ORIGINAL RESEARCH


https://doi.org/10.5005/jp-journals-10015-2256
World Journal of Dentistry
Volume 14 | Issue 6 | Year 2023

Comparative Evaluation of Cellular Toxicity of Three Heat Polymerized Acrylic Resins: An In Vitro Study


Jerry J Chokkattu1, Singamsetty Neeharika2, Indira P Brahmajosyula3, Lakshmi Thangavelu4

1,2Department of Prosthodontics, Saveetha Dental College & Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India

3Department of Prosthodontics, CKS Theja Institute of Dental Sciences and Research, Tirupati, Andhra Pradesh, India

4Department of Pharmacology, Saveetha Dental College & Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India

Corresponding Author: Lakshmi Thangavelu, Department of Pharmacology, Saveetha Dental College & Hospital, Saveetha Institute of Medical and Technical Sciences (SIMATS) (Deemed to be University), Chennai, Tamil Nadu, India, e-mail: lakshmi@saveetha.com

Received on: 04 May 2023; Accepted on: 05 June 2023; Published on: 22 August 2023

ABSTRACT

Aim: This research aims to assess the cytotoxicity of Triplex Hot, Trevalon H, and dental products of India (DPI) heat cure acrylic resins by comparing their optical density at 1, 24, and 72 hours and evaluating the residual monomer’s effect on fibroblast cells.

Materials and methods: A total of 90 heat-cured acrylic resin specimens (30 each of Trevalon H, Triplex Hot, and DPI) were divided into three groups. HT-1080 fibroblasts were cultured, and resin samples were introduced into respective wells. The inflammatory response was assessed after 1, 24, and 72 hours of incubation. Cell vitality was measured using an 3-(4,5-dimethylthiazol-2-yl)—2,5-diphenyl tetrazolium bromide (MTT) assay and spectrophotometer. Statistical analysis included one-way analysis of variance (ANOVA) and post hoc Tukey’s test.

Results: All three groups have displayed cytotoxicity at varied levels after 1 hour, 24 hours, and 72 hours, but with no significant difference among groups after 24 hours, increasing safety concerns towards their usage. H3 samples showed a higher level of cytotoxicity in general when compared with H1 and H2.

Conclusion: It has been concluded that levels of cytotoxicity were found to be higher and lower after 1 hour and 72 hours incubation period respectively, whereas all the resins displayed similar levels of cytotoxicity after 24 hours explaining that the residual monomers will percolate maximum within the first 24 hours after processing of dentures.

Clinical significance: It was discovered that resin had varying degrees of cytotoxic effects, raising concerns about the safe clinical application of these materials. Hence proper care should be taken during the selection of resin materials to prevent adverse effects on operators and patients.

How to cite this article: Chokkattu JJ, Neeharika S, Brahmajosyula IP, et al. Comparative Evaluation of Cellular Toxicity of Three Heat Polymerized Acrylic Resins: An In Vitro Study. World J Dent 2023;14(6):492-497.

Source of support: Nil

Conflict of interest: None

Keywords: Absorbance spectrum, Acrylic resins, Cytotoxicity, HT1080 fibroblasts, Optical density.

INTRODUCTION

Edentulism has emerged as a significant concern, leading to the development of various artificial substitutes in the field of dentistry. Among these substitutes, acrylic resins have been utilized for the intraoral replacement of lost hard and soft tissues, and they have undergone notable advancements over time.1 Polymethyl methacrylate (PMMA) is commonly employed in dentistry to fabricate removable dentures, temporary crowns, orthodontic appliances, denture liners, and other dental appliances.2 Acrylic resins are available in a powder (polymers) and liquid (monomer) system, often supplemented with additives. These materials are considered biomaterials as they serve as functional and morphological substitutes for missing tissues, interacting directly with the mucosa, alveolar ridge, and remaining natural teeth.3

The evaluation of acrylic resin efficacy necessitates an understanding of their physical, chemical, and biological properties, with a particular emphasis on biocompatibility. Biocompatibility refers to the appropriate host response, indicating the absence of adverse local or systemic reactions caused by the presence of such materials.4 However, due to continuous exposure to the oral environment, these polymers undergo biodegradation, leading to the release of various toxic components such as residual monomers, initiators, and byproducts. This can result in allergic reactions and irritations of the oral mucosa.5

In summary, the growing concern surrounding edentulism has driven advancements in the development of artificial substitutes in dentistry. Acrylic resins, particularly PMMA, have played a vital role in the fabrication of dental appliances. However, their biocompatibility remains a significant limitation, as the oral environment exposes these materials to biodegradation and the release of potentially toxic components. In vitro, cytotoxicity tests provide a valuable means of evaluating the safety and biocompatibility of acrylic resins. In vitro, cytotoxicity tests have been implemented as a preliminary screening method to evaluate the biocompatibility and safety of acrylic resins. These tests are relatively convenient, rapid, reproducible, and cost-effective.3 Given these considerations, this study aimed to assess the inflammatory reactions on HT1080 fibroblasts using the 3-(4,5-dimethylthiazol-2-yl)—2,5-diphenyl tetrazolium bromide (MTT) assay to examine the time-dependent toxicity of three commonly used commercially available heat-polymerized acrylic resins—Triplex Hot, Trevalon H, and dental products of India (DPI).

MATERIALS AND METHODS

This study was conducted in the Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India (Ref No: A&E/62/Trng/SVIMS/2005) for a period of 1 month (October) in the year 2022. The study was approved by the Institutional Ethics CKS Theja Institute of Dental Sciences and Research, Tirupati, Andhra Pradesh, India. Attempts were made to standardize the procedures throughout the study to minimize the effects of variable factors on the observation and the final results.

Fabrication of Heat-cured Acrylic Test Specimens

This experimental study was created in accordance with the findings of the Saravi et al. research to assess the cytotoxicity of three heat-cured acrylic resins.6 A total of 90 specimens of heat-cured acrylic resin were divided into three groups each with 30 samples—H1 (Trevalon, Dentsply India Pvt Ltd, Gurugram, Haryana, India), 30 samples—H2 (SR Triplex Hot, Ivoclar-Vivadent Marketing Pvt Ltd, India) and 30 samples—H3 (DPI Heat Cure, Dental Products of India, Mumbai, Maharashtra, India) were fabricated in the form of circular discs of 10 × 1 mm dimensions. Modeling wax (Hindustan Modelling Wax no. 2, The Hindustan Dental Products, Hyderabad, Telangana, India) of 1.5 mm thickness sheets were used and cut into circular discs using a hollow cylindrical metal cutter with a diameter of 12 mm to compensate for the loss of acrylic resin during finishing procedure and to obtain a uniform thickness of 10 × 1 mm acrylic samples.

The wax specimens were checked for required dimensions and were invested in varsity dental flasks (Varsity Flask No. 7, Jabbar & Company, Aligarh, Uttar Pradesh, India) using model plaster (Nelkon) followed by dewaxing. All three groups of acrylic resins were proportioned in volume ratio (3 parts polymer—1 part monomer), manipulated, and packed into a varsity flask in a conventional manner. Followed by bench sitting under a hydraulic press (1.25 kg) for a minimum of 30 minutes. Resins were cured in an acrylization unit (Polybath, Delta) following a long polymerization cycle (9 hours, 165˚F). Postpolymerization cycle, the flasks were opened and bench cooled followed by finishing and polishing of the samples with sandpaper abrasives of 150, 220, 320, 400 600, 800, and 1000 grit, to simulate an outcome that resembles the denture processed in a laboratory. The dimensions of each sample were measured using a metal gauge and were subjected immediately for incubation and cytotoxicity evaluation to avoid possible changes resulting from aging.

Cell Culture and Cytotoxicity Evaluation

The HT1080 fibroblast cells were acquired from the National Centre for Cell Science, Pune, Maharashtra, India (Fig. 1). To assess the cytotoxicity of acrylic resin samples, the fibroblasts (HT1080) were initially placed in the culture flasks with fresh media (3 mL), serum (30 µL), fetal calf serum (300 µL), and 100 mg/mL penicillin/streptomycin and was kept in an incubation chamber at 5% carbon dioxide, 37˚C over 90% humidity. After reaching an adequate volume, cells that were adhered (confluent cells) to the flask walls were detached with 0.25% trypsin and seeded at a density of 3 × 105 (75 µL) into each well of a 24-well cell culture plate except D4, D5, and D6 wells.

Fig. 1: Cell line used for the study HT1080 fibroblasts under an electron microscope (40× magnification)

A continuous evaluation was done under an electron microscope (40× magnification) (Motic AE31 Elite Inverted Microscope) to check the cell viability which should be over 90%. Once the viability was checked, the fibroblast suspension was seeded into 24 well plates along with a combination of Dulbecco’s Modified Eagle Medium (DMEM) media, 10% fetal calf serum, and antibiotics of streptomycin and penicillin.

The three test specimens were added into the plates in the following manner, from A1–A6: H1, B1–B6: H2, C1–C6: H3 test specimens were added. D1–D3 wells were kept empty with only the fibroblast and DMEM media suspension as a control group. D4, D5, and D6 contain combinations like media + serum, medium, and serum, respectively to check for the viability and sources of contamination in the control group (Fig. 2).

Fig. 2: Acrylic resin specimens added into fibroblast suspension wells and incubated

Following incubation in a controlled environment (Binder incubation chamber) at 37°C 90% humidity, and 5% carbon dioxide, the plates were assessed for an immediate inflammatory response after 1 hour. After an incubation period of 1 hour, cell viability is assessed following the protocol for MTT assay (Table 1).7

Table 1: Protocol for performing MTT assay
Steps Action
1. Fibroblasts were seeded in a 24-well plate each at 75 µL except D4, D5, and D6
2. Incubate for 1 hour, 24 hours, 72 hours at 37°C, 5% CO2
3. Add 45 µL of MTT reagent
4. Incubate for 4 hours at 37°C, 5% CO2 until purple precipitate is visible
5. Remove supernatant without disturbing cells/crystals
6. Add 1 mL of acidified isopropanol
7. Record optical density at 540 nm and 630 nm

The principle of MTT assay was its ability to convert the yellow water-soluble tetrazolium salt MTT into dark blue formazan crystals with the aid of mitochondrial dehydrogenase enzyme. The cytoplasm of these living test cells houses these water-insoluble crystals since the cells are mostly impervious to them. The level of the formazan product generated directly correlates with the number of surviving cells. Just before usage, MTT solution (5 mg/mL in phosphate buffer saline) was prepared. Each well-containing fibroblast cells, test samples, control group wells, and 45 L MTT dye was added. Plates were then incubated in an incubation chamber of 5% carbon dioxide at 37°C for 4 hours. After 4 hours supernatant was removed without disturbing cells or crystals and 1 mL of acidified isopropanol was added to each well which led to the appearance of blue formazan crystals (Fig. 3).

Fig. 3: Appearance of blue formazan crystals stored in the cytoplasm of living cells after an incubation period

By dissolving the MTT formazan in dimethyl sulfoxide (DMSO) (6.25% v/v 0.1 N NaOH in DMSO), the optical density (OD) was calculated. Care has been taken to agitate the plates for 5 minutes before being introduced in a microplate reader.

Spectrophotometric absorbance was measured at an optical density of 540 nm and 630 nm to prevent absorbance of false lights. To determine the concentrations of reactants and products after MTT assay, the light transmittance of the solution was tested using a spectrophotometer. The amount of light that passes through the solution was an indication of the chemical concentration absorbed by living cells that block the light. Absorbance was recorded immediately because the product was unstable. The contents of well D4 which has medium, serum, and MTT with no cells were used as a control to the plate reader. The remaining 24 well plates with acrylic specimens and fibroblast cells were also tested for optical density after an incubation period of 24 hours and 72 hours with the above-mentioned procedures. The specimens were coded during the entire process to reduce bias. Readings were obtained for each well, and a formula was used to compute the percentage of cell viability for each well in proportion to the mean absorbance of the control wells.

Cell viability was interpreted based on the amount of cell proliferation with average values for non-cytotoxic as greater than 90% cell vitality, slightly cytotoxic between 60 and 90%, moderately cytotoxic between 30 and 59% and strongly cytotoxic if it was lesser than 30%. The repeated measure analysis of variance (ANOVA) test was used to examine the effects of time and resin type. Descriptive statistics, one-way ANOVA, and post hoc Tukey honestly significant difference tests were employed to assess the variations in light absorption across the resin groups at various intervals. A significance threshold of 0.05 was used. SPSS software version 26 was used to analyze the data.

RESULTS

Study Design

The mean (±SD) light absorption for H1, H2, and H3 after 1 hour was 0.107 ± 0.012, 0.063 ± 0.012, and 0.032 ± 0.072, respectively. In terms of cytotoxicity after one hour, which represents an acute inflammatory reaction to the resins, the one-way ANOVA indicated a significant difference between the groups (p < 0.001). Additionally, the findings of the Tukey’s test, which allowed for a paired comparison between the groups, showed that the H1 group substantially absorbed less light than the H2 and H3 groups (p < 0.043, p < 0.00) (Table 2). Between H2 and H3, the test was unable to identify any significant difference.

Table 2: Post Tukey’s test within the material after 1 hour incubation period
(I) Material (J) Material Mean difference (I-J) Standard error Significance 95% confidence interval
Lower bound Upper bound
H1 H2 .04400* 0.017 0.043* 0.001 0.087
H3 .07508* 0.017 0.000** 0.032 0.118
H2 H1 −.04400* 0.017 0.043* −0.087 −0.001
H3 0.031 0.017 0.191 −0.012 0.074
H3 H1 −.07508* 0.017 0.000** −0.118 −0.032
H2 −0.031 0.017 0.191 −0.074 0.012

*The mean difference is significant at the .005 level

The mean [standard deviation (SD)] light absorption values for the H1, H2, and H3 groups after 24 hours were 0.224 ± 0.096, 0.234 ± 0.066, and 0.176 ± 0.1, respectively. The one-way ANOVA test failed to detect any differences between the groups in terms of cytotoxicity (p = 0.2), and it also failed to detect any changes between the H1, H2, and H3 groups (Table 3).

Table 3: Post Tukey’s test within materials after 24 hours incubation period
(I) Material (J) Material Mean difference (I-J) Standard error Significance 95% confidence interval
Lower bound Upper bound
H1 H2 −0.011 0.036 0.951 −0.100 0.078
H3 0.047 0.036 0.400 −0.041 0.136
H2 H1 0.011 0.036 0.951 −0.078 0.100
H3 0.058 0.036 0.255 −0.030 0.147
H3 H1 −0.047 0.036 0.400 −0.136 0.041
H2 −0.058 0.036 0.255 −0.147 0.030

The mean (±SD) light absorption for the H1, H2, and H3 groups after 72 hours of incubation period was 0.236 ± 0.061, 0.222 ± 0.041, and 0.183 ± 0.027, respectively. After 72 hours of incubation, one-way ANOVA revealed a significant difference between the groups in terms of cytotoxicity (p < 0.02) representing an inflammatory response to resin. Tukey’s test revealed a significantly less light absorption between H1 and H3 (p < 0.01) (Table 4).

Table 4: Post Tukey’s test within materials after 72 hours incubation period
(I) Material (J) Material Mean difference (I-J) Standard error Significance 95% confidence interval
Lower bound Upper bound
H1 H2 .05317 0.018 0.019* 0.008 0.099
H3 0.014 0.018 0.720 −0.031 0.060
H2 H1 −.05317 0.018 0.019* −0.099 −0.008
H3 −0.039 0.018 0.105 −0.084 0.007
H3 H1 −0.014 0.018 0.720 −0.060 0.031
H2 0.039 0.018 0.105 −0.007 0.084

The statistical analysis showed that cytotoxicity was significantly influenced by both time (p < 0.00) and resin type (p < 0.009), with each resin group exhibiting varying degrees of cytotoxicity at various times (apart from the first 24 hours) (Table 5). Results, however, did not show a clear significant relationship between time and resin type when they were combined (p < 0.057). The tests did not conclude any significant difference between H1 and H2 and H2 and H3.

Table 5: Two-way ANOVA repeated measure
Source Type III sum of squares Degree of freedom (Df) Mean square F-value Significance
Time 0.503 2 0.252 64.391 0.000**
Material 0.038 2 0.019 4.898 0.009
Time × material 0.037 4 0.009 2.373 0.057*

DISCUSSION

Oral mucosa constitutes a major part of the oral cavity which includes the alveolar mucosa, gingiva, and tongue. Due to the direct and indirect application of dental materials, the epithelial tissue of the oral mucosa, which serves as a physical barrier, is regularly exposed to numerous stimuli.8 With an exponential rise in the use of acrylic resins in dentistry it is required to constantly assess their properties and functional importance. Several exogenous factors like saliva, mastication, changes in intraoral temperature due to diet, intraoral bacteria, and endogenous factors like technique, degree of polymerization, and factors affecting the acrylic resins play a significant effect on oral tissues compromising the material.9 During the setting of heat polymerizing resins, not all monomers are polymerized which causes leaching of residual monomers like formaldehyde, methyl methacrylate, and benzoic acid into the surrounding tissues at various degrees of concentrations. These residual monomers have proven to cause various degrees of cellular toxicity both in vitro and in vivo6 causing local (irritation and inflammation to the mucosa) and systemic effects (contact dermatitis, bronchitis) to patients and dental personnel who are handling the material on a daily basis.2 In general, residual monomer concentrations in heat-polymerized acrylic resins are much lower than in chemically cured resins, and thicker sections exhibit lower residual monomer concentrations than thin bases. Hence supporting the selection of heat-cured acrylic resins with a 1.5 mm thickness for the study. According to Gautam R et al's review, Harrion and Hugett have conducted a study on various polymerization cycles in the year 1992 and have proved that maximum conversion of monomer to polymer occurs at 7 hours of curing at 70 degree celsius followed by 1 hour of curing at 100 degree celsius, hence the choice of long polymerization of cycle in our study.10 Since PMMA is proven to have poor biocompatibility and to eliminate or reduce the toxic effect, it is suggested to process heat cure acrylic resins for a longer time in boiling water to reduce the residual monomer to a permissible level.2

Cytotoxicity testing of acrylic resins in vitro is essential to establish clinical safety limits as it closely simulates physiological processes.5 With an increase in the availability of various human and non-human immortalized cell lines from different tissues and assays, it has become easier to measure the toxicity, proliferation, and viability of cells exposed to test specimens. Among all the assays available, the MTT assay has been proven to be ideal for estimating cell densities of small cultures.4 Cell line HT1080 has been used for the study according to ISO 10993 - 5:2009 recommendation for cytotoxicity testing. Additionally, fibroblasts have been shown to predominate in the gingival connective tissue and can permit penetration of residual monomer easily in the absence of inflammation, hence the choice of the cell line in our study.6 The samples fabricated were according to International Organization for Standardization/AWI 20795-1:2013 and provided more accurate results for further follow-up studies unlike the previous regulations (ISO20795–1:2013, dentistry—part 1: denture base polymers).11

The results of the present study indicated that after one hour, 24 hours, and 72 hours, all three groups showed cytotoxicity to varying degrees, although there was no significant difference between the groups after 24 hours, raising safety concerns about their use. Comparing H3 (DPI) samples to H1 (Triplex) and H2 (Trevalon) samples often revealed greater levels of cytotoxicity in H3. Levels of cytotoxicity after 1 hour and 72 hours of incubation period were found to be higher and lower, respectively, whereas, after 24 hours, all the resins showed similar levels of cytotoxicity, indicating that the residual monomers will percolate most quickly in the first 24 hours following the processing of dentures.

According to Jorge et al., the cytotoxic impact of acrylic resins was greater in the first 24 hours following curing and gradually diminished with time. His hypothesis stated that within the first 24 hours either the toxic substances are leaching into the medium or broken down, which is in comparison with our study at 24-hour intervals and is clinically insignificant after 72 hours indicating toxicity of resins can be reduced with an increase in time. The study conducted by Ebrahimi Saravi et al. evaluated the cytotoxicity of two newly launched acrylic resins (Futura Gen and GC Reline Hard) and a conventional heat cure resin (Meliodent) with mouse fibroblasts using MTT assay at varied time intervals of 1 hour, 24 hours, and 1 week. The study determined that all three resins showed significant cytotoxicity with the highest and lowest being observed after 1 hour and 1 week after immersion in water, respectively. It was advised to soak the dentures in water prior to denture delivery to avoid break down of toxic substances in the oral environment and prevent tissue hypersensitivity, based on the aforementioned research and the current study’s findings there was no significant difference in cytotoxicity among the studied materials after 24 hours.6 Koutis and Freeman explained that acrylic resins may cause clinical symptoms based on the level of exposure, but in a healthy individual, these symptoms might be rare due to intraoral factors like a continuous flow of saliva, high mucosal vascularization, lower cell-mediated immunity which prevents the prolonged contact of the toxic substances by washing away effectively in the mouth which supports the lack of affected individual percentage systemically.13 All of the acrylic resins in the current investigation showed some degree of cytotoxicity, hence more experimental and clinical testing using various techniques is needed. Researchers can learn more about the nature of the tissue irritation through additional long-term (more than a week) studies investigating the impact of various compounds linked to denture base resins.

STUDY LIMITATIONS

In vitro, cell culture represents only the initial screening method of evaluation on the biocompatibility of newer materials and those already in clinical use. It is not ideal to withdraw credible conclusions based on these studies alone. In our study, we have assessed only three commercially available heat-cured polymerized resins that have monomers. With the recent advancement in materials such as monomer-free resins, reinforced resins,2 polyetheretherketone (PEEK) materials,14 resins with modified monomers (cycloaliphatic comonomer),15 studies have shown that the level of cytotoxicity is lesser compared to conventional ones with only disadvantage of being expensive. This study should have been comparative with the inclusion of any of these recent materials.

AREAS FOR FUTURE RESEARCH

There is a need for further studies with a larger sample size to investigate the biocompatibility of acrylic resins using a combination of in vitro, in vivo, and usage tests. With an increase in modifications of monomers and polymers showing significant results in biocompatibility,15,16 further in vivo studies are essential to introduce these new materials into the field of dentistry for clinical and laboratory purposes to reduce the potential local and systemic adverse effects.

CONCLUSION

Within the scope of this investigation, it became apparent that each evaluated resin sample exhibited varying degrees of cytotoxicity on fibroblasts. This finding raises concerns regarding the safe clinical application of these materials. Notably, the H3 resin displayed higher levels of cytotoxicity compared to the other groups, with the highest and lowest levels observed after 1 hour and 72 hours of incubation, respectively. However, after 24 hours, all resins exhibited similar levels of cytotoxicity, suggesting the potential leaching of residual monomers within the initial 24-hour period following denture processing. Consequently, careful consideration must be given to the selection of resin materials, polymerization techniques, curing processes, and patient delivery to prevent adverse effects on oral health. Despite meeting standards such as ADA and ISO, the presence of residual monomers remains a significant concern, questioning the biocompatibility of these materials. Thus, future research focusing on novel materials and further investigations in clinical scenarios are required to enhance clinical performance

ACKNOWLEDGMENTS

We thank the Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India for providing us the access to conduct the project work (Ref No: A&E/62/Trng/SVIMS/2005).

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