In vitro biocompatibility of ICON® and TEGDMA on human dental pulp stem cells

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

Available online at www.sciencedirect.com

ScienceDirect journal homepage: www.intl.elsevierhealth.com/journals/dema

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 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 b

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: [email protected] (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 filling 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 addition, 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 commercially 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 biocompatibility 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 components 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 function 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 mesenchymal 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 odontoblasts. 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 homeostasis 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 identify 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 University 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 reasons 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 culture 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 maintain stem cell properties of the human dental pulp cells [23]. Cells/Explants were kept in an incubator at 37 ◦ C in humidified 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 Gladbach, Germany) and a mouse monoclonal STRO-1 antibody (R&D Systems) as recommended by the manufacturer’s protocol. Cells were propagated in culture medium containing FGF-2 up to the ninth passage and putative mycoplasma contamination was routinely verified through PCR analysis 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 stainings.

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 temperature (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 antibody (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 passage were characterized for mesenchymal stem cell markers (CD73, CD90, CD105) using flow cytometry. In brief, 106 cells/sample were incubated with the following fluorochromeconjugated 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). Fluorochromeconjugated 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 confluence. Afterwards varying differentiation protocols were used to induce adipogenic [25], osteogenic [26] or chondrogenic differentiation [27,28]. In brief, for adipogenic differentiation 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-1methylxanthine (all from Sigma–Aldrich) for a total of 5 days followed by an incubation for 2 days in adipogenesis maintenance 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 chemically 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 atmosphere 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 followed 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 followed by 5% silver nitrate solution (Merck) for 1 h at 4 ◦ C. Then, cells were washed again with distilled water (2×) and incubated 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 solution (Sigma–Aldrich) for 10 min followed by a final washing step with aqua dest. (2×) and mounting with Aquatex.

1055

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

Table 1 – Antibodies. Antibody

Manufacturer

Species

Dilution

Anti-CD 44 antibody Anti-DSPP antibody Anti-Collagen II antibody Anti-Nestin antibody Anti-Stro-1 antibody

Dako, Hamburg, Germany Santa Cruz, Heidelberg, Germany Acris antibodies, Hiddenhausen, Germany Abcam, Cambridge, United Kingdom R&D System, Wiesbaden, Germany

Mouse Rabbit Mouse Mouse Mouse

1:50 1:100 1:100 1:50 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 performed with TBS followed by blocking unspecific binding sites with 10% anti-goat serum (DAKO) in TBS for 1 h at RT. Afterwards, cells were washed once with TBS and subsequently incubated with an anti-Collagen-II antibody (Acris Antibodies, 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 washing 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 purchased from DMG (Hamburg, Germany) and used in two ways to investigate the effect of ICON® on pulpal cells. The composition 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-ethylhexyl)-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, Northampton, 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 dissolved 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-wellplates whereas the outer wells were filled with sterile water. Dental pulp cells were then incubated with the two conditioned 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 correction 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 different donors were seeded in triplicates on 12-well plates at an initial density of 50,000 cells per well. After reaching 90% confluence, cells were grown under serum free conditions for 24 h. Then, dental pulp cells were challenged with the two conditioned medium of ICON lc and ICON nc as well as different TEGDMA concentrations which were chosen due to cytotoxicity 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

Interleukin-6 Interleukin-8 Interleukin-10



Forward: 5 -CAT GGA TGA TGA TAT CGC CGC G-3 Reverse: 5 -ACA TGA TCT GGG TCA TCT TCT CG-3 Forward: 5 -ATG AAC TCC TTC TCC ACA AGC-3 Reverse: 5 -CTA CAT TTG CCG AAG AGC CC-3 Forward: 5 -ATG ACT TCC AAG CTG GCC GTG G-3 Reverse: 5 -TGA ATT CTC AGC CCT CTT CAA AAA C-3 Forward: 5 -TTA AGG GTT ACC TGG GTT GC-3 Reverse: 5 -GCC TTG CTC TTG TTT TCA CA-3 Forward: 5 -TCA CAA GGG AGA AGG GAA TGG-3 Reverse: 5 -CTT GGA CAA CAG CGA CAT CCT-3

protocol and quantified using the NanoDrop ND-1000 Spectrophotometer (NanoDrop, Technologies, Wilmington, DE, 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)primers. The mRNA expression of dentin sialophosphoprotein (DSPP), Interleukin (IL)-6, IL-8 and IL-10 was detected by realtime polymerase chain reaction (PCR) using the iCycler iQ detection system (Bio-Rad Laboratories), SYBR Green (Bio-Rad Laboratories), and specific primers (Table 3). Verification of all primers was accomplished by computer analysis for specificity with the basic logical alignment search tool (BLAST) and synthesized of high quality (Metabion, Martinsried, Germany). In addition, the specific annealing temperatures were assessed by a temperature gradient and PCR-efficiencies for primers were determined with dilution series of cloned and sequenced primer-specific PCR-products. In Table 1 the primer sequences, annealing temperatures and efficiencies are listed. For qPCR-analysis, 50 ng cDNA was added to a mastermix containing primers and iQTM SYBR Green Supermix (Bio-Rad Laboratories). Cloned PCR-products derived from the specific primers were used as positive control, whereas water served as negative control. Every set of experiment was carried out with cDNA of the same sample to compare the expression of the different genes of interest. PCR conditions were defined as follows: 5 min denaturing step at 95 ◦ C, then 50 cycles of 15 s at 95 ◦ C, 30 s at specific annealing temperatures for the primers, and 30 s at 72 ◦ C for elongation. Target gene expression was normalized to ␤-actin mRNA expression. Relative differential gene expression was calculated using the method described by Pfaffl [30].

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 determine protein levels of IL-6 and IL-8.

2.13.

Efficiency



␤-actin

DSPP

Sequence

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

Annealing temp.

94%

69 ◦ C

105%

68 ◦ C

101%

68 ◦ C

97%

65 ◦ C

92%

67 ◦ C

multiple comparison or Dunnett’s test were applied. P-values less than 0.05 were considered to be statistically significant.

3.

Results

3.1.1. Isolation and characterization of human dental pulp stem cells (hDPSCs) Isolated Stro-1 positive dental pulp cells were analyzed for the expression of mesenchymal and neural stem cell markers and their cell differentiation capacity. As revealed by immunofluorescence staining, all cells were positive for the expression of CD44, Nestin, and Stro-1 (Fig. 1A–C). CD44 (Fig. 1A) was mainly found at the cell membrane, whereas Nestin (Fig. 1B) and Stro1 (Fig. 1C) were primarily located in the cytoplasm. Data were accomplished by flow cytometric analysis for surface epitope expression of CD73, CD90, and CD105. As shown in Fig. 1D–F, cells of the ninth passage cultured in the presence of FGF-2 were still positive for CD73 (96.1%), CD90 (73.5%) and CD105 (88.2%), respectively. Moreover, the isolated dental pulp cells could be successfully differentiated into adipocyte-, chondrocyte-, and osteoblastic-like phenotypes as demonstrated by several differentiation protocols and staining procedures (Fig. 1G–M). Adipocyte differentiation was validated by Oil red O staining showing characteristic lipid packed vacuoles in the cytoplasm (red) (Fig. 1H) compared to undifferentiated control cells (Fig. 1G). Chondrocyte differentiation was shown using Toluidin blue (Fig. 1I) and Collagen II staining (Fig. 1J). Osteoblastic-like phenotype was affirmed by Alizarin (Fig. 1K and L) and von Kossa staining (Fig. 1M and N) which induce coloration of calcium precipitates.

3.1.2.

Cytotoxicity of conditioned media with ICON®

Cell viability of DPSCs challenged with conditioned media containing 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 control (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 viability (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 inflammatory 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 expression 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 control (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 expression 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 containing ICON nc elevated the transcription level of DSPP at 2 h which was even higher after 24 h compared to control (130.3times).

3.1.4.

Effect of ICON exposure on IL-6 and IL-8 secretion

The concentration of the two pro-inflammatory cytokines IL6 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 significantly 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 concentrations 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 concentrations caused pronounced cytotoxic effects (Fig. 5A). No more viable cells were detectable using the highest TEGDMA concentration (10,000 ␮M, data not shown). The calculated 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 IL6 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) dramatically 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 protein DSPP, the same dose-dependent effect was detectable. Thereby, TEGDMA induced a concentration-dependent downregulation 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 significantly compared to control supporting our mRNA findings (Fig. 7A). Interestingly, all three TEGDMA concentrations significantly 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 infiltration. 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 2hydroxyethyl 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 concentrations as high as 1.5–8 mM in the pulp and due to their lipophilic character penetrating the lipid cell layer, they are considered 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® , interactions 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 biologic adverse effects [35,36]. Therefore, the present study used isolated human dental pulp stem cells challenged with conditioned media containing ICON® as either light-cured or non-light-cured formulation. These cells and their microenvironment are known to be important regulators of pulp repair processes due to their migration towards injury and subsequent 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 chondroblasts 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 potentiating 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 situation 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® demonstrated only a modest cytotoxicity. Similar cytotoxic effects were found for the highest (10 mM) TEGDMA concentration. Assuming that ICON® contains 100% TEGDMA, our assay concentration 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 ingredients of ICON® such as initiators or additives (Table 2). Concerning the LC50 of TEGDMA (1.8 mM), our data support 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 periodontitis [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 compared 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, dosedependent effects were detectable. The values obtained for IL-8 were in part contradictory, as lower TEGDMA concentrations 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 significant 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 processes 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 modulatory effects [49]. For example, resin monomers may interfere with lipopolysaccharide (LPS)-induced pathways. LPS, a cell wall component of Gram-negative bacteria, activates Tolllike Receptor (TLR)4 which is highly expressed on immune cells. This leads to the activation of the transcription factor 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 mediators was significantly reduced by TEGDMA in LPS-stimulated mouse macrophages after long exposure periods [50]. Thus, TEGDMA may initially induce dose-dependently an upregulation of pro-inflammatory processes and after prolonged exposure the protective anti-inflammatory mechanism collapse 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 distance 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 challenged 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 concentrations 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 formula, plays an important role in the cytotoxicity of resin adhesives [11,12,15]. Even though ICON® is a successful minimal 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 exposure 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

references

[1] Kim S, Kim EY, Jeong TS, Kim JW. The evaluation of resin infiltration for masking labial enamel white spot lesions. Int J Pediatr Dent 2011;21:241–8. [2] Meyer-Lueckel H, Paris S. Improved resin infiltration of natural caries lesions. J Dent Res 2008;87:1112–6. [3] Kugel G, Arsenault P, Papas A. Treatment modalities for caries management, including a new resin infiltration system. Compend Contin Educ Dent 2009;3:1–10. [4] Paris S, Meyer-Lueckel H, Cölfen H, Kielbassa AM. Penetration coefficients of commercially available and experimental composites intended to infiltrate enamel carious lesions. Dent Mater 2007;23:742–8. [5] Arslan S, Zorba YO, Atalay MA, Özcan S, Demirbuga S, Pala K, Percin D, Ozer F. Effect of resin infiltration on enamel surface properties and Streptococcus mutans adhesion to artificial enamel lesions. Dent Mater J 2015;34:25–30, http://dx.doi.org/10.4012/dmj. 2014-078. [6] Bakopoulou A, Leyhausen G, Volk J, Tsiftsoglou A, Garefis P, Koidis P, Geurtsen W. Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth. Dent Mater 2011;27:608–17, http://dx.doi.org/10.1016/j.dental.2011.03.002. [7] Schweikl H, Hartmann A, Hiller KA, Spagnuolo G, Bolay C, Brockhoff G, Schmalz G. Inhibition of TEGDMA and HEMA-induced genotoxicity and cell cycle arrest by N-acetylcysteine. Dent Mater 2007;23:688–95. [8] Noda M, Wataha JC, Lockwood PE, Volkmann KR, Kaga M, Sano H. Sublethal, 2-week exposures of dental material components alter TNF-alpha secretion of THP-1 monocytes. Dent Mater 2003;19:101–5. [9] Michelsen VB, Moe G, Skålevik R, Jensen E, Lygre H. Quantification of organic eluates from polymerized resin-based dental restorative materials by use of GC/MS. J Chromatogr B: Analyt Technol Biomed Life Sci 2007;850:83–91. [10] Lee SY, Huang HM, Lin CY, Shih YH. Leached components from dental composites in oral simulating fluids and the resultant composite strengths. J Oral Rehabil 1998;25: 575–88. [11] Wataha JC, Rueggeberg FA, Lapp CA, Lewis JB, Lockwood PE, Ergle JW, Mettenburg DJ. In vitro cytotoxicity of resin-containing restorative materials after aging in artificial saliva. Clin Oral Investig 1999;3:144–9. [12] Bakopoulou A, Papadopoulos T, Garefis P. Molecular toxicology of substances released from resin-based dental restorative materials. Int J Mol Sci 2009;10:3861–99, http://dx.doi.org/10.3390/ijms10093861. [13] Blomgren J, Axéll T, Sandahl O, Jontell M. Adverse reactions in the oral mucosa associated with anterior composite restorations. J Oral Pathol Med 1996;25:311–3. [14] Hensten-Pettersen A. Skin and mucosal reactions associated with dental materials. Eur J Oral Sci 1998;106:707–12. [15] Krifka S, Spagnuolo G, Schmalz G, Schweikl H. A review of adaptive mechanisms in cell responses towards oxidative stress caused by dental resin monomers. Biomaterials 2013;34:4555–63, http://dx.doi.org/10.1016/j.biomaterials.2013.03.019. [16] Tadin A, Marovic D, Galic N, Kovacic I, Zeljezic D. Composite-induced toxicity in human gingival and pulp fibroblast cells. Acta Odontol Scand 2014;72:304–11, http://dx.doi.org/10.3109/00016357.2013.824607. [17] Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci USA 2000;97:13625–30.

1063

[18] Nanci A. Dentin-pulp complex. Ten Cate’s oral histology: development, structure and function. 7th ed. St. Louis, MO: Mosby Elsevier; 2008. p. 141–90. [19] Van Landuyt KL, Snauwaert J, De Munck J, Peumans M, Yoshida Y, Poitevin A, Coutinho E, Suzuki K, Lambrechts P, Van Meerbeek B. Systematic review of the chemical composition of contemporary dental adhesives. Biomaterials 2007;28:3757–85. [20] About I, Camps J, Burger AS, Mitsiadis TA, Butler WT, Franquin JC. Polymerized bonding agents and the differentiation in vitro of human pulp cells into odontoblast-like cells. Dent Mater 2005;21:156–63. [21] Costa CA, Hebling J, Hanks CT. Current status of pulp capping with dentin adhesive systems: a review. Dent Mater 2000;16:188–97. [22] Ginzkey C, Zinnitsch S, Steussloff G, Koehler C, Hackenberg S, Hagen R, Kleinsasser NH, Froelich K. Assessment of HEMA and TEGDMA induced DNA damage by multiple genotoxicological endpoints in human lymphocytes. Dent Mater 2015;31:865–76, http://dx.doi.org/10.1016/j.dental.2015.04.009. [23] Morito A, Kida Y, Suzuki K, Inoue K, Kuroda N, Gomi K, Arai T, Sato T. Effects of basic fibroblast growth factor on the development of the stem cell properties of human dental pulp cells. Arch Histol Cytol 2009;72:51–64. [24] Dominici M, Le Blanc K, Mueller I, Slaper-Cortenbach I, Marini F, Krause D, Deans R, Keating A, Prockop Dj, Horwitz E. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 2006;8:315–7. [25] Janderová L, McNeil M, Murrell AN, Mynatt RL, Smith SR. Human mesenchymal stem cells as an in vitro model for human adipogenesis. Obes Res 2003;11:65–74. [26] Kraus D, Deschner J, Jäger A, Wenghoefer M, Bayer S, Jepsen S, Allam JP, Novak N, Meyer R, Winter J. Human ␤-defensins differently affect proliferation, differentiation, and mineralization of osteoblast-like MG63 cells. J Cell Physiol 2012;227:994–1003, http://dx.doi.org/10.1002/jcp.22808. [27] Johnstone B, Hering TM, Caplan AI, Goldberg VM, Yoo JU. In vitro chondrogenesis of bone marrow-derived mesenchymal progenitor cells. Exp Cell Res 1998;238:265–72. [28] Lizier NF, Kerkis A, Gomes CM, Hebling J, Oliveira CF, Caplan AI, Kerkis I. Scaling-up of dental pulp stem cells isolated from multiple niches. PLoS ONE 2012;7:e39885, http://dx.doi.org/10.1371/journal.pone.0039885. [29] Gregory CA, Gunn WG, Peister A, Prockop DJ. An Alizarin red-based assay of mineralization by adherent cells in culture: comparison with cetylpyridinium chloride extraction. Anal Biochem 2004;329:77–84. [30] Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001;29:e45. [31] Bouillaguet S, Wataha JC, Hanks CT, Ciucchi B, Holz J. In vitro cytotoxicity and dentin permeability of HEMA. J Endod 1996;22:244–8. [32] Geurtsen and Leyhausen. Chemical-Biological Interactions of the resin monomer triethyleneglycol-dimethacrylate (TEGDMA). J Dent Res 2001;80:2046–50. [33] Schmalz G, Krifka S, Schweikl H. Toll-like receptors, LPS, and dental monomers. Adv Dent Res 2011;23:302–6, http://dx.doi.org/10.1177/0022034511405391. [34] Volk J, Engelmann J, Leyhausen G, Geurtsen W. Effects of three resin monomers on the cellular glutathione concentration of cultured human gingival fibroblasts. Dent Mater 2006;22:499–505. [35] Paris S, Hopfenmuller W, Meyer-Lueckel H. Resin infiltration of caries lesions: an efficacy randomized trial. J Dent Res 2010;89:823–6, http://dx.doi.org/10.1177/0022034510369289.

1064

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

[36] Paris S, Schwendicke F, Keltsch J, Dörfer C, Meyer-Lueckel H. Masking of white spot lesions by resin infiltration in vitro. J Dent 2013;5:e28–34, http://dx.doi.org/10.1016/j.jdent.2013.04.003. [37] Lee CP, Colombo JS, Ayre WN, Sloan AJ, Waddington RJ. Elucidating the cellular actions of demineralised dentine matrix extract on a clonal dental pulp stem cell population in orchestrating dental tissue repair. J Tissue Eng 2015;6, http://dx.doi.org/10.1177/2041731415586318, 2041731415586318. [38] Lei M, Li K, Li B, Gao LN, Chen FM, Jin Y. Mesenchymal stem cell characteristics of dental pulp and periodontal ligament stem cells after in vivo transplantation. Biomaterials 2014;35:6332–43, http://dx.doi.org/10.1016/j.biomaterials.2014.04.071. [39] Menicanin D, Bartold PM, Zannettino AC, Gronthos S. Identification of a common gene expression signature associated with immature clonal mesenchymal cell populations derived from bone marrow and dental tissues. Stem Cells Dev 2010;19:1501–10, http://dx.doi.org/10.1089/scd.2009.0492. [40] Galler KM, Schweikl H, Hiller KA, Cavender AC, Bolay C, D’Souza RN, Schmalz G. TEGDMA reduces mineralization in dental pulp cells. J Dent Res 2011;90:257–62, http://dx.doi.org/10.1177/0022034510384618. [41] Chang HH, Chang MC, Huang GF, Wang YL, Chan CP, Wang TM, Lin PS, Jeng JH. Effect of triethylene glycol dimethacrylate on the cytotoxicity, cyclooxygenase-2 expression and prostanoids production in human dental pulp cells. Int Endod J 2012;45:848–58, http://dx.doi.org/10.1111/j. 1365-2591.2012.02042.x. [42] Barton BE. IL-6: insights into novel biological activities. Clin Immunol Immunopathol 1997;85:16–20. [43] Yao X, Huang J, Zhong H, Shen N, Faggioni R, Fung M, Yao Y. Targeting interleukin-6 in inflammatory autoimmune

[44] [45]

[46]

[47]

[48]

[49]

[50]

[51]

diseases and cancers. Pharmacol Ther 2014;141:125–39, http://dx.doi.org/10.1016/j.pharmthera.2013.09.004. Baggiolini M, Clark-Lewis I. Interleukin-8, a chemotactic and inflammatory cytokine. FEBS Lett 1992;307:97–101. Barkhordar RA, Hayashi C, Hussain MZ. Detection of interleukin-6 in human dental pulp and periapical lesions. Endod Dent Traumatol 1999;15:26–7. Silva AC, Faria MR, Fontes A, Campos MS, Cavalcanti BN. Interleukin-1 beta and interleukin-8 in healthy and inflamed dental pulps. J Appl Oral Sci 2009;17:527–32. Schmalz G, Schweikl H, Hiller KA. Release of prostaglandin E2, IL-6 and IL-8 from human oral epithelial culture models after exposure to compounds of dental materials. Eur J Oral Sci 2000;108:442–8. Tokuda M, Nagaoka S, Torii M. Interleukin-10 inhibits expression of interleukin-6 and -8 mRNA in human dental pulp cell cultures via nuclear factor-kappaB deactivation. J Endod 2002;28:177–80. Krifka S, Petzel C, Hiller KA, Frank EM, Bosl C, Spagnuolo G, Reichl FX, Schmalz G, Schweikl H. Resin monomer-induced differential activation of MAP kinases and apoptosis in mouse macrophages and human pulp cells. Biomaterials 2010;31:2964–75, http://dx.doi.org/10.1016/j.biomaterials.2010.01.005. Eckhardt A, Harorli T, Limtanyakul J, Hiller KA, Bosl C, Bolay C, Reichl FX, Schmalz G, Schweikl H. Inhibition of cytokine and surface antigen expression in LPS-stimulated murine macrophages by triethylene glycol dimethacrylate. Biomaterials 2009;30:1665–74, http://dx.doi.org/10.1016/j.biomaterials.2008.09.024. Chen Y, Zhang Y, Ramachandran A, George A. DSPP is essential for normal development of the dental-craniofacial complex. J Dent Res 2015, pii: 0022034515610768.