|Year : 2021 | Volume
| Issue : 1 | Page : 13-18
Cytotoxicity of Silorane and Methacrylate based Dental Composites on Human Pulp Cells
Prashanthi S Madhyastha1, Kumar M.R Bhat2, Divya Padma3, Madhu Keshava Bangera1, Dilip G Naik4, N. Srikant5, Ravindra Kotian1
1 Department of Dental Materials, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India
2 Department of Anatomy, Ras Al Khaimah College of Medical Sciences, RAK Medical & Health Sciences University, Ras Al Khaimah, UAE
3 Manipal GoK Bio Incubator, School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India
4 Department of Periodontics, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India
5 Department of Oral Pathology and Microbiology, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal, Karnataka, India
|Date of Submission||22-Dec-2020|
|Date of Acceptance||03-Jun-2021|
|Date of Web Publication||06-Aug-2021|
Madhu Keshava Bangera
Department of Dental Materials, Manipal College of Dental Sciences, Mangalore, Manipal Academy of Higher Education, Manipal 575001, Karnataka
Source of Support: None, Conflict of Interest: None
Introduction: The ingredients from the dental restoratives are known to leach and elicit a host response. The prerequisite to deem a material biocompatible requires its toxicologic evaluation. The study was performed to analyze the probable toxicity resulting from silorane-based composite (SBC) with methacrylate-based composite (MBC). Materials and Methods: The in vitro cytotoxicity test, methyl thiazolyl tetrazolium (MTT) assay, evaluated the cell viability and proliferation rate of dental pulp cells (DPCs). The extirpated pulp was cultured in α-MEM-containing supplements and incubated at 37°C. DPCs were subjected to varying doses of SBC or MBC at different time intervals after attaining confluence and monitored for proliferation and viability via MTT assay. An independent Student t test was performed to compare the effect of composites on the DPC. The cytotoxicity levels were compared using one-way analysis of variance and posthoc Tukey multiple comparison test at 5% level of significance and P-value of <0.05. Results: DPC exposed to MBC showed higher viability than SBC. The MTT assay reported the number of viable cells as (>90%) in the first 24 hours. The count significantly reduced by the end of 48 hours (minimum 65% in 25 μg/ml) at all concentrations (P < 0.05). SBC had lower survival than MBC in all concentrations and periods. Except at 5 μg/ml concentration at 48 hours in SBC, no statistically significant values were reported. Conclusion: DPCs are prone to the cytotoxicity caused by dental composite. In contrast to MBC, the cytotoxicity of SBC declines overtime.
Keywords: Cytotoxicity, dental pulp cells, methacrylate-based composites, MTT assay, silorane-based composites
|How to cite this article:|
Madhyastha PS, Bhat KM, Padma D, Bangera MK, Naik DG, Srikant N, Kotian R. Cytotoxicity of Silorane and Methacrylate based Dental Composites on Human Pulp Cells. J Orofac Sci 2021;13:13-8
|How to cite this URL:|
Madhyastha PS, Bhat KM, Padma D, Bangera MK, Naik DG, Srikant N, Kotian R. Cytotoxicity of Silorane and Methacrylate based Dental Composites on Human Pulp Cells. J Orofac Sci [serial online] 2021 [cited 2021 Sep 29];13:13-8. Available from: https://www.jofs.in/text.asp?2021/13/1/13/323356
| Introduction|| |
Oral tissues are exposed continuously to restorative dental materials through direct contact. Tooth restorations undergo gradual compositional changes in due course, because of chemicomechanical activities. The leached constituents may lead to epithelial and connective tissue changes. It may be diagnosed as oral lichenoid reaction or anaphylactoid reaction as a result of mucosa irritation and hypersensitivity. Chronic contact with restorative materials may also lead to fibrosis., Also, the components of restorative materials are known to penetrate and diffuse through the dentin to cause irritation of the pulp tissue and may also affect the vitality of the tooth. These effects monomers present in composite resin, namely bisphenol A glycidyl methacrylate (Bis-GMA), triethyleneglycol di-methacrylate (TEGDMA), 2-hydroxy-ethylmethacrylate (HEMA), and urethane dimethacrylate (UDMA), have been extensively reviewed in the literature.,,,,, Chemical compounds such as 2,3-epoxy-2-methyl-propionic acid methyl ester, methacrylic acid, 2,3-epoxy-2-methylpropionic acid, and bisphenol-A-bis(2,3-dihydroxypropyl) ether are produced as intermediates during the metabolism further leading to monomer toxicity which is modulated by production of cellular reaction oxygen species.,, The residual monomers are known to induce inflammation. On the other hand, the congregation with the cytokines and cellular mediators are known to induce cytotoxicity. Therefore, the biocompatibility of dental materials is necessary to maintain proper oral health postrestoration. Hence, it is essential to test the biocompatibility of restorative material and report its cytotoxic effects.
The widely used methacrylate-based composites (MBCs) present with polymerization shrinkage and loss of marginal integrity in restorations, which can be attributed to the free radical addition polymerization setting reaction. Silorane-based composite (SBC; Filtek P90) polymerizes by ring-opening cationic polymerization and can benefit to overcome the shrinkage caused by the MBC. The Filtek P90 contains silorane resin matrix, fillers, an initiator system, stabilizer, and colorants. The silorane structure is made up of siloxane and oxirane functional groups. The reduced polymerization shrinkage is associated with the oxirane moiety, and other properties such as hydrophobicity and water sorption are attributed to the siloxane moiety. Our study is unique in the sense that the Filtek P90 was used to assess biocompatibility. The other studies use siloxane and oxirane as separate molecules to study biocompatibility. Also, the reports of the effects of Filtek P90 on the epithelial cell lines are very few. Toxic effect of Filtek P90 on human pulp cells in vitro was evaluated using cytotoxicity measuring parameters.
In the present study, enzymatic conversion of methyl thiazolyl tetrazolium (MTT) as a measure of cell metabolism (MTT assay) on human dental pulp cells (DPCs) was evaluated among SBC and MBC. The subsequent toxicity can result in adverse host reactions. From a clinical point of view, it is important to evaluate the silorane-based low-shrink posterior restorative on human pulp cells. The possible cytotoxic effect can culminate in various oral health problems. Hence, outcome of this study would assess the efficacy of SBC on human gingival epithelium and the supporting connective tissue. The positive results of biocompatible tests of silorane would suggest the safeties and inert nature in oral environment and hence, may contribute to clinical success of this material.
The study was designed to evaluate and investigate the effect of SBC and MBC on the viability, proliferation rate of DPCs. Hence, the outcome of the performed study would assess the efficacy of silorane restorative on epithelia of gingiva and its connective tissue. The positive results of biocompatible tests of silorane would suggest the safety of SBC in oral environment influencing the clinical success of the restoration.
| Materials and Methods|| |
Ethical approval for this study (Protocol Ref No: 09010) was provided by the Institutional Ethical Committee of Manipal College of Dental Sciences, Mangalore, on August 11, 2009. SBC (Filtek P90, 3M/ESPE, St Paul, Minnesota, USA) and MBC (Z100, 3M/ESPE, St. Paul, Minnesota, USA) were selected for the study. [Table 1] presents the details of the compositions.
Preparation of stock solution
The composites were dissolved in <0.2% concentration of dimethylsulfoxide (DMSO). The concentration used did not present a statistical variation to the findings of the control untreated cells for the measured parameters. Seven different concentrations of composites (20, 10, 5, 2.5, 1.25, 0.625, and 0.3125 µg/ml) were obtained from the stock solution (10 mg/ml) to treat the cells at the final established concentration of DMSO (<0.2%) by serial dilutions.
Isolation and cell culture method
With informed consent from the patient and as per the regulations of the Institutional Ethics Committee, the dental pulp tissue was harvested from noncarious permanent teeth, which were extracted as a part of orthodontic therapy. The extirpated pulp tissues were transported in an isolation medium containing Dulbecco modified Eagle medium (Sigma–Aldrich, Dorset, UK) accompanied with 10% fetal calf serum (Biowest Ltd., Riverside, UK), 2% l-glutamine (Sigma), 100 IU/ml penicillin (Sigma), and 100 µg/ml streptomycin (Sigma). A sterile scalpel was employed to section the tissues into the desired number. Five to six sectioned tissues were seeded into each six-well tissue culture plates. Two milliliters of cell growth medium consisting of a modified Eagle medium (α-MEM; Sigma) complemented with 10% fetal bovine serum (FBS), glutamine, and 1% polysialic acid were introduced in each well and incubated at 37°C in the humidity of 5% CO2.
Passaging and freezing of cells
On 80% confluence of the cells, a mixture of 0.02% trypsin (Sigma) and 0.02% ethylene diamine tetraacetic acid (Sigma), followed by incubation for 2 to 5 minutes at 37°C was performed to split the cells. Culture plates were flushed with DMEM containing 10% FBS. Sample pallet was made into cell suspension by vertexing or pipetting the digested tissue several times. The resultant cell suspension was replated into a fresh culture plate. The excess cell was frozen in liquid nitrogen with appropriate media containing 10% DMSO.
Cells derived from cell lines were then treated using varying doses of SBC and MBC at different time points after the required confluence was attained, followed by treatment with (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a tetrazole) MTT assay for cell proliferation and viability evaluation.
On seeding the cells into a 96-well plate at a density of 104 cells/well, they were incubated for 48 hours to let cell attachment. Following attachment, they were treated with different doses of SBC and MBC. DMSO-treated and untreated cells functioned as the control groups. Incubation of the culture was carried out for 48 and 72 hours. On completion, 20 μl of MTT stock solution was added to each well. Subsequent incubation was performed for 4 hours at 37°C. The formazan crystals were dissolved in lysis solution (1:24 ratio of hydrochloric acid:isopropanol) for 1 hour at room temperature. An enzyme-linked immunosorbent assay plate reader at a wavelength of 570 nm was used to measure the absorbance of the colored solution. Each experiment was performed in triplicates. The obtained data were analyzed using Microsoft Excel program.
The measures of central tendencies were depicted as mean and standard deviation. An independent Student t test was performed to compare the effect of composites on the DPC. The cytotoxicity levels of the composites with the different concentration and time intervals were compared using one-way analysis of variance and posthoc Tukey multiple comparison test at 5% level of significance and P-value of <0.05.
| Results|| |
[Figure 1] represents the cultured DPC. The DPC exposed to MBC showed higher viability than SBC at both the periods tested. A significant difference was seen in the time‒group interaction of MBC and SBC. The DPC presented with different cytotoxic behavior in the two materials. The cytotoxicity assay showed >90% viability, indicating excellent viability in 24 hours. It significantly decreased in 48 hours period (minimum 65% in 25 μg/ml) in all concentration (P < 0.05). This decrease was statistically significant in all concentrations of SBC and MBC except in 20 μg/ml, where the reduction was not substantial (P = 0.078). However, the proliferation rate at any given concentration was never below 90% at 24 hours for both the materials, whereas it decreased below 80% at 48 hours in SBC as well as MBC, as shown in [Figure 2]. SBC had lower survival than MBC in all concentrations and time periods except at 5 μg/ml concentration at 48 hours in SBC when compared using an independent Student t test, but without statistically significant results. Studies with the solvent DMSO used as a control in MTT assay showed an average cell survival of 75%. The percentage of cell survival given by each dose of SBC and MBC was estimated in relation to the untreated group. At 48 hours, in concentrations of 5 μg for SBC, there was a significant increase (P = 0.001), but at 2.5 μg for MBC showed higher values (P = 0.029). In conclusion, the viability was lower at 48 hours in both the materials; cell death was higher in SBC than MBC.
|Figure 1 Cultured dental pulp cells (magnification 100x) (A) clustered view (B) free and scattered fibroblasts after passaging|
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|Figure 2 Comparison of cytotoxicity of silorane-based composite (SBC) and methacrylate-based composite (MBC) at 24 and 48 hours on dental pulp cells|
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| Discussion|| |
In the current study, the viability and proliferation rate of DPC after exposure to SBC and MBC representing two composite resins based on different monomer compositions were assessed. Biocompatibility of resin composite is an essential criterion for pulp vitality after any operative restoration. The cytotoxicity evaluation of the composite resins is more clinically relevant as these restorations are in close proximity to the DPCs especially in deep cavities.,,, Cultured pulp cells are thus an accepted standard for biocompatibility investigation. The fibroblasts obtained from pulp are considered challenging to culture with low survival rate but are known to be precise. This signifies that pulp cells can be a sensitive indicator of cytotoxicity.,
Inadequate polymerization of resin monomers affects the physiochemical properties resulting in the unsatisfactory clinical performance of the composites. Interaction of the restorations with the living cells and oral fluids results in biodegradation and leaching of residual resin monomer.,,,,, Also, resin monomers such as Bis-GMA or UDMA, TEGDMA or HEMA inevitably disturb the vital cell mechanisms and are cytotoxic via apoptosis, induce genotoxic effects, promote prostaglandin E2 production to DPC, and alter the cell cycle. Additional factors which influence the pulp reactivity are the solubility of monomers, the progression of caries, and the remaining dentin thickness.
The literature on the biocompatibility of the SBC is sparse when compared with classical MBC. A study suggested minimum cellular responses of the SBC (HermesIII) when compared with MBC with a marked reduction in cytotoxicity of silorane with time. The hydrolytic stability of silorane explains the different cytotoxic profile., In the present study, at lower concentrations at 48 hours of SBC (5 μg/ml) showed similar observations, as shown in [Figure 2]. A study proved that SBC displayed less solubility, water sorption, and diffusion coefficient after short- and medium-term immersion intervals in comparison to MBC (Filtek Z250). The authors attributed the low release of unpolymerized monomers to its hydrophobic properties and reduction in residual monomers on polymerization., Besides, the low solubility of silorane monomers in water is considered responsible for the absence of cell responses. A study stated that the silorane monomers are discharged from SBC into an organic rather than an aqueous medium. Brackett et al. reported the cytotoxic effect (cell viability) of the material instantly after direct contact with cells, whereas no cytotoxicity following aging. The result obtained from this study was in conformity with another study performed by Shafiei et al. who suggested that the exposure of DPC to SBC caused a rise in the cell viability in the 14th-day extract when compared with the seventh day.
On the contrary, in the present study, the cell viability was higher in the first 24 hours and decreased with aging. According to the obtained results, MBC and SBC exhibited different cytotoxic behaviors in both the period tested. However, the cytotoxic effects of both the investigated composites were comparatively low. But comparing MBC and SBC, viability was higher in MBC than SBC, contradicting all the results in the literature. The reason for toxicity could be a lower degree of conversion after curing as discussed by Marchesi et al. who suggested that silorane had a lesser degree of conversion than MBC (Filtek Z250). A study reported human pulp fibroblasts to be more sensitive than gingival fibroblasts to the variations from the most tested substances, and this fact should not be ignored. In contrast to in vitro studies, one of the in vivo study detailed that SBC resulted in no more undesirable pulpal and periapical reactions in the deep cavities compared to MBC.
In a clinical scenario, there are concerns when performing direct pulp capping or deep cavity restoration with composites, as the resinous monomers diffuse through dentinal tubules. When the residual dentin thickness is under 1 mm, minor histologic reaction can be observed pulp as a result of contact with a dental material.,,
| Conclusion|| |
The DPCs are more susceptible to the cytotoxic effect of the composites. This study concludes that both the test materials were not cytotoxic and are regarded safe when tested for the DPCs in an in vitro study. Compared to MBC, the cytotoxic effect of SBC decreases with time. This difference is of high importance when considering the regenerative capacities of pulp in deep cavities. Also, the present study adds new information to our knowledge about the cytotoxicity of dental composite resin restorations based on different chemistries.
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Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]