d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064

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In vitro biocompatibility of ICON® and TEGDMA on

human dental pulp stem cells

Lina Gölz a , Ruth Andrea Simonis b , Joana Reichelt b , Helmut Stark b ,

Matthias Frentzen c , Jean-Pierre Allam d , Rainer Probstmeier e ,
Jochen Winter c , Dominik Kraus b,∗
a Department of Orthodontics, Center of Dento-Maxillo-Facial Medicine, University of Bonn, Bonn, Germany
b Department of Prosthodontics, Preclinical Education and Material Sciences, Center of Dento-Maxillo-Facial
Medicine, University of Bonn, Bonn, Germany
c Department of Periodontology, Operative and Preventive Dentistry, University of Bonn, Bonn, Germany
d Department of Dermatology and Allergy, University of Bonn, Bonn, Germany
e Neuro- and Tumor Cell Biology Group, Department of Nuclear Medicine, University Hospital Bonn, Bonn, Germany

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

Article history: Objectives. Resin infiltrants have been successfully used in dental medicine preventing the
Received 20 January 2016 progression of tooth decay in an early phase of caries development. ICON® is an infiltrant
Received in revised form of low-viscosity which penetrates via dentinal tubules into the lesion in dependence of the
30 April 2016 demineralization depth. Hence, we performed an in vitro study to determine the effect of
Accepted 3 June 2016 ICON® on human dental pulp stem cells (hDPSCs).
Methods. Using explant technique, primary hDPSCs were collected from extracted teeth.
Characterization and isolation were performed with typical mesenchymal stem cell markers
Keywords: (Stro-1, CD73, CD90, CD105) and hDPSCs differentiation was validated by immunofluo-
ICON® rescence and flow cytometry. HDPSCs were stimulated with light-cured ICON® (lc) and
Dental pulp stem cell non-light-cured ICON® (nc) conditioned media as well as different TEGDMA concentra-
Resin infiltration tions followed by the analysis of cytotoxicity, pro- and anti-inflammatory responses and
TEDGMA differentiation using XTT assay, RT-PCR and ELISAs, respectively.
Cytotoxicity Results. Initial analysis demonstrated that hDPSCs express characteristic mesenchymal stem
Inflammation cell markers and differentiate into adipocytes, chondrocytes and osteoblasts. Notably, ICON®
Biocompatibility nc dramatically reduced cell viability (up to 98.9% after 48 h), whereas ICON® lc showed
only a modest cytotoxicity (10%). Data were in line with cytokine expression demonstrating
increased levels of IL-6 and IL-8 as well as decreased IL-10 after ICON® nc exposure compared
to ICON® lc. ICON® lc caused almost no alterations of DSPP, whereas ICON® nc markedly
elevated DSPP mRNA levels (130.3-times). A concentration-dependent effect was observed
in TEGDMA challenged hDPSCs.
Significance. ICON® is a successful minimal invasive technique. However, clinicians should
strictly follow manufacturer’s instructions to prevent adverse effects.
© 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.

∗
Corresponding author at: Department of Prosthodontics, Preclinical Education, and Material Sciences, Center of Dento-Maxillo-Facial
Medicine, University of Bonn, Welschnonnenstraße 17, 53111 Bonn, Germany. Tel.: +49 228 287 22436; fax: +49 228 287 22385.
E-mail address: Dominik.Kraus@ukb.uni-bonn.de (D. Kraus).
http://dx.doi.org/10.1016/j.dental.2016.06.002
0109-5641/© 2016 The Academy of Dental Materials. Published by Elsevier Ltd. All rights reserved.
 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064
1. Introduction
Caries is one of the most common diseases in industrial
countries leading to tooth decay or even tooth loss. New fill-
ing techniques, materials and preventive measures have been
established during the last few decades which resulted in a
decline of caries lesions. However, demineralization or white
spots are still frequent due to acid food or impaired hygiene
ability for example during orthodontic treatment. In addi-
tion, anxieties against dental drilling are a serious problem
in dental practice leading to avoidance behavior. Recently,
minimal invasive techniques (e.g. resin infiltration) have been
developed to improve patients’ compliance and reduce the
invasiveness of conventional filling methods [1].
Resin infiltration technique has been successfully used in
dental medicine preventing the progression of tooth decay in
an early phase of caries development [1,2]. ICON® is a com-
mercially available resin infiltrant of low-viscosity consisting
of a methacrylate based resin matrix, initiators and additives.
At present it is primarily applied for white spots and initial
proximal lesions. The lesion is penetrated by the infiltrant in
dependence of its demineralization depth and might therefore
get in contact with pulpal tissue and cells via dentinal tubules
[3,4]. Recent investigations have shown that ICON® reduced
demineralized enamel roughness, increased microhardness
and inhibited bacterial adhesion [5]. Thereby, studies primarily
focused on penetration capacity [4], whereas biocompati-
bility has not been tested, even although evidence implies
that resin monomers like Triethylene-Glycol-Dimethacrylate
(TEGDMA), which is the major component of ICON® , modulate
cell metabolism as well as function [6,7] and may penetrate
into pulpal tissue [8,9]. TEGDMA and other monomers or resin
components have also been detected in saliva and dentin
structures supporting studies determining the release of resin
materials [10,11]. Notably, several in vitro investigations have
shown that monomers induce toxic effects in a variety of
cells [6,7,12]. Moreover, in vivo studies imply that resin com-
ponents may cause allergic reactions and mucosal changes
like lichenoid reactions, which may transform to malignant
tumors [13,14]. Additionally, monomers and their derivatives
may provoke apoptosis and genotoxic events, inhibit cell func-
tion and influence the innate immune system due to their high
chemical reactivity. One mechanism refers to the increase of
reactive oxygen species (ROS) which may lead to oxidative
stress and DNA damage [15]. In vitro experiments indicate that
these effects are cell line dependent supporting the idea that
periodontal or pulpal cells are more prone to acrylates than
gingival fibroblasts [16].
Pulpal cells consist of a heterogenic cell population which
may contain fibroblasts, odontoblasts, immune cells and mes-
enchymal stem cells (MSCs) or dental pulp stem cells (DPSCs)
[17]. DPSCs are pluripotent with the ability to differentiate for
example into adipocytes, osteoblasts, chondrocytes or odon-
toblasts. Therefore, these cells play a crucial role for the
regenerative capacity of the dental pulp [17,18]. Until now,
there are no data describing the influence of ICON® on these
cells. However, efforts have been made to elicit the effect
of resin monomers like TEGDMA on various cell systems,
even pulpal cells [6,7,12]. TEGDMA is a component of most
1053
resins and adhesives (including ICON® ) with varying content
(25–50%) [19]. Several studies indicate that this component
may induce inflammatory reactions, disturb cellular homeo-
stasis or modulate cell differentiation [12,20–22].
Even though ICON® is primarily for superficial lesions it is
yet unclear whether ICON® influences pulpal structures, cell
viability as well as inflammatory pathways due to its major
component TEGDMA. Therefore, the objective of the present
in vitro study was to characterize human primary dental pulp
cells and to determine the effect of ICON® in comparison
to TEGDMA on these cells. Thereby, we hypothesize that the
TEGDMA-based infiltrant induces cytotoxic effects as well as
inflammatory reactions in dependence of polymerization. The
data will improve our knowledge concerning the effects of
resin monomers on pulpal tissue, their risk of hazard and iden-
tify monomer concentrations which modify cell homeostasis
and signaling.
2. Materials and methods
2.1. Cell isolation
The study was approved by the Ethic committee of the Uni-
versity of Bonn. Pulpal tissue was obtained from healthy
caries-free teeth which have not fulfilled root formation yet.
Tooth extraction was performed because of orthodontic rea-
sons in 9–12-year-old patients. Their parents approved to the
study and gave written consent. Human dental pulp stem cells
(hDPSCs) were isolated and cultured by an outgrowth method
as follows: the tooth was scaled and disinfected using 70%
ethanol. 5 mm apical of the cement-enamel junction, a cavity
was performed using a diamond burr followed by final tooth
opening under sterile condition with a spatula. Afterwards,
dental pulp tissue was collected, dissected into pieces and
left to adhere to a 60 mm culture dish for 2 min before cell
culture medium (Dulbecco’s Modified Eagle’s Medium, DMEM,
Life Technologies, Darmstadt, Germany) was added. Cell cul-
ture medium was supplemented with 10% fetal bovine serum
(FBS), 1% antibiotic and antimycotic solution (all from Life
Technologies), and 5 ng/ml fibroblast growth factor- (FGF) 2
(R&D Systems, Wiesbaden, Germany) to enhance and main-
tain stem cell properties of the human dental pulp cells [23].
Cells/Explants were kept in an incubator at 37 ◦ C in humidi-
fied atmosphere of 5% CO2 in air and medium was changed
every 2–3 days. After one week a sufficient number of cells
were grown out of the pulpal tissue.
2.2. Cell characterization
Cells were harvested and STRO-1 positive cells were isolated
using the MACS® technology (Miltenyi Biotec, Bergisch Glad-
bach, Germany) and a mouse monoclonal STRO-1 antibody
(R&D Systems) as recommended by the manufacturer’s pro-
tocol. Cells were propagated in culture medium containing
FGF-2 up to the ninth passage and putative mycoplasma
contamination was routinely verified through PCR analy-
sis and 4 ,6-diamidino-2-phenylindole (DAPI; Sigma–Aldrich,
Munich, Germany) staining. To verify mesenchymal stem
cell properties, cells were characterized by the expression of
1054 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064
mesenchymal stem cell markers (CD73, CD90, CD105) and
their ability to differentiate into an adipocyte-, chrondocyte-
, and osteoblast-like phenotype using flow cytometry and
immunofluorescence as well as immunohistological stain-
ings.
2.3. Immunofluorescence
Cells were cultured on sterile coverslips for 24 h followed
by fixation with 4% paraformaldehyde (PFA; Sigma–Aldrich)
for 15 min, washed in phosphate buffered saline (PBS) and
treated with 0.1% Triton X-100 (Sigma–Aldrich) in PBS for
15 min. After washing with PBS, cells were blocked with 5%
goat serum (DAKO, Hamburg, Germany) for 1 h at room tem-
perature (RT) and incubated over night with anti-CD44 (DAKO,
1:50), anti-Nestin (DAKO, 1:100), and anti-Stro-1 (R&D Systems,
1:50) antibodies diluted in Tris buffered saline (TBS) containing
1% bovine serum albumin (BSA, Sigma–Aldrich) at 4 ◦ C. After
extensive washing with PBS, a Cy3-conjugated secondary anti-
body (Dianova, Hamburg, Germany) was applied for 1 h at
RT (1:250). Finally, cells were washed again with PBS, nuclear
staining was performed using DAPI (Sigma–Aldrich) for 8 min
followed by PBS washing and mounting on glass slides with
Mowiol/DABCO (Roth, Karlsruhe, Germany) for fluorescence
microscopic imaging.
2.4. Flow cytometry
Human DPSC established cultures up to the ninth pas-
sage were characterized for mesenchymal stem cell markers
(CD73, CD90, CD105) using flow cytometry. In brief, 106
cells/sample were incubated with the following fluorochrome-
conjugated mouse anti-human antibodies as recommended
by the manufacturer’s protocol: anti-CD73-PE, anti-CD90-APC,
and anti-CD105-FITC (all from Miltenyi Biotec). Fluorochrome-
conjugated isotype control antibodies were used to test
specific labelling. Finally, the cells were measured with
FACS-Canto (BD Biosciences, Heidelberg, Germany) and were
analyzed by FACSDiva (BD Biosciences) and FlowJo (TreeStar
Inc., Ashland, OR) software.
2.5. Cell differentiation
Different cell differentiation models were applied to verify
the stem cell character of our dental pulp cells [24]. Hence,
cells were seeded on 12-well-plates at an initial density
of 50,000 cells/well and were grown to 100% cell conflu-
ence. Afterwards varying differentiation protocols were used
to induce adipogenic [25], osteogenic [26] or chondrogenic
differentiation [27,28]. In brief, for adipogenic differentia-
tion two-day post-confluent hDPSCs were challenged with
an adipogenesis-inducing medium containing DMEM, 4.5 g/l
glucose, 10% FBS, 1% antibiotic and antimycotic solution
(Life Technologies) supplemented with 1 ␮M dexamethasone,
0.2 mM indomethacin, 1.7 ␮M insulin, 0.5 mM 3-isobutyl-1-
methylxanthine (all from Sigma–Aldrich) for a total of 5 days
followed by an incubation for 2 days in adipogenesis main-
tenance medium (DMEM, 4.5 g/l glucose, 1.7 ␮M insulin, 10%
FBS, 1% antibiotic and antimycotic solution). This procedure
was repeated two times for a total adipogenic differentiation
period of 21 days.
Osteoblastic differentiation was induced by incubation of
confluent hDPSCs with osteogenic medium (DMEM, 4.5 g/l
glucose, 10% FBS, 1% antibiotic and antimycotic solution
(Life Technologies), 0.2 mM l-ascorbic acid 2-phosphate, 10 nM
␤-glycerophosphate and 100 nM dexamethasone (all from
Sigma–Aldrich)) for a total of 18 days with medium change
every third day.
Chondrogenic differentiation was performed using pellet
culture technique. Briefly, 2 × 106 cells were centrifuged at
500 g in 15 ml polypropylene conical tubes and the resulting
pellets were cultured for 4 weeks. To induce chondrogenic
differentiation cells were grown in a serum-free chemi-
cally defined medium consisting of DMEM, high-glucose
(4.5 g/l; Life Technologies) supplemented with 6.25 mg/ml
insulin, 6.25 mg/ml transferrin, 6.25 mg/ml selenious acid,
5.33 mg/ml linoleic acid, 1.25 mg/ml bovine serum albumin,
1 mM sodium pyruvate, 0.1 mM l-ascorbic acid 2-phosphate,
100 nM dexamethasone (all from Sigma–Aldrich), and 10 ng/ml
transforming growth factor beta 3 (TGF-␤3; R&D Systems).
Cultures were incubated for 4 weeks at 37 ◦ C in a humid atmo-
sphere containing 5% CO2 . Medium changes were carried out
at 2–3-day intervals. Adipogenic differentiation was controlled
by oil red staining, osteogenic by Alizarin red and von Kossa
staining and chondrogenic by Toluidin blue- and Collagen-II
staining.
2.6. Oil red O staining
Cells on sterile coverslips were washed twice with PBS, fixed
with 4% PFA (Sigma–Aldrich) for 10 min at RT and rinsed with
50% ethanol (AppliChem, Darmstadt, Germany). This was fol-
lowed by oil red O staining for 30 min, a washing step with 50%
ethanol and aqua dest. Nuclear staining was performed using
Mayer’s haematoxylin (Merck, Darmstadt, Germany) staining
for 2 min followed by 5 min of washing with running water and
finally, mounting with Aquatex (Merck).
2.7. Alizarin red staining
Following a modified protocol of Gregory et al., cells were
washed twice with PBS and fixed with 4% PFA (Sigma–Aldrich)
for 20 min at RT [29]. Subsequently, cells were rinsed with
aqua dest. and incubated with 40 mM Alizarin red solution
(Sigma–Aldrich) for 20 min (pH 4.1). Finally, cells were washed
again with aqua dest. (5×) and mounted with Aquatex.
2.8. Von Kossa staining
Human pulpal cells were washed with PBS (2×), fixed with
4% PFA for 20 min and washed twice with distilled water fol-
lowed by 5% silver nitrate solution (Merck) for 1 h at 4 ◦ C. Then,
cells were washed again with distilled water (2×) and incu-
bated with 1% pyrogallol solution (Merck) for 5 min. Fixation
was performed using 5% sodium thiosulfate solution (Merck)
for 5 min at RT and subsequent washing with running water.
Nuclear staining was conducted with nuclear fast red solu-
tion (Sigma–Aldrich) for 10 min followed by a final washing
step with aqua dest. (2×) and mounting with Aquatex.
 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064 1055

Table 1 – Antibodies.
Antibody Manufacturer Species Dilution
Anti-CD 44 antibody Dako, Hamburg, Germany Mouse 1:50
Anti-DSPP antibody Santa Cruz, Heidelberg, Germany Rabbit 1:100
Anti-Collagen II antibody Acris antibodies, Hiddenhausen, Germany Mouse 1:100
Anti-Nestin antibody Abcam, Cambridge, United Kingdom Mouse 1:50
Anti-Stro-1 antibody R&D System, Wiesbaden, Germany Mouse 1:25

2.9. Toluidin blue staining
In order to verify chondrogenic differentiation, cryosections
of cell pellets were conducted and stained with toluidine blue
solution (Sigma–Aldrich) for 3 min followed by a washing step
under running water and mounting with Aquatex.
2.10. Collagen-II staining
Sections of cell pellets were fixed with 100% methanol
(AppliChem) for 8 min at −20 ◦ C. After rehydration with
PBS and 0.1% Triton X-100 (Sigma–Aldrich) containing
PBS for 10 min, pellets were treated with hyaluronidase
(Sigma–Aldrich) for 30 min at RT. Subsequently, pellets were
washed with distilled water and TBS followed by proteinase K
incubation (DAKO) for 30 min. Another washing step was per-
formed with TBS followed by blocking unspecific binding sites
with 10% anti-goat serum (DAKO) in TBS for 1 h at RT. After-
wards, cells were washed once with TBS and subsequently
incubated with an anti-Collagen-II antibody (Acris Antibod-
ies, Hiddenhausen, Germany, Table 1) overnight at 4 ◦ C. After
extensive washing with PBS, a Cy3- or AF488-conjugated
goat anti-rabbit IgG secondary antibody (Dianova, Hamburg,
Germany) was applied for 1 h at RT (1:250). Finally, sections
were washed again with PBS, nuclear staining was performed
using DAPI (Sigma–Aldrich) for 5 min followed by PBS wash-
ing and mounting on glass slides with Mowiol/DABCO (Roth,
Karlsruhe, Germany).
2.11. Assessment of cytotoxicity using XTT assay
The commercially available caries infiltrant ICON® was pur-
chased from DMG (Hamburg, Germany) and used in two ways
to investigate the effect of ICON® on pulpal cells. The com-
position of ICON® is listed in Table 2. In one group, ICON®
Table 2 – ICON® composition (according to
manufacturer).
Individual components Chief constituents
ICON-Infiltrant: Triethylenglycoldimethacrylat-
(TEGDMA) based resin matrix
(about 78%),
trimethylolpropantriacrylat (20%),
campherchinon (<1%), (2-ethyl-
hexyl)-p-dimethylaminobenzoat
(<1%),
2,6-di-tert-butyl-4-methylphenol
(<1%), initiators
was light-cured as recommended by the manufacturer (ICON®
light-cured (ICON lc)) In brief, 50 ␮l ICON® was pipetted into a
polyvinylsiloxane mold and covered with a glass slide. The
specimens were then light cured for 40 s using a dental
curing unit (Optima-10 LED, B.A. International, Northamp-
ton, UK) and measured exitance irradiance of approximately
1000 mW/cm2 to ensure complete curing. Subsequently, the
specimens were sterilized by UV radiations for 15 min in a
cell culture hood, placed in 50 ml culture medium (DMEM)
and incubated for 24 h at 37 ◦ C. In the other group, ICON®
was not light-cured (ICON nc) and 50 ␮l ICON® directly dis-
solved in 50 ml culture medium (DMEM) and also incubated
for 24 h at 37 ◦ C. Additionally, TEGDMA was purchased from
Sigma–Aldrich (Taufkirchen, Germany) and diluted in DMEM
to obtain a stock solution of 10 mM.
Cell cytotoxicity was determined using the PromoKine XTT
Assay Kit (Promocell, Heidelberg, Germany). In brief, 10,000
hDPSCs (third passage) per well were seeded in 96-well-plates
for 24 h. To avoid evaporation effects of the peripheral outer
wells, cells were only grown in the middle wells of 96-well-
plates whereas the outer wells were filled with sterile water.
Dental pulp cells were then incubated with the two condi-
tioned ICON lc and ICON nc media as well as different TEGDMA
concentrations (100 ␮M, 200 ␮M, 300 ␮M, 500 ␮M, 1000 ␮M,
2000 ␮M and 5000 ␮M). After the indicated time points, XTT
reaction solution was added to the medium for 3 h followed
by the measurement of absorbance at 490 nm with correc-
tion wavelength 670 nm in a microplate reader. The lethal
concentration 50 (LC50 ) of TEGDMA in DPSCs was calculated
using GraphPad Prism software (Version 6, GraphPad Software,
San Diego CA, USA). Experiments were performed with hDPCs
from three different donors in hexaduplicates.
2.12. Cell exposure
For in vitro experiments, third passage cells from three differ-
ent donors were seeded in triplicates on 12-well plates at an
initial density of 50,000 cells per well. After reaching 90% con-
fluence, cells were grown under serum free conditions for 24 h.
Then, dental pulp cells were challenged with the two condi-
tioned medium of ICON lc and ICON nc as well as different
TEGDMA concentrations which were chosen due to cytotox-
icity results (100 ␮M, 1000 ␮M and 2000 ␮M). Another group
which was not exposed to ICON or TEGDMA served as control.
After 2 and 24 h cells were collected for mRNA and protein
expression analysis.
2.12.1. RNA-isolation, first strand cDNA synthesis, qPCR
Total RNA was isolated using the RNeasy Plus Mini Kit
(Qiagen, Hilden, Germany) according to the manufacturer’s
1056 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064

Table 3 – Primers.
Primer Sequence Efficiency Annealing temp.
 
␤-actin Forward: 5 -CAT GGA TGA TGA TAT CGC CGC G-3 94% 69 ◦ C
Reverse: 5 -ACA TGA TCT GGG TCA TCT TCT CG-3
Interleukin-6 Forward: 5 -ATG AAC TCC TTC TCC ACA AGC-3 105% 68 ◦ C
Reverse: 5 -CTA CAT TTG CCG AAG AGC CC-3
Interleukin-8 Forward: 5 -ATG ACT TCC AAG CTG GCC GTG G-3 101% 68 ◦ C
Reverse: 5 -TGA ATT CTC AGC CCT CTT CAA AAA C-3
Interleukin-10 Forward: 5 -TTA AGG GTT ACC TGG GTT GC-3 97% 65 ◦ C
Reverse: 5 -GCC TTG CTC TTG TTT TCA CA-3
DSPP Forward: 5 -TCA CAA GGG AGA AGG GAA TGG-3 92% 67 ◦ C
Reverse: 5 -CTT GGA CAA CAG CGA CAT CCT-3

protocol and quantified using the NanoDrop ND-1000 Spec- multiple comparison or Dunnett’s test were applied. P-values
trophotometer (NanoDrop, Technologies, Wilmington, DE, less than 0.05 were considered to be statistically significant.
USA). First-strand cDNA synthesis was performed with
1 ␮g RNA and the iScriptTM Select cDNA Synthesis Kit
(Bio-Rad Laboratories, Munich, Germany) using oligo(dT)-
3. Results
primers. The mRNA expression of dentin sialophosphoprotein
3.1.1. Isolation and characterization of human dental
(DSPP), Interleukin (IL)-6, IL-8 and IL-10 was detected by real-
pulp stem cells (hDPSCs)
time polymerase chain reaction (PCR) using the iCycler iQ
detection system (Bio-Rad Laboratories), SYBR Green (Bio-Rad
Isolated Stro-1 positive dental pulp cells were analyzed for the
Laboratories), and specific primers (Table 3). Verification of all
expression of mesenchymal and neural stem cell markers and
primers was accomplished by computer analysis for speci-
their cell differentiation capacity. As revealed by immunofluo-
ficity with the basic logical alignment search tool (BLAST)
rescence staining, all cells were positive for the expression of
and synthesized of high quality (Metabion, Martinsried,
CD44, Nestin, and Stro-1 (Fig. 1A–C). CD44 (Fig. 1A) was mainly
Germany). In addition, the specific annealing temperatures
found at the cell membrane, whereas Nestin (Fig. 1B) and Stro-
were assessed by a temperature gradient and PCR-efficiencies
1 (Fig. 1C) were primarily located in the cytoplasm. Data were
for primers were determined with dilution series of cloned and
accomplished by flow cytometric analysis for surface epitope
sequenced primer-specific PCR-products. In Table 1 the primer
expression of CD73, CD90, and CD105. As shown in Fig. 1D–F,
sequences, annealing temperatures and efficiencies are
cells of the ninth passage cultured in the presence of FGF-2
listed.
were still positive for CD73 (96.1%), CD90 (73.5%) and CD105
For qPCR-analysis, 50 ng cDNA was added to a mastermix
(88.2%), respectively.
containing primers and iQTM SYBR Green Supermix (Bio-Rad
Moreover, the isolated dental pulp cells could be suc-
Laboratories). Cloned PCR-products derived from the specific
cessfully differentiated into adipocyte-, chondrocyte-, and
primers were used as positive control, whereas water served
osteoblastic-like phenotypes as demonstrated by several dif-
as negative control. Every set of experiment was carried out
ferentiation protocols and staining procedures (Fig. 1G–M).
with cDNA of the same sample to compare the expression of
Adipocyte differentiation was validated by Oil red O staining
the different genes of interest. PCR conditions were defined as
showing characteristic lipid packed vacuoles in the cyto-
follows: 5 min denaturing step at 95 ◦ C, then 50 cycles of 15 s at
plasm (red) (Fig. 1H) compared to undifferentiated control
95 ◦ C, 30 s at specific annealing temperatures for the primers,
cells (Fig. 1G). Chondrocyte differentiation was shown using
and 30 s at 72 ◦ C for elongation. Target gene expression was
Toluidin blue (Fig. 1I) and Collagen II staining (Fig. 1J).
normalized to ␤-actin mRNA expression. Relative differential
Osteoblastic-like phenotype was affirmed by Alizarin (Fig. 1K
gene expression was calculated using the method described
and L) and von Kossa staining (Fig. 1M and N) which induce
by Pfaffl [30].
coloration of calcium precipitates.

2.12.2. Enzyme-linked immunoassay (ELISA)
Supernatants were collected after 24 h of cell exposure and
analyzed using a commercially available enzyme linked
immunoassay (ELISA) kit (PeproTech, Hamburg-Uhlenhorst,
Germany) according to the manufacturer’s protocol to deter-
mine protein levels of IL-6 and IL-8.
2.13. Statistical analysis
GraphPad Prism software, Version 6 (GraphPad Software, San
Diego CA, USA) was used for statistical analysis. Mean ± SEM
were calculated and one-way ANOVA and the post-hoc Tukey’s
3.1.2. Cytotoxicity of conditioned media with ICON®
Cell viability of DPSCs challenged with conditioned media con-
taining ICON lc or ICON nc formulation was determined after
2, 24 and 48 h using XTT assay. After 2 and 24 h cell viability
remained unchanged in the ICON lc group compared to con-
trol (Fig. 2A, B). After 48 h a significant decline in cell viability of
about 10% was detectable (Fig. 2C). Notably, exposure to media
containing ICON nc induced a significant decrease in cell via-
bility (62.1%) after 2 h which was even more pronounced after
24 h (97.6%) (Fig. 2A and B). After 48 h the cell survival rate in
the ICON nc group was only 1.1% (Fig. 2C).
 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064 1057

Fig. 1 – Human dental pulp stem cells (hDPSCs) characterization and differentiation. Immunofluorescence staining of
hDPSCs revealed that all cells were positive for the expression of CD44 (A), Nestin (B) and Stro-1 (C). In addition, a
representative flow cytometric analysis for surface epitope expression of CD73 (D), CD90 (E), and CD105 (F) of dental pulp
cells of the ninth passage revealed a mesenchymal stem cell phenotype. (Scale bar: 100 ␮m.) As for hDPSC differentiation,
several differentiation protocols and stainings were performed (G–N). Adipocyte differentiation was validated by Oil red O
staining showing characteristic lipid packed vacuoles in the cytoplasm (red) (H) compared to undifferentiated control cells
(G). Chondrocyte differentiation was confirmed by Toluidin blue (I; glykosaminoglykane are stained blue) and Collagen II
(red) staining (J). Osteoblastic-like phenotype was affirmed by Alizarin (K and L) and von Kossa staining (M and N) which
induce coloration of calcium precipitates. (K and M: control cells without induction of differentiation. (L and N) hDPSCs after
successful induction of osteoblastic differentiation). (For interpretation of the references to color in this figure legend, the
reader is referred to the web version of the article.)

Fig. 2 – Cytotoxicity of ICON® . To assess ICON® cytotoxicity, cell viability of human dental pulp stem cells was determined
after exposure to (ICON® light-cured (ICON lc)) and in the other group ICON® was not light-cured (ICON nc) for 2 (A), 24 (B)
and 48 h (C) using the XTT assay. Unstimulated hDPSCs served as control. Mean ± SEM were calculated and one-way ANOVA
and the post-hoc Tukey’s multiple comparison test were applied (*P < 0.05).
1058 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064

3.1.3. Effect of ICON® exposure on mRNA expression of
IL-6, -8, -10 and DSPP
After exposure to both ICON® conditioned media, qPCR
analysis was performed to verify potential inflam-
matory reactions and differentiation processes in
DPSCs (Fig. 3A–H). Exposure to both conditioned media
decreased the IL-6 mRNA expression after 2 h significant
ly compared to control only in the ICON nc group (Fig. 3A).
However, after 24 h a significant increase in IL-6 mRNA expres-
sion after incubation with ICON nc was recorded compared
to the control group. Remarkably, IL-6 transcript expression
level in the ICON lc group was below the one detected in
untreated cells (Fig. 3B).
Notably, IL-8 mRNA expression was not significantly
altered by ICON lc nor by ICON nc after 2 h compared to con-
trol (Fig. 3C). In contrast, ICON lc induced an 8.6-times higher
IL-8 mRNA expression level compared to control after 24 h,

Fig. 3 – mRNA expression of IL-6, -8, -10 and DSPP in human dental pulp cells challenged with ICON® . To determine
inflammatory and differentiation processes in human dental pulp stem cells, mRNA expression of IL-6 (A and B), -8 (C and
D), -10 (E and F) and the extracellular matrix protein dentin sialophosphoprotein (DSPP) (G and H) were evaluated after 2 and
24 h. Mean ± SEM were calculated and one-way ANOVA and the post-hoc Tukey’s multiple comparison test were applied
(*P < 0.05).
 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064 1059

Fig. 4 – Protein secretion of IL-6 and IL-8 induced by ICON® . Protein levels of IL-6 (A) and IL-8 (B) induced by ICON®
conditioned media were determined in human dental pulp stem cell supernatants after 24 h. Mean ± SEM were calculated
and one-way ANOVA and the post-hoc Tukey’s multiple comparison test were applied (*P < 0.05).

whereas ICON nc caused an even higher gene activity of IL-8
(37.5-times) which was statistically significant (Fig. 3D).
Analyzing the expression of the anti-inflammatory
cytokine IL-10 revealed a significant increase in mRNA
expression in the ICON lc group at both time points with
the highest relative transcriptional level (9.6-times higher
than control) after 24 h (Fig. 3E and F). In contrast, ICON
nc challenge induced no IL-10 mRNA alterations compared
to control after 2 h. However, due to the relative low basal
expression level of IL-10 in DPSCS and the strong cytotoxic
effect of ICON nc, IL-10 mRNA expression were no longer
detectable after 24 h (Fig. 3F).
In order to investigate potential effects of the caries
infiltrant on pulpal cell differentiation, the mRNA expres-
sion of the extracellular matrix protein DSPP was evaluated
(Fig. 3G and H). After 2 h DSPP mRNA expression was slightly
decreased in the ICON lc group, which was no longer
detectable after 24 h. Instead, the conditioned media contain-
ing ICON nc elevated the transcription level of DSPP at 2 h
which was even higher after 24 h compared to control (130.3-
times).
3.1.4. Effect of ICON exposure on IL-6 and IL-8 secretion
The concentration of the two pro-inflammatory cytokines IL-
6 and IL-8 in cell culture supernatants was analyzed after
challenging DPSCs with both conditioned media for 24 h
(Fig. 4A and B). The basal secretion was about 430 pg/ml for
IL-6 and 850 pg/ml for IL-8. In line with our mRNA data, a
significant decrease of IL-6 (about 20%) was induced by ICON
lc. In contrast, IL-6 secretion was not significantly changed
by ICON nc (Fig. 4A). Notably, both conditioned media sig-
nificantly increased the secretion of IL-8 in DPSCs after 24 h
with highest concentration in the ICON nc group (1730 pg/ml;
Fig. 4B).
3.1.5. Cytotoxicity of conditioned media with TEGDMA
Since the resin monomer TEGDMA is the main ingredient
of the caries infiltrant ICON® , we used different concen-
trations of pure TEGDMA dissolved in cell culture medium
to compare cytotoxic effects with the results of ICON
conditioned media. As demonstrated in Fig. 5A, TEGDMA
concentrations between 0 and 500 ␮M did not alter cell
viability of DPSCs. However, 1000 ␮M induced a significant
reduction of cell viability (13.6%) and even higher con-
centrations caused pronounced cytotoxic effects (Fig. 5A).
No more viable cells were detectable using the highest
TEGDMA concentration (10,000 ␮M, data not shown). The cal-
culated median lethal concentration (LC50 ) for TEGDMA was
1784 ± 96.93 ␮M.

Fig. 5 – Cytotoxicity of TEGDMA. Since the resin monomer Triethylene-Glycol-Dimethacrylate (TEGDMA) is the main
ingredient of ICON® , different concentrations of TEGDMA were dissolved in cell culture medium and used for human dental
pulp stem cell exposure to determine cytotoxic effects using XTT. The median lethal concentration (LC50 ) was determined
using the GraphPad Prism software. Mean ± SEM were calculated and one-way ANOVA and the post-hoc Dunnett’s test were
applied (*P < 0.05).
1060 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064
3.1.6. Effect of TEGDMA exposure on pro- and
anti-inflammatory cytokines and DSPP mRNA expression
As a result of cytotoxicity investigations, exposure to three
TEGDMA concentrations (100, 1000 and 2000 ␮M) was further
evaluated. Interestingly, all three concentrations decreased IL-
6 mRNA expression after 2 and even more after 24 h which was
statistically significant compared to control (Fig. 6A and B).
As for IL-8 mRNA, lower TEGDMA concentrations (100
and 1000 ␮M) induced almost no IL-8 mRNA alterations after
2 h, whereas prolonged incubation significantly reduced IL-8
mRNA transcription levels compared to control (Fig. 6C and D).
Interestingly, higher TEGDMA concentrations (2000 ␮M) dra-
matically increased the IL-8 mRNA expression after 2 h which
was still observable after 24 h (Fig. 6C and D).
Analyzing the mRNA expression of the anti-inflammatory
cytokine IL-10 revealed a dose-dependent significant decrease
after 2 as well as 24 h (Fig. 6E and F).
Regarding the expression of the extracellular matrix pro-
tein DSPP, the same dose-dependent effect was detectable.
Thereby, TEGDMA induced a concentration-dependent down-
regulation of DSPP mRNA expression in hDPSCs after 2 and
24 h (Fig. 6G and H).
3.1.7. Effect of TEGDMA exposure on IL-6 and IL-8
secretion
Protein levels of IL-6 and IL-8 in supernatants of hDPSCs were
determined after 24 h to validate their mRNA data (Fig. 7A and
B). TEDGMA concentrations decreased IL-6 secretion signif-
icantly compared to control supporting our mRNA findings
(Fig. 7A). Interestingly, all three TEGDMA concentrations sig-
nificantly elevated IL-8 protein levels in hDPSC supernatants
(Fig. 7B) which was only in part consistent with mRNA data
(Fig. 6E and F).
4. Discussion
First experiments using dental adhesives have shown that
these materials predominately penetrate the superficial caries
lesions. Therefore, new low-viscosity resins were developed
with higher penetration coefficients to improve caries infiltra-
tion. As a consequence of the higher penetration efficiencies,
possible adverse reactions due to the release and diffusion
of monomers through dentin tubules are discussed. Many
studies have been performed demonstrating that especially 2-
hydroxyethyl methacrylate (HEMA) and TEGDMA are released
from a variety of resins [8,9]. In addition, in vitro models have
revealed that these monomers are traceable in dental pulp
chamber. Thereby, HEMA and TEGDMA may reach concentra-
tions as high as 1.5–8 mM in the pulp and due to their lipophilic
character penetrating the lipid cell layer, they are consid-
ered as monomers with the highest toxic potential [8,9,31–33].
Moreover, there is evidence that they may induce cell death
and oxidative stress via overexpression of reactive oxygen
species (ROS) and depletion of intracellular glutathione (redox
systems) [6,15,34].
Since TEGDMA is the main ingredient of ICON® , inter-
actions between the caries infiltrant and dental pulp are
plausible. However until now, investigations primarily focused
on the penetration capacity of ICON and none on its bio-
logic adverse effects [35,36]. Therefore, the present study used
isolated human dental pulp stem cells challenged with con-
ditioned media containing ICON® as either light-cured or
non-light-cured formulation. These cells and their microenvi-
ronment are known to be important regulators of pulp repair
processes due to their migration towards injury and sub-
sequent differentiation into odontoblast-like cells producing
reparative dentin [37]. In vitro and in vivo investigations have
evaluated DPSCs characteristics showing that they possess
adult mesenchymal stem cell (MSC) properties [38,39]. MSCs
in standard culture conditions are plastic-adherent, express
CD105, CD73 and CD90, lack expression of CD45, CD34, CD14 or
CD11b, CD79alpha or CD19 and HLA-DR surface molecules and
may differentiate at least to osteoblasts, adipocytes and chon-
droblasts in vitro which is in line with our findings. Moreover,
using FGF-2 as a supplement in cell culture media maintained
stem cell properties of our isolated cells up to passage nine.
This supports previous investigations which demonstrated
enhanced sternness properties by up-regulating stem cell
gene expression, increasing proliferation ability, and poten-
tiating differentiation potency of human dental pulp cells and
cells from the apical papilla under FGF-2 treatment [23].
As for the assessment of cytotoxicity, hDPSCs were exposed
to light-cured ICON® as well as non-light-cured in order
to simulate the condition in dental practice and a situa-
tion that may occur due to not following the manufacturer’s
instructions properly. Notably, the ICON® non-light-cured
conditioned medium dramatically decreased cell viability
almost immediately, whereas the light-cured ICON® demon-
strated only a modest cytotoxicity. Similar cytotoxic effects
were found for the highest (10 mM) TEGDMA concentration.
Assuming that ICON® contains 100% TEGDMA, our assay con-
centration of ICON® dissolved in cell culture medium would
result in a final concentration of 3.81 mM TEGDMA. Thus, the
increased cytotoxicity in the non-light-cured ICON® group
compared to pure TEGDMA may be caused by other ingre-
dients of ICON® such as initiators or additives (Table 2).
Concerning the LC50 of TEGDMA (1.8 mM), our data sup-
port previous studies [6,32,40,41]. They detected LC50 values
between 0.1 mM and 4 mM depending on the used human
dental pulp cells and cytotoxicity assays.
In order to determine pro-inflammatory capacities of
ICON® , IL-6 and IL-8 were evaluated. IL-6 induces the secretion
of acute phase proteins and influences various pathologies
such as rheumatoid arthritis, osteoporosis and apical peri-
odontitis [42,43]. Concerning IL-8, also known as chemokine
CXCL8, there is evidence that this mediator is responsible for
immune cell recruitment including endothelial and epithelial
cells, fibroblasts, chondrocytes, lymphocytes and neutrophils
[44]. Both cytokines are elevated in inflamed dental pulps com-
pared to healthy pulp tissues [45,46]. We demonstrated that
ICON lc induced a decrease of the pro-inflammatory cytokine
IL-6 and an increase of IL-8. In contrast, 24 h of ICON nc
exposure caused an upregulation of both mediators to an
even higher extent. Concerning TEGDMA induced IL-6, dose-
dependent effects were detectable. The values obtained for
IL-8 were in part contradictory, as lower TEGDMA concentra-
tions reduced IL-8 mRNA expression, whereas protein levels
 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064 1061

Fig. 6 – mRNA expression of IL-6, -8, -10 and DSPP in human dental pulp cells challenged with TEGDMA. To determine
inflammatory and differentiation processes in human dental pulp stem cells challenged with different TEGDMA
concentration, mRNA expression of IL-6 (A and B), -8 (C and D), -10 (E and F) and the extracellular matrix protein dentin
sialophosphoprotein (DSPP) (G and H) were evaluated after 2 and 24 h. Mean ± SEM were calculated and one-way ANOVA
and the post-hoc Dunnett’s test were applied (*P < 0.05).
1062 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064
Fig. 7 – Protein secretion of IL-6 and IL-8 induced by TEGDMA. Protein levels of IL-6 (A) and IL-8 (B) induced by different
TEGDMA concentration conditioned media were determined in human dental pulp stem cell supernatants after 24 h.
Mean ± SEM were calculated and one-way ANOVA and the post-hoc Dunnett’s test were applied (*P < 0.05).
were increased. Higher TEGDMA concentrations led to a sig-
nificant induction of both IL-8 mRNA and protein. Similar
findings were detected by Schmalz and colleagues showing
reduced IL-6 expression levels in human oral epithelial cells
incubated with non-toxic TEGDMA concentrations [47].
As for anti-inflammatory effects, the induction of IL-10
expression was determined. IL-10 plays a crucial role in
immune defense, inhibiting an increase of inflammatory pro-
cesses in order to avoid multiple organ failure and shock.
Notably, Tokuda et al. have shown that IL-10 deactivates
nuclear factor-␬B which results in reduced IL-6 and IL-8 levels
in human dental pulp cells [48]. In the present study, ICON lc
induced a significant elevation of IL-10 mRNA which was not
observed after incubation with ICON nc. TEGDMA decreased
IL-10 in a dose-dependent manner. Our data are in line with
cytotoxicity results implicating that hDPSCs challenged with
ICON nc or high doses of TEGDMA are disabled to counteract
the inflammatory reaction. This is supported by Krifta et al.
demonstrating that TEGDMA may induce immune modula-
tory effects [49]. For example, resin monomers may interfere
with lipopolysaccharide (LPS)-induced pathways. LPS, a cell
wall component of Gram-negative bacteria, activates Toll-
like Receptor (TLR)4 which is highly expressed on immune
cells. This leads to the activation of the transcription fac-
tor nuclear factor (NF)-␬B which subsequently translocates
into the cell nucleus inducing the expression of various
pro- and anti-inflammatory mediators such as the cytokines
IL-6 and IL-10. Interestingly, the release of these media-
tors was significantly reduced by TEGDMA in LPS-stimulated
mouse macrophages after long exposure periods [50]. Thus,
TEGDMA may initially induce dose-dependently an upregu-
lation of pro-inflammatory processes and after prolonged
exposure the protective anti-inflammatory mechanism col-
lapse leading to dysregulated cell function and immune
response.
Since, hDPSCs regulate the regeneration of the pulp
via their differentiation to odontoblasts which synthesize
increased amounts of DSPP, we investigated DSPP expression
after ICON® and TEGDMA exposure [18,51]. Notably, DSPP is
necessary for dentin mineralization which increases the dis-
tance between toxic stimuli and the pulp. Interestingly, ICON
lc caused almost no alterations of DSPP, whereas ICON nc
markedly elevated DSPP mRNA levels. TEGDMA induced a
dose-dependent downregulation of DSPP mRNA. Hence, our
results indicate that unpolymerized ICON® and low TEGDMA
concentrations induce odontoblast differentiation in order to
protect pulp tissue against toxicity. Data imply that cells chal-
lenged with ICON lc are not in need of high DSPP expressions
due to low toxicity, whereas high TEGDMA concentrations may
inhibit the protective machinery which was also detectable for
IL-6 expression.
We acknowledge that the exposure of interproximal or
cervical gingival tissues is also very likely and may result
in different findings as has been described by Tadin et al.
demonstrating that pulpal cells are more prone to acrylates
than gingival fibroblasts [16]. The high turn-over rate as
well as the constant exposure to various and high concen-
trations of pathogens leading to a more tolerant character
of gingival fibroblasts may explain these results. Moreover,
salivation and ingestion may have an important impact
for in vivo cytotoxicity which is difficult to simulate in
vitro. Therefore, our experiments focused on dental pulpal
tissues.
In conclusion, present data are in line with current in vitro
findings demonstrating that the monomer release, which
depends on monomer turn-over, monomer size and resin for-
mula, plays an important role in the cytotoxicity of resin
adhesives [11,12,15]. Even though ICON® is a successful min-
imal invasive technique, our in vitro results implicate that
it should be used with caution and clinicians should strictly
follow manufacturer‘s instructions to avoid side effects like
dental pulp inflammation. Moreover, further investigations
are needed to determine adverse effects after prolonged expo-
sure duration due to resin degradation.
Acknowledgements
LG and DK were supported by a grant of the BONFOR Research
Foundation of the Medical Faculty of the University of Bonn.
Part of this work is content of the doctoral thesis of RS.
 d e n t a l m a t e r i a l s 3 2 ( 2 0 1 6 ) 1052–1064
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