effect of gamma radiation sterilization versus

Effect of Gamma Radiation sterilization versus Disinfection on Removable

Partial Dentures (Microbiological study)

Thesis submitted for partial fulfillment of the requirements for master degree of Removable

Prosthodontics.

Presented by

Mostafa Abd Allah Mohammed

B.D.Sc.

Faculty of Oral and Dental Medicine,

Department of Removable Prosthodontics,

Cairo University.

2011

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الصناعية ستعاضةالإ قسم

سنانالأ و الفم طب آلية

القاهرة جامعه

ê‰ìn×†Ûa@OÝßc@óÜÇ@áÜíì@ @الصناعية ستعاضةالإ مدرس


سنانالأ و الفم طب آلية

القاهرة جامعة

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رسالة مقدمة من

صطفى عبدالله محمد م/الطبيبسنان درجة الماجستير في طب الفم و الأتمهيدا للحصول على

ستعاضة الصناعيةقسم الإ

سنانوالأ الفم طب آلية


القاهرة جامعة

2011

SUPERVISORS

Prof. Dr. SAID A. EL BASTY

Professor of Removable Prosthodontics,

Removable Prosthodontic Department,


Cairo University.

Dr. AMAL ALI SWELEM.

Lecturer of Removable Prosthodontics,

Removable Prosthodontic Department,


Cairo University.

Dr. EMAN M. FATHY EL-MAGHRABY

Assistant Professor of health radiation research department,

National Centre for Radiation Research and Technology,

Atomic Energy Authority.

Acknowledgment

Acknowledgment

First of all, thanks to Allah who gave me the power to accomplish this

research.

I’d like to express my deepest and grateful gratitude to Prof. Dr. Said A.

El Basty, professor of prosthodontic department, Faculty of Dentistry, Cairo University,

he always find the time to support, guide and encourage me. His hard work and

guidance throughout the entire research and believing in me was a motive to

work harder and without him this work wouldn’t have been possible. Special

heartful thanks to him for his great and endless support. It was a great honor to

work under his wise and supervision and I’d like to declare how much I learnt from him.

My deepest thanks and appreciation to Dr. Amal Ali Swelem, Lecturer of

prosthodontic department, Faculty of Dentistry, Cairo University, for her

heartful, great and endless support she always and still giving to me and in spite

of being abroad, she always found the time to guide and encourage me as being her

brother or even more. I have learned so much from her, and without her great

effort, this research wouldn’t have been possible.

Special and endless thanks to Dr. Eman M. Fathy El-Maghraby, Assistant

Professor of health radiation research department (Oral surgery), at National

Center for Radiation Research and Technology, Atomic Energy Authority, for

her supervision, support, heartful advices she used to give me and guiding me

honestly throughout the entire course of this research. This research would have

never been in such form without her very important and expected effort.

I’d like to express my deep gratitude to Prof. Dr. Tarif H. Sallam,

professor of clinical pathology department, Faculty of Medicine, Ain Shams University,

my soul father and advisor, for his hard work and not only for guiding me

Acknowledgment

throughout this research but also throughout my whole life. If he were my

father, he wouldn’t treat me like that.

All my respect and regards to Dr. Neveen Mostafa Nosseir, Assistant

Professor of health radiation research department (Clinical pathology), at

National Center for Radiation Research and Technology, Atomic Energy

Authority, for her heartful support and guidance in collecting and culturing

samples and also collecting data and candidal count. That part of the research

wouldn’t have been accomplished without her unlimited concern and efforts.

I’d like also to thank Dr. Sameh S. Tawfik, Assistant Professor of health

radiation research department, at National Center for Radiation Research and

Technology, Atomic Energy Authority, for his help and support throughout this

research. He was the first to teach me how to search for the needed and recent

resources either through the internet or from the dental journals.

Appreciation is extended to all members of Prosthodontic Department,

Faculty of Oral and Dental Medicine, Cairo University and Health Radiation

Research Department, National Centre for Radiation Research and Technology,

for their co-operation, help and support during this research.

I’d like to thank all my family for their unlimited support, understanding

and encouragement specially my wife, mother, brother and sister.

First of all I have the honour to dedicate this work to the soul of my father who was always beside me and who I

wished to be here today.

My mother for her support and endless love she used to give me and for standing with me and encouraging me through

all my life.

I can’t forget my dear wife, she was always there for me lifting and helping me, standing with me and giving me

confidence.

My brother and sister for supporting me and believing in me.

My soul father, uncle Tarif, who was and still there when I need him. It’s a great honour to be his nephew.

Finally I dedicate this work to my sons Ahmed & Yousef.

Dedicated to

Table of Contents

Title

page

List of Tables I

List of Figures II,III

List of abbreviations IV,V

INTRODUCTION 1

REVIEW OF LITERATURE 3

DDiissiinnffeeccttiioonn aanndd SStteerriilliizzaattiioonn ooff DDeennttaall PPrroosstthheesseess 3

DDeeffiinniittiioonnss 3

IImmppoorrttaannccee ooff ddiissiinnffeeccttiioonn 3

MMeetthhooddss ooff ddiissiinnffeeccttiioonn 7

MMeetthhooddss ooff sstteerriilliizzaattiioonn 17

IIoonniizziinngg rraaddiiaattiioonn 19

DDiirreecctt aanndd iinnddiirreecctt eeffffeeccttss ooff iioonniizziinngg rraaddiiaattiioonn 20

Mechanisms of biological effects of ionizing radiation 22

GGaammmmaa rraaddiiaattiioonn 24

MMeecchhaanniissmm ooff ggaammmmaa rraaddiiaattiioonn iinn sstteerriilliizzaattiioonn 25

AAddvvaannttaaggeess ooff ggaammmmaa rraaddiiaattiioonn sstteerriilliizzaattiioonn 25

DDiissaaddvvaannttaaggeess ooff ggaammmmaa rraaddiiaattiioonn sstteerriilliizzaattiioonn 25

OOrraall CCaannddiiddaa 29

Presence and Intra-oral distribution of Candida albicans 29

Pathogenicity of Candida albicans 30

Adherence of Candida albicans to oral mucosa 31

Adherence of Candida albicans to partial dentures 32

Factors promoting and increasing Candida adherence to dentures 32

Attempts to decrease Candida adherence to dentures 33

DDeennttuurree rreellaatteedd SSttoommaattiittiiss 34

DDeeffiinniittiioonn 34

IInncciiddeennccee 34

CCllaassssiiffiiccaattiioonn 34

SSiiggnnss aanndd ssyymmppttoommss 35

EEttiioollooggyy aanndd pprreeddiissppoossiinngg ffaaccttoorrss 36

IIssoollaattiioonn aanndd iiddeennttiiffiiccaattiioonn ooff CCaannddiiddaa aallbbiiccaannss 39

Isolation of Candida albicans from the oral cavity 39

Media and methods for the identification of Candida albicans 39

AIM OF STUDY 44

MATERIALS AND METHODS 45

RESULTS 60

DISCUSSION 67

SUMMARY AND CONCLUSION 78

REFERENCES 80

ARABIC SUMMARY 96

List of tables

I

List of tables

No. Table Legend Page

Table 1

Effect of group on mean candidal colony count

61

Table 2 Effect of time on candidal colony count in the two groups

65

List of figures

II

List of figures

No. Figure Legend Page

Figure 1 The penetrating power of different types of radiation

20

Figure 2 The phases of the mechanism of radiation effect

23

Figure 3 Preoperative photograph of the maxillary arch 47

Figure 4 Upper and lower preliminary impressions 48

Figure 5 The design drawn on the study cast 49

Figure 6 Prepared master cast 50

Figure 7 Wax pattern of the RPD on the investment cast 50

Figure 8 The RPD framework on the cast 51

Figure 9 Try-in of the RPD framework in the patients mouth

51

Figure 10 The finished upper RPD on the cast 52

Figure 11 The finished RPD checked in the patients mouth

52

Figure 12 The Fittydent super cleansing tablets 54

Figure 13 The Indian Gamma Cell 54

Figure 14 Volumetric bacteriology platinum loop 55

Figure 25 The incubator 56

List of figures

III

Figure 16 The manual contact colony counter 56

Figure 17 White candidal colonies 57

Figure 18 Creamy candidal colonies 57

Figure 19 No candidal growth 57

Figure 20 Divided petri dishes showing:

A: Candidal count before and after sterilization

B: Candidal count before and after disinfection

58

Figure 21 Candida Albicans under microscope 59

Figure 22 Effect of group on mean candidal colony count 61

Figure 23 Effect of time on candidal colony count in Group I (Disinfection group); (a) 1 month after delivery (b) Immediately after disinfection (c)

1 week after disinfection (d) 2 weeks (e) 3 weeks (f) 4 weeks (g) 5 weeks after

disinfection

63

Figure 24 Effect of time on candidal colony count in Group II (Sterilization group); (a) 1 month

after delivery (b) Immediately after Sterilization (c) 1 week after Sterilization (d) 2 weeks (e) 3 weeks (f) 4 weeks (g) 5 weeks after

Sterilization

64

Figure 25 Effect of time on candidal colony count in the two groups

66

List of abbreviations

IV


μl Microleter μm Micrometter ADA American Dental Association AIDS Acquired immunodeficiency syndrome Bleach Sodium hypochlorite C Centigrade

C.albicans Candida Albicans C60 Cobalt-60 Calgon calcium chelating dishwasher detergent CDC Centers for Disease Control Alcide LD Chlorine dioxide Co-59 Cobalt-59 Co-60 Cobalt-60 CRAs chlorine-releasing agents DNA Deoxyribonucleic acid g Gram h Hour H2O Water HIV Human immunodeficiency virus Biocide Iodophor kGy KiloGray


V

krad Kilorads MeV Mega Voltage MHz Mega Hertz Min Minute O2 oxygen P(EA-co-HEMA) poly(ethyl acrylate-co-hydroxyethyl methacrylate) PMMA Polymethylmethacrylate Rads Radiation measuring unit RPD Removable partial denture W Watt x X-ray α Alpha β Beta γ Gamma Ci Curie

Introduction

1

Introduction

It has been well established that the denture base plays a great role in

partial denture retention, stability and support. Ideal requirements of denture

bases include accuracy, sufficient strength, low specific gravity, good thermal

conductivity, biological and esthetic acceptability in addition to their ability to

be kept clean.

Previous studies have demonstrated that dental prosthesis brought into the

dental clinics for repair or adjustment are contaminated with bacteria, viruses

and fungi specially species of candida and in particular candida albicans which

is thought to be involved in the etiology of denture stomatitis.

Studies have also shown that candida and other oral microorganisms are

associated with denture plaque. Adequate maintenance of removable prosthesis

is thus needed for denture wearers to have an esthetic, odor free appliances and

good oral health. These studies supported the need to establish an effective

disinfection and sterilization protocol for any prosthetic appliance.

Denture cleaning or disinfection can be carried out by two main

approaches, chemical methods and mechanical methods or combination of both.

Studies reported that less than 60% of denture wearers use chemical cleansing

products and that 60–90% use mechanical cleansing in association with tooth

paste, soap or water.

Since some chemicals used for disinfection may become absorbed into the

material porosities and may not be completely eliminated by rinsing (i.e)

chemicals may be unintentionally introduced intra-orally, sterilization of

dentures was suggested and recommended by some investigators. Microwave

irradiation was hence introduced and was found to be a safer, simple, and

Introduction

2

inexpensive method (Dixon et al., 1999). In fact, Willcox, (2002) found that

denture sterilization using Microwave irradiation may be a more effective

method than denture disinfection by soaking in sodium hypochlorite.

Since denture sterilization by microwaving has already been

investigated, the aim of the current study was to introduce a new method of

sterilization using Gamma radiation (radio sterilization). It seemed interesting to

know whether there will be a significant difference between denture disinfection

(using a commercially available denture cleanser) and denture sterilization,

using a more sophisticated and drastic measure as gamma radiation or not?

Although the answer to this question seems to be apparent and logic, yet it was

necessary to augment it and prove it with objective proof. Hence, this study was

conducted to answer this question by comparing the effects of disinfection,

using commercially available cleansing tablets: Fittydent super cleansing

tablets, versus sterilization, using Gamma radiation, on the candidal colony

count in maxillary partial dentures.

Review of Literature

3

DDiissiinnffeeccttiioonn aanndd SStteerriilliizzaattiioonn ooff

DDeennttaall PPrroosstthheesseess

II-- DDeeffiinniittiioonnss

Disinfection: is the process of killing pathogenic organisms or rendering

them inert (Mosby, 2009).

Sterilization: Is the process of completely eliminating microbial viability

including bacteria, bacterial spores, fungi, and viruses (Academy of

Prosthodontics, 2005).

IIII-- IImmppoorrttaannccee ooff ddiissiinnffeeccttiioonn

The increasing number of patients requiring dentures and the rising

knowledge and understanding of cross infection, highlights the significance of

denture hygiene. For many years, researches have stressed upon the importance

of disinfecting dental related items; including impressions and dental

appliances. Disinfection has been, and is still and will continue to be a

fundamental topic for many investigators.

The core aim of disinfecting dental appliances is to eliminate “Denture

Plaque” with all its unwanted consequences. Denture plaque is now defined as a

dense microbial layer comprising micro-organisms and their metabolites, and it

is known to contain more than 10 organisms per gram in wet weight. Light and

electron microscopic studies have revealed that denture plaque has essentially


4

the same structure as dental plaque on natural teeth. The composition of

microbial flora of denture plaque also resembles that of dental plaque, except

for an increased number of Candida species which accompanies denture-

induced stomatitis (Nikawa et al., 1998).

Importance of disinfecting dental appliances “especially removable

partial dentures” can be summarized in the following goals:

1- To maintain supporting oral tissues healthy

Introducing a removable partial denture (RPD) into the oral cavity can

greatly change its ecological environment. This change may be quantitative,

increasing the total amount of oral micro-organisms, or qualitative, increasing a

certain part of the oral microflora (Mahonen et al., 1998).

Removable partial dentures have the physical, chemical and biological

adverse aspects which can cause caries of abutment teeth, tooth mobility,

inflammation of mucosa and residual ridge resorption. The biological adverse

aspects are thought to be serious. Wearing a removable partial denture

complicates the oral environment and restricts the flow of food and the self-

cleaning action of the buccal mucosa and tongue, which results in the

accumulation of plaque on the prosthesis and surrounding tissue (Shimura et

al., 2010).

“Dental Plaque” is a predisposing factor for caries and periodontal

disease hence endangers the supporting teeth. Similarly, “Denture Plaque” is a

predisposing factor for denture-related stomatitis and hence endangers the

supporting soft tissues .Candida albicans is the most prevalent fungus in the

human oral cavity and has been identified in the denture plaque. Due to their

high affinity to adhere to both, oral tissues and dental appliances, they have


5

become known as the primary cause for denture related stomatitis (Buergers et

al., 2008).

2- To prevent other non-oral infections

Canon et al., (1995) pointed out that there is a possibility that the

colonization of oral surfaces, including the denture fitting surface, can serve as a

reservoir for disseminated infections, such as aspiration pneumonia and

gastrointestinal infection.

Nikawa et al., (1998) also reported that the continuous swallowing or aspiration

of micro-organisms from denture plaque exposes patients, particularly the

immune-compromised host or medicated elderly, to the risks of unexpected

infections. They emphasized the importance of denture plaque control from that

point of view as it could subsequently affect the patients “quality of life”.

3- To attain and prolong the serviceability and performance of the denture

Maintaining the health of the teeth and tissues supporting a restoration will

consequently aid in prolonging the serviceability of the restoration, and

eventually achieve a long term successful treatment. Since plaque adheres to

and accumulates on dentures as they do on teeth and tissues, it is not enough to

keep the teeth and tissues clean. It becomes of utmost importance to clean and

disinfect the dentures as well if plaque control is to be achieved. Denture

hygiene is hence essential to maintain the performance and serviceability of the

denture (Nikawa et al., 1998).

4- To maintain an esthetic and odor free appliance

The fungal organisms that are most commonly associated with denture

plaque are of the genus Candida. It is present in the saliva in the majority of

denture wearers and displays an affinity to adhere to methacrylate resin. They


6

are predominantly effective at populating the craters formed by an intersection

of a monomer bubble with the polished surface of the acrylic resin from which

they are difficult to remove. Other multiple harmless, as well as, pathogenic

bacterial varieties have also been found in the denture plaque. These

microorganisms excrete volatile sulfur compounds that cause halitosis (Shay,

2000).

The adherent material “containing these fungi and bacteria” is not only

unesthetic in appearance but also unpleasant in the taste, odor and tactile

sensation. Unfortunately, the sensory receptors undergo a process called

“accommodation”. This makes the person with the unclean denture unaware of

the unpleasant smell and taste of his prosthesis. Gustatory experiments proved

impairment in taste and smell perception of external stimuli under

circumstances of poor denture hygiene (Shay, 2000).

5- To avoid cross infection and prevent health hazards

From what has been mentioned, it could be quite clear that the dental

prosthesis that is brought to the dental office for repair or adjustments constitute

a potential health hazard to the person that handles the prosthesis. At the same

time, blood and saliva-contaminated prostheses may carry high concentrations

of viruses such as those causing the acquired immune deficiency syndrome

(AIDS) and hepatitis B (Pavarina et al., 2003).

.Oral and non-oral pathogenic organisms associated with oral and

systemic disease have been cultured from contaminated prosthesis and dental

laboratory pumice. These organisms may cause infectious diseases such as

urinary tract infections, conjunctivitis, pneumonia and meningitis. A series of

eye infections have actually occurred among members of laboratory staff after

carrying out some polishing procedures. Infection control protocols and proper

disinfection of dental prostheses should therefore be applied in dental


7

laboratories. If disinfection procedures are not practiced, a cycle of cross-

contamination may occur and thereby expose personnel, as well as, patients to

infection, some of which may be serious (Pavarina et al., 2003).

For this reason, the Centers for Disease Control (CDC) and the American

Dental Association (ADA) have issued guidelines for cleaning and disinfection

of dentures and dental prostheses to assure the prevention of cross infection

between the dental clinic and the dental laboratory (Rutala and weber, 1997;

Pavarina et al., 2003).

IIIIII-- MMeetthhooddss ooff ddiissiinnffeeccttiioonn

Denture cleaning or disinfection can be carried out by two main approaches,

chemical methods and mechanical methods or combination of both. Studies

reported that less than 60% of denture wearers use chemical cleansing products

and that 60–90% use mechanical cleansing in association with tooth paste, soap

or water. Some of those who used chemical cleansing methods preferred

homemade products, as they are easy to handle, cheap and effective (Veres et

al., 1985; Arita et al., 2005; Mese and Mese, 2007).

AA --MMeecchhaanniiccaall mmeetthhooddss

11-- MMaannuuaall bbrruusshhiinngg

Shay, (2000) reported that the most common mechanical method for

denture cleaning is the use of a dental brush in the presence of either hot or cold

water. There are brushes specifically designed and sold for this purpose.


8

Tarbet, (1982) confirmed that when a vigorous cleaning regime was

instituted, the lesions improved, or disappeared, emphasizing the importance of

instituting a regular homecare cleaning program aiming at removing plaque

from tissues , as well as, denture surfaces . However, microbiological essays

and scanning electron microscope images showed that manual brushing with

water is ineffective in removing unacceptable large population of adherent

microorganisms. Since then, magnetic stirrers, agitators, sonic vibrators and

ultrasonic devices have been introduced, so as to be used in combination with

mechanical brushing (Mahonen et al., 1998; Shay, 2000).

22-- SSoonniicc cclleeaanneerrss

They are devices that convert electrical energy into mechanical energy at

the frequency of sound waves, and have been used for the removal of dental

plaque (Shay, 2000). The sonic cleaners employ vibratory energy, not

ultrasonic energy, to clean the dentures. The effectiveness of a sonic action-

cleaning program in removing calculus and stain has been investigated (Abbas,

2008).The use of sonic cleaner is considered a less common, however, an

incomparably more effective approach to denture cleaning (Shay, 2000; Abbas,

2008).

Disadvantages of mechanical methods:

Mechanical denture cleaning is simple, applicable and inexpensive,

however, evidence prove that mechanical cleaning alone is not sufficient to

completely remove denture plaque (Arita et al., 2005; Devine et al., 2007).

Kulak et al., (1997) and Webb et al., (1998) compared between

mechanical and chemical denture disinfection. They found that denture biofilm

control that uses only brushing is not effective as chemical cleansing, in


9

reducing prosthesis plaque, and also in preventing denture stomatitis associated

with Candida.

BB--CChheemmiiccaall mmeetthhooddss

Chemical cleaning methods include immersion in cleaning solutions

(such as hypochlorites, peroxides, enzymes, acids, and vinegar), exposure to O2

through air drying, and microwave radiation (Arita et al., 2005).

Advantages and Disadvantages of chemical disinfection:

All reported studies revealed that chemical disinfection proved to be more

effective than mechanical cleaning. However, chemical disinfection is

accompanied by several disadvantages. Some disinfectant solutions (such as

glutaraldehyde) may have an irritating influence on the oral mucosa, which

necessitate rinsing the dentures carefully with tap water after soaking. From the

patient’s point of view, a major drawback is that some chemicals are relatively

expensive (Mahonen et al., 1998).

Moreover, chemical disinfection has been known to affect the mechanical

properties of the denture material by reducing the hardness, dimensional

stability, flexural strength and color stability. In fact, some chemicals when not

used in the proper concentrations and timing cause damage to the denture

material (whether acrylic resin, metals or metal alloys) (Arita et al., 2005;

Buergers et al., 2008).

Some methods as air drying and microwave radiation are ineffective in

complete debridement of the dentures except when preceded by mechanical

cleaning (Shay 2000).


10

Requirements of an ideal chemical disinfectant:

Many materials have been introduced and are now available for denture

disinfection. However, an ideal chemical disinfectant should have the following

requirements (Pavarina et al., 2003; Mese and Mese, 2007; Devine et al.,

2007; Pinto de Campos et al., 2009).

1. Capable of rapidly inactivating pathogenic microorganisms; bactericidal and

fungicidal.

2. Effective at removal of organic and inorganic deposits.

3. Non-toxic and non-irritant to human and oral tissues.

4. Compatible and suitable for a wide range of denture materials.

5. Simple and easy to use.

6. Cost-effective “relatively inexpensive”.

PPrroodduuccttss uusseedd ffoorr cchheemmiiccaall ddiissiinnffeeccttiioonn

CChhlloorrhheexxiiddiinnee Chlorhexidine is a broad spectrum antimicrobial and antiseptic

disinfectant, that works by destroying the cell membrane and precipitating in the

cell cytoplasm (Pavarina et al., 2003).

SSooddiiuumm hhyyppoocchhlloorriittee

Sodium hypochlorite is considered as one of the chlorine-releasing

agents (CRAs) (McDonnell and Russell, 1999). It is also considered as the

most commonly used household solution. It is one of the commercially

available, most easily and widely used disinfectants. When diluted 1:10 in tap


11

water, this concentration is adequate for killing the adhering microorganisms

but will be ineffective against calculus build up and stains. However, it has an

unpleasant odor and causes the metallic element of removable dentures to

acquire a tenacious black stain if soaked in it for more than 10 minutes daily

(Veres et al., 1985; Dychdala, 1991; Shay 2000).

In their clinical study, Barnabe et al., (2004) evaluated the antimicrobial

activity of sodium hypochlorite as a chemical disinfectant when associated with

mechanical brushing with coconut soap. Mucosal evaluation was done

according to modified Newton's classification. The denture was cleaned

mechanically using coconut soap then immersed in 0.05% sodium hypochlorite

for 10 minutes for a period of 15 days. Their results revealed that this

disinfection protocol was effective in controlling the denture biofilm which in

turn resulted in a significant reduction in the signs and symptoms of denture

stomatitis.

Samples of acrylic resin were contaminated with a variety of bacteria and

were then placed in three different disinfecting solutions as directed by the

manufacturers. After the specific dilution and immersion time, cultures were

made from the resin samples. The only effective disinfectant was a 0.525%

solution of sodium hypochlorite at a 10-minute immersion. It disinfected not

only the surfaces but also the bacteria that penetrated the surfaces to a depth of

3 mm (Dychdala, 1991; Schwartz et al., 1994; Chau et al., 1995; Ellakwa

and El-sheikh, 2006).

Several authors have discussed effective means of surface disinfection of

dental prostheses, however, the vast majority or researches on acrylic resin

disinfection has been specific to surface contamination and not subsurface

disinfection. Saunders et al., (1998) conducted a study to evaluate in-depth

disinfection of complete denture prostheses that had been worn for periods of 15


12

to 20 years. These specimens were then fractured and cultured to determine

whether the disinfectant solutions penetrated the depth of the acrylic resin. This

study showed a dental prosthesis must be disinfected on the exterior as well as

the interior surface because of contaminating microorganisms. The conclusion

was that ten minutes immersion in full strength sodium hypochlorite (5.25

percent) more effectively disinfects dental acrylic resin than either Biocide

(Iodophor) or Alcide LD (Chlorine dioxide) disinfectant solutions (Saunders et

al., 1998). On the other hand some studies demonstrated that the Alcide LD

(Chlorine dioxide) was the most effective disinfectant when compared with

5.25% sodium hypochlorite (Brace and Plummer, 1993; Schwartz et al.,

1994; Lin et al., 1999).

The effects on Vitallium and Ticonium alloys immersed in different

concentrations of sodium hypochlorite for various immersion times were

investigated. The results of this investigation demonstrated that samples of these

metals soaked in 2% sodium hypochlorite for at least 5 minutes or in 5.25%

sodium hypochlorite for a minimum of 3 minutes were sterilized and not

tarnished or corroded. Although Vitallium alloy was more resistant than

Ticonium alloy to the deleterious effects of sodium hypochlorite, either metal

can be safely immersed for sterilization purposes under the guidelines

presented. Therefore, an effective and economical method for sterilizing

removable partial denture frameworks made of chrome-cobalt or chrome-nickel

alloys is attainable through the proper use of household bleach (McGowan et

al., 1988).

Some investigators have immersed three different types of dental acrylic

resins contaminated with 105 to 107 organisms (S. aureus, E. coli, P.

aeruginosa, and Streptococcus pneumoniae) in 0.525% sodium hypochlorite

and demonstrated complete killing of the tested bacteria on both the exterior and


13

interior surfaces of the resins within 4 min. The effectiveness of four

disinfectants (sodium hypochlorite, a phenolic, chlorine dioxide, and an

iodophor) for irreversible hydrocolloid impressions was evaluated. Sodium

hypochlorite was a much more effective disinfectant than either the phenolic or

the iodophor (Rutala and weber, 1997).

Sodium hypochlorite (Bleach), diluted 1:10 in tap water is adequate for

killing adherent organisms but will be ineffective against calculus build up and

stain. Addition of a teaspoon of calcium chelating dishwasher detergent

(Calgon) to a cup of the diluted bleach solution has been advocated as a means

for controlling calculus and the stains associated with it. A disadvantage of

bleach is that metallic elements of removable partial dentures acquire a

tenacious black stain after soaking in the bleach solution for more than 10

minutes daily (Shay, 2000).

CChhlloorriinnee DDiiooxxiiddee

The biocidal effectiveness of chlorine dioxide and 5.25% sodium

hypochlorite (diluted 1:10) on acrylic resin strips inoculated with

Staphylococcus aureus, Candida albicans, or Escherichia coli in the presence

of an organic load was investigated. Chlorine dioxide achieved complete

disinfection of all three organisms within 2 minutes. Sodium hypochlorite

achieved complete disinfection of all three organisms within 4 minutes (Bell

et al., 1989).

Alcide LD (Chlorine dioxide) is considered by some as an effective

disinfectant. A study conducted to evaluate the effectiveness of Alcide LD as a

disinfectant revealed that the majority of microorganisms from the interior and

exterior surfaces of the acrylic resin have been eliminated .However, it does not

completely eradicate all microorganisms, even on the external surfaces


14

(especially rough surfaces with crevices). In the study conducted by Lin et al.,

(1999) it was demonstrated that the Alcide LD was the most effective

disinfectant when compared with 5.25% sodium hypochlorite.

BBeerrbbeerriinnee hhyyddrroocchhlloorriiddee

Trial denture cleansers that were composed of the berberine

hydrochloride were examined as regards their effect on some candidal species

(Candida albicans, Candida glabrata) and compared to other commercial

denture cleansers. The results revealed that the trial denture cleansers removed

64% to 89% of the adherent fungal cells from the acrylic resin and also had a

little adverse effect on the other physical properties of the denture like the color

stability of the dental material and the surface roughness of the acrylic resin

plates (Nakamoto et al., 1995).

GGlluuttaarraallddeehhyyddee

Glutaraldehyde is an important dialdehyde that has found usage as a

disinfectant and sterilant, in particular for low-temperature disinfection and

sterilization of endoscopes and surgical equipment. Glutaraldehyde has a broad

spectrum of activity against bacteria and their spores, fungi, and viruses, and a

considerable amount of information is now available about the ways whereby

these different organisms are inactivated. It is considered as a traditional dental

disinfectant that is widely used for reducing biofilm accumulation on removable

prostheses (McDonnell and Russell, 1999; Buergers et al., 2008).

Ellakwa and El-sheikh, (2006) evaluated the effect of different chemical

disinfectant solutions on the transverse strength of the repaired heat-

polymerized acrylic resin. Complete dentures in that study where immersed in


15

the following disinfectants respectively: 1%, 2%, and 5.25% sodium

hypochlorite, and 2% glutaraldehyde for 10 minutes. They were removed,

washed, dried, and sectioned. Their results revealed that the transverse strength

of the dentures was not affected by any of the disinfectants.

AAllkkaalliinnee ppeerrooxxiiddee ssoolluuttiioonnss

Alkaline peroxide based chemical soaks displayed a broad antimicrobial

activity against gram-negative anaerobic rods, gram-positive facultative cocci

and gram-negative anaerobic cocci. Its ability to decrease candida albicans

biofilm activity has also been reported (Pavarina et al., 2003).

MMoonnooccaapprriinn

Monocaprin is a 1-monoglyceride of capric acid. It has antimicrobial

activity against enveloped viruses, certain bacteria and the yeast candida

albicans (Thorgeirdottir et al., 2006).

TTeettrraassooddiiuumm EEDDTTAA

Tetrasodium EDTA was found to be effective in treating biofilms

containing staphylococci, other gram-positives, pseudomonas aeruginosa and

enterobacteria. Its efficiency in virus neutralization was also determined. 16

hours immersion in Tetrasodium EDTA was also found to reduce salivary

bacterial and Candida albicans biofilm viable counts by more than 99%. It

removed most or all of the biofilm from the material surfaces as well (Devine et

al., 2007).

TTooootthh ppaasstteess

Yigit et al., (2008) investigated the anti-candidal activity of six different

toothpastes containing the following active ingredients: sodium fluoride,


16

sodium monoflurophosphate, triclosan and herbal compounds. The herbal

toothpaste exhibited good antifungal activity against all Candida species.

VViinneeggaarr ((aacceettiicc aacciidd ssoolluuttiioonn))

Vinegar is natural, readily available, easy to handle, and well priced, but there

is conflicting evidence about its effectiveness (Buergers et al., 2008). It was

found to be effective in killing the adherent microorganisms although less

effective than the bleach solution. One advantage of the vinegar over the bleach

is that inadequate rinsing after soaking in vinegar does not result in mucosal

damage. However, neither the vinegar nor the bleach has a pleasant odor, so the

patients choose to soak their dentures in certain mouthwash products due to

their more desirable odors, flavour and antibacterial claims of their

manufacturers (Shay, 2000).

OOzzoonnee TThheerraappyy

The antimicrobial efficacy of ozone as a denture cleaner was investigated.

Ozone therapy was found to be effective and can be clinically used for denture

disinfection (Murakami et al., 2002; Arita et al., 2005; Estrela et al., 2006).

The effect of ozonated water on the Candida albicans on acrylic denture

plates was investigated. The study showed that the combination of ozonated

water and ultrasonication has a strong effect on the viability of Candida albicans

adhering to acrylic resin plates. Results also revealed insignificant differences in

the antimicrobial activity between ozonated water with ultrasonication and

commercially available denture cleaners. It was hence concluded that the

application of ozonated water may be useful in reducing Candida albicans

colonization on acrylic plates (Arita et al., 2005; Nogales et al., 2008).


17

IIVV-- MMeetthhooddss ooff sstteerriilliizzaattiioonn

AA--MMiiccrroowwaavvee iirrrraaddiiaattiioonn

Some chemicals used for disinfection may become absorbed into the

material porosities and may not be completely eliminated by rinsing. Thus,

chemicals may be unintentionally introduced intra-orally. Sterilization of

dentures with microwave irradiation proved to be a safer, simple, and

inexpensive method (Dixon et al., 1999).

Dixon et al., (1999) conducted a 3-phase investigation to examine the

efficacy of microwave irradiation against C. albicans colonized on 3 soft

denture liners and 1 heat-polymerized denture base resin, as well as the effect of

this irradiation on the hardness of the materials tested. Their results revealed

that only the 5-minute irradiated specimens immersed in water were effectively

sterilized (all Candida albicans present on the materials tested were killed). The

results of this study provided a promising method for sterilizing dentures

contaminated with Candida albicans, with or without soft liners, by using an

ordinary microwave oven. This was especially significant to persons and long-

term health care facilities. The effect of heating the dentures in water on

dimensional stability and bond of the liners to acrylic resin bases was yet to be

investigated.

The effect of microwave disinfection on the dimensional stability of

denture acrylic resin seemed to be a controversial issue. Polyzois et al, (2005)

reported that dentures exposed to microwave radiation for up to 16 minutes,

presented no dimensional changes. While, Thomas and Webb, (1995)

observed unacceptable dimensional instability in acrylic resin bases after

microwave disinfection for 10 minutes at 604 W.


18

The efficacy of microwave irradiation and sodium hypochlorite soak on

denture sterilization was compared in vitro. Microbiological analyses showed

that the inoculated dentures became sterile after six min of irradiation at

medium setting (2450 MHz, 350 W). Microbiological analyses also showed that

the experimental dentures inoculated with C. albicans became sterile, but those

inoculated with S. gordonii did not become sterile after soaking for 8 hours in

either 0·02%, or 0·0125% sodium hypochlorite solution. Willcox, (2002) found

that microwaving may be a more effective method of denture sterilization than

denture soaking in sodium hypochlorite. However, compared with

microwaving, hypochlorite reduces the levels of residual non-viable

micro-organisms attached to the denture surface (Baysan et al., 1998; Willcox,

2002; Cardoso et al., 2007; Mima et al., 2008).

Neppelenbroek et al., (2008) evaluated the effectiveness of microwave

disinfection of maxillary complete dentures on the treatment of Candida related

denture Stomatitis. Patients were instructed to perform denture care and the

upper dentures were microwaved three times per week. Results revealed that

microwaving dentures was effective in treating denture Stomatitis and

recurrence of Candida dramatically.

BB-- RRaaddiioosstteerriilliizzaattiioonn

Radiosterilization or ionizing radiation (gamma rays) is used for sterilization of

dental equipments as implants, needles and gloves. According to international

atomic energy agency, ionizing radiation can be used for sterilization of

polymethylmethacrylate and acrylics (International atomic energy agency,

1973; Fu, 1988; Philippine nuclear research institute, 1996).


19

IIoonniizziinngg rraaddiiaattiioonn

All humans are continuously exposed to natural background ionizing

radiation from cosmic rays; radioactive elements in the earth’s crust; potassium-

40, and other radionuclides normally present in human tissues; as well as

inhaled radon and its daughter elements. In people residing at high altitudes, the

contribution from cosmic rays may be increased two-folds. Likewise, in regions

where the earth’s crust is rich in radium, the contribution from this radionuclide

may be similarly increased (Perez and Brady, 1998).

Radiation is classified according to its energy into non-ionizing radiation

(any radiation that has insufficient energy to produce ions in matter and tissues,

such as ultraviolet, infrared, visible light, ultrasound and radio waves) and

ionizing radiation (any radiation consisting of moving particles or

electromagnetic waves that has sufficient energy to produce ions in matter and

tissues) (Vyas et al., 1994).

According to their nature, ionizing radiations may be electromagnetic or

corpuscular radiation. Electromagnetic radiations consist of stream of high

photons and have no mass or charge. They include X-rays and gamma-rays

which can easily penetrate the body tissues and deposit their potentially harmful

energy deep in the body. Corpuscular radiations consist of particles that have an

electric charge and mass. They include α and β particles, electrons, protons and

neutrons. They are hazardous only when are inhaled, ingested, or deposited in

an open wound, because they have poor penetration power and are unable to

penetrate the outer layer of skin. However, β particles may penetrate as much as

few centimeters of tissues (Behrman et al., 2000).

Botkin and Keller, (2000) reported that the most commonly employed

radiations in occupation health are x-rays, γ-rays, α and β-particles and

neutrons. Gamma (γ)-rays have shorter wave length and higher penetration than


20

x-rays. The penetrating power of different types of radiation is illustrated in Fig.

1. They are emitted when a nucleus undergoes a transition from a higher to a

lower energy level (Yu, 2001).

Fig. (1): The penetrating power of different types of radiation

DDiirreecctt aanndd iinnddiirreecctt eeffffeeccttss ooff iioonniizziinngg rraaddiiaattiioonn

Ionizing radiation affects the biological tissues through either direct or

indirect actions. Both types of actions occur together when charged particles

pass through a cell causing cell damage by different mechanisms (Bast et al.,

1996). The direct effect is the change in the molecules that appear as result of

absorption of radiation energy by the target organ. The indirect effect is the

change in the molecules caused by the products of radiation decomposition of


21

water ions (Karcher and Jentzsch, 1972).

Direct effect:

The direct effect on the biological organic molecules can be summarized

as follows; where RH is the biological target molecule and R• is an organic free

radical (Przbyszweski et al., 1994).

RH RH+ + e ¯

RH+ R• + H+

Indirect effect:

Indirect action is when a molecule reacts with a molecule or the product

of a molecule that has undergone direct action. Since water is the major

constituent of all biological materials, it enters into the reaction that is believed

to eventually cause cell death after irradiation. The death of the cell by water is

brought about by the formation of free radicals. The free radicals OH• and H•

are highly reactive unstable species that interact with the biological organic

molecules. This organic free radical R• may give rise to permenant damage

(Fauci et al., 1998).

RH + OH• R• + H2O

RH + H • R• + H2

The highly reactive radical is responsible for most of the indirect


22

radiation damage that affects cell survival (Kachur et al., 1998). Consequently,

superoxide radicals and hydrogen peroxide are formed in irradiated solutions in

presence of dioxygen, (Bioaglow et al., 1992). The concentration of these

substances is dependant on the degree of oxygenation of the cell or tissue being

irradiated, which may explain why oxygen effectively enhances radiation

damage. In general, the more dilute the system, the more the radiation damage

is attributed to the indirect effect. In addition, the presence of oxygen increases

the radiosensitivity of the tissues (Buettner, 1993).

Mechanisms of biological effects of ionizing radiation:

All types of ionizing radiation produce injury in the same manner

regardless of the type of particle or the ray emitted. The variations are

quantitative rather than qualitative. Absorption of energy may cause molecules

in the path of the radiation to become ionized. These molecules may form

substances that alter biochemical processes within the cell or its environment

(Hall, 1994; Mettler and Williamson, 2000).

Fauci et al., (1998) reported that after absorption of ionizing radiation by

the target molecules, several changes take place through four phases. These four

phases are demonstrated in Fig. 2.


23

Fig. (2): The phases of the mechanism of radiation effect (Fauci et al.,

1998).

1- Physical phase, through which radiation energy is dissipated in the cell

within short time causing ionization and excitation of ions and molecules.

The absorption of energy by a water molecule results in the ejection of an

electron leaving a positively charged species, H2O+.

2- Physico-chemical phase, the positive ion dissociate to a hydrogen ion

(H+) and hydroxyl free radicals (OH•). The emitted electron reacts with

other molecules of water which then dissociate to hydrogen free radical

(H•) and hydroxyl ions (OHـ These free radicals are very active species .(ــ

which may undergo many reactions, for example two (OH•) radicals may


24

combine to form hydrogen peroxide, which is a powerful oxidizing agent

(Adams et al., 1968).

3- Chemical phase, the radicals react with organic molecules within the

cell. Reactions with proteins and DNA of the cell nucleus may have

particularly serious effects, ranging from loss of the cells capacity of

division to the production of mutations which may lead to cancer or the

birth of genetically damaged off springs.

4- Biological phase, during this phase the chemical changes are transformed

into cellular changes. Three types of cellular changes may be recognized,

these are early cell death, prevention or delay of cell division (mitosis)

and the production of permenant inheritable changes which may lead to

other changes which affect the whole organism. This pass of development

of radiation injury shows the longest and most variable time scale; some

effects may develop within few hours while others, for example cancer

induction may take years to be apparent (Fauci et al., 1998; Chevion et

al., 1999).

GGaammmmaa rraaddiiaattiioonn

Gamma irradiation is an electromagnetic radiation of short wavelength

emitted by radioactive isotopes as the unstable nucleus breaks up and decays to

reach a stable form. It is widely used for sterilization of medical devices, food

preservation and processing of tissue allografts and blood components,

obviating the need for high temperatures that can be damaging to such products.

Gamma radiation may be in the form of Cobalt-60 or Caesim-137. Radioactive

cobalt (Co-60) can be produced by exposure of naturally occurring Co-59 to

neutrons. The half-life of Cobalt-60 is 5.27 years. Most of the medical devices


25

are polymer based and utilize ionizing radiation for sterilization (Trampuz et

al., 2006).

MMeecchhaanniissmm ooff ggaammmmaa rraaddiiaattiioonn iinn sstteerriilliizzaattiioonn

The Microorganisms are killed by direct or indirect damage to its DNA.

The propagation of life is arrested due to DNA impairment by the ionizing

radiation. The direct effects are due to the damage to DNA may be as single

breaks, double breaks, base damage, intra-or intermolecular crosslink formation.

And the indirect effects are due the free radicals formed due to radiolysis of

water: Hydroxyl, Hydrated electrons, Hydrogen atoms, Hydrogen molecule,

Hydrogen peroxide and hydrated proton (Min et al., 2003).

AAddvvaannttaaggeess ooff ggaammmmaa rraaddiiaattiioonn sstteerriilliizzaattiioonn

γ-ray sterilization method does not require neither operation for

removal of residual toxin nor disinfection with chemicals (ethyl alcohol,

formalin, glutaraldehyde, chlorohexidine, benzalkonium chloride, etc.) and

hence, merits as sterilization method for non-heat resistant medical materials

have been widely recognized (Haque et al., 2001).

Gamma sterilization is the complete removal (inactivation) or complete

and total destruction of all forms of microbial life from the products in terms of

their inability to reproduce. It's a cold process with high penetrating power so

heat sensitive products can be sterilized effectively by this method. In most

countries a minimum or an average dose of 25 kGy is suitable for sterilization

of most of the healthcare products. It is equivalent to sterilization with saturated

steam at 121 C for 15 min or dry heat at 160 C for two hours (Sharma).. DDiissaaddvvaannttaaggeess ooff ggaammmmaa rraaddiiaattiioonn sstteerriilliizzaattiioonn

It has been warned that irradiation of a large quantity of γ-ray results in

denaturation (radical decomposition and oxidation decomposition of materials)


26

of most of rubbers and plastics to produce soluble (water-soluble) low

molecular weight deterioration products and furthermore, mechanical strength

of the materials often decreases (Haque et al., 2001).

Cross-linking of polymers is achieved through the use of ionizing

radiation. Ionizing radiation forms free radicals through the radiolytic cleavage

of C-H and C-C bonds in polymer. These free radicals recombine with each

other and form cross-links in the amorphous portion of the polymer. The free

radicals generated during irradiation in the crystalline phase, however, become

trapped and adversely affect the long-term stability of the material. Radiation

induced changes include embrittlement, discoloration, odor generation,

stiffening, softening, changes in chemical resistance & melt temperatures.

Ionizing radiation is unique and powerful tool to modify polymer properties in

the solid state unlike chemical or thermal processes. The presence of antirads-

the substances added to prevent radiation damage- makes the prediction more

difficult (Oral et al., 2006).

Data resulting from gamma radiation of polymethylmethacrylate indicate

that the clinical use of C60 irradiation of neoplastic disease in bone is not

affected by the presence of polymethylmethacrylate, nor is the

polymethylmethacrylate affected at the level of gamma radiation in clinical use

today. Polymethylmethacrylate was released in 1971 for clinical use in the

management of secondary neoplastic disease in bone. Since that time,

polymethylmethacrylate has been used frequently in the surgical management

of secondary neoplastic diseases of bone. No effect by gamma radiation on

polymethylmethacrylate was observed with 25,000 rads of radiation. The

samples were observed to yellow with 30,000 rads. An odor similar to burning

candle wax was emitted by the samples at twenty-five million rads (twenty five

megarads). After forty-five million rads (forty five megarads) the samples were

easily fractured by thumb pressure. At approximately 160 million rads (160


27

megarads) the polymethylmethacrylate sample could be pulverized between the

thumb and index finger (Murray et al., 1974).

Electron beam irradiation can be used to influence the properties of

polymers. It was the aim of some studies to investigate whether PMMA denture

base materials can benefit from irradiation in order to have increased fracture

toughness, work of fracture or hardness. Rectangular specimens of heat-and

auto-curing denture base materials were electron beam irradiated (post-cured)

with 25, 100 and 200 kGy using an electron acceleration of 10 MeV or 4.5 MeV

respectively. Fracture toughness, work of fracture, Vickers hardness and colour

changes were measured and compared with not-irradiated specimens. The

toughness, work of fracture and hardness increased using 10 MeV with a dose

of 25 kGy and with 100 kGy using 4.5 MeV. However, the clinical use may not

benefit from the observed small changes. Higher dosage (200 kGy) decreased

the values significantly. The colour changes reached a level which was found to

be not clinically acceptable. The conclusion was that PMMA denture base

materials do not benefit from post-curing with electron beam irradiation (Behr

et al., 2005).

Lately interest of researchers engaged in different fields of knowledge is

seen to be focused on determination of the action of nanomaterial addition, such

as fullerene and nanotubes, on properties of polymers and their compositions.

Incorporation of fullerene and nanotubes into chemical composition of

polymers gives one more opportunities for their study and application as

composite materials (films, fibres) serving different purposes. For all that

properties of the above polymers can be substantially changed (strength,

thermostability, gas-penetrability, electrical conduction). Effect of fullerene C60

was investigated on thermal, mechanical and optical properties of

polymethylmethacrylate (PMMA) under ionizing radiation. It was stated that

fullerene C60 behaves as an effective antirad with respect to PMMA. Fullerene


28

C60 addition raises temperature of destruction for polymer subjected to electron

radiation by 20–25 °C, lowers the rate from 4 to 4.5 times and increases the

activation barrier for radiated PMMA destruction reaction. Fullerene C60

addition promotes improvement of strength properties of PMMA: for films

containing C60 addition and else subjected to electron radiation treatment a

decrease in rupture strength is 10–15%, for samples containing no fullerene it

equals 25%. Interaction of free radicals with fullerene at radiation treatment

influences optical characteristics of PMMA films (Zhogova et al., 2005).

Gamma rays were used to cure dental-base material. The effect of the

radiation dose on the thermal and rheological properties of poly(methyl

methacrylate) (PMMA) used as a dental-base material was investigated.

Samples of PMMA were cured by gamma radiation with total doses of 7.5, 15,

22.5, 30, 45, 52.5, 360, and 2160 krad. No significant differences were observed

of total dose on molecular weights. An exothermic peak in the thermograms of

samples that were not completely polymerized and crosslinked was observed

(Usanmaz et al., 2001).

The effect of the sterilization process by gamma-irradiation on the

structure of poly(ethyl acrylate-co-hydroxyethyl methacrylate) copolymer

networks, P(EA-co-HEMA) was studied for a broad dose range (7, 15, 25 and

50 kGy). Gamma-irradiation promotes chain scission in PHEMA homopolymer

but induces new crosslinking points in PEA homopolymer. Both effects are

present in the copolymers, with a net result that depends on composition. For

copolymers with high HEMA fractions chain scission predominates, while, as

the amount of EA in the copolymer increases, the situation changes and the net

effect turns out to be an increase in the number of elastically active chains.

Further, gamma-irradiation strengthens the gamma relaxation in PHEMA

homopolymer, what suggest that the number of interchain hydrogen bonds

decreases (Diego et al., 2007).


29

OOrraall CCaannddiiddaa

Oral flora is defined as those organisms that are present in a healthy mouth as

a commensally and normal inhabitant without causing any disease. The mouth

supports the growth of a wide diversity of micro-organisms including bacteria,

yeasts, mycoplasmas, viruses and (on occasions) even protozoa (Muzyka,

2005). Candida species are normal oral commensals present in 17% to 60 % of

apparently healthy persons (Odds, 1988). Muzyka, (2005), however

demonstrated that denture wearing encourages the growth of Candida species,

staphylococci, streptococci, Neisseria and Diphtheroids.

Presence and Intra-oral distribution of Candida albicans

Candida is an obligate associate of human beings and other warm-

blooded animals. It is usually encountered as harmless commensal of the

digestive tract and constitutes a part of the normal microbial flora of the host.

Candida albicans (C.albicans) is part of normal flora and can be cultivated from

the mouth of approximately 50% of adults (Milillo et al., 2005).

Bhowate and Dubey, (2004) reported that C.albicans could be isolated

from the mouths of over 23% of normal patients having natural or artificial

teeth. Furthermore, the prevalence of Candida species in the mouths of 140

infants at birth was 5.7%, which increased to 82% at age of 1 month and

declined to 60% by 8 month of age.

C.albicans is considered the most common as well as the most

pathogenic of all Candida species followed by C. tropicalis, C. stellalodia, C.

parapsilosis, C. pseudotropicalis, C. krusie, C. guillier mondi and C. glabrata

(Budtz-Jorgensen, 1990).


30

C.albicans and related species are highly successful opportunistic

pathogens that reside in a benign state as commensally in the oral, digestive and

vaginal cavities. When the host is immunologically compromised or undergoes

physiological changes, Candidal infection can develop (Lockhart et al., 1999).

Denture wearing and patients associated factors such as changes in

environmental conditions, trauma from ill-fitting denture, denture cleanliness

and continuous denture wearing are considered the most important factors that

predispose to oral Candidal infections. Oral Candidal growth and colonization

are more pronounced in acrylic rather than metallic dentures. Wearing dentures

both by day and night is associated with increased density and frequency of

Candidal denture colonization and denture stomatitis (Budtz-Jorgensen, 1990).

C. albicans was reported to be present in the vicinity of carious lesions

(Bahn et al., 1962), plaque coated surfaces of teeth (Arendorf and Walker,

1980), sub gingival flora, gingival tissues of advanced periodontal abscesses,

and in advanced periodontitis of AIDS patients (Olsen and Tenderup, 1990).

In healthy dentate individuals, C.albicans was found not to be uniformly

distributed throughout the mouth, the dorsum of the tongue particularly its

posterior half area near circumvallate papillae as the sole oral reservoir of the

fungus. It was claimed that, the tongue papillae provide a large surface area for

adherence of microorganisms and shelter them from removal during eating and

swallowing (Arendorf and Walker, 1980; Bagg et al., 1999).

Pathogenicity of Candida albicans

Candida albicans is a unicellular eukaryotic micro-organism that

reproduces by budding. It is considered as a polymorphic fungus that can exist

as a commensal or as an opportunistic fungus that is capable of causing

superficial to life threatening infections (O’Toole et al., 2000).


31

Candida albicans was found to be more pathogenic than other species.

This was attributed to the “highly-frequent phenotypic switching” phenomenon

demonstrated by some strains of Candida albicans. This phenomenon allows for

all reversible phenotypic modifications (Morrow et al., 1994).

Candida species are assumed to cause disease by tissue invasion, by

inducing a hypersensitive state or by producing potent Candida toxins. This

phenotypic switching capability of Candida albicans and its transformation to

the hyphal pattern may facilitate multistage invasion, which in turn may initiate

the disease. This may be one of the reasons why Candida albicans are

considered more pathogenic than other organisms that are incapable of this

phenotypic switching phenomenon (Budtz-Jorgensen, 1990; Jones et al.,

1994).

“Candidal bio-film” is defined as structural microbial communities that

adhere to a surface and become encased in the matrix of an exopolymeric

material. Candidal bio-film can grow over a variety of human tissues and also a

number of biomaterials including acrylic resin dentures (Thein et al., 2007;

Cao et al., 2008).

Adherence of Candida albicans to oral mucosa

Budtz-Jorgensen, (1990) reported that the pathogenicity, colonization of

the host tissue and infective ability of Candida depend on its ability to adhere to

epithelial cells or a closely associated surface. After attachment and

colonization, Candida cells invade the epithelial cells. This invasion process, as

mentioned earlier, initiates an acute inflammatory response.

Adhesion of Candida albicans to mucosal surfaces appear to involve

lectin-like interactions between the protein of mannoprotein located in fibrils on


32

the yeast surface and glycoside receptors on epithelial cells (Budtz-Jorgensen,

1990).

Hazen, (1989) and Kulak et al., (1997) believe that the adherence of

Candida albicans may also be related to non-specific forces such as “Van Der

Waal Forces” (which is the attractive force between the molecules other than

those due to covalent bonds or to the electrostatic interaction of ions with one

another or within neutral molecules), “London forces” (which is the attraction

between two rapidly fluctuating, temporary dipoles) and “the hydrophilic

interactions” (which refers to the physical property of a molecule that can

transiently bond with water (H2O) through hydrogen bonding) .

Adherence of Candida albicans to partial dentures

Pathogenicity of Candida albicans may be due to its non specific yet high

affinity to adhere to and subsequently colonize denture acrylic resin material

and other plastics. Candida has been commonly found and isolated from both

the palatal fitting surface, as well as, the outer surface of complete dentures

(Budtz-Jorgensen, 1990, Shinada et al., 1997). Hence, complete dentures can

act as a reservoir to candidal species making it possible for such organisms to

continually re-infect the mucosal surface. This could explain the close

relationship between colonization of the denture base by Candida albicans and

denture related Stomatitis.

Factors promoting and increasing Candida adherence to dentures

(i) Rough and unpolished surfaces: Several investigators suggested that the

surface roughness and cracks within the acrylic resin encourage attachment,

penetration and colonization of Candida. Penetration is even greater if the

surface was unpolished. Even if the surface may appear smooth and polished,


33

dentures still have a pockmarked surface when viewed under microscopic

magnification. This is due to bubble formation from the un-polymerized

monomer in the course of denture processing. Adherence to these imperfections,

allows them to be furtherly protected from any washing actions (Shay, 2000,

Ramage et al., 2004).

(ii) Dietary carbohydrates: It was also found that sugars (dietary

carbohydrates) promote the adhesion of Candida to acrylic surfaces. The fungi

convert materials such as sucrose and glucose into a protective plaque covering,

under which they can thrive and proliferate even further (Shay 2000).

Attempts to decrease Candida adherence to dentures

One attempt included the addition of a silicon polymer (to which oral

microorganisms are unable to adhere), as a component of a commercial denture

cleanser. The polymer floats on the surface of the denture bath, and when the

denture is removed from the solution, a thin layer of the material coats all

surfaces of the prosthesis. The material will not be rinsed or rubbed off but will

be slowly lost over the day. Human and animal trials suggested that the material

is inert and the volume ingested is in no way harmful (Shay 2000).

Park et al., (2003) introduced a modified resin (surface – charged

polymethylmethacrylte) in an attempt to reduce adhesion of Candida albicans to

denture surfaces. The material had a negative charge that was incorporated by

co-polymerization of methacrylic acid to methyle-methacrylate. Surface contact

analysis of surface-charged resin surfaces revealed that as the amount of

methacrylic acid increased, the surface energy of the resin increased and

consequently, the surface area of adherent Candida albicans decreased. The

study supported the role of electrostatic interaction of the surface charge as an

effective method in decreasing the adhesion of Candida albicans to poly-

methyle-methacrylate.


34

DDeennttuurree rreellaatteedd SSttoommaattiittiiss

DDeeffiinniittiioonn

Denture related stomatitis is defined as an inflammatory process that

mainly involves the palatal mucosa when covered by complete or partial

dentures (Samaranayake and MacFarlane, 1990; Jacobsen et al., 2008).

It was described by Newton as an erythematous reaction that can be focal

or diffused; the mucosa may present either a smooth surface or papillary

hyperplasia (Arendorf and Walker, 1987).

IInncciiddeennccee

Denture stomatitis is the most commonly encountered intra-oral

pathological condition among denture wearers (Dorey et al., 1985). It refers to

inflammatory changes of the denture bearing mucosa, which may appear

erythematous, spongy and sometimes may show papillary hyperplasia

(Williamson, 1968; Arendorf and Walker, 1987).

Bergendal and Isacsson, (1983) observed that denture stomatitis more

often affects the maxillary than the mandibular denture bearing mucosa. Their

results demonstrated that although the condition may involve the entire upper

denture bearing area, it is more often confined to the mucosa of the palatal

vault.


35

CCllaassssiiffiiccaattiioonn

Newton classification (1962):

It has been most widely used. Newton classified the denture related

stomatitis as follows:

1) Pinpoint hyperemic foci.

2) Diffuse hyperemia of the denture supporting tissues.

3) Papillary hyperplasia.

Modified Newton Classification (2003):

The presence of denture related stomatitis was assessed according to the

modified version of Newton's classification. This classification reflects the

classic type of inflammation and the extent to which tissues are affected:

1) No Stomatitis, no evidence of palatal inflammation.

2) Newton type I, petechiae dispersed throughout all or any part of

palatal mucosa in contact with the denture.

3) Newton type II, macular erythema without hyperplasia.

4) Newton type III, diffuse or generalized erythema with papillary

hyperplasia.

SSiiggnnss aanndd ssyymmppttoommss

Frequently it is asymptomatic, however some patients may complain of

inflamed mucosa, mucosal bleeding, burning sensation, halitosis, bad taste,

discomfort and dryness of the mouth (Arendorf and Walker, 1987; Nikawa et

al., 1998).


36

EEttiioollooggyy aanndd PPrreeddiissppoossiinngg FFaaccttoorrss

Denture related stomatitis is considered to be multifactorial condition.

Possible etiological factors could be divided into systemic and local factors.

AA-- LLooccaall ffaaccttoorrss

Local (denture related) factors are the most recognized reasons for denture

related stomatitis (Pereira-Cenci et al 2008).

i- Denture Plaque and lack of denture hygiene

Microbial plaque accumulation on the fitting surface of removable

dentures plays a critical role, changing the oral flora from a commensal to a

pathogenic state (Ramage et al., 2004). Denture plaque is considered as a

source of infectious oral material. It can be calcified if not removed regularly.

The surface of the mineralized calculus enhances further plaque

accumulation. Therefore, dentures need to be cleaned regularly to prevent the

build up of microbial populations (Shay, 2000).

Patients usually face a difficulty keeping dentures clean as it is necessary

to use chemical cleansing agents to reduce the number of the active

microorganisms. Studies concluded that the denture stomatitis is associated

with the growth of Candida on denture biofilm and not the mucosa or the

palate. Therefore, the treatment would be better directed towards the denture

and not towards the mucosa (Webb et al., 1998; Ramage et al., 2004).

ii- Duration of denture wearing

Continuous denture wearing might cause denture stomatitis through

increasing not only the local injuries but also the time of mucosal exposure to

denture plaque. Wearing the denture continuously night and day was found to

increase the frequency and density of the Candida albicans in the fitting


37

surface of maxillary dentures (Arendorf and Walker, 1987; Figueiral et al.,

2007).

iii- Denture trauma

A poorly fitted denture predisposes the oral mucosa to denture related

stomatitis. Lesions occur less frequently under non traumatic or well fitted

dentures. The inflammatory exudates leaking from the traumatized palatal

mucosa might similarly enhance Candida colonization of the denture surfaces

(Budtz-Jorgensen and Bertram, 1970; Arendorf and Walker, 1987).

Reduced vertical dimension and unstable occlusion was also found to be

one of the prosthetic factors related to denture related stomatitis (Figueiral et

al., 2007).

iv- Denture base material

Denture related stomatitis is associated with bio-prosthetic surfaces (bio-

materials). The characteristic porosity of acrylic dentures acts as a suitable

media for the colonization and growth of Candida albicans. This aids in the

initiation of inflammatory reactions in the denture foundation area leading

consequently to denture stomatitis (Carr et al., 2005; Thein et al., 2007). On

the other hand metallic denture bases are more hygienic and resistant to

plaque accumulation and bacterial adherence as compared to non metal bases;

this may be due to their polished and non porous surfaces with fewer

tendencies for food accumulation (Krol et al., 1990).

Perezous et al., (2006) conducted an in- vivo investigation to compare

the oral candidal population between heat-cured acrylic resin and nickel-

chromium-beryllium alloy in maxillary complete dentures in HIV-infected

patients. The metal base proved to be effective in decreasing the fungal

growth typically present in complete dentures. Although overt clinical


38

manifestations were not present, colony counts of Candida species were high

in the acrylic resin denture bases of these patients. Their investigation

demonstrated that metal base complete dentures provide an important

alternative dental service for edentulous HIV-positive and other patients who

are particularly prone to higher incidences of fungal infections.

BB-- SSyysstteemmiicc ffaaccttoorrss

i. Dietary factors: Patients with denture stomatitis have been found to have

nutritional deficiencies especially vitamin B12 and folate deficiencies

(Arendorf and Walker, 1987).

ii. Malignancies: Candida albicans was the predominant organism isolated

from the oral swabs of patients with advanced cancers and therefore they are

more prone to denture related stomatitis and oral candidiasis (Davies et al.,

2006).

iii. Immunosuppressants: Immunosuppressed patients are more frequently

subjected to Stomatitis and Candidal infection. The immune deficiency or

the side effects of the medication received by those patients may even

amplify the intensity of denture related stomatitis (Tylenda et al., 1989;

Golecka et al., 2006).

iv. Smoking: According to a preliminary study smoking is considered as one of

the systemic predisposing factors of denture related stomatitis (Fenlon et al.,

1998).

v. Diabetes: People with type II diabetes were more likely to be affected with

denture related stomatitis as a result of their weak immune systems (Fenlon

et al., 1998).


39

IIssoollaattiioonn aanndd iiddeennttiiffiiccaattiioonn ooff CCaannddiiddaa

aallbbiiccaannss

Several methods have been developed for isolation and identification of

Candida albicans. Such methods play a key role in the diagnosis and treatment

of oral Candida infection and denture related stomatitis.

Isolation of Candida albicans from the oral cavity

Two main methods are available for the isolation of Candida from the oral

cavity. They are:

1) Oral swabs that can be obtained from the hard palate in the susceptible

area of denture stomatitis (Ramage et al., 2004).

2) Patient rinses 10 ml of sterile saline for 10 seconds and spit into a

sterile cup (Ramage et al., 2004).

Media and methods for the identification of Candida albicans

Variable techniques of yeast identification are now available and can be

used in conjunction with conventional identification procedures (Wiiliams and

Lewis, 2000).

Direct microscopic examination: Candida albicans may appear in a

tissue section as yeast, pseudohyphea (3-5μm in diameter), or

combination of both. “Pseudohyphea” are yeast cells with a single bud

(3-4μm in diameter) that remain attached to the parent cell, then

elongate and bud again. Pseudohyphea, which are a hall-mark of


40

Candida albicans, resemble true hyphea, but a slight constriction

remains where each bud begins (Elizabeth, 1997).

Culture the organism: Candida albicans usually grow in 24 to 48 hours on

most bacterial and fungal media. The organism is 5-10 μm in diameter.

Under certain conditions (in serum at 37°C), within 2-3 hours, Candida

albicans will produce germ tubes that are non-separate, mycelia-like

elongation of the yeast cell. The species can be identified by morphology of

the pseudo-mycelium and the chlamydospores that form in subculture on

cornmeal agar, by carbohydrate assimilation tests and by fermentation tests

(Elizabeth, 1997).

i- Germ tube test: This can be used for direct identification of Candida

albicans. The direct germ tube test is 87.1% sensitive and 100% specific for

the identification of Candida albicans (Sheppard et al., 2008).

ii- Agar diffusion tests:

1) Blood agar: It is an inexpensive general purpose agar base which with

the addition of 5% sterile blood can be used to cultivate a wide range of

microorganisms of clinical significance. Typical haemolysis is obtained

on this medium. For Candida albicans it should be also followed by the

germ tube test. Strains of C. albicans subcultured on either blood agar or

Sabouraud’s agar retained their ability to form germ tubes for 9 weeks.

Strains maintained on blood agar produced a higher mean number of

germ tubes throughout the 9 weeks, although the range was similar to

that of strains maintained on Sabouraud’s agar (Martin, 1979).

2) Sabouraud dextrose agar: Sabouraud dextrose agar was devised by a

French dermatologist, Raymond Sabouraud, for the cultivation of

dermatophytes in 1894 and is the preferred medium of microbiology


41

laboratories. It is a general-purpose medium that supports the growth of

most pathogenic fungi, including Candida and other yeast species.

However, Sabouraud agar is not a differential medium, and colonies of

different yeast species grown on this agar cannot easily be distinguished

from each other (Ilkit et al., 2007).

3) Chromogenic agar media: Two Chromogenic agars are widely used in

the clinical mycology laboratories for the detection and identification of

Candida species, specially the Candida albicans. They are Candida ID agar

and Chrom-agar candida (Baumgartner et al., 1996; Baradkar et al.,

2010).

Candida ID is a new chromogenic medium for the identification of

yeasts from clinical specimens. C. albicans produces blue pigmentation,

whereas pink pigmentation is produced by C. tropicalis, C. lusitaniae, C.

guilliermondii and C. kefyr; other Candida species appear white (Olver et

al., 2002; Yucesoy et al., 2005).

Albicans ID (bioMe´rieux) is a commercially available, ready to use

medium composed of (bio- Thione, 2 g; yeast extract, 6 g; sodium

hydrogenophosphate, 0.5 g; chromogenic substrate [hexosamine], 0.05 g; N-

2-acetamidoimino- diacetic acid monosodium salt buffer, 0.5 g; gentamicin

sulfate, 0.1 g; chloramphenicol, 0.05 g; and agar, 14 g; made up to 1 liter

with distilled water [pH 6.6] and which, after hydrolysis by the

corresponding enzyme, allows the specific identification of C. albicans

colonies.

Chromogenic media such as Albicans ID and Biggy agar allow rapid and

presumptive identification of yeasts on primary plates. An indicator

substance in these media allows the differentiation of different species

according to their colony colour or morphology. Bismuth sulphite glucose


42

glycine yeast { Biggy agar (Oxoid) }, also known as Nickerson’s medium, is a

selective and differential medium used in the detection, isolation and

presumptive identification of Candida species. The bismuth sulfite acts as

an inhibitory agent to suppress bacterial growth, which enables the

recovery of isolated colonies of Candida. Candida species produce sulfide

and bismuth, which combine to produce brown to black-pigmented

colonies and zones of dark precipitate in the medium surrounding colonies

of some species through a process of substrate reduction. Candida

albicans, Candida tropicalis, Candida krusei and Candida kefyr were

reported to be identifiable on biggy agar (Ilkit et al., 2007).

CHROMagar Candida (CHROMagar) is a differential isolation

medium composed of (peptone, 10 g; glucose, 20 g; agar, 15 g;

chloramphenicol, 0.5 g; and chromogenic mixture, 2 g; made up to 1 liter

with distilled water) which, after hydrolysis by the corresponding enzyme,

allows the specific identification of C. albicans colonies on the basis of

their green color, C. tropicalis colonies on the basis of their blue color

surrounded by a pink halo, and C. Krusei colonies on the basis of their

pink color and downy appearance (Baumgartner et al., 1996).

Chromagar, known recently as Brilliance Candida agar, has the

advantage of being a selective and differential medium that allows

selective isolation of yeasts and simultaneously identifies (by color

reactions and colony morphology) colonies of C. albicans, C. tropicalis,

and C. krusei with a high degree of accuracy. It facilitates the detection

and identification of yeasts from mixed cultures and can provide results

24 to 48 h sooner than standard isolation and identification procedures


43

(Odds and Bernaerts, 1994; Pfaller et al., 1996; Horvath et al, 2003;

Yucesoy et al., 2005; Baradkar et al., 2010; Alfonso et al., 2010).

A recent study used the chromogenic agar medium to detect the

Candida species, including the Candida albicans, Candida krusi, Candida

tropicalis and also the Candida glabrata. It was concluded that the

Chromagar Candida can be used as a primary media for the detection of

Candida species from urine specimens. The primary isolation by the

chromagar Candida may enable the clinicians to make early judgments on

the most appropriate antifungal therapy (Okulicz et al., 2008).

Another recent study was conducted for the purpose of rapid and

accurate identification of Candida species. In this study the investigators

tested 135 isolates of Candida species from the oropharyngeal candidiasis

in HIV and found that the chromogenic Candida agar was useful in the

presumptive identification of Candida albicans and Candida krusi

(Sivakumar et al., 2009).

Aim of the study

44

Aim of the study

The aim of this study was to compare between the effects of disinfection,

using commercially available cleansing tablets: Fittydent super cleansing

tablets, versus sterilization, using Gamma radiation, on the candidal growth in

maxillary partial dentures (A Microbiological study).

Materials& Methods

45

Materials and Methods

PPAATTIIEENNTT SSEELLEECCTTIIOONN

Fourteen partially edentulous patients age ranging from 29 to 42 years

(with an average of 32.8 years), were selected from the Outpatient Clinic of

Prosthodontics Department, Faculty of Oral and Dental Medicine, Cairo

University according to the following criteria:

1- All patients were non-smokers and free from systemic diseases,

especially those affecting candidal count in the oral flora.

2- The selected patients had partially edentulous maxillary arches

represented as Kennedy class I with the first premolars as the last

standing abutments (Fig. 3). The opposing lower arches were dentulous.

3- The last tooth extraction, in all cases, was at least six months before

starting any prosthetic treatment.

4- The edentulous ridges had normal morphology and covered with healthy

firm mucosa, free from severe bony undercuts or flabby tissue.

5- Proposed abutments had adequate bony support (at least ¾ of the root

length).

6- All patients had adequate interarch distance. Patients with supererupted

mandibular posterior teeth opposing the edentulous areas were not

included in the study.

7- Patients receiving antibiotic treatment or corticosteroid therapy were

excluded.

Materials& Methods

46

The selected patients were informed of the nature of this research work. Only

motivated patients who showed co-operation participated in the study.

PPAATTIIEENNTT EEXXAAMMIINNAATTIIOONN

Patient examination included medical history, to evaluate the general health of

the patient, dental history and examination, as well as extra-oral and intra-oral

examination.

Medical History

Patients were asked about their past and present medical condition. They were

also asked to perform special laboratory investigations to exclude any systemic

disease that could affect the Candidal count such as immunosuppression,

anemia and diabetes. These investigations included complete blood picture. The

leucocytes count and the total RBCs count were observed to exclude the

immunosuppressive and the anemic patients. Blood glucose level was also

examined to exclude diabetic patients.

Dental History and Intraoral Examination

* Each patient was asked about the cause of extraction, date of the last

extraction and about any previous experience(s) with prosthetic restorations.

* Mucosa of the edentulous areas was examined visually to detect any signs of

inflammation or any pathology. Mucoperiostium covering the edentulous areas

was palpated to detect the presence of any tissue flabbiness.

* Maxillary edentulous ridges were examined for the presence of any bony

undercuts, sharp ridge, tori or any abnormality.

Materials& Methods

47

* Oral hygiene was evaluated. Generally, upper and lower teeth were examined

for caries, gingival recession, and presence of periodontal pockets, calculus,

over-eruption or tilting.

Radiographic Examination

Panoramic radiographs were made to detect the presence of any remaining

root(s), any pathologic lesion(s) or any periapical pathosis around the abutment

teeth.

Periapical radiographs were made to evaluate the amount of bone support

around the abutments, the continuity of the lamina dura and width of

periodontal ligament space.

Figure 3: Preoperative photograph of the maxillary arch

Materials& Methods

48

SStteeppss ooff RReemmoovvaabbllee PPaarrttiiaall DDeennttuurree CCoonnssttrruuccttiioonn

For all patients, the following was carried out: Supra and sub-gingival scaling

was done to achieve a common hygienic baseline for all patients before starting

any prosthetic treatment. Carious teeth were restored or crowned when

necessary.

Upper and lower preliminary* impressions were made using irreversible

hydrocolloid impression material in a suitable stock tray (Fig. 4) and

immediately poured in dental stone** to obtain diagnostic casts.

Figure 4: Upper and lower preliminary impressions

The maxillary study cast was surveyed to determine the most acceptable path of

insertion that satisfies the requirements of sufficient guiding planes, good

retention, easy removal and insertion, as well as, good esthetics. A preliminary

design was then drawn on the surveyed study cast (Fig. 5).

* Tropicalgin, Zhermack S.p.A, Italy.

** Type III Dental stone, Lascod SPA, Sestofino (f1), Italy.

Materials& Methods

49

Figure 5: The design drawn on the study cast

The removable partial denture design constituted Combination clasps on the

first premolars (with mesial occlusal rests, palatal bracing arms and buccal

gingivally approaching clasp arms), canine extensions on the canines,

meshwork saddles and a combination complete palatal coverage major

connector.

Mesial occlusal rest seats were prepared on the first premolars. They

were prepared so as to be triangular oval in shape. The floor of the rest seat was

spoon shaped with no sharp edges and was deeper towards the center. Rest seats

were also prepared on the cingulum of the canines using an inverted cone bur.

Materials& Methods

50

An acrylic special tray with spacer was constructed on the upper study cast. A

final impression of the upper arch was made using rubber base impression

material*, then poured in type IV improved stone** to obtain the master cast.

The master cast was then surveyed and prepared (Fig. 6) for duplication

into an investment cast. Waxing up of the partial denture framework was then

carried out on the obtained investment cast (Fig. 7).

Figure 6: Prepared master cast

Figure 7: Wax pattern of the RPD on the investment cast

The wax pattern was then sprued and cast into the metallic framework (Fig. 8).

Try-in of the metallic framework was carried out in the patients' mouth (Fig. 9)

to ensure its proper and passive seating. Any occlusal interference from occlusal

rests or other parts of the framework were detected and eliminated.

An interocclusal record was made using softened baseplate wax***

on a trial

denture base made at the edentulous areas. The upper cast was mounted on a

semi-adjustable articulator using a facebow record and the lower cast was

mounted using the interocclusal record.

* Monpren transfer, Kettenbach Dental Germany.

** Lascod SpA, Sestofino (f1), Italy.

*** Cavex set-up regular, Haarlem, Holland.

Materials& Methods

51

Figure 8: The RPD framework on the cast

Figure 9: Try-in of the RPD framework in the patients mouth

Materials& Methods

52

Setting up of the artificial teeth was carried out using cross-linked acrylic

teeth**

followed by waxing-up. Occlusion was checked in centric and lateral

relations and any supra-contacts were eliminated.

The waxed-up denture was flasked, packed and processed into heat cured

acrylic resin, as usual (Fig. 10). The processed denture was checked in the

patients' mouth (Fig. 11). Any pressure areas were detected using pressure

indicating paste and eliminated. Occlusion was checked and any necessary

adjustments were made to achieve a harmonious occlusal relationship in all

mandibular excursions.

Finally, the removable partial denture was polished and delivered to the patient.

Figure10: The finished upper RPD on the cast

Figure 11: The finished RPD checked in the patients mouth

The patient was instructed to use the RPD correctly and to perform oral

hygiene measurements as follow: * Vitapan cross-linked acrylic teeth, Vita, Germany.

Materials& Methods

53

1) RPD should be removed from the mouth after each meal and cleaned

under running water to remove loose adherent remnants.

2) Natural teeth should be cleaned using tooth brush, tooth paste and dental

floss after each meal mainly proximal surfaces facing edentulous ridges.

3) RPD should be treated carefully.

4) At night the RPD should be removed and kept in cold tap water.

Patients were informed about the importance of plaque control and the

effect of its neglect. They also were instructed to return after one week

for correcting any denture complaints and were asked to contact if any

pain in teeth or soft tissues was experienced.

Patients were equally and randomly divided into two main groups.

Group I-Disinfection group

Dentures in this group were disinfected using Fittydent super cleansing

tablets*1

(Fig. 12). The denture was cleaned with water thoroughly. One tablet

was dropped in a glass with warm water. The water was enough to completely

cover the denture. The denture was left in the solution for 30 minutes (as

recommended by the manufacturer). After the specified time, the denture was

removed from the solution and well rinsed under running water.

Group II-Sterilization group

Dentures in this group were subjected to radiosterilization using the Indian

* Fittydent International GMBH A-7423 , Pinkafeld, Austria.

Materials& Methods

54

Gamma Cell (Fig. 13), 9100 Ci, Sep. 2005, Co-60*1

. Irradiation of the dentures

was performed at the National Centre for Radiation Research and Technology

(NCRRT), Cairo, Egypt, using cobalt 60.

The Indiain Gamma Cell provides a dose rate of 10 KGy (Mrad)/ 3 hr Co 60

at

the time of the experiment. Each denture was wrapped in a plastic wrapping

then exposed to a dose of 15 KGy.

Figure 12: The Fittydent super cleansing tablets

Figure 13: The Indian Gamma Cell

Swabs were taken to collect samples from the dentures at the following periods:

One month after denture delivery

*Baha Baha Research Chamber. Model No. 400 A, India. Irradiation Chamber Dimension: Diameter 16 cm,

height 20 cm.

Materials& Methods

55

Immediately after disinfection / sterilization

One week after disinfection / sterilization

Two weeks after disinfection / sterilization

Three weeks after disinfection / sterilization

Four weeks after disinfection / sterilization

Five weeks after disinfection / sterilization

The bacteriological part of this study was done in the same lab by the same

clinician. Swab technique was used as follows; sterile cotton swabs were used

to collect samples from the fitting surfaces of the dentures by moving the tip of

the swab on the whole acrylic part of the denture only once and in only one

direction. Swabs were immediately immersed in 2 ml of sterile saline for 5

minutes to extract the Candida in the swabs.

Volumetric bacteriology platinum loop (10 μl) (Fig. 14) was used to pull 1 ml

out of the saline and then culture all its content on a sabouraud’s dextrose agar

plate.

Figure 14: Volumetric bacteriology platinum loop

Materials& Methods

56

These plates were then incubated at 37 C° for 48 hours (Fig. 15).

Bacteriological candidal colony count was then performed using a manual

contact colony counter*1

(Fig. 16).

Figure 15: The incubator

Figure 16: The manual contact colony counter

Candidal colonies appeared to be white (Fig. 17) to creamy (Fig. 18) in color,

smooth and glistening. If no colonies were visible, a negative result was

recorded (Fig. 19).

* Manual contact colony counter, IUL instruments, S.A.

Materials& Methods

57

Figure 17: white candidal colonies

Figure 18: Creamy candidal colonies

Figure 19: No candidal growth

Materials& Methods

58

Some sabouraud’s dextrose agar plates were divided using a metal loop to

culture the swabs of the same patient before and after disinfection and

sterilization to show the effect of both methods on candidal count (Fig. 20).

A B

Figure 20: Divided petri dishes showing: A: Candidal count before and after sterilization B: Candidal count before and after disinfection

Smears from the colonies were prepared, stained with gram stain and

examined by oil- immersion lens of the microscope for the presence of budding

oval gram positive yeast cells and pseudohyphae of Candida (Fig. 21).

Materials& Methods

59

Figure 21: Candida Albicans under microscope

Data were tabulated and represented as mean and standard deviation.

Statistical analysis was performed with the aid of Microsoft Office 2003 (excel)

and Statgraphics Plus version 5.

t- test was used to compare between the two groups. Paired t-test was

conducted to detect the presence of any significance between the follow up

periods. Value less than 0.05 was considered as the level at which statistical

significance exists.

Results

60

Results

Results of this study were represented in tables (1 and 2) and figures (22 - 25).

A- Effect of group on candidal colony count

Generally speaking, subjecting the dentures to both methods (disinfection and

sterilization) led to a noticeable decrease in the candidal count. However this

decrease was highly and markedly evident in Group II (Gamma Sterilization

group) as compared to Group I (Disinfection group).

One month after denture delivery, mean candidal count was 114.14 and 111.71

in groups I and II respectively. After subjecting the dentures to the two

methods, mean candidal count decreased dramatically to 11.43 in group I and to

a radical 0.00 in group II.

The mean candidal count then increased gradually in group I to be 22.29 and

38.86 1 week and 2 weeks after denture disinfection, whereas in group II , the

dentures were still candidal free (No candiadal growth) as the mean candidal

count remained 0.00 1 and 2 weeks after gamma sterilization.

This was followed by a gradual increase in the mean candidal count in both

groups. After 3 weeks it was 56 and 5.57 in groups I and II respectively. After 4

weeks, it was 69 and 12.71 in groups I and II respectively. At the end of the

follow up period (after 5 weeks), it was 88.57 and 19.43 in groups I and II

respectively.

Statistical analysis revealed highly significant differences between the

two groups 3, 4 and 5 weeks after Disinfection/Sterilization. (table 1 and figure

23, 24).

Results

61

Table 1: Effect of group on mean candidal colony count

Group I Disinfection

Group II Gamma

Sterilization t-test P value

Mean 114.14 111.71 1 m after

denture delivery SD 8.41 10.70 0.472 NS

Mean 11.43 No growth Immediately

after D/ S SD 6.75 ------ ------ ------

Mean 22.29 No growth 1 week after D/ S SD 10.87 ------

------ ------

Mean 38.86 No growth 2 weeks after D/

S SD 9.39 ------ ------ ------

Mean 56 5.57 3 weeks after D/

S SD 10.12 2.99 12.648** <0.0001

Mean 69 12.71 4 weeks after D/

S SD 10.91 4.31 12.697** <0.0001

Mean 88.57 19.43 5 weeks after D/

S SD 17.63 6.02

9.819** <0.0001

D= Disinfection; S= Gamma Sterilization; SD = standard deviation; NS: non significant; p > 0.05 * Significant at P ≤ 0.05. **: very highly Significant at P ≤ 0.0001

Figure 22: Effect of group on mean candidal colony count

Results

62

B- Effect of time on candidal colony count

I- Group I (Disinfection)

There was a dramatic decrease of candidal colony count immediately after

disinfection. This was followed by a gradual increase throughout the study

period. However, even 5 weeks after disinfection the mean candidal count was

still clearly lower than the initial value (before disinfection).

1 month after delivery the mean candidal count was 114.14. After

disinfection this mean dramatically decreased to 11.43. 1 week later the mean

slightly increased to become 22.29. This was followed by a gradual steady

increase in the mean candidal count as it became 38.86, 56, 69 and 88.57 after

2, 3, 4 and 5 weeks respectively. Statistical analysis revealed significant

differences between the first period and all other follow up periods however this

significance was very high till the sixth period then it decreased slightly at the

end of the follow up period (table 2 and Figure 23, 25).

II- Group II (Gamma Sterilization)

Mean candidal colony count was radically decreased to 0.00 immediately after

sterilization. This candidal free result (0.00 value) was also recorded at the 1

and 2 weeks follow up periods. This was followed by a gradual yet slight

increase in the candidal count throughout the study period. Five weeks after

sterilization, the mean candidal count was still considerably and incomparably

lower than the initial value (before sterilization). 1 month after delivery the

mean candidal count was 111.71. After sterilization it dramatically decreased to

0.00 and remained 0.00 1 week and 2 weeks later. It then slightly increased to

5.57, 12.71 and 19.43 after 3, 4 and 5 weeks respectively. Statistical analysis

revealed highly significant differences between the first period and the fifth,

sixth and seventh periods. (table 2 and Figure 24, 25).

Results

63

Figure 23: Effect of time on Candidal colony count in Group I (Disinfection group); (a) 1 month after delivery (b) Immediately after disinfection (c) 1 week after disinfection (d) 2 weeks (e) 3 weeks (f) 4 weeks (g) 5 weeks after disinfection

a b

c d

e f

g

Results

64

Figure 24: Effect of time on Candidal colony count in Group II (Sterilization group); (a) 1 month after delivery (b) Immediately after Sterilization (c) 1 week after Sterilization (d) 2 weeks (e) 3 weeks (f) 4 weeks (g) 5 weeks after Sterilization

a b

c d

e f

g

Results

65

Table 2: Effect of time on candidal colony count in the two groups

Group I Disinfection

Group II Gamma Sterilization

Groups

Period Mean SD Mean SD

1 m after denture delivery 114.14 8.41 111.71 10.70

Immediately after D/ S 11.43 6.75 No growth

------

1 week after D/ S 22.29 10.87 No growth

------

2 weeks after D/ S 38.86 9.39 No growth ------

3 weeks after D/ S 56 10.12 5.57 2.99

4 weeks after D/ S 69 10.91 12.71 4.31

5 weeks after D/ S 88.57 17.63 19.43 6.02

First period – second period P-value <0.0001** ------

First period – third period P-value <0.0001** ------

First period – fourth period P-value <0.0001** ------

First period – fifth period P-value <0.0001** <0.0001**

First period – sixth period P-value <0.0001** <0.0001**

First period – seventh period P-value 0.005 * <0.0001**

D= Disinfection; S= Gamma Sterilization; SD = standard deviation * Significant at P ≤ 0.05. **: very highly Significant at P ≤ 0.0001

Results

66

Figure 25: Effect of time on candidal colony count in the two groups

Discussion

67

Discussion

Discussion of methodology

The importance of disinfecting dental appliances, as mentioned earlier,

has been and will continue to be a crucial issue for dental clinicians. Because of

the vitality of this subject, different new materials and methods for denture

disinfection are being continuously introduced.

Since some chemicals used for disinfection may become absorbed into

the material porosities and may not be completely eliminated by rinsing (i.e)

chemicals may be unintentionally introduced intra-orally, sterilization of

dentures was suggested and recommended by some investigators. Microwave

irradiation was hence introduced and was found to be a safer, simple, and

inexpensive method (Dixon et al., 1999). In fact, Willcox, (2002) found that

denture sterilization using Microwave irradiation may be a more effective

method than denture disinfection by soaking in sodium hypochlorite.

Since denture sterilization by microwaving has already been investigated,

the aim of the current study was to introduce a new method of sterilization using

Gamma radiation (radio sterilization). It was interesting to know whether there

will be a significant difference between denture disinfection (with a

commercially available denture cleanser) and denture sterilization, using a more

sophisticated and drastic measure as gamma radiation. Although the answer to

this question seems to be apparent and logic, yet it was necessary to augment it

and prove the hypothesis of “sterilization is more effective than disinfection”

with objective proof, and this was the core aim of this study.

The close relationship between colonization of the denture base by

Candida albicans and denture related stomatitis has been well documented in

Discussion

68

the dental literature. Candida albicans was also found to be the most prevalent

of all Candida species, both in healthy and diseased oral cavities (Budtz-

jorgensen and Bertram, 1970; Arendof and Walker, 1980; Ferracane,

1995). Also, it has been reported to be the most pathogenic member of Candida

species, capable of adhering to epithelial cells and acrylic surfaces causing

infection (Arendof and Walker, 1987; Renner et al., 1979). From this

prospective, the current study was mainly concerned with and evaluated only

the effect of both methods on candidal count (growth).

Taking that into consideration, patient selection was very critical as the

selection criteria were mainly directed towards eliminating the possible factors

that could affect Candidal count, to ensure that the findings reached in the study

were only due to the evaluated methods and not to any other factor. This was

necessary for the reliability and validity of the results. Therefore, special

investigations were made to exclude patients with systemic diseases that could

increase the Candida count such as immunosuppression, anemia and diabetes.

Patients were questioned orally and were asked to undergo some laboratory

investigations to ensure that these specific criteria were truly fulfilled. Some

systemic diseases such as, diabetes mellitus, anaemia and immunodeficiency

have been reported to cause imbalance of the oral microbial flora and to

adversely affect tissue tolerance, resulting in increased mucosal inflammation

with the use of dentures (Bell et al., 1977; Bergendal, 1982; Dorey et al.,

1985). Moreover, antibiotic treatment or corticosteroid therapy may affect the

oral microbial balance (Bergendal, 1982) especially candidal growth. The

intake of some drugs as anticholionergics, antihistamines, sedatives and

hypnotics have also shown to decrease salivary flow, providing a suitable

environment for mucosal inflammation and infection (Budtz-Jorgensen, 1978).

Accordingly, patients receiving these drugs were also not considered. To

confirm the results of the laboratory investigations thorough intraoral clinical

Discussion

69

examination was also carried out, to detect the presence of any white lesions,

hyperplastic tissues or any inflamed areas.

Patients with partially edentulous maxillary arches were selected as

Bergendal and Isacsson, (1983) observed that denture stomatitis more often

affects the maxillary than the mandibular denture bearing mucosa. Their results

demonstrated that although the condition may involve the entire upper denture

bearing area, it is more often confined to the mucosa of the palatal vault.

The RPD design used in this study was chosen according to the principles

advocated and recommended by Stewart et al., (1992) and McGivney et al.,

(2000).

Since the selected cases were free-end saddle cases, combination clasps

with buccal gingivally approaching retentive clasp arms were used to decrease

the torque on the abutments. Canine extensions were used on the canines

bilaterally to act as indirect retainers to decrease the rotational movement of the

distal extension bases. Complete palatal coverage major connector was used to

provide more support, as well as, direct and indirect retention for the RPD. The

combination type was specifically chosen where the anterior part was metallic

and the middle and posterior parts were mainly acrylic resin. This was

important for two reasons. First, this is the most commonly used type in RPDs

restoring Kennedy class I cases. Secondly, it has been reported that the

characteristic porosity of acrylic denture bases acts as a suitable media for the

colonization and growth of Candida albicans (Carr et al, 2005; Thein et al.,

2007). On the other hand metallic denture bases are more hygienic and resistant

to plaque accumulation and bacterial adherence as compared to non metal

bases; this may be due to their polished and non porous surfaces with fewer

tendencies for food accumulation (Krol et al, 1990). Therefore to obtain more

Discussion

70

evident and perceptible results from which conclusive statements can be drawn,

the parts from which the swabs were taken were made from acrylic resin.

Poor denture hygiene has been established as a significant factor in the

development of mucosal inflammation (Budtz-jorgensen and Bertram, 1970;

Renner et al., 1979; Abelson, 1981). Therefore, patients were instructed to

clean their dentures after each meal under tap water, with no other mechanical

or chemical means, to try to decrease mucosal inflammation with all its adverse

effects. Also, wearing dentures day and night increases plaque accumulation

and hence, increases the risk of developing denture related stomatitis

(Bergendal, 1982; Arendorf and Walker, 1979). For this reason, patients were

instructed to remove their dentures during sleep for 6-8 hours. This will also

give chance for tissue recovery and physiologic stimulation, which is important

for the integrity of the oral tissues.

As mentioned earlier, the aim of the study was to compare between

disinfection with a commercially available denture cleanser and sterilization

with gamma radiation. Fittydent super cleansing tablets were used in this study

as the commercial denture cleanser. These tablets are one of the products of

Fittydent International GmbH company which is an Austrian company

specializing in the manufacture and marketing of unique oral hygiene products.

According to the manufacturer these tablets can clean, whiten and freshen full

dentures, partial dentures and orthodontic braces. It can kill harmful bacteria

that cause odor, dissolve tough nicotine and tea stains, and help prevent daily

stain and plaque build up.

According to the company, the ingredients of these tablets are: Sodium

perborate, sodium bicarbonate, potassium monopersulphate, trisodium

phosphate, peg-240, sulphamic acid, pvp, taed, silica, sodium methyl oleoyl

taurate, cellulose-lactose, colour c.i. 42090 and aroma.

Discussion

71

The Fittydent Cleansing Tablets work quickly to remove stains and

hygienically clean dentures. If the user uses the fittydent cleansing tablets on a

daily basis, the denture will be sufficiently cleaned in a few minutes. If the user

uses the tablets irregularly, a cleansing time of 30 minutes is recommended. In

order to remove older residues or stains, leaving the dentures in the solvent

overnight is recommended. When fittydent cleansing tablets are used regularly,

the baking soda and peroxide formula cleans even stubborn stains like tar and

nicotine, and whitens teeth. If a thorough cleaning is necessary, leaving the

denture in the solvent overnight is recommended. Since the fittydent cleansing

tablets are alkaline, not acid, metal parts will not be affected. Therefore they are

also highly recommended for partial dentures and orthodontic braces.

As for gamma sterilization, the current study could be a pioneer in

studying the effect of this type of radiation from a biological point of view. Few

earlier studies (Usanmaz et al., 2001; Diego et al., 2007; Morejon et al., 2008)

investigated the effect of gamma radiation from other aspects. Usanmaz et al.,

(2001) investigated the effect of the radiation dose on the thermal and

rheological properties of poly (methyl methacrylate) (PMMA) used as a dental-

base material. Diego et al., (2007) studied the effect of sterilization process by

gamma-irradiation on the structure of poly (ethyl acrylate-co-hydroxyethyl

methacrylate) copolymer networks, P(EA-co-HEMA) for a broad dose range (7,

15, 25 and 50 kGy). Hence comparison between the current study with other

studies in the dental literature was somehow a “mission impossible”!

In the present study, the Indian Gamma Cell was used to sterilize

dentures at a dose rate of 10 KGy / 3hr at the time of experiment with a total

dose of 15 kGy for every denture (the source of γ radiation was Cobalt-60). The

Discussion

72

15kGy dose was particularly chosen based on the recommendations of earlier

investigations that showed that doses higher than 15kGy could lead to adverse

effects on the sterilized material, such as colour changes or mechanical

alteration as brittleness or even fracture (Murray et al., 1974; Behr et al, 2005;

Diego et al., 2007).

Dentures were wrapped in a plastic wrapping during gamma sterilization,

to prevent air from entering to the sterilized dentures as Sychterz et al., (2004)

and Stea et al., (2006) proved that polyethylene (which is a polymer) sterilized

in air showed significantly greater wear than those sterilized without air.

The initial, as well as, follow up swabs were taken from the fitting

(tissue) surface of the denture. The tissue surface of the denture is considered as

an irregular surface as it usually shows micro pits and microporosities that

harbor microorganisms that are difficult to remove by mechanical methods. The

swabs were taken, specifically from the acrylic part of the denture. This is

noteworthy, as they are the most suspected areas for candidal colonization

(Borg and Ruchel, 1988; Shinada et al., 1997; Pereira-Cenci et al., 2008).

Standardization is a key factor in obtaining reliable, valid and accurate

results. For that reason, it was of utmost importance to perform all the necessary

measures that will achieve standardization as much as possible. First of all, all

swabs were done in the same lab by the same clinician. This was essential to

eliminate human variability. The swab technique itself was consistent

throughout the study where sterile cotton swabs were used to collect samples

from the dentures by moving the tip of the swab on the whole acrylic part of the

denture only once and in only one direction. Furthermore, all swabs were

immediately immersed in 2 ml of sterile saline for 5 minutes to extract the

Candida. A volumetric bacteriology platinum loop (10 μl) was then used to pull

exactly 1 ml out of the saline and culture it on sabouraud’s dextrose agar plate

Discussion

73

so that all the loop contents were cultured on the plate (Collee et al., 1996;

Cheesbrough, 2000). This ensured that, each time, equal amounts of saline was

cultured on the plates to eliminate quantification variability. Moreover, both the

incubation temperature (37 C°) and time (48 hours) were constant for all tested

plates to eliminate procedural variability.

There are several ways to count the resultant Candidal colonies. Some

investigators divide the dish into four quadrants, count the number in one

quadrant and then multiply this number by 4. However, this method is somehow

inaccurate. A more accurate method is to count all the colonies appearing in the

dish without dividing it. For more accurate and reliable results, the latter method

was used in this study using a specific colony counter which facilitated the

counting procedure as it made visualization of the candidal colonies clearer and

easier.

To confirm that the white colonies that appeared on the plates were

candidal colonies, light microscopic examination was performed to detect the

presence of budding oval gram positive yeast cells and pseudohyphae of

Candida (Cheesbrough, 2000).

Discussion

74

Discussion of Results

Initially speaking, the present study emphasized the significance of

disinfecting dental prostheses. First of all, the results demonstrated that all

samples obtained in the initial culture (one month after denture delivery) in both

groups were highly contaminated with Candida albicans as candidal colony

count exceeded 100 in both groups. Such finding throws the light on the fact

that a dental prosthesis could be a source of infection and cross contamination

between patients and dental personnel, and as mentioned at the beginning,

emphasizes the importance of disinfection. It is noteworthy at this point to

mention that despite the patients were instructed to clean their dentures under

running water using a denture brush; still it was not enough in disinfecting the

dentures. This finding coincides with the evidence reached by other studies in

that mechanical cleansing alone is not sufficient to completely remove denture

plaque (Arita et al., 2005; Devine et al., 2007).

As regards the effect of group, the results obtained revealed that,

disinfecting dentures using fittydent super cleansing tablets and sterilizing them

using gamma rays are both effective methods in reducing the number of

Candida. However, this decrease was extremely and markedly evident in group

II (sterilization group) as compared to group I (disinfection group), throughout

the study period. Despite the fact that the two methods reduced the candidal

count significantly, yet its worthy to mention that throughout the study period,

the mean candidal count recorded for Group II (sterilization group) was always

lower than the mean candidal count recorded for group I (disinfection group -

fittydent cleansing tablets). In fact, statistical analysis revealed highly

significant differences between the two groups.

Discussion

75

Several findings supported the initial conclusion that gamma sterilization

is superior to and more efficient than disinfection using the Fittydent tablets.

First of all, the candidal count in group I decreased from 114.14 to 11.43

representing a considerable 90% reduction. However, in group II, candidal

count drastically and radically decreased from 111.71 to “0.00” (No candidal

growth) representing an outstanding 100 % reduction. Meaning that gamma

sterilization resulted in complete eradication of candida leading to a “candidal –

free denture”! This suggests that gamma sterilization had a more direct

influence on the actual viability of candida leading to the death of ALL candidal

colonies and consequently arrested their reproduction process. It has been

reported that Gamma sterilization could be defined indeed as the complete

removal (inactivation) or complete and total destruction of all forms of

microbial life (Sharma). On the other hand, the cleansing tablets seemed to

have had more of an indirect effect leading maybe to the death of most but not

all candida that colonized the denture base. This allowed and gave the

opportunity to the remaining viable candidal groups to nourish and reproduce

leading subsequently to the higher candidal counts recorded for group I.

Another explanation could be that the cleansing tablets may have had a

superficial effect; either by interfering with the adherence of candida to the

denture surface or by eliminating the superficial biofilm and killing the candida

colonizing the external surface only. As mentioned earlier, this oxygenating

cleanser contains alkaline compounds, detergents, sodium perborate and

flavoring agents that when mixed with water, sodium perborate decomposes

releasing peroxides which in turn releases oxygen. Cleansing is a result of the

oxidizing ability of the peroxide decomposition and the effervescent action of

the evolved oxygen. This effectively breaks down, dissolves, and floats away

the organic deposits and kills micro organisms. This action is mainly on the

exterior of the denture. On the other hand, the effect of gamma sterilization

Discussion

76

seemed to have extended deeper into the interior of the denture eradicating even

the well adherent and retained candida that invaded inaccessible areas in the

denture base. Hence it acted on the exterior as well as the interior structure of

the denture. Such double effect could be attributed to its high penetrating power

(Yu, 2001) that even exceeds that of X-rays. Such power allows it to penetrate

thick lead (Yu, 2001), so it seems very sensible that it could have easily

penetrated the acrylic denture base, which is much less compact and less dense

than lead.

The high penetrating power and direct effect of gamma rays could also

explain why the denture remained “candidal-free” for two whole weeks which

seems to represent the time needed by the candida in the oral cavity to re-adhere

and re-colonize on the denture surface. It also proves that gamma radiation did

in fact eradicate all the candidal colonies on the denture even those that were

sheltered in deeper areas.

It is worthy to mention that both methods had a favourable long term effect.

Despite the gradual increase in the candidal colony count that was observed in

both groups ,the values obtained at the end of the follow-up period (5-weeks)

were still lower than the initially recorded values. This could explain the highly

significant differences found between the initial and the rest of the follow up

periods in both groups. Its noteworthy however that the values recorded at the

end of the study suggest that gamma radiation, does not only have a superior

immediate effect, but also has a more superior and long term effect than the

cleansing tablets. Although final values were 22% lower than the initial values

in group I, which could still be considered as a satisfactory result, yet

interestingly enough, final values in group II were still 83% lower than the

initial values. Such observation augments and supports the “high penetrating

power and direct effects” of gamma sterilization.

Discussion

77

To sum up, the present study answered to a great extent the introductory

question. Sterilization using gamma radiation proved to have a more superior

qualitative, as well as, quantitative influence on candidal growth as compared to

disinfection with Fittydent cleansing tablets. However, it is fair to say that

despite its exceptional efficiency, it is still a more sophisticated, technique-

sensitive and more expensive procedure that requires the availability of a

specific device. On the other hand, using denture cleansing tablets is a simpler,

less sophisticated, cheaper, more applicable and an easy-to use home measure

that still is efficient enough to disinfect dentures. Hence from a biological point

of view, sterilization using gamma radiation could be considered the better and

more reliable of the two methods. However from the applicability and patient

point of views disinfection using commercially available cleansing tablets

seems a more appropriate alternative.

Summary and conclusion

78


This study was conducted on fourteen patients from the outpatients’ clinic

in the Faculty of Oral and Dental medicine (Cairo University). One upper

removable partial denture was made for each patient and all patients wore their

dentures for a period of one month to evaluate the effect of both disinfection

and sterilization on the number of Candida albicans adhering to their dentures.

Disinfection was done using commercially available Fittydent super

cleansing tablets. Whereas, the used sterilization method was Gamma radiation

where we used the Indian cell present at the National Centre for Radiation

Research and Technology (NCRRT), Cairo, Egypt, using cobalt 60. The effect

of both methods on the number of Candida albicans adhering to the dentures

was evaluated by taking seven swabs from the dentures. The swabs were before

disinfection and sterilization, immediately after disinfection and sterilization,

after one, two, three, four and finally after five weeks of disinfection and

sterilization.

Patients were divided into two groups, each group included seven

patients. In the first group, dentures were disinfected using Fittydent super

cleansing tablets. In the second group, dentures were sterilized using Gamma

radiation.

Culturing the specimen and counting the colonies was done using the

sabouraud’s dextrose agar. Data was collected and analyzed statistically using t-

test and paired t-test.


79

Gamma sterilization was much more superior and compared favourably

to disinfection using Fittydent tablets. Statistical analysis revealed highly

significant differences between the two groups after 3, 4 and 5 weeks of follow

up periods. Despite the gradual increase in the candidal colony count that was

observed in both groups ,the values obtained at the end of the follow-up period

(5-weeks) were still lower than the initially recorded values. Statistical analysis

revealed highly significant differences between the first period (1 month after

denture delivery) and all other follow up periods for disinfection group. While

for sterilization group statistical analysis revealed highly significant differences

between the first period (1 month after denture delivery) and 3, 4 and 5 weeks

of follow up periods

Within the limitations of this study, the following conclusions could be

drawn:

1- Both methods were effective in decreasing the number of Candida albicans

adhering to the dentures.

2- Sterilization using Gamma radiation had a more superior immediate, as well

as, long term efficiency as compared to disinfection using Fittydent super

cleansing tablets.

Recommendation

To achieve results similar or close to those obtained for the sterilization group,

it may be recommended to immerse the dentures in the Fittydent super

cleansing tablet solution for eight hours (overnight) daily.

References

80

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96

ت ذه أجري ة ه ى الدراس ة عل شراً أربع ن ع رددين المرضى م ى المت ادة عل العي

عمل تم .القاهرة بجامعة سنانالفم و الأ طب بكلية الصناعية الإستعاضة بقسم الخارجية

م ى طق وى (متحرك جزئ د و مريض لكل )عل م مرضى ال إستعمل ق ده الأطق شهر، لم

ى التعقيم و التطهير تأثير لتقييم وذلك دا (الأبيض الفطر عدد عل ك الملتصق ) الكاندي بتل

.الأطقم

ذه فى إستخدم وقد راص الدراسه ه ره، )Fitty dent( أق ا المطه إستخدمت بينم

ه إستخدمت حيث للتعقيم جاما آشعة ه الخلي المرآز الموجوده الهندي ومى ب و لبحوث الق

آلا تأثير بمقارنة قمنا وقد. ٦٠ الكوبلت بواسطة بالتعقيم تقوم والتى الإشعاع تكنولوجيا

ى الملتصق ) الكانديدا (الأبيض الفطر عدد على الطريقتين ة حالاسط عل م الداخلي للأطق

م ذلك سبيل وفى الجزئيه م آل من مسحات سبع أخذ ت ى المسحه . طق ل آانت الأول قب

ر يم، و التطهي ا التعق ه أم ت الثاني د فكان ر بع يم و التطهي رة التعق ا مباش ت بينم اقى آان ب

.متتالية أسابيع خمسة لمدة أسبوعياً تأخذ المسحات

وعتين الى المرضى تقسيم تم فى مرضى، سبعة من تتكون عة مجمو آل مجم

ه المجموعه فى أما للتطهير )Fitty dent( أقراص استخدم الأولى المجموعه د الثاني فق

.للتعقيم جاما آشعة إستخدمت

سبارو آجار بإستخدام معمليا العينات زرع وتم م ال ائج جمعت ث م و النت ل عمل ت التحلي

.لها الإحصائى

ى القضاء فى الطريقتين بين فاعلية آثرالأ هى آانت جاما آشعة أن وجد وقد عل

ر يض الفط دا(الأب ة) الكاندي تخدام مقارن راص بإس ره، الأق د و المطه ت ق ات أثبت العملي

الملخص العربي

97

امس ع و خ ث و راب ى ثال وعتين ف ين المجم وظ ب وى ملح رق معن ود ف صائيه وج الإح

ده التدريجيه بالرغم من الزيا. إسبوع من فترة المتابعة التى امتدت لخمسة أسابيع متتاليه

دا (فى عدد الفطر الأبيض ائج ) الكاندي وعتين إلا أن النت ى لوحظت فى آلا المجم والت

جلت ل ى س ددالت ر ع يض الفط دا (الأب صق) الكاندي ى الملت ة الأسطح عل م الداخلي للأطق

ه ى الجزئي ة ف ه نهاي ت التجرب ل آان ن أق ائج م ى النت ى سجلت الت دايتها ف د. ب أثبتت وق

ره العمليات الإحص ين الفت ى ب ائيه أيضاً وجود فرق معنوى ملحوظ فى المجموعه الأول

ه . و باقى فترات المتابعه ) بعد شهر من تسليم الأطقم (الأولى ا فى المجموعه الثاني بينم

و ثالث و رابع ) بعد شهر من تسليم الأطقم (وجد فرق معنوى ملحوظ بين الفتره الأولى

. امتدت لخمسة أسابيع متتاليهو خامس إسبوع من فترة المتابعة التى

:التالية النتائج الدراسة هذه من أستخلص

دا (الأبيض الفطر عدد خفض في فعالتين آانتا الطريقتين آلا -١ ى الملتصق )الكاندي عل

.الجزئيه للأطقم الداخلية الأسطح

عة -٢ ا آش ى جام ر ه ة الأآث ين فاعلي ريقتين ب ى الط ضاء ف ى الق ر عل الفط

يض ا) داالكاندي(الأب م أن آم ة الأطق عة المعقم ا بآش ل جام ه تظ ن خالي ر م الفط

.المطهرة الأقراص بإستخدام مقارنة التعقيم بعد أطول لمدة) الكانديدا(الأبيض

ول أثيرل للوص ساو ت ب أو م ن قري أثير م تخدام ت عة إس ا آش ى جام ذه ف ه ه صح الدراس ين

ع يومياً واحد قرص المطهره )Fitty dent( أقراص باستخدام رك م م ت فى الطق

.الليل ساعات طوال المحلول

effect of gamma radiation sterilization versus

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