Dentin-like tissue formation and biomineralization by multicellular human pulp cell spheres in vitro
Head & Face Medicine
Dentin-like tissue formation and biomineralization by multicellular human pulp cell spheres in vitro
Jrg Neunzehn 0 3
Marie-Theres Weber 2
Gretel Wittenburg 1
Gnter Lauer 1
Christian Hannig 2
Hans-Peter Wiesmann 0 3
0 Technische Universitat Dresden, Institute of Material Science , Chair for Biomaterials, Budapester Strasse 27, D-01069 Dresden , Germany
1 Department of Oral and Maxillofacial Surgery, University Hospital Carl Gustav Carus , Fetscherstrasse 74, D-01307 Dresden , Germany
2 Department of Restorative and Pediatric Dentistry, University Hospital Carl Gustav Carus , Fetscherstrasse 74, D-01307 Dresden , Germany
3 Technische Universitat Dresden, Institute of Material Science , Chair for Biomaterials, Budapester Strasse 27, D-01069 Dresden , Germany
Introduction: Maintaining or regenerating a vital pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell aggregates (spheres) were applied onto bovine and human root canal models to evaluate their potential use as pre-differentiated tissue units for dental pulp tissue regeneration. Methods: Human dental pulp cells (DPC) were derived from wisdom teeth, cultivated into three-dimensional cell spheres and seeded onto bovine and into human root canals. Sphere formation, tissue-like and mineralization properties as well as growth behavior of cells on dentin structure were evaluated by light microscopy (LM), confocal laser scanning microscopy (CLSM), scanning electron microscopy (SEM) and energy dispersive X-ray spectroscopy (EDX). Results: Spheres and outgrown cells showed tissue-like properties, the ability to merge with other cell spheres and extra cellular matrix formation; CLSM investigation revealed a dense network of actin and focal adhesion contacts (FAC) inside the spheres and a pronounced actin structure of cells outgrown from the spheres. A dentin-structure-orientated migration of the cells was shown by SEM investigation. Besides the direct extension of the cells into dentinal tubules, the coverage of the tubular walls with cell matrix was detected. Moreover, an emulation of dentin-like structures with tubuli-like and biomineral formation was detected by SEM- and EDX-investigation. Conclusions: The results of the present study show tissue-like behavior, the replication of tubular structures and the mineralization of human dental pulp spheres when colonized on root dentin. The application of cells in form of pulp spheres on root dentin reveals their beneficial potential for dental tissue regeneration.
Biomineralization; Dental pulp cells; Tissue formation; Pulp spheres; Pulp tissue regeneration
The root canal treatment is a way of preserving a tooth
by removing contaminated or injured dental tissue,
disinfecting the canal system as well as obturating and
sealing it with synthetic material. Over the past decade,
the success rate of endodontic treatments was greatly
increased due to advancements in dental materials,
antimicrobial therapy and endodontic technology [1,2].
However, endodontically treated teeth are lacking blood
and nervous system supply. This results in the loss of
sensing environmental changes such as caries progression and
distinction of temperature differences . Furthermore,
those teeth are more vulnerable to masticatory forces since
the ability to perceive tactile stimuli is diminished due to a
reduction of mechanoreceptors when removing the pulp
tissue . The lack of pulp tissue and the containing
odontoblasts inhibit reparative dentin formation, which is
particularly important for the protective self-defence-system of
the tooth .
Besides the endodontic approach, injuries of the coronal
pulp caused by trauma and deep carious lesions resulting in
pulp exposure still mark a major challenge. In consideration
of these concerns, maintaining or regenerating pulp vitality
is a preferable goal in current endodontic research.
The revascularization of the tooth during root
maturation has been investigated by several studies focussing
on trauma patients [5,6]. One promising approach is cell
therapy with pulp stem/progenitor cells directed by
morphogens . Thereby, a pronounced regenerative
potential of the pulp-dentin-complex was observed, which was
ascribed to the multipotency of pulp stem cells .
Based on their embryologic origin pulp fibroblasts and
stem cells are able to differentiate into ecto- and
mesodermal cell types causing them to be of special interest
in tissue engineering. The human dental pulp contains
cells such as stem cells, fibroblasts, undifferentiated
mesenchymal cells, odontoblasts and defence cells such
as histiocytes, macrophages, granulocytes, mast cells and
plasma cells. Additionally, an extensive vascular supply
and the nerv plexus of Rashkow are located within the
pulp chamber that have important functions in
inflammatory events and subsequent tissue repair.
In vivo studies revealed the possible use of dental pulp
stem cells in the regeneration of various tissues. Further
studies confirm that the differentiation of stem cells as
well as angiogenesis and neurogenesis are essential for
pulp regeneration .
Therefore, scaffolds, cells and bioactive molecules are
essentially needed for dental tissue engineering. A wide
variety of scaffolds such as collagen, fibrin, synthetic matrices
or other hydrogels are frequently used for this purpose
[10,11]. In this context, dentin specimens are suitable for
in vitro experiments to evaluate the efficacy of these
strategies. Pulp cells seeded onto pre-treated dentin surfaces
had a proliferation rate similar to that of pulp cells on
twodimensional controls; in addition, they exhibited multipolar
processes extending into dentinal tubules [12,13]. Another
study showed the same extension of DPC processes into
dentinal tubules, which proved their odontoblastic
phenotype after being inoculated onto dentin discs . The
studies mentioned above indicate that not only the composition
of dentin but also its topography, in this case dentinal
tubules, might play a key role in cellular differentiation of the
There are contrasting results regarding the seeding
efficiency of DPC on scaffolds. While suspension cells are
routinely used for dental cell biology in two-dimensional
systems, it is known that micromass cultures have several
advantages over suspension cells for tissue engineering
approaches. Three-dimensional and tissue equivalent cell
agglomerates, so called spheres, show similar cell
proliferation and differentiation as tissues in vivo. In different
studies micro mass cultures of osteoblasts, osteoblast-like cells
and other cell types such as stem cells [16,17] were
investigated intensively and affirmed the tissue equivalent cell
3-D cell-culture-systems and especially the use of
micromass cultures have proven themselves as a potent
method when being used as in vitro testing systems due
to their tissue-like behavior. Furthermore, these
cellculture-systems were also employed for biomaterial
testing and could probably be used directly as an already
pre-differentiated tissue unit for tissue regeneration
The use of pulp spheres containing DPC could have
an advantage over previous restoration methods, where
cells had to be connected to a scaffold in order to be
placed into a prepared root canal for pulp-tissue
engineering. Using pulp spheres, however, it is possible to
insert DPC scaffold-free into root canals. Furthermore, the
three-dimensional cultivation method of the spheres
enables a pre-differentiation of the DPC into different
kinds of tissue for a faster formation of pulp tissue
before the cells are placed into the root canal. An
application of these pre-differentiated pulp spheres into
prepared roots for tissue engineering, but also during a
partial pulp removal is conceivable.
Therefore, spheres containing DPC derived from
human wisdom teeth were applied onto bovine root dentin
and into human root canals as an in vitro test system for
the first time. The aim of this study was to investigate
the aptitude of these micromass cultures, spheres,
regarding tissue engineering and pulp regeneration on root
dentin and in root canals from a morphological and
Materials and methods
Bovine and human root canal model preparation
To investigate the behavior of human dental pulp spheres
on dentin in vitro two different root canal models were
A bovine root canal model was prepared to reveal
the spheres behavior on a vast root dentin surface
(Figure 1a). Therefore, the pulp tissue of extracted
bovine incisor roots of two-year-old cattle (Sdost Fleisch
GmbH, Altenburg, Germany) was extirpated and the
roots were divided in half, longitudinally with an average
canal length of about 1 cm, using a precision table top
cut-off machine (Accutom-50, Struers). Additionally, the
root canals were treated with a rose-bur (low-speed:
1000 rpm) to remove residual pulp tissue, denticles as
well as sclerotic zones in the root dentin and to simulate
a root canal preparation with files.
A human root dentin canal model served to examine the
sphere behavior on a more tight and physiological bases
(Figure 1b). Incisors, premolars and molars of middle-aged
donors (in the age of 2550) were extracted during routine
surgical treatment and underwent a root canal preparation
using ProTaper (Dentsply Maillefer, Ballaigues, Switzerland)
and sodium chloride 0,9% (NaCl) as irrigant. The roots
were explored and prepared up to ProTaper finishing file
F5 with a 0.50 mm diameter and a fixed taper of 5% in its
apical extent. Afterwards, the endodontically treated roots
were cut horizontally into 3 mm thick discs. The
accumulated smear layer(abrasive dust, debris) in the bovine as
well as the human root canals was removed by ultrasonic
desorption using 70% EtOH, 3% EDTA and distilled water
for one minute each, respectively. Subsequently, the bovine
Figure 1 Preparation of bovine and human root canal models. Bovine roots were sectioned longitudinally and the root canal surface was
treated with a rose-bur (a). Additionally, human roots were cut horizontally into 2 mm thick discs after root canal preparation (b). Both models
underwent ultrasonic desorption to remove the accumulated smear layer(abrasive dust, debris).
root canal specimens were sterilised to prevent a possible
contamination of the spheres.
Cultivation of human dental pulp cells
Pulp tissue derived from human wisdom teeth was
macerated enzymatically with collagenase in order to isolate
the cells from the surrounding tissue. According to facs
analysis approximately 80% of the isolated cells showed
stem cell character. The DPC were cultivated up to the
fourth passage in D-MEM (low glucose), 20% FCS, 2%
HEPES, 100 U/ml penicillin, 100 g/ml streptomycin,
50 g/ml gentamicin, 2,5 g/ml amphotericin B (all
PAA, Clbe, Germany) at 37C, 5% CO2 and 95% humid
atmosphere with a medium change two to three times
per week. The used cells were tested positive for their
potential to be differentiated in an osteogenetic and
angiogenetic way as described in literature .
Harvesting of human dental pulp cells and sphere
DPC were harvested by trypsin incubation, counted
using CASY cell counting technology (Schrfe System
GmbH, Reutlingen, Germany) and transferred to a
nonattachment environment. For this purpose, chambers of
96-well plates were prepared by applying 50 l of a
mixture of 20 mg/ml agarose (Biozym Scientific GmbH) in
DMEM (Biochrom)/HGEM per well. A population of
100 000 cells per well was seeded into the treated cell
culture dishes and incubated in the medium mentioned
above at 37C and 8% CO2. The medium was changed
twice per week. In preliminary tests it was possible to
differentiate the cultivated human pulp cell spheres in
different tissue specific ways to validate their
multipotent stem cell character as described previously .
Cultivating pulp spheres on biological environment
In order to evaluate the potential of the pulp spheres to
interact with a physiological environment, five-day-old
pulp cell spheres were aspirated with a sterile pipette
and transferred to be cultured on ten prepared halved
bovine roots and into eleven human root canals,
Depending on the root canal size, two or three spheres
were seeded onto bovine dentin. They were located far
enough from each other, so that the cells that started to
grow out of the one sphere would not disturb the cells
which grew out of the other spheres (with a distance of
3 mm between each other). In case of the human root
canals, two spheres were seeded into one canal to ensure
a contact between the spheres inside the canal to
evaluate the sphere interaction in a narrow and physiological
The sphere-seeded material was evaluated during the
whole trial period by light microscopy (LM) and after
seven days and 28 days by scanning electron microscopy
(SEM), respectively. As a reference and for LM- and
confocal laser scanning microscopical-analytics (CLSM),
five cell spheres were cultivated on polystyrene culture
dishes and were also evaluated after seven days. The
cell-seeded specimens and culture dishes were cultivated
as described above.
The pulp spheres were investigated by light microscopic
analysis (LM) during the whole trial period by the use of
a Zeiss Axiovert 40 CFL combined with a Canon
PowerShot G11. This method was mainly used during the
examination of spheres grown in root canal models to
monitor potential contamination and to notice
unphysiological cell development.
Scanning electron microscopy
For scanning electron microscopic investigation (SEM),
pulp spheres were cultivated on the dentin and fixed
with glutaraldehyde, followed by dehydration in an
ascending series of isopropanol, and chemical drying
through the iterative transfer into hexamethyldisilazane
(HMDS). The samples were fixed on SEM stubs and
sputtered with gold-palladium. Scanning electron
microscopy was carried out using a Philips ESEM XL 30 in
Hi-Vacuum mode by detecting secondary electrons for
imaging and by detecting backscattered electrons to
investigate material contrasts. For a more detailed material
investigation an energy dispersive X-ray spectroscopy
(EDX) was performed.
Confocal laser scanning microscopy
To evaluate sphere formation, cell-cell-contacts inside
the cell agglomeration and cell performance of the
outgrown cells, five-day-old pulp spheres were stained with
actin, focal adhesion contacts (FAC) and
4,6-diamidino2-phenylindole (DAPI, Sigma) for confocal laser
scanning microscopy (CLSM).
After washing and fixing, the cells were permeabilized
with 0.2% Triton-X-100 in PBS and blocked with 1%
bovine serum albumin (BSA, Sigma) for 30 min. FAC were
stained with AlexaFluor 488-Phalloidin (Invitrogen),
cytoskeletal actin was stained with AlexaFluor 546 and
cell nuclei were stained with DAPI.
Microscopy was carried out by the use of an upright
Axioscop 2 FS mot equipped with a LSM 510 META
module (Zeiss, Jena, Germany) controlling an argon-ion
(Ar+) laser, helium-neon (HeNe) laser and
NIR-femtosecond titanium-sapphire laser for 2-photon excitation
(Coherent Mira 900 F). The excitation of AlexaFluor 488
was carried out at 488 nm (Ar + laser) and the excitation of
AlexaFluor 546 at 546 nm (HeNe laser). The NIR-fs-laser
was used for the excitation of DAPI at 750 nm (2 photon
excitation) and fluorescence was recorded at 461 nm.
Light microscopic evaluation during sphere cultivation
showed cell and sphere development without any
contamination and a fast and complete formation of round
cell spheres after five days.
After sphere formation, confocal laser scanning and
light microscopic investigations illustrate a round shape
(Figure 2a) with evenly plain surface (Figure 3a). The
results of the actin and FAC staining, as well as
CLSMinvestigations showed a very high expression of these
factors inside the spheres. Both actin and FAC were
equally allocated in the spherical cell construct building
up a dense network (Figure 2a).
At the contact area between the pulp sphere and an
untreated cell culture dish surface, the cells grew out of
the sphere as described in literature . The outgrown
cells migrated on the polystyrene substrate and
presented a pronounced actin skeleton (red) with FAC
(green) and consistent round cell nuclei dyed blue
(Figure 2b and c).
Nearby lying pulp spheres in the human root canals
aligned themselves towards each other and merged into
bigger tissue-like cell formations containing multiple cell
layers (Figure 3a). Figure 3a represents the intense
contact between two adjacent spheres and the surrounding
human root dentin.
After 28 days, the two fused spheres expanded to a
homogenous tissue unit. The light microscopic
evaluation indicated a nearly complete union of the spheres
Figure 2 Confocal laser scanning microscopic evaluation. Actin (red), FAC (green) and DAPI (blue) stained cells in the upper third of a pulp
sphere (a) and of its outgrown cells (b and c) representing a dense actin formation and consistent round cell nuclei.
Figure 3 Tissue-like behavior of human pulp sphere cells. Figure (a) shows merging pulp-spheres in a human root canal model after five
days. After 28 days of cultivation the two spheres merged, cells grew out of the spheres and coated the root dentin walls (white arrows).
(b) Outgrowing cells also covered the underlying polystyrene culture dish and built up a dense cell layer (black asterisk). SEM investigation of the
merged spheres (c and d) represents the even surface of the new cell construct and the widespread coverage of the root canal walls (c) as well
as the randomly distributed different sized particles (d).
in the lumen of a human root canal (Figure 3b). The
newly built cell tissue branched out and covered the
human root canal walls (Figure 3b white arrows, Figure 3c).
Other cells, outgrown from the spheres, attached on the
polystyrene cell dish surface, migrated and formed a
dense cell layer (Figure 3b *).
SEM images in Figure 3c and d represent the
homogeneous surface of the two merged spheres with numerous
different sized particles (zooming in Figure 3d).
The SEM investigation showed a fibrillar surface
of the closed cell construct of the merged spheres
(Figure 4a) with different sized mineral like structures
on it (Figure 4b). These mineral particles seem to
be connected with the cell matrix (white arrows in
Figure 4b). The back scattered electrons (BSE)
represented a very clear, atomic number depending material
contrast between the spheres cell matrix and the mineral
particles (Figure 4c and d). Especially Figure 4c
represents the very large number of biomineralized particles.
A further result of the SEM investigation illustrates
the different localization of the mineral particles. The
black asterisks in Figure 4b and d point out the
detections of the particles with a higher atomic number as the
cell material, which is not visible in the secondary
electron image with a topographical contrast of the same
area (Figure 4b). In this image these particles are
covered with cell matrix. This effect is also seen in Figure 4c.
The shining particles are represented in two different
ways. In some areas the edges seem to be definite from
the surrounding cell material (middle part of Figure 4c).
In other regions they appear to look pale and blurred (in
the upper part of Figure 4c).
The additionally performed mineral proof by EDX
(Figure 4e-g) revealed a high concentration of phosphate
(Figure 4f ) and calcium (Figure 4g) on the newly build
SEM images of a mechanically broken pulp sphere
after seven days of micromass formation revealed the
integrity of highly aggregated pulp cells inside the sphere
(Figure 5a,b). The smooth surface of the spheres seemed
to be constituted by epithelial-like cell layers that are
superimposed by each other with distinctive cell-cell
contacts (Figure 5a). Cells in the inner region of the
spheres appeared to be less organized. These cells
seemed to interact with each other in clearly visible
cellcell-contacts and with a high formation rate of
extracellular matrix (Figure 5b).
The pulp spheres seeded onto bovine dentin showed a
very intense contact with the biological substrate.
Figure 5c and d represent the widespread contact area
between the spheres and the dentin surface. In this
specific contact area, a high number of cells grew out of the
spheres in multiple layers (Figure 5c). The whole area,
surrounding the spheres was covered by multilayered
cells. The majority of this area was covered with small,
tubule like vents (Figure 5c white arrows). On the
Figure 4 SEM and EDX investigation of merged human pulp cell spheres in a human root canal. Figure (a) shows a fibrillar surface of the
closed cell construct with different sized mineral like structures on it (Figure 4b) by detection of secondary electrons. The white arrows in figure
(b) represent a cellular connection between the mineral particles and the underlying cell layer. The images in figure (c) and (d) show the same
areas investigated by the detection of back scattered electrons with a clearly seen atomic number depending material contrast between the
spheres cell matrix and the mineral particles. Black asterisks in figure (b) and (d) denote particles with higher atomic numbers, which could only
be seen in figure (d) by the aid of the material contrast. The area of the sphere, which was investigated by EDX, is marked with black lines in
figure (e). The results concerning the higher concentration of mineral specific elements are represented in figure (f) (phosphate) and figure
dentin, the migrated cells aligned themselves on the
physiological surface, representing a very intense cell
adhesion, which is indicated by the flat spread out cell
bodies on the dentin (Figure 5d).
Furthermore, pulp cells partially extended into dentinal
tubules immediately after growing out of the cell spheres
(Figure 6a). After 28 days, a number of dentinal tubular
walls were covered by ingrown pulp cells. The cells grew
vortically into the porous structure (Figure 6b).
Especially, in the areas directly adjacent to the spheres,
the multilayered outgrown cells emulated dentin
structures with newly constructed tubule like formations
(Figure 6c). The white arrows in Figure 6c and d indicate
the formation of this dentin like structure over several
cell layers in a preparation conditioned split between cell
layer and sphere. Figure 6d also represents the fibrillar
Within the last years, cells derived from pulp-tissue were
investigated concerning their multipotent stem cell
character as well as their ability to differentiate in diverse ways
such as angiogenetic or osteogenic differentiation . In
this study, the possibility of human dental pulp cells (DPC)
serving as potential progenitors for odontoblast formation
and biomineralization initiators was investigated. Several
studies displayed the high potential of DPC regarding this
matter [22,24-26]. Besides the possibility to influence the
specific differentiation of these cells, an odontoblast-like
phenotype, structure, cell formation and differentiation
behavior were shown in in vitro and in vivo setups [25-27].
Figure 5 Scanning electron microscopic sphere and cell investigation. SEM images of the outer epithelial-like shell of a cell sphere (a) and
the less organized cells with a higher production of extracellular matrix inside the sphere (b). Figure (c) shows a pulp sphere attached onto
bovine dentin with a high number of outgrowing, multilayered cells as well as flat spread out and attached cells on bovine root dentin (d). The
white arrows in figure (4c) highlight the dentin tubule-like holes in the cell multi-layer.
The results of the present study revealed the same
differentiation characteristics of the adapted cells on the
dentin models as postulated in literature mentioned
above. The DPC used in this study proved a stem
celllike character if differentiated angiogenically and
osteogenically. Furthermore, a cultivation of the cells over
various passages without losing the differentiating
potential was possible.
During the last years, the aim in several studies was to
use cells derived from pulp tissue to induce a regeneration
of the pulp. Hitherto, the cells have been seeded on
different scaffold materials such as organic collagen, chitosan,
hydroxyapatite/tricalcium phosphate (HA/TCP) or
inorganic polymer polylactic-co-glycolic-acid (PGLA) in order
to support the organization and vascularization of the
newly formed tissue [10,28-30]. However, the unpredictable
degradation of inorganic as well as organic scaffold
materials represents a risk factor concerning wound healing and
complete tissue regeneration in vivo.
Three-dimensional micromass cultures, so called
spheres, are known to construct tissue-like formations
and are often used as in vitro cell culture systems to test
biomaterials and surfaces for their tissue tolerance.
These solid, three-dimensional constructs are also
described as a potential method for scaffold free tissue
engineering [18-21,31]. According to the sole use of
DPC-spheres and the absence of additional scaffold
materials, negative or non-physiological influences can be
excluded almost completely.
In this study, pulp spheres were generated from
cultivated cells derived from human pulp tissue. The
applicability of pulp spheres for pulp tissue reconstruction was
investigated for the first time.
In order to simulate the structure and geometry of the
physiological requirements found in vivo, sectioned
bovine roots and horizontally cut human root canal discs
were used as root canal models. After preparation, the
surface of the treated root canal models represented
evenly distributed dentinal tubules. The smear layer
resulting during the preparation was removed via
ultrasonic treatment, so that a potential cell growth into the
open tubules was possible. The cultivated pulp spheres
were distributed homogenously with a distance of
approximately 3 mm from each other onto the bovine root
canal dentin. Due to the small lumina of human root
canals, the spheres were placed within the human dentin
discs in a more physiological and tighter condition.
In this study, primarily bovine teeth were chosen for
the preparation of the root canal models because of their
good availability, processability and comparability. The
greater size of bovine roots provides a larger area for
experimental procedures . Bovine teeth from animals
in the same age category with common genetic ancestry
and diet show a more homogeneous mineral
composition of the dental hard tissue when compared with teeth
gained from humans of different ages and diverse diets.
The hardness (KHN = Knoop Hardness Numbers) of
human and bovine dentin is quite similar . Also the
Figure 6 Cell behavior on bovine dentin. Scanning electron microscopic images show the bovine dentin surface orientated migration of
outgrown pulp sphere cells (a) and the vortical ingrowth into the dentinal tubules as well as the covering of the tubular walls by these cells
(b). Multilayered cell formations with cellular emulation of three-dimensional dentin structures could be detected in the multi layered outgrowth
area adjacent to a sphere (white arrows in Figure 6c). The white arrows in figure (d) denote the fibrillar dentin tissue-like structure over numerous
cell layers (black arrow).
size and the amount of dentinal tubules per mm2 in
crown dentin are identical for human molars and bovine
incisors [34,35]. However, the diameter of dentinal
tubules in bovine root dentin is larger than the diameter of
dentinal tubules in human root dentin [34-36]. As
mentioned before, the typography of surfaces influences the
cell adherence, growth and even the differentiation
behavior of the cells. Thus the difference in tubule
diameter comparing the decreasing sizes of bovine root
dentin tubules and coronal, middle and apical human
root dentin tubules was of interest to examine the
behavior of the DPC on diverse topographic dentin
surfaces. The larger diameter of bovine dentinal tubules
could have facilitated the extension of the growing cells
into the tubules as compared with human root dentin.
Furthermore, the size of the tubule-diameter is also
dependent on dentinal sclerosis. Dentinal sclerosis which
implies a decrease in size up to a complete blocking of
dentinal tubules of root dentin is, unlike dentinal
sclerosis in crown dentin, age-related [37,38].
In this investigation, root dentin of two-year-old
cattle was used for the root canal models. Hence, the
results of this study can only be compared to young
human teeth of patients in their mid-twenties when
dentinal sclerosis of the root dentin has not yet taken
In order to observe the behavior of cell spheres in a
narrow and physiological in vivo like environment in
comparison to a flat and larger area provided by the
bovine root dentin models, the spheres were also applied
into human root canal models. After two days, the
spheres already merged and after ten days outgrown
cells covered the human dentinal walls. The structure,
form and cell orientation of the cultivated spheres, as
well as the outer sphere shell and the development of
intimate cell-cell contacts and matrix formation of the
cells inside the spheres shown in this study correspond
to the sphere-behavior of other cell types in literature
and suggest a similar and characteristic tissue-like
CLSM analysis and the proof of actin and FAC inside
all fully developed spheres proved the intimate cell-cell
contacts as shown in the literature for spheres of other
cell types such as osteoblasts and mesenchymal stem
cells [18,19,39]. Moreover, the dense network of stained
actin and FAC inside the spheres (Figure 2) confirms the
tissue-like cell interaction shown in the images of the
scanning electron microscopic investigations.
Of special interest is the ability of single cells to grow out
of the sphere after its attachment on different substrates.
This is an elementary requirement for the use of spheres as
testing method for biomaterials or as a possible 3-D tissue
equivalent cell formation for tissue engineering and tissue
regeneration similar to this study.
Actin and FAC staining of the outgrowing cells represent
a dense actin skeleton, FAC and equally round shaped cell
nuclei, which are signs for vital cell metabolism. The radial
and consistent outgrowth of the cells at the contact area
between the spheres and substrate indicates the same
developmental stage of the pulp cells, which form a fully
developed sphere. Furthermore, the outgrown sphere cells
emphasize the tissue-like character and cell vitality of the
pulp spheres. The feature of cells growing out of tissue
explants is well known and used as a cell extraction strategy
for primary cell cultures.
Further evidence for the tissue-like character of the
pulp spheres used for the first time in this study is the
ability of several spheres to merge and build new and
tissue-like cell agglomerations. As shown in the results,
these new constructs build up a common surface very
similar to the outer layer of an individual sphere that
formed a new cell structure which had the ability to
grow around and cover biomaterials .
In this study, the cell spheres were cultured on bovine
and in human root canals to analyze their reaction
towards a physiological environment.
Directly after the spheres attached onto the dentin
surface, single cells started growing out of the cell
agglomerate, spread out flat and aligned themselves on the
dentin structure (Figures 5 and 6). Following the surface
orientated migration (Figure 6a) cell processes grew into
dentinal tubules followed by a complete coverage of the
internal tubule walls (Figure 6b). This progress, from the
outgrown cells over the surface orientated migration up
to the complete dentinal coating of the root canal
surface and the walls of the dentinal tubules, clarifies the
ability of the pulp cells after being pre-differentiated into
tissue-like spheres to interact with their original
physiological environment. It seems to be a considerable
advantage that the cells grew out of the spheres in a very
high number. Shao et al. also report about a multilayered
cell construct that was formed in a two-dimensional cell
culture system on dentin slabs after four days of
cultivation . The application of spheroidal, micromass
cultures as performed in this study, showed a direct and
multilayered outgrowth of the cells from the pulp
spheres on the root canal wall as well as into the
dentinal tubules (Figures 5 and 6).
The results of the in vivo-study of Kodonas et al. showed
an odontoblast-like cell construct with an organic matrix
formation on the root canal wall surface of porcine teeth
after ten weeks . The prepared teeth had been treated
with porcine dental pulp stem cells. In the present study, a
phenomenon regarding the multilayered cell accumulation
on the root dentin surfaces occurred, which had not been
described before in studies dealing with pulp tissue
regeneration. After 28 days, a three-dimensional tubules-formation
was detected within the multilayered cell accumulation
simulating the root dentin surface underneath (Figures 5
and 6). It seems that these cell structures represent a higher
level of cell organization as previously shown in other
studies where multiple cell layers were described. The newly
three-dimensional emulated dentin-like tissue presents
tubule structures and the beginning of physiological dental
hard tissue formation. Not only multilayered cell constructs
that have been described in literature before, but also newly
build three-dimensional structures with cell-cell contacts
and extracellular matrix formation over its entire volume
were detected in the present study.
The findings of other studies have shown that dentin
structures, and the release of different bioactive agents
embedded in dentin, such as transforming growth
factor- can induce and provide an odontogenic
differentiation of cells despite the use of disinfectants during
endodontic root canal treatment . In this study, the
dentinal substrate seems to stimulate the cell growth out
of the pre-differentiated spheres to form cell-cell
contacts, to develop extracellular matrix and to create
dentin tissue-like three-dimensional structures. The use
of the pre-differentiated three-dimensional cultured cells
 may enhance the stimulation of mantle dentin or
tertiary dentin formation respectively, and might have a
substantial impact on pulp capping and partial pulp
removal in endodontics. This kind of tissue regeneration is
also promoted by the calcium ion release of different
pulp capping materials .
Interestingly, the use of the spheres in contact with the
human dentin initiated a detectable biomineral formation.
28 days after seeding the spheres into the human root
canals and the tissue like fusion of the spheres mineral
formation was proven by SEM investigation (SE- and
BSEdetection) and EDX without any additional treatment with
differentiation factors. The fact that the mineral particles
detected under the sphere surface covered by cell layers
(Figure 4) leads to the conclusion that the mineral is
formed inside the spheres and is transported out of the
spheres to its surface. Lammers et al. have shown mineral
formation starting inside the spheres by osteogenically
stimulated stem cell spheres .
The cell caused mineralization is a clear sign of a fast
and highly advanced differentiation of the pulp cells
into a mineral/dentin producing cell type. To the best
knowledge of the authors, dentin like tissue formation
and comparable mineral formation on spheres surfaces
as presented in this study have not been shown until
now. This applies for the use of human dental pulp cell
spheres in contact with dentin. The potential clinical
application of spheres appears very promising, but the
impact of disinfective irrigants on the spheres or of
residual bacteria needs to be investigated.
potential for dental tissue regeneration and scaffold free
pulp tissue engineering due to their proven ability to
emulate dentin like tissue and to initiate biomineralization.
JN performed cell culture, light microscopic analysis, SEM- and
EDXinvestigation and carried out the conception of this study and the
manuscript. MTW and GW prepared the human and bovine root specimen,
provided and cultivated the DPC and drafted and wrote parts of the
manuscript. CH, GL and HPW contributed to the conception, design and
coordination of the study and manuscript as well as the interpretation of the
data. The study was performed under supervision of HPW. All authors read
and approved the final manuscript.
The authors would like to thank the laboratory staff members of the
involved groups and Dr. R. Ali for his support concerning CLSM-analytics.
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