ORIGINAL ARTICLE Surface adherence of Candida Albicans to different Polymethylemethacrylate Denture Base Resins: An in-vitro-study

Rajani Kalla, Harikesh Rao, Sunil Kumar MV Abstract The Objective of this study is to check the adherence of Candida albicans on the different types of Polymethylemethacrylate denture base resins and to compare the adherence of cells among the various groups. In-vitro adhesion assays on heat cured (Trevalon light, DPI, & Lucitone reinforced), light cured & self cured acrylic resins specimens using the type strain of C. albicans (192887g). The adherence observed only on the intaglio surface of the specimens. Significantly greater adhesion was seen in DPI self cure polymethylemethacrylate followed by light cure polymethylemethacrylate. Adhesion was low in Lucitone199 heat cure denture base material, followed by DPI heat cure denture base resin, which was followed by Trevalon Clear heat cure denture base resin. Lucitone 199(reinforced) had the least adherence of C. albicans.

Keywords: Candida albicans, Polymethylemethacrylate, Denture base material. Introduction:

The human oral cavity is known to harbor a multitude of organisms. Among them, Candida albicans has lately become a cause of great concern to the dental profession. Candida albicans has been termed as a notorious “opportunistic pathogen” amongst the Candida species. Clinically, the most significant member of the genus is Candida albicans which can cause infections in humans (Oral Candidiosis or Thrush) (1). The conversion from commensalism to parasitism and exuberant growth is usually associated with intraoral environmental changes (e.g. unhygienic prostheses, xerostomia) and/or systemic factors such as diabetes and immunodeficiency. An essential prerequisite for successful Candidial colonization and infection is the ability of the yeast to adhere to the superficial epithelial cells as well as to the fitting surface of the denture, considering the latter as a reservoir of infection (2). A number of materials have been tried in the fabrication of denture bases. Polymethyl methacrylate (PMMA) has been widely employed in the fabrication of dentures. Adherence of C. albicans to various denture base materials has been investigated upon. Studies have shown various factors influencing adherence of C. albicans to the denture base materials like: surface free energy, van der walls forces, hydrophobic nature of the material, pervious nature of the material, water sorption property of the material. J.C. Davenport, (3) studied the oral distribution of Candida in denture stomatitis patients. He observed that Candida was found in most of the patient smears. In general, much larger cells of Candida were seen on the denture smears rather than the smears of the mucosa. He suggested that the treatment intended the reduction of the amount of Candida present on the denture surface rather on the mucosa. L.P. Samaranayake and T.W. McFarlane, (4) studied the adherence of Candida albicans to acrylic surfaces. They showed a positive correlation between yeast concentration and number of cells adherent. In the year 1983 (5) author studied the probability of acidic pH prevailing beneath the maxillary denture to potentiate the palatal inflammation associated with

denture stomatitis. The aims of this study were:

1. To check the adherence of C. albicans on the self cure denture base resin.

2. To check the adherence of C. albicans on the light cure denture base resin.

3. To check the adherence of Candida albicans on three types of heat cure denture base resins.

- Trevalon Clear heat cure denture base resin.

- DPI heat cure denture base resin.

- Lucitone 199 (reinforced) heat cure denture base resin.

4. To compare the adherence of C. albicans on the self cure, light cure and the three types of heat cure denture base resins materials.

Materials and Method

Ideal maxillary stone cast, modeling wax, Hydraulic bench press (Dentafarm, Torino, Italy), acrylizer (Intensiv Industries), Standard Light Unit (Shofu Solidilite Light unit), complete set of finishing and polishing burs, glass slides-150, Sterile Petri dishes-30( 9cm diameter)Sterile test tubes-20 (50ml capacity), Sterile Sabouraud’s broth-500ml , Sterile Distilled water-2l, Candida albicans (strain 192887g), light microscope, Grams stains, DPI self-cure polymethylmethacrylate denture base resin, Light cure (Voco company-Profibase, Germany) polymethylmethacrylate denture base resin, Trevalon Clear (Dentsply Ltd, De Tray Division, Weybridge, UK) heat cure polymethylmethacylate denture base resin, DPI heat cure polymethylmethacrylate denture base resin, Lucitone 199 (Dentsply, Intl, York, Pa) heat cure polymethlmethacrylate denture base resin.

Sample number: 30/denture base resin, sample size: 10mmx10mm (length) and 2mm (width).

Preparation of polymethylmethacrylate specimens:

Test samples were fabricated by scribing a 10mmx10mm area of incisive papilla and a part of rugae with a 2H black lead pencil using a flexible ruler on an ideal maxillary stone cast. A 2mm thick modeling wax sheet was cut with a sharp BP knife, with a dimension of 10mm x10mm and was adapted on the scribed area of the cast with gentle finger pressure after it was softened in warm water. The cast was then invested using Type III dental stone to have a good reproduction of the surface. Keeping in mind the sample size and number, the samples were fabricated by proper mixing of powder and liquid, working time and curing time according to the manufacturer instructions for all the types of polymethylmethacrylates(6). For the means of convenience, all the five types of denture base materials were designated by Groups and then compared. They were:

Group I - Self cure denture base resin

Group II - Light cure denture base resin

Group III - Trevalon Clear heat cure denture base resin

Group 1V - DPI Heat cure denture base resin

Group V - Lucitone 199 heat cure denture base resin.

These acrylized sheets were trimmed for excess. The polished surfaces of the sheets were subjected to finishing & polishing using the standard laboratory finishing & polishing techniques. While the tissue surface was left untouched, all the samples were washed and stored in sterile distilled water.

Culture preparation: The culture preparation and the growth of C. albicans on the specimens prepared was conducted in SMS Medical College and

Hospital, Jaipur, Rajasthan. Laboratory isolates of C. albicans (192887g) were used for this study. Before each adhesion experiment, 10ml of Sabouraud’s broth was incubated with C. albicans at 37° C for 24 hrs. This was then added to 90ml of Sabouraud’s broth and further incubated at 37°C for 24 hrs to attain a stationary phase . The 48 hrs broth culture with Candida albicans (100ml) was dispensed in 10 sterile petri dishes. To these, 15 specimens in each of the petri dish containing the broth were introduced. These were then incubated at 37°C for 48 hrs. After completion of the incubation period, the specimens were removed using sterile forceps to avoid any contamination, washed in sterile phosphate buffered saline (PBS: 10Mn phosphate buffer, 2.7 Mn potassium chloride, 137 Mn sodium chloride, Ph 7.4). The specimens were then fixed using sterile methyl alcohol for 1 min after which the alcohol was drained. Staining was done using Gram’s staining technique (Fig.1).

Fig. 1- Gram staining for all specimens

Specimens were washed in water and the stained smear was allowed to dry in air. A drop of cedar-wood oil was placed over the specimen that was placed on a glass slide and observed under oil immersion lens (1000x) of microscope. Microscopically, yeast cells are dark purple and show characteristic budding (Fig: 2, 3). A total of 10 random fields were viewed under the light microscope for each of 30 samples, and the fields that showed 0 cells were not included in statistical analysis. After 10 random fields, numbers of cells were counted and were tabulated as 1 for 1, 2 for 2 and 3 for 3 and so on and used for statistical analysis.

Fig 2 - Budding cells in self cure specimen

Fig 3 - Candidal cells on Lucitone

Statistical analysis:

Table no.1 shows the overall totality of cells in all the individual groups.

Table no.2 shows a mean ± Sd (Standard deviation) values for various groups.

Table no.3 shows the values of Anova test conducted.

Results:

The values of the test result thus showed that the statistical analysis was highly significant. Hence, it was refuted that the null hypothesis was at 0.1% level of significance and it was concluded that the material used in Group-I, II, III, IV & V vary significantly. The adherence of Candida albicans was seen to be significantly low in Lucitone199 heat cure denture base material, followed by DPI heat cure denture base resin, which was followed by Trevalon Clear heat cure denture base resin. Significantly high adherence was noted in DPI self cure polymethylemethacrylate denture base resin and light cure denture base material, but the self cure showing more.

Discussion:

A commonly occurring condition observed frequently is denture stomatitis secondary to candidal infection. Numerous yeasts are commonly found on the palatal surface of the denture and this lends support to the theory that the upper denture acts as a reservoir of infection. An essential prerequisite for a successful colonization & infection of host tissue by a microorganism is the ability to adhere to superficial epithelial cells or a closely associated surface (7). The highly significant increase in adhesion of species of C. albicans when incubated in sucrose suggests that the soft, carbohydrate-rich diets consumed frequently by denture wearers could induce yeasts colonize & adhere more tenaciously to denture surfaces (8). This study was carried out to evaluate the surface adherence of Candida albicans to five different types of commonly used acrylic denture base materials. Acrylics, though economical and easy to manipulate, show some features such as water sorption and permissive surface. These features may alter the degree of adherence of Candida albicans to acrylics (9).

The methodology chosen here was based on a study by John F. Miner (1973) with following modifications. An incubation period of 48 hrs was considered for all the specimen groups. Therefore, the adherence of the cells was observed only for the tissue surface of the specimens, keeping in mind of the fact that C. albicans is mainly seen thriving in this surface. The reason being, the intaglio surface of the denture base approaches a closed system favoring microbial adhesion and colonization. Beneath a denture, bacteria and fungi colonize in the tissue surface and the epithelial surface of the mucosa. The polished surface of a denture on the other hand, presents a smooth and glossy texture, which is not much capable of harboring fungi and bacteria as compared to the rough

surface(7). Moreover, the unpolished fitting surface is more capable of absorbing infected oral fluids than the polished surface, thus creating a suitable environment for the microorganisms to grow (7, 8).

The specimens were viewed under 10 random fields. The number of fields and the number of adherent cells were also compared. It was observed that there was no association between the presence of adherent cells and the fields observed. Some of the fields showed adherent Candidal cells and some did not show any cells. This can be attributed to the irregular surface due to the simulation of tissue topography. Thus after washing the specimens, the adherent cells on the elevated areas would have been removed easily compared to the cells in the depressed areas. This can be attributed to the fields without cells and fields with cells respectively.

Summary & conclusion:

1. Heat cure polymethylmethacrylate denture base resins showed less adherence of Candidal cells compared to self cure and light cure resins.

2. Among the heat cure also, Lucitone199 showed the least adherence of Candidal cells.

3. After Lucitone199, DPI heat cure denture base resin showed less adherence to yeast followed by Trevalon Clear heat cure denture base resin.

So, according to this we can say that Lucitone199 has the least adherence to Candida. This study would be very helpful for fabricating removable prostheses for those patients who are immunologically compromised, systemically affected by diabetes mellitus, blood deficiencies, malnutrition and debilitating disorders, and also for those who require removable stents and obturators in cases of carcinomas, trauma or any maxillofacial defect. As these patients have more tendencies to get infections and Candida is the most common organism of the oral flora. As such, by using Lucitone199 as a denture base material, we can of sure make maximum use of the prostheses without compromising patients health.

Limitations of the study: The study did not use patient’s saliva which could help in simulating oral environment. Saliva plays a very pivotal role in the adherence of Candida. It consists of enzymes, proteins, antibodies, Immunoglobulins (esp. IgA) and most importantly flow which controls the growth of microorganisms in oral cavity. Other limitations include small sample size, metal denture base and other types of denture base materials which were not included in the study.

Authors affiliations

1. Dr. Rajani kalla, Assistant Professor

2. Dr. Harikesh Rao, Professor

3. Dr. Sunil Kumar M.V, Professor & Head of the Department, Department of Prosthodontics, Crown & Bridge & Implantology, Jaipur Dental College, Jaipur , Rajasthan,INDIA.

References:

1. Candida- free Wikepedia, www.googlesearch.com

2. L.P.Samaranayake. “Chronic hyperplastic candidiasis/candidiosis (Candidal leukoplakia) Crit Rev Oral Biol Oral Med, 2003; 14(4): 253-267.

3. Davenport J.C. “The role of oral distribution of Candida in denture stomatitis.” Brit.Dent.J.1970; 129:151.

4. L.P.Samaranayake & T.W.MacFarlane “An in-vitro study of the adherence of Candida albicans

to acrylic surfaces.”Arch Oral Biol. 1980, Vol.25, 603-609.

5. Samaranayake L.P, Weetman D.A.Geddes D.A.M and McFarlane T.W. “Carboxylic acids and pH of denture plaque in patients with denture stomatitis.” J.Oral Path, 1983; 12:84-89.

6. Gunjan Dhir, David W. Berzins, Virendra B. Dhuru. “Physical properties of denture base resins potentially resistant to Candida adhesion.” J. Prosthodontics 2007; 16(6):465-472.

7. Stysiak Zofia Danielwicz. “Allergy as a cause of denture sore mouth.” J.Prosth.Dent. 1971; 25(1), 16-18.

8. Gruber RG, Lucatarto EM, Molnar EJ.Fungus growth on tissue conditioners and soft denture liners. Rev Dent Liban. 1968 Jul-Oct; 18(3):36-43.

9. Tony Axcell and L.P.Samaranayake. “A proposal for reclassification of oral Candidosis.” Oral Surg Oral Med Oral Path, 1997; 84; 111-112.

10. Milan Kuhar, and Nenad Funduk, “Effects of polishing techniques on the surface roughness of acrylic denture base resins”, Journal of Prosthetic Dentistry, 2005; vol.93: 76-85.

Address of correspondence Dr. Rajani Kalla Assistant Professor Department of Prosthodontics, Crown & Bridge & Implantology Jaipur Dental College, Jaipur (RAJ). Ph. No. +971558813810

E-Mail- rajnikalla209@gmail.com

Source of Funding: Nil Conflict of interest: None Declared