Evaluation of the In-Vitro Cytotoxicity of Heat Cure Denture Base Resin Modified with Recycled PMMA-Based Denture Base Resin.

Aim: The aim of the present study was to evaluate the in-vitro cytotoxicity of heat-cure denture base resin (PMMA) modified with recycled denture base resin at 10%, 20%, 30%, 40%, and 50% (w/w) concentration. Materials and
Methods: A total of 30 disk-shaped specimens were prepared and divided into six groups (n = 5). The Control group (R0) consisted of unmodified processed denture base resin, the experimental group consisted of denture base resin processed with substitution of 10% 20%, 30%, 40%, and 50% (w/w) of recycled denture base resin (R10, R20, R30, R40, and R50). Eluates were prepared using five sterile specimens of each group. The mouse fibroblast cell line (L929) was seeded in a 96-well cell plate system at a concentration of 1 × 104 cells/well in the DMEM medium with 1× antibiotic and antimycotic solution and 10% fetal bovine serum at 37°C with 5% CO2 and incubated in a CO2 incubator for 48 h. MTT assay was applied and the absorbance was measured at 570 nm using a microplate reader to assess the in-vitro cytotoxicity. One-way analysis of variance (ANOVA) along with post hoc Scheffe test was used to statistically compare the mean optical density (OD) values and cell survival/viability % amongst the groups.
Results: No statistically significant difference was observed in the mean and standard deviation of the optical density and cell viability % of the test groups that were compared.
Conclusion: Modification of denture base resin using recycled PMMA does not have a cytotoxic effect on the mouse fibroblast cell line L929. Copyright:

INTRODUCTION

Denture base resins have proved themselves to be the front runners for ideal denture base materials. Denture base resin acrylic was introduced in the early 1900s; with a boom in the plastic industry, various polymers were tried out as denture base resins. The breakthrough came in the 1930s with the invention of acrylic-type plastic denture bases by Otto Rohm. The transparent sheets of Otto's invention, polymethyl methacrylate (PMMA) were introduced along with Hass. Later on, powdered PMMA was produced by Du Dout De Nemours in 1937. This product took dentistry by storm and by 1946, more than 90% of the dentures were fabricated using PMMA. This feat was possible only because of the desirable ideal properties of PMMA. An ideal dental material must fulfill the criteria of optimum physical properties, stability in the oral environment, ready availability, and biocompatibility.[123] Biocompatibility may be defined as “the ability of a dental material to perform the desired function without causing any adverse local and systemic in response to the recipient of the dental prosthesis”.[4]

Today's commercially available heat cure denture base resin materials are supplied in a 2-component powder-liquid system. The powder consists mainly of pre-polymerized poly (methyl methacrylate) and liquid component consisting of methyl methacrylate in monomeric form as the major component. Many researchers have discussed the polymerization process of the denture base resins and its influence on the physical and biocompatibility properties of the resultant denture base. Despite overwhelming research in the area to optimize the water: powder ratio, polymerization method, and curing cycle, it has been established that the conversion of monomer to polymer is incomplete. This unreacted monomer left behind in the processed denture is called the residual monomer. It has been established that the residual monomer that can leach out of the cured denture base intraorally may cause adverse reactions.[5678] The adverse reactions due to the leach of residual monomer during service of the denture may range from mild mucosal irritation to glossodynia.[589]

It is prudent to undertake in-vitro cytotoxicity analysis to assess the levels of biocompatibility of any dental material before introducing them to the oral environment. According to the recommendations by ISO 10993-5 “Biological evaluation of medical devices- tests for in-vitro cytotoxicity,”[10] MTT assay can be used to quantify the cell viability by monitoring the metabolic activity of the cell. One of the first assays to be developed specifically for the 96-well cell plate format, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) tetrazolium reduction assay is suitable for high throughput screening. The cell viability is calculated as a function of the cell metabolism that can be monitored by its ability to convert the yellow MTT into a formazan product that is purple in color with absorbance at approximately 570 nm.[711]

The increasing demands of dental materials are depleting the natural resources, and their disposal into the environment is a major cause of land and water pollution. It has been reported by the Ministry of Environment, Forest and Climate Change in 2018–2019 that 22,904.70 TPD ended up in landfills. The annual plastic consumption in India was about 8 million tons and plastic waste generated per day was 25,940 tons.[1213] The present research aims at evaluating the effect of substitution of recycled denture base resin (r-PMMA) at the concentration of 10%, 20%, 30%, 40%, and 50% (w/w) to commercially available heat cure denture base resin on the biocompatibility of the cured denture base resin. The null hypothesis was that the substitution will not affect the cytotoxicity of the cured acrylic resin.

MATERIALS AND METHODS

Test material

The present research was carried out at the Pondicherry Centre for Biological Science and Educational Trust, Puducherry, India. Recycled heat cure denture base resin was substituted in the commercially available denture base resin powder at the concentrations of 10%, 20%, 40%, and 50% (w/w) (R10, R20, R30, R40, and R50), these served as experimental groups. Unmodified commercially available heat denture base resin (DPI® Heat Cure, Denture Base Material, DPI, India) served as the control group (R0). Triple blinding was carried out to overcome bias.

Specimen preparation

Five circular disk specimens (diameter, 22 mm; thickness, 2 mm) were fabricated of each of the six groups under aseptic conditions. The investment was done with a stainless steel metallic die, having a diameter of 22 mm and thickness of 2 mm, in the dental flask to obtain appropriate mold space. Heat cure denture base resin was manipulated according to the instructions of the manufacturer. Trial closure of the flask assembly was done at 3500 psi of pressure using a hydraulic bench press (P 400; Hydraulic Press, Sirio Dental Division) for 10 min. The excess flash was removed and curing was carried out in an electric acrylizer (Unident Instruments India Pvt. Ltd.) at 74°C for 8 h, which was followed by a period of terminal boiling at 100°C for 1 h. Following bench cooling of 30 min, the specimens were retrieved and finished emery 30 μm, followed by polishing with pumice of 10 to 20 μm for ≤1 min and wet wheel and dry wheel. A single investigator prepared all specimens.

Eluate preparation

Three specimens of each group are placed inside sterile glass Petri dishes containing 9 mL of Dulbecco's modified Eagle medium (DMEM) to prepare the eluates [Figure 1]. The DMEM was supplemented with 5% fetal bovine serum and antimicrobial and antimycotic solutions of 100 IU/mL penicillin and 100 μg/mL of streptomycin each. the entire assembly was incubated for 24 h at 37°C. The ratio of the total surface area of the specimens to the volume of elution medium was calculated as 3 cm2/mL as per the recommendations by ISO 10993-12.[14] At the end of the incubation period, the eluates were filtered through 0.22-μm cellulose acetate filters into sterile glass vials, labeled, and stored in a refrigerator [Figure 2]. Negative controls were included in the analysis as recommended by the ISO 10993-5 by incubating 9 mL of DMEM medium without disk specimens.

Figure 1

Dulbecco's Modified Eagle Medium (DMEM)

Figure 2

Preparation of Eluates

CELL CULTURE

L929 mouse fibroblast cell line was employed for determining the in-vitro cytotoxicity of denture base resin modified with recycled denture base resin. The culture medium used to maintain the cells was DMEM with supplementation of 10% fetal bovine serum (HiMedia, India) and 1× of an antibiotic and antimycotic solution containing 200 μL/mL penicillin, 200 μg/mL streptomycin, and 2 μg/mL amphotericin B, each. The medium was changed on alternate days until the cells reached the required confluence. After ensuring sufficient growth of the cell line for the present analysis, the cells were carefully plated onto 96-well culture microtiter plates at a concentration of 1 × 104 cells/well [Figure 3]. It was ensured that each well contained a cell suspension of 100 μL before incubating the microtiter plates at 37°C for 24 h under 5% CO2. This led to the formation of a monolayer of the culture. After 24 h of incubation, the used medium was discarded from every well. Next, 100 μL of prepared eluates of each test group, positive and negative controls were introduced into the 96-well microtiter plates. The entire experiment was triplicated and the cell viability was measured using the MTT assay.

Figure 3

96 Wells culture microtitre plate system

MTT ASSAY

Once the test materials were discarded from the wells, 50 μL of MTT (tetrazolium salt 3-45-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), which was dissolved in phosphate-buffered saline solution at a concentration of 5 mg/mL, was added to every well of a 96-well culture microtiter plate and incubated at 37°C for 4 h in a dark environment. The addition of 100 μL of isopropanol followed the removal of the MTT solution. The culture microtiter plates were then placed on a shaker to assist in solubilizing the purple crystal formazan formed. The quality of the formed formazan was measured by evaluating the optical density by a microplate reader using UV spectrophotometry with a 570 nm filter, and a reference wavelength of 650 nm. The workflow for the MTT assay is presented in Table 1.

Table 1

Workflow for MTT cytotoxicity assay[10]

Time (in hours)	Procedure
00:00	96-well culture microtiter plate is seeded with L929 mouse fibroblast cell line at a concentration of 1×104 cells/well
	Incubate for 24 h at 37°C under 5% CO2 to obtain a monolayer of cell culture
24:00	Remove the culture medium
	Prepared test material eluates, positive and negative controls were added to the microtiter plates.
	Incubate at 37°C for 24 h under 5% CO2
48:00	Evaluation of microscopic alterations using microscopy
	Discard the culture medium
	Add 50 µmL of MTT solution
	Incubate for 2 h at 37°C under 5% CO2
51:00	Remove MTT solution
	Add 100 mL of isopropanol and sway to dissolve formazan crystals
51:30	UV spectrometry at 570 nm to detect the absorption of purple crystal formazan

Rationale of MTT

MTT cytotoxicity assay measures cell viability by quantifying the metabolic activity in cells. Yellow water-soluble MTT is metabolized by vital cells to a purple insoluble crystal formazan. The exact mechanism of this cellular process is yet to be understood. However, the likely explanation could involve a reaction with NADH or other similar reducing molecules that may transfer electrons to MTT. Literature has also attributed the involvement of specific mitochondrial enzymes in MTT metabolism and hence MTT assay is used to predict mitochondrial activity. The formazan produced as a metabolite of MTT accumulates as insoluble crystalline precipitates inside the cell and near the cell surface. The quantity of formazan produced is presumed to be directly proportional to the number of viable cells. As the number of vital cells reduces, the capacity to metabolize MTT to formazan also decreases. The crystalline formazan has to be solubilized to be read by spectrometers. Various reagents such as isopropanol, DMSO, and sodium dodecyl sulfate (SDS) can be used to solubilize the formazan crystals. The formed formazan can be measured by a micro reader with a 570 nm filter.[15]

MTT data interpretation[10]

The viability of cells is directly correlated to their ability to convert MTT to purple formazan. This is assessed by the optical density (OD) read with a 570 nm filter. To calculate the cell viability, the following formula was used:

where

OD570e = mean optical density of the 100% extracts of the test sample;

OD570b = mean optical density of the blanks.

The lower the cell viability %, the higher the cytotoxic potential of the test item. If viability is reduced to <70% of the blank, the test material has a cytotoxic potential.

Statistical analysis

The results obtained were tabulated and analyzed using SPSS, version 26.0 (SPSS, Chicago, IL). One-way analysis of variance (ANOVA) with post hoc Scheffe tests was used to compare the mean OD values and cell viability between the groups. The obtained data were considered to be statistically significant when the P value was less than 0.05.

RESULTS

Table 2 presents the mean values obtained from the triplicate study of OD evaluated by the spectrometric analysis. One-way ANOVA of mean and standard deviation (SD) of the OD obtained are presented in Table 2. Table 3 shows the one-way ANOVA of mean and SD of the cell viability % obtained by employing the formula mentioned above. The highest and lowest ODs were obtained in the negative and positive controls, respectively. The mean cell viability % NC was 100.00 ± 1.22, which was the highest recorded in the experiment. There was no statistically significant difference observed between the test groups for OD and cell viability %. Accordingly, [Tables 2 and 3] the OD and cell viability % suggest that the substitution of recycled denture base resin was not cytotoxic at the concentrations of 10%, 20%, 30%, 40%, and 50% to the mouse fibroblast L929 used in the analysis. Tables 4 and 5 represent the values obtained by employing the post hoc Scheffe test for OD and cell viability %, respectively.

Table 2

One-way ANOVA of mean OD at 570 nm

Groups	Mean	Standard deviation (SD)	Standard error (SE)	F	P
R0	0.302	0.151	0.087	1570.682	> 0.05
R10	0.303	0.348	0.201
R20	0.303	0.848	0.490
R30	0.302	1.348	0.779
R40	0.303	1.848	1.068
R50	0.301	2.349	1.357
NC	0.327	0.003	0.002
PC	0.071	0.004	0.002

Table 3

One-way ANOVA of the mean of cell viability %

Groups	Mean	Standard deviation (SD)	Standard error (SE)	F	P
R0	92.633	1.413	0.815	1570.682	> 0.05
R10	92.726	0.983	0.567
R20	92.956	0.505	0.291
R30	92.540	1.458	0.841
R40	92.930	0.211	0.122
R50	92.313	1.189	0.686
NC	100.00	1.220	0.704
PC	21.950	1.283	0.741

Table 4

Post hoc Scheffe test of OD

Group	Comparative group	Mean difference	SE	Significance
R0	R10	-0.00030520	0.00297633	1.000
R0	R20	-0.00105730	0.00297633	1.000
R0	R30	0.00030520	0.00297633	1.000
R0	R40	-0.00097010	0.00297633	1.000
R0	R50	0.00104640	0.00297633	1.000
R0	NC	-0.02408900*	0.00297633	0.000
R0	PC	0.23113450*	0.00297633	0.000
R10	R20	-0.00075210	0.00297633	1.000
R10	R30	0.00061040	0.00297633	1.000
R10	R40	-0.00066490	0.00297633	1.000
R10	R50	0.00135160	0.00297633	1.000
R10	NC	-0.02378380*	0.00297633	0.000
R10	PC	0.23143970*	0.00297633	0.000
R20	R30	0.00136250	0.00297633	1.000
R20	R40	0.00008720	0.00297633	1.000
R20	R50	0.00210370	0.00297633	0.999
R20	NC	-0.02303170*	0.00297633	0.000
R20	PC	0.23219180*	0.00297633	0.000
R30	R40	0.00127530	0.00297633	1.000
R30	R50	0.00074120	0.00297633	1.000
R30	NC	-0.02439420*	0.00297633	0.000
R30	PC	0.23082930*	0.00297633	0.000
R40	R50	0.00201650	0.00297633	0.999
R40	NC	-0.02311890*	0.00297633	0.000
R40	PC	0.23210460*	0.00297633	0.000
R50	NC	-0.02513540*	0.00297633	0.000
R50	PC	0.23008810*	0.00297633	0.000
NC	PC	-0.25522350*	0.00297633	0.000

*Statistically significant mean difference

Table 5

Post hoc Scheffe test of cell viability %

Group	Comparative group	Mean difference	SE	Significance
R0	R10	-0.09333	0.91019	1.000
R0	R20	-0.32333	0.91019	1.000
R0	R30	0.09333	0.91019	1.000
R0	R40	-0.29667	0.91019	1.000
R0	R50	0.32000	0.91019	1.000
R0	NC	-7.36667*	0.91019	0.000
R0	PC	70.68333*	0.91019	0.000
R10	R20	-0.23000	0.91019	1.000
R10	R30	0.18667	0.91019	1.000
R10	R40	-0.20333	0.91019	1.000
R10	R50	0.41333	0.91019	1.000
R10	NC	-7.27333*	0.91019	0.000
R10	PC	70.77667*	0.91019	0.000
R20	R30	0.41667	0.91019	1.000
R20	R40	0.02667	0.91019	1.000
R20	R50	0.64333	0.91019	0.999
R20	NC	-7.04333*	0.91019	0.000
R20	PC	71.00667*	0.91019	0.000
R30	R40	-0.39000	0.91019	1.000
R30	R50	0.22667	0.91019	1.000
R30	NC	-7.46000*	0.91019	0.000
R30	PC	70.59000*	0.91019	0.000
R40	R50	-0.61667	0.91019	0.999
R40	NC	-7.07000*	0.91019	0.000
R40	PC	70.98000*	0.91019	0.000
R50	NC	-7.68667*	0.91019	0.000
R50	PC	70.36333*	0.91019	0.000
NC	PC	7.27333*	0.91019	0.000

*Statistically significant mean difference

DISCUSSION

The present research applied the MTT assay to evaluate the cytotoxicity of denture base resin that was substituted with recycled PMMA at concentrations of 10%, 20%, 30%, 40%, and 50%. MTT assay has been employed by Huang et al., Jorge et al., Campanha et al., Dahl et al., Ata et al., and Melilli et al. previously to evaluate the in-vitro cytotoxicity of denture base resins.[161718192021] The assay was carried out using continuous cell lines of mouse fibroblast L929, which is similar to previous studies by Vallittu et al., Cimpan et al., Jorge et al., Jorge et al., Campanha et al., Dahl et al., and Tanis et al.[17181922232425] In the present study, the prepared specimens were disinfected under ultraviolet light. The cytotoxicity was measured by an indirect method using eluates prepared from the samples. Though direct contact with dental materials is possible in oral environment, when the material is placed directly on the exposed dental pulp tissue or oral mucosa, this is generally not the case with denture base resins. Denture base resins are placed in close contact with the mucosa and interact with the environment through soluble products being leached out of the material. The direct method may have allowed for a comparison between aging intervals as each set of samples would have been statistically independent. In the present study, eluates of the test samples were prepared immediately after sample fabrication to avoid the loss of toxic substances released from the samples at the initial stage.

In-vivo, the denture base resin material would come in contact with various cells of the oral mucosa. The mouse fibroblast L929 was selected because it closely resembles the target cells that the material may come in contact with during function. It has been suggested that resins in close contact with the keratinized and non-keratinized oral epithelium may be able to infiltrate into the connective tissue layers and reach the connective tissue fibroblasts. Though the use of primary cell lines has not proven to be of obvious advantage, their economic disadvantage and inaccessibility precluded their use in the present study.

The test groups showed no statistically significant difference in cell viability % compared with the control in the present study. Denture base resins have been shown to display varying degrees of cytotoxicity in the expansive literature on the subject. These results may be due to the prevalence of unreacted compounds retained in the material after polymerization. Sheridan et al. reported that the longer the resin is left to eluate, the lower the cytotoxic effect exhibited. Cimpan et al., who evaluated three heat cure denture base resins reported significantly lower cell viability % than the control. However, studies by Jorge et al.,[23] Huang et al.,[16] and Jorge et al. have suggested that when polymerization was carried out according to the manufacturer's instructions, it decreased the cytotoxicity of the polymer. The literature also supports that processing of denture base resin in a water bath resulted in lower cytotoxicity. Jorge et al. compared heat-cure denture base resin against microwave-cured denture base resin and concluded that all compared materials exhibited cell viability of more than 80%. Although a long curing cycle was used to cure the heat-cure denture base resin samples, UV radiation may have affected the degree of polymerization and as a result the amount of residual monomer present in the material. This finding is in accordance with the results of the present study. While comparing auto polymerizing and heat-cure denture base resin, it was a general observation that heat-cured resin was less toxic.[2021242627] This could be attributed to the presence of monomer urethane dimethacrylate, which causes cell growth inhibition, and that auto polymerizing resins exhibit higher contents of residual methyl methacrylate than the heat-polymerized resins.

The intergroup comparison using post hoc Scheffe tests revealed no significant difference in the OD and cell viability %. This may be attributed to the lack of change in the chemical makeup of control and experimental groups. The high cell viability % found in this study could be attributed to the polymerization cycle that was followed to fabricate the samples. It has been established that varying the W: P ratio, time, and temperature, may influence the degree of polymerization. Increased rates of polymerization may reduce the amount of unreacted monomer—residual monomer present in the cured material. This study followed the long cure cycle of 74°C for 8 h, followed by terminal boiling for 1 h. Harrison and Huggett have reported that this may help in the maximum conversion of the monomer to polymer.[28] Urban et al., concluded that a shorter curing cycle with terminal boil promoted a lower amount of residual monomer (0.08%) when compared to a long curing cycle without terminal boiling (0.24%).[29] Hence, in the present study, a long curing cycle followed by a 1-h terminal boil was followed, which could have been the reason for the reduced amount of residual monomer present in the samples and reduced cytotoxicity.

The present study attempts to recycle and reuse PMMA scraps as a substitute to denture base resin polymer at various concentrations, which has not been attempted yet. The results of the presents studies may not be translated to in-vivo due to the presence of a complex environment and various confounding factors. The in-vitro cytotoxicity test nevertheless provides valuable information. They are easy to execute, economical, and reproducible. Further studies to understand the interactions of the recycled denture base resin are warranted to know its long-term effects on the oral mucosa and cellular physiology.

CONCLUSION

Within the limitations of this study, it can be concluded that heat cure denture base resin modified with recycled PMMA at concentrations of 10%, 20%, 30%, 40%, and 50% (w/w) shows no cytotoxic effects on the mouse fibroblast line L929.

Social implications

The recycling and reuse of dental materials are areas of focus for the sustainable growth of dentistry in the future. The ability to reuse a dental material will greatly reduce the burden on the environment and non-renewable natural resources that are used to produce these materials.

Financial support and sponsorship

The research work was undertaken from fundings provided for TPN NO.: 59169, Project Proposal under Waste Management Technology (WMT) Program, Department of Science & Technology (DST) Technology Development & Transfer (TDT) Division, Ministry of Science and Technology, Government of India.

Conflicts of interest

There are no conflicts of interest.