dental implant treatment depends on not only
osseointegration around the implant body, but also a soft tissue barrier that protects theunderlying hard tissue structures. Numerous surface modification techniques have been
introduced to enhance bone contact on the implant surface, but there has been little
research on the peri-implant soft tissue seal.
Purpose: This study aimed to investigate the role of epithelial and connective tissues seals
around implants with various surface finishes.
Materials and methods: Testing surfaces had been machined (Ms), roughened bysandblasting and acid etching (Rs), treated hydrothermally with CaCl2 (Cs), or anodized(As). (1) In vitro study: rat oral epithelial cells (OECs) and fibroblasts were cultured on
Ms, Rs, Cs, and As titanium plates. (2) In vivo study: implants with Ms, Rs, Cs, and As
surfaces were placed in the rat’s oral cavity.
Results: (1) In vitro study: there was less cell adherence of OECs and more collagen
expression when cultured on Rs and As plates than when cultured on Ms and Cs plates.
(2) In vivo study: Cs group had superior sealing for the implant surface, Ms and Rs
implants showed weaker sealing capacity than Cs. After 16 weeks, Rs implants exhibited
peri-implant epithelial apical down-growth and had lost bone support, presumably
because their resistance to penetration by epithelial attachment was low with this surface
type.
Conclusion: Although a smooth surface (Ms, Cs) showed better epithelial attachment,rough surfaces (Rs, As) are more suitable for binding to connective tissue. Strong
epithelial attachment to the implant surface seems to be a fundamental first-line of defense
against foreign body penetration. Thus selecting suitable surfaces to ensure strong sealing
is important for implant success.

Disrupted tongue microbiota and detection of nonindigenous bacteriaon the day of allogeneic hematopoietic stem cell transplantation
Saori Oku1,2, Toru Takeshita1,3, Toshiko Futatsuki2, Shinya Kageyama1,
Mikari Asakawa1, Yasuo Mori4, Toshihiro Miyamoto4, Jun Hata5,6,
Toshiharu Ninomiya5,6, Haruhiko Kashiwazaki2, Yoshihisa Yamashita11Section of Preventive and Public Health Dentistry, Division of Oral Health, Growthand Development, Faculty of Dental Science, Fukuoka, Japan. 2Section of Geriatric
Dentistry and Perioperative Medicine in Dentistry, Faculty of Dental Science, Fukuoka,
Japan. 3OBT Research Center, Faculty of Dental Science, Fukuoka, Japan.
4
Department of Medicine and Biosystemic Science, Kyushu University Graduate
School of Medical Sciences, Fukuoka, Japan. 5Department of Epidemiology and Public
Health, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
6
Center for Cohort Studies, Graduate School of Medical Sciences, Kyushu University,
Fukuoka, Japan.

Disruption of the intestinal microbiota caused by intensive chemotherapy, irradiation
and antibiotics can result in development of severe gut graft-versus-host disease andinfectious complications, leading to poorer outcomes among allogeneic hematopoietic
stem cell transplantation (allo-HSCT) recipients. Although the oral cavity is also
densely colonized by indigenous microorganisms, the bacterial composition in alloHSCT recipients remains unclear. We determined the tongue microbiota composition of
45 patients with hematological disorders on the day of transplantation and comparedthem to 164 community-dwelling adults. The V1–V2 regions of the 16S rRNA genesequences demonstrated that the allo-HSCT recipients had less diverse and distinct
microbiota from that of community-dwelling adults. The full-length 16S rRNA genesequences identified 146 bacterial taxa in the microbiota of allo-HSCT recipients, ofwhich 34 bacterial taxa did not correspond to bacteria primarily inhabiting the oralcavity deposited in the expanded Human Oral Microbiome Database. Notably, thedetection of Staphylococcus haemolyticus and/or Ralstonia pickettii was significantly
associated with a higher risk of mortality during the follow-up period. These results
demonstrate that the oral cavity of allo-HSCT recipients is colonized by a disrupted
microbiota on the day of transplantation and suggest that detection of specificnonindigenous taxa could be a predictor of transplant outcome.

Activated M2 Macrophages Contribute to the Pathogenesis of IgG4Related Disease via Toll-like Receptor 7/Interleukin-33 Signaling
Noriko Ishiguro-Kakizoe1, Masafumi Moriyama1,2, Katsuhiro Furusho1,3,
Takuma Shibata3, Yusuke Murakami3, Akira Chinju1, Yuka Gion4, Miho
Ohta1, Takashi Maehara1, Akihiko Tanaka1, Masaki Yamauchi1, Mizuki
Sakamoto1, Keita Mochizuki1, Yasuharu Sato4, Tamotsu Kiyoshima5,
Hidetaka Yamamoto6, Kensuke Miyake3, and Seiji Nakamura1
1

Section of Oral and Maxillofacial Oncology, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University, Fukuoka, Japan
2
OBT Research Center, Faculty of Dental Science, Kyushu University, Fukuoka, Japan
3
Division of Innate Immunity, Department of Microbiology and Immunology, Instituteof Medical Science, University of Tokyo, Tokyo, Japan
4
Department of Pathology, Okayama University Graduate School of Medicine,
Dentistry and Pharmaceutical Sciences, Okayama, Japan
5
Laboratory of Oral Pathology, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University, Fukuoka, Japan
6
Division of Pathology, Kyushu University Hospital, Fukuoka, Japan

Objectives: IgG4-related disease (IgG4-RD) is a unique inflammatory disorder inwhich Th2 cytokines promote IgG4 production. In addition, recent studies have
implicated the Toll-like receptor (TLR) pathway. This study was undertaken to examine
the expression of TLRs in salivary glands (SGs) from patients with IgG4-RD.
Methods: SGs from 15 patients with IgG4-RD, 15 patients with Sjögren’s syndrome,
10 patients with chronic sialadenitis, and 10 healthy controls were examinedhistologically. TLR family gene expression (TLR1 through TLR10) was analyzed by
DNA microarray in the submandibular glands (SMGs). Up-regulation of TLRs was
confirmed in SGs from patients with IgG4-RD. Finally, the phenotype of human TLR7
(huTLR7)-transgenic C57BL/6 mice was assessed before and after stimulation with
TLR agonist.
Results: In patients with IgG4-RD, TLR4, TLR7, TLR8, and TLR9 were
overexpressed. Polymerase chain reaction validated the up-regulation of TLR7 in IgG4RD compared with the other groups. Immunohistochemical analysis confirmed strong
infiltration of TLR7-positive cells around ectopic germinal centers (eGCs) and fibrotic
areas in the SGs of patients with IgG4-RD. Double immunohistochemical staining
showed that TLR7 expression colocalized with CD163+ M2 macrophages. After in vitro
stimulation with a TLR7 agonist, CD163+ M2 macrophages produced higher levels of
IL-33, which is a Th2-activating cytokine. In huTLR7-transgenic mice, the focus andfibrosis scores in SMGs, pancreas, and lungs were significantly higher than those inwild-type mice (P < 0.05). Moreover, the concentration of serum IgG, IgG1, and IL-33
in huTLR7-transgenic mice was distinctly increased upon stimulation with a TLR7
agonist (P < 0.05). Immunohistochemical analysis demonstrated focal accumulation ofCD206+ cells (marker for M2 macrophage in mice), IgG1+ plasma cells, and IL-33+
cells around eGCs in the SMGs from huTLR7-transgenic mice.
Conclusion. TLR7-expressing M2 macrophages may promote the activation of Th2
immune responses via IL-33 secretion in IgG4-RD. A more thorough understanding of
the role of TLR7+CD163+ M2 macrophages in IgG4-RD could lead to the establishmentof the world's first mouse model of IgG4-RD.

KOB & OBT Joint International Symposium 2021

OBT Special Lecture (2)Chaired by Dr. Zhou Wu

Glial activation and neurodegeneration in neuroinflammation
Sadayuki Hashioka.
Department of Psychiatry, Faculty of Medicine, Shimane University, JapanIn 1980’s, McGeer et al. identified both activated astrocytes and activated microglia in
affected regions of a broad spectrum of neurodegenerative disorders, such as Alzheimer’s
disease (AD), Parkinson’s disease, amyotrophic lateral sclerosis and multiple sclerosis.
They built the neuroinflammation hypothesis on these immunohistochemical findings andthe epidemiological finding that the prevalence of AD in rheumatoid arthritis patients,
who receive anti-inflammatory therapy, was sixfold spared compared to age-matchedgeneral populations. Activated astrocytes could be detrimental to neighboring neurons inneuroinflammatory process. Astrocytes exposed to certain inflammatory stimulants invitro have been shown to release potentially neurotoxic molecules, including
inflammatory cytokines, glutamate, nitric oxide and reactive oxygen species. We havepreviously demonstrated that adult human astrocytes stimulated with the inflammatory
cytokine interferon (IFN)-γ, that is predominantly produced by T cells and natural killer
cells and is detected only in the pathological brains, exert potent neurotoxicity. This IFNγ-induced neurotoxicity of human astrocytes was mediated by astrocytic activation of thesignal transducer and activator of transcription (STAT) 3 pathway with phosphorylationof STAT3 at tyrosine-705 residue. In addition, the proton pump inhibitors lansoprazole
and omeprazole, the histone deacetylase inhibitor suberoylanilide hydroxamic acid, andthe L-type calcium channel blockers nimodipine and verapamil significantly reduced theastrocytic neurotoxicity. All the reagents attenuated the IFN-γ-induced phosphorylationof STAT 3 at Tyr705, but not STAT1. Therefore, control of STAT3 activation in humanastrocytes may be a promising new therapeutic strategy for various neurodegenerativeand neuroinflammatory disorders in which activated astrocytes could contribute to thepathology.

KOB & OBT Joint International Symposium 2021

Graduate Student’s Session (2)Chaired by Muzhou Jiang

Assessment of periodontitis by next-generation sequencing method
focusing on subgingival plaque-specific bacteria in saliva
Jiale Ma1, Shinya Kageyama1, Toru Takeshita1,2, Yukie Shibata1, Michiko
Furuta1, Mikari Asakawa1, Yoshihisa Yamashita1*1Section of Preventive and Public Health Dentistry, Division of Oral Health, Growthand Development, Faculty of Dental Science, Kyushu University, Fukuoka, Japan.
2
OBT Research Center, Faculty of Dental Science, Kyushu University, Fukuoka, Japan.

Saliva contains diverse bacteria shed from various oral sites, including subgingival plaque.
Focusing on the total bacterial occupancy in saliva of subgingival plaque-specific bacteria(SUBP bacteria) that live in subgingival environments regardless of virulence isreasonable for detecting periodontitis in salivary testing. This study aimed to validate theclinical utility of the SUBP bacteria in salivary microbiota for assessment of periodontitis
by using next-generation sequencing method. We examined stimulated saliva samples
collected from 125 subjects who visited three dental clinics. The relative abundances ofpreviously identified 11 SUBP bacteria were determined using 16S ribosomal RNA genesequencing and the reference-based approach. The prediction performance was evaluatedusing the receiver operating characteristic (ROC) curve. The SUBP bacteria accounted
for a total of 0-15.4% in their salivary microbiota, and the percentage distinguished severe
periodontitis patients with ≥15 deep probing sites (≥4mm depth) with 0.90 (95%
confidence interval [CI], 0.81-0.98) of sensitivity and 0.70 (95% CI, 0.60-0.80) of
specificity (area under the ROC curve [AUC], 0.87). Among 2,047 combinations of 11
SUBP bacteria, in particular, combinations including Streptococcus constellatus,
Porphyromonas gingivalis, and Fusobacterium nucleatum subsp. vincentii demonstrated
significantly higher AUC values in the detection of severe periodontitis patients. These
results suggest that examining the SUBP bacteria in saliva may be useful for screening
severe periodontitis patients without periodontal probing.

P. gingivalis LPS Induces Inflammatory Bone destruction
and Alzheimer’s Disease-Like Brain Pathologies
in middle aged mice
Yebo Gu1, Zhou Wu2,3, Ichiro Takahashi1
1

Section of Orthodontics and Dentofacial Orthopedics, Division of Oral Health, Growthand Development, Faculty of Dental Science, Kyushu University, Fukuoka, Japan2
Department of Department of Cell Biology and Pharmacology, 3OBT Research Center,
Faculty of Dental Sciences, Kyushu University, Fukuoka, Japan

Background: As common issues affecting the elderly, Alzheimer’s disease (AD) andbone loss can be clinically exacerbated. However, the mechanism underlying thisexacerbation remains poorly understood. In the present study, we tested our hypothesisthat periodontitis is involved in the exacerbation, contributing to AD pathologies.
Objective: We tested our hypothesis that periodontitis is involved in the exacerbation,
contributing to AD pathologies.
Methods: The bone, memory, and inflammation in bone and brain were examined in 12month-old mice after systemic exposure to lipopolysaccharide from Porphyromonas
gingivalis (PgLPS) for 3 consecutive weeks.
Results: Compared with control mice, bone loss in tibia (26% decrease) and memory
decline (47% decrease) were induced in mice with a positive correlation after exposure
to PgLPS (r = 0.7378, p = 0.0011). The IL-6 and IL-17 expression in tibia was negatively
correlated with the bone volume/total tissue volume (r = –0.6619, p = 0.0052; r = –0.7129,
p = 0.0019), while that in the cortex was negatively correlated with the memory test
latency (r = –0.7198, p = 0.0017; p = 0.0351, r = –0.5291). Furthermore, the IL-17
expression in microglia was positively correlated with A42 accumulation in neurons (r =0.8635, p < 0.0001). In cultured MG6 microglia, the PgLPS-increased IL-6 expression
was inhibited by a PI3K-specific inhibitor (68% decrease), and that of IL-17 was inhibitedby IL-6 antibody (41% decrease). In cultured N2a neurons, conditioned medium fromPgLPS-stimulated microglia (MCM) but not PgLPS increased the productions of AβPP,
CatB, and Aβ42, which were significantly inhibited by pre-treatment with IL-17 antibody(67%, 51%, and 41% decrease).
Conclusion: These findings demonstrated that chronic systemic exposure to PgLPS
simultaneously induces inflammation dependent bone loss and AD-like pathologies by
elevating IL-6 and IL-17 from middle age, suggesting that periodontal bacteria induce
exacerbation of bone loss and memory decline, resulting in AD progression.

Porphyromonas gingivalis infection induces leptomeningeal cellsmediated synaptic failure in neurons
Wanyi Huang1, Junjun Ni2, Zhou Wu1,3.
1

Department of Cell Biology and Pharmacology 3OBT Research Center,
Faculty of Dental Science, Kyushu University, Japan.
2
Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology,
School of Life Science, Beijing Institute of Technology, China
Background: Synaptic failure is the earliest sign before Alzheimer’s disease (AD) onset
and closely associated with cognitive decline. Clinical studies have shown that
periodontitis is positively correlated with both the onset and pathological progression ofAD, and preclinical studies have shown that Porphyromonas gingivalis (P. gingivalis),
the key pathogen in periodontitis, and its virulence factors induced memory decline inmice. However, the mechanisms underlying the involvement of P. gingivalis in memory
decline remain unclear.
Methods: We used primary leptomeningeal cells and primary cortical neurons to evaluate
the effects of leptomeningeal cells on synaptic generation and plasticity after P. gingivalis
infection in vitro. The expression of the related molecules was examined by mRNA in
real time and by protein levels using Western blotting. Pharmacological and genetic
approaches were used to explore the mechanism underlying the involvement of
leptomeningeal cells in synaptic changes after P. gingivalis infection.
Results: NLRP3 inflammasome activation was involved in augmenting the IL-1β
secretion by primary leptomeningeal cells after P. gingivalis infection, as determined by
the knockdown of NLRP3 with siRNA. Cathepsin B (CatB) mediated the activation ofboth NLRP3 inflammasomes and NF-κB to produce IL-1β by P. gingivalis-infectedprimary leptomeningeal cells, as determined by the pharmacologically specific inhibitionof CatB. In contrast, P. gingivalis-infected leptomeningeal cells induced an IL-1βdependent decrease in synaptic molecules in primary cortical neurons, as determined by
the pharmacological blockage of the IL-1 receptor. P. gingivalis-infected leptomeningeal
cells also induced the IL-1β-dependent suppression of BDNF signaling in cultured N2aneurons, a stable mouse neural cell line.
Conclusion: The CatB-mediated IL-1β production was augmented in leptomeningeal
cells, resulting in synaptic failure and blockage of BDNF signaling in neurons during P.
gingivalis infection. These findings highlight a new mechanism underlying the
involvement of periodontitis in AD initiation and suggest that CatB may be an earlyintervention therapeutic target for delaying the onset of AD during P. gingivalis infection.

Cathepsin B inhibition blocks neurite outgrowth in cultured neurons
by regulating lysosomal trafficking and remodeling
Muzhou Jiang1, Junjun Ni2, Zhou Wu1,3
1

Department of Cell Biology and Pharmacology 3OBT Research Center,
Faculty of Dental Science, Kyushu University, Japan.
2
Key Laboratory of Molecular Medicine and Biotherapy, Department of Biology,
School of Life Science, Beijing Institute of Technology, China
Background: Lysosomes are known to mediate neurite outgrowth which is essential forneuronal network to enhance brain function. However, the principal lysosomal molecule
controlling that outgrowth is unclear.
Methods & Results: We studied primary mouse neurons in vitro and found that theynaturally develop neurite outgrowths over time and as they did so the lysosomal cysteine
protease cathepsin B (CTSB) mRNA levels dramatically increased. Surprisingly, we
found that treating those neurons with CA-074Me, which inhibits CTSB, prevented
neurites. As that compound also inhibits another protease, we evaluated a N2a neuronalcell line in which the CTSB gene was deleted (CTSB KO) using CRISPR technology andinduced their neurite outgrowth by treatment with retinoic acid. We found that CTSB KO
N2a cells failed to produce neurite outgrowths but the wild-type (WT) did. CA-074Me isa cell permeable prodrug of CA-074, which is cell impermeable and a specific CTSB
inhibitor. Neurite outgrowth was and was not suppressed in WT N2a cells treated with
CA-074Me and CA-074, respectively. Lysosome-associated membrane glycoprotein2(LAMP2)-positive lysosomes traffic to the plasma cell membrane in WT but not in CTSB
KO N2a cells. Interestingly, no obvious differences between WT and CTSB KO N2a cells
were found in neurite outgrowth regulatory proteins, PI3K/AKT, ERK/MAPK, cJUN and
CREB.
Conclusion: These findings show that intracellular CTSB controls neurite outgrowth andthat it does so through regulation of lysosomal trafficking and remodeling in neurons.
This adds valuable information regarding the physiological function of CTSB in neuraldevelopment.

KOB & OBT Joint International Symposium 2021

KOB Special Lecture
By Prof. Mieke Sylvia Margaretha

Chaired by Prof. Fusanori Nishimura

Forensic Odontology in Indonesia from A to Z
Mieke Sylvia Margaretha
Department of Forensic Odontology, Faculty of Dental Medicine, Universitas
Airlangga, Surabaya, Indonesia

Indonesia is the largest archipelago in the world with over 17,400 numbers ofisland. The intersection of three major plates, namely the Australian-indian plate, theEurasia plate, and the pacific plate have caused Indonesia to become one of the countries
which prone to natural disasters. It is also supported by the location of Indonesia in the
Pacific Circum Series and the smaller Philipine plate.
Indonesia is also a country with the largest Muslim population of all countries in
the world. In the Islamic religion, one important funeral rite should be done is the burialof the deceased has to be done immediately or within 24 hours after the time of death.
Unfortunately, in the forensic field, the process of identification of the victim needs someextra time and sometimes causes some delayed in the final burial.
There are other challenges for a forensic expert in personal identification inIndonesia due to the high-risk place and lack of ante mortem data. Most of the low society
populations in Indonesia do not aware of their oral health and does not know theimportance of medical record. Furthermore, dental care is not covered by the insurancecompany. Because of the several issues, sometimes it is hard to collect the antemortem
data in Indonesia. With this in mind, there are several suggestions that we have to solve
this problem. First, we will need a specific approach and technique for personal
identification which use the photo of the victim when alive and superimposed it with thepost mortem photo using smartphone. Second, we should build a new program to educate
the society about the importance of medical record used as an ante mortem data in “highrisk area”. With this program, we hope every citizen has a personal medical record soante mortem data can be obtained easily when needed.
Slowly but sure we also have been doing some excellent researches which canhelp in personal identification procedure in the future. The development of forensic
science studies in Indonesia itself also have been good and have become a government’s
concern.

Keywords: Indonesia, Culture, Religion, Specific Approach, Dental Education andResearch