Cytotoxicity of Resin-Based Cleansers: An In Vitro Study

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Article Information:


Group: 2010
Subgroup: Volume 12, Issue 2
Date: March 2010
Type: Original Article
Start Page: 152
End Page: 162

Authors:

  • M Vojdani
  • Department of Prosthodontic, Faculty of Dentistry, Shiraz University of Medical Sciences, Shiraz, Fars, Iran
  • M Sattari
  • Associate Professor of Immunology Department, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
  • Sh Khajehoseini
  • Department of Prosthodontic, Faculty of Dentistry, Shiraz University of Medical Sciences, Shiraz, Fars, Iran
  • M Farzin
  • Department of Prosthodontic, Faculty of Dentistry, Shiraz University of Medical Sciences, Shiraz, Fars, Iran

      Correspondence:

      Affiliation: Associate Professor of Immunology Department, School of Medicine, Shahid Beheshti University of Medical Sciences
      City, Province: Tehran,
      Country: Iran
      Tel: +98-912-2466490
      Fax: +98-21-22439970
      E-mail: mandanasattari@yahoo.com

Abstract:


Background: Cell culture has been used to study the cytotoxicity of denture base resins. Indeed, denture cleansers and their effects on the resin cytotoxicity need deep concern. The aim of the present in vitro study was to investigate the cytotoxic effect of a heat cured denture base resin treated with two denture cleansers on mouse L-929 fibroblasts.

 

Methods: Under aseptic conditions, sample disks of a heat treated resin were prepared, following manufacturers’ instruction and divided into 12 groups of ten as follows: I) soaking in 1% sodium hypochlorite, II) soaking in alkaline peroxide solution, and III) soaking in water bath for 1, 24, 72 hours and a week. Suspensions of the fibroblasts with acrylic disks were put in 24-well culture plates, and the culture media containing RPMI-1640 environment plus antibiotics and 10% fetal bovine serum were added. After incubation of the plates at considered time intervals, cytotoxicity of the resin was carried out by MTT assay.

 

Results: A significant difference was noticed for solutions in relation to the biocompatibility of the acrylic resin samples at determined time intervals. The cleansers’ soaking samples showed higher cytotoxicity in comparison to those immersed in water at each time interval. The difference between cytotoxic effects of the samples immersed in water or cleansers were significant after 72 hours and one week.

 

Conclusion: Overnight immersion in alkaline peroxide and 1% hypochlorite solutions increased the cytotoxicity of the heat cured acrylic resin, but water storage improved the biocompatibility of the material tested.

 

Keywords: Cytotoxicity; Denture base resin; Cleansers; Fibroblasts (L-929)

Manuscript Body:


Introduction

 

Acrylic resin is still the most frequently used material in denture base fabrication. Despite its satisfactory aesthetic properties, the ease of processing and accurate fit, residual methyl methacrylate (MMA), resulting from incomplete conversion of monomers into polymer, has the potential to cause irritation, inflammation, and an allergic response of oral mucosa.1-4 Clinical signs and symptoms most frequently reported include erythema, erosion and a burning sensation of the oral mucosa and tongue.4 The pathologic effects of substances such as MMA, formaldehyde, benzoic acid, methacrylic acid, dibutyl phthalate, phenyl benzoate, phenyl salicylate and dicyclonhexyl phthalate existing in the chemical composition of acrylic resin depend on the way of their entrance to the oral cavity.1,5

Residual monomer concentration varies with the methods and the conditions of polymerization.2,6,7 The variations in the chemical composition and purity of the commercially available resin systems, the degree of conversion of their constituent monomers, and manipulative variables may all affect the biological and physical properties of the acrylic resins.7,8

To ensure the safety of dental materials, in vitro cytotoxicity tests have been developed. Testing of dental materials by cell culture methods is relatively simple to perform, reproducible and cost effective, and can be carefully controlled.3 Different parameters such as cell growth inhibition, cytolysis, cytoplasmic markers and changes in metabolic activity are used to evaluate cytotoxicity of dental materials.9-12 Cytotoxic test systems vary in the way of cells employments. Most cells are transformed or are of tumor origin, like the model for cell response. Normal diploid cells differ from established or transformed cells in many ways and may respond differently to cytotoxicity challenge.13-15 However, the use of both permanent and primary cells is recommended for a better screening of the cytotoxic effects of denture base resins.16 Cell culture methods are more suitable than costly controversial animal experiments, which may have uncontrollable variables.17

Although denture storage in water could reduce the toxic agents which leach out of denture base3,8, usage of alkaline peroxide or sodium 1% hypochlorite  solutions, as two common cleansers, may change the cytotoxicity of acylic resin material tested.

Today, few data exist about the effect of different denture cleansers on the resultant biocompatibitity of acrylic resins. Therefore, the objective of this study was to determine the cytotoxic effects of denture base resin, following treatment with alkaline peroxide and 1% sodium hypochlorite as denture cleansers.

MTT assay as a common biologic test for evaluating cytotoxicity was used in this study.3

 

 

Materials and Methods

 

The material tested was a heat cured acrylic resin (Meliodent; Heraeus Kulzer, Berkshire, Germany). One hundred and twenty disk shaped specimens of acrylic resin material (2 mm thick; 5.5 mm in diameter) were fabricated according to the manufacturers’ specifications. The polymerization cycle was 7 hours at 70oc, followed by 1 hour at 100oc. Mimicking heat cured denture base fabrication, finishing and polishing were carried out. The disks were exposed to ultraviolet light for 20 minutes to kill the microorganisms which had contaminated the disk during fabrication.

Sixty acrylic disks (except for 1 hour) were immersed in fresh 1% sodium hypochlorite (Golrang-Pakshoo Co., Iran) or alkalin peroxide (Thramed Dusseldrof, Spain) solutions for 8 hours daily, and for the rest of the day, they were stored in distilled water. Forty disks were stored only in distilled water at 1, 24 and 72 hours and 1 week time intervals.  

Each disk was placed into each well of a sterile 24-well culture plate containing 0.1ml cell (3×105 cells) and 0.9 ml RPMI (Gibco BRL, Scottland) with 10% FBS (Gibco) and then placed at 37oc in Co2 incubator (5% Co2). After 1, 24 and 72 hours and one week interval, they were assessed for cytotoxic effects. The medium without disks was also considered as negative control. Another medium in which PRMI-1640 was replaced with distilled water had the role of positive control. Distilled water can cause the lysis of all cultured cells.

Mouse fibroblast cells L-929 (Pasteur Institute, Iran) were cultured in RPMI-164, supplemented with streptomycin and penicillin and 10% fetal bovine serum. They were maintained at 37oc in Co2 incubator (5% Co2).

The MTT cytotoxicity assay was first described by Mosmann18 and the results of this test are directly related to the number of viable culture cells.19 The test measures the action of the intracellular enzyme on tetrazolium salts which is converted into soluble form of formazan. The cells were subcultured and placed into 24-well culture plates (3×105 cell/ 100 microlitre of culture medium). The MTT test was done separately after one hour, 24 and 72 hours and one week of incubation. One hundred microliters of MTT solution (Sigma Chemical, USA) was added to the samples after the proper time and then incubated for 4 hours at 37oc. When the color changed, 200 microliters of HCL-Isopropanol was added to each well and mixed to dissolve the dark blue formazan crystals. Then, the Optical Density (OD) of the plates was tested with ELISA procedure (Anthous 2020, Australia). The absorbance represents the total number of viable cells. 

Statistical analysis of the data was performed, using one-way analysis of variance (ANOVA).  A p value <0.05 was considered as statistically significant.  Repeated measurement was used to rule out the possibility of re-measuring.

 

 

Results

 

The eluates of acrylic resin disks soaked in alkaline peroxide, 1% sodium hypochlorite solutions and water revealed different biocompatibilities at each time duration (Figure 1). The mean and standard deviation (SD) of optical density for control and experimental groups are shown in Table 1. Although after 1 and 24 hours, the samples which were immersed in alkaline peroxide (A), 1% sodium hypochlorite (H) and water (W) revealed cytotoxic effects, the resultant cytotoxicity by both cleansers was higher than that caused by water. At both time intervals, alkaline peroxide was in priority, followed by 1% sodium hypochlorite.  After 72 hours, the samples stored in both cleansers showed almost the same effect on fibroblasts; however, water had a lower level of cytotoxic effect in comparison to both cleansers; the effect was statistically significant (p<0.001).

 

 

Fig. 1: Mean Optical Density (OD) at each time interval

 

Table 1: Mean and standard deviation of Optical Density in three solutions and fibroblasts

 

After one hour

After 24 hours

After 72 hours

After one week

solution 

mean

SD

mean

SD

mean

SD

mean

SD

hypochlorite sodium 1%

0.244

0.050

0.173

0.036

0.158

0.056

0.235

0.096

alkaline peroxide

0.215

0.018

0.170

0.040

0.192

0.013

0.289

0.117

water

0.256

0.015

0.224

0.044

1.087

0.141

1.959

0.523

fibroblast

0.292

0.024

0.454

0.092

1.203

0.165

3.582

0.951

 

After one week, the specimens immersed in both cleansers showed approximately the same level of biocompatibility, which was significantly lower than that of the specimens stored in water (p<0.001).

After 72 hours and 1 week, the resultant cytotoxic effect of acrylic resin samples was significantly lower than that after 1 hour and 24 hours soaking in water (p<0.05).

 

 

Discussion

 

On the basis of the previous studies, it can be concluded that the cytotoxic effect of denture base resin may be related to storage time, powder to liquid ratio, polymerization method and cycle.17,20,21 To the best of our knowledge, no study has been carried out on cytotoxic effect of acrylic resins after immersion in denture cleansers. Our data revealed that cytotoxicity of denture base resin could be affected by the type of cleansers and duration of storage.

The cytotoxic effect of autopolymerized resins is greater than that of heat-activated ones. This effect is attributed to leaching of more cytotoxic components such as MMA, formaldehyde, metacrylic acid and benzoic acid.20-22 Therefore, to decrease the bias, only one common type of heat-cured acrylic resin (Meliodent) was used for all groups in our study. Since previous studies showed that long curing cycles followed by boiling at 100oc provides the maximum conversion of residual monomer3,7, we used a similar polymerization cycle for fabrication of samples. Moreover, the denture cleansers used in this study were alkaloid type and their pH was more than 7.0. It was demonstrated that leaching of cytotoxic component can occur at a range of pH from 4.0 to 6.8.23

Based on Baker et al's study, the cytotoxic effect may occur for several days after polymerization. But it can be minimized if the denture is immersed in water for 24 hours.22 According to the authors, it is recommended that dentists soak the prostheses in water for at least 72 hours before inserting them in the patient’s mouth, especially in patients with infected, inflamed or lacerated mucosa as a result of concurrent medications or nutritional problems.

Among the cleansers, the samples immersed in water showed the least cytotoxicity. It is possible that oxygen has been expelled during the immersion of resin in water and has probably reduced the oxygen effects and resulted in a higher degree of polymerization.3 But alkaline peroxide when mixed with water and sodium perborate decomposes and releases peroxide which in turn decomposes and releases O2. The cleansing effect of this product depends on the O2 release. On the other hand, 1% sodium hypochlorite affects the organic matrix by its OH- (hydroxyl) group and resists against stain.24 Perhaps, the O2 radical disturbs the methyl methacrlyate polymerization and so acrylic resin samples immersed in alkaline peroxide were more cytotoxic than those immersed in sodium hypochlorite solution.

The data of this study are relevant but cannot be directly transferred to clinical scenarios. However, the in vitro methods could play an important role in analyzing the cytotoxicity of denture base resins.3,17

Although this study showed that cytotoxicity of the resin might vary by alkaloid cleansers, future research is recommended to identify the cytotoxic effect of other cleansers and the individual components of eluates that are responsible for the cytotoxic effect. Moreover, in vivo and human clinical studies should be done to clarify the biocompatibility of acrylic resins after soaking in denture cleansers.

Within the limitation of this in vitro study, the following conclusion may be drawn. 1. Both 1% alkaline peroxide and hypochlorite solutions increase the cytotoxicity of the heat cured resin in comparison to those immersed in water and 2. The longer a denture is soaked in water, the less cytotoxic effect of acylic resin will be.

 

 

Acknowledgment

 

We would like to thank the office of Vice Chancellor of Research of Shiraz University of Medical Sciences for Financial support.

 

Conflict of interest: None declared.

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