| 46 | Smile Dental Journal | Volume 6, Issue 4 - 2011

AbstractThis study investigated adherence of Candida albicans to denture base acrylic resins and denture soft liners with varying surface roughness.

Materials and Methods: Two denture base acrylic resins (heat cured resin and cold cured resin) and two commercial soft liners (one is heat polymerized acrylic resin based and one is room temperature polymerized silicon based) having dimensions of 10X10X1.5mm for each specimen. Each material was divided into two groups: one is processed against glass slide surface and the other is processed against dental stone (10 samples for each group). Surface roughness measurements were made using a profilometer where a stylus traverses across the layer of the surface. Human saliva was collected from volunteers and the specimens were stored in human saliva which was contaminated with yeast suspension of approximately 106 Candida albicans per milliliterandincubatedfor24hrsat37C.Afterincubation,fixationoftheattachedcells was done by treating the specimens with 100% ethanol for 3s and left to dry in sterile plates. Specimens were stained using sterilized, fixated Methylene Blue stain for 1min and subsequently evaluated under optical microscope (Olympus, Japan) at X400 magnification. Visible measurement field was calculated in mm2 and the obtained data were expressed in cell/mm2.

Results: The materials processed against glass surface showed a very high significant difference in surface roughness values than those processed against dental stone surface (student t test, P

Smile Dental Journal | Volume 6, Issue 4 - 2011| 47 |

The presence of Candida albicans on the upper fitting surface of the denture is a major causative factor in denture-associated chronic atrophic candidosis (denture stomatitis), the most common form of oral candidosis.2 Candida albicans is a dimorphic fungus that is commensal in the gastrointestinal and reproductive tracts of healthy individuals. Under certain predisposing conditions, Candida albicans can convert into a pathogen capable of causing a variety of oral infections including pseudomembranous candidiasis, erythematous candidiasis and hyperplastic candidiasis, as well as Candida-associated denture stomatitis, Candida associated angular cheilitis, rhomboid glossitis and chronic mucocutaneous candidiasis.4

Denture stomatitis is an erythematous pathogenic condition of the denture bearing mucosa and is mainly caused by microbial factors, especially Candida albicans.5 The etiology is multifactorial consisting of either ill-fitting prostheses leading to mechanical irritation or poor hygiene leading to chronic infection, regardless of the initiating process Candida ablicans is the main cause of fungal origin in denture stomatitis.6

The first step implicated in denture stomatitis is adherence of Candida to acrylic or to salivary pellicles adsorbed on the surface of dental prosthesis. This is considered the most important event in the ability of Candida albicans to colonize dentures in the mouth.4 The aim of this study is to assess the ability of Candida albicans adherence to two types of acrylic resin and two types of soft lining materials with different surface roughness.

Materials and MethodsTwo commercially available denture base acrylic resins were used, one is heat cured (HC) and the other is cold cured (CC). Two liners were used, one is heat polymerized acrylic resin based (V) and the other is room temperature polymerized silicone based (M). All of these materials were listed in Table 1.

Preparation of the specimensPink modeling wax forms (10X10X1.5) mm were punched from a sheet of wax. Stone was mixed according to the manufacturers instruction in the lower half of the flask. Two types of mold were prepared in

such a manner that in the first type, one part of the mold was dental stone and the other is glass surface, while in the second type, both parts of the mold were dental stone. To produce the specimens against the glass, a glass microscope slide was pressed onto the stone mixture in the lower part of the flask. After the stone has set, wax specimens were placed on the top of the glass slide surface. The upper part of the flask was placed in position and the dental stone was poured over the wax specimens. The flasks were separated and boiled out, and the cover glass was degreased with liquid detergent. The surface of the investing dental stone was lubricated with separating media before packing of the materials. All the tested materials were processed according to the manufacturers instructions.

Sample groupingHC1: Heat cured denture base acrylic resin processed against glassCC1: Cold cured denture base acrylic resin processed against glassV1: Heat polymerized acrylic resin-based resilient liner processed against glassM1: Room temperature polymerized silicone-based resilient liner processed against glassHC2: Heat cured denture base acrylic resin processed against dental stoneCC2: Cold cured denture base acrylic resin processed against dental stoneV2: Heat polymerized acrylic resin-based resilient liner processed against dental stoneM2: Room temperature polymerized silicone-based resilient liner processed against dental stoneEstimation of surface roughness of the specimens

The surface roughness of the specimens was measured with profilometer (Talysurf 4, Taylor Hasbon, UK), where a stylus traverses across the layer of the surface. Three readings were taken for every specimens and the average was calculated. The average surface roughness values for all tested specimens are presented in Table 2. Methods of saliva collectionWhole unstimulated saliva samples were collected and pooled from 5 healthy male volunteers to eliminate sample variation,4 aged 18 22, (mean 20 years). The

Type of Material Trade Name Manufacturer Batch Number

Heat polymerized denture base acrylic resin Major base 2 (HC) ItalyISO 1567, type I class I ADA no.12

Room temperature polymerized denture base acrylic resin Major repair 2 (CC) ItalyISO 1567, type II class I ADA no.12

Heat-polymerized acrylic resin-based resilient liner Vertex Soft (V)Vertex-Dental BV, Zeist, The Netherlands

100001

Room temperature polymerized silicone-based resilient liner Mollosil (M)Detax, GmbH & Co. KG, Germany

03008

(Table 1) Materials used in this study

| 48 | Smile Dental Journal | Volume 6, Issue 4 - 2011

saliva was collected between 9.00 and 10.00 am and the volunteers had not eaten that morning. They were not taking any drugs or medications known to affect saliva production, composition, or flow within the last three months. They were not taking any antibiotics or antifungal agents.8,7,4 Saliva was centrifuged at 14000g for 15min and then it was used immediately.8

Obtaining Candida albicans Candida albicans strain ATCC 2091 was obtained as a stock culture (from Pathological Analysis Department of the College of Health and Medical Technologies, Baghdad, Iraq), and incubated on Sabouraud dextrose agar slope at 37C for 48h. Standard amounts of this culture were inoculated into 2ml of liquid Sabouraud dextrose agar and incubated at 37C for 24h. The culture was then centrifuged (Function Line, Labofuge 400 R, Hereaus

Instruments, Germany) at 3,000 rpm for 10min and the resultant cell pellet was washed twice with phosphate-buffered saline solution (0.15 M, pH 7.3). After dilution with this solution, a final yeast suspension of approximately 106 Candida albicans per milliliter was prepared.8

Adherence assaySpecimens were sterilized in autoclave at 121C for 20min. They were then placed in separate sterile tubes and incubated with 1.5ml natural saliva (pH=7) that was contaminated with Candida albicans for 24hrs.9 After incubation, in order to remove the unattached cells, the specimens were gently removed from the tubes and rinsed by dipping them into phosphate buffer solution with 0.89% NaCl at pH 7.2 for approximately 75s. Then for fixation of the attached cells, the specimens were treated with 100% ethanol for 3s and left to dry in sterile plates. Specimens were stained using sterilized, fixated Methylene Blue stain for 1min and subsequently evaluated under optical microscope (Olympus, Japan) at X400 magnification. Visible measurement field was calculated in mm2 and the obtained data were expressed in cell/ mm2.9 (as in Table 3)

ResultsSurface roughness measurementsMean and standard deviation (SD) of surface roughness values of all tested groups are illustrated in Table 2. The highest surface roughness value was obtained with M2 (7.480.345), while the lowest value was obtained with HC1 (1.310.166). In Table 4, student t test compared the mean surface roughness values of each material according to the type of surface processing. There was a very high statistically significant differences between the groups (P0.05). In Table 6, one way ANOVA test compared the Candida albicans adhesion among materials processed against dental stone surface. There was a very high statistically significant difference among them (P

Smile Dental Journal | Volume 6, Issue 4 - 2011| 49 |

significant difference test (LSD) revealed that, there is a very high significant difference in comparing HC2 versus CC2, or in comparing HC2 versus V2, or in comparing HC2 versus M2, or in comparing CC2 versus M2, or in comparing V2 versus M2 (P0.05). In Table 8, student t test comparing the Candida albicans adhesion of all groups according to the type of surface processing. It revealed that there is a very high significant difference between the Candida albicans adhesion to smooth surfaces and the adhesion of Candida albicans to rough surfaces (P

| 50 | Smile Dental Journal | Volume 6, Issue 4 - 2011

adhesion on hydrophobic material was low. This result also agrees with16 and19, these studies stated that silicon soft liner are more susceptible to Candida albicans adhesion than acrylic resin since surface porosity, texture and biologic and physical / chemical affinity between the materials and microbial cells may be an important factor.

The results of the present study showed that molosil soft liner processed against dental stone showed significantly higher adhesion than vertex soft liner and this agrees with20, a study stated that heat polymerized soft liner showed lower adhesion than room temperature polymerized soft liner. This finding is in agreement with3, a study which explained this result due to the presence of porosities inside the matrix of the room temperature polymerized material which facilitates the penetration of blastospores. This finding agrees with5,3 and19. This finding also agrees with9, a study found that the adherence of Candida albicans on room temperature polymerized surfaces is related to the polymerization method of the material tested.

In this study, there was no statistically significant difference in Candida albicans adhesion between cold cured denture base acrylic resin (CC2) and heat polymerized acrylic resin based liner (V2) polymerized against dental stone surface. This is because the chemical composition of (V2) is similar to that of the polymethyl methacrylate of cold cured denture base acrylic resin polymer.21

ConclusionRough surfaces of the denture base and soft liner promote the adhesion of Candida albicans in vitro and the surfaces that are as smooth as possible are more desirable in terms of cleanability and prevention of fungal disease. The selection of appropriate material for a given function and their fabrication may affect the performance of the material.

References1. Radford DR, Watson TF, Walter JD Stephen J. Challacombe SJ. The

effects of surface machining on heat cured acrylic resin and two soft denture base materials: a scanning electron microscope and confocal microscope evaluation. J Prosthet Dent. 1997;78(2):200-8.

2. Waters MGJ, Williams DW, Jagger RG, Lewis MAO. Adherence of Candida albicans to experimental denture soft lining materials. J Prosthet Dent. 1997;77(3):306-12.

3. Bulad K, Taylor R, Verran J, Mc Cord F. Colonization and penetration and denture doft lining materials by cardida. albicans Dent Mater. 2004;20:167-75.

4. Elguezabell N, Maza JL, Dorronsoro S, Pontou J. Whole saliva has a dual role on the adherence and C. albicans to polyethyl methacrylate. The open Dentistry J. 2008;2:1-4.

5. Nikawa H, Jintc Hamad A, Smak RA, Kumage H, Murat H. The interactions between thermal cycled resilient denture lining materials salivary and serum pellicles and Candida albicans in vitro: part II Effects on fungal colonization. J Oral Rehab. 2000;27:124-30.

6. Kulak Ozkan Y, Kazazoglu E, Arikan A. Oral hygiene habits, denture cleanliness, presence and yeasts and stomatitis in elderly people. J Oral Rehabel. 2002;29(3):300-4.

7. Moura JS, da Silva WJ, Pereira T, Bel Cury AAD, Rodrigues Garcia RCM. Influence of acrylic resin polymerization methods and saliva on the adherence of four Candida species. J Prosthet Dent. 2006;96:205-11.

8. Radford DR, Sweet SP, Challacombe SJ, WalterJD. Adherence of Candida albicans to denture-base materials with different surface finishes. Journal of Dentistry. 1998;26:577-83.

9. Vurual C, Ozdemir G, Kurtulmus H, Kumbuloglu O, Ozcan M. Comparative effects of two different artificial body fluids on Candida albicans adhesion to soft lining materials. Dent Mater J. 2010;29(2):206-12.

10. Douglass LJ. Candida biofilm and their role in infection. Trends Microbiol. 2003;11:30-6.

11. Grubb BR, Chadburn JL, Boucher CR. Cr. In vitro microdialysis for determination and nasal liquid composition, Am J Physiol. 2002;282:1423-31.

12. NevzatoluEU,zcanM,Kulak-OzkanY,KadirT.Adherenceof Candida albicans to denture base acrylics and silicone-based resilient liner materials with different surface finishes. Clin Oral Invest. 2007;11:231-6.

13. Verran J Maryan CJ. Retention of Candida abicans on acrylic resins and silicon and different surface topography. J Prosthet Dent. 1997;77:535-9.

14. Taylor R, Maryan CH, Verran J. Retention of oral microorganisms on cobalt chromium and dental acrylic resin with different surface finishes. J Prosthet Dent. 1998;80(5):592-7.

15. Henriques M, Azeredo J, Oliveira R. Adhesion of Candida albicans and Candida dubliniensis to acrylic and hydroxyapatite. Colloids and Surfaces B:Biointerffaces. 2004;33(3-4):235-41.

16. Pereira T, Cury AADB, Cenci Ms, Rodrigues Garcia RCM. In vitro Candida colonization on acrylic resins and denture linors: Influence and surface frequencu, roughness, saliva and adhering bacteria. Int J prosthodnt. 2007;20:308-10.

17. Hammoudi IM. Evaluation of the effect of polishing techniques on surface roughness and adhesion of Candida albicans to the acrylic complete denture. A thesis submitted to the College of Dentistry in partial fulfillment of the requirements of Master of Science in Prosthodontics, 2006.

18. Periera Cenci T, Del bdcury A, Crielard W Tencate JM. Development and Candida associated denture stomtitis: New in sights. J App Oral sci. 2008;16(2):86-94.

19. Bal BT, Yavuzyilmaz H, Yucel M. A pilot study to evaluate the adhesion oral microorganisms to temporary soft lining materials. J Oral Sci. 2008;50(1):1-8.

20. Gedik H, Ozkan YK. The effect of surface roughness of silicone-based resilient liner materials on the adherence of Candida albicans and inhibition of Candida albicans with different disinfectants.Oral Health Prev Dent. 2009;7(4):347-53.

21. Mese A, Guzel KG. Effect of storage duration on the hardness and tensile bond strength of silicone- and acrylic resin-based resilient denture liners to a processed denture base acrylic resin. J Prosthet Dent. 2008;99:153-9.