Biological Effects of Provisional Resin Materials on Human Dental Pulp Stem Cells
Objectives: This study investigated the in vitro cytotoxicity as well as the proinflammatory cytokine expression of provisional resin materials on primary cultured human dental pulp stem cells (hDPSCs).
Methods: Five commercially available provisional resin materials were chosen (SNAP [SN], Luxatemp [LT], Jet [JE], Revotek LC [RL], and Vipi block [VB]). Eluates that were either polymerizing or already set were added to hDPSCs under serially diluted conditions divided into three different setting times (25% set, 50% set, and 100% set) and incubated for 24 hours with 2× concentrated culture media. Cell cytotoxicity tests were performed by LDH assay and live and dead confocal microscope images. The expression of proinflammatory cytokines in SN and VB was measured using cytokine antibody arrays. Data were analyzed using repeated measures analysis of variance (ANOVA) or ANOVA followed by the Tukey post hoc test at a significance level of p<0.05.
Results: Cytotoxicity greater than 30% was observed in the 50% diluted culture in SN, LT, and JE in the already set stage (p<0.05), while it was detected in SN and LT in early or intermediate stage samples. The cytotoxicity of SN, JE, and LT was greater with eluates from the polymerizing phase compared to that from already set samples (p<0.05), as observed by live and dead images. On the other hand, RL and VB did not exhibit cytotoxicity greater than 30%. Proinflammatory cytokines were not detected in 12.5% diluted culture with eluates from VB and early set stage SN.
Conclusions: The eluates from chemical-activated provisional resin materials during polymerization (SN, LT, and JE) were cytotoxic to hDPSCs and may adversely affect pulp tissue.SUMMARY
INTRODUCTION
Provisional resin materials are widely used for interim restorations in operative dentistry to maintain the marginal integrity of prepared teeth, protect the pulp tissue, ensure occlusal function, and provide an acceptable esthetic appearance until the adjustment of final restorations.1 They present easy handling, suitable mechanical properties, and low cost.2,3
Provisional resin materials are classified into five categories based on how the restoration is obtained during processing: 1) chemical-activated, 2) heat-activated, 3) light-activated, 4) dual-activated, and 5) computer-aided design and computer-aided manufacture (CAD/CAM) acrylic resins.4,5 There are several commercially available provisional resin materials with various types of monomers: 1) polymethyl methacrylate (PMMA), 2) polyethyl methacrylate (PEMA), 3) urethane dimethacrylate (UDMA), 4) other types or combinations of unfilled methacrylate resins, and 5) composite resins of bisphenol A–glycidyl methacrylate (Bis-GMA) or Bis-acryl.5-7
For initial biocompatibility testing of dental materials, cytotoxicity tests with various types of cells derived from the oral tissue have been investigated and adverse cellular effects are considered to be the result of released toxic components.8-13 Many researchers have used oral fibroblasts and keratinocytes to investigate the cytotoxicity of provisional resin materials on oral mucosa.5,14,15 However, there have been no reports concerning their cytotoxicity with regard to dental pulp cells even though material eluants may diffuse into pulp tissue via open dentinal tubules. When the exposed dentin of prepared teeth is exposed to provisional resin materials, toxic material components can reach the pulp tissue via open dentinal tubules and damage these tissues, which are more highly sensitive to such components than is oral mucosa.16-20 Even worse, provisional resin materials undergoing polymerization accelerate adverse effects to pulp tissue. When being polymerized, provisional resin materials are able to release more unreacted monomers or toxic eluates when placed on prepared teeth.21-23
In addition to cytotoxicity, dental pulp stem cells are adversely affected by chemicals to produce numerous proinflammatory cytokines/chemokines, including interleukin-6 (IL-6), IL-8, chemokine (C-C motif) ligand 2 (CCL2), and C-X-C motif ligand 12 (CXCL12), which increase the likelihood of irreversible pulp inflammation. Chemokines, such as CCL2 and CXCL12, are a family of small cytokines and act as chemoattractants to guide the migration of immune cells and induce an inflammatory reaction.24-26 Therefore, proinflammatory cytokines/chemokines from dental pulp stem cells are investigated after stimulation by eluates from provisional resin materials.5,14
In this experiment, we performed cytotoxicity tests to determine the effects on primary cultured human dental pulp stem cells (hDPSCs) observed during the early or intermediate stage of polymerization with the provisional resin materials compared to the already set stage counterpart. In addition, the proinflammatory cytokine expression of eluates from provisional resin materials was tested to reveal the possible adverse response. The null hypothesis was that the cytotoxic effects on hDPSCs of extracts from the polymerizing materials and those from the already set materials would not differ. The second null hypothesis was that proinflammatory cytokine expression from provisional resin materials did not show any difference compared to controls under less cytotoxic concentrations.
METHODS AND MATERIALS
Provisional Resin Materials
Five provisional resin materials were used in this study: chemical-activated polyethyl methacrylate resin (SN; Snap, Parkell Inc, Edgewood, NY, USA), chemical-activated bis-acryl composite resin (LT; Luxatemp, DMG, Englewood, NJ, USA), chemical-activated polymethyl methacrylate resin (JE; Jet, Lang Dental, Wheeling, IL, USA), light-activated urethane dimethacrylate resin (RL; Revotec LC, GC America, Alsip, IL, USA), and CAD/CAM fabricated polymethyl methacrylate resin blocks (VB; Vipi block, Madespa, Río Jarama, Toledo, Spain). Table 1 shows their general chemical compositions.
Preparation of Specimens
Each provisional resin material was placed within a Teflon mold 10 mm in diameter and 2 mm in height. When the mixed material reached the early “dough” stage, it was packed into a mold and covered with a cover glass. The polyethyl methacrylate resins (SN) and polymethyl methacrylate resins (JE) were measured, hand mixed, and autopolymerized. The powder was weighed using an electronic balance (Explorer, Ohaus Corporation, Parsippany, NJ, USA), and the liquid was measured by volume. For each material, the manufacturer-recommended powder-to-liquid ratio was used. For LT, the material was mixed using the dispensing syringe. The material was dispensed into the mold and allowed to autopolymerize. RL was hand molded and placed into the Teflon molds, and LED light (Litex 695, Dentamerica, City of Industry, CA, USA) was applied for polymerization. Each group was divided into three different setting times (25% set, 50% set, and 100% set) and was immediately placed in distilled water (DW). RL was placed in the Teflon mold and the specimen was either unpolymerized or polymerized by a light-curing unit (Litex 695) for 10 or 20 seconds. It was immediately placed in DW for extraction. The PMMA block (VB) was CAD/CAM processed according to the manufacturer's protocol and was sterilized using ultraviolet light for 1 hour after the application of ethylene oxide gas. The specimen was placed into DW. The experimental conditions are summarized in Table 2.
Collection of Resin Extract
Each provisional resin specimen was extracted at a ratio of 3 cm2/mL following the recommendations of ISO 10993-12.27 Because the surface of the sample was 2.2 cm2, they were incubated in 0.73 mL of medium. For each provisional resin material except VB, nine specimens were fabricated and divided into three groups for a different extraction starting point. Three specimens were used for extracting VB. To simulate the clinical environment, eluates of all specimens were collected for 24 hours at 37°C in a shaking incubator (120 rpm). All procedures were performed on a sterilized clean bench to prevent any possible contamination. Extract was freshly gathered independently for each set of cytotoxicity and microarray tests.
Culture of hDPSCs
The hDPSCs were used at a low passage (below 10) throughout the experiments. After the experimental protocol was approved by the Institutional Review Board, the hDPSCs were gathered from extracted third molars from human adults. The cells were cultured in α-MEM supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA), 1% penicillin/streptomycin (Invitrogen, Carlsbad, CA, USA), 2 mM of GlutaMAX (Gibco), and 0.1 mM of L-ascorbic acid (Sigma, St. Louis, MO, USA) in a humidified atmosphere at 37°C with 5% CO2. All culture systems adhered to the above conditions.
hDPSCs Potency Analysis by Flow Cytometry
The hDPSCs grown at confluence in a six-well culture plate were detached with Accutase (Invitrogen), washed twice with phosphate-buffered saline (PBS; Welgene, Daegu, Korea), and then resuspended to 1 × 105 cells/mL. The harvested cells were fixed with 4% paraformaldehyde for 10 minutes and washed with PBS. Then each sample was treated with 0.2% Triton X-100 (Sigma) for 5 minutes and blocked using 1% bovine serum albumin (Sigma), and the cells were probed with primary antibodies, such as goat anti-CD105 and anti-CD73 antibodies and rabbit anti-CD 106, CD-34, and CD-45 antibodies at 1:100 dilution overnight at 4°C. The cells were then incubated with fluorescein isothiocyanate (FITC)-labeled goat secondary anti-goat or anti-rabbit antibodies at 1:100 dilution for 2 hours at room temperature in the dark, after which they were washed with PBS three times and kept on ice until analysis. All primary antibodies were obtained from Santa Cruz Ltd (Dallas, TX, USA). All FITC-labeled secondary antibodies were obtained from Invitrogen. Data were acquired using a sterile clean capped fluorescence-activated cell sorting (FACS) tube (5 ml) (BD Bioscience, San Jose, CA, USA) with 10,000 cells using FACS (Calibur Flow Cytometer, BD Bioscience) after all channels were calibrated by BD FACS caliber beads (BD Bioscience). Data were analyzed using Cell Quest-Pro software (BD Biosciences).
Cytotoxicity Tests and Cell Viability
Cytotoxicity tests were performed according to ISO 10093-5.28 All specimens from each provisional resin group were cultured in a 96-well plate (SPL Life Sciences, Pocheon-si, Gyeonggi-do, Korea) at 1 × 104 cells/well with 100 μL of supplemented media for 24 hours at 37°C. After being washed with PBS, the cells were cocultured for another 24 hours with 2× supplemented media (50 μL), and serially diluted extract with DW (50 μL) was added. The percentages of the final concentrations of extracts in the culture media were 50%, 25%, 12.5%, 6.25%, and 3.125%. Samples in which only DW was added to hDPSCs served as the control group.
The potential cytotoxic effects on hDPSCs were evaluated by the quantity of extracellularly released cytosolic lactate dehydrogenase (LDH) using the CytoTox 96 Non-Radioactive Cytotoxicity Assay (Promega, Madison, WI, USA). After the cell cultures were exposed to the material extracts for 24 hours, the cell culture supernatants were collected. The cell supernatants were centrifuged at 250g for 4 minutes to remove cell debris, and 50 μL of supernatants were transferred into an optically clear 96-well plate, followed by the addition of 50 μL of reagent. After incubation for 30 minutes at room temperature, a stop solution of 50 μL was added, and absorbance at 490 nm was measured using a microplate reader (SpectraMax M2e, Molecular Devices, Sunnyvale, CA, USA). The cytotoxicity results are expressed as percentage of extracellularly released LDH activity, calculated against the total (intracellular + extracellular) LDH activity, which was obtained after cell lysis buffer was added to hDPSCs. This percentage corresponds to the relative quantity of dead cells among the total cells in culture.
To evaluate live and dead cells, confocal images were obtained on a Zeiss LSM700 laser scanning microscope (Carl Zeiss, Thornwood, NY, USA). Cell viability was assessed with calcein AM (0.5 μM) and an ethidium homodimer-1 (4 μM) (Molecular Probes, Eugene, OR, USA) solution for 30 minutes, and they were examined under a confocal laser microscope (LSM700, Carl Zeiss). Green fluorescence was observed from the live cells and red fluorescence from the dead cells.
Cytokine Analysis
The 12.5% eluates from SN1 (early stage 1) and VN (already set stage 1), which showed less than 20% cytotoxicity, were selected for cytokine analysis because the 25% or 50% eluates may have cytotoxicity-induced cytokines. DW and 4 μg/mL of lipopolysaccharide (from Escherichia coli; LPS, Sigma) in DW were added to the same quantity of 2× supplemented media for negative and positive controls, respectively, to synchronize the total DW volume in total media. The Bio-Rad Protein Assay kit (Bio-Rad Laboratories, Hercules, CA, USA) was used to determine the total protein concentrations for each of the conditioned media from the eluates. The same amount of total protein of the conditioned media was used for cytokine/growth factor analysis. A Proteome Profiler human cytokine array detection kit (Proteome Profiler, R&D Systems, Minneapolis, MN, USA) was used to detect cytokine/growth factor expression from the primary human dental pulp cells after exposure to the eluates from provisional resin specimens, as described by the manufacturer. Briefly, each membrane was blocked for 1 hour, incubated with the conditioned media at 4°C for 12 hours, washed, incubated with horseradish peroxidase–conjugated streptavidin for 30 minutes at room temperature, and finally washed before adding the detection agents for 1 minute according to the manufacturer's instructions. The signal from each membrane was examined with a chemiluminescence imager (ImageQuant LAS 4000, GE Healthcare, Little Chalfont, UK). The cytokine array test was repeated three times, and each cytokine was present twice on each membrane. The density of the protein spots was measured using a densitometry program (ImageQuant TL, Ver. 7.0., GE Healthcare). The positive control spots on the membranes were used to normalize the intensity between the different membranes to allow for comparison of the signal intensities.
Statistical Analysis
The cytotoxicity data from different extraction starting points were statistically analyzed by repeated measures analysis of variance (ANOVA). ANOVA was used for cytotoxicity comparison among serially diluted extract groups (50%, 25%, 12.5%, 6.25%, 3.125%, and 0%) within the same product and extract starting point. The Tukey post hoc test was used at levels of significance of p<0.05. The SPSS PASW version 23.0 software program (SPSS Inc) was used.
RESULTS
Stem Cell Plasticity of hDPSCs
Positive expression of CD73, CD105, and CD106 was detected in over 90% of hDPSCs, while they showed negative expression of CD34 and CD45 (not shown).
Cytotoxicity
The cytotoxicity test results for the LDH assay are shown in Figure 1. Extracellularly released LDH from hDPSCs of various provisional resins with three different states of polymerization was measured.
Citation: Operative Dentistry 42, 2; 10.2341/16-137-L
An early polymerizing state, intermediate polymerizing state, and final polymerized state were used to investigate the cytotoxicity to hDPSCs. Cell viability was determined by the leakage of LDH, an intracellular enzyme, from extract-conditioned groups compared to that from the total lysis group, which represented 0% cell viability. According to ISO 10993-5, 30% of cytotoxicity compared to the control was indicated as the cytotoxic threshold.28
Overall, after incubation with 50% extract, all extracts from SN, LT, JE, and RL showed significant differences in cytotoxicity compared to the DW-treated 0% control (Figure 1a-d, p<0.05). In contrast, cell cytotoxicity was not significantly observed in the VB (Figure 1e, p>0.05), which was considered a negative control. All extract conditions from SN, JE, and LT except JE3 showed greater than 30% cytotoxicity, while cytotoxicity was significantly higher in the early or intermediate state than in the already set state for SN, JE, and LT (Figure 1a-c, p<0.05). RL showed relatively low cytotoxicity (10%-20%, Figure 1d).
After incubation with 25% extract, all extract conditions from SN, LT, JE, and RL, except LT3, JE3, and RL3, showed significant differences in cytotoxicity compared to the DW-treated 0% control (Figure 1a-c, p<0.05). Cell cytotoxicity over 30% was shown only in SN1, and there was a significant difference between SN1 (eluates from the early set stage) and SN3 (eluates from the already set stage) (Figure 1a, p<0.05). After incubation with 12.5%, 6.25%, and 3.125% extract, only 12.5% SN1, LT1, JE1, and JE2 exhibited significant cytotoxicity compared to 0% (Figure 1a-c, p<0.05), while cytotoxicity over 30% was not observed in any of the groups.
Severe cytotoxicity from 50% cultured conditions was reevaluated by confocal microscopic images after live and dead staining. The results are shown in Figure 2, in which live cells appear green and dead cells appear red. A significant number of dead cells but few live cells appeared in the early set, intermediate set, and already set stages of SN, JE, LT, and RL, whereas similar numbers of viable cells appeared in VB compared to the control. The early set and intermediate set stage showed dead cells with few live cells compared to the already set state in SN, JE, LT, and RL (Figure 2).
Citation: Operative Dentistry 42, 2; 10.2341/16-137-L
Proinflammatory Cytokines
Cytokine expression in conditioned media from extract-treated hDPSCs was compared to DW-treated hDPSCs in Figure 3. There was no significant difference in cytokine/chemokine expression in 12.5% SN1 and VB extract-treated hDPSCs compared to their DW-treated counterparts (p>0.05, Figure 3e), which showed MIF and SerpinE1expression. Additionally, the cytokine/chemokine expression of IL-8, CCL2, and CXCL12/SDF-1 was significantly observed in the LPS-treated positive group (p<0.05, Figure 3d,e).
Citation: Operative Dentistry 42, 2; 10.2341/16-137-L
DISCUSSION
Based on these results, the first null hypothesis that there would be no differences in the levels of cytotoxicity between materials that were in the process of polymerizing and those that had already set was rejected. However, we accepted the second null hypothesis that there would be no differences in the levels of proinflammatory cytokines from provisional resin materials compared to controls under less cytotoxic conditions.
In vitro cytotoxicity testing has been widely used to evaluate the biocompatibility of dental materials because it facilitates the reproducibility of testing results and provides highly reliable data from standardized protocols in a fast, inexpensive way.28 In vitro cytotoxicity testing can be divided into two methods, direct and indirect, depending on the method of contact with mammalian cells. Direct contact exposes a dental material or an extract from a dental material directly to mammalian cells, whereas indirect contact involves a barrier, such as agar, an artificial membrane filter, or dentin.
In this experiment, direct in vitro cytotoxicity tests were performed using extracts from five provisional resin materials and hDPSCs to mimic the clinical circumstances in which extracts from provisional resin materials could adversely affect pulp tissue via open dentinal tubules. The indirect cytotoxicity test using natural dentin (from human or bovine teeth) or artificial dentin (ie, Millipore filter) is considered to successfully reflect the intervention of dentin when pulp tissue is subjected to extracts from provisional resin materials. In this study, direct in vitro cytotoxicity tests were chosen to avoid natural dentin– and artificial dentin–dependent bias, which cause a lack of accessibility or of similarity to natural dentin structure, respectively. Gathering intact human dentin from adult tooth is possible only when third molar or premolar teeth are intentionally extracted for therapeutic reasons. In addition, established artificial dentin does not exist even though many attempts have been made to mimic the natural dentin structure and its biological/mechanical function.29 Direct in vitro cytotoxicity testing using extracts also has many drawbacks, such as the inability to fully reproduce dentinal fluid and dentin structure. However, in this study, direct in vitro cytotoxicity tests using extracts provided an initial investigation for showing differences of cytotoxicity based on the extract starting time because of the advantage of the test's reproducibility. Further study using natural human dentin disks with artificial chambers is needed to confirm the extract starting time–dependent cytotoxicity for achieving clinical relevance.
In the development of cytotoxicity tests for dental materials, many attempts were made to mimic the clinical conditions of application. For example, the cytotoxicity of elastomeric impression materials was evaluated using human gingival fibroblasts with extract from the polymerizing and already set states, which revealed differences in cytotoxicity.30 Other dental materials, such as zinc oxide eugenol, root canal sealers, resin adhesives, and resin-reinforced glass ionomer cements, have also been evaluated during and after setting, and all results showed an increase in cytotoxicity in the setting state compared to the already set state.31-34 These cytotoxicity tests are considered to successfully mimic the clinical changes of polymerizing/setting dental materials which accelerate putative leaching or the entry of dissolved substances resulting in contact with oral tissues.
Provisional resin materials can affect pulp tissue via open dentinal tubules when they are applied onto exposed dentin during polymerization. After the provisional materials have set, the eluates from provisional resins and temporary cementation material continuously make contact with dentin and possibly affect pulp tissue. Therefore, to mimic the application of provisional resin on prepared teeth with exposed dentin and to evaluate the possible adverse effects on the pulp tissue during the fabrication of provisional resin prostheses, extracts from provisional resin materials during and after polymerization were cultured with hDPSCs, which make up dental pulp tissue and determine the pulp's regenerative potential.
In vitro cytotoxicity testing using single cells with extract has many drawbacks, such as less clinical relevance compared to indirect human dentin barrier tests and an inability to be used in the tissue complex.35 However, as an initial screening test, in vitro testing using affected tissue-specific cell lineage with extract from material is necessary to investigate the cytotoxicity of materials before conducting further clinically relevant in vitro, in vivo, or clinical studies, which sometimes take more time, cost more money, or raise ethical problems.28 In this study, three different extraction starting times (early, intermediate, and already set stages) after the start of mixing were used for assessing clinical relevance in the polymerizing process. Within the limitation of the in vitro cytotoxicity test, results showed that polymerizing provisional resin materials (especially chemical-activated products) showed the potential to adversely affect dental pulp cells. To confirm these findings and to achieve more clinical relevance, in vitro cytotoxicity testing using human dentin samples with simulated dentinal fluid flow, in vivo animal tests, and clinical testing is recommended for further investigation.36 From the above studies, the adverse effects from provisional materials on pulp tissue can finally be characterized in detail. Of course, it is also important to note that in vivo animal testing has the limitation of having unreliable accuracy in assessing pulpal compatibility for humans due to anatomical and biological differences.37
The hDPSCs are the main source of cells for maintaining the homeostasis of pulp tissue and accelerating the regeneration of pulp tissue.38 Their natural function in the production of odontoblasts to create reparative dentin supports the regeneration of dentin structures by the production of the dentin/pulp-like complex as a protective barrier to the pulp tissue.39 In addition, hDPSCs play a role in the immune response of pulp tissue against pulp damage by cytokine/chemokine production.40,41 After confirming the stem cell plasticity of hDPSCs with positivity for mesenchymal stem cell markers (CD73, CD105, and CD106), which represent the potency to differentiate into osteogenic, chondrogenic, and adipogenic lineage,42 and negativity for the hematological markers CD34 (early hematopoietic stem cell marker) and CD45 (hematolymphoid cell marker),42-44 in vitro cytotoxicity studies were performed.
LDH is a natural cytosolic enzyme present in mammalian cells. When intact plasma membrane is damaged by the external environment, intracellular LDH is released into the cell culture media, which is recalled as extracellular LDH, an indicator of cytotoxicity. The extracellular LDH in the media is quantified by a series of enzymatic reactions in which LDH catalyzes the conversion of lactate to pyruvate via the reduction of NAD+ to NADH. Diaphorase uses NADH to reduce a tetrazolium salt to a red formazan product, which can be quantitatively measured at 490 nm. Under defined conditions, such an enzyme reaction to detect extracellular LDH reflects the levels of cytotoxicity.45 Therefore, the LDH assay is a widely performed cytotoxicity test to evaluate dental materials.46-48 We also used this assay to investigate the cytotoxicity of provisional resin materials.
Among the five products evaluated, the chemical-activated products (SN, LT, and JE) were more cytotoxic to hDPSCs than were the light-activated (RL) and CAD/CAM-fabricated (VB) products. Chemical-activated PEMA (SN1: ∼87%, SN2: ∼53%, and SN3: ∼52%) and Bis-acryl (LT1: ∼78%, LT2: ∼65%, and LT3: ∼45%) showed higher levels of cytotoxicity with 50% extract concentration than did the chemical-activated PMMA (JE1: ∼49%, JE2: ∼36%, and JE3: ∼2%) when considering cytotoxicity numeric values in the same extract condition (p<0.05). Based on the composition of the five tested products, EMA (from SN), Bis-acryl (from LT), MMA (from JE and VB), or UDMA (from RL) is considered a major extractable monomer that induces cytotoxicity in each product. The monomers or eluates released from provisional resin materials were toxic to various types of oral cells, including hDPSCs.49-51 It was reported that the major substances to induce cytotoxic effects from provisional resin materials consisting of acrylic resin were resin monomers.50,52 Along with previous cytotoxicity tests, which used various monomers contained in dental resin materials, EMA and Bis-acryl, which were possibly released from SN and LT, respectively, were more cytotoxic than MMA, which was possibly released from JE.53,54 This may explain why SN and LT were more cytotoxic to hDPSCs than JE in this study.
Chemical-activated products are older provisional resin materials, which lack biocompatibility and required more than 10 minutes for polymerization.1,55 Recently, to overcome those characteristics, light-cure resins containing UDMA and CAD/CAM PMMA resins were introduced as provisional resin materials, and they showed enhanced biocompatibility in accordance with this study.7,56
Cytokines/chemokines are important mediators of cellular activities and significantly contribute to inflammatory responses.57 Cytokines such as IL-6 and IL-8 are well known to induce proinflammatory functions.58,59 Chemokines such as CCL2 and CXCL12 recruit immune reaction-related cells, such as monocytes, memory T cells, or dendritic cells, to the sites of inflammation produced by either pulp tissue injury or infection.60,61
In this study, of five types of products, the most cytotoxic provisional resin (SN) at a 50% dilution was chosen to analyze cytokine expression along with the noncytotoxic CAD-CAM acrylic resin block (VB) as a negative control. VB is not thought to increase cytokine/chemokine expression because it is provided in the fully polymerized state from the manufacturer. As an experimental group, SN1, which starts to extract DW from the early set stage, was considered more clinically relevant than SN2 and SN3 in mimicking the impact of provisional resin materials during polymerization. There are concerns about cell death–induced increases in proinflammatory cytokines along with the related mRNA gene expression.62,63 Therefore, the 12.5% diluted extract from SN (SN1), which first showed less than 30% cell cytotoxicity among serially diluted conditions (50%, 25%, 12.5%, and 6.25%), was selected to analyze cytokine/chemokines to minimize the cell death–induced expression of biomolecules.
The relative cytokine/chemokine expression levels of conditioned media from 12.5% of SN1 and VB eluate-treated hDPSCs were not significantly greater than the control, which suggested that the less cytotoxic concentration (12.5%) of SN1 and VB eluates did not induce a severe inflammatory reaction in hDPSCs. Only MIF and SerpinE1 acted as positive regulators of the inflammatory state, along with the DW-treated negative control. The LPS-treated positive control showed significant expression of the IL-8, CCL2, and CXCL12 proteins.
In summary, within the limitations of this study, extracts from polymerizing (early stage or intermediate stage) provisional resin materials, especially chemical-activated ones (SN, LT, and JE), had more cytotoxicity than the already set counterpart, meaning that exposing a prepared tooth to provisional resin materials in the polymerizing stage could be dangerous to pulp tissue. Otherwise, light-activated ones and CAD/CAM-fabricated resin blocks did not show severe cytotoxicity against hDPSCs (less than 30%) regardless of polymerizing stage, meaning that biocompatibility to pulp tissue is relatively higher in light-activated ones and CAD/CAM-fabricated resin blocks compared to the chemical-activated counterparts. Other adverse effects in terms of inflammation-related cytokine/chemokine expression were not detected under less cytotoxic concentrations of extract in culture media, meaning that a severe inflammatory reaction from hDPSCs is limited after excluding cytotoxicity-induced inflammation. However, dentin-pulp complex characterizations in terms of three-dimensional cellular structure with dentinal tubules, cell diversity in pulp tissue, and three-dimensional extracellular matrix in the dentin-pulp complex were not considered in this study which was a two-dimensional in vitro investigation using direct extract treatment. Further investigation using an artificial pulp chamber incubating system with co-culture of various pulp cells or in vivo studies is necessary to confirm the above findings.
CONCLUSIONS
It was concluded that extracts obtained from provisional resin materials during polymerization, especially chemical-activated ones (SN, LT, and JE), showed cytotoxic effects on hDPSCs, while they do not induce the expression of proinflammatory cytokines at less cytotoxic concentrations. Therefore, the possible pulp damage from released toxic components should be considered, particularly when chemical-activated provisional resin materials are applied to extensively prepared teeth.
Cell cytotoxicity results of five provisional resins according to the extract conditions (early, intermediate, and already set stage) and the concentration of extract in culture media with hDPSCs (0%, 3.125%, 6.25%, 12.5%, 25%, or 50%). Different letters indicate significant differences among the same extract concentration (p<0.05). # indicates a significant difference in cytotoxicity compared to 0% (p<0.05). Representative means ± standard deviations (n=6) are shown after experiments independently run in triplicate.
Live (green) and dead (red) cells in 50% extract. Live and dead cells in media supplemented with 50% distilled water (DW) are shown as a control. Representative images are shown after experiments independently run in triplicate.
Proinflammatory cytokine/chemokine expression in conditioned media from eluate-treated hDPSCs. After hDPSCs were treated with distilled water (DW) (a), 12.5% of SN1 (b), 12.5% of VN extract (c), or 4 μg/mL of LPS (d) for 24 hours, cytokine array experiments were performed. After normalizing the cytokine intensity from experimental groups to that from the DW-treated control group, the relative cytokine expressions are shown as means ± standard deviations (e). Means ± standard deviations are shown of experiments independently run in triplicate. Asterisks indicate significant differences compared to controls (p<0.05).
Contributor Notes
Jung-Hwan Lee, DDS PhD, Institute of Tissue Regeneration Engineering, Cheoanan, Dankook University, Republic of Korea