13 th Congress of theInternational Society ofBone Morphometry
ISBM
2015

Program Book
Date
Venue
President

April 27-29, 2015

Tokyo Garden Palace Hotel, Tokyo
Masaki Noda

Tokyo Medical and Dental University

http://www.procomu.jp/isbm2015/

13 th Congress of theInternational Society ofBone Morphometry

April 27-29, 2015 ● Tokyo Garden Palace Hotel

Welcome to the XIIIth Congress of the International Society ofBone Morphometry
Masaki Noda, M.D., Ph.D.
President
Department of Molecular Pharmacology
Medical Research InstituteTokyo Medical and Dental University,
Tokyo, JapanAs a president, I would like to welcome all of you to the 13th congress of The International Society of BoneMorphometry (ISBM) in Tokyo. ISBM was founded in 1973, and incorporated as a non-profit organization in 1996.
The mission of ISBM is to emphasize the importance of traditional morphometric techniques in the field of bone
research, to embrace new technologies that result in refinements and advances in bone morphometric analyses, andto facilitate the education and training of basic scientists and clinicians in all aspects of bone morphometry. Many
preclinical and clinical studies utilize bone morphometry as an essential technique to assess changes in bone structureand function in response to a variety of influences such as hormones, cytokines, growth factors, mechanical loading,and bone-active drugs. Bone morphometry is also essential for skeletal phenotyping of knockout and transgenic
mice. Recent rapid advances in human bone imaging are emphasizing the importance of bone morphometry as a tool
for patient evaluation and monitoring drug efficacy in clinical practice.
This meeting, the 13th Congress of the Society, will be held on April 27-29, 2015 in Tokyo. We will begin the
congress by a wonderful keynote speaker, Jack Martin. Following his presentation, there will be main six sessions
where distinguished invited scientists in the field of skeletal diseases and basic research will present spectacularseries of talks. We are also welcoming delegations from 12 countries. Their papers will be presented in oral or poster
presentation sessions.
I express my sincere gratitude to Professor Masako Ito, the President of Japanese Society for Bone Morphometry
for her very kind and full support for this meeting. I am also very much thankful to Dr. Hideaki Takahashi, Professor
Emeritus of Niigata University, the past president of the 5th Congress of ISBM held in Japan, who has been
wonderfully leading this field of bone morphomety for many years.
The congress would not be possible without the generous supports of our sponsors, exhibitors, and donators.
Special thanks are in order for these companies whose names are listed in a knowledgement page.
ISBM congress serves as a unique opportunity for graduate students, postdocs, and young and senior scientists
for innovation in clinical and basic research based on a merging of molecular biology, preclinical and clinicalpathophysiology, and future development of bone-active drugs. Please join and enjoy ISBM Tokyo 2015 to become
more innovative, connected and interactive in the field of skeletal clinical and basic sciences.
April 3, 2015i

13th Congress of the International Society of Bone MorphometryiiApril 27-29, 2015 ● Tokyo Garden Palace Hotel

Table of Contents

Welcome to the XIIIth Congress of the International Society ofBone Morphometry… ……………………………………………………………

i

Officers and Board Members of the International Society ofBone Morphometry… ……………………………………………………………

v

To Participants… ………………………………………………………………… vi
To Poster Session Chairpersons and Speakers… ………………………………… viii
Floor Plan… ……………………………………………………………………… ix
Poster & Exhibition Layout… …………………………………………………… ix
Venue for Receptions………………………………………………………………

x

Special Thanks to our Congress Sponsors and Exhibitors………………………… xi
Program at a Glance… …………………………………………………………… xii
Congress Agenda… ……………………………………………………………… xv
Keynote Lecture……………………………………………………………………

1

Main Sessions… …………………………………………………………………

5

Work Shop………………………………………………………………………… 23
Luncheon & Evening Seminars…………………………………………………… 27
Oral Presentations… ……………………………………………………………… 39
Poster Sessions… ………………………………………………………………… 63iii

13th Congress of the International Society of Bone Morphometryiv

April 27-29, 2015 ● Tokyo Garden Palace Hotel

Officers and Board Members of the International Society ofBone Morphometry
President : Masaki Noda, M.D., Ph.D. (Tokyo Medical & Dental University)
Past President : Thomas J. Wronski, Ph.D. (University of Florida)
ISBM Board Members :Jillian Cornish, Ph.D. (University of Auckland)
Christopher J. Hernandez, Ph.D. (Cornell University)
Urszula T. Iwaniec, Ph.D. (Oregon State University)
Michaela Kneissel, Ph.D. (Novartis Institutes for Biomedical Research)
Natalie A. Sims, Ph.D. (St. Vincent’s Institute of Medical Research)
D. Rick Sumner, Ph.D. (Rush University)
Sara H. Windahl, Ph.D. (Gothenburg University)
Access ＆ Map

HOTEL,BANQUET & RESTAURANT Tokyo Garden Palace
Address:1-7-5 Yushima, Bunkyo-ku, Tokyo 113-0034 Japan Telephone 03-3813-6211

Facsimile 03-3818-6060

To Yushima Shrine

N

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o

Tokyo Garden Palace

St.

Marunouchi Lin

ine
aL

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ky
To

Och
an
Sta omizu
tionoMTokyo Medical and Dental
University

JR Chuo Line
/Sobu Line

1

YushimaTemple2

Ochanomizubashi BridgeodKanda Shrine

Kanda Riverhiy

Juntendo University

i St.

KuramaebashoCTokyo Metro

Hongo Kyusuijo Park

Japan Football Museum

Ho

Ochanomizubashi exitSo

JR Ochanomizu Sta.

tob

B1

Japan Football Museum

shi

eba

a
ram

Ku

St.

Sotobori St.

Tokyo Metro
Marunouchi Line
Ochanomizu Sta.

uo

To Akihabara

Lin

To TokyoeShoheibashi
BridgeTo Tokyo

Yushima Temple
Hijiribashi Bridge● Please get off at Ochanomizu Station (JR Chuo Line). Tokyo
Garden Palace is a 5-minute walk from the Hijiribashi exit.
● Please get off at Shin-Ochanomizu Station (Tokyo Metro ChiyodaLine). Tokyo Garden Palace is a 5-minute walk from the station.
● Please get off at Ochanomizu Station (Tokyo Metro MarunouchiLine). Tokyo Garden Palace is a 5-minute walk from the station.

By carToei Shinjuku Line

JR
Ochanomizu
Sta.
NikolaiCathedral

Hongo St.

Meidai St.

Ochanomizubashi
BridgeJR Sobu LineCh

By train (from Tokyo Station)
Kanda Shrine

Kanda River
To Shinjuku

St.

= Subway exit

Hongo St.

Tokyo Medical
and Dental University-Ocha
Sta nomizu
.

St.
Ho

ngo

Tokyo Garden Palace

JR

Shin

ai S
Me
id
= JR wicket

200m

ori

B2

t.

Hijiribashi exit

To TokyoTokyo Metro
Chiyoda Line
Shin-Ochanomizu Sta.

Yasukuni St.

Surugadai-shita● From Shibuya/Shinjuku area
From the Kandabashi IC, direct access to Hongo Street is prohibited.
Please follow the arrow to Hongo Street.
● From Ginza/Chiba area
From the Kandabashi IC exit, direct access to the Hongo
Street/Hijiribashi Bridge area is prohibited. Please follow the arrow tomake a detour to the Hongo Street/Hijiribashi Bridge area.

Ogawamachi

Kandabashi IC
To Ginza/Chiba area

To Shibuya/Shinjuku area

Metropolitan Expressway
KDDI
Otemachi Bldg.

NTT
Otemachi
Bldg.

Underground parking lot
Tokyo Garden Palace

Exit

Entrance

Hongo St.

● Parking fee: ¥300 for the first 30 min.
(¥300 added every 30 min.)● Overnight stay: ¥1,000/night (incl. tax)
● Banquet/meeting: Free of charge for 3 hours● Wedding: Free of charge for 4 hours● Restaurant: Free of charge for 2 hoursv

13th Congress of the International Society of Bone Morphometry

To Participants
1. Main Reception Desk
The registration/information desk is located in the lobby of the Banquet Hall “Takachiho” in the TOKYOGARDEN PALACE
Dates and Times: April 27 (Monday) –April 29 (Wednesday) 8:00 a.m. - 5:00 p.m.
2. On-Site Registration
On-site registrants must fill out a registration form and pay their fees. At registration, participants will be given
a badge on which to write their name and affiliation. These badges should be worn at all times during theconference. Students must present their student ID cards at registration.
3. Registration Fees
Member: JPY 30,000 Non-Member JPY 32,000
Post Graduate Student/Resident: JPY 27,000
4. During question-and-answer and discussion sessions, please follow the chairman's directions and state yourname and affiliation when you first approach the microphone.
5. Smoking is not permitted in any of the conference venues.
6. Mobile phones should be turned off or in silent mode.
7. Lunch will be provided at luncheon seminars. Participants also may dine at the hotel's restaurants and coffee
shops.
8. The recording of all or part of any scientific presentation or poster presentation is prohibited.

To Invited Speakers of Main Sessions
Presentation data and PC<Presentation time>
50minutes for presentation and 10 minutes for Q&A<Presentation equipment>
1. All presentation materials should be prepared in Microsoft PowerPoint 2010 or 2013 for Windows PC.
2. You can bring your presentation data on a USB memory stick or CD-R in the above format.
3. You are advised to bring your own laptop PC if your presentation materials including video/movies cannot be
prepared in the above mentioned format.
4. Please make sure that your laptop is equipped with mini D-sub (15pins). If you are using Macintosh, please
make sure to bring AC adaptor and connector to mini D-sub (15pins).
5. Please deactivate your screensaver and power saving settings.
6. Please bring your own power cable. Presentations using batteries may result in problems.
7. To avoid losing important data saved on the PC used for your presentation, be sure to backup such data
beforehand.

viApril 27-29, 2015 ● Tokyo Garden Palace Hotel

To Speakers of Oral Presentations
Presentation registration
After registering for the conference at the reception desk of the site, all speakers for the oral sessions are asked tocome to the “PC Center” to check in their presentation data. You are advised to come to the PC Center before 30
minutes prior to their presentations.
New investigator award winners need to attend the closing ceremony on April 29th 2015 to receive the awards.
<Presentation time>
9 minutes for presentation and 3 minutes for Q&A<Presentation equipment>
1. All presentation materials should be prepared in Microsoft PowerPoint 2010 or 2013 for Windows PC.
2. You can bring your presentation data on a USB memory stick or CD-R in the above format.
3. You are advised to bring your own laptop PC if your presentation materials including video/movies cannot be
prepared in the above mentioned format.
4. Please make sure that your laptop is equipped with mini D-sub (15pins). If you are using Macintosh, please
make sure to bring AC adaptor and connector to mini D-sub (15pins).
5. Please deactivate your screensaver and power saving settings.
6. Please bring your own power cable. Presentations using batteries may result in problems.
7. To avoid losing important data saved on the PC used for your presentation, be sure to backup such data
beforehand.

vii

13th Congress of the International Society of Bone Morphometry

To Poster Session Chairpersons and Speakers[Presentation time]
1 minutes for presentation and 2 minutes for Q&A(There will be a chairperson in front of your poster. Please follow his instruction and present your poster for 4
min at the indicated date and time shown above. There will be 2 min discussion (Q & A) period just after yourpresentation.)[Venue]Tenku A (2nd floor)[Poster dimension]
Poster maximum size:
Height: 210 cm
Width: 90 cm[Poster setup time]
April 27th (Mon.) 8:30-12:00[Poster presentation time]
April 28th (Tues.) 13:00-14:00[Poster presentation session frame]
April 28th (Tues.) Poster Presentation 1 13:10-13:46
April 29th (Wed.) Poster Presentation 2 13:10-13:46*Please stand by your poster 5 minutes before the session starts.
*Please follow the instruction given by your chairperson[Poster removal time]
April 29th (Wed.) 14:00-18:00
Note : All posters should be taken down from the boards by the above time. Conference secretariat will not assume
responsibility for possible loss of posters left on the board , in particular posters left after the last day of thecongress.
For Poster Session Speakers
1. Poster Board Size
90cm wide by 210cm high, including 20cm for title, name and affiliation.
In preparing your presentation, we ask that you keep your name, title, and affiliation at 70cm wide by 20cm
high. The poster number (20cm by 20cm) will be added by the secretariat.
The poster display space is 190cm high by 90cm wide. If more space is needed, use the space allotted for thename, title, and affiliation, as information in the lower portion of the poster board will be difficult to see.
2. Ribbons and push-pins for the speakers will be at the poster session reception desk. Speakers also should wear
their ribbons during the poster presentation.
New investigator award winners need to attend the closing ceremony on April 29th 2015 to receive the awards.

viiiApril 27-29, 2015 ● Tokyo Garden Palace Hotel

Floor Plan

Secretariat
Main Session
Halls

Hall A（TACHIBANA）

Rest
Room

Hall B

Poster Session&
Exhibition

1F Main
Lobby（TAKACHIHO A） （TAKACHIHO B）（TENKU）

Reception
Rest
Room

Rest
Room

PC Center
Anteroom
For Seminar

Anteroom
For Seminar（FUYO）（BOTAN）

Poster Session / Exhibition（Tenku A）

P2-5

P1-6

P2-6

P2-12 P1-12 P2-11

P1-5
P2-4

P1-11

E

F

P1-10

P1-4

P2-9

P2-3
P1-3

P2-10

C

D

Exhibitor
A： Shimadzu

Corporation.

P1-9

P2-2

P2-8

B： RATOC Systems, Inc.

P1-2

P1-8

C： Summit Pharmaceuticals

P2-1

A

B

P1-1

P2-7
P1-7

Exhibition

International Corporation.
D： MEGMILK SNOW BRANDGroup

E： SHOSHIN EM Corporation.
F ： TOYO Corporation.

Poster Group A

Poster Group B

1-A( April 28th)

1-B( April 28th)2-A(April 29th)

2-B(April 29th)ix

13th Congress of the International Society of Bone Morphometry

Venue for Receptions
April 27, 2015 (Monday)
Social Evening 7:00 p.m. 〜 8:30 p.m.
Venue : Garden Palece Hotel (Takachiho B)
Tokyo Night View from Sky Lounge (26F) 8:30 p.m. 〜 9:00 p.m.
Venue : Tokyo Medical and Dental University MD-Tower, 26F Floor

April 28, 2015 (Tuesday)
International Evening (Bus will depart from the Hotel at 6:10 p.m.)
Venue : Symphony Cruise (Departing at 7:00 p.m.)
Hinode Pier in Tokyo Bay
Access & Map● JR Ochanomizu Sta.

Tokyo Garden Palace Hotel
JR Chuo Line

Hongo St.

● JR Tokyo Sta.

26F
Sky
LoungeYushimaTempleElevators

Tokyo Medical and Dental
University
Ochanomizubashi Bridge

JR Yamanote Line● JR Shinbashi Sta.

26F

Kanda River

Yurikamome Line

JR Chuo Line

To Tokyo

Ochanomizubashi exit

Hijiribashi exit

● Hinode Sta.

JR Ochanomizu Sta.
● Hinode Pier

26F
Sky Lounge
MD Tower
TMDU

EntrancexApril 27-29, 2015 ● Tokyo Garden Palace Hotel

Special Thanks to our Congress Sponsors and Exhibitors
Gold Sponsors :Luncheon Seminar
・CHUGAI PHARMACEUTICAL Co., Ltd.
・Taisho Toyama Pharmaceutical Co., Ltd.
・Asahi Kasei Corporation.
・Pfizer Inc.
・DAIICHI SANKYO COMPANY, LIMITED.
・MSD K.K. a subsidiary of Merck & Co., Inc.
Evening Seminar・Hisamitsu Pharmaceutical Co., Inc.
・Eli Lilly Japan K.K.

Silver Sponsors :Contribution
・Sumitomo Dainippon Pharm Co., Ltd・Taisho Toyama Pharmaceutical Co., Ltd.
・Novartis Pharma K.K.
・Osteometrics, Inc.
・Astellas Pharma Inc.
Exhibition
・SHIMADZU CORPORATION.
・RATOC Systems, Inc.
・Summit Pharmaceuticals International Corporation.
・Bean Stalk Snow Co., Ltd. (MEGMILK SNOW BRAND Group)・SHOSHIN EM Corporation.
・TOYO Corporation.

Bronze Sponsors :Advertisement
・SHIMADZU CORPORATION.
・Astellas Pharma Inc.
・Eisai Co., Ltd.
・RATOC Systems, Inc.
・Hologic Japan,Inc
・ZEISS・DAIICHI SANKYO COMPANY, LIMITED・RIKAKEN CO., LTD.
・John Wiley & Sons (Asia) Pte. Ltd.
・Summit Pharmaceuticals International Corporation.

xi

13th Congress of the International Society of Bone Morphometry

at a Glance
Program at aProgram
GlanceApril 27(Mon)

April 28(TueHall A(2F)Hall B (2F)

Poster (3F)Hall A(2F)

Room ”Takachiho A”

Room ”Takachiho B”

Room ”Tenku A”

Room ”Takachiho A”

Hall B (2F

Room ”Takachih

8:00

8:30

9:00

8:40 Opening Remark
8:45 Keynote Lecture

8:30
Session 3 : Fracture and Bone
Structure

8 30-12:00

How concepts of bone
remodeling influence
therapeutic development inosteoporosis
Jack Martin

Assessment of bone structure in
relation to biomechanicalproperties

Masako Ito

9:45

Session 1 : New Treatment andPoster setup
time

Diagnosis for
Osteoporosis

10:00

New imaging techniques inthe diagnosis of bone diseases

Ralph Muller
10:30 Oral Presentation 3

Thomas Link
11:00

Hierarchical imaging ofbone failure

Targeting cathepsin k forthe treatment of osteoporosis

O3-1-O3-6

Le Duong
12:00

11:45 Group Picture
12:00 Luncheon Seminar

Novel modes of action of anti-boneresorptive drugs revealed byintravital two-photon bone imaging
technology

Locomotive syndrome –Measures for increasing
healthy life expectancy

Masaru Ishii

Kozo Nakamura

Chugai Pharmaceutical Co., Ltd

Taisho Toyama PharmaceuticalCo., Ltd.

13:00

12:00 Luncheon Seminar

12:00 Luncheon Seminar

12:00 Luncheon Sem

Morphological examination
on anabolic effects ofintermittent administrationof PTH in bone
Norio Amizuka

Bone histomorphom
orthopedic clinical p

Noriaki Yama
Pfizer Inc.

Asahi Kasei Corporation.

13:00

Oral Presentation 1

O1-1-O1-5
14:00

14:00

14:00
Session 4 : Bone Impairment
and Clinical Bases

Session 2 : Bone Diseases andCell Regulation
Cell communication in
osteoimmunology

Clinical management of
osteoporosis : new options

Hiroshi Takayanagi

15:00

Robert Recker

Effects of sclerostin antibody on
different bone surfaces with shorterand longer- term treatment

Genetic and molecular bases ofosteolytic/osteoporotic disorders

Yoichi Ezura

David Ke

16:00

Cartilage and meniscus
regenerationby synovial stem
cells : from bench to clinic

PosterExhibition

Ichiro Sekiya

17:00

17:00

Rapid and reproducible ex vivo
imaging of mouse articularcartilage using confocal microscopy

Matthew Warman

17:00 Evening Seminar

Oral Presentation 2

Anabolic therapy forthe skeleton with PTH– now and the futureJack Martin

O2-1-O2-5

Eli lilly japan K.K.
18:00

18:00
E v e n in g S e m in a r

18:10 Transportation to Intern

Geometry of lower extremities
and atypical femoral fractures
Muneaki Ishijima
19:00(Bus will depart from Gard

Hisamitsu Pharmaceutical Co., Inc.
19:00-20:30

Social Evening
Garden Palace Hotel
20:30-21:00
21:00

xii

TMDU MD Tower 26F
Sky Lounge

19:00-21:00

International Evening on
at Hinode Pier in T

April 27-29, 2015 ● Tokyo Garden Palace Hotel

Program at a Glance
April 27(Mon)

April
April 28(Tu

28(Tues)27(Mon)
April
April

Hall B (2F)

Poster (3F)

Hall
HallA(2F)A(2F)

HallBB(2F)
Hall
(2F)

(3F)
PosterPoster(3F)

A(2F)
Hall
Hall
A(2F)

Room ”Takachiho B”

Room ”Tenku A”

Room
Room”Takachiho”Takachiho A”
A”

Room
Room”Takachiho”TakachihoB”B”

Room
Room”Tenku”TenkuA”A”

Room
Room
”Takachiho”TakachihoA”A”(2F
Hall BHal

Room Room”Takachi
”T

8:00

8:30

8 30-12:00

8:30 Opening Remark
8:40
and Bone
Session3 : Fracture
KeynoteLecture8:45
Structure

8:30
8:30
Bone
SessionSession5 :3 Instability: Fracture and Skeletal
Structure
Diseases

8 30-12:00

How concepts of bone
remodeling influence

9:00

Assessment of bone structure intherapeutic development inrelation to biomechanicalosteoporosis
properties

Assessmentof bonestructurein
AFF:
Clinical andhistologicalfeaturesofrelation
to biomechanicalatypicalsubtrochantericand diaphyseal
properties
femoralfractureJack Martin
MasakoIto

9:45

Poster setup
time

Masako Ito
Naoto Endo

Hierarchical imaging of

Session 1 :bone
Newfailure
Treatment andPoster setup
time

Diagnosis for
MullerRalph
Osteoporosis

10:00

New imaging techniques in
the Oraldiagnosisof bone diseases
3
10:30
Presentation

MullerRalph
Chun
Jang-Soo

Presentation
43
10:30Oral
Oral
10:30
Presentation

Thomas Link

11:00

Hierarchicalimaging ofRegulationof the catabolic
cascade in
failureosteoarthritisbone
by thezinc·ZIP8·MTF1 axis

PosterExhibition

Targeting cathepsin k forO3-1-O3-6
the treatmentof osteoporosis

O3-1-O3-6
O4-1-O4-6

Le Duong
12:00

2:00 Luncheon Seminar

11:45 Group Picture
12:00 Luncheon
LuncheonSeminar
SeminarMorphological examination

Locomotive syndrome –Measures for increasing
healthy life expectancy

Novel modes of action of anti-boneon anabolic effects ofresorptive drugs revealed byintermittent
administration
intravital
two-photonbone imaging
of PTHin bonetechnology

Norio
Amizuka
Ishii
MasaruKozo Nakamura

Bone histomorphomertry in
Locomotive
syndrome
orthopedic
clinicalpractice –Measures for increasing
Noriaki Yamamoto
healthy life expectancy
Pfizer Inc.

MSD K.K.
Merck

Daiichi Sankyo Company, Ltd.

13:10-13:46
Poster Presentation 1-A, 1-B

13:00

Oral Presentation 1

Talk & Discussion
P1-1-P1-12

O1-1-O1-5
14:00

TanakaAsahi Sakae
Kasei Corporation.

Taisho Toyama PharmaceuticalCo., Ltd.

13:00

12:00 12:00
Luncheon
Sem
LuncheoImaging analysis of the effects ofdenosumab on bone structural
Bone histomorphom
Morphological examination
Bone microa
properties
orthopedic
clinicalon anabolic effects ofalendronat
MasakoIto
Noriaki Yamaintermittent
administration
osteoporof PTHbone
Pfizer Inc
Denosumab
forinthetreatment of
Robe
Norio
Amizukaosteoporosis

Kozo Nakamura

Asahi Kasei Corporation.
Chugai Pharmaceutical Co., Ltd

Taisho Toyama PharmaceuticalCo., Ltd.

12:00
12:00Luncheon
Luncheon
SeminarSeminar

12:00
Seminar
12:00Luncheon
Luncheon
Seminar

14:00
SessionS
ession42: :Bone
BoneImpairment
Diseases andandClinical
Bases
Cell
Regulation

14:00
p
14:00Workshop
Session 4 : Bone Impairment
Proper bone histomorphometry
and Clinical Basesin rats

Thomas Wronski
Cell communication
Clinicalmanagement inofosteoimmunology
osteoporosis: new options

Clinical management ofHiroshiReckerRobertTakayanagi

Bonehistomorphometryosteoporosis: new optionsin mice:
Getting
it right!
ReckerRobertEffects andof sclerostin
antibody
Genetic
molecularbases ofon
differentbone surfaces withshorterosteolytic/osteoporotic
disorders
and longer- term treatment

Genetic and molecular bases of
15:00
osteolytic/osteoporotic
disordersfor Skeletal
Session
6 : Molecular BasesNatalie Sims
15:00

Pathology
Yoichi Ezura

Yoichi Ezura
David Ke

16:00

PosterExhibition

Rapid and reproducible ex vivo
Cartilage and meniscus
imaging of mouse articularregenerationby synovial stem
cartilage using confocal microscopy
cells : from bench to clinic

PosterExhibition

Matthew
SekiyaIchiro Warman

17:00

1
7:00 Evening Seminar
17:00

17:00 Evening Seminar

Investigating the dynamic biology
Anabolic therapy forof osteocytes and bone extracellular
the skeleton with PTH
matrix assembly using live cell– now and the future
imaging approaches
Jack Martin
Sarah
DallasEli lilly
japan
K.K.

Oral
Presentation
2 forAnabolic
therapy
the skeleton with PTH– now and the futureO2-1-O2-5
Jack Martin
Eli lilly japan K.K.
18:00

18:00 Award Ceremony
Closing Remark

18:00
E v e n in g S e m in a r

18:extremities
10 Transportation to International Evening Venue
Geometry of lower
and atypical femoral fractures(Bus will depart from Garden Palace Hotel)
Muneaki Ishijima

9:00-20:30

19:00

Social Evening
Garden Palace Hotel
TMDU MD Tower 26F
Sky Lounge

19:00-21:00

19:00-20:30

20:30-21:00

at Hinode Pier in Tokyo Bay
21:00

18:10 Adjourn

18:10 Transportation to Intern(Bus will depart from Gar

Hisamitsu Pharmaceutical Co., Inc.

Social Evening
Hotel
EveningPalace
International Garden
on Symphony
Cruise0:30-21:00

Dynamic live imaging of bones:
opening a new era with ‘Bone
Rapid and reproducible ex vivo
histodynametry’imagingof mouse articularcartilage using
confocalmicroscopy
MasaruIshii
Matthew Warman
Histological aspects on the biological
function of osteocytes
Norio Amizuka

TMDU MD Tower 26F
Sky Lounge

19:00-21:00

International Evening on
at Hinode Pier inxiii

13th Congress of the International Society of Bone Morphometry

Program at a Glance
April 29(Wed)April 27(Mon)

April 28(Tues)

Hall B (2F)m ”Takachiho B”

April 28(Tues)

Poster (3F)

A(2F)
Hall
A(2F)
Hall

Hall BHall(2F)B (2F)

Poster (3F)
Poster (3F)Hall A(2F)Hall B (2F)

Room ”Tenku A”

Room
Room
”Takachiho”Takachiho
A” A”

Room ”Takachiho
Room ”Takachiho
B”
B”

Room ”Tenku
Room A””Tenku A”

Room ”Takachiho A”

Room ”Takachiho B

8:00

8:30

8:30 Opening Remark
8:40
Session5 : Instability
KeynoteLecture and Skeletal
8:45
Diseases

8:30
Session 3 : Fracture and Bone
Structure

8 30-12:00

How concepts of bone
remodeling influence

9:00

Assessment of bone structure in
relation to biomechanicalproperties

AFF:
Clinical andhistological featuresoftherapeutic
developmentinatypical subtrochanteric and diaphyseal
osteoporosis
femoral fractureJack Martin
Naoto Endo

Masako Ito

9:45
Regulationof thecatabolic cascadein
Session1 : New
andTreatment
10:00

Osteoporosis
ChunJang-Soo

New imaging techniques in
the Oraldiagnosisof bone diseases
Presentation
4
10:30

PosterExhibition

Ralph Muller
10:30 Oral Presentation 3

Thomas Link

11:00

Hierarchical imaging ofbone failure

Poster setup
time

osteoarthritis
by the zinc·ZIP8·MTF1
axis
Diagnosis
forTargeting cathepsin k forO4-1-O4-6
the treatmentof osteoporosis

O3-1-O3-6

Le Duong
12:00

uncheon Seminar

11:45 Group Picture
12:00 Luncheon
LuncheonSeminar
SeminarSeminar
12:00 12:00
Luncheon
SeminarLuncheon

12:00 Luncheon Seminar

Imaging analysis of the effects ofdenosumab on bone structural
Novel modes ofproperties
action of anti-bone
Locomotive
syndrome –Bone microarchitecture
after
resorptive drugs revealed byalendronate
treatment of
Measuresfor increasing
Masako Ito
intravital two-photon bone imaging
women
healthy osteoporotic
life expectancy
Denosumab
for the treatment oftechnology
Robert Recker
osteoporosis
Masaru Ishii
Kozo Nakamura
MSD K.K. A subsidiary of
Sakae Tanaka

histomorphomertry inpedic clinical practiceoriaki Yamamoto
Pfizer Inc.

13:00

13:10-13:46
Poster Presentation 1-A, 1-B

Chugai Pharmaceutical Co., Ltd
Daiichi Sankyo Company, Ltd.

Bone histomorphomertry
orthopedic clinical practic

Noriaki Yamamoto
Pfizer Inc.

Asahi Kasei Corporation.

13:00-13:46
Poster Presentation 2-A, 2-B

Oral Presentation 1

Talk & Discussion
P2-1-P2-12

O1-1-O1-5
14:00

Morphological examination
on anabolic effects ofintermittent administrationof PTH in bone
Norio Amizuka

Merck& Co., Inc.
Taisho Toyama
PharmaceuticalCo., Ltd.

13:00

Talk & Discussion
P1-1-P1-12

12:00 Luncheon Seminar

14:00 Workshop
p

14:00-18:00

Session2 :boneBone
Diseases andProperhistomorphometry
CellinRegulation
rats

14:00
Session 4 : Bone Impairment
and Clinical Bases

Thomas Wronski
Cell communication in

Clinical management of
osteoporosis : new options

osteoimmunology
Bone histomorphometry
in mice:
HiroshiTakayanagi
Getting
it right!

Robert Recker

Natalie Sims
15:00

16:00

17:00

18:00

on to International Evening Venue

Genetic and molecular bases ofosteolytic/osteoporotic disorders

Yoichi Ezura

David Ke
Dynamic live imaging of bones:
opening a new era with ‘Bone
Cartilage
and meniscus
histodynametry’regenerationby synovial stem
MasaruIshii
cells : from bench toclinicPoster removal time
PosterExhibition

SekiyaIchiroHistologicalaspects
on the biological
function of osteocytes
Norio Amizuka
17:00

Rapid and reproducible ex vivo
imaging of mouse articularcartilage using confocal microscopy

Matthew Warman

17:00 Evening Seminar

Investigating the dynamic biology
Oral Presentation 2of osteocytes and bone extracellularmatrix assembly using live cell
imaging
approaches
O2-1-O2-5
Sarah Dallas

Anabolic therapy forthe skeleton with PTH– now and the futureJack Martin
Eli lilly japan K.K.

18:00 Award Ceremony
18:00
Closing RemarkEv e n ing S e m in a r
18:10
Adjourn

18:10 Transportation to Internation

Geometry of lower extremities
and atypical femoral fractures
Muneaki Ishijima

rt from Garden Palace Hotel)
19:00(Bus will depart from Garden P

Hisamitsu Pharmaceutical Co., Inc.
19:00-20:30

Social Evening
Garden Palace Hotelvening on Symphony Cruisede Pier in Tokyo Bay

20:30-21:00
21:00

xiv

Effects of sclerostin antibody on

15:00
different bone surfaces with shorterBases for Skeletal
Session: Molecularand6longerterm treatment
Pathology

TMDU MD Tower 26F
Sky Lounge

19:00-21:00

International Evening on Sym
at Hinode Pier in Toky

April 27-29, 2015 ● Tokyo Garden Palace Hotel13th Congress of the International Society of Bone Morphometry
2015.4.27 Monday
8:40amOpening Remark

8:45am-9:45am

Keynote Lecture

Chair : Masaki Noda

・Jack Martin : How concepts of bone remodeling influence therapeutic development(p3)

in osteoporosis
9:45am-11:45am

Session 1

New Treatment and Diagnosis for Osteoporosis (Takachiho A)

Chairs : Tom Wronski, Natalie Sims
9:45am-10:45am

・Thomas Link : New imaging techniques in the diagnosis of bone diseases(p7)

10:45am-11:45am

・Le Duong : Targeting cathepsin k for the treatment of osteoporosis(p8)

11:45am-11:50am

Group Picture (Takachiho A)

12:00pm-13:00pm

Luncheon Seminar Speaker : Masaru Ishii (Takachiho A)(p29)

Dynamic live imaging of bones : opening a new era with bone histodynametry
Sponsored by CHUGAI PHARMACEUTICAL Co., Ltd
Luncheon Seminar Speaker : Kozo Nakamura (Takachiho B)(p30)

Locomotive syndrome –measures for increasing healthy life expectancy
Sponsored by Taisho Toyama Pharmaceutical Co., Ltd.
1:00pm-2:00pm

Oral Presentation 1 Chairs : Jean-Paul Roux, Akira Nifuji (Takachiho A)

1:00pm-1:12pm

O1-1

Yanfei Linda Ma : Histomorphometric analyses of cortical effects of teriparatide		

in postmenopausal women with or without long-term alendronate therapy

1:12pm-1:24pm

Ko Chiba : In vivo bone microstructural analysis of osteoporosis

O1-2

(p41)
(p42)

		

using HR-pQCT

1:24pm-1:36pm

Nobuhito Nango : Measurement of bone strength and medication evaluation can be

O1-3

(p43)

		

achieved using medical CT images

1:36pm-1:48pm

J. Ignacio Aguirre : Comparative analysis of the development of periodontal disease

O1-4

		

in rice rats and lewis rats

1:48pm-2:00pm

Akira Nifuji : G9a deficiency leads to impaired osteoblastic differentiation

O1-5

		

2:00pm-5:00pm

in mice

Session 2

(p44)
(p45)

Bone Diseases and Cell Regulation (Takachiho A)

Chairs : Matthew Warman, Yoichi Ezura
2:00pm-3:00pm

・Hiroshi Takayanagi : Cell communication in osteoimmunology

(p9)

xv

13th Congress of the International Society of Bone Morphometry

3:00pm-4:00pm

・David Ke : Effects of sclerostin antibody on different bone surfaces with shorter-

and longer- term treatment
4:00pm-5:00pm

・Ichiro Sekiya : Cartilage and meniscus regeneration by synovial stem cells : frombench to clinic
5:00pm-6:00pm

Oral Presentation 2 Chairs : Linda Ma, Aguirre Ignacio (Takachiho A)

5:00pm-5:12pm

O2-1

murine bone

5:12pm-5:24pm

Andreas Johnathan Truessel : Quantitative local in vivo environment (live)

O2-2

		

Imaging of osteocytes during dynamic bone remodeling

5:24pm-5:36pm

Guangyi Li : Influence of subchondral bone plate integrity on subchondral bone

O2-3

(p11)

Christina Vrahnas : Osteocytic ephrinB2 restrains mineralisation of

		

(p46)
(p47)
(p48)

		

homeostasis in osteoarthritis

5:36pm-5:48pm

Hiroshige Sano : Intravital bone imaging by two-photon excitation microscopy to

O2-4

(p49)

		

identify osteocytic osteolysis in vivo

5:48pm-6:00pm

Smriti Aryal : Nck adaptor proteins control osteoblastic migration, bone formation

O2-5

		

6:00pm-7:00pm

and bone mass

Evening Seminar Speaker : Muneaki Ishijima (Takachiho A)
Geometry of lower extremities and atypical femoral fractures
Sponsored by Hisamitsu Pharmaceutical Co., Inc.

xvi

(p10)

7:00pm-9:00pm【Social Evening】 Garden Palace Hotel (Takachiho B)

8:30pm-9:00pm

Night vewing of Tokyo (MD Tower 26th Floor)

(p50)
(p36)

April 27-29, 2015 ● Tokyo Garden Palace Hotel2015.4.28 Tuesday
8:30am-10:30am

Session 3

Fracture and Bone Structure (Takachiho A)

Chairs : Robert Recker, Thomas Link
8:30am-9:30am

・Masako Ito : Assessment of bone structure in relation tobiomechanical properties
9:30am-10:30am

・Ralph Muller : Hierarchical imaging of bone failure

10:30am-11:45am

Oral Presentation 3 Chairs : Urszula Iwaniec, Norio Amizuka (Takachiho A)

10:30am-10:42am

O3-1

Anders Palmquist : Bone-implant interface: morphology and ultrastructure

10:42am-10:54am

O3-2

Paksinee Kamolratanakul : Comparative histological study in maxillary sinus

(p12)
(p13)

(p51)

		

augmentation using coarse or fine particle of xenograft		materials (preliminary report)(p52)

10:54am-11:06am

O3-3

Phil Salmon : Is it time to say goodbye to SMI?

(p53)

11:06am-11:18am

O3-4

Omar Omar : Labeling and seeding of MSCs on hydroxyapatite bone substitute :		cell behavior and in vivo tracking

11:18am-11:30am

Furqan A. Shah : Apatite orientation corresponds to the shape of the osteocyte

O3-5

		

lacuna in sheep trabecular bone

11:30am-11:42am

Urszula T. Iwaniec : Housing mice at room temperature results in premature

O3-6

		

12:00pm-1:00pmbone loss : implications for the mouse as a model of osteoporosis

Luncheon Seminar Speaker : Norio Amizuka (Takachiho A)

(p54)
(p55)
(p56)
(p31)

Morphological examination on anabolic effects of intermittent administrationof PTH in boneSponsored by Asahi Kasei Corporation.
Luncheon Seminar Speaker : Noriaki Yamamoto (Takachiho B)(p32)

Bone histomorphomertry in orthopedic clinical practiceSponsored by Pfizer Inc.
1:00pm-2:00pm

Poster Presentation 1-A Chair : Noriaki Yamamoto (Tenku A)

1:10pm-1:16pm

P1-1

Eunji Kim : Alveolar bone healing after the removal of temporary		anchorage devices

1:16pm-1:22pm

Nam Cong Nhat Huynh : Inhibition of histone deacetylases enhances

P1-2

		

the osteogenic differentiation of human periodontal ligament cells

1:22pm-1:28pm

Igor Zakharov : Principles of selection methods of radiation diagnosis of

P1-3

		

postmenopausal osteoporosis

(p65)
(p66)
(p67)

xvii

13th Congress of the International Society of Bone Morphometry

1:28pm-1:34pm

P1-4

Makiri Kawasaki : TGF-beta impairs cilia morphology via suppression

		of Ift88 expression in chondrocytic ATDC5 cells

1:34pm-1:40pm

Takuya Notomi : Membrane depolarization regulates intracellular RANKL

P1-5

(p68)
(p69)

		

transport in non-excitable osteoblasts

1:40pm-1:46pm

Sakie Katsumura : Poldip2 is expressed in osteoblasts and its expression is enhanced

P1-6

		

by osteogenic differentiation medium containing beta glycero-phosphate and ascorbic

		acid or BMP(p70)

Poster Presentation 1-B Chair : Sadakazu Ejiri

1:10pm-1:16pm

P1-7

Maureen Watson : Bone regeneration is increased with local application of

(p71)

		

lactoferrin in critical-sized rat calvarial defects

1:16pm-1:22pm

Yayoi Izu : Collagens vi and xii form complexes mediating osteoblast interaction

P1-8

		

during osteogenesis

1:22pm-1:28pm

Tomoki Uehara : A rankl-binding peptide accelerates BMP-induced bone

P1-9

		

1:28pm-1:34pm

in osteoblastic MC3T3-E1 cells
growing female mice

(p75)

P1-12 Koji Mineta : Osteocytes in primary cultures express higher levels of

		

β2-adrenergic receptor mRNA while MLOY4 cells exhibit culture period		

dependent regulation of the expression levels

2:00pm-5:00pm(p74)

P1-11 Urszula T. Iwaniec : Paradoxical effects of partial leptin deficiency on bone in		

1:40pm-1:46pm(p73)

P1-10 Chantida Pawaputanonna Mahasarakham : BMP-2 induction of LGR4 expression

		

1:34pm-1:40pm

regeneration in murine maxilla by subperiosteal injections(p72)

Session 4(p76)

Bone Impairment and Clinical Bases (Takachiho A)

Chairs : Sarah Dallas, Naoto Endo
2:00pm-3:00pm

・Robert Recker : Clinical management of osteoporosis : new options

3:00pm-4:00pm

・Yoichi Ezura : Genetic and molecular bases of osteolytic/osteoporotic disorders (p15)

4:00pm-5:00pm

・Matthew Warman : Rapid and reproducible ex vivo imaging of mouse articularcartilage using confocal microscopy
5:00pm-6:00pm

Evening Seminar Speaker : Jack Martin (Takachiho A)
Anabolic therapy for the skeleton with PTH – now and the future
Sponsored by Eli Lilly Japan K.K.

xviii

6:10pm-Transportation to International Evening Venue. Hinode Pier in Tokyo Bay

7:00pm-9:30pm【International Evening】Symphony Cruise Dinner

9:30pm-10:00pm

Transportation back to Garden Palace Hotel

(p14)

(p16)
(p37)

April 27-29, 2015 ● Tokyo Garden Palace Hotel2015.4.29 Wednesday
8:30am-10:30am

Session 5

Instability and Skeletal Diseases (Takachiho A)

Chairs : Le Duong, Ralph Muller
8:30am-9:30am

・Naoto Endo : AFF: clinical and histological features of atypical subtrochanteric(p17)

and diaphyseal femoral fracture
9:30am-10:30am

・Jang-Soo Chun : Regulation of the catabolic cascade in osteoarthritis by the(p18)

zinc·ZIP8·MTF1 axis
10:30am-11:45am

Oral Presentation 4 Chairs : Watson Maureen, Mika Ikegame (Takachiho A)

10v30am-10:42am

O4-1

Jean-Paul Roux : Texture analysis improvement in cortical evaluation :

(p57)

		

influence of osteophyte in femoral neck in osteoarthritis

10:42am-10:54am

Miyuki Takasu : Accuracy of clinical computed tomography for measurement

O4-2

(p58)

		

of bone quality : a study in cadavers

10:54am-11:06am

Mika Ikegame : Melatonin suppresses the microgravity-induced activation of

O4-3

		

osteoclasts in cultured goldfish scale

11:06am-11:18am

Michael Dray : Histomorphometric analysis of bone to assess safety and

O4-4

		

mechanical performance of low dose fluoride

11:18am-11:30am

Hitoshi Amano : Craniofacial anomalies in cartilage calcification

O4-5

		

insufficient (CCI) rat

11:30am-11:42am

Yasuhiro Sawada : Temporal association of tensin 1 and p130Cas at focal

O4-6

		

12:00pm-1:00pm

(p59)
(p60)
(p61)
(p62)

adhesions links actin inward flux to cell migration

Luncheon Seminar Speakers (Takachiho A)(p33-34)

Masako Ito

Imaging analysis of the effects of denosumab on bone structural properties
Sakae Tanaka

Denosumab for the treatment of osteoporosis
Sponsored by DAIICHI SANKYO COMPANY, LIMITED.
Luncheon Seminar Speaker : Robert Recker (Takachiho B)(p35)

Bone microarchitecture after alendronate treatment of osteoporotic womenSponsored by MSD K.K. a subsidiary of Merck & Co., Inc.
1:00pm-2:00pm

Poster Presentation 2-A Chair : Takashi Teramoto (Tenku A)

1:10pm-1:16pm

P2-1

Mari Sato : Potential role of osteocyte in the fracture healing: comparison of		

fishes with and without osteocytes

1:16pm-1:22pm

Tadayoshi Hayata : Dullard/Ctdnep1 regulates endochondral ossification via

P2-2

		

suppression of TGF-β signaling

(p77)
(p78)
xix

13th Congress of the International Society of Bone Morphometry

1:22pm-1:28pm

P2-3

Huayue Chen : Masticatory activity affects bone remodeling in mice

1:28pm-1:34pm

P2-4

Shuichi Moriya : β2-adrenergic receptor expression in osteoblastic

		

MC3T3-E1 cells is regulated by PTH

1:34pm-1:40pm

Wataru Yano : Quantification analysis of cranial bones development among

P2-5

(p79)
(p80)

		

primates species using geometric morphometrics method(p81)

1:40pm-1:46pm

Wanting Lin : Deletion of Pfn1 has an effect on bone formation

(p82)

P2-6

Poster Presentation 2-B Chair : Omar Omar (Tenku A)

1:10pm-1:16pm

P2-7

Hiroyuki Nakano : A novel measurement method for the morphology of the

(p83)

		

mandibular ramus using homologous modeling

1:16pm-1:22pm

Takayuki Yamada : BMP-induced alkaline phosphatase expression in osteoblast-like

P2-8

		

MC3T3E1 cells is suppressed by β2 adrenergic receptor activation

1:22pm-1:28pm

Akira Minematsu : Voluntary exercise prevents deterioration of trabecular

P2-9

		

1:28pm-1:34pm

bone architecture in diabetes mellitus model rats
linked to FUCCI-indicated cell cycle under the influence of PTH and		mechanical flow signals
microarchitecture in ovariectomized rats

(p87)

P2-12 Jumpei Shirakawa : PTH-induced proliferation in osteoblasts requires		

2:00pm-2:30pm(p86)

P2-11 Yasue Nishii : Influence of magnet implant on tibial trabecular bone		

1:40pm-1:46pm(p85)

P2-10 Jumpei Shirakawa : Morphological and dynamic analysis of migration		

1:34pm-1:40pm(p84)

ubiquitin specific peptidase 2 (Usp2)(p88)

Workshop (Takachiho A)
Thomas Wronski : Proper bone histomorphometry in rats(p25)

2:30pm-3:00pm

Natalie Sims : Bone histomorphometry in mice : Getting it right!

(p26)

3:00pm-6:00pm

Session 6

Molecular Bases for Skeletal Pathology (Takachiho A)

Chairs : Jack Martin, Jang-Soo Chun
3:00pm-4:00pm

・Masaru Ishii : Dynamic live imaging of bone: opening a new era with bone

histodynametry
4:00pm-5:00pm

・Norio Amizuka : Histological aspects on the biological function of osteocytes

5:00pm-6:00pm

・Sarah Dallas : Investigating the dynamic biology of osteocytes and bone

extracellular matrix assembly using live cell imaging approachesxx

6:00pmAward Ceremony & Closing Remark

6:10pmAdjourn

(p19)
(p20)
(p21)

April 27-29, 2015 ● Tokyo Garden Palace Hotel

Keynote Lecture1

13th Congress of the International Society of Bone Morphometry2April 27-29, 2015 ● Tokyo Garden Palace HotelHow concepts of bone remodelling influence therapeutic
development in osteoporosis
T. John Martin
St Vincent’s Institute of Medical Research ;University of Melbourne Department of Medicine
Remodelling of bone takes place throughout life asynchronously in “basic multicellular units” (BMUs) throughout
the skeleton, providing the mechanism for repair of micro-damage, removal of old bone, and adaptation tomechanical stress. Essential to remodelling is that at each BMU, equal volumes of bone are removed by osteoclasts
and replaced by osteoblasts. Within any individual BMU the volume of bone replaced by formation is approximately
equal to that removed by resorption, a mechanism referred to as “coupling.
All approaches to prevention and treatment of osteoporosis are underpinned by the cellular and molecular control of
the above events. Antiresorptives (bisphosphonates, anti-RANKL, estrogen, SERMs) reduce the rate of remodelling,either by inhibiting osteoclast formation or activity, hastening their death, or combinations of these.
With all of these drugs, inhibition of resorption is inevitably accompanied by inhibited bone formation within
the BMUs, because of the coupling of the two processes. With increased understanding of coupling, attempts arebeing made to develop drugs that inhibit the volume of bone resorbed by each BMU and increasing the volume
of bone formed by each BMU – in other words uncoupling bone formation from resorption. The first of these,using a cathepsin K inhibitor, arose from the finding that genetic blockade of cathepsin K resulted in decreasedbone resorption without decline in formation. Preclinical studies indicated that cathepsin K null osteoclasts that
had diminished resorptive capacity, were able to produce activity that promoted bone formation – in that casesphingosine-1-phosphate, one of a number of candidate coupling factors that are the subject of much current interest,
and these will be discussed in depth.
Anabolic treatment with PTH increases the number of active BMUs upon intracortical, endocortical and trabecularsurfaces and deposits new osteoid. PTH promotes differentiation of committed osteoblast precursors, activation of
osteoclasts that produce coupling activities, promotes survival of osteoblasts and osteocytes, and inhibits sclerostin
production by osteocytes. The possible involvement of local coupling activities in the anabolic action of PTH is ofmuch interest. PTH treatment results in an increased amount of bone which is at first inadequately mineralized, so its
enhancement of BMD is on an entirely different basis from the increase with anti-resorptive treatment.
Antibody against sclerostin has proven effective in preclinical and current clinical studies, showing clear evidence
of increased bone formation. Anti-sclerostin treatment was associated with a transitory increase in formation marker
P1NP, that decreased to control levels, whereas the resorption marker, CTX, declined rapidly and remained so.
The reason for the decrease in bone formation marker after an initial rise is unknown. The decreased resorption
marker might be explained by a change in the osteoblast lineage population as a result of profoundly increasedbone formation, with lesser availability of cells that stimulate RANKL production and therefore osteoclasts. These
aspects of anti-sclerostin action draw attention to the fact that the mechanisms involved in sclerostin blockade are
new to us. In rodents and non-human primates the tissue level mechanism by which anti-sclerostin increases bone ispredominantly through quiescent surfaces – thus a modelling effect.
The intercellular communication mechanisms provide the challenges that bone presents to drug development. There
are specific ways in which current and prospective anti – resorptive drugs differ from one another in their actions.
There is a special need for effective, safe stimulators of bone formation, to provide for those who have undergone
substantial bone loss. Exciting possibilities of new anabolic therapies are evident, the result of new insights emerging
through mouse and human genetics

313th Congress of the International Society of Bone Morphometry

4April 27-29, 2015 ● Tokyo Garden Palace Hotel

Main Sessions

5

13th Congress of the International Society of Bone Morphometry

6April 27-29, 2015 ● Tokyo Garden Palace Hotel

New imaging techniques in the diagnosis of bone diseases
Thomas M. Link, MD, PhD
Department of Radiology and Biomedical Imaging,
UCSF, San Francisco, CA, USAStandard imaging techniques to quantitatively assess metabolic bone diseases include Dual X-ray Absorptiometry–DXA- and Quantitative Computed Tomography –QCT-, but it is well-known that these technologies to measurebone mineral density –BMD- have limitations in predicting fracture risk. To improve prediction of fracture risk the
FRAX score was developed, allowing improved assessment of hip and major osteoporotic fracture risk by including
BMD and clinical risk factors.
BMD, however, only provides information on bone mass but bone strength is also governed by the architectureof the trabecular and cortical bone. This prompted the Bone Quality Initiative by the National Institutes of Healthapproximately a decade ago and a number of new technologies have been investigated and used in clinical studies
since then. Among the most promising candidates for measuring bone quality are high resolution peripheral QCT-HR-pQCT-, high resolution Magnetic Resonance Imaging –MRI-, MR Spectroscopy and QCT with finite element
analysis:
1. HR-pQCT: HR-pQCT was developed for imaging of trabecular and cortical bone architecture of the distal radius
and tibia. The HR-pQCT system is produced by a single manufacturer (XtremeCT, Scanco Medical AG, Brüttisellen,
Switzerland) and has a substantially higher spatial resolution compared to standard cross-sectional imaging
techniques. The reconstructed voxel size is 82 μm for the standard patient HR-pQCT protocol and newer generation
HR-pQCT systems have a voxel size down to 41 μm. The effective radiation dose is low with <3 microSv. The
system allows acquisition of BMD, trabecular and cortical bone architecture as well as finite element assessment.
Previous clinical studies have shown promising results in differentiating postmenopausal females and older men withand without fragility fractures and in monitoring therapeutic interventions.
2. High Resolution MRI: High resolution MRI can be used for the distal radius, tibia, knee and proximal femur.
With higher field strength (3 and 7 Tesla) excellent visualization of the proximal femur trabecular bone architecture
has been achieved allowing finite element assessment. Previous studies have shown the technique’s potential indifferentiating patients with and without osteoporotic fractures also in those patients that do not differ by BMD.
3. MR Spectroscopy: Proton magnetic resonance spectroscopy allows to measure bone marrow fat non-invasively
and previous studies have shown that bone marrow fat increases with decreasing BMD and is significantly elevatedin postmenopausal females and older men. This technology may in particular allow to assess and monitor metabolic
diseases such as Type 2 diabetes that have an increased risk of fragility fractures.
4. QCT with finite element analysis: Standard CT can be used to derive bone strength data through finite element
modeling and this has been shown to provide improved monitoring of treatment effects compared to DXA andgreater sensitivity in fracture risk assessment at the proximal femur and lumbar spine.
Also some additional technologies have been established and optimized which include Quantitative Ultrasound ofBone, Positron Emission Tomography and Texture analysis of DXA spine images (Trabecular Bone Score).
In summary in the last decade a number of new imaging technologies to measure trabecular bone structure andquality have been established that, though currently used mostly for research purposes, have shown to provide
additional information beyond BMD in characterizing fracture risk and monitoring therapy.

7

13th Congress of the International Society of Bone Morphometry

Targeting cathepsin k for the treatment of osteoporosis
Le T. Duong
Bone Biology, Merck Research Laboratories,
West Point, Pennsylvania, PA, USA
Bone resorption requires cellular secretion of acid for dissolution of mineral as well as enzymes for degradation ofcollagen. The lysosomal cysteine protease cathepsin K (CatK), one of the most active collagenases, is responsiblefor degradation of bone matrix proteins. CatK is predominantly but not exclusively expressed by osteoclasts. Lossof-function mutations in the CatK gene lead to pycnodysostosis, a rare autosomal recessive disorder in humans
associated with osteosclerosis with increased risk of non-traumatic fractures, short stature, acro-osteolysis and skull
deformities. CatK gene deletion in mice results in high bone mass phenotype due to defective osteoclastic resorption
activity. However, the number of osteoclasts, osteoblasts and the bone formation rate (BFR) are all elevated in CatK
knock-out mice. Several small molecular weight orally available inhibitors of CatK with varied selectivity profiles
toward other human cathepsins have advanced into clinical development. Odanacatib (ODN) is a selective andreversible CatK inhibitor currently in Phase III for the treatment of postmenopausal osteoporosis. This inhibitor has
been shown to reduce bone resorption efficiency without impairing osteoclast differentiation or survival in vitro.
Daily administration of ODN clearly increases spine and hip bone mineral density (BMD) in ovariectomized (OVX)
skeletally mature rabbits and monkeys. In rabbits, ODN reduces bone resorption while allowing bone remodeling as
determined by BFR to continue in trabecular and endocortical bones. In monkeys, ODN reduces remodeling-based
bone formation, while dose-dependently enhancing modeling-based bone formation in endocortical and periosteal
femoral surfaces, leading to significantly increased cortical thickness and improved bone strength. In a phase II
clinical trial extended over 5-years, postmenopausal women with osteoporosis receiving ODN 50mg once-weekly
present a sustained reduction of bone resorption markers (NTx), whereas the bone formation markers (P1NP) aremodestly reduced and demonstrate an early return toward baseline. Consequently, areal BMD of spine and total hip
increased continuously up to 5 years. Consistent with the mechanism of CatK inhibition observed from preclinicalstudies, trabecular mineralizing surface and BFR in transilial biopsies from patients on ODN are not different
from those on placebo. After treatment for 2-years, hip QCT and radius/tibia HR-pQCT further show a gain oftrabecular and cortical vBMD, and modest but significant improvement of cortical thickness at these sites. On theother hand, following discontinuation of ODN after 2-years of therapy, bone mass in these subjects reverses back tobaseline within a year. The ODN fracture prevention clinical trial included ~16,000 PMOP for 3 years, with ~8000
of them continuing on in a 5-year extension with maintained randomization. ODN 50mg demonstrated significant
reduction of vertebral, non-vertebral and hip fractures. In summary, the CatK inhibitor ODN has been demonstrated
to be an effective therapy for the treatment of osteoporosis via a molecular mechanism distinct from the standard
antiresorptives.

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April 27-29, 2015 ● Tokyo Garden Palace Hotel

Cell communication in osteoimmunology
Hiroshi Takayanagi
Department of Immunology, Graduate School of Medicine and Faculty of Medicine
The University of Tokyo, Japan,
Bone cells and hematopoietic cells share the same microenvironments in the bone marrow, communicating throughvarious factors. Osteoblasts, osteoclasts and osteocytes are not only degrading or forming bone but may have distinct
roles in the immune regulation. However, the molecules involved in the cell communication in the bone marrow arepoorly identified. IGF-1 and TGF-b are among the classical coupling factors important in the transition phase. OPG,RANKL and sclerostin are important modulators of bone remodeling. Osteocytes are the critical source of thesefactors, suggesting that osteocytes regulate bone remodeling in response to mechanical stress utilizing these factors.
Recent studies suggest that semaphorins play an essential role in the regulation of bone remodeling but they functionin the distinct bone remodeling phases. I will discuss the recent progress in the field of osteoimmunology focusing
on the molecules controlling the cell communication in the bone marrow as well as T cell interaction with osteoclasts
in autoimmune arthritis.

913th Congress of the International Society of Bone Morphometry

Effects of sclerostin antibody on different bone surfaces withshorter- and longer-term treatment
Hua Zhu (David) Ke
Bone Research, New Medicines Therapeutics,
UCB Pharma, Slough, England.
Wnt/beta-catenin signaling plays an important role in bone formation and resorption. Sclerostin (Scl) binds to coreceptors Lrp5/6 and inhibits wnt/beta-catenin signaling in osteoblast lineage, results in decreased bone formation.
Inactivation of sclerostin leads to substantially increased bone mass in humans and in genetically manipulated
animals. A series of shorter-term preclinical studies demonstrated that monoclonal antibody targeting sclerostin (SclAb) increases bone formation on all bone surfaces, decreases bone resorption on trabecular and endocortical surface,
and increases BMD and bone strength in multiple skeletal sites in animal models of osteoporosis. In shorter-term (4-6weeks) studies, it has been showed that increased bone formation induced by Scl-Ab was primary stemmed from
activating bone formation on the quiescent bone surface (modeling-based bone formation) and prolong the formationof new bone on pre-existing bone remodeling surface in animal models. Scl-Ab treatment in OVX rats decreased
osteoclastogenesis in bone marrow culture in vitro, and decreased osteoclast number and surface in vivo, suggestingthat Scl-Ab treatment decreases bone resorption. In a longer-term (26 weeks) OVX rat study, despite continuously
increased BMD and bone strength, trabecular and periosteal bone formation temporally increased and gradually
returned to control level, while endocortical bone formation remained significantly elevated at end of the study.
However, in this longer-term study, strong correlation between bone mass and bone strength was remained at the endof the study, indicating normal quality of bone was induced by longer-term Scl-Ab treatment, as observed from those
shorter-term studies. Similarly, a humanized Scl-Ab, romosozumab, has been shown to simultaneously increasebone formation markers and decrease bone resorption markers, leading to a significant increase in BMD giving bymonthly s.c. injection for 12 months. In this clinical study, serum bone formation markers were temporally increased
and returned to baseline within 6 to 12 months while serum bone resorption markers remained decreased throughout
the 12 months duration. The mechanism for time-dependent changes in bone formation and bone resorption ondifferent bone surfaces with longer-term Scl-Ab treatment is not completely understood. Local bone mechanicalsignal and other feedback pathways may be involved. These data may indicate that the early bone gains with SclAb treatment were accompanied by increased cortical and trabecular bone formation and reduced osteoclast activity,
whereas later gains were attributed to residual endocortical and trabecular osteoblast stimulation and persistently low
osteoclast activity.

10April 27-29, 2015 ● Tokyo Garden Palace Hotel

Cartilage and meniscus regeneration by synovial stem cells :from bench to clinic
Ichiro Sekiya
Center for Stem Cell and Regenerative Medicine
Tokyo Medical and Dental University
Tokyo, Japan
According to our previous studies, the number of mesenchymal stem cells (MSCs) in synovial fluid increase inknees with anterior cruciate ligament injury (Rheumatology 2008), meniscus injury (Clin Orthop Relat Res 2014),
and osteoarthritis (J Orthop Res 2011). The morphology and gene profiles in synovial fluid MSCs were more similar
to those in synovial MSCs than in bone marrow MSCs. Principal component analysis of gene profiles for various
mesenchymal tissues-derived MSCs and chondrocytes demonstrated that MSCs from intraarticular tissues andchondrocytes were closer to each other than MSCs from extraarticular tissues (J Orthop Res 2008). Synovium may
be a reservoir for MSCs to contribute to the intraarticular tissue repair. After intraarticular tissues, such as cartilage,
meniscus, and ligament are injured, MSCs may be mobilized from synovium to synovial fluid, adhere to the injured
site, and contribute to the repair. However, the number of MSCs is limited, therefore, the injured tissues cannot be
healed in the natural course. Transplantation of enough number of synovial MSCs to the injured tissues will promote
intraarticular tissue healing.
MSCs are attractive cell source for cartilage and meniscus regeneration. Our in vitro and in vivo chondrogenic assay
demonstrated that synovial and bone marrow MSCs had a higher chondrogenic ability than adipose and muscle
MSCs (Arthritis Rheum 2005, Cell Tissue Res 2007, Cell Tissue Res 2008). Human synovial MSCs expanded morein human serum than bone marrow MSCs (Arthritis Rheum 2008). In rat, rabbit, and pig studies, transplantationof synovial MSCs promoted cartilage and meniscus regeneration (Stem Cells 2007, Stem Cells 2009, Cytotherapy
2012, Osteoarthritis Cartilage 2012, J Bone Joint Surg Am 2012, Biochem Biophys Res Commun 2013). Also,transplantation of synovial MSCs promoted healing after meniscal repair with induction of synovium into thelongitudinal tear in the avascular zone of meniscus in pigs (Osteoarthritis Cartilage 2015).
Current cell therapy for cartilage and meniscus regeneration requires invasive procedures. We have developed
a novel implantation procedure with synovial MSCs. Cartilage or meniscus defect is filled with synovial MSC
suspension for 10 minutes. According to our in vitro and in vivo studies, more than 60% cells adhered to the defect,
and promoted cartilage and meniscus regeneration (Arthritis Res Ther 2008, J Orthop Res 2013).
Transplantation of MSCs is one possible strategy to achieve articular cartilage repair. We investigated whether
transplantation of synovial MSCs regenerated cartilage defect. Patients with a symptomatic single cartilage lesion of
the femoral condyle were indicated and 10 patients were enrolled in this study. Our method has such advantages thatno periosteal coverage or scaffold were required and that transplantation is possible arthroscopically. For this small,initial case series, transplantation of synovial MSCs improved clinical outcomes (Clin Orthop Relat Res 2015). We
are also trying to regenerate osteoarthritis of the knee with osteotomy or meniscus centralization (Arthrosc Tech2012) by using synovial MSCs. We have started a clinical trial using transplantation of synovial MSCs to promotehealing after meniscal suture repair.

1113th Congress of the International Society of Bone Morphometry

Assessment of bone structure in relation to biomechanicalproperties
Masako ItoCenter for Gender Equality,
Nagasaki University
Nagasaki, Japan
With the development of noninvasive imaging techniques such as computed tomography (CT), micro-CT, and highresolution peripheral quantitative CT (HR-pQCT), imaging of the bone architecture provides important information
about age- and disease-related changes in bone microstructure and estimates of bone strength.
The pathogenesis of osteoporosis has been focused on dynamic trabecular bone loss due to menopause, which isstrongly related to vertebral fractures, on the other hand relatively less attention given to non-vertebral fracturesand cortical bone loss. However, since 80% of fractures are non-vertebral and occur at predominantly cortical sites.
Recently, the researches have investigated the deterioration of microstructure of the cortical bone in relation to therisk of non-vertebral fracture, furthermore in relation to the diseases associated with adult lifestyle habits (type 2
diabetes mellitus, and chronic kidney disease), and to the effect of osteoporotic agents. In Japan, it is difficult tofulfill a clinical trial large enough to obtain high-level evidence to reduce the risk of hip fracture. So that, useful
methods are required to evaluate the effect of agents on bone biomechanical properties, especially the effect ofpreventing femoral fracture.
In this lecture, I would like to review the articles of the treatment effects on the microarchitecture of the trabecularand cortical bone, and bone geometry.

12April 27-29, 2015 ● Tokyo Garden Palace Hotel

Hierarchical imaging of bone failure
Ralph Müller
Institute for Biomechanics,
ETH Zurich, Zurich, Switzerland
Aging is on the verge of a new era. Humans are approaching old age in unprecedented numbers as a result of large
baby boom cohorts born in the middle of the 20th century that are approaching traditional retirement ages. Increases
in the prevalence of age-related disease, frailty, and disability are visible signs of the potential costs and social
burdens arising from this historic demographic shift. One disease that will be affected dramatically by this shift will
be osteoporosis and with it there will be marked increase in osteoporotic bone fractures. With recent advances inmolecular medicine and disease treatment in osteoporosis, the development of diagnostic and monitoring tools must
therefore be in the focus of aging and health-related research in order to allow early detection and control of thedisease.
A number of new microstructural imaging modalities have been put forward over the last two decades allowing
quantification with high precision and accuracy. Biomedical imaging technology such as MRI or CT is readily
available, but few attempts have been made to expand the capabilities of these systems by integrating quantitative
analysis tools as an integrative part of biomedical information technology and by exploring structure–function
relationships in a hierarchical fashion over the different length scales. Nevertheless, such quantitative endpoints
have become an important factor for success in basic research and the development of novel therapeutic strategies inbiomedicine and clinical practice. Where the 20th century was the century of image formation, success in the 21th
century will be judged by our ability to extract meaningful quantitative information from these images. Micro- andnano-computed tomography is key to these developments being an approach to image and quantify trabecular bonein three dimensions and providing multi-scale biological imaging capabilities with isotropic resolutions ranging froma few millimeter (clinical CT), to a few micrometers (microCT) down to one hundred nanometers (nanoCT).
As part of the presentation, new strategies for advanced hierarchical quantification of bone and their structure
function relationship will be presented. The focus will be on hierarchical micro- and nano-imaging as well as imageguided biomechanics. At the microscopic level, bone microstructure is known to influence bone strength and failure
mechanisms significantly. Biomechanical imaging allows direct time-lapsed visualization and computation of local
displacements and strains for better quantification of bone fracture initiation and progression. Recent introduction ofhighly brilliant synchrotron radiation X-ray sources now permits to explore bone on the nanoscopic level to uncover
the ultrastructure of bone including vascular and cellular structures and to investigate their role in the developmentof bone failure.
In conclusion, hierarchical microimaging is well suited to investigate quantitative structure function relationships
and failure in both trabecular and cortical bone. The approach can help improve predictions of bone failure, clarify
the pathophysiology of skeletal diseases, and define the response to therapy. We expect these findings to improve ourunderstanding of bone failure and with that to also allow improved quality control and more successful outcomes instudies dealing with pharmacological treatment of bone.

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13th Congress of the International Society of Bone Morphometry

Clinical management of osteoporosis : new options
Robert R. Recker, M.D., M.A.C.P., F.A.C.E.
Head of Creighton University’s Osteoporosis Research Center
Omaha, NE, USA
Osteoporosis became recognized in the early 20th century as skeletal weakness due to loss of bone mass. In 1947
Fuller Albright recognized that bone removal and replacement (bone remodeling) occurred throughout life, andthat loss of estrogen in women at menopause resulted in increased bone removal without a similar increase in boneformation. This resulted in a net loss of bone tissue at menopause which continued as time passed, and resulted inrisk of major fractures. Thus, for most of the past 60 years osteoporosis was considered a disease of low bone mass
and weakened skeleton. This idea was strengthened by the invention of dual photon-absorptiometry (DPA) in the late
70s followed by dual x-ray absorptiometry (DXA) in the 80s. With these inventions we were able to measure bonemass, and express the results as a two-dimensional density. Soon thereafter epidemiologic data became available
which showed that fracture risk could be indexed to the T-score (DXA), which was the Standard Deviation of bonedensity in a healthy population in their 20s. We then began to focus on ways to increase bone mass, and/or to stopbone loss. The first successful intervention to stop bone loss was hormone (estrogen) replacement therapy whichbecame widely used in the 1940s and 1950s in the United States and Europe, even in the absence of solid antifracture efficacy data.
In ~1995 the first bisphosphonate that slowed bone remodeling, slowed bone loss and reduced the risk of fracture
became available. This was Alendronate the first effective anti-fracture treatment for osteoporosis. Subsequently,
three other bisphosphonates were approved for use in the United States; Actonel, Ibandronate, and Zoledronic acid.
At nearly the same time a selective estrogen receptor modulator, raloxifene, became available. All of these treatments
reduce the risk of fracture by about 50%. They have been, and still are, used extensively throughout the world.
However the ideal medical “cure” of osteoporosis would need to reduce the risk of fracture to perhaps 10 or 15%. It
is unlikely that the risk of fracture can be completely eliminated; however, recent discoveries hold some promise that
we might be able to reduce the risk of fracture in osteoporosis patients by 75 to 95%. This arises from the discoveryof an autosomal dominant mutation in LRP5, (Lipoprotein-Related Receptor Protein 5). LRP5 activates the wnt
signaling pathway that when linked with its co-receptor, Secreted Frizzled Protein (SFP), signals bone forming cells
to form bone. The affected individuals in the family in which this mutation was discovered had T-scores of +5 or
higher. The 19 affected individuals in that family had never had a fracture in lifespans ranging from childhood to age
86. The wnt pathway has at least two inhibitors that bind LRP5, sclerostin and DKK-1. Very recently an antibody tosclerostin has been formulated, and is being tested by two pharmaceutical companies. Preliminary results in humans
show very remarkable gains in bone density of more than 15%, i.e., a more than one standard deviation increase in
the T score. Soon we may be able to offer our osteoporosis patients something resembling a “cure”.

14April 27-29, 2015 ● Tokyo Garden Palace Hotel

Genetic and molecular bases of osteolytic/osteoporotic disorders
Yoichi Ezura, Masaki Noda
Department of Molecular Pharmacology
Medical Research InstituteTokyo Medical and Dental University
Tokyo, Japan
Osteolysis or regional bone loss is a serious complication in patients in orthopedics, dentistry, or rheumatology as thelesions around the implants, peri-articular erosion in chronic arthritis, and osteomyelitis or infectious bone diseases.
Inflammatory responses for pathogens, materials, and autoantigens may be the triggers, whereas the metastatic or
primary bone tumors could be the cause. Shared pathogenic mechanisms could be involved in osteoporosis (OP) insome extent, because OP also represents rapid bone loss in some instances. Indeed, genetic association studies for OP
have partially implicated such possibilities. Here, I would like to discuss about such possibilities from three aspectsof the molecular and genetic understandings of the inherited disorders associated with osteolysis.
First, a number of inherited human conditions are known to be caused by a defect in osteoclasts. In general,mutations leading to ineffective osteoclasts cause osteopetrosis, whereas mutations leading to osteoclast activationor production may cause osteolysis. For example, dominant negative mutations in osteoclast inhibiting factor, v-maf
musculoaponeurotic fibrosarcoma oncogene ortholog B gene (MAFB) causes multicentric carpo-tarsal osteolysis(MCTO). Constitutive active mutations of the receptor activator of the nuclear kappa B gene (RANK) cause familial
expansile osteolysis (FEO). Therefore, the repeatedly detected strong association of genetic variations in RANK
locus, as well as RANKL and OPG loci by genome-wide association studies (GWAS) for low bone mineral density(BMD) in general population implies that acceleration of osteoclastogenesis may be involved in OP pathogenesis inpart. The association of RANK polymorphisms in the 3’-untranslated region, for which we have shown a bindingof the deadenylation complex forming Cnot3 by mouse study, may indicate the involvement of regulation in mRNAstability.
Secondly, the osteolysis could also be the result of accelerated inflammation. Cherubism is an autosomal dominant
hereditary disorder characteristically presented by osteolytic lesions in facial bones of children accompanied byballooning appearance. The disease is known to be caused by constitutive active mutations of the SH3-domainbinding protein 2 gene (SH3BP2). The mutations results in enhanced TNF-a receptor and TLRs signaling by
getting lid of the regulation through poly(ADP-ribose) polymerizing enzyme tankyrase, but the pathogenesis andthe mechanisms explaining an interesting feature of this disease, the remission of the osteolysis after puberty is not
fully understood yet. The accelerated inflammation is also the case in familial cold auto-inflammatory syndrome(FCAS1), caused by mutations in a gene encoding inflammasome protein Cryopyrin (also named as NLRP3).
Osteolytic lesions in the leg are prevalent in the FCAS patients. We assume the accelerated inflammation could be
involved in susceptibility for many other disorders including OP, as well as osteolysis causing rheumatoid arthritis
and osteomyelitis.
Lastly, several osteolytic disorders are paradoxical or mysterious on its pathogenesis. Pycnodysostosis is causedby homozygous mutations in cathepsin K, and its major bone phenotype is systemic osteosclerosis. However, thepatients sometimes represent paradoxical osteolysis in fingertips (acro-osteolysis). The acro-osteolysis is a symptom
observed in several diseases. Sometimes it is explained by peripheral circulation and/or neurotic disorders, butbasically its pathogenesis is not understood well. Paradoxical osteolysis is also the case in multicentric osteolysis
nodulosis and arthropathy (MONA) that is caused by loss of function mutations in MMP2 gene, encoding abone collagen degrading enzyme. The Nasu-Hakola disease (NHD, also known as polycystic lipomembranous
osteodysplasia with sclerosing leukoencephalopathy; PLOSL) is also mysterious because the causative homozygous
mutations are found in genes encoding transmembrane receptor molecules TREM2 and DAP12 that are known to be
essential for osteoclastogenesis. Clarification of the mechanisms for such conditions is needed.

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13th Congress of the International Society of Bone Morphometry

Rapid and reproducible ex vivo imaging of mouse articularcartilage using confocal microscopy.
Matthew L. Warman, M.D.
Investigator, Howard Hughes Medical Institute
Professor of Genetics and Orthopaedic Surgery
Boston Children’s Hospital and Harvard Medical School Boston, MA, USA
Mice are commonly used to study biologic pathways involved in articular cartilage development, homoeostasis, anddegeneration. However, the small size of articular cartilage surfaces in mice makes it challenging to evaluate theeffects of genetic or environmental perturbations on cartilage structure and function. Current methods for evaluatingmouse cartilage are time-consuming, labor intensive, and dependent upon the laboratory’s expertise in obtaining
appropriate images. I will describe a 3-D confocal microscope imaging technique Dr. Minjie Zhang has beendeveloping in my laboratory that is easily applied to different mouse articular cartilage surfaces. This technique
can rapidly and reliably measure chondrocyte number and distribution. It can also identify chondrocytes thatincorporated EdU in vivo, an indicator of cell division, and chondrocytes that express cell-type-specific fluorescentproteins. I will show the utility of this imaging method in experiments we designed to determine if chondrocyte
death causes cartilage surface damage and, conversely, if cartilage surface damage causes chondrocyte death.
Confocal imaging offers advantages over other methods of evaluating mouse cartilage, in terms of the speed of dataacquisition, the sample sizes needed to obtain statistically meaningful results, and the ability to provide new insights
about articular cartilage biology.

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April 27-29, 2015 ● Tokyo Garden Palace Hotel

AFF: clinical and histological features of atypical subtrochantericand diaphyseal femoral fracture
Naoto Endo
Division of Orthopedic Surgery, Niigata University Graduate School of Medical and Dental Sciences,
Niigata, Japan
AFF, atypical subtrochanteric and diaphyseal femoral fracture have been reported in relation to SSBT (severely
suppressed bone turnover) due to bisphosphonate treatment. Recently several cases with AFFs in denosmab treatment
also were reported. This fracture site is atypical, such as subtrochanteric or diaphyseal area of femoral shaft, whereas
osteoporosis-related hip fractures are located in the neck or trochanteric region of femur in aged population. Bothfractures most often occur with minimal trauma. Since AFFs occur at very low rate, data on these patients with AFFs
are limited. The mechanism and risk factors associated with AFFs are still unknown.
1. According to ASBMR Task Force 2014 (J Bone Miner Res 2014:29:1-23), these patients with AFF experienced
thigh pain before fracture and had cortical thickening of femur. This is the unusual radiographic features,
suggesting periosteal and/or endosteal stress reaction. This is the critical point to distinguish AFFs fromosteoporotic fractures.
2. The data on the patients with AFFs in Japan reported from Japanese Orthopedic Association. More than 300 cases
were reported in each year in 2010 − 2013.
3. Clinical and histological features in patients with AFFs reported by Niigata University showed that
histomorphometric analysis indicated low ~ normal bone-turnover in patients with AFFs.
4. Treatment: Since AFFs are difficult to unite in most cases, surgical treatment for fracture with plate or nailand enhancement for fracture healing with bone grafting, bone-stimulating agents (teriparatide), and so on arerecommended.

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13th Congress of the International Society of Bone Morphometry

Regulation of the catabolic cascade in osteoarthritis
by the zinc·ZIP8·MTF1 axis
Jang-Soo Chun
Cell Dynamics Research Center and School of Life Sciences,
Gwangju Institute of Science and Technology, Gwangju, Korea.
Osteoarthritis (OA), primarily characterized by cartilage degeneration, is caused by an imbalance between anabolicand catabolic factors. Here, we investigated the role of zinc (Zn2+) homeostasis, Zn2+ transporters, and Zn2+-dependenttranscription factors in OA pathogenesis. Among Zn2+ transporters, the Zn2+ importer ZIP8 was specificallyupregulated in OA cartilage of humans and mice, resulting in increased levels of intracellular Zn2+ in chondrocytes.
ZIP8-mediated Zn2+ influx upregulated the expression of matrix-degrading enzymes (MMP3, MMP9, MMP12,
MMP13, and ADAMTS5) in chondrocytes. Ectopic expression of ZIP8 in mouse cartilage tissue caused OA cartilage
destruction, whereas Zip8 knockout suppressed surgically induced OA pathogenesis, with concomitant modulationof Zn2+ influx and matrix-degrading enzymes. Furthermore, MTF1 was identified as an essential transcription factor
in mediating Zn2+/ZIP8-induced catabolic factor expression, and genetic modulation of Mtf1 in mice altered OA
pathogenesis. We propose that the zinc·ZIP8·MTF1 axis is an essential catabolic regulator of OA pathogenesis.

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April 27-29, 2015 ● Tokyo Garden Palace Hotel

Novel modes of action of anti-bone resorptive drugs revealed byintravital two-photon bone imaging technology
Masaru Ishii
Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences,
Osaka University, and JST, CREST
Osaka, Japan
Intravital two-photon microscopy adopted for visualizing live bone tissues has undoubtedly revolutionized theresearch field of bone biology. Now that we can grasp the real mode of migration, interaction and function ofdifferent bone cell types in vivo, we could also dissect actual pharmacological actions of several drugs treatingbone diseases. For example, we have recently identified that active vitamin D and its therapeutic analogues, whose
therapeutic mechanisms had still been elusive, exerted their anti-bone resorptive activity by altering migratory
behaviors of circulating monocytoid cells, limiting the number of osteoclast precursors on the bone surfaces.
Bisphosphonates and RANKL blocker were shown to have distinct properties for inhibiting osteoclastic bone
destructions. These findings which could only be derived from intravital bone imaging technology enable us todeepen our understanding on current therapeutic regimens as well as to develop conceptually novel lines of treatment
against bone diseases in future.

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13th Congress of the International Society of Bone Morphometry

Histological aspects on the biological function of osteocytes
Norio Amizuka, Tomoka Hasegawa, Hiromi Hongo, Tomomaya Yamamoto
Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine,
Hokkaido University
Sapporo, Japan
Osteocytes build up functional syncytia, i.e., the osteocytic lacunar-canalicular system (OLCS). The osteocytes areconnected each other by means of their cytoplasmic processes interconnected through gap junctions, which passthrough narrow passageways referred to as osteocytic canaliculi. There are two possible pathways for moleculartransport, one formed by the cytoplasmic processes connected by gap junctions, and the other being the pericellularspace in the osteocytic canaliculi. The transport of minerals and small molecules carried through the OLCS serves
as a fundament for osteocytes intimately involved in maintaining bone minerals, mechanosensing and regulatingbone remodeling. In order to achieve these putative functions, a geometrically well-organized OLCS in maturebone seems to be efficiently functional. Recently, osteocyte-derived molecules, sclerostin, dentin matrix protein1(DMP-1), fibroblast growth factor 23 (FGF23), have been highlighted because these molecules may reflect
osteocytic function responding to mechanosensing, regulation of bone remodeling and so forth. In our observations,
FGF23 and sclerostin synthesis seemed to be associated with the spatial regularity of the OLCS, while DMP-1 was
not related to the geometrical arrangement of OLCS.
More recently, the idea of “osteocytic osteolysis” has revisited, which is respondent to elevated concentrationof parathyroid hormone (PTH) or low calcium diet, was proposed by Bélanger in 1960’s. In order to clarify theoccurrence of osteocytic osteolysis, we have examined osteocytes and their lacunae after the PTH administrationin the tibial metaphysis and cortical bone of mice. At six hours after the PTH administration, enlarged osteocytic
lacunae were observed mainly in the cortical bone, but not in the metaphysis. Transmission electron microscopy andvon Kossa staining demonstrated broadly demineralized bone matrix surrounding osteocytes in the cortical bone.
Intense immunoreactivity of vacuolar type H-ATPase was found in many osteocytes in the PTH-treated corticalbone, indicating that proton secreted by osteocytes might erode the surrounding bone minerals. Unlikely, the control
bone without PTH administration did not reveal such histological alteration of osteocytic lacunae. In order to exclude
the possible involvement of osteoclastic bone resorption after the PTH administration, we have examined OLCS
in Rankl-/- mice which lack osteoclasts. As a consequent, the osteocytic lacunae were enlarged as was seen in the
PTH-administered wild-type mice. Thus, it seems likely that osteocytes in cortical bone, rather in metaphysis, would
participate, at least, in part, in controlling serum concentration of calcium and phosphate by responding to PTH, i.e.,
osteocytic osteolysis in bone.
This review will introduce our recent studies on the biological function of osteocytes.

20April 27-29, 2015 ● Tokyo Garden Palace Hotel

Investigating the dynamic biology of osteocytes and bone
extracellular matrix assembly by live cell imaging approaches
Sarah L. Dallas
University of Missouri, Kanasas City, MO, USA

Much of our understanding of skeletal biology comes from static imaging approaches, such as light and electron
microscopy, combined with biochemical analysis and/or molecular genetic approaches. However, the biologicalprocesses occurring in the skeleton, such as bone development, remodeling and repair are highly dynamic events that
include the dimension of time. To gain new insight into bone cell function, we have used live cell imaging techniquesthat enable visualization of temporal changes in living specimens, such as cells, tissues and intact animals and allow
the quantitation of cellular, subcellular and tissue behavior as a function of time.
To gain insight into the process of osteocyte differentiation and embedding, we have generated transgenic miceexpressing a membrane targeted GFP under control of the dentin matrix protein-1 promoter (Dmp1-memGFP mice).
Live imaging in neonatal calvarial organ cultures and primary mineralizing osteoblast cultures from these mice
as well as in live mice has revealed that osteocytes are highly dynamic cells. The Dmp1-memGFP transgene isswitched on in a surface motile cell type prior to embedding that appears to be a non-dividing cell already committed
to becoming an osteocyte. As this cell embeds and transitions to an osteocyte, it extends and retracts “exploratory
dendrites”, which explore the entire area around the cell and appear to play a role in positioning/spacing of theosteocyte. Our studies have shown that the embedding and recently embedded osteocyte is highly dynamic and thatits dendrites extend and contract, making transient dendritic connections between adjacent osteocytes. Osteocytes
also showed deformations/undulating motions of their cell bodies within their lacunae and extensive membrane
ruffling. A surprising finding was that osteocytes shed extracellular vesicles from their cell body and dendrites during
embedding. These microvesicles contain a cargo of protein, mRNA and miRNA and may provide a mechanism forintercellular communication through delivery of this cargo to target cells to alter their function.
To investigate the dynamic processes of bone extracellular matrix assembly we have generated transgenic miceexpressing a GFP-collagen fusion protein in which GFP replaces the N-terminal propeptide of α2(I)-collagen.
Live imaging in primary osteoblasts from these mice showed that collagen assembly is a highly dynamic process
involving extensive cell motions which stretched and contracted the fiber network during assembly. Upon additionof ascorbate, GFP-collagen translocated to vesicle-like structures which showed extensive intracellular movement tolocalized regions within the cell and appeared to be involved in the assembly process. During assembly, cells wereable to physically reshape the collagen fiber network by pushing collagen outwards to generate holes resembling
lacunae. Live imaging of cells from double transgenic mice expressing GFP-collagen together with a membrane
TdTomato reporter enabled visualization of collagen assembly and cell membrane dynamics. Imaging of these cells
seeded at low densities on wild type osteoblasts showed GFP-collagen deposition only in areas in which the cellbody or cell processes had been, suggesting active placement of collagen by cells rather than diffusion of collagenaway from the producer cell. Live imaging of collagen assembly in a wound healing assay showed that osteoblasts
can assemble collagen ”on the move”, that multiple cells contributed to the assembly of the collagen fiber networkand that cells actively engaged in collagen assembly were able to divide and then resume assembly of collagen.
Together these studies are revealing the highly dynamic nature of bone cells and discovering novel aspects ofosteocyte biology that were not previously appreciated. They have also shown that the bone matrix assembly process
is highly dynamic, that multiple cells contribute to assembly of collagen fibers and that cells exert considerable
forces on ECM fibrils during their assembly.

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13th Congress of the International Society of Bone Morphometry

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13th Congress of the International Society of Bone Morphometry

24April 27-29, 2015 ● Tokyo Garden Palace Hotel

Proper bone histomorphometry in rats
T.J. Wronski and J.I. Aguirre
Department of Physiological Sciences,
University of Florida, Gainesville, FL, USA
Bone histomorphometry is an important technique for evaluation of changes in bone structure and levels of boneformation and resorption in response to a variety of influences such as bone-active drugs, hormones, growth factors,
and mechanical forces. Recommendations for proper bone histomorphometry in rats will be presented, including:1) embedding undecalcified bone, 2) identification of osteoblasts and osteoclasts in stained histologic sections, 3)
optimal regimens for fluorochrome labeling of cancellous bone in young and aged rats, 4) standardization of theregion of interest for histomorphometric data collection in cancellous and cortical bone, and 5) the most importantmeasurements of bone structure, formation, and resorption. These recommendations are intended to improve thequality of bone histomorphometry in preclinical studies of rat models.

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13th Congress of the International Society of Bone Morphometry

Bone histomorphometry in mice: getting it right!
Natalie A Sims
St. Vincent’s Institute of Medical Research, University of Melbourne,
Melbourne, Australia
Genetically altered mice are common tools used for discovery of novel pathways that regulate bone structure andbone cell function. In addition, the use of mice as models of bone and joint disease for testing pharmacologicalinterventions is increasing because of their low cost compared to larger animals. Although information on structure
can now be derived by micro-computed tomography, histomorphometry remains the best method for determining
the changes in bone formation and resorption that lead to the changes in bone structure. However, differencesin measurement methods, reporting of histomorphometric markers and selection of controls sometimes lead toconfusion in the literature within the bone field when studies have been repeated in multiple laboratories.
This talk will focus on practical and theoretical issues of how to carry out histomorphometry on mouse models. It
will consider issues such as selection of measurement sites, including methods for measuring cortical bone, the useof appropriate controls, including controls for Cre-directed genetic manipulation, and the ways that diet, genetic
background, age, and sex modify bone remodelling parameters. Practical issues such as fluorochrome labelling
protocols, paraffin vs plastic embedding techniques will be explored. Guidance will be given on how to reportyour histomorphometric data in a way that is accurate and meaningful and point out some limitations of how tomeasure and interpret standard histomorphometric parameters. It will also explore ways in which histomorphometricmethods have been applied to more unusual features of bone structure, including analysis of growth plate dynamics,
breast cancer metastasis, focal bone erosions, and the use of histomorphometric techniques for measuring
immunohistochemical markers. Limitations of the mouse model compared to larger mammals will also be explored.

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Luncheon & Evening Seminars

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Dynamic live imaging of bones : opening a new era with‘bone histodynametry’
Masaru Ishii
Osaka University Graduate School of Medicine and Frontier Biosciences, Japan,
JST, CREST, Osaka, Japan
During the last decade, multi-photon fluorescent microscopy has launched a new trend in the field of biology.
By using this advanced imaging technique we have established a new system for visualizing in situ behavior ofa diversity of living cells within intact tissues and organs. Among them, we succeeded in visualizing the various
dynamic phenomena within bones, a mysterious organ where various kinds of hematopoietic and immune cells areproduced and functioning although poorly analyzed by conventional methodology such as histological analyses
with decalcified bones. Especially we have focused on the behavior of osteoclast, a kind of specialized macrophage
contributing to physiological bone remodeling as well as to bone destruction in arthritis and osteoporosis, andhave been revealing novel mechanisms controlling migration and function of osteoclasts in situ. This novel ‘bone
histodynametrical’ methodology is sure to, collaborating with conventional histomorphometical analyses, contributeto opening a new era in the field of bone and mineral researches in future.

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Locomotive syndrome –measures for increasing healthy life
expectancy
Kozo Nakamura
President
National Rehabilitation Center for Persons with Disabilities
Saitama, Japan
Japan is now facing the advent of a super-aged society very rapidly.The difference between average life expectancy
and healthy life expectancy is about nine years for men and twelve years for women, which is not short. The numberof elderly who need nursing care service is increasing, and the increase has become an issue of great concern in oursociety.
The reasons for which the elderly aged 65 or older needed nursing care services include cerebrovascular disorders(21.5%), dementia (15.3%), and importantly, problems related to the locomotive organs, such as falls/fractures(10.2%) and joint disorders (10.9%). Most people recognize cerebrovascular disorders and dementia as a reason for
nursing care services, but unfortunately not locomotive organ disorders.
Locomotive organ disorders hamper individuals in standing and walking. Since most elderly have plural disorders oflocomotive organs, including bones, joints, cartilage, intervertebral discs, and muscles, we have to pay attention totheir general walking function as well as their local symptoms.
This is a new affair in a super-aged society. We therefore need a new concept. The Japanese Orthopaedic Association(JOA) proposed locomotive syndrome in 2007. Locomotive syndrome refers to a decrease in walking function due toproblems of the locomotive organs. The syndrome increases risk of requiring nursing care service.
Because gait speed and chair stand time are correlated with such risks, patients with locomotive syndrome areassessed using brief methods such as the two-step test, which involves dividing the maximum stride length by theheight of the patient, and the stand-up test, which involves standing on one or both legs at different heights.
The syndrome is also evaluated by “locomo-25”, the self reporting questionnaire, which consists of 25 items.
The goal of countermeasure against locomotive syndrome is to enable people to stand-up and walk independently
even in old age. JOA recommends two exercises as a basic training. They are possible to do at home without any
special equipment, and we have to avoid excess burden on already degenerated lumbar spine and knee joint.
JOA calls this training as locomotion training, “loco-tra” for short. They are “one leg standing” and “squatting”.
This concept has been accepted and has spread quite rapidly in Japan. The syndrome has been included in National
Health Promotion Movement (2013-2022).

30April 27-29, 2015 ● Tokyo Garden Palace HotelMorphological examination on anabolic effects of intermittent
administration of PTH in bone
Norio Amizuka
Department of Developmental Biology of Hard Tissue, Graduate School of Dental Medicine,
Hokkaido University
Sapporo, Japan
In order to clarify the behavior of osteoblastic cells after intermittent PTH(1-34) treatment and to elucidate therole of osteoclasts on the mediation of PTH-driven bone anabolism, we have administered PTH intermittently towild-type and c-fos-/- mice. Structural and kinetic parameters related to bone formation were increased in PTHtreated wild-type mice, whereas in the osteoclast-deficient c-fos-/- mice, there were no significant differences
between groups. In wild-type and knockout mice, PTH administration led to significant increases in the number ofpreosteoblasts. Ultrastructural examinations showed two major preosteoblastic subtypes: one rich in endoplasmic
reticulum (ER), the hypER cell, and other with fewer and dispersed ER, the misER cell. The latter constituted themost abundant preosteoblastic phenotype after PTH administration in the wild-type mice. In c-fos-/- mice, misER
cells were present on the bone surfaces but did not seem to be actively producing bone matrix. We concluded thatthe absence of osteoclasts in c-fos-/- mice might hinder PTH-driven bone anabolism and that osteoclastic presence
may be necessary for full osteoblastic differentiation and enhanced bone formation seen after intermittent PTH
administration.
In Japan, daily and once-weekly administration of PTH(1-34), teriparatide, is approved treatments for osteoporosis.
Both regimens enhance bone mineral density and bone mass in osteoporotic patients. To provide evidence of how
the different intervals of PTH administration increase bone in vivo, we have examined the histology of mice femora
subjected to different PTH administration intervals. Mice were randomly allocated to control or to three treatmentgroups (80μg/kg of PTH per dose, 80μg/kg of PTH daily, or 20μg/kg of PTH daily). For each group, PTH wasinjected once every other day, once daily, twice daily and four times/day during two weeks. In the 80μg/kg daily
and in the 20μg/kg daily groups, more frequent PTH administration increased metaphyseal trabecular number.
However, trabecular histology was different across the groups: thin metaphyseal trabeculae were common in the fourtimes/day regimen, while the once daily PTH regimen formed thicker trabeculae. Cortical bone tended to change
its compact profile into cancellous with the four times/day regimen. Higher PTH administration frequency (fourtimes/day) showed a thick preosteoblastic cell layer and many osteoclasts, and the resultant bone matrix included
scalloped cement lines that indicate osteoclastic bone resorption prior to new bone deposition. These findings
suggest accelerated bone remodeling. In contrast, lower PTH administration frequency (once daily) did not induce
the formation of a thick preosteoblastic cell layer, without any clear separation between the bone marrow and mature
osteoblasts. These mature osteoblasts were occasionally localized atop mildly convex bone surfaces that featured
smooth cement lines, identical to arrest lines. These findings suggest mini-modeling-based bone formation. Thus,
it appears that higher PTH administration frequency rapidly increases bone mass by forming thin trabeculae throughaccelerated bone remodeling. On the other hand, lower PTH administration frequency forms thicker trabeculae not
only through bone remodeling but also through mini-modeling.

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Bone histomorphomertry in orthopedic clinical practiceNoriaki Yamamoto, Hideaki Takahashi
Niigata Bone Science Institute, Niigata, Japan
Department of Orthpedic Surgery, Niigata Rehabilitation Hospital, Niigata, Japan
Bone histomorphometry is a method for direct examination of bone metabolism, which obtains several informationof bone remodeling.
Niigata bone science institute started since 1999 in Niigata rehabilitation hospital, and mainly conducted clinicalhistomorphometric work with orthopedic doctors in Japan. We have developed the new device for iliac bone biopsy
more safety and easy way. Also we have published new text book of bone Histomorphometry to inform the bone
histomorphometry method and standard knowledge to young doctors. In Japan we have a special group of bone
histomorphimetry, and annual workshop was held to have technical consensus.
In clinical work, we orthopedic doctors experience atypical cases of fracture patients. Bone histomorphometry
can provide the important information about bone metabolism and bone tissue, such as SSBT, osteomalacia, acutedestructive joints and others. In this talk, I demonstrate some special clinical cases with iliac bone biopsy to show
histomorphpometric analysis.
Another topic of this talk is the concept of minimodeling which means new bone formation without previous
bone resorption in trabecular surface. We had reported minimodeling was observed in hip osteoarthritis patients.
Recently minimodeling measurement studies were indicated in some reports, but the definition of minimodeling is
not established yet. We have been observed many minimodeling like structure in human trabecular bone, and wish topropose the final definition of minimodeling.
A last topic is Osteocyte evaluation in histomorphometry. Recently it was suggested that osteocytes play a key rolein the regulation of bone remodeling. Bone histomorophometery demonstrate osteocyte density and empty lacunadensity. We had some studies osteocyte measurement in human cortical and cancellus bone.
I strongly endorse the bone histomorphometry in clinical work to orthopedic doctors who handle the bone tissue
directly.

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Imaging analysis of the effects of denosumab on bone structuralproperties
Masako ItoCenter for Gender Equality
Nagasaki University
Nagasaki, Japan
Denosumab is a human monoclonal antibody that blocks the binding of RANK ligand (RANKL) to its receptor
and selectively inhibits the activity of RANKL, thereby suppressing increased bone resorption in patients withosteoporosis. The effects of denosumab to reduce the risk of fracture have been demonstrated in clinical studies.
There has been a progress in the elucidation of structural properties, mainly in non-clinical studies including theanalysis of bone specimens collected primarily from experiments with ovariectomized (OVX) cynomolgus monkeys,
as well as in clinical studies of human iliac bone biopsy specimens using micro-CT, and of in vivo radius and tibia
morphometric analysis using high-resolution peripheral quantitative CT (HR-pQCT).
The effects of denosumab on cancellous bone microstructure have been reported. The results of studies on bone
tissue from cynomolgus monkeys and iliac biopsy specimens from humans showed that BV/TV is maintained orincreased with decreasing osteoblast surface/bone surface (%), osteoclast surface/bone surface (%), and eroded
surface/bone surface (%). Moreover, with regard to the effects of denosumab on the cortical bone, a reduction incortical porosity has been observed in human iliac biopsy specimens, and in cortical components of the radius andtibia using HR-pQCT. A number of studies have reported an increase of the cortical thickness due to suppression ofendosteal resorption, and these effects have been shown to be greater than those of bisphosphonates, according tocomparative studies with alendronate. Denosumab has been shown to have a great effect on improving cancellous
and cortical bone microstructure, relative to the treatment with teriparatide, which is a bone formative agent.
Furthermore, a recent study observed fluorochrome labeling, which showed that after treatment with denosumab,
the endosteum of the cortical bone in the upper part of the femoral neck and the lower periosteum were markedly
labeled over 6 - 16 months, whereas the cancellous bone was only slightly labeled. It indicates that modeling-based
osteogenesis have been reported, which may explain the increase BMD during more than 7 years treatment withdenosumab.
Clinically, the effects of denosumab have also been demonstrated by hip structure analysis (HSA) based on dualenergy X-ray absorptiometry (DXA) and imaging evaluation with radial-femoral quantitative CT.
In this lecture, I will discuss the effects of denosumab on bone structural characteristics and the mechanism of
associated improvements in bone fragility based on imaging analysis data.

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Denosumab for the treatment of osteoporosis
Sakae Tanaka
Department of Orthopaedic Surgery,
Faculty of Medicine, The University of Tokyo
Tokyo, Japan
Osteoclasts are multinucleated giant cells primarily responsible for bone resorption. Osteoclasts are differentiatedfrom monocyte/macrophage-lineage precursor cells in the presence of receptor activator of NF-κB (RANKL)
and macrophage colony-stimulating factor. A number of studies have shown that RANKL-RANK pathways playessential roles in the pathologic bone resorption such as osteoporosis, rheumatoid arthritis, cancer bone metastasis
and periodontal diseases. Denosumab, a fully human IgG2 monoclonal antibody that binds human RANKL with ahigh affinity, potently suppresses osteoclast development and reduces bone resorption. In a study of postmenopausal
osteoporosis patients (FREEDOM), denosumab significantly reduced the risk of new vertebral fractures at 3 years
by 68%, non-vertebral fractures by 20% and hip fractures by 40% relative to placebo. To examine the anti-fracture
efficacy and safety of denosumab (60 mg subcutaneous injection every 6 months [Q6M]) in Japanese patients withprimary osteoporosis, a randomized, double-blind, placebo-controlled trial with an open-label referential arm wasconducted (DIRECT trial). Denosumab reduced the risk of new or worsening vertebral fracture, with incidences of
3.6% in the denosumab group and 10.3% in the placebo group in 24 months without increasing the risk of adverse
events of interest. These results suggest that anti-RANKL therapy is effective in reducing osteoporosis fractures.

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Bone microarchitecture after alendronate treatment of
osteoporotic women
Robert R. Recker, M.D., M.A.C.P., F.A.C.E.
Head of Creighton University’s Osteoporosis Research Center
Omaha, NE, USA
Alendronate was the first bisphosphonate to be approved for treatment of postmenopausal osteoporosis in the
U.S. Preliminary studies demonstrated that it attached to bone surfaces, and markedly inhibited bone resorption
by osteoclasts. Originally it was thought (hoped?) that during treatment with Alendronate, bone formation would
continue, positive bone balance would ensue, bone mass/density (BMD by DXA) would increase and fractures
would be prevented. The early studies did demonstrate increased BMD on treatment, but the increase reached amodest plateau at about 1½ years of continuous treatment. Clinical trials showed remarkable reduction in fracturesof ~50% on treatment. At about the same time it became obvious from serum biomarker data that bone formation
decreased to about the same extent as bone resorption. The bone resorption markers declined very precipitously
after starting treatment, while the formation markers decreased gradually, reaching a plateau at about 6 months after
starting treatment. Further, dynamic bone histomorphometry data from the human transiliac bone biopsies showed
reduction in formation surfaces that mirrored the reduction in circulating bone formation markers. Thus, there was a
relatively brief “uncoupling” of bone resorption from bone formation. However, the histomorphometry data showedthat it was the bone resorption phase of bone remodeling that was affected by Alendronate, and that the activation
frequency of new remodeling sites was decreased. Since the resorption phase at remodeling sites (Basic Multicellular
Units, BMUs) is much shorter than the formation phase, it took much longer for the “remodeling space” to be filled,
and this explained the delay in the plateau of positive bone balance. It was the reduction in activation frequency(Ac.f) of remodeling sites that was responsible for the gain in BMD. However, it was doubtful that this gain in
BMD, ~6-8%, was enough to account for the reduction in fractures, since the reduction in BMD prior to treatment
was much greater, ~15-20% or more. More refined anatomic measurements of trabecular microarchitecture(histomorphometry, Micro-CT) then suggested that the loss of trabecular volume, numbers, thickness, connectivity
exaggerated the weakening due to bone loss beyond the reduction of overall BMD. This stimulated several reportsof the effect of Alendronate treatment on microarchitecture, using histomorphometry and Micro-CT of trabecularbone in transiliac biopsies, as well as in vivo high resolution imaging. The findings generally showed increases intrabecular bone volume (BV/TV), thickness (Tb.Th), connectivity (Tb.C), number (Tb.N) and connectivity indices,along with reductions in trabecular spacing (Tb.Sp). The changes were +20%, +11%, +11% for BV/TV, Tb.N, andTb.Th, respectively, and -15% for Tb.SP. These were regarded as respectable benefits to microarchitecture, but were
they enough to account for the ~50% reduction in fractures, along with the rather modest gains in BMD? There
are other potential explanations for more of the anti-fracture effect of Alendronate. For example, the high bone
remodeling rates in the usual osteoporotic patient would reduce the overall age of the bone tissue, perhaps making
it less mechanically competent. The reduction in Ac.f by Alendronate treatment would reverse this effect, and might
contribute to the anti-fracture effect. Another benefit might accrue due to continued periosteal formation (which is
not preceded by activation of resorption) coupled with inhibition of endocortical resorption. This might increase themoment of inertia of most bones in the skeleton, yielding an increase in mechanical strength out of proportion to
the gain in BMD. As of yet, we do not have a complete explanation of the mechanism of the anti-fracture benefit of
Alendronate, beyond its ability to reduce the remodeling space and thus result in some gain in bone mass.

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Geometry of lower extremities and atypical femoral fractures
Muneaki Ishijima, M.D., Ph.D.
Department of Medicine for Orthopaedics and Motor Organ
Juntendo University Graduate School of Medicine, Tokyo, Japan
Osteoporotic fractures, most notably hip fractures, are associated with significant morbidity and mortality. Althoughosteoporosis had been thought to be a natural part of aging among women, it is no longer considered age- or sexdependent. It becomes preventable due to the remarkable progress in the scientific understanding of its causes,
diagnosis, and treatment. As bone strength primarily reflects the integration of bone mass and bone quality, treatmentof osteoporosis is to increase the bone mass and, hopefully, to improve the bone quality. As bone is a remodelingorgan and the recent development of bone biology, we are recently able to increase bone mass by modulating a bone
remodeling processes. However, the goal of therapy is fracture prevention.
The bisphosphonates (BPs) are the current cornerstone of osteoporosis treatment and prevention. Treatment with
BPs reduces the rate of bone resorption and increases bone mass, improving bone strength and reducing fracture risk.
Denosumab, a fully human monoclonal antibody against the receptor activator of nuclear factor-κB ligand (RANKL),
prevents the interaction of RANKL with RANK, its receptor, on osteoclasts and their precursors, thereby blocking
the formation, function, and survival of osteoclasts. These anti-bone resorbing agents are only drugs currently
available, those can reduce hip fractures in patients with osteoporosis. However, the treatments by these agents must
be continued for 3 to 4 years for reducing the risk of these fractures in osteoporotic women.
In spite of its clinical benefits, the long-term treatment of BPs has been implicated in the rare but serious adverse
events, such as atypical femoral fractures (AFFs). Although the recent studies with radiographic adjudication haveindicated the significant associations between them, the pathogenesis of AFFs is not completely understood.
AFFs are characterized by unique radiographic features and also unique clinical features, such as prodromal pain andbilaterality, those resemble stress fractures or reactions. The ASBMR task force reported the original case definition
to highlight the unusual radiographic features that distinguish AFFs from ordinary osteoporotic typical femoral
fractures (TFFs). Although AFFs appear to be more common in patients who have been exposed to long-term BPs,
usually for more than 3 years, every series includes patients who have not been treated with BPs, suggesting that the“background factors” of AFF in osteoporosis patients is not zero. The majority of studies have found a significant
association with glucocorticoid use or duration. Although the relative risks of AFFs are very high in patients on BPs,
their absolute risk is extremely low. Thus, these fractures are rare, particularly when considered against the incidenceof common osteoporotic fractures of all types and ordinary TFFs, all of which have been proven to decrease with BP
therapy. In addition, it has also been increasingly reported the cases suggesting the association between denosumab
and AFFs. Therefore, it becomes more important to know the “background factors” of AFF to treat patients withosteoporosis who are susceptible for osteoporotic fractures.
We recently reported that the incidence of AFFs in the Japanese population was similar to that of those in Caucasians,
and taking BPs and glucocorticoids and suffering from collagen diseases were the risk factors for developing AFFs
(J Bone Miner Metab, 2014). We also reported that the fracture sites of AFFs are associated with the standing lower
limb alignment and that the lower limb alignment is suggested to be one of the risk factors for AFFs (Bone, 2014).
In this lecture, I’d like to focus on the “background factors” of AFF in addition to summarizing the epidemiology,
risk factors and pathology of AFFs.

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Anabolic therapy for the skeleton with PTH– now and the future
T.J. Martin
St Vincent’s Institute of Medical Research;
University of Melbourne Department of Medicine
Melbourne, Australia
Anti-resorptive agents do not reconstruct the skeleton, but a new approach to achieving that has come with thedevelopment of PTH as an anabolic therapy, approved in several countries as PTH(1-34) and PTH(1-84). The
anabolic effectiveness of PTH requires that it be administered intermittently, and this has been achieved with the useof daily injections which rapidly achieve a peak level in blood which is not maintained.
The pharmacokinetics required for this effect are that a peak of circulating PTH is required, returning to control
levels within 3 hours. Prolongation of elevated levels brings into play the stimulation by PTH of osteoclast formation
and bone resorption . This resorption effect is enhanced greatly with infusion of PTH over some hours, or with theconsistently elevated PTH of primary hyperparathyroidism.
Studies of PTH pre- and post-treatment bone biopsies in women indicated that the predominant PTH effect is toincrease remodelling, with some lesser effect on modelling. Current views of the anabolic action of PTH are thatit increases the activation of BMU’s in remodelling, that it acts on committed osteoblast precursors to promote
their differentiation, inhibits osteoblast and osteocyte apoptosis and inhibits the production of the bone formation
inhibitor, sclerostin. There is also much interest in the possibility that PTH treatment results in transient activationof osteoclasts, that in turn produce activities that enhances the osteoblast differentiation effect. The latter may be
independent of resorption, and may result from the release of growth factors (TGFβ, IGF-1) in the resorption thatenhance the availability of mesenchymal stem cells (TGFβ), or their differentiation in the osteoblast lineage (IGF-1).
When PTH treatment is stopped, the accrued bone is likely to be lost through resorption over a period of time, socourses of PTH treatment are likely to require treatment with anti-resorptive drugs to retain the newly formed bone.
This is being actively investigated, as also is the question whether PTH could usefully be administered together with
anti-resorptive? Some studies, but by no means all, indicate that co-treatment with bisphosphonate impairs the PTH
effect. Much of this work is confounded by the use of BMD measurement as end-point. New methods of assessing
bone quality will be needed to answer this question. There seems a sound logic in such combination, and this will be
discussed.
We might now regard PTH in its application as a skeletal anabolic therapy as an attempt to reproduce the local actionof PTHrP. This sharing of function between a hormone and its related cytokine is reminiscent of the relationshipbetween growth hormone and IGF-1. If this is so, the pharmacological challenge comes from the fact thatphysiologically, PTHrP is generated and acts within those BMU’s that require it. Pharmacological administrationof PTH on the other hand, is presented globally to the skeleton, and able to activate BMU’s generally. This might
explain the increased bone resorption that inevitably begins after several weeks’ treatment with daily PTH.
What does the future offer in relation to PTH in therapy? Attempts continue to find low molecular weight peptide
or even non-peptide mimics that can activate through the specific G protein – coupled receptor PTHR1. Maybe
alternative methods of delivery can be developed - e.g. intranasal or intrapulmonary, and perhaps PTH secretagogues,that provide a sharp burst of PTH release from the parathyroid gland, sufficient to promote the anabolic response,
but not so prolonged that the resorption effect predominates. Efforts are being made to find such drugs by seeking
antagonists of the calcium -sensing receptor. Such an approach, developing PTH secretagogues as anabolic agents,
will have to satisfy the pharmacokinetic requirement of transiently increasing PTH in patients to the desired level.
Reproducing the physiological function of PTHrP presents very much greater difficulty.

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Oral Presentations

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13th Congress of the International Society of Bone Morphometry

40April 27-29, 2015 ● Tokyo Garden Palace HotelHistomorphometric analyses of cortical effects of teriparatide in
postmenopausal women with or without long-term alendronate
therapy
Yanfei L. Ma, Qing Q. Zeng , Alan Y. Chiang 1), David Burr 2), Jiliang Li 3),
Harald Dobnig , Astrid Fahrleitner-Pammer 4), Dana Michalská 5),
FernandoMarin , Imre Pavo 6), Jan J. Stepan 7)1) Lilly Research Lab, Indianapolis, IN, USA
2) Dept of Anatomy and Cell Biology, Indiana University, Indianapolis, IN, USA
3) Dept of Biology, Indiana University Purdue University, Indianapolis, IN, USA
4) Dept of Internal Medicine, , Medical University of Graz, Graz, Austria5) 3rd Dept. of Internal Medicine, Charles University Faculty of Medicine 1, Prague, Czech Republic
6) Lilly Research Center, Windlesham, United Kingdom
7) Institute of Rheumatology, and Charles University Faculty of Medicine 1, Prague, Czech Republic
Purposes:
Osteoporotic and age-related bone loss is characterized by a progressive thinning of cortical bone and increasing
in intracortical porosity, consequently leading to increased risk of fracture. We assessed the cortical effects ofteriparatide (TPTD) 20 μg/day sc treatments in postmenopausal women with osteoporosis previously receiving
alendronate (ALN) treatment for a mean duration of 63.6 months or being treatment naïve (TN).
Materials and methods:
Twenty-nine ALN and 16 TN paired tetracycline-labeled transiliac crest bone biopsies obtained from patients atbaseline and after 24 months TPTD treatment were evaluated for bone formation activity at the periosteal, endosteal
and intracortical osteonal bone surfaces using dynamic histomorphometry.
Results:
At baseline, bone turnover was lower in patients previously treated with ALN compared to the TN patients. Double
label prevalence in endocortical and periosteal surfaces were 28% and 3% in the ALN group compared to 50% and
38% in the TN group. The numbers of specimens without any tetracycline label on endocortical or periosteal surfaces
were 38% and 59% in the ALN group compared to 6% and 13%, respectively, in the TN group. The percentageof quiescent osteons was also higher in the ALN (75%) specimens than TN (83%). Endocortical and periosteal
mineralizing surface (MS/BS%), periosteal bone formation rate (BFR/BS), mineral apposition rate (MAR) andthe number of intracortical forming osteons were significantly lower in specimens from the ALN-group than fromthe TN group. Following teriparatide treatment, the frequency of endocortical and periosteal unlabeled biopsies
decreased in both ALN and TN groups; the percentage of quiescent osteons were reduced while forming osteons
were increased in the ALN group. Teriparatide treatment resulted in significant increases of endocortical MAR in
ALN- group, and periosteal MS/BS% in the periosteal compartment in both ALN and TN groups. Most indicesof bone formation remained lower in the ALN-pretreated group compared with the TN group at study end. In thepooled analysis, percent changes in periosteal and endorcortical MS/BS and BFR/BS; total cortical area, cortical
thickness, and porosity area increased significantly after 24 months TPTD treatment compared to baseline. These
results suggest that 24-month teriparatide treatment increases cortical bone formation activities in patients who were
either treatment-naïve or had lower bone turnover initially due to previous alendronate therapy. The increased bonebuilding activities were associated with thicker cortex, increased cortical turnover in both patient groups.

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13th Congress of the International Society of Bone Morphometry

In vivo bone microstructural analysis of osteoporosis using
HR-pQCT
Ko Chiba, Masako Ito, Makoto Osaki 1),
Andrew J. Burghardt, and Sharmila Majumdar 2)
1) Nagasaki University School of Medicine, Japan
2) University of California, San Francisco, USA
Introduction:
It had been difficult to evaluate the bone microstructure of living human bones accurately using clinical imaging
modalities. Due to the development of imaging technology in recent years, in vivo analysis of patients' bone
microstructure has been attempted using various clinical modalities such as CT and MRI.
HR-pQCT is a clinical high resolution CT dedicated to patients' extremities. Using this CT, cancellous bone structuresuch as trabecular number (Tb.N) and trabecular separation (Tb.Sp), and cortical bone microstructure such as cortical
thickness (Ct.Th) and cortical porosity (Ct.Po) can be measured. This CT is now widely used for the research ofpathophysiology and drug development for osteoporosis.
Methods:
A male osteoporosis study using HR-pQCT has been conducted at the University of California, San Francisco.
The subjects consisted of 80 male volunteers. The distal radius and tibia were scanned at the voxel size of 82 um,
and Tb.N, Tb.Sp, Ct.Th, and Ct.Po, etc. were measured. Possible correlations between these parameters wereanalyzed, along with: age, height, weight, body mass index, smoking/drinking habits, serum calcium, vitamin D andtestosterone value, bone mineral density value of the lumbar vertebra, proximal femur, distal radius measured bydual-energy x-ray absorptiometry (DXA).
Results:
We obtained mainly two results: 1) It was impossible to estimate the existence of cortical porosity by DXA, althoughsome micro-structural parameters of cancerous bone had correlations with DXA value. 2) The most significant factor
that was associated with the formation of the cortical porosity was aging.
Discussion:
HR-pQCT was developed in 2004 and now used in over 50 institutions all over the world, but not in Japan. In 2014, a
new model of HR-pQCT was released, and it has been decided to install the CT in Japan this year. Research projects
in 5 fields are under contemplation: 1) osteoporosis, 2) rheumatoid arthritis, 3) fracture healing, 4) osteoarthritis, and 5)
anatomy. It is expected that new findings will emerge from these projects.

42April 27-29, 2015 ● Tokyo Garden Palace Hotel

Measurement of bone strength and medication evaluation can beachieved using medical CT images
Nobuhito Nango 1), Shogo Kubota 1), Yusuke Horiguchi 1), Kazutaka Nomura 1)
and Masafumi Machida 2)
1) Ratoc System Engineering Co., Ltd.
2) National Hospital Organization Murayama Medical Center
Introduction:
Prevention of fractures is a global issue in aging societies. Bone strength is determined by bone density and quality(NIH Consensus Statement 2000), and bone microstructure is a component comprising bone quality. Prediction ofbone strength by measuring bone density and microstructure is an important step forward in preventing vertebral
fractures. We can contribute to enhancing incentives for treatment by quantifying the effect of osteoporosis
medications.
Purposes:
We investigated whether human bone morphometry can be achieved using clinical CT (MDCT) images. Althoughhuman trabecular bone has a width of 0.1–0.3 mm, the MDCT scan has a slice thickness of 0.5 mm. Therefore, we
examined the validity of numerical values obtained by measurement using MDCT images.
Materials and methods:
Specimens were T12, L1, L2, and L3 extracted vertebrae. We scanned them with MDCT using FOV of 100–150mm and thickness of 0.5 mm. Specimens were scanned using µCT with a resolution higher than the trabecular bonewidth. Both CT images were converted to TMD images using an HA phantom. Volume position adjustment was
performed for MDCT and µCT images, and trabecular bone and cortical shell were extracted from both the images
to measure trabecular and cortical bones. We then ran the bone strength simulation of compression load and bonemorphometric measurement.
Results:(1) The BMD value correlation between MDCT and µCT showed an R2 of >0.7. (2) The morphometry result showed
an R2 of >0.6 for correlation among indexes such as BV/TV, Tb.Th, V*m.space, and V*tr. (3) The shear stress wascorrelated with an R2 of >0.5 (p < 0.00).
Discussion:
In MDCT, we could not separate trabecular bones individually, but its macroscopic bone shape was consistent withthat on the µCT images. Based on the abovementioned results, trabecular bone morphometric values obtained using
MDCT images may represent the state of bone accumulation Thus, we suggest that MDCT images can be used forhuman bone morphometry measurement.

MDCT CT images (left), and µCT Images (right)
Trabecular bone and cortical shell were extracted.
MDCT macroscopic bone shape was consistent with that on the µCT images.
Comparison of TMD images obtained using MDCT and µCT.

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13th Congress of the International Society of Bone Morphometry

Comparative analysis of the development of periodontal disease inrice rats and lewis rats
J. Ignacio. Aguirre1, Stephanie La2, Jonathan G. Messer2, Joshua F. Yarrow3,
Mohammed P. Akhter4, Lakshmyya Kesavalu5, Deborah E. Kipp2, Donald B. Kimmel4,
Thomas J. Wronski1
1) Department of Physiological Sciences, University of Florida (UF), Gainesville, FL
2) Department of Nutrition, UNC Greensboro, Greensboro, NC
3) Research Service, Department of Veterans Affairs Medical Center, North Florida/South Georgia Veterans Health
System, Gainesville, FL and Department of Applied Physiology and Kinesiology, UF
4) Osteoporosis Research Center, Creighton University, Omaha NE
5) Department of Periodontology and Oral Biology, College of Dentistry, UF
Purposes:
Periodontal disease (PD) is a chronic dental disease resulting from a persistent immune-response to chronic infectionin the mouth of humans/domestic animals. Identification of PD as a predisposing factor in the development of antiresorptive-related osteonecrosis of the jaw (ARONJ) has generated interest in animal models of this disease. We havestudied two rodent models of PD: 1)the rice rat (Oryzomus palustris), a species susceptible to spontaneous PD, which
can be accelerated by feeding the rats with a diet high in sucrose/casein (H-SC), and 2)the Lewis rat, a strain used to
study Th1-mediated-autoimmune diseases. We aim to compare the development of PD and coincident alveolar boneloss (ABL) in these two species.
Materials and methods:
Three groups of rats (N=8-20) of each species were used. A group of rats of each species was euthanized at weaningage (baseline). The other two groups of each species were fed standard (STD) or H-SC diets for a period of 23/24
wks after weaning. Mandibles and maxillae were processed to assess vertical(v) and horizontal(h) ABL, and degreeof inflammation (PD score), by MicroCT, histometry and qualitative histology, respectively.
Results:
We found that compared to baseline, rice rats had greater vABL, hABL and PD scores (moderate/severe) in
mandibles and maxillae of groups fed either the H-SC or the STD diet for 23/24wks. In Lewis rats, there were no
significant differences between baseline and STD groups for any parameter. However, slightly worse PD score (slight/
mild) and significantly worse mandibular and maxillary vABL were observed in Lewis rats fed the H-SC diet for
23/24 wks.
Conclusion:
These findings suggest that after 23/24 wks of dietary treatments: 1)STD rice rats develop PD comparable to H-SC
rice rats with a moderate/severe degree; 2)no significant age-related PD develops in Lewis rats (STD); 3)the H-SC
diet causes slight to mild PD and alveolar bone loss in Lewis rats; and 4)the H-SC diet-related PD develops more
slowly in Lewis rats than in rice rats. These findings support use of rice rats with PD for preclinical studies of
ARONJ, but the greater availability of Lewis rats merit further evaluation as a rodent model for PD.

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April 27-29, 2015 ● Tokyo Garden Palace HotelG9a deficiency leads to impaired osteoblastic differentiation inmice
Akira Nifuji 1), Hisashi Ideno 1), Akemi Shimada 1), Koichiro Komatsu 1),
Taichi Kamiunten 1), Yoshiki Nakamura 1), Kazuhisa Nakashima 1), Hiroshi Kimura 2),
and Makoto Tachibana 3)1) Tsurumi University School of Dental Medicine, Japan
2) Tokyo Institute of Technology, Japan3) The University of Tokushima, Japan
Purposes:During development, histone H3K9 methyltransferases are expressed in many tissues and regulate celldifferentiation. In this study, we investigated expression and function of an H3K9MTase, G9a, during osteoblastic
differentiation,
Materials and methods :We investigated G9a expression during the differentiation of MC3T3E1 cells and primary calvarial cells. To examine
G9a expression during embryonic bone formation, in situ hybridization (ISH) was carried out on sections preparedfrom mouse embryos at day 16.5(E16.5). To investigate functions of G9a, we performed G9a knockdown by usingsiRNA against G9a. We also isolated primary osteoblasts from G9a(fl/fl) mice and deleted G9a by infection of creexpressing adenovirus. To examine functions of G9a in skeletogenesis in vivo, mice carrying a floxed allele of G9awere crossed with mice expressing Cre recombinase under the control of the Sox9 promoter
Results and conclusion:
G9a was expressed during osteoblastic differentiation in MC3T3E1 cells and primary calvarial cells. ISH revealed
that G9a expression was observed in cells residing on bone surfaces. Knockdown or deletion of G9a in osteoblastic
cells resulted in decreased cell proliferation rate and suppression of osteoblastic marker expression such as alkaline
phosphatase. Conditional knockout mice showed impaired bone formation in cranial bones. Taken together,the H3K9MTase, G9a, is expressed during osteoblastic differentiation and its expression is required for properdevelopment of bone.

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13th Congress of the International Society of Bone Morphometry

Osteocytic ephrinB2 restrains mineralisation of murine bone
Christina Vrahnas1,2, Ingrid Poulton1, Huynh Nguyen3, Mark Forwood3, Keith Bambery4,Mark Tobin4, T John Martin1,2, Natalie Sims1,2
1) St. Vincent’s Institute of Medical Research, Melbourne, Australia
2) University of Melbourne, Australia3) Griffith University, Gold Coast, Australia
4) Australian Synchrotron, Melbourne, Australia
Parathyroid hormone (PTH) rapidly upregulates expression of EphrinB2 in the osteoblast lineage. EphrinB2
deletion in osteoblasts and osteocytes (OsxCre.EphrinB2 f/f) caused a phenotype of compliant bones due to delayedmineralization and osteoblast apoptosis. In contrast, ephrinB2 deletion in osteocytes (Dmp1Cre.EphrinB2 f/f) results
in increased bone brittleness, suggesting a different function for EphrinB2 in osteocytes. However, no defect inhistomorphometric parameters of bone formation was observed. We have used synchrotron-based Fourier-Transform
Infrared Microscopy (sFTIRM) to determine aspects of mineralisation that contribute most strongly to bone strength
in the Dmp1Cre.EphrinB2 f/f mouse.
3µm tibial sections from 12 week old female Dmp1Cre.EphrinB2 f/f mice and littermate Dmp1Cre controls (w/w)(n = 13 per group) were analysed in 3 x 15 µm square regions commencing 1.5mm below the growth plate at theperiosteal edge of the anterofibular cortex and concluding in the older mature bone. Regression analysis with 3-point
bending data of femoral bones from the same mice was also conducted.
Our sFTIRM analysis revealed that the brittle bones from Dmp1Cre.EphrinB2 f/f mice had higher mineral/matrix(by 12%, p<0.05) and carbonate/mineral (by 37%, p<0.05) ratios than controls, even in the newly formed bone at theperiosteal edge. This indicates that the greater brittleness of this bone occurred due to greater primary mineralization,rather than prolonged secondary mineralization. There was no significant change in collagen content (amide I band).
The bone in this same region had altered collagen orientation (lower amide I/amide II ratio) (by 13%, p<0.001)
which may underly the greater deposition of mineral in Dmp1Cre.EphrinB2 f/f mice. Regression analysis showedthat while the energy absorbed to failure in 3-point bending tests correlated with the amount of collagen in control
mice (r2= 0.40, p<0.05), this relationship failed in Dmp1Cre.EphrinB2 f/f mice. Instead, ultimate strength of
Dmp1Cre.EphirnB2 f/f bones was determined by the carbonate/mineral ratio (r2= 0.50, p<0.05). sFTIRM analysis of
the compliant OsxCre.EphrinB2 f/f bones showed no significant changes.
Deletion of ephrinB2 in osteocytes therefore accelerates the initial step of mineralisation on the periosteal surface,suggesting that osteocytic ephrinB2 restrains initial mineral deposition and may regulate collagen orientation.

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April 27-29, 2015 ● Tokyo Garden Palace Hotel

Quantitative local in vivo environment (live) Imaging of osteocytes
during dynamic bone remodeling
Andreas J. Trüssel 1), Felicitas R. Flohr 1), Gisela A. Kuhn 1) and Ralph Müller 1)
1) Institute for Biomechanics, ETH Zurich, Zurich, Switzerland
Purposes:
The maintenance and adaptation of the morphology of bone results from orchestrated remodeling processes. These
processes are locally coordinated by osteocytes with biochemical signals that result in increased or decreased boneformation or resorption activities. To better understand the morphology, we therefore have to understand how
osteocytes determine dynamic morphometric parameters within their local microenvironment.
We developed a local in vivo environment (LivE) imaging technique using in vivo micro-computed tomography(microCT) in combination with histology and image processing. LivE imaging allows us to quantify the mechanicaland remodeling in vivo microenvironment of hundreds of individual osteocytes for several weeks prior tohistological processing. Here, we used LivE imaging in trabecular mouse bone to show that dynamic morphometry is
locally linked to quantitative single-cell gene expression.
Materials and Methods:
The 6th caudal vertebrae of adult female C57BL/6 mice (n=9) were imaged three times over a period of two weeks
by in vivo microCT. 3D dynamic morphometric parameters were assessed using advanced registration techniques.
The local stain energy density (SED) was calculated for each timepoint by micro-finite element analysis performed
on a supercomputer (Cray XT5, CSCS, Lugano, Switzerland). Cryosections were cut and registered into 3D
microCT data. Osteocytes were identified, mapped into the microCT coordinate system, tracked back in time throughLivE imaging and grouped according to their SED values within volumes around mineralizing, quiescent or eroding
surfaces. By laser capture microdissection, osteocytes (n=720) were then isolated in subpopulations containing ten
osteocytes on average and their gene expression was analyzed.
Results and Discussion:
LivE imaging showed that osteocytes around eroding surfaces blocked bone formation activities by Sfrp1, a WNT
antagonist and increased local bone resorption by MMPs. Osteocytes in areas of high SED increased not only WNT
signaling by expression of β-catenin and connexin43 to enhance bone formation but also increased the expression
level of the extracellular matrix protein Col1a2 indicating active remodeling of their perilacunar matrix.
Conclusion:
LivE imaging allows bridging the gap between dynamic morphometry and biochemical signaling and thereby help
us to understand how osteocytes contribute to the dynamic remodeling of bone morphology on the molecular level.

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13th Congress of the International Society of Bone Morphometry

Influence of subchondral bone plate integrity on subchondral bonehomeostasis in osteoarthritis
Guangyi Li 1, 2), Tak Cheng 2), Nathan Pavlos 2), Changqing Zhang 1) and Minghao Zheng 2)
1) Department of Orthopaedic Surgery, Shanghai Jiao Tong University Affiliated Sixth People's Hospital, Shanghai,
China2) Centre for Orthopaedic Research, School of Surgery, The University of Western Australia, Perth, Australia
Purposes:
To investigate the influence of subchondral bone plate (SBP) integrity on the homeostasis of underlying subchondral
trabecular bone (STB) in osteoarthritis (OA).
Materials and methods:
Subchondral bone cylinders were extracted in the load-bearing region of femoral heads from 110 patients with latestage OA after hip replacement surgery. SBP integrity, STB microarchitecture and subchondral bone cysts (SBCs)
were examined by micro-CT. Bone remodeling in STB and pathological changes inside SBCs were assessed byhistology.
Results:
All specimens were observed with SBP breach. 73 specimens displayed full-thickness breach of SBP with exposureof the underlying STB, while the remaining 37 specimens showed partial-thickness breach of SBP without exposureof STB. STB with full-thickness breach of SBP exhibited more sclerotic microarchitecture with higher bone
remodeling level and SBCs frequency, as compared to those with partial-thickness breach of SBP. In samples withpartial-thickness breach of SBP, microarchitecture and bone remodeling in STB did not correlate with either the
thickness or porosity of SBP. However, in samples with full-thickness breach of SBP, microarchitecture and bone
remodeling in STB were significantly associated with SBP defect size. A mixed pathology was detected within SBCs,
including fibrous tissue, abnormal blood vessels, fibrocartilaginous tissue, hyaline cartilaginous tissue, remnantbone fragments and adipose tissue. The trabecular region immediately adjacent to SBCs exhibited more sclerotic
microarchitecture and higher bone remodeling level, compared with distal regions.
Conclusion:
SBP plays a pivotal role in the homeostasis of underlying STB in the progression of OA. It may act as abiomechanical and biochemical shield between the synovial space and STB.

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April 27-29, 2015 ● Tokyo Garden Palace Hotel

Intravital bone imaging by two-photon excitation microscopy toidentify osteocytic osteolysis in vivo
Hiroshige Sano 1,2), Junichi Kikuta 1), Masayuki Furuya 1), Naoki Kondo 2), Naoto Endo 2),
and Masaru Ishii 1)
1) Department of Immunology and Cell Biology, Graduate School of Medicine and Frontier Biosciences, Osaka
University, Osaka Japan
2) Department of Orthopedic Surgery, Graduate School of Medical and Dental Sciences, Niigata University, Niigata,Japan
Purpose:Bone is a highly dynamic organ in which several cell types function cooperatively. Among these,
osteocytes have recently emerged as an important regulator of bone homeostasis. Moreover, it is revealed that
osteocytes have various functions such as endocrine organ, although most of their mechanisms are still unclear.
Recent circumstantial evidences support the enigmatic concept, ‘osteocytic osteolysis’. However, no direct evidences
have yet been presented. The aim of this study is to identify this phenomenon in vivo.
Materials and methods:
Ten-week-old female C57BL/6 mice were subjected to unilateral sciatic neurectomy, and another side sham surgery(day 0) under isoflurane-induced anesthesia. Intravital bone imaging by two-photon excitation microscopy allowed
us to directly visualize lacuno-canalicular systems in cortical bone of tibiae. Osteocyte lacunae and the canalicular
network were detected by calcein (s.c.) staining, and local acidification areas in these structures were monitored
using a topically applied pH sensor. Finally, we could identify ‘osteocytic osteolysis’, or resorption of bone in the
lacuno-canalicular system by osteocyte in vivo.
Results and conclusion:
We demonstrated that sciatic neurectomy causes significant acidification around osteocytic lacunae and enlargementof the areas. These results provide strong evidences for ‘osteocytic osteolysis’, and demonstrate that two-photon
excitation microscopy is useful for analyzing the function of osteocytes in vivo.

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13th Congress of the International Society of Bone Morphometry

Nck adaptor proteins control osteoblastic migration, boneformation and bone mass
Smriti Aryal A.C1, Yoichi Ezura1, Kentaro Miyai1, Izu Yayoi1, Tony Pawson2,
Masaki Noda1
Department of Molecular Pharmacology, Tokyo Medical and Dental University, Tokyo, Japan
Mount Sinai Hospital, Samuel Lunenfeld Research Institute, Toronto, Canada
Osteoblastic migration and spreading are key events in the maintenance of bone remodeling and bone mass and they
are under the control of cytoskeletal activity in osteoblasts through molecular mechanisms, which are incompletely
understood. Ncks (non- catalytic region of tyrosine kinase) are SH2 SH3 domain containing adaptor proteinsthat play a major role in regulating actin cytoskeleton and cell migration. However the role of Nck in osteoblast
cytoskeleton organization and migration is unknown.
Here, we present genetic evidence that osteoblast specific Nck conditional double knock-out mice (ob-Nck-dko)
generated under the control of 2.3kb type I collagen promoter showed significant reduction in trabecular bonevolume (BV/TV) and trabecular number (Tb.N/BS) resulting in osteoporosis, defect in primary osteoblast migration,
suppression of repair and spreading after bone injury in vivo compared to control (Ct). Bone histomorphometry
revealed that the mineral apposition rate (MAR), bone formation rate (BFR) and mineralized surface per bone surface(MS/BS) were all reduced in the trabecular bone of Ob-Nck-dko mice compared to Ct suggesting that the individual
osteoblast activity and function were affected in the trabecular bone in the absence of Nck. Nck deficiency-induced
reduction of bone formation was independent from the alteration in osteoclastic activity. Mechanistically, Nck
regulates cytoskeletal reorganization and migration of osteoblasts. Nck also controls expression of genes encoding
proteins related to osteoblastic phenotypes. These data indicate that Nck adaptors are playing a role in controlling
cytoskeletal organization and bone formation to determine the levels of bone mass.

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April 27-29, 2015 ● Tokyo Garden Palace Hotel

Bone-implant interface: morphology and ultrastructure
Anders Palmquist1,2, Furqan A. Shah1,2, Kathryn Grandfield3, Peter Thomsen1,2
1) Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Sweden
2) BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Sweden3) Department of Materials Science and Engineering, McMaster University, Canada
Introduction:
The osseointegration of titanium has revolutionized the treatment of edentulousness. The underlying mechanisms
for a direct bone-implant interface include both material and host related factors. However, until recently thevast majority of ex-vivo analyses of bone tissue have been restricted to the micrometer level and the light optical
microscope. Bone is a highly hierarchical tissue with well-defined structures ranging from the nanoscale tothe macroscale. Further, intentional implant surface modification at the nanoscale is emerging in the field of
implantology, however, the specific role of this length-scale is unknown. The aim of the current paper is to provide a
correlative methodology for multidimensional and multiscale evaluation of the bone-implant interface.
Materials and methods:
The test material had a combination of micro- and nano-scale modifications created by laser ablation, confined to thethread valleys of a screw-shaped titanium implant. Bone-implant samples were retrieved from experimental animalstudies in rabbits (8 weeks and 6 months healing), as well as retrieved clinical dental implants (2.5 months and 3.9
years healing). Samples were formalin fixed and resin embedded prior to analysis. Multiscale analyses included
biomechanical testing, microCT, histology, backscattered electron SEM, FIB, TEM, and electron tomography.
Results:
Significantly higher removal torque was needed to unscrew the modified implants as compared to unmodified
controls, together with a different failure mechanism. Load-deformation plots indicated a fracture in the bone rather
than at the interface, which was histologically confirmed. Comparable high amounts of bone had formed aroundboth implant types, with increasing amounts after longer healing times. Clinically retrieved samples showed a highdegree of osseointegration with > 80% bone-implant contact after 3.9 years in function. Well-defined osteonal bone
occupied the thread area with osteocytes aligned with the implant surface and the bone lamellae. At the nano-scale,a chemical intermix of calcium and phosphorous was found with the nano-rough surface oxide, creating a gradedinterface. Osteocyte processes were frequently observed close to the implant surface.
Conclusion:
The results clearly demonstrate the importance of using complementary analytical techniques ranging across thelength-scales in order to increase the knowledge of the structural aspects of osseointegration and bone bonding.

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13th Congress of the International Society of Bone Morphometry

Comparative histological study in maxillary sinus augmentation
using coarse or fine particle of xenograft materials(preliminary report)
Paksinee Kamolratanakul DDS, Ph.D
Associate Prof. Pornchai Jansisyanont, DDS, MS
Chulalongkorn University, Thailand
Purposes:
To compare the histology of the bone healing in maxillary sinus augmentation using coarse grained particle or finegrained particle of xenograft materials.
Materials and methods:
Double blinded, clinical trial study was performed in 10 patients with posterior maxillary edentulous ridge
associated with sinus pneumatization. Patients were subjected to lateral window technique of sinus lift procedureusing xenograft material commercially prepared as coarse particle grafting (1-2mm) in first group and fine particle(0.25-1mm) in second group. Bone specimens were harvested after 6 months of augmentation and subjected tohistomorphological analysis for newly-formed bone, residual particle and fibrous tissue formation.
Results:
The preliminary data initially showed more newly-formed bone significantly (p value=0.002) in the group of sinus
lift using coarse particle of xenograft (mean of percentage area=22.47%,) compared to the group using fine particleof xenograft(mean of percentage area=11.26%). Residual particle in the group of coarse particle xenograft (meanof percentage area=6.19%) was more than in the group of fine particle xenograft (mean of percentage area=1.88%)
without the statistic significance. The group of fine particle xenograft revealed abandon of fibrous tissue surrounding
the space of particle (mean of percentage area=86.85%) significantly compared to the group of coarse particle
xenograft ( mean of percentage area=71.38%) with p value=0,021.
Results and conclusion:
Coarse particle of xenograft material trends to promote more newly-formed bone in maxillary sinus augmentation
which is possibly due to the more space between particle that provides enough space for ingrowth bone cell, celladhesion and migration of neovascularization. We further study the immunohistochemistry to evaluate the bonehealing and microstructure of newly-formed bone will be analyzed using micro-computerized tomography.

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April 27-29, 2015 ● Tokyo Garden Palace HotelIs it time to say goodbye to SMI?
Phil Salmon (1), Michael Doube (2), Asa Tivesten (3)1) Bruker-microCT, Kontich, Belgium2) Royal Veterinary College, University of London, UK
3) Sahlgrenska Institute, Goteborg, Sweden
Purposes:
Osteoporotic deterioration of trabecular bone involves a transition from plate-like to rod-like architecture. The
structure model index (SMI) [1] measures convex curvature and has long been used to quantify this transition. But
it has a significant short-coming: it is confounded by porosity. Both high porosity and high percent volume result in
extensive concave curvature, whose existence is ignored in the standard narrative of the shape-dependence of SMI.
Traditionally SMI ranges from 0 (plate) to 3 (rod); however in real bone samples negative SMI routinely arises fromhigh porosity. Different combinations of trabecular plate-rod architecture and porosity can give the same SMI. To
improve upon SMI two alternatives are presented.
Materials and methods:
The first is the Ellipsoid Factor (EF), a development of the 3D local thickness algorithm [2], but with fitted
ellipsoids in place of spheres [3]. The ellipsoid has freedom to assume a spectrum of shapes from oblate (flat discus
shaped, EF→-1) to prolate (long javelin shaped, EF→1). Thus EF ranges from -1 (ideal plate) to +1 (ideal rod).
The EF algorithm calculates both a mean value of EF and a 3D spatial map of local EF values. However EF is nota discretization method artificially dividing the trabecular continuum into connecting pieces in contrast to otherproposed methods.
The second alternative to SMI is a much simpler parameter; the "unplate index" (uPi) is introduced which is
the ratio of the trabecular "direct" thickness measured in 3D by sphere-fitting, Th(d), to that calculated from thehistomorphometric plate model (2BV/BS). Thus uPi = Th(d).BS/2BV. For an ideal plate uPi = 1 and with departurefrom plate shape the value increases above 1.
Results and conclusion:
MicroCT datasets of rat femoral trabecular bone from an OVX study into the efficacy of sex steroid drugs [4] is used
to compare the values of SMI, EF and uPi. This study is a case where high BV/TV from anabolic drug treatment
resulted in negative SMI. Both EF and uPi are shown to be free of this problem of out-of-range values arisingfrom porosity which compromises SMI, and thus represent a potential step toward less ambiguous measurement oftrabecular architecture.
1. Hildebrand T, Ruegsegger P (1997) Comp. Meth. Biomech. Biomed. Eng. 1: 15-23.
2. Remy E, Thiel E (2002). Pattern Recognition Letters 23: 649–661.
3. Doube M (2015) Frontiers in Endocrinology / Bone Research, Vol. 6 article 15. doi: 10.3389/fendo.2015.00015
4. Tivesten A et al.(2004) J. Bone Miner. Res. 2004; 19 (11):1833-1839.

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13th Congress of the International Society of Bone Morphometry

Labeling and seeding of MSCs on hydroxyapatite bone substitute:
cell behavior and in vivo tracking
Omar Omar1, 2, Ibrahim Elgali1, 2, Anders Palmquist1, 2, Anna Johansson1, 2, Birgitta
Norlindh1, 2 and Peter Thomsen1, 2
1 Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Sweden
2 BIOMATCELL, VINN Excellence center of Biomaterials and Cell Therapy, Gothenburg, Sweden
Purposes:
The role of biomaterial properties for bone regeneration is not fully understood. This process includes materialinduced effects on inflammatory cells and communication between monocytes/macrophages and mesenchymal
stromal cells (MSCs). Further, priming of bone substitutes with MSCs has been suggested to trigger the recipientbone microenvironment and promote bone regeneration, but the mechanisms are largely unknown. This is partly due
to the lack of few in vivo models that allow the tracking of the MSCs and their spatial distribution in the augmented
defect. This study aimed to develop a model for seeding MSCs on hydroxyapatite (HA) granules, with possibilities
to track the implanted cells in vivo, but yet without jeopardizing their osteogenic potential.
Materials and methods:
Rat MSCs were labeled with iron oxide beads at different bead concentrations, and the effect of labeling wasevaluated in vitro after 1, 6 and 28d. Thereafter, labeled MSCs were seeded on HA granules at different seeding
densities. To determine the possibility to track the labeled MSCs in vivo, the combined MSCs-HA granules were
implanted in rat femur defects and samples were retrieved, after 1h and 3d, for histology, BS-SEM and EDS
analyses.
Results:
After 1d, the viability was slightly lowered with the high bead concentration compared to medium and low. With
FGF, the number of MSCs was doubled after 6d for all groups. Under osteogenic condition, higher Ca and P wererecorded, after 28d, for the medium and high bead concentration groups. Viability of labeled MSCs was slightly
lower in the high (5×106) seeding density group compared the medium (5×105) and low (2.5×105) seeding densities.
In vivo, it was possible to detect the darkly stained cells, due iron content, using Prussian Blue staining. Detection ofiron signal was also possible using BS-SEM and elemental mapping with EDS.
Conclusion:
A model was developed consisting of a labeling procedure and in vivo implantation of combined MSCs-HA
granules, which allowed for tracking of the implanted MSCs. In combination with morphological and molecularanalytical tools, the model will enable detailed studies on the interactions between implanted MSCs, properties of theimplanted material and the recipient local environment.

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April 27-29, 2015 ● Tokyo Garden Palace Hotel

Apatite orientation corresponds to the shape of the osteocyte
lacuna in sheep trabecular bone
Furqan A. Shah 1, 2, Ezio Zanghellini 3, Aleksandar Matic 3, Peter Thomsen 1, 2,Anders Palmquist 1, 2
1 Department of Biomaterials, Sahlgrenska Academy at University of Gothenburg, Göteborg, Sweden
2 BIOMATCELL VINN Excellence Center of Biomaterials and Cell Therapy, Göteborg, Sweden
3 Department of Applied Physics, Chalmers University of Technology, Göteborg, Sweden
Introduction:
Osteocyte lacunae (Ot.Lc) in lamellar bone are aligned parallel to the collagen fibre direction. While the organic
matrix guides the mineral directionality, it is not entirely known what causes the organic phase to become organised
and whether a parallel relationship exists between osteocytes and the mineral phase. Here, the ultrastructural
relationship between the shape of the Ot.Lc and the orientation of nanoscale mineralised structures at the Ot.Lc floor
was evaluated.
Materials and methods:
2 mm thick longitudinal slices of trabecular bone obtained from the tibia and femur of sheep were deproteinisedby soaking in NaOCl, followed by dehydration in a graded ethanol series. Scanning electron microscopy, energy
dispersive x-ray spectroscopy (EDX), and Raman spectroscopy were utilised for structural characterisation andchemical analysis. The OrientationJ plugin for ImageJ,was used for image analysis.
Results:
Following deproteinisation, the Ca/P ratio was 1.69±0.1 at the Ot.Lc floor and 1.68±0.05 externally. Raman
spectra only showed peaks associated with carbonated apatite, while signals from collagen and other organic
components were mostly absent. Compared to untreated bone, the apatite-to-collagen ratio of deproteinisedbone was considerably higher; the mineral crystallinity and the carbonate-to-phosphate ratios however remained
similar. Relative to the Ot.Lc long axis, the mean orientation (Fig. 1) of mineral structures at the Ot.Lc floor was
19.01±14.35° (tibia) and 19.79±13.92° (femur). The mean coherency was 37±7% (tibia) and 38±9% (femur). Linear
regression analysis showed poor correlation between the coherency and orientation. Of all the Ot.Lc evaluated,
69.37% (tibia) and 74.77% (femur) exhibited a mean orientation of mineralised structures between 0–25°. The
highest fraction of lacunae showed mineral orientation in the narrow 15–20° range; 17.12% (tibia) and 19.8% (femur).

Fig. 1: (a) The shape of the osteocyte lacuna observed by scanning electron microscopy, and (b) the correspondingorientation/directionality analysis.
Conclusion:
The directionality of the mineralised structures at the Ot.Lc floor closely follows the shape of the Ot.Lc suggestingthat collagen production is a spatially and temporally synchronised process.

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13th Congress of the International Society of Bone Morphometry

Housing mice at room temperature results in premature bone loss:
Implications for the mouse as a model of osteoporosis
Urszula T. Iwaniec1, Kenneth A. Philbrick1, Carmen P. Wong1, Dawn A. Olson1,
Adam J. Branscum2, and Russell T. Turner1
1 Skeletal Biology Laboratory, School of Biological and Population Health Sciences, Oregon State University,
Corvallis, OR, 97331, USA
2 Biostatistics Program, School of Biological and Population Health Sciences, Oregon State University, Corvallis,
OR, 97331, USA
Purpose:Mice, because of their small size, short lifespan and ease of genetic manipulation, are a mainstay for cutting edge
research in fundamental skeletal biology. However, in marked contrast to age-related cancellous bone loss inhumans, cancellous bone loss in weight bearing long bones in mice begins during growth, even as cortical bone
continues to be accrued. As a consequence, mice exhibit low cancellous bone volume fraction prior to skeletal
maturity (4 months of age). Unlike humans who are homeotherms, mice are facultative daily heterotherms,
experiencing cyclic changes in core temperature and bouts of torpor when subjected to cold stress (temperatures
below thermoneutral; ~30°C for mice) and/or caloric restriction. Activation of sympathetic signaling is importantfor mice to successfully adapt to low temperature and suppression of sympathetic tone has been reported to increase
cancellous bone in mice. We therefore hypothesized that premature age-related cancellous bone loss commonly seenin mice may be due to temperature stress induced by housing the animals at room temperature (20-24°C).
Materials and methods: 2-month-old female C57BL6/J mice were randomized to 1 of 2 temperatures (22°C or
32°C; n=10/group) and maintained for 2 months. Femora were evaluated by microcomputed tomography andhistomorphometry.
Results:
Mice housed at 32°C consumed less food than mice housed at 22°C. However, significant differences in body weight
were not detected with temperature. Furthermore, there were minimal temperature-dependent effects on corticalbone. In contrast, mice housed at the warmer temperature maintained higher cancellous bone volume fraction in
the distal femur metaphysis. The sparing effect of thermoneutral housing on cancellous bone was associated withincreased bone marrow adiposity, higher bone formation and no change in bone resorption.
Conclusions: Our findings support the conclusion that mild cold stress has a major negative impact on cancellous
bone mass in mice and that early onset cancellous bone loss in this species is due, in part, to cold adaptation. The
findings also suggest that studies using mice as a preclinical model to investigate interactions between energy
homeostasis and bone or the etiology of age-related bone loss need to account for confounding species differences inthermoregulation and energy metabolism.

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Texture analysis improvement in cortical evaluation: influence ofosteophyte in femoral neck in osteoarthritis.
Jean-Paul Roux, Gustavo Davi Rabelo, Nathalie Portero-Muzy, Stéphanie Boutroy,
Roland Chapurlat, Pascale Chavassieux.
INSERM UMR 1033 and Université de Lyon, Lyon, France
Purposes:
Osteoarthritis (OA) is a multi-factorial disease characterized by cartilage and bone lesions. In hip OA, thesubchondral plate is thickened and subchondral bone becomes sclerotic with the presence of osteophytes. It is usuallyacknowledged that OA might have a protective effect on fragility fracture and osteophytes have been suggested toplay a role in the bone strength.
Many studies have shown the importance of the evaluation of cortical bone by itself, bone resistance to rupture
depends on 40 to 90% of cortical bone at femoral neck. Cortical porosity is one major determinant of bone strength.
However, the limit of resolution of clinical or laboratory computerized tomography underestimates of the corticalporosity and overestimates the pore diameter, principally due to partial volume effect. To overcome this resolution
limitation, we propose the use of texture analysis.
Materials and methods:
Femoral neck samples were obtained during arthroplasty for hip osteoarthritis in postmenopausal women (n=18;
aged 66± 8 years). Microarchitecture was measured using a high-resolution peripheral quantitative computed
tomography system (XtremeCT, Scanco Medical, Switzerland), with a nominal isotropic voxel size of 82 μm. The
following cortical parameters were measured: thickness (Ct.Th), thickness heterogeneity (Ct.ThSD), porosity (Ct.
Po), Fractal Dimension indicating how much an object fills up the space using threshold or unthreshold-based
methods (respectively Ct.FDbin, Ct.FDgray) and Lacunarity (Ct.Lac), a scale dependent measure of heterogeneity of
texture.
These approaches of evaluation were performed due to the possibility of the use of texture analysis by threshold or
unthreshold-based methods, which allowed the identification of some extra characteristics of bone channels, related
to their distribution and their spatial organization.
Results:

Results and conclusion:
The osteophyte area was characterized by a higher Ct.FD than the opposite quadrant of the femoral neck. Similar
results were observed with the two methods (Ct.FDbin and Ct.FDgray). Ct.Th and Ct.ThSD were significantly
higher in the osteophyte quadrant, than in the opposite part. Lacunarity and porosity were not significantly different
between osteophyte and opposite quadrant. Osteophyte was characterized by a higher complexity than the other partof femoral neck. This may influence the mechanical properties of the femoral head.

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13th Congress of the International Society of Bone Morphometry

Accuracy of clinical computed tomography for measurement ofbone quality: a study in cadavers
Miyuki Takasu1), Chikako Fujioka2), Masao Kiguchi2), Eiji Nishimaru2),
Koumei Takauchi2), Masafumi Machida3), Nobuhito Nango4), Kazuo Awai1)
1) Department of Diagnostic Radiology, Hiroshima University, Japan
2) Department of Radiology, Hiroshima University, Japan
3) National Institute of Musculo-Skeletal Disorders, Murayama Medical Center, Tokyo, Japan
4) Ratoc System Engineering Co., Ltd, Tokyo, Japan
Purpose:The application of clinical multidetector CT (MDCT) in evaluating bone quality has become a clinical research toolfor in vivo studies. The purpose of this study was to determine the accuracy of MDCT for architectural measurements
through a comparison with micro computed tomography (micro-CT) as the gold standard.
Materials and Methods:
L1 and L2 vertebrae of 10 fresh human cadavers were scanned by 64-section MDCT (LightSpeed VCT;
reconstruction kernel, BONEPLUS; collimation, 64 × 0.625 mm), 16-section MDCT (LightSpeed Ultra16;
BONEPLUS, 16 × 0.625 mm), 320-section MDCT (Aquilion One; FC30, 80 × 0.5 mm), 80-section MDCT (Acquilion
One Vision Edition; FC30, 80 × 0.5 mm), 16-section MDCT (Aquilion LB; FC30, 16 × 0.5 mm), 32-section MDCT
(Biograph mCT-S(64); B75f, 64 × 0.6 mm), and by micro-CT (TOSCANER 30000). The reconstructed voxel sizes
were 0.2×0. 2×0.16 mm for MDCT and 50×50×80 mm for micro-CT. Trabecular structural parameters and BMC/TVof the central 10-mm thickness portion of the vertebrae were calculated. Relationships between MDCT-derived andmicro-CT-derived trabecular indices were compared using Spearman’s correlation coefficient. The Scheffe’s post hoc
test was used to test for significant differences among the different MDCT systems and the micro-CT measurements.
Results:
BMC/TV measured by MDCTs significantly correlated with micro-CT measures (ρ=0.84-0.88). Seven of nine ofmorphological parameters measured by MDCTs significantly correlated with micro-CT measures. The correlation
coefficient was the highest for bone volume/tissue volume (BV/TV, ρ=0.89-0.91). Conversely, degree of anisotropy(ρ=0.14-0.6) showed the lowest correlation with micro-CT. Trabecular thickness and trabecular width did not show
significant correlation with micro-CT measures. The mean values of trabecular spacing, fractal dimension, and BV/
TV did not differ from those of micro-CT. BMC/TV was significantly higher in five of six MDCTs than in microCT. For three out of nine morphological parameters, 80-section MDCT had the highest correlation with micro-CT
measurements.
Conclusion:
The validation here of MDCT against gold-standards micro-CT suggests that indices of bone quality including BV/
TV may be appropriate for clinical monitoring of bone quality by MDCT. The degree of over- or underestimation oftrabecular parameters is important when absolute values are needed.

58April 27-29, 2015 ● Tokyo Garden Palace Hotel

Melatonin suppresses the microgravity-induced activation of
osteoclasts in cultured goldfish scale
Mika Ikegame 1), Atsuhiko Hattori 2) , Tatsuki Yamamoto 3), Kei-ichiro Kitamura 4),
Yoshiaki Tabuchi 5), Masaki Nakano 2), Sachiko Yano 6), Toshio Yamamoto 1),
and Nobuo Suzuki 3)
1) Okayama University Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Japan
2) College of Liberal Arts and Sciences, Tokyo Medical and Dental University, Japan
3) Institute of Nature and Environmental Technology, Kanazawa University, Japan
4) Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Japan
5) Life Science Research Center, University of Toyama, Japan, 6) Japan Aerospace Exploration Agency, Japan
Purposes:
Melatonin is a hormone secreted from the pineal gland that controls circadian rhythms and that has recently beenshown to affect various biological activities in peripheral tissues, including bone. In particular, melatonin has beenreported to suppress osteoclast differentiation. Although previous studies indicate that microgravity influences
osteoclast activity, the effect of melatonin on osteoclasts under microgravity conditions is unknown. Teleost scale isa calcified tissue similar to mammalian bone. Building upon our previous study showing that melatonin suppresses
osteoclastic activity in goldfish scales, the present study investigated the effects of melatonin on scale osteoclasts
under modeled microgravity.
Materials and methods:
Microgravity was modeled by a three-dimensional clinostat, in which two axes rotate simultaneously to randomize
the gravity vector. Regenerating goldfish scales in 96-well plates were set on the clinostat and rotated for 4 days withor without melatonin in the culture medium. Plates with control scales were incubated in the same environment but
were not set on the clinostat. At the end of the culture period, scales were fixed with paraformaldehyde. We detected
tartrate-resistant acid phosphatase activity as a marker of osteoclasts and assessed the presence of actin rings thatform at the matrix adherent site of resorbing osteoclasts. We also examined the presence of melatonin receptors in
the regenerating scales by immunohistochemistry.
Results and conclusion:
Modeled microgravity significantly increased the number of nuclei and the formation of actin rings in scale
osteoclasts. Melatonin significantly suppressed the microgravity-induced multinucleation and actin ring formation
but not to the same extent as that in the control condition. Melatonin receptors were detected in osteoblastic cellson the periphery of the osseous layer and the surface of the fibrillary plate. Therefore, melatonin appears to exert asuppressive effect on the differentiation and activation of osteoclasts under modeled microgravity by interacting withosteoblastic cells in the scales.

5913th Congress of the International Society of Bone Morphometry

Histomorphometric analysis of bone to assess safety andmechanical performance of low dose fluoride
Michael Dray 1), Andrew Grey 2), Karen Callon 2), Maureen Watson 2),
Dharshini Sreenivasan 3), Justine Fernandez 3) and Jillian Cornish 2)
1) Histology Department, Waikato Hospital, New Zealand2) Department of Medicine, University of Auckland, New Zealand
3) School of Engineering Sciences, University of Auckland, New ZealandPurposes:
To investigate the skeletal effects of low dose fluoride
Materials and methods:
A cohort of 24 post-menopausal women from a larger group of 180 participants with osteopenia in a double-blind,placebo-controlled randomized trial volunteered for transiliac bone biopsy after tetracycline double labeling. Prior
to preparation of undecalcified sections for histomorphometric analysis the alcohol fixed cores of bone underwent
micro-CT scanning. Histomorphometric and micro-CT derived parameters relating to mineralization and trabecularbone structure were submitted for further computational modeling.
Results:
23 of 24 biopsies were evaluable. Placebo, 2.5, 5, and 10mg fluoride per day groups showed differences inmineralization. Osteoid surface tended to be higher in the fluoride treated group, P=0.08, with values in 4 participants
exceeding the published normal range. Osteoid volume was not different, P=0.17, although the same 4 participants
exceeded the normal range. Osteoid thickness, mineralization lag time, and mineral apposition rate did not differ.
Pooled data from the fluoride treated group showed an increase in osteoid surface P=0.02, and osteoid volume
P=0.03, but no difference in other parameters. By comparison participants in an earlier trial who received 20mg/day
for 4 years showed hyperosteoidosis and prolongation of mineralization lag time.
Computational modeling of the micro-CT structural data enabled estimation of bone strength, assessment of bone
surface density, trabecular separation and intersection surface, in addition to percentage bone volume, trabecular
thickness and trabecular number. This 3D data was compared to the measured 2D histomorphometric data.
Percentage bone volume was observed to be a strong surrogate for mechanical strength R2=0.83, while bone
surface density R2=0.6, trabecular thickness R2=0.5, and intersection surface R2=0.6, showed moderate correlation.
Trabecular separation R2=0.31, and trabecular number R2=0.35, were only mildly correlated. Substituting the 3D
indices with 2D measurements decreased the correlation by up to 58%.
Results and conclusion:
Even small doses of fluoride show mild impairment of mineralization and a tendency towards hyperosteoidosis
without showing an anabolic effect on structural parameters. Micro-CT examination can give good structural
data upon which to base computational modeling and assess bone strength. Dynamic mineralization and osteoidparameters to assess drug safety remain an important role of 2D histomorphometric analysis.

60April 27-29, 2015 ● Tokyo Garden Palace Hotel

Craniofacial anomalies in cartilage calcification insufficient(CCI) rat
Hitoshi Amano1), Kakei Ryu 2), Minoru Watanabe 3), Motohiko Nagayama 4),
Shunichi Shibata 5), Junichi Tanuma 4), Masami Tanaka 6), Kiyoshi Ohura 1)
1) Osaka Dental University, Japan
2) Showa University School of Medicine, Japan
3) Inst. Animal Exp.St. Marianna University, Japan
4) Asahi University School of Dentistry, Japan
5) The University of Aizu, Japan
6) Tokyo Medical and Dental University, Japan
Purposes:
The cartilage calcification-insufficient (CCI) rats were established as a naturally occurring dwarf mutant caused byan autosomal recessive mutation. Sprague-Dawley rat-derived CCI rats show impaired development accompaniedby short limbs, tail and overall size reduction as systemic changes. In this study, we further characterize thecraniofacial skeletal phenotype of the CCI rat strain.
Materials and methods:
Carcasses of 4, 8 and 13-week-old rats were fixed in 4% neutral buffered glutaraldehyde solution after the skin andvisceral organs had been removed [1]. The head and the bilateral femurs and tibias were excised. Radiograms wereacquired using soft X-ray apparatus. Specimens were embedded in Epon 812 without decalcification. Two µm-thicksections were stained with toluidine blue and von Kossa stain. Other specimen were decalcified and embedded inparaffin using standard procedures. Serial 5 µm-thick sections were used for immunohistochemistry.
Results:
CCI rats are characterized by systemic ossification deficiency of cartilage observed in soft X-ray and histologicalanalyses. An immunohistochemical examination detected an aggrecan synthesis disorder, which might have led todelayed calcification and increased growth plate thickening in the dwarf rats. CCI rat exhibit that the mandibularbone moved forward as compared with maxillary bone, and an edge-to-edge bite phenotype associated withcraniofacial dysmorphism.
Results and conclusion:
We hypothesized that the principal characteristics of the CCI rats were systemically induced by insufficient cartilage
calcification in their skeletal growth.

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13th Congress of the International Society of Bone Morphometry

Temporal association of tensin 1 and p130Cas at focal adhesions
links actin inward flux to cell migration
Zhihai Zhao1), Song Hui Tan1), Hiroaki Hirata1) and Yasuhiro Sawada1), 2)
1) National University of Singapore, Singapore
2) National Rehabilitation Center for Persons with Disabilities, Japan
Purposes:
p130Cas (Crk-associated substrate; hereafter Cas) has been reported to be involved in various cellular processes,
including the mechano-sensing by which physical information is converted to a biochemical event (Sawada et al.,Cell 2006). However, the molecular mechanism of how Cas relates to force-bearing or –generating cell machinery,i.e., actomyosin system, is poorly understood. We have recently reported that actomyosin contraction displaces
Cas molecules from focal adhesions depending upon their phosphorylation, thereby promoting cell migration(Machiyama et al., J. Cell Sci. 2014). The purpose of this study is to further delineate the mechanism underlying theCas dissociation from the adhesion complexes.
Materials and methods:
An actin-binding protein tensin1 is tested as a candidate that links actin cytoskeletons to phosphorylated Cas at focal
adhesions. We analyzed the co-localization of Cas and tensin1 with particular reference to the phosphorylation of
Cas and the domain specificity of tensin1. We biochemically examined the phosphorylation-dependent, domainspecific interaction between Cas and tensin1 by GST-pull down or co-immunoprecipitation experiments. Conducting
FRAP (fluorescence recovery after photobleaching) analysis using a TIRF (total internal reflection fluorescence)microscope, we examined whether tension1 regulated the turnover of Cas molecules at focal adhesions.
Results:
Analysis of the subcellular distribution reveals that tensin1 co-localizes with Cas, but not its phosphorylationdefective mutant at the cell lamellipodia. While a truncation mutant of tensin1 that lacks the phosphotyrosine-binding
region, SH2PTB domain, does not co-localize with Cas, bacterially expressed recombinant tensin1-SH2PTB protein
binds to Cas in vitro in a Cas phosphorylation-dependent manner. Furthermore, the exogenous expression of tensin1SH2PTB, which is devoid of the actin-interacting motifs, interferes with the Cas-driven cell migration, slows down
the inward flux of Cas molecules, and impedes the displacement of Cas molecules from focal adhesions.
Results and conclusion:
Tensin1 transmits actomyosin contractility to adhesion sites by temporally binding to phosphorylated Cas, providing
a clutch that supports cell migration.

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Poster Sessions

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13th Congress of the International Society of Bone Morphometry

64April 27-29, 2015 ● Tokyo Garden Palace Hotel

Alveolar bone healing after the removal of temporary anchorage
devices
Eunji Kim, Yoon Jeong Choi, Kyung-Ho Kim, and Chooryung J. Chung
Department of Orthodontics, Gangnam Severance Dental Hospital, College of Dentistry, Yonsei University, Korea
Purposes:
Temporary anchorage devices (TADs) such as miniscrews are frequently used for contemporary orthodontic
treatment. However, visible scarring is noted at the removal sites and the bone healing process after the removal of
TADs is still unknown. Therefore, the purpose of the study is to evaluate the course of alveolar bone healing after theremoval of orthodontic miniscrews.
Materials and methods:
Total of 32 orthodontic miniscrews (1.6mm in diameter) were inserted in the interdental bone (both maxilla andthe mandible) of two beagle dogs. Miniscrews were removed serially and calcein/ tetracycline was systemicallyinjected for florescent labeling. Micro CTs were taken just after, 4,7, and 10 weeks after the removal the miniscrews(n=4). Bone volume (BV)/ tissue volume (TV) of the miniscrew removal sites were measured and compared to
the neighboring interdental bone (control) followed by histologic evaluation of mineral apposition rate and boneformation rates of the interdental bone after the removal of miniscrews.
Results:
Just after the removal, BV/TV of the removal sites was significantly reduced compared to the control interdentalbone (p<0.05). BV/TV was gradually increased and reached the levels of the control bone after 7 weeks in both themaxilla and the mandible. However, in some mandibular removal sites, rough irregular cortical surfaces were notedeven after 10 weeks.
Results and conclusion:
Because of the small diameter and atraumatic insertion and removal procedures of TADs, many clinicians expect
complete healing after its removal. However, the removal of TADs leaves a transit full thickness defect penetrating
the oral cavity, soft tissue and the underlying alveolar bone. Fortunately, BV/TV reached similar levels to the control
after the period of 7 weeks in both the maxilla and the mandible indicating natural healing of the alveolar bone.
However, some cortical surface roughness remained in the mandible even after 10 weeks. Thus, long term bone
remodeling of the TAD removal sites should be monitored carefully in the clinical setting.

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13th Congress of the International Society of Bone Morphometry

Inhibition of histone deacetylases enhances the osteogenic
differentiation of human periodontal ligament cells
Nam Cong-Nhat Huynh 1) 4), Vincent Everts 2), Prasit Pavasant 1) 3),
Ruchanee Salingcarnboriboon Ampornaramveth 4)
1) Mineralized Tissue Research Unit, Faculty of Dentistry Chulalongkorn University, Bangkok, Thailand.
2) Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA), University of Amsterdam
and VU University Amsterdam, Research Institute MOVE, Gustav Mahlerlaan 3004, 1081 LA Amsterdam, The
Netherlands.
3) Department of anatomy, Faculty of Dentistry Chulalongkorn University, Bangkok, Thailand.
4) DRU in Oral Microbiology, Microbiology department, Faculty of Dentistry Chulalongkorn University, Bangkok,
ThailandThe purpose of this study was to investigate the possible role of histone deacetylases (HDACs) in osteogenic
differentiation of human periodontal ligament (hPDL) cells.
Methods:
The activity of HDACs was blocked by using the inhibitor trichostatin A (TSA). The effect of TSA on proliferationand cytotoxicity of primary hPDL cells was tested by MTT assay. Osteogenic and adipogenic differentiation were
induced in the presence of various concentrations of TSA. Gene expression was assessed by semi-quantitive and realtime RT-PCR. ALP activity and mineral deposition assays were used to assess osteoblast phenotype. Western blotting
was used to investigate the effect of TSA on histone acetylation and the expression of different HDACs.
Results:
During the course of osteogenic differentiation in the presence of the HDAC inhibitor, osteoblast-related gene
expression was increased by hPDL cells. ALP activity as well as bone nodule formation were also enhanced underthese conditions. Inhibition of HDACs did not induce differentiation into the adipocyte lineage. hPDL highly
expressed HDACs of both class I (HDAC 1, 2, 3) and class II (HDAC 4, 6). During osteogenic differentiation
HDAC 3 expression gradually decreased. This was apparent in the absence and presence of the inhibitor. The levelof acetylated Histone H3 was increased during osteogenic differentiation. Inhibition of HDAC activity induced
hyperacetylation of Histone H3 therefore demonstrating Histone 3 as a candidate target molecule for HDAC
inhibition in our model. In conclusion, hPDL cell express a distinguished series of HDACs and these enzymes appear
to be involved in the osteogenic differentiation; their inhibition promotes the latter process.

66April 27-29, 2015 ● Tokyo Garden Palace Hotel

Principles of selection methods of radiation diagnosis of
postmenopausal osteoporosis
Zakharov IS, Kolpinskiy GI, Ushakova GA, Shkaraburov AS, Popova OP
Russia, Kemerovo State Medical Academy
The main characteristic of osteoporosis is a decrease in bone mineral density (BMD). BMD determined radiation
methods – dual energy X-ray absorptiometry (DXA) and quantitative computed tomography (QCT). Studies,including a comparative assessment of the diagnostic value of the QCT and the DXA in identifying postmenopausal
osteoporosis, few (Bansal SC, 2011; Pickhardt PJ, 2011), and the question about the benefits of a particular method
remains open. Given that most actively decreased bone mineral density in women occurred at the age of 50 years,
comparing the results of different modalities of diagnostic imaging in the evaluation of BMD in postmenopausal
women is relevant.
The aim of the study was a comprehensive assessment of indicators of bone mineral density in women aged 50 years
and older by dual-energy X-ray absorptiometry and quantitative computed tomography.
Material and methods:
The study included 210 postmenopausal women who are in the range of 1–2 weeks densitometry was performed bydual-energy X-ray absorptiometry and quantitative computed tomography. Depending on patient age were divided
into four groups: I group (50–59 years, n=73), II group (60–69 years, n=58), III group (70–79 years, n=53), IV group(80 years and older, n=26).
Results:
Assessment of BMD in women in the age group 50-59 years did not reveal significant differences in the results ofdensitometry. In carrying out the DXA osteoporosis was diagnosed in 15.1%, with QCT – 20.5% of the patients.
From the age of 60 years and older incidence of osteoporosis by QCT was higher than in the DXA (44.8% and24.1%, respectively, p=0.001). In assessing the correlation was determined positive correlation of moderate intensity
between the results of the DXA and the QCT, which decreased with increasing age, with a significant difference wasobserved with 70 years (p=0.02). Probably, these results are due to age-related degenerative involutional processes inthe vertebrae, causing occurs overstatement of the BMD during the DXA.
Conclusions:Thus, with increasing age of the women surveyed noted the discrepancy densitometry results depending on themethod used. Women after 60 years of age is recommended densitometry by QCT to improve the quality of diagnosisof osteoporosis.

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13th Congress of the International Society of Bone Morphometry

TGF-beta impairs cilia morphology via suppression of Ift88
expression in chondrocytic ATDC5 cells
Makiri Kawasaki, Tadayoshi Hayata, Tetsuya Nakamoto, Takuya Notomi,
Shuichi Moriya, Takayuki Yamada, Yayoi Izu, Yoichi Ezura, Masaki Noda
Tokyo Medical and Dental University, Japan
Purposes:
Osteoarthritis is a serious disease affecting over 25 million patients, while molecular and cellular bases are still
incompletely understood. Cilium is a critical structure for chondrocytes and cartilage. However, how this structure
is controlled by cytokines that play critical roles in osteoarthritis is not well understood. Therefore, we examined theeffects of TGF beta on the morphology of cilia in a chondrocytic cell line, ATDC5.
Materials and methods:
We cultured ATDC5 cells under the presence of various concentrations of recombinant human TGF beta. For
immunocytochemistry, primary cilia were stained with anti-acetylated alpha-tubulin. Real-time PCR was performed
using SYBR Green after RNA extraction and subsequent reverse transcription.
Results:
TGF beta treatment reduced the number of cilia positive cells. TGF beta treatment not only reduced the cilia positive
cell population size but also suppressed the average length of cilia. Analysis of cilia length variation indicated thatTGF beta treatment reduced mode, median and minimum lengths of cilia. Interestingly, maximum length of cilia
was not affected indicating the heterogeneity in the sensitivity of length parameters to TGF beta treatment. As amechanism for this action, TGF beta suppressed Ift88 mRNA expression in ATDC5 cells. TGF beta suppressed thelevels of Ift88 mRNA in a dose-dependent manner starting from as low as 0.5 ng/ml and reaching maximal at around2 ng/ml. TGF beta suppressed the protein levels of Ift88. TGF beta suppression was not affected by the presence ofa protein synthesis inhibitor and a transcriptional inhibitor. In contrast, TGF beta treatment suppressed the levelsof Ift88 mRNA stability. Knockdown of Ift88 by siRNA enhanced TGF beta-induced increase in type II collagenmRNA expression revealing the suppressive role of Ift88 on TGF beta-induced regulation of extracellular matrix
protein expression.
Results and conclusion:
TGF beta impairs cilia morphology via suppressing Ift88 posttrascriptionally and this suppression leads to further
enhancement of TGF beta-induced increase in the expression of extracellular matrix protein.

68April 27-29, 2015 ● Tokyo Garden Palace Hotel

Membrane depolarization regulates intracellular RANKLtransport in non-excitable osteoblasts
Takuya Notomi 1), Miyuki Kuno 2), Yoichi Ezura 3), Kiyoshi Ohura 1), and Masaki Noda 3)
1) Osaka Dental University, Japan2) Osaka City University, Japan
3) Tokyo Medical and Dental University, Japan
Parathyroid hormone (PTH) are important factors in Ca2+ homeostasis, and promote osteoclastogenesis bymodulating receptor activator of nuclear factor kappa-B ligand (RANKL) mRNA expression. However, theircontribution to RANKL intracellular transport, including the trigger for RANKL lysosomal vesicle fusion to the cell
membrane, is unclear. In neurons, depolarization of membrane potential increases intracellular Ca2+ level ([Ca2+]i)and promotes neurotransmitter release via fusion of synaptic vesicles to the cell membrane. To determine whether
membrane depolarization also regulates cellular processes such as RANKL intracellular transport in MC3T3-E1
osteoblasts (OBs), we generated a light-sensitive OB cell line and developed a system for altering their membranepotential via delivery of a blue light stimulus. In the membrane fraction of RANKL-overexpressing OBs, PTH
increased membrane-bound RANKL 10 min after application without affecting the mRNA expression level,and depolarized the cell membrane while transiently increasing [Ca2+]i. In our novel OB line stably expressing
channelrhodopsin, wide receiver blue light-induced depolarization increased membrane-bound RANKL, which wasreversed by blockers for voltage-gated Ca2+ channels and Ca2+ release from endoplasmic reticulum. In co-cultures ofosteoclast precursor-like RAW264.7 cells and light-sensitive OBs overexpressing RANKL, light stimulation induced
an increase in tartrate-resistant acid phosphatase activity and promoted osteoclast differentiation. These results
indicate that depolarization of the cell membrane is a trigger for RANKL lysosomal vesicle fusion to the membrane,
and that membrane potential contributes to the function of non-excitable OBs. Elucidating the mechanism of
RANKL intracellular transport regulation by PTH will be useful for the development of drugs to prevent bone loss inosteoporosis and other bone diseases.

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13th Congress of the International Society of Bone Morphometry

Poldip2 is expressed in osteoblasts and its expression is enhancedby osteogenic differentiation medium containing beta
glycero-phosphate and ascorbic acid or BMP.
Sakie Katsumura, Yoichi Ezura, Yayoi Izu, Takayuki Yamada, Kiyoshi Harada,
Kathy Griendling, Masaki Noda
Tokyo Medical and Dental University, Japan
Emory University School of Medicine, Atlanta, USA
Purposes:
Osteoporosis is a bone disease that is characterized by a decrease in bone mass and density. This decrease is due tothe imbalance of bone formation by osteoblasts and bone resorption by osteoclasts. Osteoblastic cells are under thecontrol of oxidative stress, cell adhesion, aging and cell cycle progression. One of the molecules related to thesemodulating components is Poldip2. Poldip2 binds to p22phox and activates NADPH oxidase 4 (Nox4). This Nox4
is required for BMP2-induced alkaline phosphatase as well as BMP2 gene expression in osteoblasts. However, it
is not known whether Poldip2 is expressed in osteoblasts and if so, whether expression of this gene is regulated inosteoblasts. Therefore, we examined these points by using osteoblast-like cell line, MC3T3E1 cells.
Materials and methods:
Osteoblast like MC3T3E-1 were cultured in α-medium in the presence or the absence of BMP2. Some cultures wereconducted in the presence or the absence of ascorbic acid and β-glycerophosphate. The expression levels of Poldip2
and osteoblast-related mRNAs were examined based on real-time PCR.
Results and Conclusion:
We first examined the expression of Poldip2 mRNA in the cell cultures of MC3T3E1 cells and found that these cells
express Poldip2 mRNA constitutively. When the cells were cultured in the presence of beta glycerophosphate andascorbic acid (osteogenic medium) to induce differentiation, Poldip2 mRNA levels were increased along with timecourse in conjunction with the increase of alkaline phosphatase mRNA. These observations indicate that Poldip2 is
not only expressed in osteoblasts but its expression is regulated developmentally in these cells. As it was reported
that one of the factors that could play a role in such osteogenic medium would be BMP, we further examined whether
BMP alone regulates Poldip2 mRNA expression in these osteoblasts. BMP treatment enhanced the expression of
Poldip2 mRNA in these osteoblasts. This BMP effect was time dependent with a peak at 24 hours of the treatment.
We further examined if BMP effects on Poldip2 is dependent on dosage of BMP. Treatment with 50ng/ml BMP did
not significantly enhance Poldip2 mRNA expression while 100ng/ml BMP significantly enhance the expressionof Poldip2 mRNA in these osteoblastic cells. These data indicated that Poldip2 is one of the BMP target genes inosteoblasts.

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Bone regeneration is increased with local application of
Lactoferrin in critical-sized rat calvarial defects
Maureen Watson1, Ryan Gao1, Karen Callon1, Donna Tuari1, Jacob Munro2,
David Musson1, Jillian Cornish1
1) University of Auckland, Auckland, New Zealand2) Department of Orthopaedic Surgery, Auckland District Health Board, New ZealandPurposes:
Lactoferrin is an iron-binding glycoprotein which is present in milk and exocrine secretions in mammals. In vitro,
lactoferrin functions as a potent bone anabolic factor by increasing the proliferation, differentiation and survivalof osteoblasts and by inhibiting osteoclastogenesis. In vivo, local injection of lactoferrin above the hemicalvaria ofadult mice results in substantial increases in the dynamic histomorphometric indices of bone formation and bone
area. The objective of this study was to assess the ability of bovine lactoferrin to increase bone regeneration in a rat
critical-sized calvarial defect model.
Materials and methods:
Critical-sized defects (5mm diameter) were created in the right parietal bone of 60 male Sprague-Dawley rats withthe left parietal bone used as a contralateral control. The rats were randomised into three groups: Group 1- empty
defect; Group 2-collagen gel and Group 3- collagen gel containing 10μg bovine lactoferrin. The rats were sacrificed
at 4 or 12 weeks post-operatively and the calvaria were imaged by micro-CT (Skyscan 1172) with a 12μm voxel
size. Cylindrical volumes of interest were created and the percentage of bone formed in the defect was calculated
and referenced to the contralateral control.
Results:
The relative percentage of new bone formation in the defects, at both 4 and 12 weeks was significantly greater in thegroup treated with lactoferrin compared with other groups. The relative percentage of new bone formation in groups 1,
2 and 3 was 42.7±4.2%, 35.9±5.9% and 62.6±2.5%, respectively, at 4 weeks (P=0.0019); and 41.3±5.2%, 45.8±4.8%
and 74.6±4.3%, respectively, at 12 weeks (P<0.0001).
Results and conclusion:
This study demonstrated that local application of lactoferrin significantly increased bone regeneration in a rat criticalsized calvarial defect model. These findings suggest that lactoferrin has therapeutic potential for the treatment ofbony defects and fracture non-union.

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13th Congress of the International Society of Bone Morphometry

Collagens VI and XII form complexes mediating osteoblast
interaction during osteogenesis.
Yayoi Izu 1), Yoichi Ezura 1), Manuel Koch 2), David E. Birk 3), Masaki Noda 1)
1) Tokyo Medical and Dental University, Japan
2) University of Cologne, Germany3) University of South Florida, USA
Purposes:
Ullrich congenital muscular dystrophy (UCMD) and Bethlem myopathy (BM) are diseases representing overlapping
phenotypes of connective tissue and muscle. Both diseases are caused by genetic mutations in the genes encoding
collagen VI. Recently COL12A1 gene mutations were identified in UCMD and BM-like patients harboring no COL6
mutations, indicating shared functions of these collagens for connective tissue homeostasis. We have previously
demonstrated that the genetic deletion of Col6a1 or Col12a1 in mice cause bone mass reduction and fragility. The
purpose of this study is to determine whether collagens VI and XII have coordinate regulatory role (s) during boneformation.
Materials and methods:
Primary osteoblasts were obtained from neonatal mouse calvariae and were cultured in a medium supplemented
with 10% FBS or osteogenic medium. The cells were harvested on day 2, 4, and 11 and then analyzed based on
immunofluorescence microscopy using antibodies against collagen I, VI, and XII.
Results:
The double staining analysis revealed that co-localization of collagen VI and XII was detected in the matrix bridges
between adjacent cells on day 2 and 4 when osteoblasts were still considered in migration and proliferation phase. On
the other hand, collagen I was localized in a region close to the cell surface where it colocalized with collagen XII.
The percentage of the cells harboring collagens bridges was analyzed based on the immunostaining, demonstratingthat matrix bridges were were made via collagen VI and XII but not via collagen I.
Conclusion:
Our data indicate that collagen VI and XII have a novel coordinate function in the formation of cell communicating
networks at bone forming sites during bone formation.

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April 27-29, 2015 ● Tokyo Garden Palace HotelA rankl-binding peptide accelerates BMP-induced boneregeneration in murine maxilla by subperiosteal injections.
Tomoki Uehara1), Setsuko Mise2), Yuki Arai3), Yasutaka Sugamori2), Genki Kato2),
Yukihiko Tamura2), Yasuhiko Tabata4), Ramachandran Murali5), Noriyuki Wakabayashi3),
Michiyo Miyashin1), Kazuhiro Aoki2)
1) Department of Pediatric Dentistry, Division of Oral Health Sciences, Graduate School, Tokyo Medical and Dental
University (TMDU), Japan
2) Section of Pharmacology, Department of Bio-matrix, Graduate School, TMDU, Japan
3) Section of Removable Partial Prosthodontics, Department of Masticatory Function Rehabilitation, Graduate
School, TMDU, Japan
4) Department of Biomaterials, Field of Tissue Engineering, Institute for Frontier Medical Sciences, Kyoto
University, Japan
5) Department of Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
Purposes:
Although the beneficial effects of recombinant human BMP-2 on bone regeneration have been shown in many
clinical cases, recent evidences clearly revealed that BMP-2 increased cancer risk. To decrease the cancer risk, theBMP-2 amount needs to be reduced. Several cytokines has already known to be effective to reduce a BMP amountto accelerate BMP-2-induced bone tissue, but the protein drug is tend to cause sometimes life-compromising side
effects. We have recently developed peptide X, which binds to receptor activator of NF-κB ligand (RANKL),
thereby having the bone resorption inhibitory activities. Since the other RANKL-binding peptide WP9QY canaccelerate bone formation, we hypothesized that this peptide X could have the same effect. The aim of this study
is to investigate whether peptide X can promote BMP-2- induced bone formation in murine maxilla. In this study,
we have developed a unique drug delivery system to induce bone formation at the site of injection toward a clinicalapplication using this peptide X.
Materials and methods:
Twenty-four eight-week-old male C57BL/6J mice were used. Gelatin hydrogel was used as a carrier of the peptide
X and BMP-2. Mice were divided into 4 groups (n=6) depending on materials incorporated in the carrier; 1) BMP2(1 µg), 2) BMP-2(1 µg) + peptide X (0.56 mg), 3) BMP-2(1 µg) + control peptide (0.56 mg) or 4) vehicle. Those
carriers were subperiosteally injected into the maxillary diastema between the right incisor and the first molar.
Calcein and demeclocycline were subcutaneously injected on day 12 and 26, respectively. The mice were sacrificed 4
weeks after the subperiosteal injections, and the local bone formation was analyzed radiologically and histologically.
Results and conclusion:
Micro-CT analyses showed the highest radio-opacity at the site of injection in the group 2) compared with theother experimental groups. Bone mineral density and bone mineral content measured by pQCT confirmed theseobservations. Histomorphometric analyses revealed that demeclocycline-labeled area was significantly increasedin the group 2) compared with the other 3 groups. Interestingly, calcein-labeled area was observed outside of thedemeclocycline-labeled area. These results suggest that peptide X could accelerate the bone regenerating efficiency
induced by BMP-2.

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13th Congress of the International Society of Bone MorphometryBMP-2 induction of LGR4 expression in osteoblastic
MC3T3-E1 cells.
Chantida Pawaputanon na mahasarakham1,2), Makiri Kawasaki1), Shuichi Moriya1),
Takayuki Yamada1), Yayoi Izu1), Yoichi Ezura1), Katsuhiko Nishimori3)
Yuichi Izumi2), Masaki Noda1)
1) Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Japan
2) Department of Periodontology, Tokyo Medical and Dental University, Japan
3) Graduate School of Agricultural Science, Tohoku University, Japan
Purposes:
The leucine-rich repeat containing G protein-coupled receptor4 (LGR4) is a subfamily of GPCRs that plays animportant role in stem cell/progenitor proliferation. A nonsense mutation of LGR4 was found to be associated withseveral human diseases. Strikingly, this mutation is strongly associated with low BMD and osteoporotic fractures.
Consistently, the deletion of LGR4 in mice have delayed embryonic and postnatal bone formation and downregulated Runx2, Atf4, Ocn and Bsp. LGR4 is also thought to play a potential role in osteoblast differentiation
and bone formation. However, the regulation of LGR4 expression in osteoblasts is not known. The present study
therefore examines the effects of BMP2 on LGR4 expression in osteoblast-like MC3T3-E1.
Materials and methods:
Osteoblast like MC3T3-E1 cells were cultured in medium supplemented with or without BMP2. The expression
levels of LGR4 and osteogenic-related mRNAs were examined based on real-time RT-PCR. Furthermore, thefunction of LGR4 on BMP signaling-induced transcription was analysed by examining the influence of the down
regulation of LGR4 on BMP response element (BRE)-luciferase activity.
Results and conclusion:
We examined the response of LGR4 in Osteoblast like MC3T3-E1 cells. These cells expressed LGR4 mRNA and its
baseline levels slightly declined along with time course. BMP2 treatment enhanced the expression of LGR4 mRNAlevel. This enhancement started at 10hours after BMP2 treatment in MC3T3-E1 osteoblastic cells and peaked at
24 hours and then, maintained for 3 days. BMP2 enhanced LGR4 mRNA levels in these cells in a dose-dependent
manner starting at 10ng/ml and saturated at 50-100ng/ml. BMP2 enhancement of LGR4 expression was no longer
observed in the presence of a transcriptional inhibitor, DRB, and a protein synthesis inhibitor, CHX. Therefore,
LGR4 is regulated in a manner at least in part through transcriptional events and requires new protein synthesis.
The knockdown of LGR4 down-regulated the mRNA expression of OSX, ALP and BSP. Finally, LGR4 mRNA
knockdown also enhanced the BRE-reporter expression.
Conclusion:
Our data reveal for the first time that LGR4 is a novel target for BMP in these cells.

74April 27-29, 2015 ● Tokyo Garden Palace HotelParadoxical effects of partial leptin deficiency on bone in growing
female mice
Kenneth A. Philbrick1, Russell T. Turner1, Adam J. Branscum2, Carmen P. Wong1,
and Urszula T. Iwaniec1
1) Skeletal Biology Laboratory, School of Biological and Population Health Sciences, Oregon State University,
Corvallis, OR, 97331, USA
2) Biostatistics Program, School of Biological and Population Health Sciences, Oregon State University, Corvallis,
OR, 97331, USA
Purpose:Morbidly obese, leptin-deficient ob/ob mice display low bone mass, mild osteoclast-rich osteopetrosis, and increasedbone marrow adiposity. Partial leptin deficiency results in increased weight. However, the skeletal manifestationsof partial leptin deficiency are not well defined. We therefore analyzed femora and lumbar vertebrae in growing(7-week-old) female C57BL/6 wildtype (WT) mice, partial leptin-deficient ob/+ mice, and leptin-deficient ob/ob
mice.
Materials and methods:
Bones were evaluated by dual energy absorptiometry, microcomputed tomography, and histomorphometry.
Results:
As expected, ob/+ mice were heavier, had more white adipose tissue, and lower serum leptin than WT mice, butwere lighter and had less white adipose tissue than ob/ob mice. Bone mass, density and indices of bone architecturein ob/+ mice were not intermediate between WT mice and ob/ob mice. In general, cancellous bone architecture, cell(osteoblast, osteoclast, and adipocyte), and dynamic measurements were similar between WT and ob/+ mice. In
contrast, ob/ob mice differed from WT and ob/+ mice in that ob/ob mice had lower cancellous bone volume fraction
and higher bone marrow adiposity in the femur metaphysis, and higher cancellous bone volume fraction in lumbar
vertebra. Paradoxically, ob/+ mice had greater femoral cross-sectional bone volume, cortical volume, marrow
volume, and polar moment of inertia than WT mice and longer femora and higher bone mineral content and bonevolume compared to either WT or ob/ob mice. There was a positive correlation between body weight and femur
volume in all three genotypes. However, the positive effect of body weight on bone volume occurred at much lower
body weight in mice that produced leptin.
Conclusions:
The paradoxical differences in bone size among WT, ob/+, and ob/ob mice may be explained if leptin, in addition
to stimulating bone growth and cancellous bone turnover, acts directly or indirectly to lower the set-point at which
increased body weight leads to a commensurate increase in bone size.

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Osteocytes in primary cultures express higher levels of β2adrenergic receptor mRNA while MLOY4 cells exhibit culture
period dependent regulation of the expression levels.
Koji Mineta , Yoichi Ezura ,Yayoi Izu , Masaki Noda
Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical & Dental University, Japan
Introduction
Osteocytes are becoming more considered to play key roles in regulation of bone metabolism in addition to theactions of osteoblasts and osteoclasts. Osteocytes have been reported to express RANKL, FGF23, Phex, Sost andDMP-1. Although osteocytes are consisting about 90% of the cells in bone, full repertoire of its actions is still
incompletely understood. One of the functions of osteocytes is considered to be the mediation of mechanical stimuli
to bone. Based on the unloading-induced bone loss model using tail suspension, sympathetic tone has been suggested
to be related to such unloading-induced bone loss. However, the profile of sympathetic signaling-related moleculesin osteocytes is not well understood. Sympathetic tone suppresses bone formation and enhances bone resorption in
way similar to unloading-induced modulation of each arms of bone metabolism. In particular, β2 adrenergic receptor(Adrb2) is a major entry point of sympathetic tone. Isoproterenol binds toβ2 adrenergic receptor and enhancesosteopontin expression while loss of osteopontin attenuates β2 adrenergic receptor related events. Since osteopontin
is also required for the unloading-induced bone loss on both side of suppression of bone formation and enhancementof bone resorption and it is expressed in osteocytes, we focused on the expression of β2 adrenergic receptor in
osteocytes.
Methods
Primary osteocytes were obtained from 7-week-old mouse long bones. The cells were separated into early fractions(2-4) and late fraction (5-9). The cells were cultured for 10 days. These cells were subjected to the analyses ofmRNA expression of osteocyte markers (Sost, Dmp1, Phex, Fgf23), osteoblast markers (Alp, Col1a1) and Adrb2 by
real-time PCR. MLO-Y4 cells were also used.
Results and Conclusion
We found that mRNA of Adrb2 was expressed in primary osteocytes obtained from 7-week-old mice. When
comparing diverse fractions, we observed that cells in late fractions (5-9) demonstrated high levels of mRNA of
Adrb2 in association with high levels of osteocyte markers including Dmp1 and Fgf23. In contrast, cells in early
fractions (2- including 4) expressed lower levels of mRNA of Adrb2 in association with high levels of osteblast
markers alkaline phosphatase. We also found that an osteocytic cell line MLO-Y4 expressed mRNA of Adrb2. The
levels of Adrb2. mRNA expression in MLO-Y4 altered in a culture period dependent manner. When MLO-Y4 cells
were cultured for longer period of time, the levels of expressed mRNA of Adrb2 were declined. These result suggestthat primary cultures of osteocytes and osteocytic cell line MLO-Y4 express β2 adrenergic receptor.

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Potential role of osteocyte in the fracture healing:
comparison of fishes with and without osteocytes
Mari Sato1, Tatsuya Shimizu1, Takashi Okubo2, Takuya Hosoki3, Masato Tamura1,
Itsuro Koizumi2
1) Biochemistry and Molecular Biology, Graduate School of Dental Medicine., Hokkaido Univ., Japan,
2) Animal Ecology, Graduate School of Environmental Earth Science, Hokkaido Univ., Japan,
3) School of Fisheries Science, Hokkaido Univ., Japan
Purposes:
The human have acquired bones with osteocytes through evolution. However, it is a mystery why our bones need
osteocytes. Interestingly, many fishes have bone that lack osteocytes, whereas all other vertebrates have osteocytes.
However, it remains unclear when and why some fishes have acquired or lost osteocytes. Bone fracture caused by
predatory attacks and aggressive competitions may be one of the lethal factors for fishes, living in hard environment
exposed various mechanical stress such as big waves or rapid flows. In human and some other mammals osteocytes
contribute to bone homeostasis by converting mechanical stress to biological signals. Therefore, osteocytes may be
involved in the acceleration of fracture healing by mechanical stimuli including ultrasound stimulation. In this study,
we examined the effect of low-intensity pulsed ultrasound (LIPUS) for the fracture healing in two types of fish whichhave bones with or without osteocytes. Our goal is to understand the adaptive significance of osteocytes, especiallysolving the enigma of fish bones without osteocytes.
Methods:
Zebrafish and medaka were our study species that have bones with and without osteocytes, respectively. We fractured
the bones in caudal fin rays using sharp-edged tungsten needles. 1 day after fracture, fishes were stimulated by
LIPUS (Teijin) every day. The process of fracture healing was determined by Alizarin red and Alcian blue staining.
Results and conclusion:
We decided the point at which the fracture healing finished was when we could observe Alizaline red-positive
calcified smooth new bone. Medaka required around 24 days to complete fracture healing, whereas zebrafish
required only around 18 days. LIPUS stimulation accelerated the healing periods significantly only in zebrafish.
These results suggest that bone with osteocytes may have an advantage in the bone fracture healing and accelerationof it by LIPUS in fishes, although we cannot rule out other possibilities (e.g. higher metabolic rate). In future studies,
we will increase sample sizes and also use other fish species with or without osteocytes. We will also investigate thecorrelations between fishes with/without osteocytes and their habitats, ecology, and evolutionary histories.

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13th Congress of the International Society of Bone Morphometry

Dullard/Ctdnep1 regulates endochondral ossification via
suppression of TGF-β signaling
Tadayoshi Hayata1,2), Yoichi Ezura3), Makoto Asashima4), Ryuichi Nishinakamura5),
Masaki Noda3)
1) Department of Biological Signaling and Regulation, Faculty of Medicine, University of Tsukuba, Japan
2) Ph.D. Program in Life Science Innovation, University of Tsukuba3) Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Japan
4) Research Center for Stem Cell Engineering, National Institute of Advanced Industrial Science and Technology(AIST), Japan
5) Department of Kidney Development, Institute of Molecular Embryology and Genetics, Kumamoto University,
Japan
Transforming growth factor (TGF)-β signaling plays critical roles during skeletal development and its excessive
signaling causes genetic diseases of connective tissues including Marfan syndrome and acromelic dysplasia.
However, the mechanisms underlying prevention of excessive TGF-β signaling in skeletogenesis remain unclear.
We previously reported that Dullard/Ctdnep1 encoding a small phosphatase is required for nephron maintenanceafter birth through suppression of bone morphogenetic protein (BMP) signaling. Unexpectedly, we found that
Dullard is involved in suppression of TGF-β signaling during endochondral ossification. Conditional Dullarddeficient mice in the limb and sternum mesenchyme by Prx1-Cre displayed the impaired growth and ossificationof skeletal elements leading to postnatal lethality. Dullardwas expressed in early cartilage condensations and laterin growth plate chondrocytes. The tibia growth plate of newborn Dullard mutant mice showed reduction of theproliferative and hypertrophic chondrocyte layers. The sternum showed deformity of cartilage primordia and delayed
hypertrophy. Micromass culture experiments revealed that Dullard deficiency enhanced early cartilage condensationand differentiation, but suppressed mineralized hypertrophic chondrocyte differentiation, which was reversedby treatment with TGF-β type I receptor kinase blocker LY-364947. Dullard deficiency induced upregulation ofprotein levels of both phospho-Smad2/3 and total Smad2/3 in micromass cultures without increase of Smad2/3
mRNA levels, suggesting that Dullard may affect Smad2/3 protein stability. The phospho-Smad2/3 level was also
upregulated in perichondrium and hypertrophic chondrocytes in Dullard-deficient embryos. Response to TGF-βsignaling was enhanced in Dullard-deficient primary chondrocyte cultures at late, but not early, time point. Moreover,perinatal administration of LY-364947 ameliorated the sternum deformity in vivo. Thus, we identified Dullard as a
new negative regulator of TGF-β signaling in endochondral ossification.

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Masticatory activity affects bone remodeling in mice
Huayue Chen1), Takao Senda1), and Kin-ya Kubo2)
1) Gifu University Graduate School of Medicine, Japan
2) Seijoh University, Japan
Purposes:Mastication is very important not only for food intake, but also for preserving and promoting the general health.
We hypothesize that mastication affects bone health. To test this hypothesis, we examine the effects of masticatory
activity on bone remodeling in mice.
Materials and methods:
We used male senescence-accelerated mouse strain P8 (SAMP8). For masticatory dysfunction experiment, mice
were divided into control and toothless groups. All upper molar teeth were removed at 1 month of age in toothlessgroup. Bone response was evaluated at 2, 5 and 9 months. For active mastication experiment, mice were divided into
control, stress, and stress/chewing groups. Mice in the stress and stress/ chewing groups were placed in a ventilated
restraint tube for 60minutes, twice a day for 4weeks. The restrained mice were simultaneously subjected daily to
one of the following stressors: water immersion, physical shaking and flashing lights. Mice in the stress/chewing
group were allowed to chew a wooden stick during the experimental period. After experiments, bone response was
evaluated using quantitative micro computed tomography, bone histomorphometry, and biochemical markers.
Results and conclusion:
Tooth loss caused an increase in circulating corticosterone level with age. Bone formation was suppressed, bone
resorption was activated, and trabecular bone volume fraction of vertebra and femur was decreased in the toothlessmice with age. Chewing under chronic stress prevented the increase in circulating corticosterone level. Exposure ofmice to chronic stress resulted in a reduction of bone formation and an activation of bone resorption. Chewing underchronic stress improved reduced bone formation, activated bone resorption and trabecular bone loss. The present
findings indicated that tooth loss induced masticatory dysfunction, impaired dynamic homeostasis of bone formation
and bone resorption, leading to reduced bone strength with age. Active mastication can ameliorate chronic stressinduced bone loss in mice.

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13th Congress of the International Society of Bone Morphometry

β2-adrenergic receptor expression in osteoblastic MC3T3-E1 cells
is regulated by PTH
Shuichi Moriya1,2), Tadayoshi Hayata1), Takayuki Yamada1), Jumpei Shirakawa1),
Makiri Kawasaki1), Kazuo Kaneko2), Yoichi Ezura1), Masaki Noda1)
1) Tokyo Medical & Dental University, Medical Research Institute, Japan
2) A Department of Orthopaedic Surgery, Juntendo University School of Medicine, Japan,
Purposes:
The molecular bases underlying the regulation of bone mass and remodeling are still incompletely understood. Beta2
adrenergic receptor and a receptor for parathyroid hormone (PTH) are co-expressed in osteoblasts. PTH acts as ananabolic agent for bone used for osteoprosis. However, whether beta2 adrenergic receptor is under the regulation by
PTH or no is not still to be elucidated. We therefore examined the effects of PTH in beta2 adrenergic receptor gene
expression in osteoblastic cells.
Materials and methods:
Osteoblastic cell line, MC3T3E1 were used for all experiments. si-RNA for Adrb2 or control si-RNA, RNAs from
cells were subjected to quantitative RT-PCR. cyclic AMP response element Luciferase assay and Western blot for
cyclic AMP response element binding protein (CREB) antibody and phosphorylation CREB antibody of analyses
were carried out.
Results:
The expression levels of beta2 adrenergic receptor mRNA was immediately suppressed by PTH treatment. This
PTH-induced Adrb2 mRNA suppression was observed in a dose-dependent manner. A transcriptional inhibitor,
DRB, inhibited such PTH effects while by a protein synthesis inhibitor, cycloheximide did not, suggesting directtranscription control. In terms of function, we observed that knockdown of beta2 adrenergic receptor enhanced
PTH-induced expression of c-fos, an immediate early response gene. With respect to molecular bases for thisphenomenon, knockdown of beta2 adrenergic receptor enhanced PTH induced transcriptional activity of cyclic AMP
response element-luciferase construct in osteoblasts. Knockdown of beta2 adrenergic receptors tended to enhance
caPPR (constitutive active mutant of PTH receptor, H223R)-induced luciferase expressions. Finally, knockdown of
beta2 adrenergic receptor enhanced base line level as well as PTH-induced phosphorylation of cyclic AMP response
element binding protein (CREB).
Conclusion:
Our data reveal that PTH regulates beta2 adrenergic receptor as a hormonal target suppresses beta2 adrenergic
receptor in osteoblasts.

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Quantification analysis of cranial bones development among
primates species using geometric morphometrics method
Wataru Yano 1), Takeshi Nishimura 2), Ryuta Watanabe 1), Kazuhiko Satoh 1),
Sadakazu Ejiri 1)
1) Asahi University, School of Dentistry, Japan
2) Primates Research Institute, Kyoto University, Japan
Cranial morphology plays a particularly important role in the primate evolution. Although the previous studies1,2
elucidated cranial variation among extant primates, it is unknown the timing and location in which cranial bonecomponents accelerate/retard. The knowledge is, however, essential in linking the morphological evolution ofprimates skull with potential physical and physiological factors inducing them. The growth pattern of each bones,
yet, were not easily quantified with traditional linear measurements due to the complex configuration and articulationof cranial bones. New morphological techniques are anticipated to quantify cranial bone configuration in highdimensions.
Purposes:
We aimed to identify the timing and location of bone growth crucial for the phylogenetic variation by quantificationof cranial form of three primates species (Chimpanzee, Japanese macaque and Baboon) using the Geometricmorphometrics method (GM).
Materials and methods:
Dried crania of three primates species (Chimpanzee (n=8), Japanese macaque (n=18) and Baboon (n=16)) wereused. The specimens are CT-scanned and external surface models are reconstructed. 31 anatomical landmarks and 46
semilandmarks were dotted on the sagittal plane to trace the outline of 12 bone parts. By defining the total varianceof landmark coordinates as a quantity of form difference, the form information of each specimen was projected onto
the high dimensional shape space. Subsequently, they were decomposed into scaling, rotation, transition, and nonaffine deformation factors. The relative growth score is calculated by standardizing them with the score of maximum
adult specimens. Then, the temporal change is induced by regressing these scores by the size of whole cranium astime proxy.
Results and conclusion:
The original GM method successfully quantified the shape of cranial bones in high dimensions. Based on relative
growth scores, we identified crucial change of developmental pattern which leads to the cranial shape evolution,
specifically relative retardation of palatine bone growth and accelerated development of premaxilla in Chimpanzees.
The method and data of this study will underlie the future study on human craniofacial evolution.

1

Yano, Wataru, et al. Primates 51.3 (2010): 263-271.

2

Nishimura, Takeshi D., Yano, Wataru, et al. Anthropological Science 122.2 (2014): 101-113.

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13th Congress of the International Society of Bone Morphometry

Deletion of Pfn1 have an effect on bone formation
Wanting Lin 1), 2), Yoichi Ezura 1), Yayoi Izu 1), Keiji Moriyama 2), Masaki Noda 1)
1) Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University,
Tokyo, Japan
2) Department of Maxillofacial Orthognathics,Tokyo Medical and Dental University, Tokyo, Japan
Purposes
Bone development is a dynamic process that requires cell motility and morphological adaptation under the control
of actin cytoskeleton. This actin cytoskeleton system is regulated by critical modulators including actin-binding
proteins. Among them, profiling 1(Pfn 1) is a key player to control actin fiber structure and it is involved in a
number of cellular functions such as migration. BMPs are multifunctional cytokines involved in cell proliferationand differentiation. BMP2, an osteoinductive factor, is clinically approved for use in bone regeneration and repair.
Although multiple transcription factors and secreted proteins are known to mediate BMP signal intensity or duration,less is known about mechanisms determining lineage specificity in response to BMP. In this study, we examined therole of Pfn1 in BMP-induced osteogenic activities
Materials and Methods.
As for cells , we used MC3T3E1 cells and MLO-Y4 cells. These cells were cultured as reported previously. To
estimate the levels of gene expressison, RNAs were isolated and were subjected to real-time RT-PCR. For knock
down of Pfn1, siRNAs were used. To examine transcriptional activity, luciferase reporter linked to BMP response
element (BRE) was used.
Results and Conclusion.
In MC3T3E1 cells , Pfn1 mRNA is expressed constitutively and BMP treatment of these cell suppressed the levelsof Pfn1. This effect was time dependent and suppression was more when the BMP treatment was continued fora longer period of time. Along with this time course, alkaline phosphatase mRNA levels were enhance in a timedependent manner as described before. To address the functional role of Pfn1 during BMP-induced differentiation of
MC3T3 cells, we knocked down Pfn1 via siRNA technique. The efficiency of Pfn1 knock down (KD) by si-RNA wasconfirmed by quantitative RT-PCR. The result shows that about 80% expression of Pfn-1 was observed. Interestingly,
Pfn1 KD enhanced basal levels of alkaline phosphatase (Alp) activity in MC3T3-E1 cells. When Pfn1 KD was donein the cells treated with BMP, BMP-induced expression of Alp activity in MC3T3-E1 cells were further enhanced.
Surprisingly, such Pfn1 KD enhancement of Alp was still observed even when the BMP concentration was increased
to 200ng/ml. Pfn1 KD enhancement of Alp was time-dependent. As for underlying mechanism, Pfn1 KD enhanced
BMP-induced Alp mRNA expression. For transcription, Pfn1 KD enhanced BMP-induced enhancement of BREluciferase activity. In osteocytic MLO-Y4, Pfn1 KD decreased the number of cellular processes and disorganized thecytoskeletal patterns. These results demonstrated that the Pfn1 is a novel target of BMP and acts as an endogenous
suppressor of BMP actions via at least in part transcriptional events.

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April 27-29, 2015 ● Tokyo Garden Palace HotelA novel measurement method for the morphology of themandibular ramus using homologous modeling
Hiroyuki NAKANO1), Kazuya Inoue2), Tomoki SUMIDA1), Naruto OTAWA2),
Naoyuki FUKUDA2), Tomohiro YAMADA3), Katsuhiro MINAMI4), Katsuaki Mishima5),
Makiko Kouchi6), Yoshihide MORI7)
1) Senior Assistant Professor, Section of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University, Higashi-ku, Fukuoka, Japan
2) Assistant Professor, Section of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University, Higashi-ku, Fukuoka, Japan
3) Associate Professor, Section of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic and Surgical
Sciences, Faculty of Dental Science, Kyushu University, Higashi-ku, Fukuoka, Japan4) Senior Assistant Professor, Division of Research and Treatment for Oral and Maxillofacial Congenital Anomalies,
Faculty of Dentistry, Aichi-Gakuin University, Chikusa, Nagoya, Aichi, Japan
5) Associate Professor, Department of Oral and Maxillofacial Surgery, Yamaguchi University Graduate School of
Medicine, Minamikogushi, Ube, Yamaguchi, Japan
6) Digital Human Research Center, National Institute of Advanced Industrial Science and Technology, Koto-ku,
Tokyo, Japan
7) Professor and chairman, Section of Oral and Maxillofacial Surgery, Division of Maxillofacial Diagnostic andSurgical Sciences, Faculty of Dental Science, Kyushu University, Higashi-ku, Fukuoka, Japan
Abstract
Some three-dimensional (3D) studies on facial asymmetry have reported differences in linear and angle
measurements between the deviated and contralateral sides in asymmetric mandibles. However, these studies cannot
clarify the 3D morphology of ramus. In this study, we aimed to evaluate the mandibular ramus between the deviatedand contralateral sides in asymmetric mandibles using traditional and homologous model methods.
Fifteen Japanese females with jaw deformities treated by orthodontic surgery were enrolled. 3D computed
tomography (CT) images were reconstructed and 13 landmarks identified on the model surface. Homologous ramus
models were constructed for each sample.
Principal component analysis revealed that >80% ramus morphology was suggested by principal components 1–8.
The 3 principal components were significantly different between the deviated and contralateral sides. The t-values
for the mandibular ramus height, posterior mandibular ramus height, condylar width, condylar head height, coronoid
process height, horizontal and vertical dilated ramus angles, and ramus angle were 2.45, 2.30, 3.48, 2.36, −2.24,−2.08, −4.67, and 2.19, respectively. The F-values for the mandibular ramus height, condylar width, and horizontal
and vertical dilated ramus angles were 18.34, 15.56, 11.94, and 16.88, respectively. The squared multiple correlation
coefficient adjusted for the degrees of freedom was 0.815. Visual simulation revealed a larger condylar head on thecontralateral side, a larger coronoid process on the deviated side, and a mandibular angle that was turned inwards toa greater extent on the deviated side. In conclusion, we successfully created a homologous model of the mandibular
ramus and demonstrated the effectiveness of this model in the 3D comparison of ramus morphology between
contralateral and deviated sides in asymmetric mandibles.

83

13th Congress of the International Society of Bone MorphometryBMP-induced alkaline phosphatase expression in osteoblast-like
MC3T3E1 cells is suppressed by β2 adrenergic receptor activation.
Takayuki Yamada 1), 2), 3), Yoichi Ezura 1), Tadayoshi Hayata 1), Shuichi Moriya 1) ,Junpei Shirakawa1), 3), Takuya Notomi 1), 3), Smriti Arayal 1), Makiri Kawasaki 1),
Yayoi Izu 1), Kiyoshi Harada 2), Masaki Noda 1), 3)
1) Department of Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University,
Tokyo, Japan
2) Department of Oral and Maxillofacial Surgery, Tokyo Medical and Dental University, Tokyo, Japan
3) Global COE program, Tokyo Medical and Dental University, Tokyo, Japan
Purposes:In vivo β adrenergic stimulation suppresses bone formation while its actions in osteoblastic differentiation are notfully understood. Terefore, we examined the effects of β2 adrenergic stimulation on osteoblast-like MC3T3-E1 cells
focusing on BMP-induced alkaline phosphatase (ALP) expression in vitro.
Materials and methods:
For cell culture, osteoblast-like cell line, MC3T3E-1 was used. ALP activity measurement and protein determination
were performed according to PNPP method. For ALP staining, we used BM purple (Roche), and analyzed the ALPpositive area using Image-J software. For Real-time PCR, StepOneTM system (life technology) was used to gene
expression analysis. For luciferase assay, reporter activity was measured by using a LIMAT LB9507 (Berthold). The
data was standardized in Renilla luciferase reporter activity. Differences between groups were analyzed by Student's
t-test. P values less than 0.05 or 0.01 were considered to be significant.
Results:
In terms of morphology, isoproterenol (ISO) treatment reduces BMP-induced increase in the numbers of small
ALP-positive foci in the cultures of MC3T3-E1 cells. With respect to biochemical effects, ISO treatment inhibits
BMP-induced enzymatic activity of ALP in a dose-dependent manner. Isoproterenol treatment suppressed BMPinduced ALP activity even at higher dosage of BMP was used. Regarding cell density, ISO treatment tends to inhibit
BMP-induced increase in ALP activity compared to those at lower cell density. In addition, we observed that ISO
treatment did not affect cell proliferation in MTT assay. In terms of treatment protocol, continuous ISO treatment iscompared to cyclic treatment. Continuous ISO treatment is more suppressive against BMP-induced increase in ALP
activity than cyclic regimen. At molecular level, ISO treatment inhibits BMP-induced enhancement of ALP mRNA
expression. With regard to the mode of ISO action, ISO inhibits BMP-induced BRE-luciferase activity. These datasuggest that ISO regulates BMP-induced ALP expression in osteoblast-like MC3T3E1 cells.
Conclusion:
ISO treatment suppresses BMP-induced ALP expression in osteoblast-like MC3T3-E1 cells. BMP-induced
enhancement of osteoblastic differentiation may be one of the target points of sympathetic tone-dependent
suppression of osteoblastic activity, such phenomena may be relevant to those in disuse osteoporosis.

84April 27-29, 2015 ● Tokyo Garden Palace Hotel

Voluntary exercise prevents deterioration of trabecular bonearchitecture in diabetes mellitus model rats
Akira Minematsu, Tomoko Hanaoka, Yoshihiro Takada, Shunji Okuda, Hidetaka
Imagita, and Susumu Sakata 1).
1) Kio University, Japan
Purpose:Type 2 diabetes mellitus (DM) is well-known to cause bone fragility and increase in fracture risk, although bonemass remains unchanged. In patients with type 2 DM, regular physical exercise may improve not only DM symptom
but also bone fragility that appears to be caused by poor bone quality. The purpose of this study is to examine theeffects of voluntary wheel-running on trabecular bone architecture of lower limb in type 2 DM model Otsuka LongEvans Tokushima Fatty (OLETF) rats.
Materials and methods:
Five-week-old male OLETF rats were used as experimental animals and male Long-Evans Tokushima Otsuka(LETO) rats as control animals. Half of the OLETF rats performed daily voluntary wheel-running throughout theexperimental period of 16 months (OLETF-EXE), and the remainder of the OLETF and LETO rats did not performthe wheel-running. After 16 months, left distal femur and proximal tibia were scanned with micro-CT, and wereanalyzed for trabecular bone architecture.
Results:
Long-term voluntary wheel-running prevented the deterioration of trabecular bone architecture in the femur andtibia, where bone volume, bone volume fraction, trabecular number, and connectivity density were significantly
lower in the OLETF than in the OLETF-EXE and LETO rats. In addition, the OLETF-EXE rats showed a significant
decrease in tissue volume compared with the OLETF and LETO rats.
Conclusion:
Although trabecular bone architecture of femur and tibia was deteriorated in type 2 DM model OLETF rats aged
17 months, this deterioration was prevented by daily voluntary wheel-running that started at the age of 1 month.
Based on these results, we conclude that regular physical exercise would be a potent strategy for preventing thedeterioration of trabecular bone architecture in patients with type 2 DM.

85

13th Congress of the International Society of Bone Morphometry

Morphological and dynamic analysis of migration linked to
FUCCI-indicated cell cycle under the influence of PTH andmechanical flow signals
Shirakawa J(1,2,3, Ezura Y(1, Moriya S(1, Kawasaki M(1, Yamada T(1, Notomi T(1,Hayata T(1, Miyawaki A(3, Omura K(2, Noda M(1.
1) Dept. Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Japan
2) Dept. Oral and Maxillofacial Surgery, Tokyo Medical and Dental University, Japan
3) Laboratory for Cell Dynamics, Brain Science Institute, RIKEN, Japan
Bone Cellular responses to hormones and mechanical stress may be linked to the cell morphology and migration.
Osteoblasts are responding to Parathyroid hormone (PTH) and also mechanical stress. However, how these signals
regulate cell migration and cell cycle when the two signals are together isstill incompletely understood. To address this point, we subjected osteoblasts to PTH and mechanical signaling andmonitored the behavior by using fluorescent ubiquitination-based cell cycle indicator (FUCCI) reporter system. ThisFUCCI system allows simultaneous monitoring of cell migration and cell cycle as G1 as well as S/G2 /M phase canbe detected due to the marker expression specific to each cell cycle stage. We found that osteoblastic cells in S/G2 /M phase migrate faster than those in G1 phase. Parathyroid hormone as an anabolic agent also regulates migration
velocity of the osteoblasts. Flow induced mechanical stress increased migration speed. The presence of both PTH
and mechanical stress, the migration velocity got back to the base line levels. These observations suggest that cell
migration is influenced by the PTH and mechanical flow and these two signals my influence each other. Our data
show that morphological monitoring of osteoblasts could reveal complex interactions of PTH and mechanical stressin osteoblasts.

86

April 27-29, 2015 ● Tokyo Garden Palace Hotel

Influence of magnet implant on tibial trabecular bone
microarchitecture in ovariectomized rats.
Yasue Nishii 1), Hidetaka Imagita and Akira Minematsu 2)
1) Graduate school of Health Science, Kio University, Japan
2) Department of Physical Therapy, Faculty of Health Science, Kio University, Japan
Purposes:Static magnetic field (SMF) was reported to inhibit reduction of bone mineral density (BMD) in ovariectomized(OVX) rats. Those were assessed by dual energy X-ray absorptiometry and there are few studies to investigate theeffects of SMF on bone structure. The purpose of this study is to examine the influence of SMF on trabecular bone
microarchitecture (TBMA) in OVX rats.
Materials and methods:
Female SD rats (mean body weight 376.8±10.5 g) were used. They underwent either ovariectomy (OVX, n=14)
or sham-operation (SHAM, n=6). Half of OVX rats (OVX+MG group) were surgically implanted disk-shaped
neodymium magnet and stainless steel in lateral part of the right and left knee joints, respectively. The remainder of
the OVX rats (OVX group) and SHAM rats (SHAM group) were surgically implanted stainless steel in lateral part ofthe both knee joints. The right knee joints were harvested after 20-week intervention. Then, the proximal tibias were
scanned using x-ray micro-computed tomography and were analyzed for TBMA with 3D bone analysis software.
Results:
Final body weights of the OVX+MG and OVX groups were significantly heavier than the SHAM group. TBMA wasremarkably deteriorated by OVX. The ratio of decrease for bone volume fraction was 81% in the OVX group and
69% in the OVX+MG group compared with the SHAM group. Parameters of TBMA in the OVX+MG group tended
to be maintained compared with the OVX group, for trabecular thickness and width were significantly higher in the
OVX+MG group than in the SHAM group. In addition, trabecular star volume (V*tr) as osteoporotic index was
higher in OVX+MG group than in the OVX and SHAM groups.
Results and conclusion:
OVX caused deterioration of TBMA. Compared with the OVX group, the degree of deterioration in TBMA was
smaller and V*tr was significantly higher in the OVX+MG group. These results suggested that SMF was likely toinhibit deterioration of TBMA, especially trabecular thickness and width, in OVX rats. We need to consider the SMF
conditions, the timing and term of interventions, and histomorphological study.

87

13th Congress of the International Society of Bone Morphometry

PTH-induced proliferation in osteoblasts requires ubiquitin
specific peptidase 2 (Usp2)
Jumpei Shirakawa 1), Yoichi Ezura1), Yayoi Izu1), Masaki Noda1)
1) Dept. Molecular Pharmacology, Medical Research Institute, Tokyo Medical and Dental University, Japan,
Purposes:
Osteoporosis is a common disease that affects mainly the elderly individuals by increasing the fragility fracturerisk. Teriparatide (PTH) is the only agent that induces anabolic action of bone forming osteoblastic lineage cells.
Though the molecular bases the anabolic action of PTH has been investigated from various aspects, the mechanismof its action is still incompletely understood. We were interested in the genes directly induced by cAMP pathway as
possible downstream targets leading to osteoblast proliferation. Ubiquitin specific peptidase 2 (Usp2, also named as
Ubp41) was suggested be one of the direct target genes of PTH. However, the functional role of Usp2 gene in PTH
effects on osteoblast proliferation is not known.
Materials and methods:
Murine osteoblast-like MC3T3-E1 cells and primary calvarial osteoblasts isolated from newborn double Fuccitransgenic mice were used. To examine the role of Usp2 enhanced by PTH treatment we transfected si-Usp2. Protein
levels were determined by western blot. We also used real time PCR and live imaging.
Results:
PTH1-34 induced Usp2 expression in MC3T3E1 cells. Usp2 has three isoforms and all of them were expressed andinduced by PTH in osteoblasts. Cycloheximide (CHX) treatment did not affect the Usp2 induction by PTH treatment.
To examine if the Usp2 expression could be induced by cyclic AMP-PKA pathway, MC3T3E1 cells were treatedwith forskolin. Forskolin induced Usp2 expression through PKA pathway. These results suggested that PTH induced
Usp2 expression through cyclic AMP pathway directly. Then we analyzed whether knocking down of Usp2 would
influence the proliferation-inducing effect of PTH on osteoblasts, using live imaging of the calvarial osteoblasts
obtained from Fucci-transgenic mice. PTH induced osteoblast proliferation by increasing the numbers of cells thatenter the S/G2/M phase from G0/G1 phase. However, the cells transfected with si-Usp2 did not show significant
increase of the proliferating cells that enter the S/G2/M phase after PTH treatment. These results indicated that Usp2
play a role in regulation of proliferation in osteoblasts after PTH treatment.
Conclusion:
Usp2 gene is a target of PTH is involved in PTH-induced regulation of proliferation in osteoblasts.

88

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International Society of Bone Morphometry