An In vivo Model for Short-Term Evaluation of the Implantation Effects of Biomolecules or Stem Cells in the Dental Pulp.
ABSTRACT The continuously growing rodent incisor is a widely used model to investigate odontogenesis and mineralized tissue formation. This study focused on evaluating the mouse mandibular incisor as an experimental biological tool for analyzing in vivo the capacity of odontoblast-like progenitors or bioactive molecules to contribute to reparative dentinogenesis. We describe here a surgical procedure allowing direct access to the forming part of the incisor dental pulp Amelogenin peptide A+4 adsorbed on agarose beads, or dental pulp progenitor cells were implanted in the pulp following this procedure. After 10 days A+4 induced the formation of an osteodentin occluding almost the totality of the pulp compartment. Implantation of progenitor cells leads to formation of islets of osteodentin-like structures located centrally in the pulp. These pilot studies validate the incisor as an experimental model to test the capacity of progenitor cells or bioactive molecules to induce the formation of reparative dentin.
The Open Dentistry Journal, 2008, 2, 67-72 67
1874-2106/08 2008 Bentham Open
An In Vivo Model for Short-Term Evaluation of the Implantation Effects
of Biomolecules or Stem Cells in the Dental Pulp
Sally Lacerda-Pinheiro1, Arnaud Marchadier2, Patricio Donãs3, Dominique Septier3,
Laurent Benhamou2, Odile Kellermann1, Michel Goldberg3,* and Anne Poliard1
1Laboratoire de Différenciation Cellulaire, Cellules Souches et Prions, IFR- 2937 CNRS, Villejuif, France; 2Laboratoire
de Caractérisation du Tissue Osseux par Imagerie, INSERM U658, Orléans, France and 3Laboratoire Régénération et
Réparation des Tissus Cranio-faciaux, EA2496, Faculté de Chirurgie Dentaire, Université Paris - Descartes, Mon-
Abstract: The continuously growing rodent incisor is a widely used model to investigate odontogenesis and mineralized
tissue formation. This study focused on evaluating the mouse mandibular incisor as an experimental biological tool for
analyzing in vivo the capacity of odontoblast-like progenitors or bioactive molecules to contribute to reparative dentino-
genesis. We describe here a surgical procedure allowing direct access to the forming part of the incisor dental pulp
Amelogenin peptide A+4 adsorbed on agarose beads, or dental pulp progenitor cells were implanted in the pulp following
this procedure. After 10 days A+4 induced the formation of an osteodentin occluding almost the totality of the pulp com-
partment. Implantation of progenitor cells leads to formation of islets of osteodentin-like structures located centrally in the
pulp. These pilot studies validate the incisor as an experimental model to test the capacity of progenitor cells or bioactive
molecules to induce the formation of reparative dentin.
Key Words: Mouse incisor, dental pulp progenitor cells, amelogenin, experimental model.
the central part of the pulp to the periphery of the embryonic
pulp where the last cell division occurs . When one of the
daughter cells comes in contact with the basement membrane
(BM) the cell becomes an odontoblast, whereas the other
daughter cell located some distance away from the BM is
incorporated in the Höehl’s cell layer . Odontoblasts and
Höehl’s cells constitute a specific subpopulation of neural
crest-derived and paraxial derived mesenchymal-cells. After
terminal differentiation they are responsible for dentin pro-
duction (physiological and reactionary dentin). If the odon-
toblasts are injured or destroyed during a carious lesion or its
treatment, Höehl’s cells may be reactivated and differentiate
into new odontoblasts. Upon more severe injury, when the
odontoblasts and Höehl’s sub-odontoblastic cell layer are
irreversibly destroyed, dentin formation can be mediated by
odontoblast-like cells recruited among pulpal cells during the
process known as reparative dentinogenesis . However, it
is well known that the natural capacity for healing of a tooth
is limited and that therapies involving calcium hydroxide are
not always successful. Therefore the search for strategies
stimulating or mimicking the natural healing properties of
the dental pulp is important. Two types of such therapeutic
strategies can be conceived today depending on the severity
of the lesions. Firstly, implantation of bioactive molecules
used as new pulp capping agents that would accelerate
and/or induce the formation of a bridge of reparative dentin
. Secondly, implantation in an exposed pulp of purified
*Address correspondence to this author at the Faculté de Chirurgie Dentaire,
1 rue Maurice Arnoux , 92120, Montrouge, France; Tel: +33 158076808;
Fax: +33 158076806; E-mail: firstname.lastname@example.org
During dentinogenesis, pre-odontoblasts migrate from
stem/progenitor cells that would promote the formation of a
Pulp implantation models have been developed over the
years in different species [rat, rabbit, ferret [5-7], monkey [8,
9], dog , essentially to evaluate the long-term effects of
bioactive molecules used as pulp capping agents. Mouse
models have never been described, very likely due to their
small size and hence difficulties in manipulating their teeth.
However, the recent development of stem cell research, with
all the data collected on mouse systems, makes it important
to have access to a kin model.
The continuously erupting mandibular incisor has been
extensively used to study the cellular and extracellular events
involved in odontogenesis, because all the successive stages
of development can be found in a single tooth, which exhib-
its many similarities with human tooth formation [11, 12]. A
niche of stem cells was identified in the apical end region
(cervical loop) of continuously growing teeth [13-15]. In the
rat incisor, some of the stem cells continuously differentiate
into odontoblasts, becoming functional, mature, old and fi-
nally degenerate . Ever-growing teeth (hypselodont)
such as the rodent incisors, maintain this function through a
rich population of stem cells, whereas molars display limited
growth (brachyodont) in rodents, and consequently have a
reduced number of stem cells. Therefore, the murine incisors
may provide a model to first dissect the cellular and molecu-
lar events underlying the regenerative processes.
In the present study, we have investigated whether the
mouse mandibular incisor could be exploited as an experi-
mental model for analyzing the potential of dental pulp pro-
genitors cells or bioactive molecules to promote the forma-
tion of reparative dentin. A surgical technique was devel-
68 The Open Dentistry Journal, 2008, Volume 2 Lacerda-Pinheiro et al.
oped to gain an easy access to the apical forming end of the
incisor by a limited perforation and to make a pulp exposure.
An amelogenin peptide (A+4), which has been shown to act
as a signaling molecule [17-19], and a dental pulp-derived
odontoblast progenitor clone (17IA4) , were implanted
into the pulp to validate the efficiency of the surgical ap-
proach. In both types of implantation, formation of a os-
teodentin-like neodentin was observed. Our data thus point
to the validity of the mouse incisor as a tool to evaluate the
potential of different types of stem cells or biomolecules for
MATERIALS AND METHODS
(3 months old-25g) (Charles River - Lyon, FR) were used in
these experiments. All experiments were performed under an
institutionally approved protocol for animal research.
Thirty lower incisors from 15 adults males C57Bl/6 mice
Agarose Bead, Molecules and Cells Preparation
Labs, Hercules, CA) were used as a carrier for bioactive
molecules and to facilitate the localization of the implanta-
tion sites. They were rinsed several times in PBS and dis-
tributed into a 12 microwell plate (?10 beads/ wells) with or
without (control beads) 2 ?g of A+4. The plate was then
incubated for 24h at 37°C to allow complete adsorption of
the proteins at the surface of the beads.
Affi-gel agarose beads (75-150?m in diameter, Biorad
embryonic (ED18) pulp and cultured as described . Ap-
proximately, 2.5 x 105 cells combined or not with the aga-
rose beads were distributed in Eppendorf tubes and centri-
fuged (1000 rpm / 1min) to form pellets which were thereaf-
ter implanted in the incisor.
17IA4, an odontoblast progenitor clone was derived from
toneal injection of a solution of ketamine 20% (Imal-
gène®500, Bayer Pharma, Puteaux, Fr) and xylazine 5%
(Rompun®2%, Merial, Lyon Fr) (10ml/kg). An incision of
about 1cm long was made through the skin with fine scissors
to access the subjacent muscle layer, along a theoretical line
joining the auditory meatus and the lip commissure (Fig.
1A). The masseter muscle was incised along its longitudinal
axis with a scalpel blade. The periosteum was scrapped with
a curette and the bone surface was exposed between the pos-
terior angle of mandible and the molars block, approximately
1mm above of the mandibular basal border. The point where
the outer oblique line crosses the apical loop of incisor was
selected as a site for the pulp exposure (Fig. 1B, 1C). A low-
speed dental drill equipped with a round tungsten-carbide
burr size 6 (Dentisply-Maillefer, Ballaigues, Switzerland)
was used to create a hole through the bone and tooth 9 (Fig.
1D). The mice were divided into 5 groups of 3 animals each.
They received group 1- agarose beads only, group 2- A+4 +
agarose beads, group 3- 17IA4 cells + agarose beads (this
group was performed as a control for cell implantation),
group 4- 17IA4 cells alone, and group 5- sham (surgery
without implantation). After implantation, the muscular layer
was closed with one drop of cyanoacrylate and cutaneous
At day 0, the animals were anesthetized with an intraperi-
layer was sutured with Vicryl-Monocryl* 4-0 (Ethicon –
Jonhson&Johnson, Piscataway, NJ, USA). All the animals
received 10 mg/kg ketoprofen (Profenid, Paris, France) as
analgesic immediately after surgery.
The mice were killed 10 days after implantation by cervi-
cal dislocation. Immediately after, the two hemi-mandibles
were dissected out and fixed in a 4% paraformaldehyde solu-
tion overnight at 4°C (Fig. 1E).
Fig. (1). Surgical procedure for implantation in the incisor pulp.
Incision was done along a theoretical line joining the auditory mea-
tus and the lip commissure (A), bone exposure (B) and perforation
(C). Cells or agarose beads were implanted (D) and the mandibles
were dissected after 10 days (E). Arrow points to the perforation
micro-CT (Skyscan®, Kontich, Belgium). The radiographic
projections (n = 413) were acquired at 70 kV and 90 ?A with
a fixed exposure time of 4 seconds. Four frames were aver-
aged for each rotation increment of 0.45° to increase the sig-
nal to noise ratio. The acquisition time was 2 hours. One
thousand slices were reconstructed with the manufacturer
reconstruction software (NRecon, Skyscan®) based on a
modified Feldkamp algorithm. The resulting 3D dataset con-
tained 1024 x 1024 x 1000 3.12 ?m voxel elements. The 3D
surface rendering of the mineralized tissues was made using
the manufacturer visualization software (CtVol, Skyscan®)
with a global threshold chosen halfway between grey level
of soft tissues and hard tissues. The 3D grey level dataset
The mandibles were scanned using a 1072 Skyscan®
An In Vivo Model for Short-Term Evaluation The Open Dentistry Journal, 2008, Volume 2 69
and the 3D surface rendering allowed evaluating the opacity
and the spatial distribution of the dentin formed.
tion. After dehydration in graded ethanol, the tissues were
embedded in Paraplast plus (Kendall, Mansfield, USA). Sag-
ittal (7 μm) sections were collected on SuperFrost slides
(Menzel-Glaser®, Braunschweig, Germany). The slides were
dewaxed, rehydrated and stained either with hematoxylin-
eosin or Masson’s trichrome.
The samples were demineralized, in 4.13% EDTA solu-
infection or tissue necrosis was detectable in the treated
mandibles, and despite the surgery, the soft tissues were in-
volved in a healing process. 17/18 incisors were implanted
successfully. In the two control groups 1 and 5, implanted
with agarose beads alone or after surgical preparation with-
out any implantation, respectively reparative dentin forma-
tion was limited to area located around the beads or occlud-
ing the pulp exposure site (Fig. 2A, 2B). Implantation with
A+4-soaked agarose beads (group 2) led to the formation of
a diffuse mineralized structure within the pulp as evidenced
by histological (Fig. 2C) and microscanner analysis (data not
shown). The newly formed tissue is reminiscent of osteoden-
Ten days after implantation, no inflammatory process,
tin, since the cells were trapped into osteoblast-like structure.
Implantation of the progenitors cells (17IA4), alone or in
association with agarose beads (groups 3 and 4), also pro-
moted, in both cases, the formation of islets of osteodentin-
like structures in the pulp (Fig. 2D-F). To assess the degree
of mineralization of the newly formed dentin, 3D analyses
by X-ray microscanner of the hemimandibles were per-
formed (Fig. 3A-C). The pulps of the control and sham
groups were radiolucent (Fig. 3D, 3F). In contrast, those
implanted with the progenitor cells (Fig. 3E, 3G) or the bio-
active molecules (data not shown) displayed a radiopacity
very similar to dentin or bone. The microscanner analysis
allowed determining that 10 days after implantation, the
zone of mineralized neodentin has extended within the pul-
pal compartment over a distance of roughly 1milimeters.
that allows a direct access to the pulp of the forming mouse
incisor. As no vital organ is concerned, the animals recover
promptly from the surgery and it is possible to evaluate the
biological effects of implantations of cells or molecules over
a period of 1 to10 days. Afterwards, endogenous aging proc-
esses, and odontoblast and pulp degeneration [16, 21], may
interfere with the biological reaction under investigation.
We have developed an in vivo experimental approach
Fig. (2). Formation of neodentin ten days after implantation of the A+4 peptide or 17IA4 dental pulp progenitor cells in the pulp.
Control groups showed a limited formation of neodentin  around the beads (A) or occluding the pulp exposure site (B). Implantation of
A+4 charged beads induced the formation of neodentin in the pulp compartment (C). Implantations of the 17IA4 cells with (D) or without
agarose beads (E) induced also the formation of osteodentin-like structures (F). Pulp (p), dentin (d), enamel (e), alveolar bone . Bar
70 The Open Dentistry Journal, 2008, Volume 2 Lacerda-Pinheiro et al.
Previous studies have used a similar surgical approach by
drilling a cavity through the basal bone to gain access to the
enamel organ of the rat incisor [22, 23]. Another surgical
attempt to selectively target the odontogenic organ was de-
scribed, using an osmotic minipump connected to a bony
window overlying the apical end of the rat incisor .
These approaches were mainly aimed at studying the effect
of pharmacologic agents on the formation of dental mineral-
ized tissues. Our strategy was focused on gaining easy access
to the dental pulp, without perturbation of the enamel organ
and of the stem cells niche located at the apex of the incisor
[13, 25]. The present results establish our method as a useful
and reliable approach in evaluating the capability of progeni-
tors cells or bioactive molecules of promoting dentin regen-
eration or repair.
The Control Groups
present by itself a capacity of self-repair [6, 7, 26, 27]. Scaf-
folds of gelatin, collagen, alginate or agarose beads have
been used as carriers for molecules implanted in the exposed
pulps and may not be completely biological [9, 28]. The con-
trol groups did not display any reparative mineralization in
the pulp, and showed only a limited amount of reparative
dentin at the exposure site as expected.
The rodent incisors as other pulpal implantation models,
The Effects of the A+4 Implantations
uct (A+4) has been identified as a molecule expressed by the
odontoblasts. It appears to function as a signaling molecule
[18, 19]. A+4 had been shown to promote chondrogenesis
The small molecular weight amelogenin gene spice prod-
and osteogenesis either in vitro or when implanted in ectopic
sites [19, 29, 30]. When used as capping-agent in rat molars,
A+4 induce the formation of a true dentinal bridge, occlud-
ing the pulp exposure in 15 to 30 days . After implanta-
tion in the pulp of molars, A+4 stimulates the recruitment
(commitment) of cells that proliferate and differentiate into
osteo/odontoblast-like progenitors, which after terminal dif-
ferentiation will form a reparative osteodentin-like structure.
The bioactive potential of A+4 was confirmed in the present
study in the incisor model since formation of a significant
amount of neodentin is promoted by this peptide in the den-
tal pulp. In this context, as a continuously growing tooth
containing an identified stem cell niche , the incisor may
constitute a sensitive tool, to study the early stages of the
dentinal repair process promoted by the amelogenin peptide,
namely the recruitment steps of the pulp progenitors.
The Effects of the Progenitors Cells Implantations
human dental pulp. Significant effort is devoted to determine
their therapeutic potential. After ectopic transplantation (sub-
lingual region, subcutaneous), they produced a bone or den-
tin matrix [32-34]. However, up to now, no report provides
data on the ability of these cells to directly contribute to re-
parative dentin formation after direct implantation into an
injured dental pulp. The experimental approach used here
provides some hints that such would be the case. Indeed,
implantation of the clonal progenitor cells, 17IA4, into the
pulp leads to the production of a large amount of reparative-
like dentin. In contrast to the diffuse dentin formed after A+4
implantation, the 17IA4 cells promote the formation of a
Stem cells have been isolated and characterized in the
Fig. (3). Micro-scanner analyzes of incisor implanted with the 17IA4 cells. The red block indicates the zone analyzed (A) and the asterisk
(*) corresponds to the perforation site in the bone (B), and in the tooth (C). Ten days after implantation in the pulp, no mineralization was
observed in the control group (D,F) in contrast, the 17IA4 group formed a large mineralized structure within the pulp (E,G).
A B C
D E F G
An In Vivo Model for Short-Term Evaluation The Open Dentistry Journal, 2008, Volume 2 71
dentin mostly located in the central zone of the pulp. Mi-
croscanner analysis confirms the histological data on
neodentin formation further demonstrating the mineraliza-
tion process and its extent within the pulpal compartment.
The formation of neodentin in the pulp may be explained
by either the 17IA4 cells differentiating toward an odonto/
osteoblast phenotype and forming a osteodentin-like struc-
ture, or by these cells producing signals that stimulate the
commitment, proliferation and differentiation of some resi-
dent progenitors cells. These alternative hypotheses are now
under evaluation and preliminary results suggest that the
implanted progenitor cells are indeed capable of producing
neodentin (data unpublished).
of the mouse incisor model as a tool to obtain a better under-
standing of the mechanisms of reparative dentin formation
stimulated by biomolecules or cell implantations. Therefore,
it should also allow the study of the efficiency of new thera-
peutic agents that may be used as pulp capping agents. Fi-
nally, our results of cell implantation in the pulp show the
capacity of dental pulp progenitors of contributing to dentin
formation in an injured tooth, paving the way for initiating
studies on cell therapy approaches of dentin injuries.
In conclusion, the data presented herein validate the use
Lacerda-Pinheiro was supported by Coordenação de Aper-
feiçoamento de Pessoal de Nivel Superior (CAPES), Brazil.
This research was supported by Fondation de l’Avenir,
l’Institut Français pour la Recherche Odontologique (IFRO)
and an Inserm “Somatic Stem Cells Research Grant”
We thank Arthur Veis for his generous gift A+4. Sally
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Received: December 12, 2007
Accepted: March 27, 2008
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