Practical whole-tooth restoration utilizing autologous bioengineered

tooth germ transplantation in a postnatal canine model

Mitsuaki ONO, PhD1,2*, Masamitsu OSHIMA, PhD1,3,4*, Miho OGAWA, PhD3,4,5, Wataru SONOYAMA,

PhD1, Emilio Satoshi HARA, PhD1, Yasutaka OIDA, PhD1, Shigehiko SHINKAWA, PhD1, Ryu NAKAJIMA,

PhD1, Atsushi MINE, PhD1, Satoru HAYANO, PhD6, Satoshi FUKUMOTO, PhD7, Shohei KASUGAI,

PhD8, Akira YAMAGUCHI, PhD9,10, Takashi TSUJI, PhD3,4,5 & Takuo KUBOKI, PhD1†

1Department of Oral Rehabilitation and Regenerative Medicine, Graduate School of Medicine, Dentistry

and Pharmaceutical Sciences, Okayama University, Okayama, 700-8525, JAPAN2Department of Molecular Biology and Biochemistry, Graduate School of Medicine, Dentistry and

Pharmaceutical Sciences, Okayama University, Okayama, 700-8525, JAPAN3Research Institute for Science and Technology, Tokyo University of Science, Noda, Chiba, 278-8510,

JAPAN4RIKEN Center for Developmental Biology, Kobe, Hyogo, 650-0047, JAPAN5Organ Technologies Inc., Tokyo, 105-0001, JAPAN6Department of Orthodontics, Graduate School of Medicine, Dentistry and Pharmaceutical Sciences,

Okayama University, Okayama, 700-8525, JAPAN7Division of Pediatric Dentistry, Tohoku University Graduate School of Dentistry, Sendai, Miyagi, 980-

8575, JAPAN8Section of Oral Implantology and Regenerative Dental Medicine, Graduate School of Tokyo Medical and

Dental University, Bunkyo-ku, Tokyo, 113-8549, JAPAN9Section of Oral Pathology, Department of Oral Restitution, Graduate School of Tokyo Medical and

Dental University, Bunkyo-ku, Tokyo, 113-8549, JAPAN10Oral Health Science Center, Tokyo Dental College, Chiyoda-ku, Tokyo, 101-0061, JAPAN

*These authors contributed equally to this work.†To whom correspondence may be addressed: Takuo Kuboki, DDS, PhD.

SUPPLEMENTARY INFORMATION

Supplemental Figure 1, Ono et al.

Deciduous molar

tooth germ

A Epithelial cell

Mesenchymal cell

Epithelial tissue

Mesenchymal tissue

Organ culture

for 2 days

BDeciduous molar tooth germ

C Epithelial tissue and

Mesenchymal tissue

Epithelial tissue and

Mesenchymal cell

Epithelial cell and

Mesenchymal tissueNatural tooth germ

100 μm

E

D

E

D

Epi

Mes

Epi

Mes

Epi

Mes Mes

Epi

Epi

Mes

Scar bar：1 mm

Epi

Mes

Epi

Mes

Isolated from 55

day prior to birth

Transplantation into

SRC for 4W

Immunodeficient mice

2 mm2 mm2 mm2 mm

Supplemental Figure 2, Ono et al.

#2

#3

#1

A

B

Cross sectional views

8w8w2 mm

Micro-CT image (3D)

HE staining

HE staining

Photograph

Photograph

#4

8w

8w

8w

8w

Higher magnification

ED

D

kidney

kidney

kidney

kidney

kidney

2 mm

2 mm5 mm

1 mm

1 mm

2 mm 2 mm 300 μm

8w

Da

y 3

0 a

fte

r b

irth

Da

y 9

0

aft

er

birth

Da

y 1

50

a

fte

r b

irth

Scale bar: 10 mm

Da

y2

10

a

fte

r b

irth

dM3

M1

dM1 dM2

P4P2

P3

dM3

M1

dM1 dM2

P4P2 P3P1

P4P2 P3 M1

P4P2 P3 M1P1

Supplemental Figure 3, Ono et al.

Supplemental Figure 4, Ono et al.

Toluidine blue staining

P2P4

M1

M1

dM3dM1 dM2

P3

P4P3

dM3dM1 dM2

10 mm

Deciduous molar

Permanent Premolar & Molar germ

5 mm

B Micro CT image

C

P2 5 mm

P1

A Photograph

dM3dM1 dM2

5 mm

Supplemental Figure 5, Ono et al.

Day60 Day120 Day180Day0

Sa

gitta

l im

age

Fro

nta

l im

age

Day180

Scale bar : 10 mm

Scale bar : 5 mm

Legends to Supplemental Figures

Supplemental Fig. 1. Generation of the bioengineered tooth under the various reconstructing conditions.

(A) Schematic representation of various reconstructing conditions for bioengineered tooth germ. (Illustration by R.N.) (B)

Photograph and histological image of deciduous molar tooth germ. Epi, epithelial tissue; Mes, Mesenchymal tissue. (C)

Histological analysis of the bioengineered tooth at 4 weeks after subrenal capsule transplantation under the various

reconstructing conditions. Boxes indicate the area shown at higher magnification in the lower panels. E, enamel; D,

dentin; Epi, epithelial tissue; Mes, mesenchymal tissue.

Supplemental Fig. 2. Generation of bioengineered tooth in the reconstructing condition of epithelial cells and

mesenchymal cells.

(A) The frequency of bioengineered tooth generation was low (16.7%) in the reconstructing condition of epithelial cells

and mesenchymal cells. In a few successful samples, the bioengineered tooth germ developed into subrenal capsule

(upper, white arrow) and showed tooth-crown formation in micro-CT analysis (upper). Histological analysis of the

bioengineered tooth showed the tooth tissue structure, including the enamel and dentin, which were equivalent to those of

the natural tooth (lower). Boxes indicate the area shown at higher magnification. E, enamel; D, dentin.

(B) The majority of samples (83.3%) in the reconstructing condition of epithelial cells and mesenchymal cells did not show

the development of the bioengineered tooth into the subrenal capsule (left panels, red arrow). These samples did not

show tooth tissue structures such as enamel, dentin, pulp or PDL (centre and right panels). Boxes indicate the area

shown at higher magnification.

Supplemental Fig. 3. CT images at various tooth developmental stages in the canine mandible.

CT images of canine tooth development in the mandible at postnatal days 30, 90, 150 and 210. White arrowhead,

deciduous molars (dM1, dM2 and dM3); Red arrowhead, permanent premolars (P1, P2, P3 and P4) and permanent first

molar (M1).

Supplemental Fig. 4. Micro-CT and histological analysis of the canine mandible at postnatal day 30.

Photograph (A), micro-CT image (B) and histological image obtained by toluidine blue staining (C) of the canine mandible

at postnatal day 30. All deciduous molars (dM1, dM2 and dM3) erupted into the oral cavity, and the permanent premolar

germs (P2, P3, and P4) were at a developmental stage suitable for the reconstruction of bioengineered tooth germ.

Supplemental Fig. 5. CT images of the bioengineered tooth development.

Continual CT images of bioengineered tooth development at 0, 60, 120 and 180 days after autologous transplantation

into the mandible. White arrowhead, bioengineered tooth.