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

Abstract

Objectives

Resin infiltrants have been successfully used in dental medicine preventing the progression of tooth decay in an early phase of caries development. ICON ® is an infiltrant of low-viscosity which penetrates via dentinal tubules into the lesion in dependence of the demineralization depth. Hence, we performed an in vitro study to determine the effect of 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 (Stro-1, CD73, CD90, CD105) and hDPSCs differentiation was validated by immunofluorescence and flow cytometry. HDPSCs were stimulated with light-cured ICON ® (lc) and non-light-cured ICON ® (nc) conditioned media as well as different TEGDMA concentrations followed by the analysis of cytotoxicity, pro- and anti-inflammatory responses and differentiation using XTT assay, RT-PCR and ELISAs, respectively.

Results

Initial analysis demonstrated that hDPSCs express characteristic mesenchymal stem cell markers and differentiate into adipocytes, chondrocytes and osteoblasts. Notably, ICON ® 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.

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 .

Resin infiltration technique has been successfully used in dental medicine preventing the progression of tooth decay in an early phase of caries development . 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 . Recent investigations have shown that ICON ® reduced demineralized enamel roughness, increased microhardness and inhibited bacterial adhesion . Thereby, studies primarily focused on penetration capacity , 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 and may penetrate into pulpal tissue . TEGDMA and other monomers or resin components have also been detected in saliva and dentin structures supporting studies determining the release of resin materials . Notably, several in vitro investigations have shown that monomers induce toxic effects in a variety of cells . Moreover, in vivo studies imply that resin components may cause allergic reactions and mucosal changes like lichenoid reactions, which may transform to malignant tumors . 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 . 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 .

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) . 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 . 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 . TEGDMA is a component of most resins and adhesives (including ICON ® ) with varying content (25–50%) . Several studies indicate that this component may induce inflammatory reactions, disturb cellular homeostasis or modulate cell differentiation .

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.

Materials and methods

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 . Cells/Explants were kept in an incubator at 37 °C in humidified atmosphere of 5% CO 2 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.

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 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.

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.

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, 10 6 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.

Cell differentiation

Different cell differentiation models were applied to verify the stem cell character of our dental pulp cells . 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 , osteogenic or chondrogenic differentiation . 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 μM dexamethasone, 0.2 mM indomethacin, 1.7 μM 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 maintenance medium (DMEM, 4.5 g/l glucose, 1.7 μM 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 β-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 × 10 6 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-β3; R&D Systems). Cultures were incubated for 4 weeks at 37 °C in a humid atmosphere containing 5% CO 2 . 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.

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).

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 . 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.

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.

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.

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).

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

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 ® was light-cured as recommended by the manufacturer (ICON ® light-cured (ICON lc)) In brief, 50 μl 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/cm 2 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 μl 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.

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

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 conditioned ICON lc and ICON nc media as well as different TEGDMA concentrations (100 μM, 200 μM, 300 μM, 500 μM, 1000 μM, 2000 μM and 5000 μM). 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 (LC 50 ) 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.

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 μM, 1000 μM and 2000 μM). 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.

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 protocol and quantified using the NanoDrop ND-1000 Spectrophotometer (NanoDrop, Technologies, Wilmington, DE, USA). First-strand cDNA synthesis was performed with 1 μg RNA and the iScript™ 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 real-time 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.

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

For qPCR-analysis, 50 ng cDNA was added to a mastermix containing primers and iQ™ 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 β-actin mRNA expression. Relative differential gene expression was calculated using the method described by Pfaffl .

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.

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

Materials and methods

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 . Cells/Explants were kept in an incubator at 37 °C in humidified atmosphere of 5% CO 2 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.

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 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.

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.

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, 10 6 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.

Cell differentiation

Different cell differentiation models were applied to verify the stem cell character of our dental pulp cells . 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 , osteogenic or chondrogenic differentiation . 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 μM dexamethasone, 0.2 mM indomethacin, 1.7 μM 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 maintenance medium (DMEM, 4.5 g/l glucose, 1.7 μM 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 β-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 × 10 6 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-β3; R&D Systems). Cultures were incubated for 4 weeks at 37 °C in a humid atmosphere containing 5% CO 2 . 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.

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).

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 . 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.

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.

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.

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).

Nov 23, 2017 | Posted by in Dental Materials | Comments Off on In vitro biocompatibility of ICON ®and TEGDMA on human dental pulp stem cells
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