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Dental regenerative therapy: Stem cell

transplantation and bioengineered tooth
replacement
ARTICLE in JAPANESE DENTAL SCIENCE REVIEW JULY 2008
DOI: 10.1016/j.jdsr.2007.11.001

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Japanese Dental Science Review (2008) 44, 7075

a v a i l a b l e a t w w w. s c i e n c e d i r e c t . c o m

journal homepage: www.elsevier.com/locate/jdsr

MINI REVIEW

Dental regenerative therapy: Stem cell

transplantation and bioengineered tooth replacement
Kazuhisa Nakao a,b, Takashi Tsuji a,b,*
a

Department of Biological Science and Technology, Faculty of Industrial Science and Technology,
Tokyo University of Science, Noda, Japan
b
Tissue Engineering Research Center, Tokyo University of Science, Noda, Japan
Received 1 November 2007; received in revised form 6 November 2007; accepted 6 November 2007

KEYWORDS
Dental regenerative
therapy;
Stem cells;
Bioengineered tooth;
Cell aggregate method;
Tooth germ

Summary For clinical treatment of tooth defects and tooth loss, nonbiotechnological
approaches, such as the use of prostheses and implants, have generally been employed. Dental
regenerative therapies which restore or replace defective teeth using autologous explants are
being investigated using current understandings of developmental biology, stem cell biology, and
regenerative medicine. Recently, dental tissue stem/progenitor cells, which can differentiate
into dental cell lineages, have been identified in both impacted and erupted human teeth, and
these cells can be used to regenerate some dental tissues. Tissue engineering using scaffold and
cell aggregate methods may also be used to produce bioengineered teeth from dissociated cells
for therapeutic applications of whole tooth replacement. Recent breakthroughs in single cell
manipulation methods for the reconstitution of bioengineered tooth germ and the investigation
of in vivo development of artificial tooth germ in the adult oral environment have been reported.
These researches and developments will ultimately lead to the realization of dental regenerative
therapies for partial repair by stem cell transplantation and for whole tooth replacement using
bioengineered tooth germ.
# 2008 Japanese Association for Dental Science. Published by Elsevier Ireland. All rights reserved.

1. Introduction
Regenerative medicine is attracting much attention because
it promises new therapeutic techniques for the repair and
replacement of tissues and organs that have lost functions
due to ageing, disease, damage, and congenital defects
* Corresponding author at: Department of Biological Technology,
Faculty of Industrial Science and Technology, Tokyo University of
Science, Yamazaki 2641, Noda, Chiba 278-8510, Japan.
Tel.: +81 4 7122 9711; fax: +81 4 7125 1841.
E-mail addresses: t-tsuji@nifty.com, t-tsuji@rs.noda.tus.ac.jp
(T. Tsuji).

[1,2]. Clinical applications have already begun to repair a

wide variety of tissues, such as blood, skin, cornea, cartilage,
and bone. In dentistry, current treatments for tooth defects
are prostheses, implants, and tooth transplantation. However, these treatments have undesirable effects, such as
applying loads on neighboring tissues and causing malalignment due to subsequent growth of jaw bone. In contrast,
dental regenerative therapy promises to provide living, functional, biocompatible tissues. Recently, the identification of
stem/progenitor cells in dental tissue has encouraged active
research in cell transplantation therapies for dental defects.
Furthermore, advances have been made in the development
and replacement of entire teeth in adult oral environments.

1882-7616/$ see front matter # 2008 Japanese Association for Dental Science. Published by Elsevier Ireland. All rights reserved.
doi:10.1016/j.jdsr.2007.11.001

71

Biotechnological approaches in dental regenerative medicine

The feasibility studies of whole tooth regenerative therapies
will provide substantial contributions to the research and
development of bioengineered organ replacement strategies
and regenerative therapies for a wide variety of organs. In
this review, we outline current understanding of dental
stem/progenitor cells and bioengineering techniques, and
we discuss future directions in tooth regenerative therapies.

2. Tooth development
Almost all organs arise from organ germs, which are induced by
reciprocal interactions between epithelium and mesenchyme
in the developing embryo [35]. Tooth development is
initiated when presumptive dental epithelium and neural
crest-derived mesenchyme reciprocally regulate gene expression via extracellular signaling factors on embryonic day 10 (ED
10) (Fig. 1). On ED 11, the dental epithelium thickens and
collapses to the mesenchymal side to form a dental placode
(Fig. 1). Ambient mesenchymal cells aggregate at a signal from
the dental placode, which then extends to wrap around the
mesenchyme (Fig. 1). During ED 1315, a tooth germ forms,
at which time a region of epithelial cells called the enamel
knot acts as a signal center and controls ambient epithelial and
mesenchymal cells to regulate tooth generation and morphology (Fig. 1). From ED 15 to 18, ameloblasts, which will later
produce enamel, differentiate from the dental epithelium.
Odontoblasts, which will form dentin, dental pulp, and periodontal tissue comprising cementum, alveolar bone, and the
periodontal ligament also differentiate from the dental
mesenchyme in this stage (Fig. 1). Blood vessels and nerve
fibers penetrate into the dental pulp from the apical foramen.
A tooth is ultimately formed as a result of the integration of
spatiotemporal cellular responses, such as proliferation, apoptosis, differentiation, movement, and polarization. The number of tooth germs formed during the embryonic period then
determines the number of teeth in life. These understandings
suggest that it should be possible to regenerate specific tissues
and to even grow replacement teeth by controlling cytodifferentiation and organogenesis.

3. Dental tissue stem/progenitor cells

In most organs, such as the blood, skin, liver, and brain, there
are tissue stem/progenitor cells that appear to have limited

abilities to differentiate and perform maintenance and

restoration of these organs. It has been anticipated that dental
tissue stem/progenitor cells would be found in adult tissues
because spontaneous repair of dentin and periodontal ligaments has often been observed. Dental tissue stem/progenitor
cells would be particularly useful in dentistry for the development of cell transplantation therapies for the repair of
damaged dentin and for periodontal disorders, and the cells
are therefore currently the subjects of extensive research.
Somatic stem cells which can differentiate into odontoblasts
have been identified in pulp tissue of human permanent teeth,
exfoliated deciduous teeth, and impacted teeth (Table 1) [6
8]. In addition, the side population fraction, based on the
efflux of fluorescent dye Hoechst 33342, of human dental pulp
cells, and the periodontal tissue stem cells derived from
human extracted teeth can partially regenerate dentin and
periodontal tissue by cell transplantation into surgically created defects in adult teeth (Table 1) [9,10]. These studies
suggest that stem cell transplantation for partial tissue repair
using autologous dental tissue stem/progenitor cells is possible. These cells are thought to be dental tissue stem/progenitor cells which are already committed to dental cell lineages
because they can form dental tissues without epithelial
mesenchymal interactions. Moreover, multipotent stem cells
that can differentiate into non-dental cell lineages, such as
hepatocytes and neurons, exist in dental papilla of human
impacted tooth germ (Table 1) [11]. Due to the ease with which
teeth can be obtained and the low levels of risk, such as risk of
tumorgenesis, in stem cell transplantation, these somatic
dental stem cells will probably become a source of cells that
will be useful not only in dentistry but also in many other
regenerative therapies.

4. Whole tooth regeneration by in vitro cell

manipulation
Research on whole tooth regeneration is also advancing using
a strategy of transplanting artificial tooth germ and allowing
it to develop in the adult oral environment. To that end, it
will first be necessary to develop cell manipulation techniques to create artificial tooth germ.
Tissue engineering builds a tissue, such as skin, bone, and
cartilage, by seeding cells onto a scaffold [1]. Research
on the fabrication of teeth from dissociated cells was first

Figure 1 Schematic diagrams of tooth development. AM: ameloblasts, BO: alveolar bone, DE: dentin, DP: dental pulp, DF: dental
follicle, ED: embryonic day, EN: enamel and OD: odontoblasts.

72
Table 1

K. Nakao, T. Tsuji
Potential cell source for dental regenerative therapy

Origin

Cell

Clinical application

Reference

From dental tissues

Human impacted third molar

Dental pulp stem cells (DPSCs)

Stem/progenitor cell source for

cell transplantation therapy.
Restoration of partial dentin
by cell transplantation.
Generation of root/periodontal
complex by tissue engineering.
Restoration of partial periodontal
tissue by cell transplantation.
Generation of root/periodontal
complex by tissue engineering.
Stem/progenitor cell source for
cell transplantation therapy.
Partial liver regeneration by cell
transplantation.

[6]

Pulp SP cells
Stem cells from apical papilla
(SCAP)
Periodontal ligament stem cells
(PDLSCs)

Human exfoliated deciduous

teeth
Human impacted tooth germ
From non-dental tissues
Murine adult tibiae and femora

Stem cells from human

exfoliated deciduous teeth (SHED)
Tooth germ progenitor cells
(TGPCs)
Bone-marrow-derived cells

performed using tooth germ cells. When explants, seeded

with porcine third impacted tooth bud cells, made of polyglycolate/poly-L-lactate (PGA/PLLA) or poly-L-lactateco-glycolate (PLGA), were transplanted for 2030 weeks into
omentum, bioengineered teeth were visible within the
explant [12]. The cultured molar bud cells increased in
number and were also able to form bioengineered teeth
[13]. These raised the possibility that tissue engineering
techniques using a scaffold could be used in dental regenerative therapies. However, it seems that improvement is
necessary in the success rate of tooth formation and in the
morphology of regenerated teeth in tissue engineering methods using scaffolds. Recently, it has been reported that use of
a collagen sponge scaffold formed teeth with a higher rate of
success than use of biodegradable polymer [14]. In addition,
arranging epithelial cells and mesenchymal cells within the
scaffold by seeding them sequentially, instead of the conventional method of seeding a mixture of epithelial cells and
mesenchymal cells, improved the tissue arrangement in
bioengineered teeth [15].
On the other hand, cell aggregate methods have also been
used to achieve a more or less complete regeneration of
tissues, from dispersed cells of a particular origin, under
controlled culture conditions [16]. Artificial tooth germ has
been produced by combining a dental epithelial tissue and a
cell pellet of mesenchymal cells isolated from E13.5 mice
molar tooth germ, and the resulting artificial tooth germ was
then able to generate a correct tooth structure by transplantation into a subrenal capsule [17]. The bioengineered tooth
germ regenerated between the dissociated epithelial and
mesenchymal cells of the E14.5 molar germ also generated
teeth surrounded by periodontal-like tissue [18,19]. These
showed that the cell aggregate method is capable of creating
a bioengineered tooth germ that can be used in tooth regeneration therapy. It has also been reported that a cell aggregate reconstituted epithelial cells and mesenchymal cells
isolated from ED13.5 mice-derived molar tooth germ without
cell compartmentalization at high cell density and was also

Mesenchymal cell source of whole

tooth regeneration.

[9]
[8]
[7]

[10]
[11]

[24]

capable of regenerating a complete molar tooth [20]. Interestingly, spatial auto-cell sorting and inductive potentials in
molar-derived epithelial cells were observed. This agrees
with Steinbergs theory concerning cell movement and organization in early organogenesis [21]. In contrast, incisor
epithelial cells did not exhibit inductive potential after
autologous cell sorting [20]. These results indicate that
the auto-cell sorting and inductive potentials to organize
an organ germ differ among types of organ germs. It is also
anticipated that bioengineering methods can be developed
that can replicate organogenesis in the embryo and can be
adapted to produce a wide variety of organ germs.
We thought that it had been better to use tissue engineering method using scaffold and cell aggregation method properly. Cell aggregate methods can mimic cell-to-cell and
epithelialmesenchymal interactions in tooth germ in early
inductive stages through high cell density in bioengineered
tooth germ (Fig. 2a). In contrast, tissue engineering methods
using scaffold should be utilized for the formation of differentiated tooth or tooth germ in late stages of differentiation
which has cell polarization of mineralized tissue, and in
odontoblast/ameloblast cell lineages in organ architecture,
because they can manipulate a spatial configuration of several types of cells and scaffolds (Fig. 2b). In fact, an artificial
tooth root can be formed by combination of dental pulp stem
cells and periodontal ligament stem cells seeded onto separate scaffolds [8]. In addition, micromanipulation techniques
of tissue organization from dissociated cells have been developed in nanotechnology [22]. Cell manipulation techniques
to form differentiated teeth by tissue engineering methods
using scaffold will become more important to rapidly grow
bioengineered teeth.
Recently, we developed a novel bioengineered method,
which is a cell aggregate method, to reconstitute a threedimensional organ germ in an early stage, termed a bioengineered organ germ method [23]. The distinctive feature
of this method is that a bioengineered tooth germ can be
reconstituted between epithelial and mesenchymal cells

Biotechnological approaches in dental regenerative medicine

73

Figure 2 Strategies for building a bioengineered tooth from dissociated single cells. The cell aggregate method is a technique for
creating undifferentiated tooth germ using tooth germ cells, and tissue engineering aims to build differentiated teeth by seeding
several kinds of differentiated cells in scaffold.

with cell compartmentalization at high cell density, equivalent to that in the embryo, in a collagen gel drop (Fig. 3a).
This bioengineered tooth germ has the ability to generate
teeth having a correct tooth structure in a subrenal capsule,

and this occurred with a high rate of success (Fig. 3a).

Furthermore, this bioengineered tooth germ was then cultured in vitro, the epithelial cells invaginated and multiplied
prominently in mesenchymal aggregates, and then gave rise

Figure 3 Regeneration of a whole tooth from bioengineered tooth germ in vitro and in vivo. (a) Phase contrast and histological images of
the bioengineered tooth germ before and after 14 days of transplantation in a subrenal capsule. am: ameloblasts, BO: alveolar bone, bv:
blood vessels, PD: pre-dentin, DE: dentin, E: epithelial cells, EN: enamel, M: mesenchymal cells, od: odontoblasts, p: pulp cells and PDL:
periodontal ligaments. Scale bar: 100 mm. (b) Time course images of a bioengineered incisor (upper panel) and molar (lower panel) tooth
germ in an in vitro organ culture. Scale bar: 500 mm. (c) Separated primordia from bioengineered tooth germ that had been cultured for 2
days (left), and bioengineered tooth generated after 14 days of transplantation in extracted tooth cavity (right). Scale bar: 250 mm.

74
to multiple primordia from the periphery of the boundary
surface between the epithelial and mesenchymal cells
(Fig. 3b). These primordia reproduced the normal patterns
of gene expression in tooth development, and each grew into
an individual tooth (Fig. 3b). In addition, this method can be
used to grow various types of teeth, such as incisors and
molars (Fig. 3b). Direct cell-to-cell interaction induced by
high cell density and cell compartmentalization is essential in
tooth organogenesis and possibly in that of other organs as
well [23]. Our model improves understanding of principles by
which organ reconstitution can be achieved with tissues that
have been bioengineered in vitro and increases the potential
for bioengineered organ replacement in the future.

5. Regeneration of teeth in adult oral

environment
Another hurdle in the establishment of dental regenerative
therapies is the proper growth of bioengineered tooth germ in
the adult oral environment. While natural tooth germ develops
together with neighboring tissues that secrete various signaling factors in the embryo, such neighboring tissues are already
formed in the adult. It has been reported that a natural tooth
germ can grow in the adult oral environment when transplanted into the diastema between the incisors and the molars
[24]. However, it has not been demonstrated that a bioengineered tooth germ can develop in the adult oral environment.
Additionally, the penetration of nerve fibers into the dental
pulp of a bioengineered tooth is desirable in order to provide
normal sensation. Recently, we also reported that a bioengineered tooth germ primordium, which was isolated from a
bioengineered tooth germ, could be transplanted and could
develop into a tooth with correct tooth structure having
enamel, dentin, root, dental pulp, periodontal ligament,
blood vessels, and alveolar bone, in a tooth cavity after the
extraction of a mandibular incisor (Fig. 3c) [23]. These findings
suggest the possibility of successful generation of whole teeth
by transplantation of bioengineered tooth germs into the adult
oral environment [23]. Further studies for long-term transplantation of bioengineered tooth germ should be undertaken.

6. Future perspectives on dental

regenerative medicine
Previous research in dental regenerative medicine showed
the feasibility of not only stem cell transplantation, but also
organ replacement, earlier than for other organs. Currently,
it is necessary to find an effective and easily accessible
source of a patients own cells to avoid rejection. Somatic
dental tissue stem/progenitor cells are promising candidates
for the formation of whole teeth as well as for stem cell
transplantation. In addition, it has been shown that useful
cell sources are also present in non-dental tissues such as
adult bone marrow (Table 1) [24]. Furthermore, it will be
necessary to elucidate the mechanisms of tooth morphogenesis to control the size and shape of bioengineered teeth
[19,25]. Additional research to coordinate advances in various fields, such as clinical medicine, biology, materials
science, nanotechnology, and chemistry, will increase in
importance in the advancement of regenerative therapies.
The bioengineering technologies developed for tooth regen-

K. Nakao, T. Tsuji
eration will make substantial contributions to understanding
the developmental process and will encourage future organ
replacement by regenerative therapies in a wide variety of
organs, such as the liver, kidney, and heart.

Acknowledgements
This work was supported in part by an Academic Frontier
Project for Private Universities (to Yasuhiro Tomooka and
Takashi Tsuji.): a matching fund subsidy from MEXT Japan
(20032007). This work was also supported in part by a
Grant-in-Aid for Scientific Research in Priority Areas, System Cell Engineering by Multiscale Manipulation (No.
18048039), to Takashi Tsuji from MEXT Japan.

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