EFFICACY OF BIOADHESIVE COATING ON ACRYLIC RESIN …

“EFFICACY OF BIOADHESIVE COATING ON ACRYLIC RESIN

DENTURES IN PEVENTING ADHESION OF CANDIDA

ALBICANS- A COMPARATIVE IN-VITRO STUDY”

By

Dr. ANUPAMA. N

Dissertation Submitted to the

Rajiv Gandhi University of Health Sciences, Karnataka, Bengaluru

In partial fulfilment

Of the requirements for the degree of

Master of Dental Surgery

In

PROSTHODONTICS INCLUDING CROWN & BRIDGE AND

IMPLANTOLOGY

Under the guidance of

Dr. SUNIL DHADED, MDS

Professor and Head

Department of Prosthodontics

AME’S Dental College and Hospital

Raichur –584103, Karnataka, India.

2015-2018

List of abbreviations

VIII

LIST OF ABBREVIATIONS

ADA American Dental Association

ANOVA Analysis Of Variance

ATCC American Type Cell Culture

BHI Brain Heart Infusion

C.albicans Candida albicans

CFU Colony Forming Unit

FRC Fiber Reinforced Composites

µm Micrometer

µl Microliters

NT Nano Technology

PMMA Poly Methyl Methacrylate

PSI Pounds Per Square Inch

SDA Sabouraud’s Dextrose Agar

SEM Scanning Electron Microscopy

UV Ultraviolet

List Of Tables

LIST OF TABLES

Table No. Contents Page No.

1 Groups for Testing Candida albicans adhesion 24

2 Direct Culture Test Values (CFU Count) 25

3 Spectrophotometric Analysis (Optical Density

Values)

25

4 Pairwise Comparison Of Direct Culture Test

Results Between The Six Groups

26

5 Pairwise Comparison Of Optical Density Values

Between The Six Groups

27

6 Overall Comparison of Mean Optical Density

Values And Standard Deviation Between the Six

Groups

28

List Of Photographs

LIST OF PHOTOGRAPHS

Figure No. Contents Page No.

1 Denture Base Materials 54

2 Adhesives used in the study 54

3 Artificial Saliva 54

4 Sabouraud’s dextrose broth 54

5 Metal die 55

6 Wax patterns 55

7 Flasking of wax patterns 55

8 Moulds after dewaxing 55

9 Fabricated fiber reinforced heat cure and

conventional heat cure acrylic samples

56

10 C.albicans ATCC 2091 strain 56

11 Prepared SD broth 56

12 Microscopic image of C.albicans - Wet mount 56

13 Microscopic image of C.albicans - Simple staining 57

14 Specimens immersed in artificial saliva containing

Candida albicans

57

15 Incubator 57

16 Plain SDA plate 57

17 Growth of Candida on SDA plate 58

18 Vortex Mixer 58

19 Centrifuge 58

20 Groups I to VI after centrifugation 59

21 Spectrophotometer 59

Abstract

Efficacy Of Bioadhesive Coating On Acrylic Resin Dentures In Preventing Adhesion Of

Candida Albicans- A Comparative In-Vitro Study

ABSTRACT

Background and Objectives: Removable dentures provide edentulous patients with the

rehabilitation of masticatory and esthetic functions .However one of the consequences of the

continual use of removable dentures is the adhesion of microorganisms and biofilm formation

in the base of the prosthesis. Candida species are opportunistic pathogens that are .frequently

isolated from the oral cavity and its biofilms are often associated with oral candidiasis .

Candida albicans that adhere to the surfaces of prostheses are essential for the pathogenesis of

denture stomatitis. Therefore to control denture stomatitis, it is important to control the

adhesion of Candida albicans on the surface of the prosthesis. Hence this in-vitro study

evaluated and compared the efficacy of coating two different adhesives on conventional heat

cure acrylic resin and fiber reinforced heat cure acrylic resin in preventing adhesion of

Candida albicans .

Methods : In this study one hundred and forty four specimens were prepared with

dimensions 10x10x2 mm. Seventy two specimens of reinforced heat cure acrylic resin and

seventy two of conventional heat cure acrylic resin. Two types of adhesives were used in this

study, Isoamyl 2 –Cyanoacrylate (BIOCRYL) and Prime & Bond NT.The specimens were

grouped from I to VI, Group I- untreated fiber reinforced heat cure resin plates (Control) ,

Group II- fiber reinforced heat cure resin with bioadhesive coating, Group III- fiber

reinforced heat cure resin with Prime and bond NT coating , Group IV- untreated

conventional heat cure acrylic resin plates (control) , Group V- conventional heat cure with

bioadhesive coating, Group VI- conventional heat cure with Prime and bond NT

coating.The sterile specimens coated with respective adhesives were immersed in artificial

saliva containing Candida albicans and incubated for 15 days.They were then removed and

Abstract

washed with distilled water and each of them were incubated in test tubes containing

sabaroud’s dextrose broth for two days. After incubation C. albicans colonies were separated

from the acrylic resin using a vortex mix for 10 minutes and then centrifuged at 4500

rpm for 5 minutes. The concentrated pellet obtained after centrifugation was used for

evaluating the candida adhesion using two methods: Direct Culture test and Serial dilutions

followed by Spectrophotometric analysis.

Results: This study was primarily done to evaluate and compare the efficacy of coating two

different adhesives on acrylic resin plates in preventing candida adhesion . Two adhesives

were used and their efficacy against candida albicans was evaluated on two different denture

base materials. Results showed that on both conventional and reinforced heat cure acrylic

resin the adhesion of Candida albicans was greatly reduced following the application of both

the adhesives when compared to the control groups(p<0.0001). When both the adhesives

were compared with one another for their efficacy against candida adhesion there was no

statistical significance in the results however when compared proportionately Iso amyl 2-

cyanoacrylate bioadhesive prevented candida adhesion slightly better than Prime and Bond

NT and the effect was in the same order for both the denture base materials.When all the six

groups were compared using a one way ANOVA the results were shown to be highly

significant(p<0.0001)

Interpretation and Conclusion : The results of the study showed that both the adhesives

used in the study were significantly effective. However there was no significant difference

between the effect of the two adhesives in preventing adhesion of C.albicans when compared

with one another. Hence within the limitations of the study it was concluded that both the

adhesives used in this are efficacious in preventing candida adhesion on acrylic resin

surfaces.

Abstract

KEYWORDS: Candida albicans, Isoamyl 2- cyanoacrylate, Prime & Bond NT, bioadhesive,

denture stomatitis, candidiasis, adhesion.

Introduction

Page 1

Efficacy Of Bioadhesive Coating On Acrylic Resin Dentures In Preventing Adhesion Of Candida

Albicans- A Comparative In-Vitro Study

The complete edentulous situation is most often witnessed in the geriatric individuals

and the elderly. In such individuas various changes associated with aging like progressive

loss of sensitivity to thermal, chemical and mechanical stimuli , chronic atrophic changes of

the residual ridges , increased xerostomia etc also need to be considered during fabrication of

complete dentures.

In human mouth dentures are indwelling medical device which prepare an optimal

environment for adhesion and multiplication of both pathogenic and non-pathogenic

organisms. 1Therefore increase in the number of removable denture in service has stimulated

interest in measures to control the deposits and stains that normally collect on them. For

esthetics the use of teeth with more natural contours and the trends towards stippled surfaces

tend to provide more recessed areas for accumulation of plaque, stain, debris and

consequently increase the patient’s cleaning problems.2

Denture hygiene is essential to prevent malodour, and accumulation of plaque/calculus

and consequent deleterious effects on the mucosa. One of the sequels of wearing denture is

the associated denture stomatitis. Its prevalence has been reported to be at 15-65%.3

Denture stomatitis was described as 'denture sore mouth' by Cahn (1936) but this term

was replaced by 'denture stomatitis' as discomfort is often absent (Cawson, 1963). Other

names used have been 'chronic denture palatitis' (Pryor, 1936), 'stomatitis venenata', used by

Fisher (1956) to describe a proposed tissue reaction to allergens in the denture base, 'chronic

atrophic candidiasis', which included angular cheilitis as well as denture stomatitis (Lehner,

1966), 'denture-related candidiasis' (Nairn, 1975), and 'stomatitis prothetica' and

'stomatopathia prothetica' (Nater et al., 1978). The term 'denture stomatitis' seems preferable

Introduction

Page 2

since no predominant cause other than the presence of a denture has been universally

accepted.4

Denture stomatitis is an inflammatory reaction of the palatal and alveolar mucosa

underlying the prosthesis. This stomatitis is more commonly seen in the maxillary mucosa.5

This disease has a multifactorial etiology of which poor hygiene and continuous denture

wearing has been the most frequent factor. Denture induced stomatitis is the pathogenic

reaction and one of the most common state of chronic candidiasis. 6

Candida albicans is the most common opportunistic fungal pathogen that is believed to

cause a variety of Candida infections in the oral cavity. 7

Denture stomatitis develops with the

adherence of Candida albicans to denture base surfaces.8

Candida albicans that adhere to the surfaces of prostheses are essential for the

pathogenesis of denture stomatitis.9 Therefore to control denture stomatitis, it is important to

control the adhesion of Candida on the surface of the prosthesis.

Tissue surfaces of dentures usually show microporosities which harbour

microorganisms difficult to remove by mechanical or chemical cleaning. Such yeasts adhere

to the denture surfaces and act as reservoirs of microorganisms. Several investigators have

analyzed the adherence of Candida albicans to acrylic resin surfaces.10-14

Candida albicans has the capability to stick and proliferate through the hard and soft

tissues of the oral cavity and produce a complex and heterogenous bacterial biofilm and are

therefore essential for the pathogenesis of Denture Stomatitis.15

The mechanisms of microorganism adhesion have been studied extensively. The basic

mechanism of microbial adhesion to either hard or epithelial surfaces is still unclear; however

Introduction

Page 3

four phases are normally recognized: phase 1—transport to the surface; phase 2-initial

adhesion; phase 3-attachment; phase 4-colonization.The four aforementioned stages are based

on the surface free energy and surface roughness. The substratum surface energy is important

in initial adhesion, although surface roughness provides a larger surface area for attachment

and a protected environment until firm attachment is completed in phase 3. 16

Larger amounts of Candida albicans have been reported to adhere to surfaces with

increased surface wettability (i.e. surface energy), due to its hydrophilic nature.10

Surface

roughness directly influences the initial surface adherence of microorganisms, biofilm

development, and colonization of Candida albicans. Materials with the roughest surfaces

usually exhibit higher yeast counts.17-20

A reported alternative to prevent microbial adhesion is the application of a protective

coating made of a pure poly (e.g.,dimethyl) siloxane. This self-bonding polymer provides an

inert and acid-resistant mono-molecular layer which can inhibit microbial attachment and

growth. This thin coating changes the surface chemistry and provides a chemically stable,

non-sticky surface which can last as long as the substrate to which it is bonded. 21,22

The use of bioadhesives for inhibition of Candida albicans adhesion to fiber reinforced

heat cure acrylic resin denture bases has not been reported till date.

Hence a study was conducted to determine the effect of coating two different

adhesives on heat cure acrylic resin and fiber reinforced heat cure acrylic resin in prevention

of Candida albicans adhesion in acrylic resin dentures.

Objectives

Page 4

AIMS:

1. To evaluate and compare the efficacy of coating two different adhesives on

conventional heat cure acrylic resin in preventing adhesion of Candida albicans.

2. To evaluate and compare the efficacy of coating two different adhesives on fiber

reinforced heat cure acrylic resin in preventing adhesion of Candida albicans

OBJECTIVES:

1. To evaluate the effect of coating heat cure acrylic resin plates with Iso Amyl 2-

Cyanoacrylate bioadhesive and Prime & Bond NT adhesive in preventing the

growth of Candida albicans

2. To evaluate the effect of coating fiber reinforced heat cure acrylic resin plates

with Iso Amyl 2-Cyanoacrylate bioadhesive and Prime & Bond NT adhesive in

preventing the growth of Candida albicans.

3. To compare the effects of coating two different adhesives on conventional heat

cure acrylic resin and fiber reinforced heat cure acrylic resin in preventing the

growth of Candida albicans.

Review Of Literature

Page 5

G. M. Brauer, J. A, Jackson, And D. J. Termini studied the bonding of acrylic

resins to dentin with 2-Cyanoacrylate Esters in which, strength and durability of the

dentin-acrylic resin joint cemented with 2-cyanoacrylate esters were evaluated. The

study concluded that, maximum adhesion is obtained with isobutyl 2-cyanoacrylate

after 1% acid pretreatment of the dentin. Hydrolytic stability is somewhat improved

by addition of polymer to the adhesive or coating around the joint.23

Shogo Minagi, Yoichiro Miyake,et al., carried out a study to evaluate the

hydrophobic interaction in Candida albicans and Candida tropicalis adherence to

various denture base resin materials in which the effects of hydrophobicities of

substrate surfaces on microbial adherence were examined by using Candida albicans

and Candida tropicalis and 21 denture base resin materials. With increasing surface

free energy of resin plates, increasing adherence of Candida albicans and decreasing

adherence of Candida tropicalis were observed. The study concluded that the surface

free energy of Candida albicans is higher than that of all resin material surfaces, and

Candida tropicalis has surface free energy lower than that of all materials used. In

calculation of the changes of free energy accompanying the adherence, the higher

adherence tendency was accompanied by a lower value for the free energy change in

both species. From a different standpoint, the closer the surface free energy of the

substrate surface and the microorganism, the higher was the probability of

adherence.10

Mutsuo Yamauchi et al., examined the effects of various denture base resin

surface textures on the adherence of microorganisms. S. sanguis and B. gingivalis

adhered in greater amounts to the denture base resin than the other microorganisms

tested. As to bacterial adherence according to polishing state, S. oralis, B. gingivalis


Page 6

C-101, and B. intermedius C-001 more adhered to the No.400 paper-polished surface

than to the buff-polished and smoothening-treated surfaces. S. sanguis less adhered to

the smoothening-treated surface. S. mitis and C. albicans, on the other hand more

adhered to the smoothening-treated surface. For the other microorganisms tested, no

relationship was observed between surface texture and bacterial adherence. The fall-

off test revealed no remarkable differences in the fall-off of S. sanguis and B.

gingivalis C-101 by the types of surface treatment. However the fall-off of C. albicans

was poorest from the No.400 paper-polished surface. These results indicate that

smoothening the denture base surface is important for denture plaque control.24

A comparative study was done by Joanna Verran and Christopher J. Maryan on

the retention of Candida albicans on smooth and rough acrylic resin and silicone

surfaces after a washing procedure to determine the effect of surface roughness on

prosthesis infection and hygiene. Standardized cell suspensions of C. albicans were

incubated with smooth and rough acrylic resin and silicone surfaces for 1 hour at 24 °

C. After washing, cells that had been retained on the surface were stained with

acridine orange and examined with incident beam fluorescent microscopy. The results

showed that there was no significant difference in cell numbers on either of the

smooth surfaces. Significantly higher numbers of cells were observed on roughened

surfaces (silicone > acrylic resin) than on smooth surfaces. The fitting surface of the

maxillary denture was not polished. The study concluded that as the silicones used in

prostheses were processed against dental stone the resultant surface roughness may

have facilitated microbial retention and infection .It should therefore be kept to a

minimum.18


Page 7

D.R. Radford et al., assessed the in vitro adherence of candida albicans to heat-

cured hard and soft denture-base materials with varying surface roughness, and

observed the effect of a mixed salivary pellicle on candidal adhesion to these surfaces.

The results showed significantly greater adhesion of C. albicans to rough rather than

smooth surfaces , as well as increased adhesion to the machined soft lining materials

compared with acrylic and pre-coating denture-base materials with saliva reduced

candidal adhesion on all materials. It was concluded in this study that rough surfaces

on denture-base materials promote the adhesion of C. albicans in vitro. However,

saliva reduces adhesion of C. albicans and thus diminishes the effect of surface

roughness and free surface energy differences between materials.17

A study by Rebecca Taylor et al., was done to compare the retention of

Streptococcus oralis, Actinomyces viscosus, and Candida albicans on polished,

sandblasted (fine and coarse) and electrobrightened cobalt-chromium alloy and dental

acrylic resin and to assess in vitro the effect of such techniques on prosthesis

contamination. Results showed that the retention of bacterial cells was substantial

irrespective of surface finish. Maximal retention was observed on cobalt-chromium

alloy that had undergone fine sandblasting and electrobrightening . For C albicans, an

increase in surface roughness resulted in an increase in retention. The study concluded

that the cell size and the type of roughening significantly affected the retention of

microorganisms on surfaces. Electrobrightening of cobalt-chromium alloy did not

reduce the surface roughness or subsequent cell attachment.25

The aim of this in vitro study by Tuomas Waltimo et al., was to compare the

adherence of Candida albicans to the surface of denture-base polymer and to E-glass

Fibers. Test specimens were made from an autopolymerized denture-base resin


Page 8

(Palapress) reinforced with preimpregnated unidirectional E-glass fibers, which were

exposed at the surface. The study concluded that, the adherence of C albicans to E-

glass fibers was lower than to polymer matrix in the denture composite. If fibers are

exposed only during polishing of the composite, the reinforcing material appears not

to increase the adherence of this common oral yeast. However, areas with

permanently exposed fibers may provide mechanical retention for yeast cells at the

interface of the components. 26

T.D. Morgan and M. Wilson investigated the effects of surface roughness and

type of denture acrylic on the early development of a Streptococcus oralis biofilm in a

constant depth film fermentor (CDFF).Streptococcus oralis was incubated with acrylic

of known surface roughness in the CDFF. Adherent Strep. oralis were enumerated by

viable counting. Cold-cure acrylic was rougher than heat-cure acrylic after polishing

with abrasive paper of any given grit-grade. Heat-cure acrylic was colonized by fewer

bacteria than cold-cure acrylic at any given surface roughness. The number of bacteria

adhering to heat-cure and cold cure acrylic increased linearly with mean surface

roughness after 2 hour incubation, the increase being greater for the cold-cure

compared with the heat-cure acrylic. However, after 4 hour incubation surface

roughness appeared to have no effect on the number of adherent bacteria. It was

concluded by this study that the type of acrylic used and its roughness affect the early

stages of biofilm formation by Strep.oralis.27

Sang E. Park, Antony Raj Periatham, et al., investigated the ability of a new

surface-modified denture resin to reduce adhesion of Candida albicans to the denture

surface. This material has a negative charge incorporated by copolymerization of

methacrylic acid to methyl methacrylate. An in vitro system was designed to assess


Page 9

the adhesion of C. albicans to surface-charged poly(methyl methacrylate) (PMMA)

surfaces.The results showed that,as the ratio of incorporated methacrylic acid to

PMMA increased the surface area of adherent C.albicans decreased. Analysis of data

revealed a significant decrease in C. albicans adhesion to the resin blocks (p < 0.0001)

when the methacrylic acid was present at 10% of the PMMA. There also existed a

correlation between the surface area of adherent C. albicans and the contact angle

measurement as the ratio of methacrylic acid changed. Surface contact angle analysis

of resin surfaces revealed that as the amount of methacrylic acid increased the surface

energy of the resins increased.21

Gordon Ramage et al.,carried out a study to assess the contribution of Candida

biofilms to the etiology of denture stomatitis. Samples of denture acrylic were

retrieved from patients with denture stomatitis and subjected to scanning electron

microscopy (SEM) analysis. Oral swab and swish samples were taken from the same

group of patients and representative C albicans isolates recovered were used to

investigate the kinetics of biofilm development in vitro. Candida biofilms could be

visualized by scanning electron microscopy directly from denture samples from

patients with denture stomatitis. These biofilms showed a propensity to adhere along

cracks and imperfections of the denture acrylic. C albicans clinical isolates were able

to form biofilms in vitro, although differences in the extent of biofilm formation were

observed for different isolates recovered from the same patient. Susceptibility testing

indicated that the resulting biofilms showed increased resistance to antifungal

treatment. Presence of serum and saliva conditioning films increased the initial

adherence of selected isolates but had little effect in overall biofilm formation. The

study concluded that Candida biofilms play a role in denture stomatitis.28


Page 10

Erdem U. Nevzatoglu et al., evaluated the surface roughness and Candida

albicans adherence on denture base acrylic resins and silicone-based resilient liners

with different surface finishes and concluded that in all types of surface finishes C.

albicans adhesion on denture base acrylics was significantly less than those of silicone

liners.19

A study by Tatiana Pereira et al.,was done to determine the influence of surface

roughness , surface free energy , saliva and bacteria on Candida adhesion to denture

materials. The roughness and surface free energy of 2 acrylic resin specimens and 2

denture liner specimens were measured and assayed in a flow chamber for bacteria

culture perfusion plus Candida albicans or C glabrata cultures. Adhesion was

determined by counting under light microscopy. Candida adhesion showed significant

differences depending on the factors involved. The overall colonization was

significantly decreased by saliva and influenced by bacteria. Candida adhesion was

strongly affected by roughness, saliva, and bacteria, but not by surface free energy.20

A review by Tatiana Pereira-Cenci et al., was done to critically discuss several

key factors controlling the adhesion of Candida species which are relevant to

denture-associated stomatitis. A comprehensive fundamental understanding is

hampered by conflicting findings due to the large variations in experimental

protocols, while other factors have never been thoroughly studied. Surface free energy

and surface roughness control the initial adherence, but temporal changes have not

been reported.29

Yuka Yoshijima et al., examined the role of hydrophobic interactions in

candidal adherence to acrylic surfaces, particularly that of the hyphal form of Candida


Page 11

albicans. Candida clinical isolates were used. Acrylic plates coated with carrageenan

and hydrocolloid were used as a hydrophilic substratum. The results of this study

suggest that hydrophilic coating of denture surfaces could be a potent method for

reduction of the adherence of relatively hydrophobic fungal cells particularly hyphal

C. albicans which causes denture stomatitis and related infections.30

Carine Ervolino de Oliveira et al.,evaluated the presence of Candida albicans

on the internal surface of the maxillary denture, and in the blood of patients with

denture stomatitis. Although the results did not show C. albicans in the bloodstream

of patients, a strong relationship between denture stomatitis and Candida species was

confirmed for both the palatal mucosa and maxillary denture31

Aiman A. Ali et al.,determined the effectiveness of coating acrylic resin

dentures on preventing Candida adhesion in which specimens were coated with 2-

Octyl Cyanoacrylate and Adper Single Bond adhesives and were immersed separately

in containers filled with artificial saliva that contained Candida albicans and

incubated for 11 days at 37 0 C. Three methods of evaluation were used to count the

adhered Candida and concluded that coating the acrylic resin dentures with Adper

Single Bond adhesive was effective in reducing Candida albicans adhesion to

dentures while coating with 2-Octyl Cyanoacrylate adhesive completely inhibited

such adhesion.16

Buket Akalın-Evren et al., studied Candida albicans adhesion on reinforced

poly methyl methacrylate denture resin and the effect of fibre architecture and

exposure to saliva where fiber-reinforced composites (FRC) were used to reinforce

the prosthetic and restorative appliances. This study evaluated the adhesion of


Page 12

Candida albicans to a denture base resin reinforced with E-glass FRCs with different

architectures [unidirectional and woven], when exposed to either saliva or distilled

water. They concluded that polymethylmethacrylate without FRCs collected less C.

albicans and the presence of saliva seems to reduce the adhesion32

Yoshiaki Kamikawa et al.,evaluated the adherence of Candida

albicans and Candida glabrata on a heat-cured Acron resin piece supported by silver

nanoparticles(AgNPs) by low-vacuum scanning electron microscopy (SEM) and

measuring colony-forming units. C. albicans and C. glabrata increasingly adhered to

the resin surface of the control piece over time, but the adhesion of both

Candida species to the AgNP-coated surface was significantly inhibited . Low-

vacuum SEM revealed that C. albicans and C. glabrata on the resin surface of control

pieces appeared as oval colonies, with a major axis of 3-4 𝜇m and a smooth cell wall,

but those on the AgNP-coated resin surface were less abundant than the control and

showed swollen yeast features, with a major axis of more than 5 𝜇m and a corrugated

cell wall. The results of the study suggested that a way to prevent denture-associated

oral candidiasis was by using denture base materials processed by AgNPs.33

A double blind randomised controlled clinical trial of Isoamyl-2-cyanoacrylate

with N-Butyl cyanoacrylate by S T Balamurali was done to ascertain the efficacy of

Isoamyl-2-cyanoacrylate on incisional, lacerated and visceral wounds . It was

concluded that Isoamyl 2 cyanoacrylate proved to be an efficacious material for

wound closure without producing any local allergic or other reactions.34

Jeremy L Rushbrook et al., conducted a study that aimed to establish whether

the Dermabond(2-Octyl Cyanoacrylate) adhesive demonstrated any intrinsic


Page 13

antimicrobial properties. Solidified pellets of Dermabond were placed on

standardised agar plates cultured with a variety of pathogens. Inhibition of growth

was demonstrated against Gram-positive bacteria. Culture swabs taken from the

inhibition rings demonstrated no growth, suggesting that Dermabond has a

bactericidal mechanism of action. Based on the design of this study, the results

suggest that Dermabond demonstrates bactericidal properties against Gram-positive

bacteria. Its use for wound closure following surgical intervention may reduce

postoperative wound infection by Gram-positive organisms.35

Fernanda Emiko Izumida et al.,conducted a study to evaluate the effect of

experimental coatings, containing zwitterion or hydrophilic monomers, on the

adherence of Candida albicans, Candida glabrata, and Streptococcus mutans to an

acrylic resin. Acrylic samples (smooth or rough surfaces) were left untreated

(control) or coated with one of the following experimental coatings: 3

hydroxypropylmethacrylate (HP) or sulfobetaine methacrylate (S), at concentrations

of 25, 30, or 35%. Half of the specimens were coated with saliva. The adhesion test

was performed by incubating specimens in C. albicans, C. glabrata, and S. Mutans

suspensions at 37°C for 90 min. It was concluded in this study that, the experimental

coatings significantly reduced the adherence of C. albicans, C. glabrata and S. mutans

to acrylic resin.36

Parimala Sagar et al., studied the effect of long chain cyanoacrylate as an

adhesive for intraoral wound closure and also to explore its hemostatic and

antibacterial effects. Isoamyl-2-cyanoacrylate (AMCRYLATE) was used as the

adhesive in the study. In conclusion isoamyl cyanoacrylate can be used for intraoral

wound closure, as an alternative to sutures for gluing the mucoperiosteum to bone, for


Page 14

example, after impaction removal, periapical surgeries, and cleft repair. Also,

Isoamyl-2-cyanoacrylate was the only cyanoacrylate, which could be used intraorally

and cost-effective when compared to others. Octyl cyanoacrylate could not be used as

it is not indicated in areas subject to frequent moisture; hence isoamyl-n-

cyanoacrylate was chosen for the study .37

Amal Nawasrah et al., measured the antifungal effect of henna against

Candida albicans adhered to acrylic resin as a possible method for prevention of

denture stomatitis. One-hundred-eighty acrylic plates were prepared of heat-cured

acrylic denture resin. The specimens were divided into six groups of 30 samples each.

The first group was only polymer and monomer following the conventional

manufacturer instruction for processing complete dentures. The other five groups

were processed by adding different concentration of Yamani henna powder to the

polymer in a concentration of henna: polymer 1%, 2.5%, 5%, 7.5% and 10%,

respectively. Samples were incubated in artificial saliva rich with Candida albicans at

37 0C, and the effect of henna on Candida albicans was evaluated. It was concluded

in the study that, adding henna to acrylic resin denture could be effective in

controlling Candida albicans proliferation on the denture surface; however, its

effects on the physical properties of acrylic resin denture need further studies.38

The goal of this study by Ahmad M. Al-Thobity et al., was to assess the

inhibitory effect of thymoquinone incorporated in the polymethyl methacrylate

denture base material against Candida albicans. Eighty acrylic resin specimens were

fabricated and divided into eight groups according to thymoquinone concentrations

of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, and 5% of acrylic powder. Two methods were

used to evaluate the effect of thymoquinone on Candida albicans: the slide count and


Page 15

the serial dilution test. The results of this study suggested that the incorporation of

thymoquinone into the acrylic resin denture base material might be effective in

preventing Candida albicans adhesion.39

Methodology

Page 16

Armamentarium:

1. Metal die with standard dimensions 10x10x2mm

2. Dental Flasks and Clamps

3. Test tubes

4. Measuring Jar

5. Petri dishes

6. Inoculation Tubes

7. Incubator (Labotech- Deluxe model: BDI-54)

8. Vortex Mixer{(BR-2000) BIO-RAD}

9. Laboratory Centrifuge ( Remi R-4C)

10. Light Microscope{(Binocular version) Olympus}

11. Colony Counter (Himedia)

12. Spectrophotometer ( BIO-RAD)

Materials:

1. Conventional Heat Cure Acrylic Resin Specimens(DPI- Heat cure)

2. Fiber Reinforced Denture Base Acrylic Resin Specimens(Acralyn-H)

3. Modelling wax

4. Cold Mold Seal

5. Iso amyl 2-Cyanoacrylate Biodhesive (BIOCRYL)

6. Prime & Bond NT adhesive (DENTSPLY)

7. Salivary substitute

8. Sabouraud‟s Dextrose Broth (Himedia-M033-1004)

9. Distilled water

Methodology

Page 17

Ethical Clearance:

The institutional ethical committee of AME‟s Dental College and Hospital reviewed

this proposed study and clearance was obtained.

Method of study:

The methodology was divided into following parts:

1. Fabrication of specimens.

2. Sterilization of the specimens.

3. Application of adhesives onto the specimens

4. Culture of Candida albicans

5. Exposure of the specimens to Candida albicans.

6. Preparation Of Sabouraud‟s dextrose broth

7. Separation Of Candida albicans colonies

8. Counting of the Candida cells.

9. Spectrophotometric Analysis

Fabrication of specimens:

In this study 144 specimens of fiber reinforced heat cure acrylic resin and

conventional heat cure resin were fabricated with dimensions 10x10x2 mm. Each of the

denture base materials consisted of 72 specimens respectively which were further

subdivided into three groups of 24 specimens each. The specimens were grouped from I

to VI, Group I- untreated fiber reinforced heat cure resin plates (Control) , Group II- fiber

reinforced heat cure resin with bioadhesive coating, Group III- fiber reinforced heat cure

resin with Prime and Bond NT coating , Group IV- untreated conventional heat cure

Methodology

Page 18

acrylic resin plates (control) , Group V- conventional heat cure with bioadhesive coating,

Group VI- conventional heat cure with Prime and Bond NT coating .

A stainless steel metal die having the dimensions 10x10x2 mm was used to

prepare the wax pattern. The wax patterns were flasked according to conventional

technique ensuring complete closure between the counter parts of the flask. Dewaxing

was carried out. The moulds formed were immersed in hot water to remove any traces of

petroleum or wax and also facilitate the application of separating medium. The mould

cavities thus obtained were used for the fabrication of acrylic resin specimens according

to ADA specification No.12.

The monomer and polymer were mixed and left to polymerize for 7 to 11 minutes

at 22°-24°C until dough stage was reached. For conventional and fiber reinforced heat

cure, the mixing ratio was 1:3 by weight. After the dough stage was reached it was

kneaded with fingers and placed in the molds in the metal flask. The flasks were placed in

the pneumatic press for trial packing of the denture base resin, initially at 1500 psi and

finally at 3500psi maintained for 30 minutes. The flask was clamped and left for one hour

before curing. After 1 hour, the flask was immersed in automatic polymerization tank for

1 hour and 30 minutes at 740 C and for 1 hour at 100

0C. After bench curing for 30

minutes, polymerization was carried according to the manufacturer‟s instructions. The

flask was allowed to cool to room temperature before being opened. After deflasking and

superficial cleaning, finishing was done by a standardized method using progressively

smoother sandpapers and polished on wet rag wheel with pumice slurry.

Sterilization of acrylic resin specimen:

The acrylic resin specimens were sterilized in ultraviolet light chamber for 5

minutes. As ultraviolet light does not penetrate opaque materials, the specimens were

Methodology

Page 19

overturned using sterile forceps and the other side was sterilized for 5 minutes. 24 acrylic

resin specimens each were then placed in six airtight plastic containers and sterilized in

the same chamber.

Culture of Candida albicans:

Candida albicans were obtained from ATCC 2091(American type culture

collection) standard Candida albicans strains. A streak of candida albicans was picked up

with a sterile inoculator loop and inoculated on the blood agar which was incubated for

48 hours at 30°C. Then a Brain Heart Infusion (BHI) was prepared and candida colonies

were introduced into it with an inoculator loop.

Preparation of Sabouraud’s dextrose broth:

65g of 50 ml medium was suspended in one litre of purified water and heated with

frequent agitation and boiled for one minute to completely dissolve the medium. Then it

was autoclaved at 121°C for 15 minutes. After which it was allowed to cool at room

temperature

Coating Of Specimens with adhesives:

The sterile specimens were introduced into a laminar flow cabinet consisting of

an Ultraviolet-C germicidal lamp in order to prevent contamination of specimens during

the application of adhesives. Ampoules of Isoamyl 2- cyanoacrylate were cut open and

the adhesive was loaded in the syringe provided by the manufacturer. Sterile dental

applicator tips were used to coat the specimens with the bioadhesive on all the surfaces.

The second adhesive used in the study, Prime and Bond NT was coated onto the

specimens in a similar manner .

Methodology

Page 20

Exposure of specimens to Candida albicans:

24 specimens contained in each of the six main groups were further divided into 4

subgroups containing 6 specimens each in order to ensure even distribution of organisms

on all the resin plates and to avoid any bias in the study. The specimens were then

immersed in test tubes with artificial saliva containing 3x105

cells of ATCC 2091

Candida albicans matched with a 0.5 McFarland standard , which were then incubated at

370 C for 15 days..

Incubation in Sabouraud’s Dextrose Broth:

After 15 days the samples were removed from the respective test tubes and

washed with distilled water and introduced in tubes containing 5ml of sabouraud‟s

dextrose broth . They were then incubated at 370 C for 48 hours.

Removal of Candida for evaluation:

After incubation C. albicans colonies were separated from the acrylic resin plates

using a hard vortex mix for 10 minutes from each group and then centrifuged at 4500 rpm

for 5 minutes. After centrifuging, acrylic resin plates were removed from their tubes and a

concentrated pellet was collected from each group for evaluation.

Direct Culture Test for counting of candida cells:

Ten microliters (10 μl) of the centrifuged pellet were taken from each group and

cultured in plates filled with Sabouraud‟s dextrose agar and incubated for 24 hours at

37◦C. After incubation, colonies of Candida albicans were counted in the plates using a

colony counter.

Methodology

Page 21

Serial Dilutions and Spectrophotometry:

Four tubes were filled with sabouraud‟s dextrose broth and four dilutions were

carried out at 1:25, 1: 50, 1:75 and 1:100. The fifth tube was undiluted or neat. The

turbidity of the serial dilutions was utilized for obtaining optical density values for each

of the five tubes using a spectrophotometer set at a wavelength of 490 nm.

Statistical analysis:

The data obtained was compiled systematically and further subjected to statistical

analysis. A master table was prepared and the total data was subdivided and distributed

meaningfully. Then the data was presented as individual tables along with graphs.

Statistical procedures were carried out in two steps:

1. Data compilation and presentation.

2. Statistical analysis.

Statistical analysis was done using the software „Statistical Package of Social

Science‟ (SPSS Version 22; Chicago Inc., USA). Data comparison was done by applying

specific statistical tests to find out the statistical significance of the comparisons.

Statistical tests employed for the obtained data:

Analysis of Variance (ANOVA):

One way ANOVA was used to compare the mean between the groups. It tests the

significance of difference between the means of more than two groups. It compares group

means by analysing comparisons of variance estimates. Assumptions in one way ANOVA

are dependent variables and should be normally distributed. Samples drawn from the

population should be random and cases of the samples should be independent.

Methodology

Page 22

Chi Square Test (X2):

The chi-squared test is used to determine whether there is a significant difference

between the expected frequencies and the observed frequencies in one or more categories.

Post hoc Tukey test:

Post hoc means “after the fact.” These tests are used to compare means of groups

that have been determined to have some overall statistically significant differences. The

post hoc tests are follow-ups to examine particular differences which seem worthwhile

exploring after initial statistically significant results. The post hoc tests will help to

determine if the particular pairs of values are significantly different from each other. Post

hoc tests generally require three groups to compare and the original overall analysis must

have been significant. Post hoc tests are a means for pin-pointing the source of particular

significant differences among group means and is a strong test.

Results of Spectrophotometric analysis were presented as mean and standard

deviation. Unpaired „t‟ test was used for comparison between two groups followed by

Kruskal Walli‟s ANOVA test which was used for multiple groups and followed by

Tukey‟s test for overall comparison between main groups. Chi square test was used to

compare the results of the direct culture test. For all tests a p-value of 0.05 or less was

considered for statistical significance.

Sample Size of Estimaion

Page 23

Sample size:

Sample size was calculated for outcome variable like adhesion of Candida albicans,

using the standard formula,

Where,

n=Sample size

p=prevalence of candida albicans=60%

4

q=(100-p)=40

L=Allowable error=20% of p(60)=(20x60)/100=12

Therefore ,

n=(4×60×40)/(12×12)

≃ 2

With 80% statistical power at 0.05% level of significance, the sample size for 2 groups is

72( 24 in each sub group) and since six groups are included in the study , a total of 144

samples will be used.

n=4pq/L2

n=4pq/L2

n=4pq/L2

Results

Page 24

Table 1: Groups for Testing Candida albicans adhesion

Denture Base

Materials

Group Sample Size Adhesive Used

Fiber

Reinforced

Heat Cure

I 24 Control (Untreated

specimens)

II 24 Iso amyl 2 -

Cyanoacrylate

III 24 Prime & Bond NT

Conventional

Heat Cure

IV 24 Control ( Untreated

specimens)

V 24 Iso amyl 2 -

Cyanoacrylate

VI 24 Prime & Bond NT

Results

Page 25

Table 2 : Direct Culture Test Values (CFU Count)

Table 3 : Spectrophotometric Analysis (Optical Density Values)

Dilutions ↓ Group I Group II Group III Group

IV

Group V Group VI

Neat

(Undiluted)

0.912 0.351 0.873 0.901 0.370 0.563

1:25 0.642 0.348 0.652 0.744 0.337 0.477

1:50 0.610 0.319 0.561 0.713 0.308 0.321

1:75 0.589 0.211 0.340 0.657 0.222 0.284

1:100 0.612 0.135 0.284 0.625 0.147 0.227

Test Tube Group I Group II Group III Group IV Group V Group IV

1 30x103

200 290 10x105

180 280

2 40x103

200 280 15x 104

200 241

3 55x104

200 280 10x105 180 250

4 50x105

250 300 25x105

215 255

Results

Page 26

Table 4 : Pairwise Comparison Of Direct Culture Test Results Between The

Six Groups

Overall Chi Square Value: 746100, P<0.0001, Highly Significant

Chi Square test Χ2 value ‘P’ value Significance

Group I & Group II 16070 <0.0001 HS

Group I & Group III 22720 <0.0001 HS

Group I & Group IV 720900 <0.0001 HS

Group I & Group V 14720 <0.0001 HS

Group I & Group VI 21760 <0.0001 HS

Group II & Group III 0.3893 0.9424(>0.05) NS

Group II & Group IV 2583 <0.0001 HS

Group II & Group V 2.251 0.5219(>0.05) NS

Group II & Group VI 1.399 0.7039(>0.05) NS

Group III & Group IV 3661.3 <0.0001 HS

Group III & Group V 1.865 0.6009(>0.05) NS

Group III & Group VI 1.380 0.7130(>0.05) NS

Group IV & Group V 2326 <0.0001 HS

Group IV & Group VI 3570 <0.0001 HS

Group V & Group VI 5.476 0.1401(>0.05) NS

Results

Page 27

Table 5 : Pairwise Comparison Of Optical Density Values Between The Six Groups

Unpaired ‘t’ test ‘t’ value ‘P’ value Significance

Group I & Group II 5.408 0.0006 HS

Group I & Group III 0.3177 0.31477 NS

Group I & Group IV 0.7134 0.4959 NS

Group I & Group V 5.443 0.0006 HS

Group I & Group VI 3.429 0.009 HS

Group II & Group III 2.334 0.0499 S

Group II & Group IV 7.078 <0.0001 HS

Group II & Group V 0.0676 0.9477 NS

Group II & Group VI 1.337 0.2181 NS

Group III & Group IV 1.585 0.1516 NS

Group III & Group V 2.315 0.0493 S

Group III & Group VI 1.350 0.214 NS

Group IV & Group V 7.170 <0.0001 HS

Group IV & Group VI 4.475 0.0021 HS

Group V & Group VI 1.304 0.2284 NS

Results

Page 28

Table 6 : Overall Comparison Of Mean Optical Density Values And Standard

Deviation Between The Six Groups

Groups Mean Standard

Deviation

ANOVA

Group I 0.67 0.13

P<0.0001,HS

Group II 0.27 0.1

Group III 0.54 0.24

Group IV 0.73 0.11

Group V 0.28 0.09

Group VI 0.37 0.14

Post-hoc Tukey test , p<0.001 is Highly Significant

Results

Page 29

Graph 1: CFU count from Direct culture test

Graph 2: OD values from the Spectrophotometric analysis

0

500000

1000000

1500000

2000000

2500000

3000000

3500000

4000000

4500000

5000000

Group I GroupII

Group III Group IV Group V Group IV

CO

LO

NY

FO

RM

ING

U

NIT

DIRECT CULTURE TEST

1

2

3

4

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Group I Group II Group III Group

IV

Group V Group

VI

0.67

0.27

0.54

0.73

0.28

0.37

0.13 0.1

0.24

0.11 0.09 0.14

OP

TIC

AL D

EN

SIT

Y

SPECTROPHOTOMETRIC

ANALYSIS

Mean

Sta.dev

Results

Page 30

RESULTS

This study was primarily done to evaluate and compare the efficacy of coating two

different adhesives on acrylic resin plates in preventing candida adhesion . Two adhesives

were used and their efficacy against candida albicans was evaluated on two different

denture base materials.

In this present study 144 specimens were used, out of which 72 of each fiber

reinforced heat cure resin and conventional heat cure resin material were prepared.

Specimens were divided into six groups, Group I to III for fiber reinforced heat cure resin

and Groups IV to VI for Conventional heat cure acrylic resin . Group I and IV served as

control, Groups II & IV and Groups III & VI were coated with bioadhesives and bonding

agent respectively.(Table 1)

Table II and Graph I : Direct Culture Test Values (CFU Count)

The direct culture test values showed a reduced number of colony forming units of

C.albicans in Group II and Group III when compared to the control group (Group I).

Similarly, there was a reduction in CFU count of C. albicans in Group V and Group VI

when compared to the control group (Group IV).

Table III : Spectrophotometric Analysis (Optical Density Values)

The results obtained following Spectrophotometric analysis showed a decrease in

the optical density in Group II and Group III when compared to the control group (Group

I). Similarly, optical density was reduced in Group V and Group VI when compared to

the control group (Group IV). In addition, the optical density values also reduced in each

group with subsequent dilutions.

Results

Page 31

Table IV : Pairwise Comparison Of Direct Culture Test Results Between The Six

Groups

This table depicts the intergroup comparison of CFU count which was

performed using a Chi square test. The comparison showed that p value was highly

significant (<0.0001) for group I and group II, group I and group III, group I and group

IV, group I and group V, group I and group VI, group II and group IV, group III and

group IV, group IV and group V and group IV and group VI, Whereas, intergroup

comparison between group II and group III(0.9424), group II and group V(0.5219), group

II and group VI(0.7039), group III and group V(0.6009), group III and group VI(0.7130),

group V and group VI(0.1401) showed that p value is not significant.

Table V : Pairwise Comparison Of Optical Density Values Between The Six

Groups

Pairwise intergroup comparison of optical density values following

Spectrophotometric analysis was done using unpaired t test. The comparison showed that

p value was highly significant ( <0.001) for group I and group II, group I and group V,

group I and group VI, group II and group IV, group IV and group V, group IV and group

VI. Comparisons of p value between group II and group III(0.0499), group III and group

V(0.0493) was found to be moderately significant. However, p value was found to be not

significant for intergroup comparisons between group I and group III(0.31477), group I

and group IV(0.4959), group II and group V(0.9477), group II and group IV(0.2181),

group III and group IV(0.1516), group III and group VI(0.214), group V and group

VI(0.2284)

Results

Page 32

Table VI and Graph II : Overall Comparison Of Mean Optical Density Values And

Standard Deviation of The Six Groups

An overall comparison of mean optical density values and standard deviations of

the six groups was done using one way ANOVA and the mean optical density of fiber

reinforced heat cure acrylic resin plates coated with bioadhesive iso amyl 2-cyanoacrylate

was found to be least(0.27) and the highest optical density was seen with untreated

conventional heat cure acrylic resin (control group).

Discussion

Page 33

The increased awareness of patients regarding dentistry and about the

relationship between poor oral hygiene and systemic disease has stimulated interest in

patients in improving the oral hygiene status.40

Infections of the oral cavity by various

microorganisms including Streptococcos salivarius, S.mutans, Lactobacilli,

Actinomyces, yeast like fungi (Candida), P. gingivalis , P . intermedia etc., are a matter

of major concern . Drake et al found that denture plaque containing Candida albicans

plays a major role in the pathogenesis of denture stomatitis.41

C. albicans is a diploid fungus, a normal inhabitant of skin, gastrointestinal

tract, oral and vaginal cavities. It is a polymorphic fungi i.e., it can grow as both yeast

and as filamentous cells. C.albicans is an ovoid or spherical budding yeast cell, 3-5µm

in diameter. They produce pseudohyphae that help to them to invade deeper tissue and

are often implicated in the causation of denture stomatitis and related infections.42

The prevalence of denture stomatitis among those wearing partial dentures is

markedly lower than among complete denture wearers, whose rank goes from 10% to

70%. No racial or sex predilection exists although some authors have described higher

prevalence among women depending on the population studied. Newton A V in the year

1962 classified denture stomatitis into three types based on their clinical appearance.In

Newton's type I denture stomatitis, which shows pinpoint hyperemia or localized simple

inflammation, trauma is often implicated as the causative factor. In Newton's types II

and III denture stomatitis where the denture-bearing mucosa is diffusely involved, most

workers assert that the etiology is multi-factorial. There is increasing evidence to prove

that Candida albicans colonization on the fitting surface of the prosthesis in many cases

results in denture stomatitis which is usually promoted by continuous denture wearing.43

Discussion

Page 34

In addition to poor hygiene constant denture wearing maintains relatively

anaerobic and low pH conditions between the denture base and the mucosa which can

promote opportunistic overgrowth of pathogenic yeasts such as Candida.44

Although use of mechanical and chemical denture cleansers and antifungal

treatment can eradicate C. albicans contamination, dentures must be decontaminated

and their cleanliness maintained. Without proper management the recurrence of

stomatitis is very likely after antifungal therapy is discontinued. Thus many researchers

are exploring other modalities for the prevention and management of denture

stomatitis.16,30,45

Research has proved that denture materials differ in their ability to harbour

colonies and biofilms of oral bacteria and fungi , due to areas of surface roughness and

the hydrophobicity of denture surfaces which in turn reflects on the higher or lower

susceptibility of incidence of denture stomatitis.30

Such studies have focused on

modifying the surface of acrylic resin base denture materials to make them more

resistant to adhesion by Candida. The majority of previous studies have focused on

smoothening and polishing of different acrylic resin base materials, to prevent adhesion

of candida.16

The current study proposed a novel technique in which the denture surface was

modified by coating it with a biocompatible adhesive that formed a very thin and

smooth layer. The results of this study showed that surface modification of the acrylic

resin base denture material by coating it with Isoamyl 2 cyanoacrylate or Prime & Bond

NT adhesive reduced the ability of C. albicans to adhere to its acrylic resin base surface.

The bioadhesive Isoamyl 2 cyanoacrylate (BIOCRYL) used in this study

belongs to the class of long chain cyanoacrylates which has been proven to be

Discussion

Page 35

successfully used in extraoral and intraoral wound closures thereby presenting as an

effective alternative to the use of sutures.37

It is available as 0.25ml/0.5ml/1ml Iso amyl

2- cyanoacrylate in liquid state in sterile ampoules. It solidifies within 5-10 seconds ,

solidifies rapidly in alkaline media but slowly in acidic media and is an inert,

biocompatible, hemostatic and bacteriostatic material.57

Iso amyl 2- cyanoacrylate can be used for intraoral wound closure, as an

alternative to sutures for gluing the mucoperiosteum to bone. For example it can be used

after impaction removal, in periapical surgeries, and in repair of clefts. Octyl

cyanoacrylate (DERMABOND) which is the most common tissue adhesive used for

medical and veterinary purposes , is not indicated in areas subject to frequent moisture .

Iso amyl 2- cyanoacrylate is a monomer and gets converted into polymer when it comes

in contact with moisture and therefore can be effectively used in the mouth. It is

believed to be the only cyanoacrylate, which can be used intraorally and is also easy to

use and cost effective when compared to others.37

The second adhesive used in the study, Prime & Bond NT, is a dental adhesive

which is generally used to bond a restorative material such as composite or compomer

to enamel and dentine. It is formulated by incorporating an extremely fine filler

(nanoscale filler = nanofiller) allowing production of an adhesive strengthened by a

filler but keeping a very low viscosity and therefore good penetration properties. It has a

film thickness as low as 4-6µm. It has been proved to be non toxic and biocompatible.46

Prime & Bond NT consists of PENTA which is an adhesion promoter, wetting

aid and cross linker; urethane dimethacrylate resin; nanofiller for increasing the

strength and crosslinking; initiators; stabilisors ; cetylamide hydrofluoride and acetone

which is the solvent.46

A Turkish study by Taskonak et al. (2000) investigated the effects

Discussion

Page 36

of saliva contamination on bond strengths of one-bottle bonding systems. The results

indicated that saliva contamination has no adverse effect on the bonding efficacy of

Prime & Bond NT. The results of this study proved that Prime & Bond NT remains

stable in the presence of saliva.

In the present study, a comparative analysis of results of direct culture test and

spectrophotometric analysis between group I(control) and groups II & III showed that

coating fiber reinforced heat cure acrylic resin base dentures with Iso amyl 2

Cyanoacrylate and Prime & Bond NT adhesive reduced drastically the adhesion of

Candida albicans. Further comparison of results between group II & III did not show

any significance statistically as the difference in values between the two groups was

very less, however when the results are proportionally compared , fiber reinforced heat

cure plates coated with iso amyl 2- cyanoacrylate prevented C.albicans adhesion

slightly better than Prime & Bond NT Adhesive.

A comparison between group IV( control) and groups V & VI showed that

coating conventional heat cure acrylic resin plates with Iso amyl 2 Cyanoacrylate and

Prime & Bond NT adhesive reduced the adhesion of Candida albicans. Further a

comparison of results between group V & VI did not show any significance statistically;

however when the results are proportionally compared , conventional heat cure plates

coated with iso amyl 2- cyanoacrylate prevented C.albicans adhesion slightly better

than Prime & Bond NT adhesive.

The mechanism via which the coating agents used in this study reduced adhesion

by C. albicans may be that these agents smooth the surface and fill cracks on the acrylic

resin surface and/or modify the surface hydrophilicity/hydrophobicity of the acrylic

resin. Ramage et al used scanning electron microscopy to demonstrate the propensity of

Discussion

Page 37

Candida biofilms to adhere along imperfections and cracks on denture surfaces.

Filamentous forms of Candida species were found to become deeply embedded within

these deformities.28

Von Fraunhofer and Loewy reviewed factors involved in microbial attachment and

colonization of denture surfaces, confirming that surface cracks and surface roughness

facilitate attachment of microorganisms and development of the biofilm. Furthermore,

these authors noted that prolonged brushing of denture acrylic resin with a toothbrush

and abrasive dentrifices can create surface scratches that can enhance bacterial

attachment and biofilm growth.9

Such surface cracks and roughness can be prevented by painting the acrylic resin

surfaces with iso amyl 2 cyanoacrylate which fills these cracks and irregularities and

thereby reduces the adhesion of candida.

The contribution of both electrostatic and hydrophilic/ hydrophobic forces on the

adherence of candida are important in the initial resistance or adherence of yeasts. If

should adherence occur, there is an opportunity for further bonding and formation of

denture plaque. Several factors such as saliva, other microorganisms, serum, differences

in surface texture, and chemistry may influence this complex process.10

Increasing the free surface energy of the resin material is known to increase the

surface adherence of the hydrophilic species C. albicans. At the same time, increasing

the free surface energy of the resin material decreased the adherence of the hydrophobic

species C. tropicalis. Higher adherence of microorganisms to the material is seen when

the organism had a surface free-energy closest to that of the resin.27

Discussion

Page 38

Surface hydrophobicity has been proposed to selectively increase the propensity of

hyphal forms of C. albicans to colonize denture surfaces. Adhesive agents used in this

study can coat acrylic resin dentures with a thin and glossy layer, which tends to modify

the contact angle and affect the hydrophobicity of the surface to reduce adhesion by C.

albicans.30

A comparative analysis of results between the six groups showed that painting

fiber reinforced heat cure acrylic resin base dentures with Iso amyl 2 Cyanoacrylate

adhesive had the least number of Candida adherence followed by conventional heat cure

acrylic plates painted with bioadhesive ( Iso amyl 2 cyanoacrylate).Also the efficacy of

both the adhesives used in the study was consistent in both fiber reinforced heat cure

acrylic & heat cure acrylic ,that is, the effect of the adhesives was in the same order for

both denture base materials.

Smooth acrylic resin surfaces can lead to decreased bacterial biofilm formation.24

This could be a possible explanation for the reduction of Candida adhesion ,as coating

the acrylic resin plates with Isoamyl 2- Cyanoacrylate and Prime & Bond NT adhesive

smoothened the surfaces of the acrylic resin.

Yamauchi et al demonstrated an increase in C. albicans adherence to smoothening-

treated surfaces compared to polished surfaces.24

This finding suggests that coating of

acrylic resin surfaces in this study may have mimicked the effect of polishing but not of

smoothened surfaces.

Polishing of the intaglio surfaces of dentures affects the fitting of the denture;

however, Iso amyl 2-Cyanoacrylate adhesive forms a very thin biocompatible layer,

which does not interfere with the denture fit. Similarly, Prime and Bond NT having a

Discussion

Page 39

film thickness as low as 4-6µm has negligible effect on the retention and fit of the

denture.

The two adhesives used in this study have been proven to be effective in

reducing the adhesion of Candida albicans with the bioadhesive , Isoamyl 2-

cyanoacrylate having an upper hand. However, the durability of these agents and the

effectiveness of periodic recoating of dentures with the same agents warrants further

investigation.

This study was carried out using clinically relevant C. albicans. However, it is

difficult to extrapolate the findings to the in vivo situations as the study was carried out

under laboratory conditions.

Studies simulating the application of adhesives in in vivo conditions for varied

periods of time would yield more accurate information. Neither of the two adhesives

used in the study were able to inhibit completely the adhesion of Candida cells, which

means that these adhesives alone are not sufficient. Re colonization may occur over a

period of time as candida cells remain on the surface and as the adhesive coating wears

off .

Conclusion

Page 40

Since the study was carried out under in vitro conditions, it is difficult to

extrapolate the results in vivo situations. The durability of the two adhesives, the

requirement of periodic recoatings and the effects of the denture cleaning methods

employed by the patients require further investigations to draw definitive conclusions.

Within the limitations of the study, the following conclusions were drawn:

1. Both the adhesives used in the study were highly effective in reducing the

adhesion of C.albicans on the denture base materials

2. Among the adhesives used in the study , the bioadhesive isoamyl 2- cyanoacrylate

was proven to be slightly more effective than Prime & Bond NT in preventing

Candida albicans adhesion to denture bases. However the difference was

statistically insignificant.

3. Two denture base materials were used in the study; Conventional and fiber

reinforced heat cure acrylic resin both of which are said to have different surface

topography as per the literature, owing to the incorporation of fibers in the latter.

However in this study there was no significant difference in candida adhesion on

both the materials after the application of adhesives.

4. When all the six groups were compared together, fiber reinforced heat cure acrylic

resin plates coated with bioadhesive showed least adhesion of candida , proving

that irrespective of the surface topography, isoamyl 2- cyanoacrylate was very

effective in preventing candida adhesion.

5. The efficacy of adhesives used in the study was consistent in different denture

base materials; that is, bioadhesive was slightly more effective in both

conventional and fiber reinforced heat cure resin followed by Prime & Bond NT.

6. Smoothening or polishing of the intaglio surfaces of denture affects the fitting of

the denture; however, iso amyl 2 – Cyanoacrylate adhesive forms a very thin

Conclusion

Page 41

biocompatible layer, which does not interfere with the denture fit and therefore

can serve as an effective alternative to prevent candida adhesion.

Summary

Page 42

Literature pertaining to the relationship between denture plaque, surface

topography of the denture base resins, oral pathology and novel techniques to inhibit

adhesion of microorganisms on denture surface has been reviewed. The literature reports

indicate that ;

1. Plaque on the tissue surface side of the denture is unquestionably a major

etiological factor in the pathogenesis of denture stomatitis, inflammatory papillary

hyperplasia and chronic candidiasis.

2. The surface topography of the resins has a significant contribution to microbial

adhesion; therefore altering the same could be conducive to prevent candida

adhesion.

3. Candida albicans adhere to the intaglio surfaces of the denture, which cannot be

smoothened or polished, hence requiring novel techniques to prevent adherence of

candida.

The objective of this study was to evaluate and compare the efficacy of coating

two adhesives on conventional and fiber reinforced heat cure acrylic resins in

preventing adhesion of candida albicans. The results of the study showed that both the

adhesives used in the study were notably effective. The study indicated that iso amyl 2-

cyanoacrylate was the better one of the two adhesives due to its ability to fill the cracks

and surface irregularities on the denture surface better than prime & bond NT, however

the latter was highly effective in inhibiting Candida adhesion when compared to the

control.The method of study was simple, fabricated specimens of conventional and fiber

reinforced heat cure resin were divided into six groups , coated with adhesives , immersed

in artificial saliva containing candida albicans and incubated simulating 15 days of use .

They were then removed and washed with distilled water , and each of them were

Summary

Page 43

incubated in test tubes containing sabouraud’s dextrose broth for two days. After

incubation, C. albicans colonies were separated from the acrylic resin using a vortex mix

for 10 minutes and then centrifuged at 4500 rpm for 5 minutes. Further the concentrated

pellet collected from each group after centrifugation was subjected to direct culture test

and serial dilutions followed by spectrophotometry.

The results showed that both the adhesives effectivey reduced the adhesion of candida

albicans, however there was no significant difference between the effect of the two

adhesives. It can be attributed to the fact that the adhesives used in the study could

effectively fill all the surface irregularities of the denture base materials and mimick the

effect of polishing thereby proving as efficacious alternatives in preventing candida

adhesion to denture base surfaces.

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Consent Form

Not Applicable

Ethical Committee Clearance Certificate

Page 52

Proforma

Not Applicable

Master Chart

Page 53

Photographs

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Figure 1: Denture Base materials: A) Conventional Heat Cure Resin B) Reinforced Heat Cure Resin

Figure 2: Adhesives used in the study:

A) Iso amyl 2 –Cyanoacrylate (BIOCRYL) B) Prime & Bond NT ( Dentsply)

Figure 3: Artificial saliva Figure 4: Sabouraud’s Dextrose Broth

Photographs

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Figure 5: Metal Die Figure 6: Wax Patterns

Figure 7: Flasking Of Wax Patterns

Figure 8: Moulds After Dewaxing

Photographs

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Figure 9: Specimens a) Reinforced heat cure b) Conventional heat cure resin

Figure 10 :C.albicans ATCC 2091 strain Figure 11: Prepared SD broth

Figure 12 : Microscopic image of C.albicans - Wet mount

Photographs

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Figure 13 : Microscopic image of C.albicans - Simple staining

Figure 14 :Specimens immersed in artificial saliva containing Candida albicans

( Group I to Group VI Incubated in 4 test tubes each in a similar manner)

Figure 15 :Incubator Figure 16 : Plain SDA plate

Photographs

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Figure 17 :Growth of Candida on Figure 18 : Vortex Mixer

SDA plate

Figure 19 : Centrifuge

Photographs

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Figure 20 : Groups I to VI after centrifugation

Figure 21 : Spectrophotometer

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