Indian Journal of Dental Research

ORIGINAL RESEARCH
Year
: 2011  |  Volume : 22  |  Issue : 4  |  Page : 526--529

In vitro evaluation of cytotoxicity of denture adhesives


Samarth Kumar Agarwal1, G Praveen1, Saurabh Gupta1, Renu Tandon1, Sameer Gupta2,  
1 Department of Prosthodontics, Kothiwal Dental College and Research Centre, Moradabad, Uttar Pradesh, India
2 Department of Pathology, Kothiwal Dental College and Research Centre, Moradabad, Uttar Pradesh, India

Correspondence Address:
Samarth Kumar Agarwal
Department of Prosthodontics, Kothiwal Dental College and Research Centre, Moradabad, Uttar Pradesh
India

Abstract

Aim: The aim of the study is to assess and compare the cytotoxicity of commercially available four denture adhesives ex-vivo. Materials and Methods: Four commercially available denture adhesives namely Metrodent powder, Fixon powder, Dentiro powder and Fixon cream were selected. Normal saline was used in control group. To evaluate the cytotoxicity of denture adhesives, macrophages were isolated from peritoneal cavity of Swiss albino mice and cell integrity/cell viability method was done by using trypan blue dye. Results: Viable cells were counted and subjected to statistical analysis. ANOVA, F and «SQ»t«SQ» test were performed, which showed statistically significant values (P < 0.001). The mean percentage of viable cells was highest in the control group (95%) and lowest in Fixon powder (55.66%), with Dentiro powder the mean percentage of viable cells was 63.66%, with Metrodent powder 67.6% while with Fixon cream it was 69.33%. Conclusion: All tested denture adhesives showed varied degree of cytotoxicity that is statistically significant. The degree of toxicity was more in Fixon powder followed by Dentiro powder and Metrodent powder with least in Fixon cream.



How to cite this article:
Agarwal SK, Praveen G, Gupta S, Tandon R, Gupta S. In vitro evaluation of cytotoxicity of denture adhesives.Indian J Dent Res 2011;22:526-529


How to cite this URL:
Agarwal SK, Praveen G, Gupta S, Tandon R, Gupta S. In vitro evaluation of cytotoxicity of denture adhesives. Indian J Dent Res [serial online] 2011 [cited 2019 Oct 17 ];22:526-529
Available from: http://www.ijdr.in/text.asp?2011/22/4/526/90285


Full Text

Denture adhesive is a material used to adhere a denture to the oral mucosa. It may be required under various circumstances of dental practice. Such circumstances may be the difficult and demanding patient, poor ridge anatomy and relationships, a complete denture opposed by natural teeth, or the necessity of optimal denture retention. [1] Denture adhesives are used by few denture wearers for their satisfaction, comfort and retention. In a survey on complete denture wearers, it was concluded that 12% of women and 10% of men were using or had used denture adhesives. [2],[3] They are commercially available soluble materials in the form of powder, cream, adherent bandages, paste or liquid that is applied to the tissue surface of the denture to enhance denture retention, stability and performance. One of the most important property of any material that is to remain indefinitely in contact with vital tissues is that it must be non irritant. [4] Denture adhesives have adverse effect on tissue due to their leachable components ingested and absorbed by the oral mucosa. [5],[6],[7]

Hence, the present study has been conducted ex-vivo to assess and compare the cytotoxicity of four commercially available denture adhesives. The null hypothesis of the present study is that the denture adhesives are cytotoxic.

 Materials and Methods



Four commercially available denture adhesives, Metrodent powder, Fixon powder, Dentiro powder, Fixon cream [Table 1], [Figure 1] were selected. Study was divided into two groups, experimental group and control group. Normal saline was used in the control group. Six Swiss albino mice that weighed 140-160 g were used in the study. All the procedures were undertaken in accordance with the institutional research ethics committee.{Figure 1}{Table 1}

To evaluate the cytotoxicity of selected denture adhesives, macrophages were isolated and cell integrity/cell viability method was used with trypan blue dye. [8],[9],[10],[11] The viable and non viable cells from three separate experiments were recorded, averaged and mean percent of viable cells were taken.

The macrophages were obtained from the peritoneal cavity of the six Swiss albino mice and were transiently exposed to each prepared denture adhesive (0.5 mg mixed in 2 ml of distilled water). The cytotoxic response elicited by each brand of denture adhesive was observed by trypan blue exclusion test and compared with the control group. Standard materials [Table 2], [Figure 2] and equipments were used to perform the experiment.{Figure 2}{Table 2}

Cells and medium

The various media, solutions and cells that were used in study are as follows:

Minimal essential medium

11.7 g of minimum essential medium (MEM) with Hank's salt, phenol red and 2% sodium bicarbonate were dissolved in 1 l of distilled water. Penicillin (200 IU/ml) and streptomycin (200 μg/ml) were added to the solution and the pH was adjusted to 7.2. The resultant solution was then filtered through 0.22 micron Millipore assembly and stored at 4°C.

Trypan blue dye solution

0.1% solution of trypan blue dye was made in phosphate buffered saline. The pH of solution was adjusted to 7.2.

Method for isolation of macrophages

Macrophages were obtained from the peritoneal cavity of six Swiss albino mice. The ventral surface of each animal was scrubbed with alcohol to make them sterile. 4 ml of MEM with 10% fetal bovine serum and antibiotics was injected into the peritoneal cavity of the mice using a disposable syringe. The peritoneal fluid was collected with a pipette [Figure 3]. This peritoneal fluid contained mainly macrophages and very little amount of lymphocytes, polymorphonuclear leucocytes and fibroblast cells. [12] {Figure 3}

To isolate the macrophages, peritoneal fluid was overlayed on plastic petridishes and incubated for 30 min at 37°C in an incubator. Supernatant was discarded and adherent cells were scraped with the help of Policeman rod in MEM. Resultant fluid was centrifuged at 2000 rpm for 10 min in cold centrifugal machine [Figure 4]. Cell pellet was resuspended in MEM. More than 90% of these cells were macrophages as judged by morphology. The final concentration of cells was adjusted to 1 × 10 7 cells/ml. 0.2 ml of these cells was distributed in five Eppendorf tubes. In one tube, 0.1 ml of normal saline was mixed and treated as the control group, while other four tubes were treated with 0.1 ml of each prepared denture adhesive. The tubes were incubated at 37°C for 10 minutes. Then cell viability was evaluated by trypan blue exclusion test.{Figure 4}

Determination of cell viability by trypan blue exclusion test

Percentage of cell viability was screened by trypan blue exclusion test. The viable and nonviable cells were counted under light microscope under (×10) magnifications. The number of viable cells from three separate experiments was recorded, and mean percentage of viable cells was taken. Obtained values were subjected to statistical analysis (ANOVA, F ratio and 't' test).

 Results



The mean percentage of viable cells was found to be highest in control, that was 95% and least in Fixon powder that was 55.66%. The mean percentage of viable cells in Dentiro powder was 63.66%, in Metrodent powder it was 67.6%, while in Fixon cream it was 69.33%.

The mean percentages of viable cells in different adhesives differ significantly. It showed that cytotoxicity is maximum in Fixon powder and least in Fixon cream [Graph 1]. Analysis of variance revealed statistically significant differences among different groups in the study, which is reflected as P < 0.001 [Table 3]. In inter group comparison, 't' test was highly significant (P < 0.001) except when we compared Metrodent powder and Fixon cream (P < 0.1)[Table 4].{Table 3}{Table 4}

[INLINE:1]

The results from the cell viability assay in mice clearly demonstrated that all the tested denture adhesives proved to be cytotoxic that differed statistically.

 Discussion



The denture adhesive used by the denture wearer should be biocompatible, non toxic and non irritant. We accept the null hypothesis that denture adhesives are cytotoxic because this was proved in the study. The ex-vivo method was chosen because these are standard screening tests commonly applicable. [13] To evaluate the cytotoxicity of selected denture adhesives, macrophages were isolated and cell integrity/cell viability method was used with trypan blue dye. The Swiss albino mice were used for this experiment because they are less susceptible to post operative infection, easily available and economical. Past studies recommend use of macrophages for immuno cytotoxicity testing, because they permit the measurement of the cytotoxic response directly in the cell culture and have the ability to maintain immunological functions in the presence of different agents. These cells can be activated by bacterial components, cytokines and chemicals. [9]

To evaluate the cytotoxicity of denture adhesives, cell viability method is used because it measures more subtle effects of materials on cells by determining whether cells treated with a potential cytotoxic agent have become damaged but not killed, resulting in a slower replication rate. [10] The trypan blue is a nonvital dye that penetrates into the nonliving cells and appears blue in color, while the living cells do not take up the dye.

Results showed that all denture adhesives used for the experiment proved to be toxic to some extent. Literature review provides little information on cytotoxicity of denture adhesives. Only a few studies have shown that certain denture adhesives containing zinc caused serious neurological problems. Further research needs to be conducted on why these denture adhesives proved to be toxic and which component in them is responsible for the toxicity.

 Conclusions



From the present study, following conclusions were drawn:



All four tested denture adhesives proved to be toxic in nature.The degree of toxicity was more in Fixon powder followed by Dentiro powder. Metrodent powder showed the least toxicity of all the powder forms of denture adhesives tested, while the least was evident in Fixon cream.

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