The aim of this study was to investigate the effects of resinous monomers on the odontogenic differentiation and mineralization potential of apical papilla stem cells (SCAP).
Cultures were established from developing third molars of healthy donors aged 14–18 years-old and were extensively characterized for proliferation rate, colony forming unit efficiency and expression of stem cell markers (STRO-1, CD146, CD34, CD45, CD105, CD117-c-Kit, CD24, CD90, Nanog, Oct3/4), in order to select those with enhanced stem cell and odontogenic differentiation properties. SCAP enriched cultures were then induced for odontogenic differentiation in the continuous presence of low concentrations (0.05–0.5 mM) of the monomers 2-hydroxy-ethyl-methacrylate-HEMA and triethylene-glycol-dimethacrylate-TEGDMA for 3 weeks (long-term exposure). Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were evaluated.
The results showed that both types of monomer-exposure significantly delayed the odontogenic differentiation and mineralization processes of SCAP cells. A down-regulation followed by recovery in the expression of differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP was recorded. This was accompanied by reduction of the mineralized matrix produced by monomer-treated-compared to non-treated contol cultures. Furthermore, a concentration-dependence was observed for both monomers during long-term exposure, whereas the effects of HEMA were evident at much lower concentrations compared to TEGDMA.
These findings suggest that resinous monomers can delay the odontogenic differentiation of SCAP cells, potentially disturbing the physiological repair and/or developmental processes of human permanent teeth.
Residual substances released from resin-based dental restorative materials have been well-documented with respect to cytotoxicity and genotoxicity in various cell culture systems . These effects have been attributed to the release of residual monomers or other substances derived from incomplete polymerization or resin degradation over time. Some of these compounds, such as the monomers 2-hydroxy-ethyl-methacrylate (HEMA) and triethylene-glycol-dimethacrylate (TEGDMA) have been also found able to diffuse through the dentinal tubules and reach the pulp tissue at significantly high concentrations in the low millimolar range, even in the presence of an intact dentin barrier . These concentrations are able to cause significant cytotoxicity in oral fibroblasts through mechanisms associated with oxidative stress, expressed via production of Reactive Oxygen Species (ROS), depletion of intracellular glutathione (GSH), cell cycle delays and finally induction of cell death, mainly via apoptosis .
However, there is until now only fragmentary information available on the biological effects of resinous substances released at low concentrations for extended periods of time during the long-term clinical service of these materials on primary recipient tissues, such as the dental pulp. Two previous in vitro studies have shown that long-term exposure to non-toxic concentrations of resinous monomers is able to cause significant disturbance of the differentiation processes of dental pulp cells . In addition, in vivo studies on pulp responses have shown that direct capping of exposed pulps with resin adhesives causes a persistent chronic inflammatory reaction associated with lack of mineralized barrier formation , whereas application of resinous materials into deep cavities can be also associated with slight to moderate inflammation depending on the materials used and the remaining dentin thickness . These clinical data suggest that resinous materials may be a continuous and critical source of xenobiotics leaching into the pulp cavity. However, only scarce data exist on the possible underlying mechanisms leading to a compromised pulp repair response, due to continuous exposure of pulp cells to resinous substances.
Most recently, we have shown that two frequently and in high amounts eluted resin comonomers (HEMA and TEGDMA) at very low (non-toxic) concentrations were able to significantly disturb or completely inhibit the physiologic migration, odontogenic differentiation and mineralization processes of human pulp stem/progenitor cells derived from deciduous teeth . Taking a step forward, we further evaluated in this study the long-term effects of non-toxic concentrations of these monomers on a highly proliferative and clonogenic stem cell population derived from the apical papilla of human permanent developing teeth. These cells were extensively characterized with respect to stem cell properties and presented significant expression of mesenchymal and embryonic stem cell markers, high colony forming efficiency and most importantly pronounced odontogenic differentiation and mineralization potential. The rationale of this study was to give more insight into the effects of very low concentrations of resinous monomers on dental pulp stem cell populations possessing a very high regenerative potential and that could play a significant role on the repair/regeneration of the dentin/pulp complex as a response to external stimuli . Our research hypothesis was that non-toxic concentrations of resinous monomers commonly diffusing into the pulp space are able to disturb the physiological differentiation processes of highly potent stem/progenitor cells, therefore jeopardizing the physiological repair and/or developmental processes of human permanent teeth.
Materials and methods
Chemicals and reagents
The monomers TEGDMA and HEMA were gifts from VOCO (Cuxhaven, Germany). The culture medium (α-Modification of Eagle’s Medium-α-MEM with nucleosides) and the enzyme collagenase type I were purchased from Gibco/Invitrogen (Karlsruhe, Germany), whereas the enzyme dispase from Roche Diagnostics GmbH (Mannheim, Germany). Trypsin/EDTA and penicillin/streptomycin/amphotericin B were purchased from Biochrom AG (Berlin, Germany) and Fetal Bovine Serum (FBS) from Lonza (Verviers, Belgium). The chemicals MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], dexamethasone disodium phosphate, monopotassium phosphate, β-glycerophosphate, l -ascorbic acid, Alizarin Red S, neutral buffered formalin, cetylpyridinium chloride, Naphtol-AS-MX Phosphate, N,N Dimethylformamide, Fast Blue BB Salt and Tris-(hydroxymethyl)-aminomethane were purchased from Sigma–Aldrich (Taufkirchen, Germany). The mouse anti-human antibodies phycoerythrin (PE)-conjugated CD146, allophycocyanin (APC)-conjugated CD34, PE-conjugated CD45, Peridinin-Chlorophyll-Protein-cyanin 5.5 dye (PerCP-Cy5.5)-conjugated CD117, Fluorescein isothiocyanate (FITC)-conjugated CD105, PE-conjugated Nanog and Alexa Fluor 647-conjugated Oct3/4 were purchased from BD Biosciences (Heidelberg, Germany). The fixation, permeabilization and staining buffers for flow cytometry were also purchased from BD Biosciences. The mouse anti-human antibody FITC-conjugated STRO-1 was obtained from Santa Cruz Biotechnology, Inc. (California, U.S.A.) and the mouse antihuman APC-conjugated CD24 and FITC-conjugated CD90 from BioLegend (San Diego, U.S.A.). The NucleoSpin RNA II isolation kit was purchased from Macherey-Nagel (Düren, Germany) and the Robus T I RT-PCR kit (F-580L) from Finnzymes (Espoo, Finland). The primers used for the RT-PCR analysis were synthesized by Biozym Scientific GmbH (Hess. Oldendorf, Germany). The Bicinchoninic Acid (BCA) Protein assay was obtained from Thermo Fisher Scientific (Schwerte, Germany).
The human SCAP cultures used in this study were established from the apical papilla of normal impacted third molars at the stage of root development (two thirds of the root completed). The teeth were extracted from young healthy donors aged 14–18 years for orthodontic reasons or due to lack of adequate space for eruption. The collection of the samples was performed according to the guidelines of the Institutional Review Board and all donors or their parents signed an informed consent form. Cell cultures were established using the enzymatic dissociation method, as described in previous publications by our group . Briefly, teeth were washed, disinfected and the apical papilla was retrieved through the apical part of the incomplete roots. The tissue was minced into small segments and digested in a solution of 3 mg/ml collagenase type I and 4 mg/ml dispase for 1 h at 37 °C. Single-cell suspensions were obtained by passing the cells through a 70 μm cell strainer. Cells were seeded at a density of 10 4 /cm 2 using α-MEM culture medium, supplemented with 15% FBS, 100 μM l -ascorbic acid phosphate, 2 mM l -glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin and 0,25 mg/ml Amphotericin B (=complete culture medium – CCM) and incubated at 37 °C in 5% CO 2 . After reaching 80–90% confluency, cells were collected by treatment with 0.25% trypsin/0.25 mM EDTA and then continuously passed for further experiments. Cultured SCAP cells in passage numbers from 2 to 6 were used for all the experiments with similar results.
Characterization of SCAP cultures
Before any experiment, SCAP cultures used in this study were extensively characterized with respect to growth characteristics, colony forming unit fibroblasts (CFU-F) efficiency and expression of mesenchymal, as well as embryonic stem cell markers using flow cytometry. A total number of 10 SCAP cultures ( n = 10) derived from different donors were analyzed with respect to the above mentioned characteristics in order to select the cultures with the most enhanced stem cell properties, i.e. highly proliferative and clonogenic potential and increased expression of stem cell markers to be used for further experiments.
Cell growth analysis
For cell growth analysis, cells were seeded at 5 × 10 4 cells/well in 6-well plates. The cell number was assessed every 24 h with a hemocytometer, after harvesting the cells of the corresponding wells (6 replicates for each time point of 24, 48, 72, 96, 120 and 144 h respectively) with trypsinization and the corresponding growth curves were calculated.
Colony forming unit fibroblasts (CFU-F) efficiency assay
The assay was carried out by plating early passage (p.2) SCAP cells in 6-well plates at densities ranging from 100 to 200 cells/well in three replicates ( n = 3). After plating, the dishes were placed in an incubator (37 °C, 5% CO 2 ) and left there for 10 days before being fixed with 10% neutral buffered formalin (NBF) for 1 h at room temperature (RT). Crystal violet-stained colonies were macroscopically counted. A CFU-F was defined as a group of at least 50 cells. For each sample, colony forming unit efficiency (%) was calculated as follows: (mean number of colonies/total number of seeded cells) × 100. The CFU-F assay was repeated in three independent experiments. When more than 200 cells/well were initially seeded then too many individual colonies were overlapping and no counting could be performed.
Flow cytometric analysis of stem cell markers
For flow cytometric analysis cells were first harvested by trypsinization and washed two times with ice-cold PBS. For surface epitope analysis, 10 6 cells/tube were first Fc-blocked with 1 μg of human IgG for 10 min at RT and subsequently stained by incubation with the fluorochromes conjugated mouse anti-human antibodies STRO-1-FITC, CD146-PE, CD34-APC, CD45-PE, CD117-PerCP-Cy5.5, CD105-FITC, CD24-APC and CD90-FITC in various combinations for 20 min in the dark at RT. In addition, for intracellular staining for the embryonic markers Nanog and Oct3/4, cells were first Fc-blocked and subsequently fixed with a 4% paraformaldehyde buffer, permeabilized with a saponin 0.1% (w/v) buffer and then stained with each of the fluorochrome conjugated mouse anti-human antibodies Oct3/4-Alexa Fluor 647 and Nanog-PE, as described above. After staining, cells were washed twice with 1 ml flow cytometry staining solution (dPBS + 1%BSA + 0.1%NaN 3 ) and centrifuged for 5 min at 230 × g . Supernatant was removed, cells were re-suspended in 200 μl staining solution and analyzed with a BD LSR II Flow Cytometer (BD Biosciences). A total of 100,000 events were acquired for each sample. Data were analyzed using Summit 5.1 software (Beckman Coulter, Inc., U.S.A.). After characterization of SCAP cultures derived from 10 different cell donors, SCAP cells with enhanced stem cell properties (proliferation rate, CFU-F efficiency and high expression of stem cell markers) were selected for the subsequent cytotoxicity and mineralization assays in the presence of the resinous monomers.
Cytotoxicity of HEMA and TEGDMA on highly proliferative and clonogenic human SCAP cells
TEGDMA and HEMA were dissolved in absolute ethanol and sequentially diluted to obtain different concentrations of stock solutions. The monomers were freshly diluted in culture medium prior to each experiment. The final concentration of ethanol did not exceed 0.25% (v/v). Cells incubated with medium containing 0.25% ethanol served as control. For the assessment of cytotoxicity, SCAP cells were seeded in 96-well plates (5000 cells/well) and allowed to grow for 24 h. Subsequently, SCAP were treated with HEMA (0.1–8 mM) and TEGDMA (0.05–5 mM), for 24, 48 or 72 h. Cell viability was assessed using the MTT assay to determine the mitochondrial dehydrogenase activity. Briefly, at the end of each incubation period the culture medium was discarded and 100 μl of 5 mg/ml MTT in PBS was added to each well. The cells were incubated in the dark for 3 h at 37 °C and 5% CO 2 . Then, the MTT solution was discarded and the insoluble formazan was dissolved with DMSO for 30 min at RT. The absorbance was measured against blanks (DMSO) at a wavelength of 570 nm by a microplate reader (Spectra Max 250, MWG Biotech).
Induction of odontogenic differentiation of SCAP cells in the presence of HEMA and TEGDMA
Before proceeding to the odontogenic differentiation experiments in the presence of the monomers, well-characterized (as described above) SCAP cultures derived from at least 3 different donors were also additionally screened with respect to their odontogenic differentiation and mineralization potential using the Alizarin Red S (AR-S) Method (see below), in order to select the most potent cells with respect to these properties for further analysis of monomer long-term cytotoxicity.
For the odontogenic differentiation experiments, SCAP cells were exposed to concentrations of 0.05, 0.1 and 0.5 mM of HEMA and TEGDMA, which were found in the MTT analysis to have minimal or no cytotoxicity to the cells (cell viability ≥85% for both monomers after 72-h exposure). Both control and monomer treated cultures were induced for odontogenic differentiation by being exposed to a-MEM complete culture medium (CCM), supplemented additionally with 0.01 μM dexamethasone disodium phosphate (Dexa), 1.8 mM monopotassium phosphate (KH 2 PO 4 ) and 5 mM β-glycerophosphate (β-GP) (=differentiation medium). Cells were treated for a total period of 3 weeks with the differentiation medium containing the different concentrations of the monomers being changed every 3–4 days (long-term exposure). Cultures exposed to normal CCM without the additional supplements for the same 3-week period served as negative control (uninduced-control cultures), whereas cultures exposed for 3 weeks to differentiation medium without the presence of the monomers served as positive control (induced-control cultures).
In a second series of experiments, SCAP cultures were exposed only once to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM), which were found in the MTT assay to reduce cell viability by almost 35% after a 72 h-exposure. Then, the medium with the monomers was washed out with PBS and replaced by differentiation medium without monomers that was changed every 3–4 days for the same period of three weeks (short-term exposure). This second series of experiments was performed in order to assess whether a single exposure to these monomers would be able to irreversibly affect their normal differentiation processes. At the end of each week, control and monomer-treated cultures of both long-term and short-term experiments were evaluated for mineralization by AR-S staining.
Alizarin red S mineralization assay
For the assessment of in vitro mineralization, cell cultures were washed twice with PBS (−) (without Ca 2+ and Mg 2+ ) and fixed with 10% NBF for 1 h at RT. Then, cultures were stained with 1% AR-S (pH 4.2) for 20 min at RT, followed by rinsing three times with de-ionized water. Mineralized nodules were photographed using an inverted microscope (Olympus Optical Co., Ltd., Japan) equipped with a digital camera (Olympus E-410, Olympus Optical Co., Ltd., Japan). Quantification of the total mineralized tissue produced per well was performed by extracting the AR-S from the stained sites by adding 2 ml of cetylpyridinium chloride (CPC) buffer (10%, w/v) in 10 mM Na 2 HPO 4 (pH = 7) for 12 h at 37 °C. Subsequently, 200 μl aliquots were transferred to a 96-well plate and the OD 550 nm was measured using a microplate reader (Spectra Max 250, MWG Biotech). Mineralized nodule formation was represented as nmol AR-S per μg of total cellular protein, determined by Bicinchoninic Acid (BCA) Protein assay.
Histochemical detection of alkaline phosphatase (ALP) activity
One week after induction of odontogenic differentiation, cells in 6-well-plates were washed twice with PBS (−) and fixed with 10% NBF, as described in the AR-S protocol. ALP activity was visualized by incubating the cells for 2 h at 37 °C with 0.1 mg/ml Naphtol-AS-MX Phosphate in N,N Dimethylformamide and 0.6 mg/ml Fast Blue BB Salt in 0.2 M Tris-(hydroxymethyl)-aminomethane buffer (pH 8.9). The cells were rinsed with dH 2 O and evaluated for ALP activity under an inverted microscope (Olympus Optical Co., Ltd., Japan).
Semi-quantitative reverse transcription/polymerase chain reaction (RT)-PCR analysis
Total RNA was extracted from cells with NucleoSpin RNA II kit at days 7 and 14 after induction of differentiation. For the RT-PCR reactions 0.5 μg of total RNA was diluted in a 25 μl PCR reaction of 1× PCR reaction buffer containing 1.5 mM MgCl 2 /200 mM each of dNTP/0.04 units/μl of DyNAzyme EXT DNA Polymerase)/0.1 units/μl of AMV Reverse Transcriptase (RT) and 10 pmol of each human-specific primer sets: bone sialoprotein (BSP) (sense: 5′-ATGGAGAGGACGCCACGCCT-3′, antisense: 5′-GGTGCCCTTGCCCTGCCTTC-3′), osteocalcin (OCN) (sense: 5′-GACTGTGACGAGTTGGCTGA-3′, antisense: 5′-AAGAGGAAAGAAGGGTGCCT-3′), dentin sialophosphoprotein (DSPP) (sense: 5′-GGG ACACAGGAAAAGCAGAA-3′, antisense: 5′-TGCTCCATTCCCACTAGGAC-3′ and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (sense: 5′-GAAGGTGAAGGTCGGAGT-3′, antisense: 5′-GAAGATGGTGATGGGATTTC-3′). The reactions were performed in a PCR thermal cycler (Bio-Rad iCycler, Munich, Germany) at 50 °C for 30 min for cDNA synthesis, 94 °C for 2 min for one cycle and then 94 °C/(45 s), 56 °C/(60 s), 72 °C/(60 s) for 30 cycles, with a final 10-min extension at 72 °C. RT-PCR products were analyzed by 1.5% (w/v) agarose gel electrophoresis and visualized by ethidium bromide staining.
Each experiment was performed with 3–6 replicates and repeated at least three times. Values were expressed as means ± SD. Statistical analysis of the data was performed using one-way analysis of variance (ANOVA). Follow-up comparisons between groups were then carried out using the Tukey multiple comparison test ( p < 0.05).