Journal of Oral Biosciences 64 (2022) 26e36

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Journal of Oral Biosciences

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

Review

Biological characteristics of dental pulp stem cells and their potential

use in regenerative medicine
Masaki Honda a, Hayato Ohshima b, *
a
Department of Oral Anatomy, Aichi Gakuin University School of Dentistry, Nagoya, Japan
b
Division of Anatomy and Cell Biology of the Hard Tissue, Department of Tissue Regeneration and Reconstruction, Niigata University Graduate School of
Medical and Dental Sciences, Niigata, Japan

a r t i c l e i n f o a b s t r a c t

Article history: Background: Regenerative medicine has emerged as a multidisciplinary field with the promising po-
Received 30 November 2021 tential of renewing tissues and organs. The main types of adult stem cells used in clinical trials are
Received in revised form hematopoietic and mesenchymal stem cells (MSCs). Stem cells are defined as self-renewing clonogenic
30 December 2021
progenitor cells that can generate one or more types of specialized cells.
Accepted 6 January 2022
Available online 11 January 2022
Highlight: MSCs form adipose, cartilage, and bone tissue. Their protective and regenerative effects, such
as mitogenic, anti-apoptotic, anti-inflammatory, and angiogenic effects, are mediated through paracrine
and endocrine mechanisms. Dental pulp is a valuable source of stem cells because the collection of dental
Keywords:
Adult stem cells
pulp for stem cell isolation is non-invasive, in contrast to conventional sources, such as bone marrow and
Cell differentiation adipose tissue. Teeth are an excellent source of dental pulp stem cells (DPSCs) for therapeutic procedures
Cell proliferation and they can be easily obtained after tooth extraction or the shedding of deciduous teeth. Thus, there is
Dental pulp increased interest in optimizing and establishing standard procedures for obtaining DPSCs; preserving
Odontoblasts well-defined DPSC cultures for specific applications; and increasing the efficiency, reproducibility, and
safety of the clinical use of DPSCs.
Conclusion: This review comprehensively describes the biological characteristics and origins of DPSCs,
their identification and harvesting, key aspects related to their characterization, their multilineage dif-
ferentiation potential, current clinical applications, and their potential use in regenerative medicine for
future dental and medical applications.
© 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
2. Structure of the dental pulp . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
3. Role of DPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
4. Regenerative capacity of pulp tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
5. MSCs in supernumerary teeth . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
6. Location-dependent characteristics of dental papilla/pulp cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
7. Characteristics of two populations obtained from dental papilla . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
8. Characteristics of dental pulp cells depend on their location to the crown and root portion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
9. Allogenic use of MSCs in dental pulp and a cell banking system . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
10. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Ethical statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
CRediT authorship contribution statement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34

* Corresponding author. Division of Anatomy and Cell Biology of the Hard Tissue,
Department of Tissue Regeneration and Reconstruction, Niigata University Grad-
uate School of Medical and Dental Sciences, 2-5274 Gakkocho-dori, Chuo-ku, Nii-
gata 951-8514, Japan. Fax: þ81-25-227-0804.
E-mail address: histoman@dent.niigata-u.ac.jp (H. Ohshima).

https://doi.org/10.1016/j.job.2022.01.002
1349-0079/© 2022 Japanese Association for Oral Biology. Published by Elsevier B.V. All rights reserved.
M. Honda and H. Ohshima Journal of Oral Biosciences 64 (2022) 26e36

Conflicts of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

1. Introduction Nestin-enhanced GFP (EGFP) mice, in which the EGFP gene was
inserted into the second intron enhancer involved in the regulation
The dental pulp is a loose connective tissue existing in a vacant of the Nestin gene, type VI intermediate filaments, expressed in
space (pulp cavity) surrounded by dentin or hard tissue. The outer neural stem cells of the central nervous system [3]. Interestingly,
shape of the pulp tissue almost matches the outer dentin shape, Nestin-GFP-positive cells were only present in the SOBL of the
although a shrinkage in space occurs with age because of a coronal pulp and not in the root pulp. Although Nestin protein is
continuous secondary dentinogenesis (Fig. 1aec). The pulp tissue is known as a differentiation marker for odontoblasts [4], Nestin-GFP-
placed in a special circumstance to communicate with the outside positive cells do not express endogenous Nestin mRNA and protein
primarily through the apical foramina. Although the bone marrow despite the occurrence of Nestin gene transcription. When odon-
is also a loose connective tissue surrounded by bone or hard tissue toblasts die after exogenous injury such as tooth grinding (cavity
(Fig. 1d and e), the pulp cavity is much smaller compared with the preparation), Nestin-GFP-positive cells express Nestin mRNA and
bone marrow cavity. Once inflammation occurs in the pulp tissue, protein to differentiate into odontoblast-like cells (OBLCs). Thus, it
pulpal pressure readily increases to cause deteriorative effects on is possible that the precursor cells, which are ready to differentiate
the tissue. This harmful condition persists and results in chronic into OBLCs, are arranged in the SOBL in advance [5]. This organi-
inflammation. This is the reason why the dental pulp is called a zation of the dental pulp is reasonable from the view point of
low-compliance tissue [1]. Histological observation of the coronal establishing a biological defense.
pulp reveals four distinct layers from the periphery to the core: the
odontoblast layer, cell-free and cell-rich zones (these two zones 2. Structure of the dental pulp
correspond to the subodontoblastic layer [SOBL]), and the central
pulp tissue [2] (Fig. 1f). In contrast, the presence of a cell-rich zone The dental pulp contains a wide variety of cells including
is unclear in the root pulp. Recently, we demonstrated that green odontoblasts forming dentin, dental pulp cells forming the pulp
fluorescent protein (GFP)-positive cells are localized to the SOBL in tissue, or fibroblasts that produce and break down collagen fibers,

Fig. 1. Comparison between a human mandibular first molar (aec) and a human femur (d, e) and the structure of dental pulp (f). a. The external appearance of an extracted molar. b.
Three dimensional-reconstructed image of a trimmed molar using m-computed tomography. c. Schema showing the structure of a tooth and its supporting tissue. d. The external
appearance of the part of a femur. e. The internal appearance of the part of a femur after sagittally cutting in half. f. Schema showing the mature coronal dental pulp containing three
defense layers including the odontoblast layer (OBL: frontline), subodontoblastic layer (SOBL: rearguard), and central pulp tissue (CP: main force). AB, alveolar bone; BMC, bone
marrow cavity; Ce, cementum; CL, capillary lumen; Com, compact bone; Cr crown; D, dentin; DC, dendritic cell; DP, dental pulp; E, enamel; G: gingiva; H, head; Mac, macrophage;
N, neck; P, periodontium; PC, pulp cavity; PD, predentin; R, root; S, shaft; SP, spongy bone; *, secondary dentin. Scale bars: 25 mm in (d) and 5 mm in (a).

27
M. Honda and H. Ohshima
and mesenchymal stem cells (dental pulp stem cells [DPSCs]).
There are also macrophages, lymphocytes, and dendritic cells (DCs),
which are immune cells [2] (Fig. 1f). The process of dentinogenesis
in more detail includes fenestrated capillaries that are located in
the odontoblast layer during active dentin formation [6,7]. The
positional and ultrastructural changes of these capillaries correlate
with the activity of dentin matrix deposition and calcification [8].
DCs associate with these fenestrated capillaries [9] (Fig. 2a).
Although DCs play an important role in the initial immunological
response to foreign bodies, it is possible that DCs wait for foreign
bodies to invade the microenvironment along with essential amino
acids and minerals supplied by the circulatory system for active
dentin formation. In human dental pulp, DCs are located in the
predentin and they make contact with multiple odontoblast pro-
cesses to rapidly detect conformational abnormalities of odonto-
blasts caused by exogenous stimuli [10]. In addition, in the coronal
dental pulp of aged rats where DCs are densely distributed in the
predentin along with the SOBL [11], the coronal pulp may possess
the capability of protecting the pulp tissue against exogenous
stimuli.
The primary dentin is formed during tooth development and
comprises the majority of the tooth. After completion of the root, a
Fig. 2. Schemata showing immature, mature, and resting odontoblasts during dentinogenesis (a) and the distribution of label-retaining cells (LRCs) during tooth development (b).
CL, capillary lumen; D, dentin; DC, dendritic cell; E, enamel; MD, mantle dentin; OB, odontoblasts; PAB, preameloblast; PD, predentin.
28
Journal of Oral Biosciences 64 (2022) 26e36
small amount of dentin is slowly added throughout the life to the
primary dentin. This is referred to as secondary dentin (Fig. 1b and
c). In contrast, the dentin, which is produced reactively when it is
subjected to exogenous injuries such as attrition, abrasion, caries,
and tooth drilling, is referred to as tertiary dentin [2]. The dentin
produced by newly differentiated OBLCs that replaces dead odon-
toblasts damaged through odontoblast processes in the dentinal
tubules, is known as reparative dentin, whereas the dentin pro-
duced by preexisting odontoblasts activated by stimuli is known as
reactionary dentin [12]. The fact that reparative dentin is formed
following tooth injury indicates the occurrence of adult stem cells
in the dental pulp [13]. This issue will be discussed in detail below.
Although the relationship between slow-cycling (nucleoside
label-retaining) cells and stem cells is controversial, it has been
investigated in detail in studies using continuously growing mouse
incisors [14]. In vivo pericyte labeling using Ng2-Cre revealed that
odontoblasts are derived from pericytes, suggesting that some, but
not all, DPSCs are derived from pericytes [15]. According to in vivo
stem cell labeling using Thy1-Cre, Thy1-positive cells contribute
approximately 10%e20% of odontoblasts and pulp cells, and slow-
cycling cells are Thy1-positive [14]. Labeling using two different
glial cell ERT2-Cre drivers, Plp1 and Sox10, revealed that 50% of
M. Honda and H. Ohshima
pulp cells and odontoblasts are derived from glial cells; however,
Plp1/Sox10 positive glial cells are also located in the slow-cycling
cell population [16]. Furthermore, stem cells near odontoblasts
differentiate into odontoblasts, whereas stem cells in the central
pulp differentiate into pulp cells. Thus, in the mouse incisor pulp,
DPSCs localize to areas where perivascular pericytes and peri-
neuronal Schwann cells are present [17].
The localization of DPSCs in the teeth with limited growth, such
as murine molars and human teeth, remained to be determined.
This is attributed to the fact that, unlike continuously growing
mouse incisors, the pulp of mature teeth with completed roots is a
tissue that rarely divides and the methodology for identifying slow-
cycling cells has not been well-established. We succeeded in
identifying slow-cycling cells by administering 5-bromo-20 -deox-
yuridine (BrdU) to prenatal rats and mice through their mothers
(prenatal BrdU labeling method) [18,19]. This method can distin-
guish LRCs with a densely-labeled nucleus (dense LRCs) from LRCs
with a granularly-diluted nucleus (granular LRCs) (Fig. 2b). Stem
cells have the following characteristics: (1) they remain undiffer-
entiated, (2) undergo unlimited cell division, and (3) after cell di-
vision, one daughter cell becomes a stem cell, whereas the other
becomes a transit-amplifying cell and divides actively [20]. Thus,
the cells that adsorbed thymidine-analogous BrdU during DNA
synthesis were labeled, whereas transit-amplifying cells that
actively divide exhibited a sparsely-labeled nucleus, resulting in
dense labeling of the stem cells. Therefore, dense LRCs labeled by
this prenatal labeling method are considered to be DPSCs and these
cells are localized to the perivascular niche in the central pulp.
These results suggest that DPSCs in the teeth exhibiting limited
growth also localize in areas where pericytes are present similar to
that of the mouse incisor pulp. These dense LRCs express stem cell
markers, such as STRO-1 and CD146 [19]. However, there are
inherent problems with prenatal labeling methods using BrdU: (1)
static stem cells that do not divide are not labeled, (2) living BrdU-
positive cells cannot be isolated for functional assays, (3) BrdU
exhibits toxic effects on living cells, and (4) differentiated cells
(odontoblasts) are also labeled because of the timing of BrdU
administration during cell differentiation [13]. To overcome these
problems, we investigated slow-cycling cells using TetOP-histone
2B (H2B)-GFP-modified mice (doxycycline ingestion at E14.5 or
E15.5), in which the cells turn GFP-positive following doxycycline
ingestion [21e24]. Identifying slow-cycling cells reveals that dense
LRCs are localized in odontoblasts and SOBL as well as the central
pulp. These findings suggest that the cells destined to become
odontoblasts and SOBL cells cease cell division early in develop-
ment. Furthermore, cells in the SOBL exhibit strong IGFBP5/Igfbp5
expression, suggesting that IGFBP5 plays a role in regulating the
survival and apoptosis of DPSCs during odontogenesis and pulpal
healing after exogenous stimuli [22]. Taken together with the
findings in Nestin-EGFP mice described above [5], the SOBL may
play a role as DPSCs or progenitor cells that are capable of differ-
entiating into OBLCs.
3. Role of DPSCs
DPSCs form and repair the dentin-pulp complex and maintain
pulpal homeostasis [13]. Tooth development is a sequential process
of induction, morphogenesis, cell differentiation, and matrix
secretion, with spatiotemporal precise regulation of cell prolifera-
tion and differentiation by epithelial-mesenchymal interactions
between the oral mucosal epithelium (ectoderm) and the
29
Journal of Oral Biosciences 64 (2022) 26e36
underlying cranial neural crest-derived mesenchyme (ectomesen-
chyme) [25e27]. During this process, ectomesenchymal cells are
characterized as stem cells. It is likely that MSCs gradually decrease
in number and become present in localized niches during the
progression of tooth development [13]. The major signaling mole-
cules that contribute to tissue interaction are bone morphogenetic
proteins, fibroblast growth factors (FGFs), sonic hedgehog, tumor
necrosis factor, and wingless-type MMTV integration site (Wnt)
family members [27,28]. These signals are considered toolkit genes
because they are also used during other organogenesis [29]. Tooth
development proceeds through three stages including the bud, cap,
and bell stages. The ectomesenchyme is divided into the dental
papilla, which becomes the dental pulp, and the dental follicle,
which differentiates into periodontal tissue. In the bell stage tooth
germ, cell differentiation proceeds from the cusped area, and
odontoblasts and ameloblasts initiate dentinogenesis and amelo-
genesis, respectively [2].
Differentiation of odontoblasts begins at the site of the future
cusped tip of the bell stage tooth germ, whereas dentinogenesis
proceeds through epithelial-mesenchymal interactions. Dentino-
genesis commences by trapping growth factors and signaling
molecules, such as members of the transforming growth factor b
superfamily, insulin-like growth factors, Wnts, and FGFs, secreted
by inner enamel epithelial cells in the basement membrane [30].
When dental papilla cells that have completed their last cell divi-
sion encounter the basement membrane, they begin to express
growth factor receptors. These cells probably contact the basement
membrane and receive trapped growth and other factors to
differentiate into odontoblasts [31]. Dental papilla cells in contact
with the basement membrane become preodontoblasts upon po-
larization, and odontoblasts when the predentin becomes visible.
Odontoblasts, which are columnar in shape with the development
of Golgi apparatus and rough endoplasmic reticulum, exhibit
multiple fine cell projections at their distal end (basement mem-
brane side). The odontoblasts involved in the formation of mantle
dentin are referred to as immature odontoblasts [4]. As they secrete
the matrix, the odontoblasts retreat toward the central pulp while
maintaining their cell layer, pushing each other. Thus, the cell layer
represents a pseudostratified epithelium-like arrangement. During
this period, odontoblasts develop a single thick cell process, and
fenestrated capillaries among the odontoblast layer are located
near the predentin and are responsible for the rapid supply of
amino acids and minerals [8]. These cells are known as mature
odontoblasts and are involved in circumpulpal dentin formation.
Odontoblasts that decrease in height to commit themselves into
static status are referred to as resting odontoblasts [4] (Fig. 2a).
Recent stem cell studies suggest that in tissues with high
turnover, including hair follicles, intestinal crypt, and bone marrow,
there are two types of stem cells: quiescent stem cells that are out
of the cell cycle in a low metabolic state, and active stem cells that
are cycling [32]. Two types of stem cells are also presumed to be
present at the apical end of continuously growing mouse incisors
[23,33]. Recent studies have proposed the concept that stem cells
divide symmetrically to become new stem cells, whereas quiescent
stem cells are thought to divide asymmetrically [34]. As mentioned
above, if thymidine-analogous BrdU is administered to animals at
the appropriate time, slow-cycling long-term LRCs may be identi-
fied and considered quiescent stem cells. DPSCs are considered
quiescent stem cells, because they rarely proliferate in adult tissues
under physiological conditions [13,19,35]. Bone tissue constantly
undergoes resorption and formation, or remodeling, in which old
M. Honda and H. Ohshima
bone is replaced by new bone. In contrast, dentin is a non-
remodeling tissue, but there are no available results showing the
extent to which pulp cells, including fibroblasts, and odontoblasts
are replaced by new cells throughout the life cycle.
4. Regenerative capacity of pulp tissue
Organisms have the ability to heal from physical damage such as
trauma and amputation, and can repair and regenerate their tissue
according to the location of the injury [36]. The dentin-pulp com-
plex also regenerates and irregular dentin (tertiary dentin) is
formed in response to exogenous stimuli as described above. The
wound healing process of skin and oral mucosa is an efficient
process for repairing structure and function (protection and de-
fense) after tissue damage. The healing process begins with blood
clot formation resulting from bleeding, fibrin deposition, and
platelet aggregation and coagulation, followed by acute inflam-
matory reactions by neutrophils and macrophages and damaged
tissue repair [2]. The epithelial tissue repair proceeds with active
cell proliferation and restoration of epithelial tissue continuity,
followed by connective tissue repair. Compared with the wound
healing process of skin and oral mucosa, the healing process of the
dentin-pulp complex is more complex and several factors
contribute to the healing process [13]. These factors include the
type, extent, degree and duration of stimulation, the diversity of
dentin structure because of age-related changes and previous in-
juries, and differences in individual-specific immune responses. For
example, slowly progressive attrition or abrasion and cavity prep-
aration elicit different pulpal responses, and bacterial infections
such as dental caries are more complex because of host-parasite
interactions [2]. The term “regeneration” is referred to as “repair
and restoration according to the location of the injury” [36]. With
respect to teeth, the term “scar formation” is more accurate than
“regeneration” because of the fact that the defects in hard tissues
do not repair themselves, but trigger a protective response known
as tertiary dentin formation. However, we postulate that the term
“regeneration” can be used at the cellular level, since new odon-
toblasts (or more accurately, OBLCs, since their cellular character-
istics differ from those of the original odontoblasts) replace the
degenerated odontoblasts after tooth injury. To understand the
response of the dentin-pulp complex to exogenous injury, we
evaluated the pulpal response to tooth injury using different animal
experimental models established in our laboratory [13].
Cavities cause localized damage to the dental pulp by directly
injuring the odontoblast processes present in the dentin [37].
Damage to the pulp is localized to the odontoblasts, the processes
of which are injured from cutting, leading to death of the odonto-
blasts. Although it had been thought that the cell dynamics during
the pulp repair process after cavity preparation involves the
sequential steps of cell division, migration, adhesion, and differ-
entiation, our animal model revealed that the dynamics differ
depending on the degree of injury. In an experimental cavity
preparation model using rats [38], after mechanical damage and
degeneration of odontoblasts, the migration and differentiation of
odontoblast-lineage cells begin on postoperative day 1, and OBLC
differentiation is almost completed on day 2. In contrast, the in-
crease in cell proliferation over a wide area of the damaged pulp
tissue continues up to 5 days after operation. Namely, the cell dy-
namics proceed in the following order: cell migration, adhesion,
differentiation, and cell proliferation [38]. Therefore, the fact that
cell proliferation in the central part of the pulp increases after the
OBLCs arrange in the area that used be the odontoblast layer after
cavity preparation suggests the existence of progenitor cells that
differentiate into OBLCs without cell proliferation and DPSCs sub-
sequently organize to repair the whole pulp tissue [19]. In contrast,
30
Journal of Oral Biosciences 64 (2022) 26e36
in an experimental model of cavity preparation using mice, damage
to the pulp tissue is extremely severe because of the small tooth
size, which results in a wide range of damage to influence the SOBL
and initiate OBLC differentiation 3 days after the operation, which
continues until day 5 [35]. Since the peak of cell proliferation in
mice is 3 days after the operation, it is reasonable that DPSCs in the
central part of the pulp tissue proliferate and differentiate into
OBLCs after the degeneration of odontoblasts in this model,
although the majority of OBLCs are derived from the SOBL based on
our evidence [5] (Fig. 3a). Reactionary dentin formation is also
induced on the pulpal floor in this experimental model and both
reparative and reactionary dentin formation may be analyzed
simultaneously [35,39].
Tooth replantation is a severe model of tooth damage compared
with cavity preparation since nerves and blood vessels entering
into the pulp cavity are amputated at the apical root foramina
during tooth extraction, thus transiently disrupting blood circula-
tion and innervation [40,41]. As a result, most odontoblasts die after
tooth replantation because of hypoxic conditions. Although some
odontoblasts may survive at the root apex and the pulpal floor,
depending on the presence of the medullary canal and the timing of
blood circulation restoration, most odontoblasts die throughout the
pulp cavity in addition to the SOBL cells. Therefore, since the
cascade of apoptosis, cell proliferation, migration, adhesion, and
differentiation proceeds in the pulp, the timing of OBLC differen-
tiation is at postoperative 5e7 days, which is common with rat and
mouse models [40,41] (Fig. 3b). When tooth replantation is per-
formed in rodents labeled by the embryonic labeling method,
DPSCs proliferate and differentiate into OBLCs, since OBLCs are
labeled [19,42]. Wound healing patterns after tooth replantation
are variable including tertiary dentin formation, bone tissue for-
mation, mixed types of both, replacement with fibrous tissue, and
inflammatory responses [40e43]. This is attributed to the fact that
occlusal trauma after tooth replantation affects the wound healing
pattern. When the counterpart tooth is extracted after tooth
replantation, the percentage of tertiary dentin formation increases
from 43% to 60% [41]. Survival of DPSCs is a decisive factor that
defines the pulp healing pattern after tooth replantation. Tooth
replantation using mice labeled by the embryonic labeling method
demonstrated that bone tissue formation occurs when dense LRCs,
putative DPSCs, are lost from the pulp cavity [19].
5. MSCs in supernumerary teeth
Adult MSCs can be isolated from different tissues including bone
marrow, adipose tissue, and dental pulp [44e46] and they have
been used in regenerative medicine because of their trilineage
(osteoblasts, chondrocytes, and adipocyte) differentiation potential
[47,48]. MSCs also have the ability to migrate to the site of injury,
where they modulate the inflammatory response and mobilize
intrinsic cell reservoirs through paracrine mechanisms [49,50].
MSCs in human dental pulp were first described in permanent
teeth approximately 20 years ago [46]. A distinct population of
MSCs has been isolated from the dental pulp of permanent, de-
ciduous [51], developing [52], and supernumerary teeth [53].
Importantly, DPSCs are derived from the ectomesenchymal neural
crest and share the biological features of both MSCs and neural
crest-derived cells. Dental pulp has the advantage of being readily
isolated from teeth without causing secondary damage or ethical
controversies associated with harvesting MSCs. Among the types of
teeth, supernumerary teeth are a potential source of MSCs since
they are normally discarded. To date, there are two reports with
respect to MSCs in mesiodents, supernumerary teeth in the central
portion of the upper and lower jaw [53,54]. MSCs isolated from
mesiodents exhibited colony-forming unit-fibroblast (CFU-F) and
M. Honda and H. Ohshima Journal of Oral Biosciences 64 (2022) 26e36

Fig. 3. Schemata showing chronological changes in the periphery of pulp tissue following cavity preparation (a) and tooth replantation (b) using mouse molars. CL, capillary lumen;
CP: central pulp tissue; D, dentin; OB, odontoblasts; OBLC: odontoblast-like cell; PD, predentin; SOBL, subodontoblastic layer.

the capacity to differentiate into multiple cell types [53], whereas

MSCs isolated from 3 mesiodents were comparable to stem cells
from exfoliated deciduous teeth (SHEDs) in another study [54].
Although both cell types show similar stem cell characteristics,
MSCs in mesiodents have a disadvantage in growth rate after long-
term storage compared with SHEDs. Since our previous study re-
ported the characteristics of DPSCs in 10 mesiodents compared
with those in three permanent teeth [55], we describe the char-
acterization of dental pulp cells in mesiodents.
Supernumerary teeth are an additional complement of decidu-
ous and permanent dentition (Fig. 4). The prevalence of supernu-
merary teeth ranges from 0.5% to 5.3% of the permanent dentition
[56]. The prevalence of mesiodents is 0.15%e1.9% in the general
population and they are more frequent in males than females [57].
It is widely accepted that mesiodents arise from local and inde-
pendent hyperactivity of the dental lamina, in which the lingual
extension of an additional tooth bud forms a tooth [56,58]. Another
theory, known as the dental germ dichotomy theory, suggests that
the tooth bud splits into two equal parts or into one normal tooth
and a dysmorphic one [59].
In our study, mesiodents were obtained from 10 5e8-year-old
patients and admitted for orthodontic treatment at the Nihon
University Dental Hospital [55]. The apex of the tooth root was
carefully observed in all subjects when it was extracted and the root
was confirmed to be completed and not resorbed. Mesiodents
varied significantly in size and shape. Six mesiodents appeared
Fig. 4. Radiograph showing an inverted and conical mesiodente used in this study. The
conical crown-shaped, resembling the microdont, whereas four mesiodente can be seen between the central incisors.

31
M. Honda and H. Ohshima
mesiodents appeared canine-crown-shaped. Nine mesiodents from
permanent dentition showed delayed root development compared
with that of adjacent incisors and were found unerupted in the
palatal bone. First, we attempted to culture the isolated dental pulp
obtained from the 10 mesiodents. All cell populations succeeded in
proliferating using a standard culture method. Subsequently, we
examined whether the cells exhibited the characteristics of MSCs.
All cell populations showed a colony-forming potential, the ability
to differentiate into osteogenic cell-lineages, and exhibited a very
low adipogenic potential. These results indicate that MSCs are
present in mesiodents.
6. Location-dependent characteristics of dental papilla/pulp
cells
We have been interested in the location-dependent character-
istics of dental pulp cells. A decade ago, MSCs from apical papilla
(SCAP), with a greater capacity than DPSCs from permanent teeth
were discovered in the pulp region, within the apical portion of the
root [52]. Cellular changes occur from crown to root formation,
resulting from changes in the cervical loop to Hertwig's epithelial
root sheath (HERS) during tooth root development. Dental pulp is
known as dental papilla during crown formation, but it is called
dental pulp after crown formation is completed. Regarding tissue-
forming capability, the dentinogenic potential of dental papilla
during crown formation differs from that of dental pulp during root
formation. Odontoblasts contribute to the formation of enamel
during crown formation and to the formation of cementum during
root development. Therefore, it is possible that dental pulp cells are
diverse in character, and their function depends on their location.
Based on these considerations, we focused on the differences in
the cellular characteristics of dental papilla and dental pulp that
depend on their location [60]. We first investigated the character-
istics and tissue-forming ability of two cell populations located in
different areas of the dental papilla (horn and core regions) (Fig. 5).
Second, after the dental pulp was obtained from the extracted
Fig. 5. Schema showing a third molar tooth germ of a six-month-old pig used in this experiment. Enamel (E) and dentin (D) are partly generated at this stage. The cervical loop
epithelium (CL), dental papilla from the horn chamber (PHC), and dental papilla from the core chamber (PCC) are separated and the three cell types are obtained from each tissue.
32
Journal of Oral Biosciences 64 (2022) 26e36
permanent, deciduous teeth and mesiodents, the crown and root
portions were separated at the boundary of the cement-enamel
junction [55,61,62]. We describe the cellular characteristics from
each pulp below.
7. Characteristics of two populations obtained from dental
papilla
Here, we describe the first study using porcine third molar tooth
buds at late bell stage [60]. To determine the location-dependent,
tissue-forming capability of dental papilla cells, the two-tissue
transplantation assay for tooth-tissue engineering using the
sequential cell seeding method was performed as previously
described [60]. Briefly, tooth buds were mechanically separated
into three areas: (1) cervical loop epithelium; (2) dental papilla
from the horn chamber; and (3) dental papilla from the core
chamber (Fig. 5). The cells were obtained from the chamber in each
area by using a standard method [63,64]. Each dental papilla cell
population (horn or core) was combined with cervical loop
epithelial cells in biodegradable scaffolds. The two-tissue trans-
plantation sets (either the cervical loop epithelial cells plus horn
papilla cells transplant or the cervical loop epithelial cells plus core
papilla cells transplant) were inserted into the omentum of
immunodeficient rats. The in vivo results revealed interesting
characteristics of the location-specific dental papilla cells. Although
the two-tissue sets produced hard tissue after 15 weeks, their
histological appearances were completely different. Core papilla
cells were involved in the production of enamel-dentin complex
structures, whereas horn papilla cells were primarily involved in
the formation of dentin-cementum tissue complexes. These find-
ings indicate that mesenchymal cells determine the type of hard
tissue. In other words, horn papilla cells lose the capacity to induce
the differentiation of dental epithelial cells into ameloblasts,
whereas core papilla cells maintain the capability for inducing
enamel formation. This concept is completely different from the
conventional theory that different epithelial cell types, ameloblasts
M. Honda and H. Ohshima
or HERS determine the enamel-dentin complex or cementum-
dentin complex. To investigate the tissue-forming capability of
each specific tissue individually (the cervical loop epithelium re-
gion, the papilla from the horn chamber, and the papilla from the
core chamber), each tissue was individually transplanted into the
omentum of immunodeficient rats for 15 weeks. The dental papilla
tissue implants from the horn and core areas exhibited no calcifi-
cation. In addition, when the cells from each papilla area (horn or
core) were transplanted alone, they only formed dentin tissue (and
not enamel or cementum). The decision to form a tooth crown or
tooth root may depend on the differentiation stage of the dental
papilla cells. To support our hypothesis, we examined gene
expression patterns in the dental papilla cells. The horn cells
showed 200-fold higher dentin sialophosphoprotein (Dspp: a gene
involved in dentinogenesis) expression compared with the core
cells. The genomic data demonstrated that the horn cells were
more mature than the core cells. Our study indicated that the cells
residing in a specific location of the dental papilla possess a tissue-
specific formation potential when combined with cervical loop
epithelial cells. This notion is supported by a previous study using
Fgf10-deficient mice [65]. In mouse, rat, and human molars, the
expression of Fgf10 in the dental papilla disappeared when crown
morphogenesis shifts to root formation [66,67]. The disappearance
of Fgf10 signaling induces the cervical loop to become the HERS
instead of ameloblasts, resulting in root formation [65]. Therefore,
Fgf10 signaling is a candidate for determining the competency of
dental papilla cells to induce ameloblast differentiation.
8. Characteristics of dental pulp cells depend on their
location to the crown and root portion
Several studies have suggested that DPSCs are present in the
dental pulp; however, it is unknown whether MSCs from coronal
and root pulps exhibit different characteristics. The characteristics
of MSCs derived from the coronal and root portions of deciduous,
mesiodents and permanent teeth were assessed by the CFU-F
population, growth potential, expression of surface antigens, and
osteogenic or adipogenic potential [55,61,62]. SHEDs have already
been identified in coronal pulp from human exfoliated deciduous
teeth [51]. However, it is not known whether MSCs are present in
the root portion. Therefore, we first examined the presence of MSCs
in the root pulp (root pulp cells [dRPCs]) from deciduous teeth. We
isolated cells separately from the coronal (coronal pulp cells
[dCPCs]) and root portion of dental pulp of deciduous first incisor
teeth extracted from patients undergoing orthodontic treatment
[61] (Fig. 6). Flow cytometry revealed a similar expression pattern
of MSC markers between coronal and root cells. Osteogenic and
adipogenic potential was observed for dCPCs and dRPCs. Interest-
ingly, the CFU-F population and proliferative potential were greater
in dRPCs compared with dCPCs. To support the growth potential
results, the expression of 10 genes related to cell cycle progression
was measured in dRPCs and dCPCs by primer array analysis.
Although RT-PCR analysis shows a similar expression pattern based
on ES cell markers and odontoblast markers, such as ALP, DMP1, and
DSPP, dRPCs expressed KLF4 at significantly higher levels compared
with those of dCPCs. Another study suggested that the endogenous
expression of reprogramming factors in cells is positively correlated
with their reprogramming efficiency [68e71]. We induced RPCs
into iPS cells and compared their reprogramming efficiency with
that of dCPCs. Consistent with our hypothesis, dRPCs exhibited a
higher reprogramming efficiency than dCPSs. Taken together, our
results demonstrate that MSCs are present in the root pulp of de-
ciduous teeth and the characteristics of dRPCs differ from those of
dCPCs. As noted above, DPSCs are present in mesiodents. To
determine whether mesiodente coronal pulp cells (mCPCs) and
33
Journal of Oral Biosciences 64 (2022) 26e36
mesiodente root pulp cells (mRPCs) differ from one another, they
were isolated and their characteristics were compared [55]. More
than 85% of both cell types were positive for CD13, CD44, CD73,
CD90, and CD146. In addition, both cells exhibited CFU-Fs and the
ability to differentiate into osteogenic cell lineages. Interestingly,
there were considerable differences in the proportion of CD105þ
cells between the mCPSs and mRPCs. In six mesiodents, the pro-
portion of CD105þ cells was higher in mRPCs compared with
mCPCs. In two mesiodents, the proportion of CD105þ cells was
higher in mCPCs compared with mRPCs, whereas two mesiodents
and three permanent teeth showed no differences in the propor-
tion of CD105þ cells between mCPCs and mRPCs. Next, we exam-
ined whether there was a relationship between the number of
CD105þ cells and the characteristics of MSCs. In both mRPCs and
mCPCs, the high percentage of CD105þ cells was associated with
high CFU-F number, proliferative capacity, and osteogenic differ-
entiation capacity. To further examine the association between
CD105 expression and MSCs in dental pulp, we separated and
compared CD105þ cells and CD105- cells. CD105þ cells exhibited a
higher CFU-F number and proliferative capacity than CD105- cells.
Taken together, these results indicate that the proportion of
CD105þ cells is an important factor for characterizing MSCs in
mesiodents. In the case of permanent teeth, there were no differ-
ences between the number of CD146- or CD105-positive cells and
the characteristics of MSCs of coronal and root pulp cells.
9. Allogenic use of MSCs in dental pulp and a cell banking
system
As mentioned above, dental pulp cell-based therapy has been
regarded as one of the most attractive applications in regenerative
medicine. For current cell-based therapeutic strategies, an autolo-
gous cellular source is used for transplantation to avoid the risks of
immunological rejection and pathogen transmission. However,
autologous MSC (auto-MSC) transplantation has some potential
limitations in clinical applications. First, the treatment to obtain
mesoderm-derived MSCs from human body tissues is deleterious
to the patients. Unlike other mesoderm tissues, dental pulp is
readily isolated from extracted teeth without causing secondary
damage since extracted teeth are usually discarded. Second, MSCs
isolated from elderly donors have decreased biological activity,
including differentiation and regenerative potential [72e74].
Moreover, even if dental-pulp derived MSCs are isolated from
young donors, there is high individual variation in the character-
istics of MSCs, resulting in disappointing treatment outcomes.
Therefore, it is unpredictable whether auto-MSC therapy will lead
to a therapeutic success. Third, auto-MSC therapy cannot be applied
to treat acute diseases such as spinal cord injury and myocardial
infarction. Also, it is not off-the-shelf therapy at the present time. To
succeed, cellular therapy requires a certain number of cells;
therefore, the isolated small quantities of MSCs from dental pulp
have to undergo extensive proliferation prior to their use in
transplantation. Two approaches should be considered to solve the
issues of auto-MSC therapy. First, a cell banking system for cell
isolation and long-term storage of a patient's own cells should be
considered if treatment is required in the future. The dental pulp
cell bank is the ideal solution for future patient-tailored medicine.
Thus, individuals may deposit their own tooth-derived MSCs to
cover their future medical needs [75]. Dental pulp cell banks have
received considerable attention as a new biological resource for
both research and clinical applications around the world. In Japan,
two dental cell banks are currently under operation, including the
Dental Cell Bank™ of The Nippon Dental University and Cell
Technology Company [75]. Teeth are obtained for the dental pulp
cell collection from registered dental clinics around the country.
M. Honda and H. Ohshima
Fig. 6. Schema showing a method for collecting dental pulp cells from the coronal and root portions in a human deciduous tooth. After cutting the tooth in a sagittal direction, the
dental pulp is pulled out from the pulp cavity (PC). The pulp tissue is divided into coronal and root portions and then dental pulp cells are isolated from the tissue at each location. D,
dentin; E, enamel.
After obtaining informed consent, the extracted teeth are stored in
preservation solution in registered dental clinics and are then sent
to their respective cell banks. The dental pulp is isolated and dental
pulp cells are preserved until they can be used. However, a signif-
icant difficulty in achieving successful cell bank operations is the
cost for the isolation and long-term preservation. Therefore, since
isolation and long-term preservation of patient-specific auto-MSCs
is costly and time-consuming, cell therapy using allogenic MSCs
should be considered. In this case, obtaining allo-MSCs from dental
pulp as starting material is a reasonable approach, because they are
easily obtained by using non-invasive techniques compared with
other adult MSCs. Furthermore, several reports have suggested that
exfoliated deciduous teeth are also useful [75e77]. Since there are
normally twenty deciduous teeth in human, there are twenty op-
portunities to obtain MSCs from deciduous teeth. The Cell Tech-
nology Company in Tokyo has already created an allo-dental cell
bank. The teeth for MSC collection are obtained from registered
dental clinics. After obtaining informed consent for allo-MSCs cell
therapy, extracted teeth are stored in a preservation solution in
registered dental clinics and are sent to their respective cell banks.
The dental pulp is then isolated and the dental pulp cells are pre-
served. However, the use of allo-MSCs is generally costly and time-
consuming because a human leucocyte antigen (HLA) test is
required to identify HLA-matched donors and this is also laborious.
An alternative approach is to make use of HLA haplotype ho-
mozygous donors to provide HLA-matched material to a significant
number of patients. Several studies have been conducted to
determine the number of donors needed to cover the population of
our country. Nakatsuji et al. have calculated that 15,000 individuals
would be required to identify the top 50 HLA-homo individuals at
the HLA-A, -B, and eDR antigen levels, which would accommodate
82.2% of the Japanese population [78]. Furthermore, HLA haplotype
iPS transplantation from iPS banking has already been established
in Japan [79]. Dental pulp cells could be the ideal solution for HLA-
matched cell banks for clinical translation. One report has indicated
that two million deciduous teeth are extracted in dental clinics per
year [80]. This number excludes the number of naturally dropped
deciduous teeth. If all dental clinics were to cooperate, 200,000
individual deciduous teeth could be obtained in one year. Thus,
200,000 HLA types would be obtained and 65 haplotype homo
individuals may be identified that covers 90% of the Japanese
population.
34
Journal of Oral Biosciences 64 (2022) 26e36
10. Conclusions
Dental pulp is a valuable source of stem cells because the
collection of dental pulp for stem cell isolation is non-invasive in
contrast to conventional sources, such as bone marrow and adipose
tissue. Teeth are an excellent source of DPSCs for therapeutic pro-
cedures and can be easily obtained after tooth extraction or shed-
ding of deciduous teeth. The comprehensive knowledge regarding
the biological characteristics and origin of DPSCs, their identifica-
tion and harvesting, key aspects related to their characterization,
their multilineage differentiation potential, and current clinical
applications would contribute to their potential use in regenerative
medicine for future dental and medical applications.
Ethical statement
All human and animal experiments were conducted in compli-
ance with the protocol reviewed by the Ethics Committee and the
Institutional Animal Care and Use Committee of the authors’ uni-
versities in accordance with the Code of Ethics of the World Medical
Association (Declaration of Helsinki) for experiments involving
humans and ARRIVE guidelines for animal experiments. Tooth and
femur samples (Fig. 1) are stored in the Faculty of Dentistry, Niigata
University, for the educational purpose in compliance with the
“Preservation of Autopsy” act.
CRediT authorship contribution statement
Masaki Honda: literature search, writing. Hayato Ohshima:
literature search, writing.
Conflicts of interest
The authors declare no competing interests.
Acknowledgments
The authors cordially thank Enago (www.enago.jp) for the
English language review. This work was supported by Grants-in-
Aid for Scientific Research (B) (no. 17H04366 to HO) and Chal-
lenging Research (Pioneering) (no. 20K21672 to HO) from the
Japan Society for the Promotion of Science. Images in Figs. 1e3, 5,
M. Honda and H. Ohshima
and 6 have been drawn by Dr. H. Ohshima colored with Photoshop
(Ver.23.0.0, Adobe Inc, https://www.adobe.com/jp/products/
photoshop.html).
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