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Table of Contents   
ORIGINAL RESEARCH  
Year : 2011  |  Volume : 22  |  Issue : 4  |  Page : 613
The effectiveness of magnesium oxide combined with tissue conditioners in inhibiting the growth of Candida albicans: An in vitro study


Department of Prosthodontics and Crown and Bridge, A. B Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka, India

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Date of Submission22-Dec-2010
Date of Decision25-Jan-2011
Date of Acceptance03-Mar-2011
Date of Web Publication26-Nov-2011
 

   Abstract 

Context : The presence of Candida albicans on the fitting surface of the denture is a major causative factor in denture stomatits. A treatment method is by combining tissue conditioner and antifungal agents.
Aims : The main objective of this study is to test the efficacy of magnesium oxide combined with two tissue conditioners (Viscogel and GC Soft), in inhibiting the growth of Candida albicans.
Settings and Design : Microbiological study was done in the Department of Microbiology, K S Hegde Medical Academy, Nitte University, Mangalore.
Materials and Methods : A total of 154 plates were prepared using Muller Hilton with Glucose and Methylene Blue dye medium and inoculated with 24-hr old standard Candida culture. Plates were divided into control and combination. Test discs with different concentrations of MgO were equidistantly placed in MgO Control, while sterile discs embedded with respective tissue conditioner were equidistantly placed in Viscogel and GC Soft controls. For combination groups, the tissue conditioners were mixed and the discs with MgO (1%, 3%, 5%, and 7%) were embedded in the mix. After 24 h of incubation, inhibition diameters were noted.
Statistical Analysis Used : The data was analysed using Mann Whitney U Test, ANOVA, Tukey HSD test.
Results : The inhibition effect of magnesium oxide 1% combined with tissue conditioners (VGC and GCC) is not significant in both the groups. The inhibition effect of MgO 5% and 7% combined with tissue conditioners (VGC and GCC) is very highly significant ( P < 0.001).
Conclusions : Magnesium oxide in combination with tissue conditioners are effective against Candida albicans; GC soft with magnesium oxide showed a better result than Viscogel with magnesium oxide; Increasing the concentration of magnesium oxide increases the zone of inhibition of Candida albicans.

Keywords: Antifungal sensitivity sterile test discs, Candida albicans, magnesium oxide powder, tissue conditioner

How to cite this article:
Kanathila H, Bhat AM, Krishna PD. The effectiveness of magnesium oxide combined with tissue conditioners in inhibiting the growth of Candida albicans: An in vitro study. Indian J Dent Res 2011;22:613

How to cite this URL:
Kanathila H, Bhat AM, Krishna PD. The effectiveness of magnesium oxide combined with tissue conditioners in inhibiting the growth of Candida albicans: An in vitro study. Indian J Dent Res [serial online] 2011 [cited 2019 Sep 22];22:613. Available from: http://www.ijdr.in/text.asp?2011/22/4/613/90324
Denture stomatitis is a common inflammatory condition that affects denture wearers and it may be present in upto 50% of upper complete denture wearers and its prevalence increases with age. [1] Microbial infection of oral tissues is one of the most important factors in denture stomatitis. Among the microorganisms, Candida albicans is considered most important for the development of denture stomatitis. Acrylic resin is the universal denture base material and the adhesion and colonization of yeasts and bacteria are found which may irritate the underlying tissues. Soft lining materials are frequently used to coat the dentures on a permanent or semi-permanent basis, and tissue conditioners are sometimes applied for periods of days to weeks. These materials also interact with oral microbes and their surface texture makes efficient mechanical cleaning difficult. [2]

Tissue conditioners that improve the health of abused denture-bearing tissues are more susceptible to colonization by microorganisms than denture base resins. The maintenance of tissue conditioners and the prevention of biofilm formation are important for oral hygiene. Mechanical and chemical cleaning methods may cause clinical problems such as deformation or surface degradation of tissue conditioners. [2],[3]

Several studies have attempted to incorporate antibacterial agents into tissue conditioners for plaque control and treatment of denture stomatitis. A method of treatment by combining tissue conditioner and antifungal agents was suggested by Douglas and Walker in 1973. in vitro studies on ketoconazole, miconazole and nystatin combined with various tissue conditioners reported 100% efficacy in inhibiting C. albicans.

In recent years, the use of inorganic antimicrobial agents has attracted the interest for the control of microorganisms. The key advantages of inorganic antimicrobial agents are improved safety and stability, which are lacking in organic antimicrobial agents. Studies conducted on the antimicrobial activities of metallic oxide powders (MgO, CaO, and ZnO) have shown them to be active against both bacteria and fungi and were found to have a broad antimicrobial spectrum. [4]

This in vitro study tests the efficacy of tissue conditioners (Viscogel and GC Soft) combined with magnesium oxide powder incorporated test discs in inhibiting the growth of Candida albicans.


   Materials and Methods Top


Microbiological study was done in the Department of Microbiology, K S Hegde Medical Academy, Nitte University, Mangalore. [Table 1] shows test materials used in the study.
Table 1: Test materials used in the study

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Preparation of the inoculums for susceptibility test procedure

Sabourauds dextrose agar medium was prepared. This medium was poured into a sterile plate. The sterile plate was then inoculated with C. albicans using a standard ATCC strain no.10231. After the inoculation, the plate was incubated for 24 h at 35 ± 2°C and 1-day old culture was obtained. Inoculum was prepared by picking five distinct colonies of approximately 1 mm from 24-h old culture grown on Sabouraud dextrose agar and incubated at 35 ± 2°C. The suspension thus obtained was vortexed and the turbidity was adjusted to 0.5 Mcfarland standard.

Preparation of the magnesium oxide discs

Sterile susceptibility test discs were used. Solutions were prepared of 1%, 3%, 5% and 7% of magnesium oxide by mixing 0.1 gm, 0.3 gm, 0.5 gm, and 0.7 gm of magnesium oxide in 100 ml of sterile saline (0.9%), respectively, and then the sterile discs were kept in the solution for ten minutes, so that the magnesium oxide was absorbed by sterile discs.

Susceptibility test procedure

Muller Hilton agar and 2% glucose with 0.5 mug/ml Methylene blue dye, i.e. GMB medium was prepared for carrying out the study. The prepared medium was poured into 154 plates to an approximated depth of 4 mm. The plates were kept in the incubator and allowed to dry for 15 minutes.

A sterile non-toxic cotton swab on a wooden applicator was dipped into the standardized inoculum. The soaked swab was rotated firmly against the upper side wall of the tube to express excess fluid. Then the entire media surface of the agar plate was streaked three times. The inoculum with the lid in place was allowed to dry.

Four plates served as the indicator of pure growth of the C. albicans. In another 20 plates magnesium oxide test discs, i.e. 1%, 3%, 5%, and 7% were placed (5 plates each) using sterile tweezers. Four discs were equidistantly placed on each plate [Figure 1].
Figure 1: Magnesium oxide 1%, 3%, 5%, and 7%

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A total of 10 plates for tissue conditioner controls (Viscogel and GC Soft) were prepared (5 plates for each tissue conditioner). Tissue conditioners were mixed in a sterile dappen dish following the recommended water powder ratios by the manufacturers and sterile discs were completely embedded in the mix. The discs were carefully taken up from the mix with the help of a sterile tweezer and placed gently over the agar plate such that the disc is completely embedded in the mix from all the sides. Care was taken not to disturb the shape of the discs while handling. Four discs were placed on each plate [Figure 2] and [Figure 3].
Figure 2: Viscogel control

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Figure 3: GC soft control

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The remaining 120 plates were divided into 8 equal groups, i.e. Viscogel with 1% MgO [Figure 4], Viscogel with 3% MgO, Viscogel with 5% MgO, Viscogel with 7% MgO [Figure 5], GC Soft with 1% MgO [Figure 6] and GC Soft with 3% MgO, GC Soft with 5% MgO, GC Soft with 7% MgO [Figure 7]. Using the same technique the test discs were completely embedded in the tissue conditioner mix and gently placed on the agar plates such that the disc is completely covered by the mix from all the sides. Care was taken to use an aseptic technique in all the steps and four discs were placed on each plate.

All the plates were placed back into the incubator within 15 min of placing the discs. The plates were incubated at 35 ± 2°C for 24 hrs. Inhibition diameters were noted.
Figure 4: Viscogel with 1% MgO

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Figure 5: Viscogel with 7% MgO

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Figure 6: GC soft with 1% MgO

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Figure 7: GC soft with 7% MgO

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   Results Top


[Table 2] shows that there is absolutely no inhibition of C. albicans observed in Viscogel and GC Soft controls and magnesium oxide in varying concentrations showed inhibition. For the magnesium oxide controls, the inhibition diameter values ranged from a minimum of 6 mm (MgO 1%) to a maximum of 21 mm (MgO 7%). The mean inhibition diameter were 7.6 mm for MgO 1%, 12.8 mm for MgO 3%, 14.4 mm for MgO 5%, and 18.7 mm for MgO 7%.
Table 2: Comparison of Viscogel, GC soft and magnesium oxide control groups

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[Table 3] shows that the inhibition effect of magnesium oxide 1% combined with tissue conditioners [Viscogel tissue conditioner (VGC) and GC Soft tissue conditioner (GCC)] are not significant in both the groups. The inhibition effect of MgO 3% with VGC is also not significant as they showed no inhibition when compared to the inhibition effect of MgO 3% with GCC, which is highly significant (P < 0.001). The inhibition effect of MgO 5% and 7% combined with tissue conditioners (VGC and GCC) is highly significant ( P < 0.001).
Table 3: To test the effectiveness of magnesium oxide combined with two tissue conditioners (Viscogel and GC soft), in inhibiting Candida albicans

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[Table 4] and Graph 1 shows the effectiveness in inhibiting Candida albicans by using different tissue conditioner type (Viscogel and GC soft). Viscogel group gives a value of 193.75 ( P < 0.001) and GC soft gives a value of 384.94 ( P < 0.001), which shows high significance.
Table 4: Comparison of the effectiveness of the tissue conditioner type with magnesium oxide in inhibiting Candida albicans

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[Table 5] shows that in Viscogel group, MgO 3% and 5% compared to higher concentrations gave a P value < 0.001, which shows high significance. Whereas, MgO 1% when compared to MgO 3% the P value is 1, which shows it is not significant. And when compared to MgO 5% and 7%, the P value is < 0.001, which is highly significant. In GC soft group, MgO 1%, 3%, and 5% when compared to their higher concentrations gave a P value of < 0.001, which shows high significance.
Table 5: To determine whether the effectiveness of inhibiting Candida albicans varies significantly by varying the concentration of magnesium oxide

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   Discussion Top


C. albicans is the most common of the Candida species found within the oral cavity. Candida species, as commensals, form part of the normal human microbial flora and, as such, they are not overt pathogens. However, in patients with dentures and those who are medically compromised or immunosuppressed, Candida infection can lead to the establishment of Candidiasis, which can be superficial or disseminated. [5]

The presence of C. albicans on the upper fitting surface of the denture is a major causative factor in denture-associated chronic atrophic candidiosis (denture stomatitis), the most common form of oral candidosis. [6] The relatively acidic and anaerobic environment under the denture provides an ideal microclimate for yeast growth. Treatment of Candida in denture wearers should include treatment of the appliance. [7]

More recently, it was found that incorporating antimicrobial agents into biomaterials is feasible. As a result inorganic antimicrobial agents have received more recognition in the antimicrobial product market. [8] The major advantages of inorganic antimicrobial agents are improved safety and stability, which are said to be lacking in organic antimicrobial agents. [4] Previous studies [4],[9],[10] have shown the antimicrobial properties of metallic oxide powders like magnesium oxide, calcium oxide, and zinc oxide. Coating agents with TiO­­ 2­­ photocatalyst can be effective for the maintenance of tissue conditioners against Candida when dentures were removed during sleep. This method although found to be efficacious, showed no photocatalystic effect on non radiation. [3]

Magnesium oxide incorporated into anti-fungal sensitivity sterile test discs were used in this in vitro study instead of powdered form of magnesium oxide itself. The possible advantages of using these discs over powdered form are by placing the disc in an area, the effect can be possibly localized and by placing the disc equidistantly, a near uniform concentration can be achieved.

In this study, there is absolutely no inhibition of C. albicans seen in Viscogel and GC Soft controls. The results obtained are in accordance with a previous study by Thomas et al., [11] who observed that Viscogel alone was completely inert and therefore would not be used without antifungal agents in the treatment of denture stomatitis where a yeast infection was present. Also, in an in vitro study conducted by Chow et al., [12] to know the efficacy of antifungal agents in tissue conditioners in inhibiting C. albicans, samples containing only tissue conditioners (Viscogel, Coe soft and Fitt) did not exhibit significant fungicidal activity as compared to combinations of antifungal agents plus tissue conditioners.

For MgO (1%, 3%, 5%, 7%) controls, all the inhibition diameters for C. albicans obtained in this study fall within the range of 6 mm-21 mm accordingly in the increasing order of concentration. Previous studies on the antifungal activities of MgO, CaO, ZnO powders quantitatively by indirect conductimetric assay have shown that MgO, CaO powders exhibited the antimicrobial activities against C. albicans, Saccharomyces cerevisiae, Aspergillus niger, and Rhizopus stolonifer. [4] Another study mentions about the antifungal properties of magnesium oxide against C. albicans, where it has helped to reduce the contamination of impression materials. [10] This suggests that magnesium oxide has an antifungal activity against C. albicans and also shows the sensitivity of the strain used to different concentrations of magnesium oxide.

In this present study, the combination of MgO 1% and 3% with Viscogel and 1% GC Soft showed minimal inhibition. This could be due to the interaction between the tissue conditioners and also the lower concentration of MgO (i.e. 1% and 3%) rendering the MgO completely ineffective. The inhibition effect of MgO 5% and 7% combined with tissue conditioners (VGC and GCC) was found to be very effective against C. albicans, which shows high significance (P > 0.001). An in vitro study by Yoshikawa et al., [4] which evaluated the antifungal activities of MgO, CaO, ZnO powders quantitatively by indirect conductimetric assay, demonstrated complete inhibition of C. albicans IFO 1060 at a concentration of 1.58 mg/ml of magnesium oxide. This suggests that the combination of magnesium oxide with tissue conditioners was effective, but differed with the concentration used. The difference shown in this study from the previous study could be due to different methodologies used or due to the difference in the C. albicans strain used.

In this study, it was found that the magnesium oxide in combination with tissue conditioners showed lesser zone of inhibition when compared to magnesium oxide controls. Magnesium oxide was found to be more effective combined with GC soft when compared to a combination of magnesium oxide with Viscogel, which showed a narrow zone of inhibition. The difference in the present study might be due to a component in Viscogel that does not allow magnesium oxide in the disc to leach out as readily as GC soft. Also this study utilizes the antifungal agent in the discs instead of the powdered form thereby creating an interphase between the antifungal agent and the tissue conditioner. This interaction demands further study.

In this study, in both VGC and GCC groups it was found that lower concentration of magnesium oxide showed a smaller/narrow inhibition diameter than higher concentrations. In Viscogel group, MgO 3% and 5% compared to higher concentrations showed very high significance ( P < 0.001). Whereas, MgO 1% when compared to MgO 3% showed no significance ( P = 1) and when compared to MgO 5% and 7% showed high significance ( P < 0.001). In GC soft group, MgO 1%, 3%, and 5% when compared to their higher concentrations showed a high significance (P < 0.001). A study conducted by Yoshikawa et al., [4] on the antifungal activity of metallic oxide showed 50% suppression of C. albicans with 0.94 mg/ml of MgO and complete inhibition with 1.58 mg/ml. This suggests that an increasing concentration of magnesium oxide with tissue conditioners increases the antifungal activity against C. albicans. The results obtained were in accordance with previous studies.

In an in vitro study, generation of active oxygen such as superoxide anion has been observed from the MgO and CaO powder slurry which has suggested that active oxygen is one primary mechanism of their antimicrobial activity. [4] In this study, the inhibition of C. albicans by the metallic oxide effect by embedding it into tissue conditioners is significant (P < 0.001) in most of the combinations. The probable cause of this inhibition is mainly due to the action of the magnesium oxide combined with the tissue conditioner.

The key advantages of the metallic oxides in tissue conditioners as a method of drug delivery are reduced patient compliance, simultaneous treatment of injured denture bearing tissue and Candidal infection and reduced application frequency.

This study can be of clinical significance, as incorporation of magnesium oxide sensitivity disc into GC soft and Viscogel shows good inhibition according to the increasing concentration of MgO used, and can be recommended for clinical use as well as for the incorporation of metallic oxide in tissue conditioners by the manufacturers.

Although previous studies [13],[14] proved that the addition of magnesium oxide drastically increased the mechanical properties of ceramics without altering biocompatibility and the addition into poly(methyl methacrylate) implant material had an effect on the compressive and transverse strength, the mechanical properties and biocompatibility of the combinations in this present study have not been evaluated. Hence, they are to be determined before being subjected to any clinical usage. This necessitates further studies in these aspects and if found to be effective, it could be recommended for the incorporation of magnesium oxide in tissue conditioners by the manufacturers as it can be a preventive measure against Candidal infection.


   Conclusions Top


  • Inhibition of Candida albicans varies significantly by varying the concentration of magnesium oxide.
  • The effectiveness in inhibiting Candida albicans varies by varying tissue conditioner type (Viscogel and GC soft). GC soft showed a better result than Viscogel.
  • Increasing the concentration of magnesium oxide increased the zone of inhibition.


 
   References Top

1.Wilson J. The aetiology, diagnosis and management of denture stomatitis. Br Dent J 1998;185:380-3.  Back to cited text no. 1
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2.Okita N, Orstavik D, Orstavik J, Ostby K. in vivo and in vitro studies on soft denture materials: Microbial adhesion and tests for antibacterial activity. Dent Mater 1991;7:155-60.  Back to cited text no. 2
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3.Akiba N, Hayakawa I, Keh ES, Wantabe A. Antifungal effects of a tissue conditioner coating agent with titanium dioxide photocatalyst. J Med Dent Sci 2005;52:223-7.  Back to cited text no. 3
    
4.Yoshikawa T, Sawai J. Quantitative evaluation of antifungal activity of metallic oxide powders(MgO, CaO and ZnO) by an indirect conductimetric assay. J App Microbiol 2004;96:803-9.  Back to cited text no. 4
    
5.Webb BC, Willcox MD, Thomas CJ, Harty DW, Knox KW. The effect of sodium hypochlorite on potential pathogenic traits of Candida albicans and other Candida species. Oral Microbiol Immunol 1995;10:334-41.  Back to cited text no. 5
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8.Fang M, Chen JH, Xu XL, Yang PH, Hildebrand HF. Antibacterial activities of inorganic agents on six bacteria associated with oral infections by two susceptibility tests. Int J Antimicrob Agents 2006;27:513-7.  Back to cited text no. 8
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9.Sawai J, Igarashi H, Hashimoto A, Kokugan T, Shimizu M. Evaluation of growth inhibitory effect of ceramics powder slurry on bacteria by conductance method. J Chem Eng Japan 1995;28:288-93.   Back to cited text no. 9
    
10. Casemiro LA, Martins CH, de Souza Fde C, Panzeri H, Ito IY. Bacterial, fungal and yeast contamination in six brands of irreversible hydrocolloid impression materials. Braz Oral Res 2007;21:106-11.  Back to cited text no. 10
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11.Thomas CJ, Nutt GM. The in vitro fungicidal properties of Viscogel, alone and combined with nystatin and amphotericin B. J Oral Rehabil 1978;5:162-72.  Back to cited text no. 11
    
12.Chow CK, Miiteai DW, Lawrence HP. Efficacy of antifungal agents in tissue conditioners in treating candidiasis. Gerodontology 1999;16:110-8.  Back to cited text no. 12
    
13.Chang P. Polymer Implant materials with improved X-ray opacity and biocompatibility. Biomaterials 1981;2:151-5.  Back to cited text no. 13
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14.Ryu HS, Hong KS, Lee JK, Kim DJ, Lee JH, Chang BS, et al. Magnesia doped HA/beta-TCP ceramics and evaluation of their biocompatibility. Biomaterials 2004;25:393-401.  Back to cited text no. 14
[PUBMED]  [FULLTEXT]  

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Correspondence Address:
Hema Kanathila
Department of Prosthodontics and Crown and Bridge, A. B Shetty Memorial Institute of Dental Sciences, Mangalore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0970-9290.90324

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    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]

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