The Effect of Different Polishing Systems on the Surface Roughness of the Tooth Coloured Restorative Materials

THE EFFECT OF DIFFERENT POLISHING SYSTEMS ON THE SURFACE ROUGHNESS OF THE TOOTH COLOURED RESTORATIVE MATERIALS

Zaid Hameed Mohammed Ali nema

Thesis submitted in fulfillment of the requirements for the degree of Master of Science

December 2010 DECLARATION

I certify that this dissertation (Surface roughness of nanofilled and microfilled glass- ionomer polished with three polishing systems and confocal analysis after application of

Streptococcus mutans) is my own work (practical experiments,research and written

thesis), except where indicated by referencing and that I have followed the university

regulations regarding authenticity.

Signature

ACKNOWLEDGMENT

Firstly, I offer my sincerest gratitude to my father, mother, brother and sisters who

supported and encouraged me throughout my studies and to my fiancé for her kind

patience and encouragement.

I would like to express my deepest gratitude to:

My supervisor Dr. Zuryati Ab. Ghani for her priceless guidance, support and unceasing efforts throughout my study.

My co-supervisors Dr. Dasmawati binti Mohamad and Assoc. Prof. Dr. Sam’an Malik

Masudi for their support, encouragement, continual inspiration and giving me advice throughout my study.

Once again I owe my sincere thanks to my supervisors for their unlimited knowledge, hard working, patience on reading and correcting my numerous revisions.

I would like to extend my gratitude to the Universiti Sains Malaysia for the financial

support via the short term grant no. 304/PPSG/61310036

Zaid Hameed Mohammed Ali Nema

III

TABLE OF CONTENT

1 INTRODUCTION ...... 1

1.1 Background of the study ...... 1

1.2 Statement of the problem ...... 5

1.3 Justification of the study ...... 5

1.4 Objectives of the study ...... 6 1.4.1 General Objective:- ...... 6 1.4.2 Specific Objectives:- ...... 6

1.5 Hypotheses ...... 7

2 LITERATURE REVIEW ...... 8

2.1 Glass-ionomer and composite ...... 8 2.1.1 Conventional glass-ionomer cement ...... 8 2.1.2 Resin Modified Glass-ionomer ...... 11 2.1.3 Nano glass-ionomer ...... 16 2.1.4 Nano-composite ...... 19

2.2 Surface topography ...... 25 2.2.1 Polishing and finishing ...... 26 2.2.2 Polishing systems ...... 29 2.2.3 Surface roughness analysis ...... 30

2.3 Oral microbes ...... 33 2.3.1 Streptococcus mutans biofilm ...... 34 2.3.2 Confocal laser scanning microscopy (CLSM) ...... 37

3 MATERIALS AND METHODS ...... 40

3.1 Study Design ...... 40

3.2 Sample Size Calculation ...... 40

3.3 Test materials ...... 41

3.4 Specimen Preparation...... 45

3.5 Specimen preparation for objective 1 ...... 46

3.6 Specimen preparation for objectives 2, 3 ...... 49

3.7 Research tools ...... 53

3.8 Data entry and analysis ...... 54

3.9 Flow charts of the methodology ...... 55

4 RESULTS ...... 58

4.1 Comparison of the surface roughness of the test materials cured against Mylar strip before and after application of S.mutans biofilm for 30 days...... 58

4.2 Comparison of the surface roughness between the test materials polished with three different polishing systems after 30 days of S.mutans biofilm culture...... 59

4.3 Comparison of S.mutans biofilm thickness cultured for 14 days between the test materials polished with three different polishing systems...... 67

4.4 Comparison of S.mutans biofilm thickness cultured for 30 days between the test materials polished with three different polishing systems...... 75

4.5 Comparison of S.mutans biofilm thickness between 14 days and 30 days of culture on test materials polished with three different polishing systems...... 83

5 DISCUSSION ...... 87

5.1 General overview ...... 87

5.2 The surface roughness of the test materials cured against Mylar strip before and after 30 days S.mutans biofilm culture...... 89

5.3 Comparison of the surface roughness between the test materials polished with three different polishing systems after 30 days S.mutans biofilm culture...... 91

5.4 Evaluation and comparison of the S.mutans biofilm thickness cultured for 14 days and 30 days between the test materials polished with three different polishing systems.94

6 CONCLUSIONS AND RECOMMENDATIONS ...... 99

6.1 Conclusions ...... 99

6.2 Limitations of the study ...... 100

6.3 Recommendations ...... 100 6.3.1 Clinical recommendations ...... 100 6.3.2 Recommendations for future research ...... 101

REFERENCES ...... 102

APPENDICES 110

LIST OF FIGURES

Figure 2.1 : Differences in surface roughness resulting from the loss of large and small filler particles (Albers, 2002)...... 27 Figure 2.2: Diagram showing the concept of atomic force microscopy (Yao and Wang, 2005)...... 32 Figure 2.3: Basic setup of confocal microscope (Fellers, 2007)...... 39 Figure 3.1: Test materials...... 42 Figure 3.2: Polishing systems...... 44 Figure 3.3: Acrylic mold...... 45 Figure 3.4: AFM (AMBIOS Technology Inc., USA)...... 46 Figure 3.5: Specimen culturing conditions...... 48 Figure 3.6: CLSM (Leica HC laser microscope, Germany)...... 52 Figure 4.1 : Comparison of mean (SD) surface roughness Ra (nm) between the test materials and three different polishing systems...... 62 Figure 4.2 A, B, C and D : Atomic Force Microscopy 3D images surface roughness of test material polished with different polishing techniques...... 63 Figure 4.3: Comparison of S.mutans biofilm thickness (µm) cultured for 14 days between the test materials polished with Mylar and three different polishing systems...... 70 Figure 4.4 A, B, C and D: Confocal laser microscopy 3D images of 14 days S.mutans biofilm thickness of test materials polished with different polishing techniques. .... 71 Figure 4.5: Comparison of S.mutans biofilm thickness (µm) cultured for 30 days between the test materials polished with Mylar and three different polishing systems...... 78 Figure 4.6 A, B, C and D: Confocal laser microscopy 3D images of 30 days S.mutans biofilm thickness of test material polished with different polishing techniques...... 79 Figure 4.7 A, B, C and D: comparison between 14 and 30 day S.mutans biofilm thickness on all test materials polished with Mylar, Enhance/Pogo, Astropol and Sof-Lex...... 85

VII

LIST OF TABLES

Table 3.1 Test materials...... 41 Table 3.2 polishing systems...... 43 Table 4.1 Comparison of the mean (SD) of surface roughness Ra (nm) between the test materials before and after application of S.mutans biofilm over a period of 30 days...... 58 Table 4.2 Comparison of the mean (SD) surface roughness Ra (nm) between the test materials polished with three different polishing systems after 30 days S.mutans culture...... 62 Table 4.3 Comparison of the mean (SD) S.mutans biofilm thickness (µm) cultured for 14 days between the test materials polished with three different polishing systems. .... 70 Table 4.4 Comparison of the mean (SD) S.mutans biofilm thickness (µm) cultured for 30 days between the test materials polished with three different polishing systems. .... 78 Table 4.5 Comparison of the mean (SD) S.mutans biofilm thickness (µm) between 14 days and 30 days on test materials polished with three different finishing/polishing systems...... 86

VIII

Kesan Agen-Agen Gilapan ke Atas Kekasaran Permukaan bahan-bahan tampalan gigi yang sama dengan warna gigi

Abstrak

Kajian ini bertujuan mengkaji kekasaran permukaan simen glass ionomer terisi nano berbanding dengan simen glass ionomer terisi mikro dan komposit terisi nano, dengan menggunakan tiga jenis teknik gilapan yang berbeza. Kajian ini juga mengkaji ketebalan biofilem yang terbentuk di atas bahan – bahan ujikaji selepas 14 dan 30 hari pembiakan Streptococus mutans (S.mutans), dan juga kekasaran permukaan selepas 30 hari pembentukan biofilem. Acuan akrilik digunakan untuk menyediakan cakera bahan ujikaji berdimensi 5 mm x 2 mm. Bahan ujikaji dipadatkan di dalam acuan untuk membentuk 21 cakera (Set A) yang berdimensi sama, tujuh cakera untuk setiap bahan ujikaji, iaitu simen glass ionomer terisi nano, simen glass ionomer terisi mikro dan komposit terisi nano. Semua cakera yang mengandungi bahan ujikaji diliputi dengan jalur Mylar dan dikeraskan dengan menggunakan sebuah unit ’light curing’ biasa, mengikut aturan pengilang. Selepas itu, semua cakera ujikaji tadi diperiksa menggunakan mikroskop daya atom (AFM) di bawah mod sentuhan untuk mengkaji kekasaran permukaan ketiga – tiga bahan ujikaji tadi. 10 µml titisan ampaian bakteria

(S.mutans) dititiskan ke atas setiap sampel, dan diletakkan di dalam bekas mengandungi

50 ml infusi hati dan otak (IHO), 0.2 units/ml media bacitracin, dan 20% sukrosa.

Sampel-sampel diinkubasikan pada 37oC untuk membenarkan pembentukan biofilem selama 30 hari. Selepas 30 hari, kekasaran permukaan ketiga – tiga bahan ujikaji tadi

IX diperiksa sekali lagi dengan AFM. Set B mengandungi 84 cakera yang berdimensi sama dengan Set A; 28 cakera untuk setiap bahan ujikaji. Setiap bahan ujikaji dibahagikan kepada tiga kumpulan rawatan dan satu kumpulan kawalan; setiap kumpulan mengandungi tujuh sampel (n=7). Tiga kumpulan rawatan masing-masing digilap dengan Enhance / PogoTM, Astropol® dan Sof - LexTM mengikut aturan pengilang. Kumpulan kawalan menggunakan jalur Mylar. Selepas digilap, bahan – bahan ujikaji dikultur dengan S.mutans selama 14 hari. Selepas 14 hari, ketebalan biofilem S.mutans di atas bahan – bahan ujikaji tadi diukur menggunakan mikroskop imbasan laser konfokal (CLSM). Sebanyak 84 lagi cakera (Set C) digilap dengan teknik gilapan yang sama dengan Set B, tetapi dikultur dengan S.mutans selama 30 hari.

CLSM digunakan untuk mengukur ketebalan biofilem Set C seperti dalam Set B. Ini bagi membolehkan perbandingan ketebalan biofilem kultur S.mutans antara 14 dan 30 hari. Selepas itu, semua cakera – cakera Set C dikaji kekasaran permukaannya menggunakan AFM. Data dimasukkan ke dalam perisian SPSS dan dianalisa menggunakan satu – hala ANOVA di mana P < 0.05 dianggap signifikan secara statistik. Selepas 30 hari pengkulturan S.mutans, simen glass ionomer terisi nano dan simen glass ionomer terisi mikro yang digilap dengan cakera Sof - Lex menunjukkan kekasaran permukaan paling sedikit, manakala komposit terisi nano menunjukkan kekasaran permukaan paling sedikit apabila digilap dengan Astropol. Selepas 30 hari pengkulturan, ketebalan biofilem di atas simen glass ionomer terisi mikro lebih nipis berbanding simen glass ionomer terisi nano dan juga komposit terisi nano. Cakera Sof –

Lex menghasilkan biofilem S.mutans yang lebih nipis berbanding Enhance/Pogo dan

Astropol. Namun, tidak semua keputusan ini signifikan secara statistik dalam kumpulan

– kumpulan perbandingan. Biofilem S.mutans yang berusia 14 hari adalah lebih nipis

X berbanding yang berusia 30 hari di atas semua bahan-bahan ujikaji tanpa mengira sebarang sistem gilapan.

The effect of different polishing systems on the surface roughness of the tooth coloured restorative materials

ABSTRACT The aim of this study was to evaluate the surface roughness of nanofilled glass- ionomer compared to microfilled glass-ionomer and nanofilled composite using three different types of polishing techniques. This study also evaluated the biofilm thickness of the test materials after 14 and 30 days growth Streptococcus mutans (S.mutans) and the effect of surface roughness after 30 days biofilm formation. An acrylic mold was used to prepare 5 mm x 2 mm disk specimens. The test material was packed into the mold forming 21 similar disks (Set A), seven of each test material; nanofilled glass- ionomer, microfilled glass-ionomer and nanofilled composite respectively. Then the disks were covered with a Mylar strip and photo-polymerized using a conventional light- curing unit, according to manufacturer’s instruction. The disks were then examined in contact mode under Atomic Force Microscopy to evaluate the surface roughness of those three dental materials. After that a 10 µml drop of bacterial suspension (S. mutans) was placed on each sample. The samples were then placed in a container with

50 ml of brain heart infusion (BHI) broth supplemented with 0.2 units/ml of bacitracin and sucrose present in an amount of about 20 percent by weight then incubated at 37˚C to allow the formation of a 30 day-old biofilm. After 30 days, all disks were reexamined in contact mode under Atomic Force Microscopy to evaluate their surface roughness.

Set B consisted of 84 disks of the test materials with similar dimension to Set A; with 28 disks for each test material. Each test material were divided into three treatment groups and one control group; each group consisting of seven samples (n=7). The treatment

XII groups were polished with Enhance/ PogoTM, Astropol® and Sof-lexTM, according to manufacturer’s instruction. The control group was cured against Mylar strip. After polishing procedure, the samples were cultered with S.mutans for 14 days. At the end of

14 days, the S.mutans biofilm thickness on the test materials was measured using

Confocal Laser Scanning Microscopy (CLSM). Another 84 disks of the test materials were made (Set C), polished with the same technique as in Set B, however, they were cultured with S.mutans for 30 days, for the comparison of the biofilm thickness between

14 days and 30 days of S.mutans culture. Furthermore, all disks in Set C were also examined in contact mode under Atomic Force Microscopy for surface roughness evaluation. Data was entered into SPSS software and analyzed using one-way ANOVA where

P < 0.05 was considered statistically significant. Nanofilled glass-ionomer and microfilled glass-ionomer polished with Sof-Lex disks showed the least surface roughness, while nanofilled composite showed the least surface roughness when polished with Astropol after 30 days of S.mutans culture. After 30 days S.mutans biofilm culturing, microfilled glass-ionomer displayed less S.mutans biofilm thickness compared to nanofilled glass- ionomer and nanofilled composite. Sof-Lex disks produced less S.mutans biofilm thickness compared to Enhance/Pogo and Astropol, but not all results were statistically significant within the comparative groups. The thickness of the S.mutans biofilm after

14 days of culture was less than after 30 days of culture in all test materials regardless of the polishing systems.

XIII

1 INTRODUCTION

1.1 Background of the study

Glass-ionomers are part of a large group of materials that set through an acid base

reaction in the presence of water. Historically, these materials have been referred to as

cements, and they are involved in a variety of powders and liquids. Glass-ionomers

originally acquired their name from the glass filler and ionic polymer matrix which are

used to make them (Albers, 2002).

They are available in essentially two types, conventional glass-ionomer and resin-

modified glass-ionomer. The latter was created in order to improve the physical

properties and decrease the water sensitivity of the conventional materials (Ersin et al.,

2006).

The applications of the glass-ionomer filling materials express the advantage of their

adhesive nature coupled with an inherent brittleness and fluoride release in spite of the

low aesthetic quality. The main use of glass-ionomer is to restore root and teeth abrasion

cavity, fissure sealants, as a filling in deciduous teeth and in tunnel preparation technique

(McLean, 1992).

Glass-ionomers are required to meet physical, chemical, biological and esthetic

requirements, akin to all materials used in the mouth. Requirements for use include

adequate strength, abrasion resistance, resilience and dimensional stability during processing and subsequent use. In order to match the appearance of the oral hard tissue

being replaced, translucency or transparency is also required. In addition, good color stability and resistance to oral fluids with which they are in contact (Culbertson, 2001).

Nanotechnology is used to provide some value added features not typically associated with glass-ionomer restorative materials. Generally, glass-ionomer restoratives can contain a broad range of particle sizes. Filler particle size can influence strength, optical properties, and abrasion resistance. By using bonded nanofillers and nanocluster fillers, along with Aluminum fluorosilicate glass, nanofilled glass-ionomer restorative has improved esthetics, yet still provides the benefits of glass-ionomer chemistry, such as fluoride release (Malsch, 2005).

Furthermore, with nanotechnology, a dental restorative material system will offer high translucency, high polish and polish retention similar to those of microfills while maintaining physical properties and wear resistance equivalent to several commercial hybrid materials. The combination of two types of nanofillers results in the best combination of physical properties, such as superior esthetics, long-term polish retention and other optimized physical properties (Mitra et al., 2003).

One of these physical properties is the surface roughness. The surface texture of dental materials has a major influence on plaque accumulation, discoloration, wear and the aesthetical appearance of both direct and indirect restorations. Increasing roughness is correlated with increased deposition of plaque and roughness is also a determining factor for staining. Furthermore, an increased surface roughness accelerates the wear of dental materials (Heintze et al., 2006).

The critical surface roughness threshold established for bacterial adhesion is 0.2 μm. Any

increase in surface roughness above 0.2 μm results in a simultaneous increase of plaque

accumulation and the risk of caries and periodontal inflammation (Chung, 1994).

Polishing is another factor that affects the surface roughness beside filler size. Proper

finishing and polishing are important steps in clinical restorative dentistry that enhance

both esthetics and longevity of restorations. The smoothness of restorations is also

influenced by the internal structure such as size and arrangement of the filler content

(McCabe and Walls, 2008). This study will evaluate the effect of various finishing/polishing techniques on the surface roughness of different types of glass-

ionomer which are classified according to their filler size; nano and micro filler.

As mentioned earlier, one of the outcomes of the surface roughness is dental plaque formation. Dental plaque adheres better and accumulates more quickly on rough surfaces

(Ono et al., 2007). Dental biofilm harboring cariogenic bacteria are among the virulence

factors associated with the progression of tooth decay and periodontal diseases.

Streptococci mutans (S.mutans) are among the bacteria proliferating in the dental biofilm.

Their virulence is mainly due to their high adhesion capability, acidogenicity and aciduric

properties. These S.mutans characteristics could be responsible for surface damage to

restorations, since this microorganism can be found on any hard surface in the oral cavity,

such as enamel, implants, orthodontic appliances or restorative materials. Microscopic

examination of early plaque formation on teeth shows that the bacteria adhere along

cracks and pits in the enamel, suggesting an effect of the surface structure. In addition,

the surface free energy affects the accumulation of tooth biofilm. Thus, rough surfaces

and surfaces with high surface free energy are more prone to plaque formation. Similarly,

the surface characteristics of dental materials will affect biofilm formation (Carlen et al.,

2001).

It was also reported that the effects of S.mutans biofilm on the surface properties and

microstructure are material-dependent (Fucio et al., 2008). Studies on resin composites showed that’s the amount of biofilm accumulation varies according to the particle size of fillers and monomer components of the resin matrix (Ono et al., 2007) and it was proven

by another study that the nanofilled composite cause a reduction in the biofilm formation

(Hannig et al., 2007).

Other studies on resin composites showed that S.mutans growth increases surface roughness. This change in surface integrity may further increase biofilm accumulation

(Beyth et al., 2008).

Fillers and matrices of dental resin composites also influence the growth of bacterial biofilm. Resin composite restorations tend to accumulate more dental plaque compared with other restorations (Imazato et al., 2001). It was reported that polymerization of resin composites is incomplete, as indicated by the low degree of conversion (Imazato et al.,

2001) and the finding that unpolymerized monomers can be extracted and used to

accelerate the growth of cariogenic bacteria. In addition, it was shown that polymerized

resin composites accelerate S.mutans growth in vitro (Matalon et al., 2004).

This study evaluated the interaction between surface roughness of nanofilled glass-

ionomer and bacterial biofilm. Furthermore, this study also measured the S.mutans

biofilm thickness and the interaction between polishing methods and nanofilled glass-

ionomer.

1.2 Statement of the problem

The new nanofilled glass-ionomer has not been studied comprehensively with regards to its surface polish. The surface polish will affect the surface roughness, which in turn will affect the plaque accumulation. Therefore, this study was conducted to evaluate the surface roughness of nanofilled glass-ionomer compared to microfilled glass-ionomer and nanofilled composite using three different types of polishing techniques. This study also evaluated the biofilm thickness of the test materials after 14 and 30 days growth of

S.mutans and the effect of surface roughness after 30 days biofilm formation.

1.3 Justification of the study

This study will show the ability of nanofilled glass-ionomer to resist the surface roughness caused by S.mutans biofilm, which help us to specify the appropriate usage of nanofilled glass-ionomer. Furthermore, this study will be able to show the most appropriate polishing technique for nanofilled glass-ionomer. The end results will help in determining the benefit of nanotechnology in relation to the glass-ionomer product.

1.4 Objectives of the study

1.4.1 General Objective:-

The general objective is to evaluate the surface roughness of nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled composite polished with three different polishing systems after 30 days of S.mutans culture and to measure the biofilm thickness after 14 and 30 days of S.mutans culture.

1.4.2 Specific Objectives:-

1. To compare the surface roughness of nanofilled glass-ionomer, microfilled glass-

ionomer and nanofilled composite cured against Mylar strip before and after 30

days of S.mutans biofilm culturing.

2. To compare the surface roughness of nanofilled glass-ionomer, microfilled glass-

ionomer and nanofilled composite between the three polishing techniques after 30

days of S.mutans culture.

3. To evaluate and compare the S.mutans biofilm thickness cultured for 14 days and

30 days on nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled

composite polished with three different polishing techniques.

1.5 Hypotheses

1. Microfilled glass-ionomer cured against Mylar strip will be the roughest surface

after 30 days of S.mutans culturing compared to nanofilled glass-ionomer and

nanofilled composite.

2. The surface roughness is less in nanofilled glass-ionomer compared with

microfilled glass-ionomer and nanofilled composite after culturing S.mutans

biofilm for 30 days regardless of the polishing technique.

3. The biofilm thickness of S.mutans formed on the surface of nanofilled glass-

ionomer is less than on microfilled glass-ionomer and nanofilled composite after

14 days and 30 days of S.mutans culture. The biofilm thickness of S.mutans

formed in 14 days is more than S.mutans biofilm thickness formed in 30 days in

all test materials regardless of polishing techniques.

2 LITERATURE REVIEW

2.1 Glass-ionomer and composite

2.1.1 Conventional glass-ionomer cement

Glass-ionomer cements is a combination of polyacrylic acid liquid with silicate cement

powder, yielding a material that demonstrates the best properties of both (Albers, 2002).

Conventional glass-ionomer is supplied as a powder and liquid or as a powder mixed with water. The powder/liquid in the materials consists of a sodium aluminosilicate glass

with about 20% CaF and other minor additives. The liquid may consist of an aqueous

solution of acrylic acid or of a maleic acid/acrylic acid copolymer. Tartaric acid, which is

used to control setting characteristics, is also included in the liquid component by many

manufacturers. The powder/water materials are of two types; both consist of a powder

which contains glass powder and vacuum-dried polyacid (acrylic, maleic or copolymers).

2.1.1.1 Properties

The major applications of the glass-ionomer filling materials reflect the advantage of

their adhesive nature coupled with an inherent brittleness and a less than perfect aesthetic

quality. They are mainly used to restore root and teeth abrasion cavity, fissure sealants, as a filling in deciduous teeth and in tunnel preparation technique (McLean, 1992).

The conventional glass–ionomer cements have some disadvantages, such as the long

setting time and the sensitivity to water during the early stages of setting of these

materials (Sepet et al., 1997;McKenzie et al., 2003).

2.1.1.2 Fluoride release

Glass-ionomer cement contains significant amounts of fairly mobile fluoride ions. The mobile fluoride ions diffuse to the surface of the cement; they are washed away with saliva or reacted with the surrounding tooth substance. Fluoride ions replace hydroxy groups in the apatite structure and this change makes the apatite more resistant to acid attack. The presence of glass-ionomer cements reduce the chance of caries developing in the surrounding tooth substance. The cement can be considered as applying a long term topical fluoridation effect on the tooth substance with which it is in contact. Fluoride can

also absorb from an aqueous medium which has a high fluoride concentration. Hence the

level of fluoride in the cement can be ‘topped up’ as it absorbs ions released from

toothpastes, mouthwashes, and drinking water (Nicholson, 1998).

2.1.1.3 Finishing and polishing

The methods for finishing and polishing recommended by manufacturers are similar to

those used for composite resins. In previous studies, conventional glass-ionomer obtained

the smoothest surface with mylar (Mount, 1994;Liberman and Geiger, 1994).

The decrease in the particle size of the abrasive can give a superior surface. The grit in

the polishing material should be smaller than the particle size of the restorative material

that is being polished in order to produce better results (Weinstein, 1988). The smoothest

surfaces were obtained after 24 hours of placement of the restoration.

It was also observed that a rougher surface was obtained when finishing and polishing

was carried out at an early stage after placement (Matis et al., 1988). A study by Pedrini

et al. (2003) showed that the best finishing and polishing technique for conventional

glass-ionomer was obtained through Sof-Lex disks, which was independent of time.

2.1.1.4 Color stability

Glass-ionomers offer a reasonable match for the natural tooth, although most authorities agree that a better match is achieved with resin matrix composites. The translucency of the restorative cements is achieved through the presence of unreacted glass cores which are able to transmit light. Attempts to improve the properties of glass ionomers have involved changes to the composition of the glass in order to enhance reactivity with the acid component. The aesthetic nature of the materials, although not perfect, has improved significantly since the time when the materials were first introduced. An increase in surface roughness can also be responsible for alterations in light reflection that can turn material surface opaque. It has been shown that a surface is considered reflective when imperfections are well below 1 μm (Warren et al., 2002).

2.1.2 Resin Modified Glass-ionomer

Resin modified glass-ionomer materials are a combination of the best properties of composite resins and glass-ionomers. Some cariostatic properties, a low thermal expansion and the hydrophilic qualities are the same as with the glass-ionomer cements.

The polymerizing resin matrix of resin modified glass-ionomers improves the fracture toughness, wear resistance, and polish of these materials compared with conventional glass-ionomers (McCabe and Walls, 2008).

In 1992, Mitra added the first auto cured resin capabilities to resin modified glass- ionomer cements. In addition to chemical initiators which allow the resin to polymerize without a presence of light curing unit. These materials are available in auto- and dual- cure forms. The dual-cured materials have three setting reactions: photo cure, auto cure, and acid base reaction between the glass-ionomer powder and the polyacid.

The resin modified glass-ionomer cements develop strength more rapidly because of the resin polymerization component of their setting reaction. The modified poly acrylic acid is less soluble in water. This is considered a problem with these materials. Therefore, hydroxyethyl methacrylate (HEMA) must be added as a co-solvent to avoid phase separation of the resin from the glass-ionomer components. When HEMA and similar hydrophilic monomers are added to glass-ionomer, the set material can swell in water, increase in volume and weaken. In general, the greater the amount of HEMA incorporated into a material, the greater the swelling and reduction in strength (Nicholson et al., 1992).

In brief, resin modified glass-ionomer cements, known as glass-ionomer hybrid cements, set through a combination of acid base reaction and photochemical polymerization.

“Resin-modified” refers to all cements in which the acid base reaction of true glass- ionomer cements is supplemented by a light cure polymerization reaction (McLean et al.,

1994).

2.1.2.1 Compositions

Resin modified glass-ionomer consists of powder and liquid which require mixing prior to activation of polymerization. The powder consists primarily of an ion-leachable glass.

The liquid contains four main ingredients; methacrylate resin which enables setting to

occur by polymerization, a polyacid which reacts with the ionleachable glass to bring

about setting by an acid–base mechanism, hydroxyethylmethacrylate (HEMA) and

hydrophilic methacrylate which enables both the resin and acid components to co-exist in

aqueous solution. Other minor components include polymerization activators and

stabilizers. The most convenient of the restorative type resin-modified glass-ionomers are

provided in an encapsulated form in which the powder/ liquid ratio is determined by the

manufacturer and the mixing is carried out mechanically in only a few seconds (McCabe

and Walls, 2008).

2.1.2.2 Properties

Resin modified ionomers have a different usage like: - bases, luting agents, restoratives,

and bonding agents. They are appropriate for Class V restorations and for older patients

and those at high risk for caries because they have long term fluoride release. One

unusual benefit of resin modified ionomers is their capacity to take up topically applied

fluoride. Both toothpaste with fluoride and topical neutral fluoride solutions have been

proven to recharge the fluoride depleted from glass-ionomers (Burgess et al., 1994).

Their major limitations are reduced stiffness and high wear, as well as poor dimensional

and color stability. When these limitations are weighed against good adhesion and good

caries inhibition properties, resin modified ionomers offer good service for treating the

aging dentition in non stress bearing areas.

2.1.2.3 Fluoride release

Resin modified glass-ionomers behavior is similar to the conventional glass- ionomers in

terms of both the pattern of release and the daily amount of fluoride released. Key factors

in the rate of fluoride release from resin-modified materials are the extent to which the

acid–base reaction occurs during setting and the presence of HEMA, which results in the

formation of a polymeric hydrogel through which water can diffuse quite rapidly (Kosior

and Kaczmarek, 2006). The rate of fluoride release becomes very low after the first few

days of initial fluoride burst. However, a long-term release promoted by a continual

release, recharge and re-release may well provide the positive therapeutic effects. On the

other hand, studies showed that the materials which have the greatest initial fluoride

release also have the greatest ability to be recharged (Itota et al., 2004;Markovic et al.,

2008).

The effect of fluoride released from glass-ionomers on plaque and bacteria have been examined by many in vitro and in vivo studies. Some of them showed that the long term release inhibits bacterial growth (Marczuk-Kolada et al., 2006;Koo et al.,

2005;Hayacibara et al., 2003). Mount in 1994 showed that the glass-ionomer fluoride release curve showed an initial spike followed by a nearly flat but steadily declining release. Furthermore, the move of the material along the spectrum from glass-ionomer to resin polymerization, the less fluoride is available.

2.1.2.4 Finishing and polishing

Resin modified ionomers are smoother when finished than conventional glass-ionomers.

However, resin modified ionomers should be left undisturbed for 10 minutes after the

initial set, to allow the silicate gel setting reaction to progress far enough which will

stabilize the filler and the polyacid components of the polyacid matrix. Susceptibility to

dehydration is one of the resin modified glass-ionomers properties. Therefore resin

modified glass-ionomers should be finished with a water spray after setting is complete.

Although resin modified ionomer restorations are more water stable than conventional

glass-ionomers, small defects can be noticed when the resin modified ionomers are

coated with an unfilled resin. This will decrease fluoride release and may inhibit later

fluoride uptake by the resin modified ionomer. Therefore, applying unfilled resin to the

surface is recommended only for patients with a low risk of caries (Burgess et al., 1994).

Previous study showed that the use of carbides and one-step rubber abrasive system for finishing/polishing of resin modified glass-ionomer is not recommended. Graded abrasive disk or two-step rubber abrasive systems should be used instead (Yap et al., 2002).

2.1.2.5 Color stability

Composite resins are hydrophobic and set via a polymerization reaction. They have the

best long term color stability of any direct placement material. On the other hand,

traditional glass-ionomers are hydrophilic and set via acid base reactions with moderate

color stability. However, resin modified glass-ionomers have poorer color stability than

either of the original materials in spite of its setting reaction which is through a

polymerization reaction usually with poly HEMA and an acid base reaction with poly

acid and glass particles (McCabe and Walls, 2008).

The color of resin modified materials has been reported to vary with the finishing and

polishing techniques used (Heintze et al., 2006). A recent study showed that color changes after cola exposure compromised both color stability and esthetics in the resin- modified glass ionomer cement in all shades and in composites and compomers in the darkest shade (Mohan et al., 2008). Furthermore, the color stability of the resin-modified glass ionomer is shade dependent which is directly related to the glass-ionomer composition and filler size (Yap et al., 2001). Discoloration of the material over time especially in root areas, which may be self limiting, may have little clinical significance.

However, in Class V restorations on maxillary central incisors, color stability may be a major concern regardless of the fact that the placement technique is affecting the

postoperative discoloration of resin modified glass-ionomers in the long term (O'Brien,

2002).

2.1.3 Nano glass-ionomer

Nanotechnology is the ability to measure, design and manipulate at the atomic, molecular

and supramolecular levels on a scale of about 1 to 100 nm in an effort to understand,

create and use material structures, devices and systems with fundamentally new

properties and functions attributable to their small structures (Malsch, 2005).

Nanodentistry made possible the maintenance of comprehensive oral health by

employing nanomaterials, including tissue engineering and dental nanorobots.

Nanoproducts Corporation has successfully manufactured nonagglomerated discrete

nanoparticles that are homogeneously distributed in resins or coatings to produce nano-

composites. The nanofiller used include an Aluminosilicate powder having a mean

particle size of 80 ran and a 1:4 M ratio of alumina to silica and refractive index of 1.508

(Rybachuk et al., 2009). In resin modified light cure glass-ionomer, recent technical

development combine the benefits of a resin modified light cure glass-ionomer and

bonded nano filler technology to provide some value added features not typically

associated with glass-ionomer restorative materials.

2.1.3.1 Compositions

Nano glass-ionomer consists of de-ionized water, blend including HEMA, a methacrylate

modified polyalkenoic acid and surface modified fluoroaluminosilicate (FAS) glass,

nanomers and nanoclusters. The filler content of the system consists of an acid reactive

FAS glass and a unique combination of nanofillers. It comes in two part system aqueous

paste (acidic polyalkenoic acid, reactive resins and nano fillers) and non aqueous paste

(FAS glass, reactive resins, and nano fillers). The filler loading is approximately 69% by

weight 27% FAS glass and 42% methacrylate functionalized nano fillers. All of the nano

fillers are further surface modified with methacrylate silane coupling agents to provide

covalent bond formation into the free radically polymerized matrix. The FAS glass is

radiopaque with an approximate particle size of less than 3 microns (average particle size

approximately 1 micron) and provides the basis for the glass-ionomer reaction and

extended fluoride release in the presence of water and a polycarboxylic acid functional

polymer. In addition, the nanofilled glass-ionomer contains a unique combination of two types of surface treated nanofillers (approximately 5-25 nm) and nanoclusters

(approximately 1.0 to 1.6 microns) (Douglas and Tantibirojn, 2007).

2.1.3.2 Properties

Nano glass-ionomer restoratives can contain a broad range of particle sizes. Filler particle

size can influence strength, optical properties and abrasion resistance by using bonded

nanofillers and nanocluster fillers, along with FAS glass. Nano glass-ionomer restoratives

have improved esthetics, yet still provide the benefits of glass-ionomer chemistry, such as

fluoride release. Some in-vitro studies have also demonstrated the addition of nanofillers

provides enhanced surface wear and polish relative to some other commercially available

dental materials (Douglas and Tantibirojn, 2007). The surface roughness of nano glass- ionomer is low compared to resin modified glass-ionomer after polishing with Sof-Lex disks.

Fluoride release is measured in-vitro in buffer solutions using a fluoride ion specific electrode. Nano glass-ionomer showed a high fluoride release compared to resin modified glass-ionomer. However, this study was conducted by the nano glass-ionomer manufacturer (Douglas and Tantibirojn, 2007). More neutral and comprehensive studies need to focus on nano glass-ionomer physical and chemical properties.

2.1.4 Nano-composite

A composite material is a product which consists of at least two distinct phases normally

formed by blending together components having different structures and properties. The

purpose of this was to produce a material having properties which could not be achieved

from any of the individual components alone. To understand nanotechnology and its relation to the dental material, the concept of filler size and its affect on the properties of

materials should be explained. Composite is one of the materials in which nanotechnology had an application.

2.1.4.1 Compositions

All dental composites consist of organic polymer matrix, inorganic filler particles, coupling agent and the initiator-accelerator system. The organic polymer matrix in most composites is either an aromatic or urethane diacrylate oligomer. Oligomers are viscous liquids, the viscosity of which is reduced to a useful clinical level by the addition of a diluent monomer. The inorganic particles may consist of several inorganic materials such as glass or quartz (fine particles) or colloidal silica (microfine particles). Methods used to characterize materials are based upon the technique used to activate polymerization of the resin and on the particle size distribution of filler. Resins: The nature of the resin may alter slightly from one product to another, although, essentially they all contain a modified methacrylate or acrylate. Fillers: The type, concentration, particle size and particle size distribution of the filler used in a composite material are major factors controlling properties (McCabe and Walls, 2008).

Fillers commonly used include quartz, fused silica and many types of glass including

Aluminosilicates and borosilicates, some containing barium oxide. A conventional composite contains 60–80%, by weight of quartz or glass in the particle size range of 1–

50 µm. The particle size distribution may vary, from one product to another. The filler particles are subjected to a special pretreatment prior to blending with the resin. This involves laying down a surface coating of a coupling agent on the particles to enhance bonding between the filler and resin matrix (Craig and Powers, 2002).

A microfilled composite is the next generation of dental composite after conventional composite. It contains silica particles in the range 0.01–0.1 µm with a typical mean diameter of 0.04 µm. The very small particle size produces a massive increase in available surface area for a given volume of filler. Consequently, it is not possible to incorporate very high filler loadings of this small particle size and products which are available contain only 30–60% filler by weight. Even at these lower levels, calculations showed that many filler particles must be present as agglomerates and not as individual particles surrounded by resin (Albers, 2002).

A third series of dental composite consist of conventional glass or quartz particles blended together with some submicron (silica). These products are referred to as hybrid composites. Using filler loadings of about 75% conventional size (1–50 µm) and 8% submicron size (0.04 µm average), a total filler content of 83% or greater can be achieved. Some hybrid products contain a blend of at least three different particles sizes of filler. These allow efficient packing of filler into the smallest possible volume and enable filler loadings of up to 90% by weight to be achieved (Albers, 2002).

Modern formulations of composites often involve new methods of manufacture of fillers

in which the particle size and shape is controlled and in which the traditional boundaries

between conventional microfilled and hybrid products can no longer be clearly identified.

Composites produced using very small particles of less than one µm average diameter are

‘nano-composites’, using both nanoparticle and nanocluster fillers. The combination of

nanomer sized particles to the nanocluster formulations reduces the interstitial spacing of the filler particles. This provides for increased filler loading, better physical properties and improved polish retention when compared to composites containing only nanoclusters (McCabe and Walls, 2008).

2.1.4.2 Properties

The resin phase and the reinforcing filler are the two main components of composite filling materials. The beneficial properties contributed by the resin are the ability to be molded at ambient temperatures coupled with setting by polymerization achieved in a conveniently short time. While, the beneficial properties contributed by the filler are hardness, strength, rigidity, a lower value for the coefficient of thermal expansion and markedly lowers setting contraction if the filler occupies a significant proportion of the volume of a composite material (McCabe and Walls, 2008).

The effect of filler depends on the type, shape, size and amount of filler incorporated and the existence of efficient coupling between the filler and resin. Other thermal and mechanical properties may vary in a similar way. Strength and modulus of elasticity generally increase with addition of filler. The same is true with abrasion resistance, probably as a result of increased surface hardness. If the added filler is translucent, the optical properties of the resin are improved and a more lifelike appearance produced.

Also, the filler shape strongly affects the color of composite resins and other filler

properties such as filler particle size and filler content exerted significance influence. In the context of aesthetic properties, it certainly seems beneficial and expedient to improve the color appearance of composite resins by focusing on filler shape (Arikawa et al.,

2007).

Briefly, in dental restorative composite system the use of nanotechnology offers high translucency, high polish and polish retention similar to those of microfills while maintaining physical properties and wear resistance equivalent to several commercial hybrid composites. The best combination of physical properties can be achieved by combinations of two types of nanofillers. Offering superior esthetics, long-term polish retention and other optimized physical properties (Mitra et al., 2003).

2.1.4.3 Fluoride release

Several methods have been used to provide fluoride sources for resin-based dental restorative materials. In the 1970s and 1980s, soluble free salts, such as NaF, KF, SrF2 or

SnF2, were added to sealants and composites (Arends and van der Zee, 1990). These soluble free salts can boost the initial high fluoride release. Because these inorganic salts cannot be distributed homogeneously in the monomer and they leave voids in the material after they dissolve and reduce the mechanical properties and wear resistance. In the late 1980s to the 1990s, fluoride-releasing glass fillers, such as fluoroaluminosilicate glass, or sparingly soluble inorganic salts, such as YbF3, were used in fluoride releasing composites (Xu and Burgess, 2003). Previous studies regarding the fluoride-releasing composite showed that fluoride release was found to be less than for glass-ionomer materials. However, an early burst of fluoride release was observed, but over time,

22 considerably less fluoride was released than from a glass ionomer (Glasspoole et al.,

2001). A recent study showed that the improvement of the monomers, the photoinitiators and the incorporation of ceramic whiskers or nanofibers can significantly reduce water sorption and increase mechanical properties of the composite. The improved fluoride- releasing composite can achieve significantly higher fluoride release and recharging capability and physical and mechanical properties similar to those of conventional non- fluoride-releasing dental composites (Ling et al., 2009).

2.1.4.4 Finishing and polishing

The surface of a composite material is initially very smooth and glossy due to contact with a matrix strip during setting. The surface layer is initially richer in resin than the bulk of the material and few filler particles are exposed at the surface. Any process of abrasion, however, has a tendency to cause surface roughening as the relatively soft resin matrix is worn leaving the filler particles protruding from the surface (McCabe and

Walls, 2008).

The longevity and the maintenance of a restoration can be achieved by having highly polished surface than a rough one. Moreover, it can reduce plaque accumulation, minimizing patient’s discomfort from gingival irritation, surface staining, and development of secondary caries (Jefferies, 1998).

The surface roughness of any material is the result of the interaction of multiple factors.

Some of them are intrinsic that are related to the material itself, such as the filler (type, shape, size, and distribution of the particles), the type of resinous matrix as well as the ultimate degree of cure reached and the bond efficiency at the filler/matrix interface

(Jefferies, 1998). Therefore, a different finishing and polishing technique been assigned

23 for each type of composite. Previous study showed that Sof-Lex disks and Jiffy points produced the smoothest surfaces for the packable resin composites (Borges et al., 2004).

On the other hand, Pogo produced smoothest surface compared to Super Snap for minifill-hybrid composite, and it was equivalent to Super Snap for packable composites

(Bashetty and Joshi, 2010). Furthermore, Pogo was considered the best polishing system used for flowable composite (Ozel et al., 2008). Another study showed that Super-snap abrasive discs produced a smoother surface than the Astropol polishers for composite and ormocer-based restorative materials (Baseren, 2004).

2.1.4.5 Color stability

Color stability of current composites has been studied by artificial aging and by immersion in various stains (coffee, tea, cranberry/grape juice, red wine, sesame oil).

Composites are resistant to color changes caused by oxidation but are susceptible to staining. Previous study showed that the optical properties of dental composite resins are directly affected by surface roughness. Surface texture controls the degree of scattering or reflection of the light striking on the natural tooth or the material. An increasingly roughened surface will reflect the individual segment of the specula beam at slightly different angles (Lee et al., 2002). Color differences among composite resins were related to the size of filler particles exposed on the surfaces following polishing procedures. The degree of surface roughness after polishing increases with the increase in filler particle size, and the amount of light reflection also changes accordingly (Sarac et al., 2006).

2.2 Surface topography

Roughness refers to the surface texture of a material. There are two types: the smoothness

resulting from a finishing process, referred to as applied or acquired smoothness, and the

smoothness of an unpolished material, referred to as inherent smoothness. Inherent

smoothness depends on the filler particle size of the material. A finished material will

always return to its inherent smoothness. For example, if a material has filler particles of

1 to 10 µm, it will always return to a smoothness of 10 µm; therefore, if it is polished to 5

µm, its roughness will double over time. Roughness is measured in microns or in grit. A

reading of less than 1 µm or a grit greater than 600 is considered as smooth as enamel

(Albers, 2002).

Finishing refers to all of the procedures associated with contouring, eliminating excess at

the margins, and polishing.

Smoothness is both the subjective appearance and the objective measurement of a polish.

Appearance is related to texture and the nature of the material being polished.

Polish relates to a number of other terms, such as surface smoothness, luster, or gloss.

These terms imply greater light reflection from the restored surface. Polish also implies refinement or improvement of a restored surface. But finishing improving polish is not always associated with an improved restorative surface (Craig and Powers, 2002).

2.2.1 Polishing and finishing

Polishing is an inherently destructive process. The purpose of finishing and polishing should be to achieve the best possible surface with the least restorative damage and marginal leakage. There is a point of diminishing returns. Polishing high stress bearing surfaces to the inherent polish might provide a longer lived restoration than extended polishing to gain an acquired polish (O'Brien, 2002).

Finishing is dependent on the hardness and polishability of the matrix and fillers that compose the material, that for most direct dental restoratives. Because auto set glass- ionomers have a soft matrix and relatively large filler particles, the surface smoothness achievable through polishing is poor compared with composites that have a more durable

polymerized resin matrix. Composites that contain macrofillers also have large particles,

but because their matrices are more durable than that of an auto cured glass-ionomer,

they can be finished to a higher polish immediately after placement (Yap et al., 2002) .

There is a direct relation between the surface roughness and filler size. Tooth colored dental material with large filler size are difficult to maintain a smooth finish because any loss of filler particles at the surface leaves a rough finish, whereas a filler with a smaller particle size reduces this disadvantage (Albers, 2002), as shown in Figure 2.1

Figure 2.1 : Differences in surface roughness resulting from the loss of large and small filler particles (Albers, 2002).

Resin ionomers have matrices that are stronger than those of auto set glass-ionomers and have an initial polish that is better than that of an auto set glass-ionomer but not as good as that of a macrofilled composite (Craig and Powers, 2002).

Dental composite that achieves a high acquired polish tends to pick up fewer stains, accumulate less plaque and show better wear resistance (McCabe and Walls, 2008).

Information on the average particle size of a composite is of little value in determining its polish, as surface polish is dependent on the size of the largest inorganic filler particle.

Therefore, to improve the material’s strength, large particles have been added to the composite to increase filler loading but that will decrease its inherent polish (Albers,

2002).

The effectiveness of surface finishing and polishing procedures is of fundamental importance for any restoration (Ozgunaltay et al., 2003). These procedures are commonly required after placement of direct restorations because they minimize the retention of plaque and stains and other problems resulting from the exposure of rough surfaces to the oral environment. Removal of excess material and re-contouring of restorations are frequently necessary, even if care is taken in the placement of the matrix. However, these procedures will increase surface roughness (Jung, 2002).

The factors determining the micro morphology of the surface of dental materials after finishing and polishing depend on many characteristics such as size, hardness, type and amount of filler particles. The chemical composition of the material is also important.

Resin based systems have residual components that seem to attract plaque, whereas glass- ionomers release fluoride, which is toxic to plaque (Albers, 2002).

Chung, 1994 stated that the critical surface roughness threshold established for bacterial adhesion is 0.2 μm, any increase in surface roughness above 0.2 μm results in a simultaneous increase of plaque accumulation and the risk of caries and periodontal inflammation. The rough surface of a restoration increases plaque accumulation, which may result in gingival inflammation, superficial staining, secondary caries and color change (Jefferies, 1998). Therefore, maintaining the smooth surface of a restoration is important for its success (Joniot et al., 2006).

2.2.2 Polishing systems

Many techniques have been developed to obtain highly finished and polished surfaces of dental restorations. It can be classified into four categories; coated abrasive disks and strips, cutting carbide, diamond, and stones; rubberized abrasives; and loose particulate abrasives in the form of polishing pastes and powders. Previous studies showed that smoother surfaces are obtained when the material was cured against a polyester matrix

(Roeder et al., 2000;Yap et al., 1997;Hondrum and Fernandez, 1997;Tate and Powers,

1996;Bassiouny and Grant, 1980).

With regards to the dental composite, many studies showed that multiple step polishing systems were more effective than one step polishing systems (Lu et al., 2003;Ozgunaltay et al., 2003;Baseren, 2004;Barbosa et al., 2005;Sarac et al., 2006;Uctasli et al., 2007).

However, some other studies showed that one step polishing systems, Pogo produced smoothest surface compared to multiple steps polishing systems, Sof-Lex disks (Turkun and Turkun, 2004;St-Georges et al., 2005;Da Costa et al., 2007;Scheibe et al., 2009). On the other hand, studies regarding glass-ionomer showed that multiple step polishing produce less surface roughness compared to one step polishing systems (Yap et al.,

2002).

Polishing and composition of the direct resin restorations strongly influenced bacterial adherence, but did not show similar potency in resisting biofilm formation. Therefore, it was concluded that appropriately performed polishing could reduce the bacterial adherence. S.mutans biofilms were also somewhat affected by the chemical composition of the fluoride releasing resin dental material, probably due to the effect of released fluoride (Ono et al., 2007).

2.2.3 Surface roughness analysis

Surface roughness can be measured by different imaging methods like optical microscopy, profilometer, scanning electron microscopy (SEM) and atomic force microscopy (AFM). Many studies used profilometer to measure the surface roughness,

for example, Yazici et al. (2010) conducted a comparison study of the surface roughness between flowable resin composite, a hybrid resin composite and a polyacid modified resin composite using laser profilometer. Another study measured the surface of dental composite by using contact profilometer combined with SEM (Marghalani, 2010), however, it is still efficient to use profilometer alone (Bashetty and Joshi,

2010;Kameyama et al., 2008). Other studies used SEM to measure the surface roughness of different flowable composites and a microhybrid composite (Ozel et al., 2008;Fucio et al., 2008;Senawongse and Pongprueksa, 2007). Studies using AFM were conducted by various researches to analyze the surface roughness especially for nano-composites

(Janus et al., 2010) or other nano scales materials (Santos et al., 2008;Beyth et al., 2008).

SEM images the sample surface by scanning it with a high-energy beam of electrons. The

electrons interact with the atoms that make up the sample producing signals than contain

information about the sample’s surface topography, composition and other properties

(Paluszynski and Slowko, 2009).

Optical microscopy and scanning electron microscopy give an accurate image of the

surface compared to AFM but they do not provide quantitative information about surface

roughness. Moreover, the measurement scales is a basic factor to determine the most

appropriate use for the microscopy. Profilometer is used to measure a surface’s profile

and it quantifies the surface roughness. It can measure vertical features ranging in height

from 10 nanometres to 1 millimetre. However, it is unsuitable for measuring a nano-

scales dental material like nano composite and nano glass-ionomer where the filler size

range 1-50 nm (Amelinckx et al., 1997).

2.2.3.1 Atomic Force Microscopy

AFM is a very high-resolution type of scanning probe microscope. It has a demonstrated resolution of fractions of a nanometer (1000 times better than the optical diffraction limit) and is an important tool for imaging, measuring and manipulating matter at the nano scale. Information is gathered by "feeling" the surface with a mechanical probe; therefore, a true 3D surface profile is recorded. Forces that can be measured include mechanical contact force, Van der Waals forces, capillary forces, chemical bonding, electrostatic forces, and magnetic forces. Samples viewed by AFM do not require any special treatments such as metal/carbon coatings that would irreversibly change or damage the sample and it can work perfectly well in ambient air or even a liquid environment, making it possible to study biological macromolecules and even living organisms (Yao and Wang, 2005).

AFM provides a number of advantages over conventional microscopy techniques. AFMs

probe the sample and make measurements in three dimensions, x, y, and z (normal to the

sample surface), thus enabling the presentation of three-dimensional images of a sample

surface. This provides a great advantage over any microscope available previously. With

good samples (clean, with no excessively large surface features), resolution in the x-y

plane ranges from 0.1 to 1.0 nm and in the z direction is 0.01 nm (atomic resolution).

AFMs require neither a vacuum environment nor any special sample preparation and they

can be used in either an ambient or liquid environment. With these advantages AFM has

significantly impacted the fields of materials science, chemistry, biology, physics, and the

specialized field of semiconductors.

Contact mode AFM is one of the more widely used scanning probe modes and operates

by rastering a sharp tip (made either of silicon or Si3N4 attached to a low spring constant

cantilever) across the sample. An extremely low force (~10-9 N, inter-atomic force range) is maintained on the cantilever, thereby pushing the tip against the sample as it raster’s. Either the repulsive force between the tip and sample or the actual tip deflection is recorded relative to spatial variation and then converted into an analogue image of the

sample surface (Blanchard, 1996).

In this study, AFM was used for measuring surface roughness of the dental materials

after 30 days incubation with S.mutans.

Figure 2.2: Diagram showing the concept of atomic force microscopy (Yao and Wang, 2005).

2.3 Oral microbes

Most oral diseases are induced as a result of direct or indirect involvement of oral bacteria. That has been confirmed with more advanced bactcriologic and immunologic tests. Based on present knowledge, it appears that most changes in pulpal and periradicular tissues are of bacterial origin and have to be deal with as infectious

processes (Cohen and Hargreaves, 2006).

Bacteria are essential for the development of dental caries and periodontal diseases. It is present in dental plaque which is found on most tooth surfaces. Bacterial enzymes and various toxins can probably cause tissue injury and destruction directly without an immediate host response. Bacteria products including hyaluronidase, chondroitin sulfatase, proteolytic enzymes as well as cytotoxins in the form of organic acids, ammonia, hydrogen sulfide and bacterial endotoxins can be demonstrated in tissues

(Rateitschak et al., 1985).

Dental plaque can be defined as a complex microbial community, with greater than 1010

bacteria per milligram. The formation of pellicle is the first step in plaque formation. It

include albumin, lysozyme, amylase, immunoglobulin A, proline-rich proteins and

mucins. The pellicle-coated tooth surface is colonized by Gram-positive bacteria such as

Streptococcus sanguis, Streptococcus mutans, and Actinomyces viscosus which

considered the "primary colonizers" of dental plaque. The secondary colonizers include

Gram-negative species such as Fusobacterium nucleatum, Prevotella intermedia, and

Capnocytophaga species. After one week, the tertiary colonizers include Porphyromonas gingivalis, Campylobacter rectus, Eikenella corrodens, Actinobacillus actinomycetemcomitans (Haake, 2010).

2.3.1 Streptococcus mutans biofilm

Streptococcus mutans is an acidogenic, Gram positive, facultative anaerobe, which is

found in the oral cavity of humans. Epidemiological studies have implicated S.mutans as

a significant cariogenic organism in childhood caries, caries in young adults and of root

caries in the elderly, and nursing caries in infants (Sladek et al., 2007). Their virulence is

mainly due to their high adhesion capability, acidogenicity and aciduric properties. These

characteristics are responsible for surface damage to restorations keeping in mind, this

microorganism can be found on any hard surface in the oral cavity, such as enamel,

implants, orthodontic appliances or restorative materials (Fucio et al., 2008).

Biofilm formation occurs on all hard surfaces, e.g. the tooth surface, restorative materials

and implant components. Microscopic examination of early plaque formation on teeth

shows that the first bacteria adhere along cracks and pits in the enamel, suggesting an

effect of the surface structure. In addition, the surface free energy affects the

accumulation of tooth biofilm. Thus, rough surfaces and surfaces with high surface free

energy are more prone to plaque formation (Quirynen, 1994). In the process of plaque

formation on solid substrate surfaces such as tooth and restorative materials, the initial adhesion of early colonizing bacteria to the surface is an important step (Takatsuka et al.,

2000).

It is apparent that resin composites have surface characteristics different from those of teeth. Unlike tooth surfaces, the surface properties of resin composite materials related to bacterial adhesion and biofilm formation are affected by a myriad of factors such as mechanical surface properties, material components such as filler particles and resin

34 matrix and curing conditions. In particular matrix monomers might influence the growth of some cariogenic bacterial species (Imazato et al., 2003).

Unpolished and polished surfaces of resin composites have been characterized in terms of surface roughness, chemical composition, and surface free energy. On the influence of surface structure on bacterial adhesion, a poorly finished indirect restoration might initiate biofilm adherence onto the surface and its adjoining areas in the oral cavity. The unpolished glass-ionomer surfaces were rougher and bound more protein and bacteria compared to unpolished composite resin (Carlen et al., 2001).

Polishing the glass-ionomer surface, on the other hand, produced little effect on surface roughness and did not much affect protein or bacterial binding. Glass-ionomer with a rougher and more inorganic, positively charged surface character, bound more protein and bacteria than composite resin. More binding could be due to either of these surface properties, or a combination of the two. In addition, the effect of polishing, which for the composite resin led to an increase in bound proteins and bacteria, can be explained by a change in surface roughness and electrostatic interactions between the substrate and salivary components (Carlen et al., 2001).

Various studies investigated the antibacterial properties of commercial resin composites

and their constituents. It has been reported that polymerization of resin composites is incomplete and that unreacted monomers stimulate growth of caries associated bacteria

(Hansel et al., 1998).

Resin composite restorations suffer from degradation owing to oral environmental conditions and to their physical and chemical properties. Part of the problem is the attachment and adhesion of microorganisms to the surface, as well as biofilm formation.

Secondary caries is the most frequent cause for the replacement of resin composite restorative materials (Wilson et al., 1997).

The nanometric surface changes caused by bacteria will increase surface roughness, which in turn, encourages more bacteria to attach to the resin composite and colonize.

With time the roughness continues to increase. This paradigm is a “vicious cycle” caused by the bacteria–surface interaction, which may cause restoration failure. S.mutans growth on resin composites increases surface roughness without affecting micro-hardness. This change in surface integrity may further increase biofilm accumulation (Beyth et al.,

2008). Recent studies show that surface roughness was influenced by biofilm/material interaction for glass-ionomer but not for dental composite. Therefore, the effects of

S.mutans biofilm on the surface properties and microstructure are material-dependent

(Fucio et al., 2008).

2.3.2 Confocal laser scanning microscopy (CLSM)

Biofilm thickness can be evaluated by different techniques. One of these techniques is by

measuring biofilm optical density as intensity reduction of a light beam transmitted

through the biofilm, correlates with biofilm mass, measured as total carbon and as cell

mass. The method is more sensitive and less labor intensive than other commonly used

methods for determining extent of biofilm mass accumulation. Biofilm optical thickness,

measured by light microscopy, is translated into physical thickness based on biofilm refraction measurements (Bakke et al., 2001). Another study used SEM a combined with

light microscopy. However, sample preparation for microscopic analysis is labour

intensive (Kinnari et al., 2009;Tsang et al., 1999).

Confocal laser scanning microscopy (CLSM) is a technique for obtaining high-resolution optical images. The key feature of confocal microscopy is its ability to produce in-focus images of thick specimens, a process known as optical sectioning. Images are acquired point-by-point and reconstructed with a computer, allowing three-dimensional reconstructions of topologically-complex objects. The principle of confocal microscopy was originally patented by Marvin Minsky 1957, but it took another thirty years and the development of lasers for CLSM to become a standard technique toward the end of the

1980s (Pawley, 2006).

The major application of CLSM is for imaging either fixed or living tissues that have usually been labeled with one or more fluorescent probes. It provides a slight increase in both lateral and axial resolution, although it is the ability of the instrument to eliminate the "out-of-focus" flare from thick fluorescently labeled specimens (Paddock, 1999).

Therefore, it has been used in many studies to evaluate the bacterial biofilm composition and thickness (Islam et al., 2009;Pereira-Cenci et al., 2008;Al-Naimi et al., 2008;Al-

Ahmad et al., 2008;Mohle et al., 2007;Kara et al., 2007;Thurnheer et al., 2006;Maeyama et al., 2004).

CLSM provides the capacity for direct, noninvasive, serial optical sectioning of intact, thick, living specimens with a minimum of sample preparation as well as a marginal improvement in lateral resolution. Because CLSM depends on fluorescence, a sample usually needs to be treated with fluorescent dyes to make objects visible. However, the actual dye concentration can be low to minimize the disturbance of biological systems; some instruments can track single fluorescent molecules. Also, transgenic techniques can create organisms that produce their own fluorescent chimeric molecules (such as a fusion of GFP, green fluorescent protein with the protein of interest) (Fellers, 2007).

In a confocal laser scanning microscope, a laser beam passes through a light source aperture and is then focused by an objective lens into a small (ideally diffraction limited) focal volume within a fluorescent specimen. A mixture of emitted fluorescent light as well as reflected laser light from the illuminated spot is then recollected by the objective lens. A beam splitter separates the light mixture by allowing only the laser light to pass through and reflecting the fluorescent light into the detection apparatus. After passing a pinhole, the fluorescent light is detected by a photodetection device, transforming the light signal into an electrical one that is recorded by a computer.

The detector aperture obstructs the light that is not coming from the focal point, as shown

by the dotted gray line in the image. The out-of-focus light is suppressed: most of their returning light is blocked by the pinhole, resulting in sharper images than those from conventional fluorescence microscopy techniques, and permits one to obtain images of various z axis planes (also known as z stacks) of the sample, Figure 2.3.

Figure 2.3: Basic setup of confocal microscope (Fellers, 2007).

3 MATERIALS AND METHODS

3.1 Study Design

This was a single blinded experimental study. The disks were placed in a sealed envelope

and later assigned to four different groups; each group contained the three types of tooth

colored restorative dental materials.

3.2 Sample Size Calculation

PS software (Dupont & Plummer, 1997) was used to calculate the sample size based on comparing two means.

Seven samples were needed in each group to detect the difference of 0.007 µm Ra with

80% power and alpha 0.05 (SD was estimated 0.23 µm Ra (Koh et al., 2008)).

Seven sample were needed in each group to detect the difference of 0.1 μm biofilm thickness with 80% power and alpha 0.05 (SD was estimated 0.35 μm biofilm thickness,

(Rozen et al., 2001)).

• Objective 1:- Seven samples were needed for each of the three test groups with

the total sample size of 7x3=21 samples.

• Objectives 2, 3:- Seven samples were needed in each test group with the total

sample size of 7x12=84 Samples for 14 days S.mutans biofilm culture. Another

84 samples were needed for 30 days S.mutans biofilm culture. Therefore the total

sample size for objectives two and three were 168.

3.3 Test materials

The commercial glass-ionomers and nanofilled composite were tested for surface changes

after polishing and also following the growth of biofilm. The materials used are shown in

Table 3.1 and Figure 3.1.

Table 3.1 Test materials.

Material Manufacturer Filler Composition size • Blend including HEMA, Vitrebond Copolymer. Ketac™N100 3M ESPE 5-25nm • a methacrylate modified polyalkenoic acid • FAS,Nanomers, and Nanoclusters. (Nano Light Curing Glass Ionomer Restorative)

• Powder: fluoro-alumino-silicate glass Fuji II™ LC GC 20-40 nm • Liquid: polyacrylic acid, 2-hydroxyethyl International methacrylate (HEMA), dimethacrylate, (Resin Modified Glass camphorquinone and water. Ionomer Restorative)

• BIS-GMA, BIS-EMA, UDMA with small Filtek™ Z350 3M ESPE 5-20 nm amounts of TEGDMA resins. • The fillers are combination of aggregated (Universal Restorative) zirconia/silica cluster filler with an average cluster particle size of 0.6 to 1.4 microns with primary particle size of 5-20 nm and a nonagglomerated/non-aggregated 20 nm silica filler. The inorganic filler loading is about 78.5%.

A. Nanofilled composite (Z350)

B. Nanofilled glass-ionomer (Ketac N100)

C. Microfilled glass-ionomer (Fuji II LC)

Figure 3.1: Test materials.

Three polishing systems were used as shown in Table 3.2 and Figure 3.2.

Table 3.2 polishing systems.

Systems Finishing and polishing technique Manufacturer

Enhance/Pogo Enhance:- Polymerized Urethane Dimethacrylate Resin Dentsply/Caulk, Milford,DE, USA Aluminum Oxide, Silicon Oxide and Plastic Latch-type mandrel

Pogo:- Polymerized Urethane Dimethacrylate Resin

Fine Diamond Powder, Silicon Oxide and Plastic Latch- type mandrel

Astropol The polishers consist of silicone rubber, silicon carbide, Ivoclar Vivadent, Schaan, aluminium oxide, titanium oxide, and iron oxide. Liechtenstein Astropol HP additionally contains diamond dust.

Astropol F gray finishing cup (40 μm),

Astropol P green polishing cup (20-40 μm),

Astropol HP pink high-gloss polishing cup (10 μm)

Sof-Lex Coarse aluminum oxide disks (55micrometers) 3M ESPE, St. Paul, Minn

Medium aluminum oxide disks (40 μm)

Fine aluminum oxide disks (24 μm)

Ultra fine aluminum oxide disks (8 μm)

A. Enhance/Pogo

B. Astropol

C. Sof-Lex disks

Figure 3.2: Polishing systems.

3.4 Specimen Preparation

An acrylic mold was used to prepare 5 mm x 2 mm disk specimens, as shown in Figure

3.3. The mold was placed on a clean glass slab and the test material was packed into the mold forming similar disks, which was then covered with a Mylar strip and pressed flat with a microscopic glass slide. The disks were photo-polymerized according to manufacturer instruction, using a conventional light-curing unit, (Elipar™ Free Light 2

LED, 3M ESPE, USA) with a light intensity of 1000mW/cm2. Following light curing, the specimens were placed into 37˚C humidor for 24 hours (Ab-Ghani et al., 2007).

Figure 3.3: Acrylic mold.

3.5 Specimen preparation for objective 1

A set of 21 disks (set A) were used for the comparison between the surface roughness at day 0 and day 30 of S.mutans culture cured against Mylar strip. The disks were assigned into three groups of 7 disks containing nanofilled glass-ionomer, microfilled glass- ionomer and nanofilled composite. All disks were examined in contact mode under AFM

(AMBIOS Technology Inc., USA) (Figure 3.4) to measure the surface roughness of those three dental materials prior to S.mutans culture.

Figure 3.4: AFM (AMBIOS Technology Inc., USA).

• Bacteria and growth conditions

S.mutans (ATCC# 27351) was cultured overnight at 37˚C in brain heart infusion (BHI)

broth supplemented with 0.2 units/ml bacitracin (Sigma-Aldrich, Inc.) and sucrose

present in an amount of about 20 % by weight (Gold et al., 1973). The top 4 ml of the suspension were harvested into a fresh test tube and centrifuged for 10 minutes at 3175×g to isolate bacteria in the mid exponential phase. The supernatant was discarded and the bacteria were re-suspended in 5 ml of phosphate buffered saline (PBS) containing 0.2 units/ml of bacitracin. An 800 µml volume of the culture were diluted to 106 cells /ml.

A 10 µml drop of bacterial suspension was placed on each sample, which was then incubated at 37˚C for 1 hour to evaporate excess water. The samples were placed in a container with 50 ml of brain heart infusion (BHI) broth supplemented with 0.2 units/ml of bacitracin and sucrose present in an amount of about 20 percent by weight then incubated at 37˚C to allow the formation of 30 days-old biofilm. The pH of around 5.5 was recorded. The broth was replaced every 48 hours (Figure 3.5). Before replacing the broth, the disks were vortexed for 10 seconds and the broth was decanted without disturbing the biofilm (Beyth et al., 2008).

Figure 3.5: Specimen culturing conditions.

After 30 days of S.mutans culture, the disks were washed three times for 30 seconds with

double-distilled water (DDW) and placed in test tubes containing 5ml of DDW

supplemented with 0.02% azide, at 4˚C. Bradford Protein staining was performed on each

disk to ensure that all biofilm residues had been removed (Beyth et al., 2008).

All disks were then examined in contact mode under AFM (AMBIOS Technology Inc.,

USA) to measure the surface roughness of those three dental materials. Each sample was measured in three different locations, the mean of these measurements was considered as a surface roughness of each sample, as shown in flow chart 1.

3.6 Specimen preparation for objectives 2, 3

Two sets of 84 disks (set B, set C) containing 28 disks of nanofilled glass-ionomer, 28 disks of nanofilled composite and 28 disks of microfilled glass-ionomer in each set were prepared with technique used in section 3.4 of specimen preparation. These two sets of test materials were used to achieve objectives two and three. Set B was only used to achieve the measurement of the biofilm thickness after 14 days of S.mutans culture. Set C was used to achieve the measurement of both the biofilm thickness and the surface roughness after 30 days S.mutans culture.

• Finishing and polishing

Convenient random sampling was used to further divide each test material into three treatment groups and one control group with n=7.

The treatment groups in both sets B and C were finished with three different polishing systems. The polishing systems and control group used were as follows:

Group 1:- Enhance/ PogoTM (Dentsply/Caulk, Milford, DE, USA). Each sample was finished with Enhance finishing discs (brown mandrel) using light pressure for ten seconds, then polished with Pogo polishing discs (gray mandrel) using light buffing motion for ten seconds without water. Conventional contra- angled slow speed handpiece

(10,000 rpm) was used. Enhance and Pogo discs were discarded after every three samples.

Group 2:- Astropol® (Ivoclar Vivadent, Amherst, NY, USA). Each sample was finished with Astropol F (grey) disc for ten seconds, polished with Astropol P (green) disc for ten seconds and high gloss polished with Astropol HP (pink) disc for ten seconds.

Astropol disc were used with slow speed handpiece (10,000 rpm) with water. Light pressures were applied on each sample. A new disc was used after every three samples.

Group 3:- Sof-LexTM (3MTM ESPETM, St. Paul, MN, USA). Four steps were used with

Sof-Lex discs (Coarse, Medium, Fine and Ultra fine) with slow speed handpiece (10,000 rpm). Light pressures were applied on each sample, ten seconds for each polishing step.

Sof-Lex discs were used with water. The manufacture suggested dry polishing with Sof-

Lex discs. However, water was used with Sof-Lex in this study as dry polishing produced cracks on the samples surface as shown in the pilot study. A new disc was used after every three samples.

Group 4:- Control group specimen cured against Mylar strip.

The same slow speed handpiece (10,000 rpm) was used for all systems. The polishing procedure used consisted of repetitive strokes, ten seconds per step of the system, to prevent heat buildup and formation of grooves. A conscious effort was made to standardize the strokes, downward force and the number of strokes for each polishing procedure. According to manufacturers’ instructions, Sof-Lex™ and Pogo/Enhance ™ used dry polishing. Astropol® was used with water (Koh et al., 2008).

• Bacteria and growth conditions

S.mutans (ATCC# 27351) was cultured overnight at 37˚C in brain heart infusion (BHI)

broth supplemented with 0.2 units/ml bacitracin (Sigma-Aldrich, Inc.) and sucrose present in an amount of about 20 % by weight (Gold et al., 1973). The same steps of culture preparation in objective one were used for objective two and three.

Once the polishing stage of the test materials was completed, a 10 µml drop of bacterial suspension was placed on each sample of the four groups in both set B and set C. All samples were then incubated at 37˚C for 1 hour to evaporate excess water. The samples were placed in a container with 50 ml of brain heart infusion (BHI) broth supplemented with 0.2 units/ml of bacitracin and sucrose present in an amount of about 20 percent by weight then incubated at 37˚C to allow the formation of a 14 day-old biofilm for set B and a 30 day-old biofilm for set C. The pH of around 5.5 was recorded. The broth was replaced every 48 hours. Before replacing the broth, the disks were vortexed for 10 seconds and the broth was decanted without disturbing the biofilm (Beyth et al., 2008).

The S.mutans biofilm thickness in set B and C were measured using confocal laser scanning microscopy (CLSM) (Leica HC laser microscope, Germany) (Figure 3.6). The biofilm thickness for set B was measured using CLSM after 14 days of culture, as shown in flow chart 2. The biofilm was fixed with 4% paraformaldehyde in 0.1 M merphosphate buffer (pH 7.4), at 48°C for 4 hours and stained with propidium iodide 95% (Sigma-

Aldrich, Inc.), to visualize the highest cell clusters of the biofilm (Brana et al., 2002).

Immediately after the staining procedure, one drop of fluorescent mounting medium was used on each sample; the biofilm was then analyzed using a CLSM.

Figure 3.6: CLSM (Leica HC laser microscope, Germany).

The biofilm thickness was measured microscopically by focusing on the substratum and

moving until the upper biofilm cells are in focus. This procedure was repeated at several

sites per single sample until the thickest point of the uneven biofilm surface was found.

Then optical sections of one µm were scanned through the biofilm from the highest cell clusters. Biofilm thickness was determined by summing up the number of sections. Each sample was measured in three different locations, the mean of these measurements was considered as a biofilm thickness. Three dimensional (3D) images were recorded for each sample.

The disks were then washed three times for 30 seconds with DDW and placed in test tubes containing 5ml of DDW supplemented with 0.02% azide, at 4˚C. Bradford Protein staining was performed on each disk to ensure that all biofilm residues had been removed

(Beyth et al., 2008).

The samples in set C were subjected to CLSM and AFM analyses after 30 days of

S.mutans culture, as shown in flow chart 3. The AFM analysis was carried out after

CLSM analysis. Set C was examined in contact mode under AFM to measure the surface roughness of those three dental materials after 30 days of S.mutans culture. Each sample was measured in three different locations, the mean of these measurements was considered as a surface roughness of each sample.

3.7 Research tools

• Microbiology safe cabinet.

• Incubator (SANYO Electric Biomedical Co., Ltd., Canada) to incubate the disks

with BHI broth at 37 ºC.

• Confocal laser scanning microscope (Leica HC laser microscope, Germany) to

measure the S.mutans biofilm thickness after 14 and 30 days culturing.

• Atomic force microscope (AMBIOS Technology Inc., USA) to evaluate the

surface roughness after 30 days of S.mutans culture.

3.8 Data entry and analysis

Data was entered and analyzed using SPSS version 16.0.1 (SPSS Inc., Chicago,IL,USA,

2007).

The mean and standard deviation of surface roughness (Ra) was determined. In the first

objective, Data was analyzed using T-test to calculate the differences between surface roughness of test materials at day 0 and day 30 of S.mutans biofilm culture.

The determination of the mean and standard deviation of surface roughness (Ra) was

made in objective two. Data was analyzed using One-way ANOVA and post hoc Tukey test to calculate the difference between surface roughness of the test materials according

to different polishing techniques.

The mean and standard deviation of biofilm thickness was determined in objective three.

Data was analyzed using One-way ANOVA and post hoc Tukey test to calculate the differences of bacterial biofilm thickness between the test materials after 14 days and 30

days of S.mutans biofilm culture. T-test was used to calculate the differences of bacterial biofilm thickness in each test material between 14 and 30 days of S.mutans biofilm culture.

P < 0.05 was considered different for all three statistical analyses.

3.9 Flow charts of the methodology

Flow chart 1 (Set A)

Research committee USM approval

The source population: (5 mm diameter, 2 mm thickness) disks formed from test materials

Study sample

21 specimens of the test materials were divided into three test groups with equal number of specimens, all specimens cured against Mylar strip.

Nanofilled GIC (7) Microfilled GIC (7) Nanofilled composite (7) specimens specimens specimens

Each disk was scanned with AFM in three different areas to obtain the mean Ra value for each specimen at day 0 of S.mutans culture.

S.mutans cultured by BHI supplement with 0.2 units/ml of bacitracin and sucrose present in 20 percent by weight, all groups incubated at 37ºC for 30 days

Each disk was scanned with AFM in three different areas to obtain the mean Ra value for each specimen at day 30 of S.mutans culture.

Data analysis/Results

Flow chart 2 (Set B)

Research committee USM approval

The source population: (5 mm diameter, 2 mm thickness) disks formed from test materials

Study sample

84 specimens of the test materials were divided into three test groups with each group containing an equal number of specimens

Nanofilled GIC (28) Microfilled GIC (28) Nanofilled composite (28) specimens specimens specimens

Group1 (n=7) Polished Group2 (n=7) polished Group3 (n=7) polished Group4 (n=7) cured with Enhance/Pogo with Astropol with Sof-Lex against Mylar

S.mutans cultured by BHI supplement with 0.2 units/ml of bacitracin and sucrose present in 20 percent by weight, all the groups incubated at 37ºC for 14 days

The biofilm was fixed with 4% paraformaldehyde in 0.1 M merphosphate buffer (pH 7.4) at 48°C for 4 hours and stained with propidium iodide 95%

S.mutans biofilm thickness after 14 days of incubation was evaluated by using CLSM

Data analysis/Results

Flow chart 3 (Set C)

Research committee USM approval

The source population: (5 mm diameter, 2 mm thickness) disks formed from test materials

Study sample

84 specimens of the test materials were divided into three test groups with each group containing an equal number of specimens

Nanofilled GIC (28) Microfilled GIC (28) Nanofilled composite specimens specimens (28) specimens

Group1 (n=7) Polished Group2 (n=7) polished Group3 (n=7) polished Group4 (n=7) cured with Enhance/Pogo with Astropol with Sof-Lex against Mylar

S.mutans cultured by BHI supplement with 0.2 units/ml of bacitracin and sucrose

present in 20 percent by weight, all the groups incubated at 37ºC for 30 days

The biofilm was fixed with 4% paraformaldehyde in 0.1 M merphosphate buffer (pH 7.4) at 48°C for 4 hours and stained with propidium iodide 95%

S.mutans biofilm thickness after 30 days of incubation was

measured by using CLSM

Each disk was scanned with Atomic force microscope AFM in three different areas to

obtain the mean Ra value for each specimen

Data analysis/Results

4 RESULTS

4.1 Comparison of the surface roughness of the test materials cured against Mylar strip before and after application of S.mutans biofilm for 30 days.

At day 0 of S.mutans culturing, the mean of surface roughness Ra were 32.4 nm

(nanometer) for nanofilled glass-ionomer, 44.4 nm for microfilled glass-ionomer and

24.7 for nanofilled composite. After 30 days of S.mutans culturing, the means of surface

roughness Ra were 37.8 nm for nanofilled composite, 43.7 nm for nanofilled glass-

ionomer and 120.7 nm for microfilled glass-ionomer. Statistical analysis revealed no

significant differences between means surface roughness Ra for nanofilled glass-ionomer

(P=0.491) and nanofilled composite (P=0.324) cured against Mylar strip before and

after S.mutans culturing for 30 days. However, the mean surface roughness of

microfilled glass-ionomer before S.mutans culturing was significantly less after S.mutans

culturing (P = 0.001), as shown in Table 4.1.

Table 4.1 Comparison of the mean (SD) of surface roughness Ra (nm) between the test materials before and after application of S.mutans biofilm over a period of 30 days.

Mean Ra with Mylar Mean Ra with Mylar strip P value Materials strip at day 0 of S.mutans after 30 days S.mutans culture (SD) culture (SD)

Nanofilled Composite 24.7( 1.5) 37.8(5.7) 0.324

Nanofilled glass-ionomer 32.4(2.6) 43.7 (6.9) 0.491

Microfilled glass-ionomer 44.4(3.3) 120.7(27.3) 0.001*

* Significant at P < 0.05, pair T-test

4.2 Comparison of the surface roughness between the test materials polished with three different polishing systems after 30 days of S.mutans biofilm culture.

Comparison of the means surface roughness of nanofilled glass-ionomer, microfilled

glass ionomer and nanofilled composite polished with Enhance/PogoTM, Astropol®, Sof-

LexTM and Mylar as control group is shown in Figure 4.1, and analyzed by using One- way ANOVA and post hoc Tukey test as shown in Table 4.2.

The mean surface roughness (Ra) of the samples cured against Mylar strip were 43.7 nm

for nanofilled glass-ionomer, 120.7 nm for microfilled glass-ionomer and 37.8 nm for

nanofilled composite. Statistically, the surface roughness of nanofilled glass-ionomer was

significantly less than microfilled glass-ionomer (P≤0.001), while there was no

significant difference between nanofilled glass-ionomer and nanofilled composite

(P=0.789), and nanofilled composite was significantly less than microfilled glass- ionomer (P ≤ 0.001) Figure 4.2 A.

The mean Ra of the samples polished with Enhance/Pogo were 174.5 nm for nanofilled

glass-ionomer, 547.1 nm for microfilled glass-ionomer and 114.5 nm for nanofilled

composite. The surface roughness of nanofilled glass-ionomer was significantly less than

microfilled glass-ionomer (P = 0.001), while there was no significant difference between

nanofilled glass-ionomer and nanofilled composite (P=0.674). However, the surface

roughness of nanofilled composite was significantly less than microfilled glass-ionomer

(P ≤ 0.001), Figure 4.2 B.

The mean Ra of the samples polished with Astropol were 72.4 nm for nanofilled glass-

ionomer, 560.7 nm for microfilled glass-ionomer and 83.0 nm for nanofilled composite.

The surface roughness of nanofilled glass-ionomer was significantly less than microfilled glass-ionomer (P=0.001), while there was no significant difference between nanofilled glass-ionomer and nanofilled composite (P=0.976). The surface roughness of nanofilled composite was significantly less than microfilled glass-ionomer (P ≤ 0.001), Figure 4.2

The mean Ra of the samples polished with Sof-Lex were 64.2 nm for nanofilled glass- ionomer, 263.4 nm for microfilled glass-ionomer and 176.0 nm for nanofilled composite.

The surface roughness of nanofilled glass-ionomer was significantly less than microfilled glass-ionomer (P ≤ 0.001), and significantly less than nanofilled composite (P=0.002).

The surface roughness of nanofilled composite was significantly less than microfilled glass-ionomer (P =0.010), Figure 4.2 D.

Regarding to the polishing methods and comparison between them; within the nanofilled composite the mean surface roughness (Ra) were 37.8 nm for Mylar, 176.0 nm for Sof-

Lex, 83.0 nm for Astropol and 114.5 nm for Enhance/Pogo. Statistical analysis showed that Sof-Lex produced significantly less surface roughness than Astropol (P < 0.001) and significantly more than Enhance/Pogo (P=0.011). There was no significant difference between Enhance/Pogo and Astropol (P=0.313). Mylar produced significantly less surface roughness than Astropol (P =0.023), Enhance/Pogo (P ≤ 0.001) and Sof-Lex (P ≤

0.001).

Within the nanofilled glass-ionomer the mean Ra were 43.7 nm for Mylar, 64.2 nm for

Sof-Lex, 72.4 nm for Astropol and 174.5 nm for Enhance/Pogo. Statistical analysis of the mean surface roughness revealed no significant difference between Sof-Lex and Astropol

(P=0.934), while Enhance/Pogo was significantly more than Astropol (P ≤ 0.001) and

Sof-Lex (P ≤ 0.001), respectively. Mylar showed significantly less surface roughness than Enhance/Pogo (P ≤ 0.001). However, there was no significant difference with

Astropol (P =0.186) and Sof-Lex (P =456).

Within the microfilled glass-ionomer the means Ra were 127.7 nm for Mylar, 263.4 nm for Sof-Lex, 560.7 nm for Astropol and 547.1 nm for Enhance/Pogo. Statistical analysis of the mean surface roughness showed Sof-Lex was significantly less than Astropol

(P=0.003) and Enhance/Pogo (P=0.005). However, there was no significant difference between Enhance/Pogo and Astropol (P=0.998). Mylar showed significantly less surface roughness than Astropol and Enhance/Pogo (P ≤ 0.001). However, there was no significant difference between Mylar and Sof-Lex (P=0.257).

Figure 4.1 : Comparison of mean (SD) surface roughness Ra (nm) between the test materials and three different polishing systems.

Table 4.2 Comparison of the mean (SD) surface roughness Ra (nm) between the test materials polished with three different polishing systems after 30 days S.mutans culture.

Material Polishing system Mean (SD) Mylar strip Enhance/PogoTM Astropol® Sof-LexTM

NC 37.8 (5.7)Aa 114.5 (14.5)Ab 83.0 (7.9)Ab 176.0 (64.5)Bc

NG 43.7 (6.9)Aa 174.5 (48.5)Ab 72.4 (12.3)Aa 64.2 (10.0)Aa

MG 120.7 (27.3)Ba 547.1 (221.5)Bb 560.7 (163.5)Bb 263.4 (55.1)Ca

*Within a column, values with the same upper-case superscript letter are not significantly different (p > 0.05, Tukey test). Within a row, values with the same lower-case superscript letter are not significantly different (p > 0.05, Tukey test). NG: - nanofilled glass-ionomer (Ketac™N100), MG: - microfilled glass- ionomer (Fuji II™ LC) and NC: - nanofilled composite (Filtek™ Z350)

A. Atomic Force Microscopy 3D images of the three test materials cured against Mylar stip.

Figure 4.2 A, B, C and D : Atomic Force Microscopy 3D images surface roughness of test material polished with different polishing techniques.

B. Atomic Force Microscopy 3D images of the three test materials polished with Enhance/Pogo.

C. Atomic Force Microscopy 3D images of the three test materials polished with Astropol.

D. Atomic Force Microscopy 3D images of the three test materials polished with Sof-Lex disks.

4.3 Comparison of S.mutans biofilm thickness cultured for 14 days between the test materials polished with three different polishing systems.

Comparison of the means of S.mutans biofilm thickness cultured for 14 days between

nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled composite polished

with Enhance/Pogo, Astropol, Sof-Lex and Mylar as control group is shown in Figure

4.3, analyzed with One-way ANOVA and post hoc Tukey test as shown in Table 4.3.

The means of S.mutans biofilm thickness for 14 days culturing of the samples cured

against Mylar strip were 118.5 µm for nanofilled composite, 129.2 µm for nanofilled

glass-ionomer and 122.5 µm for microfilled glass-ionomer. Statistical analysis for

S.mutans biofilm thickness showed that nanofilled glass-ionomer was significantly more

than nanofilled composite (P=0.003), and there was no significant difference (P= 0.062)

between nanofilled glass-ionomer and microfilled glass-ionomer. There was no

significant difference between nanofilled composite and microfilled glass-ionomer (P

=0.336), Figure 4.4 A.

The means of S.mutans biofilm thickness after 14 days culturing of the samples polished

with Enhance/Pogo were 111.0 µm for nanofilled composite, 129.8 µm for nanofilled

glass-ionomer and 122.5 µm for microfilled glass-ionomer. S.mutans biofilm thickness

after 14 days culturing on nanofilled glass-ionomer was significantly more than

nanofilled composite (P ≤ 0.001) and microfilled glass-ionomer (P ≤ 0.001). Nanofilled

composite was significantly less than microfilled glass-ionomer (P ≤ 0.001), Figure 4.4

The means of S.mutans biofilm thickness cultured for 14 days of the samples polished with Astropol were 114.7 µm for nanofilled composite, 127.4 µm for nanofilled glass- ionomer and 123.1 µm for microfilled glass-ionomer. S.mutans biofilm thickness cultured for 14 days on nanofilled glass-ionomer was significantly more than nanofilled composite

(P = 0.037), while there was no significant difference between nanofilled glass-ionomer and microfilled glass-ionomer (P= 0.642). There was also no significant difference between nanofilled composite and microfilled glass-ionomer (P =0.202), Figure 4.4 C.

The means of S.mutans biofilm thickness cultured for 14 days of the samples polished with Sof-Lex were 106.1 µm for nanofilled composite, 117.1 µm for nanofilled glass- ionomer and 122.0 µm for microfilled glass-ionomer. S.mutans biofilm thickness cultured for 14 days culturing on nanofilled glass-ionomer was significantly more than nanofilled composite (P ≤ 0.001) and significantly less than microfilled glass-ionomer (P= 0.041).

Nanofilled composite was significantly less than microfilled glass-ionomer (P ≤ 0.001),

Figure 4.4 D.

Regarding the polishing methods and comparison between them, within the nanofilled composite, the means of S.mutans biofilm thickness cultured for 14 days were 118.5 µm for Mylar, 106.1 µm for Sof-Lex, 114.7 µm for Astropol and 111.0 µm for

Enhance/Pogo. After 14 days of culture, statistical analysis showed significantly less

S.mutans biofilm thickness with Sof-Lex compared to Astropol (P=0.008), and no significant difference with Enhance/Pogo (P=0.213). There was no significant difference between Enhance/Pogo and Astropol (P=0.433). The biofilm thickness was significantly less with Mylar compared to Enhance/Pogo (P=0.022) and significantly more with Mylar

compared to Sof-Lex (P ≤ 0.001). However, there was no significant difference between

Mylar and Astropol (P=0.401).

Within the nanofilled glass-ionomer, the means of S.mutans biofilm thickness cultured

for 14 days were 129.2 µm for Mylar, 117.1 µm for Sof-Lex, 127.4 µm for Astropol and

129.86 µm for Enhance/Pogo. There was no significant difference in the mean of

S.mutans biofilm thickness with Sof-Lex and Astropol (P=0.102) while Sof-Lex produced significantly less biofilm thickness compared to Enhance/Pogo (P=0.031).

There was no significant difference between Enhance/Pogo and Astropol (P=0.940).

Meanwhile, Mylar produced significantly more biofilm thickness compared to Sof-Lex

(P = 0.041), but showed no significant difference compared to Enhance/Pogo (P=0.999) and Astropol (P=0.972).

Within the microfilled glass-ionomer, the means S.mutans biofilm thickness cultured for

14 days were 122.6 µm for Mylar, 122.0 µm for Sof-Lex, 123.1 µm for Astropol and

123.0 µm for Enhance/Pogo. There was no significant difference in the mean of S.mutans biofilm thickness after 14 days of culture when the microfilled glass-ionomer was polished with Sof-Lex and Astropol (P=0.906) and also with Enhance/Pogo (P=0.987).

There was also no significant difference between Enhance/Pogo and Astropol (P=0.987).

Mylar showed no significant difference from Enhance/Pogo (P=0.998), Sof-Lex

(P=0.987) and Astropol (P=0.987).

Figure 4.3: Comparison of S.mutans biofilm thickness (µm) cultured for 14 days between the test materials polished with Mylar and three different polishing systems.

Table 4.3 Comparison of the mean (SD) S.mutans biofilm thickness (µm) cultured for 14 days between the test materials polished with three different polishing systems.

Material Polishing system Mean (SD) Mylar Enhance/Pogo Astropol Sof-Lex

NC 118.5 (7.0)A,a 111.0 (2.2)A,bc 114.7 (4.7)A,ab 106.1 (2.3)Ac

NG 129.2 (2.8)B,a 129.8 (4.3)B,ab 127.4 (14.3)B,abc 117.1 (4.9)Bc

MG 122.6 (4.7)AB,a 122.5 (2.3)Ca 123.1 (2.7)AB,a 122.0 (2.4)Ca

*Within a column, values with the same upper-case superscript letter are not significantly different (p > 0.05, Tukey test). Within a row, values with the same lower-case superscript letter are not significantly different (p > 0.05, Tukey test). NC: - nanofilled composite (Filtek™ Z350), NG: - nanofilled glass- ionomer (Ketac™N100) and MG: - microfilled glass-ionomer (Fuji II™ LC).

A. Confocal laser microscopy 3D images 14 days S.mutans biofilm thickness of the three test materials cured against Mylar strip.

Figure 4.4 A, B, C and D: Confocal laser microscopy 3D images of 14 days S.mutans biofilm thickness of test materials polished with different polishing techniques.

B. Confocal laser microscopy 3D images 14 days S.mutans biofilm thickness of the three test materials polished with Enhance/Pogo.

C. Confocal laser microscopy 3D images 14 days S.mutans biofilm thickness of the three test materials polished with Astropol.

D. Confocal laser microscopy 3D images 14 days S.mutans biofilm thickness of the three test materials polished with Sof-Lex disks.

4.4 Comparison of S.mutans biofilm thickness cultured for 30 days between the test materials polished with three different polishing systems.

Comparison of the means of S.mutans biofilm thickness cultured for 30 days on nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled composite polished

with Enhance/PogoTM, Astropol®, Sof-LexTM and Mylar as control group is shown in

Figure 4.5, and analyzed using One-way ANOVA and post hoc Tukey test as shown in

Table 4.4.

The means of S.mutans biofilm thickness cultured for 30 days of the samples cured against Mylar strip were 189.7 µm for nanofilled composite, 197.8 µm for nanofilled glass-ionomer and 183.5 µm for microfilled glass-ionomer. Statistical analysis showed that S.mutans biofilm thickness for 30 days of culture on nanofilled glass-ionomer, was

significantly more than nanofilled composite (P=0.016), and was also significantly more

than microfilled glass-ionomer (P < 0.001). While there was no significant difference

between nanofilled composite and microfilled glass-ionomer (P=0.075), Figure 4.6 A.

The means of S.mutans biofilm thickness cultured for 30 days of the samples polished

with Enhance/Pogo were 215.1 µm for nanofilled composite, 202.8 µm for nanofilled

glass-ionomer and 183.5 µm for microfilled glass-ionomer. Statistical analysis showed

that S.mutans biofilm thickness for 30 days of culture on nanofilled glass-ionomer was

significantly less than nanofilled composite (P < 0.008) and significantly more than

microfilled glass-ionomer (P ≤ 0.001). There was however, no significant difference

between nanofilled composite and microfilled glass-ionomer (P ≤ 0.001), Figure 4.6 B.

The means of S.mutans biofilm thickness cultured for 30 days when the samples were

polished with Astropol were 183.8 µm for nanofilled composite, 211.7 µm for nanofilled

glass-ionomer and 173.7 µm for microfilled glass-ionomer. Statistical analysis showed no

significant difference in S.mutans biofilm thickness for 30 days of culture between

nanofilled glass-ionomer and nanofilled composite (P=0.056), while nanofilled glass-

ionomer was significantly more than microfilled glass-ionomer (P=0.008), and nanofilled composite revealed no significant difference from microfilled glass-ionomer (P=0.642),

Figure 4.6 C.

The means of S.mutans biofilm thickness for 30 days of culture when the samples were polished with Sof-Lex were 169.8 µm for nanofilled composite, 195.1 µm for nanofilled glass-ionomer and 174.8 µm for microfilled glass-ionomer. Statistical analysis showed

S.mutans biofilm thickness for 30 days of culture on nanofilled glass-ionomer was significantly more than nanofilled composite (P ≤ 0.001), and microfilled glass-ionomer

(P ≤ 0.001) respectively, while nanofilled composite revealed no significant difference from microfilled glass-ionomer (P=0.642), Figure 4.6 D.

Regarding the comparison between the means of S.mutans biofilm thickness cultured for

30 days based on polishing method for each test material; in the nanofilled composite group, the mean of S.mutans biofilm thickness were 189.7 µm for Mylar, 169.8 µm for

Sof-Lex, 183.8 µm for Astropol and 215.1 µm for Enhance/Pogo. Statistical analysis

showed no significant difference in the means of S.mutans biofilm thickness for 30 days

of culture between Sof-Lex and Astropol (P=0.508) and significantly less in the Sof-Lex

group compared to Enhance/Pogo (P ≤ 0.001), while Enhance/Pogo was significantly

more than Astropol (P=0.021). Mylar revealed no significant difference with Astropol

(P=0.935), Enhance/Pogo (P= 0.077) and Sof-Lex (P=0.218), respectively.

Within the nanofilled glass-ionomer, the means of S.mutans biofilm thickness for 30 days culturing were 197.8 µm for Mylar, 195.1 µm for Sof-Lex, 211.7 µm for Astropol and

202.8 µm for Enhance/Pogo. After 30 days of S.mutans culture, statistical analysis showed significantly less biofilm thickness with Sof-Lex compared to Astropol (P ≤

0.001) and Enhance/Pogo (P=0.049) respectively. Enhance/Pogo showed significantly less biofilm thickness compared to Astropol (P=0.020). Mylar showed significantly less biofilm thickness compared to Astropol (P ≤ 0.001), however there was no significant difference with Enhance/Pogo (P= 0.300) and Sof-Lex (P=0.765).

Within the microfilled glass-ionomer, the mean S.mutans biofilm thickness for 30 days of culture were 183.5 µm for Mylar, 173.8 µm for Sof-Lex, 174.7 µm for Astropol and

179.5 µm for Enhance/Pogo. There was no significant difference in the mean of S.mutans biofilm thickness between Sof-Lex and Astropol (P=0.950), and Enhance/Pogo

(P=0.154). There was also no significant difference between Enhance/Pogo and Astropol

(P=0.054). Mylar showed significantly less biofilm thickness than Astropol (P ≤ 0.001) and Sof-Lex (P =0.002), however there was no significant difference with Enhance/Pogo

(P= 0.271).

Figure 4.5: Comparison of S.mutans biofilm thickness (µm) cultured for 30 days between the test materials polished with Mylar and three different polishing systems.

Table 4.4 Comparison of the mean (SD) S.mutans biofilm thickness (µm) cultured for 30 days between the test materials polished with three different polishing systems.

Material Polishing system Mean (SD) Mylar strip Enhance/Pogo Astropol Sof-Lex

NC 189.7 (6.9)Ba 215.1 (5.6)Ba 183.8 (5.8)AB,ab 169.8 (5.1)B,ab

NG 197.8 (2.5)Aa 202.8 (8.9)Aa 211.1 (3.6)Ab 195.1 (3.1)Aa

MG 183.5 (4.3)Ba 179.5 (4.8)C,ab 174.7 (3.4)Bb 173.8 (3.4)Bb

A. Confocal laser microscopy 3D images 30 days S.mutans biofilm thickness of the three test materials cured against Mylar strip.

Figure 4.6 A, B, C and D: Confocal laser microscopy 3D images of 30 days S.mutans biofilm thickness of test material polished with different polishing techniques.

B. Confocal laser microscopy 3D images 30 days S.mutans biofilm thickness of the three test materials polished with Enhance/Pogo.

C. Confocal laser microscopy 3D images 30 days S.mutans biofilm thickness of the three test materials polished with Astropol.

D. Confocal laser microscopy 3D images 30 days S.mutans biofilm thickness of the three test materials polished with Sof-Lex.

4.5 Comparison of S.mutans biofilm thickness between 14 days and 30 days of culture on test materials polished with three different polishing systems.

Comparison between the means of S.mutans biofilm thickness after 14 days and 30 days

of culture on nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled

composite polished with Enhance/Pogo, Astropol, Sof-Lex and Mylar as control group is

shown in Figure 4.7, analyzed with T-test, as shown in Table 4.5.

The means of S.mutans biofilm thickness cultured for 14 days when the samples were

cured against Mylar strip were 129.2 µm for nanofilled glass-ionomer, 122.5 µm for

microfilled glass-ionomer and 118.5 µm for nanofilled composite. While the means

S.mutans biofilm thickness cultured for 30 days were 197.8 µm for nanofilled glass- ionomer, 183.5 µm for microfilled glass-ionomer and 189.7 µm for nanofilled composite.

Statistical analysis revealed the means of S.mutans biofilm thickness cultured for 14 days of nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled composite were significantly less than the means of S.mutans biofilm thickness cultured for 30 days (P ≤

0.001).

The means of S.mutans biofilm thickness cultured for 14 days when the samples were polished with Enhance/Pogo were 129.8 µm for nanofilled glass-ionomer, 122.5 µm for microfilled glass-ionomer and 111.0 µm for nanofilled composite. While the means of

S.mutans biofilm thickness cultured for 30 days were 202.8 µm for nanofilled glass- ionomer, 179.5 µm for microfilled glass-ionomer and 215.1 µm for nanofilled composite.

Statistical analysis revealed the means of S.mutans biofilm thickness cultured for 14 days of nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled composite were

significantly less than the means of S.mutans biofilm thickness cultured for 30 days (P ≤

0.001).

The means of S.mutans biofilm thickness cultured for 14 days when the samples were

polished with Astropol were 127.4 µm for nanofilled glass-ionomer, 123.1 µm for

microfilled glass-ionomer and 114.7 µm for nanofilled composite. While the means of

S.mutans biofilm thickness cultured for 30 days were 211.7 µm for nanofilled glass-

ionomer, 174.7 µm for microfilled glass-ionomer and 183.8 µm for nanofilled composite.

Statistical analysis revealed the means of S.mutans biofilm thickness cultured for 14 days on nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled composite were significantly less than the means of S.mutans biofilm thickness cultured for 30 days (P ≤

0.001).

The means of S.mutans biofilm thickness cultured for 14 days when the samples were polished with Sof-Lex were 117.1 µm for nanofilled glass-ionomer, 122.0 µm for microfilled glass-ionomer and 106.1 µm for nanofilled composite. While the means of

S.mutans biofilm thickness cultured for 30 days were 195.1 µm for nanofilled glass- ionomer, 173.8 µm for microfilled glass-ionomer and 169.8 µm for nanofilled composite.

Statistical analysis revealed the means of S.mutans biofilm thickness cultured for 14 days on nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled composite were significantly less than the means S.mutans biofilm thickness cultured for 30 days (P ≤

0.001). (Figure 4.7)

A. Mylar B. Enhance/Pogo

C. Astropol D. Sof-Lex

Figure 4.7 A, B, C and D: comparison between 14 and 30 day S.mutans biofilm thickness on all test materials polished with Mylar, Enhance/Pogo, Astropol and Sof-Lex.

Table 4.5 Comparison of the mean (SD) S.mutans biofilm thickness (µm) between 14 days and 30 days on test materials polished with three different finishing/polishing systems. polishing materials Mean biofilm thickness (µm) of Mean biofilm thickness (µm) of P systems 14 days S.mutans culture (SD) 30 days S.mutans culture (SD) value Mylar NC 118.5 (7.0) 189.7 (6.8) 0.001 NG 129.2 (2.8) 197.8 (2.5) 0.001 MG 122.5 (4.6) 183.5 (4.2) 0.001

Enhance/Pogo NC 111.0 (2.1) 215.1 (5.6) 0.001 NG 129.8 (4.3) 202.8 (8.8) 0.001 MG 122.5 (2.3) 179.5 (4.7) 0.001

Astropol NC 114.7 (4.7) 183.8 (35.8) 0.001 NG 127.4 (14.2) 211.7 (3.6) 0.001 MG 123.1 (2.6) 174.7 (3.4) 0.001

Sof-Lex NC 106.1 (2.3) 169.8 (5.0) 0.001 NG 117.1 (4.9) 195.1 (3.1) 0.001 MG 122.0 (2.3) 173.8 (3.4) 0.001

NG: - nanofilled glass-ionomer (Ketac™N100) MG: - microfilled glass-ionomer (Fuji II™ LC) NC: - nanofilled composite (Filtek™ Z350) *Significant at P < 0.05, T-test

5 DISCUSSION

5.1 General overview

The esthetic and longevity of tooth colored restoratives are highly dependent on their surface characteristics and chemical composition (O'Brien, 2002). Residual surface

roughness of restorations encourages biofilm accumulation, which may result in gingival

inflammation, superficial staining, and secondary caries. The surface roughness of the

studied materials is determined by finishing and polishing techniques, but could be

affected by biological and chemical degradation in the oral environment. Although

restoratives materials that are cured against a matrix are not without surface

imperfections, they present the smoothest surfaces possible (Hotta et al., 1995;Baseren,

2004;Beun et al., 2007).

Nanofilled glass-ionomer is a new modification of glass-ionomer. The idea is that

decreasing the size of filler particles to nano scale will improve certain properties such as

esthetics properties. Materials with fillers of larger sizes generally show more surface

roughness than those with fillers of smaller size (Yap et al., 1997). The surface roughness

has been recognized as a parameter of high clinical relevance for wear resistance, plaque

accumulation, gingival inflammation, material discoloration and surface gloss (Larato,

1972;Shintani et al., 1985). Many studies have shown that the smoothest surface

imparted to restorative materials occurs when they have been allowed to set against a

Mylar strip, the smoothest polished materials should have a surface roughness

comparable to the surface against matrix (Yap et al., 1997). These surfaces against matrix

were smoother than polished surfaces because the unpolished surfaces are composed of

more polymer matrix than fillers (Bassiouny and Grant, 1980). However, this surface is

polymer rich, making it relatively unstable (Morgan, 2004).

After polishing, some composites presented greater surface roughness than the

unpolished surfaces to varying degrees. While in nanofilled resin composites there was no difference in surface roughness between polished and unpolished surfaces. This is because a nanofilled composite has an average particle size less than that of microhybrid or microfilled resin composites (Mitra et al., 2003).

The interaction between surface roughness and dental plaque discussed in many studies, indicate that the smooth surfaces with a low surface-free energy is necessary to minimize plaque formation, thereby reducing the occurrence of caries and periodontitis (Quirynen and Bollen, 1995). A clear relationship exists between the number of dental caries and

S.mutans inhabiting the plaque, therefore, plaque control plays a significant role in the prevention of caries (Reichart and Gehring, 1984). S.mutans is among the bacteria proliferating in the dental biofilm. Their virulence is mainly due to their high adhesion capability, acidogenicity and aciduric properties. These S.mutans characteristics could be

responsible for surface damage to restorations, since this microorganism can be found on

any hard surface in the oral cavity, such as enamel, implants, orthodontic appliances or

restorative materials (Auschill et al., 2002;Eick et al., 2004).

The filler size, polishing technique and plaque accumulation in addition to glass-ionomer

fluoride release all interacted together. Change in the filler size and the type of the

polishing technique will change the result of surface smoothness. The plaque

accumulation will increase or decrease depending on surface roughness in addition to

fluoride release.

5.2 The surface roughness of the test materials cured against Mylar strip before and after 30 days S.mutans biofilm culture.

The first objective was to compare the surface roughness between nanofilled glass-

ionomer, microfilled glass-ionomer and nanofilled composite, cured against Mylar strip,

subjected to 30 days of S.mutans biofilm culture. This would show the effect of S.mutans

biofilm on the surface texture of the test materials. These materials are different in filler size and fluoride release, Table 4.1.

After 30 days S.mutans biofilm culture, S.mutans biofilm did not affect the surface roughness of nanofilled glass-ionomer and nanofilled composite. However, it significantly changed the surface roughness of microfilled glass-ionomer.

It has been reported that the surface cured against Mylar strip is the smoothest surface for most direct tooth colored restorations. However, this surface is polymer rich, making it relatively unstable (Senawongse and Pongprueksa, 2007). Surface roughness is due to the deterioration of the resin surface, which is caused by degradation of the filler matrix bonds and the subsequent elution of matrix constituents. The surface of dental resin materials is usually more complex and may be very inhomogeneous due to fractions of filler and different matrix constituents that are present on the surface of a test material

(Hahnel et al., 2009). The unpolished glass-ionomer surfaces were rougher and bound more bacteria compared to unpolished composite resin (Carlén et al., 2001;Ahmad et al.,

2006). Based on these facts, it can be deduced that the increase in the surface roughness of microfilled glass-ionomer is fundamentally down to a large filler size compared to nanofilled glass-ionomer and nanofilled composite. The reaction between S.mutans biofilm and matrix of microfilled glass-ionomer leave the surface with big holes due to

the breaking down of the bond between matrix and fillers, regardless of the fluoride effect on the S.mutans. Previous studies showed that fluoride affects the growth rate of the bacteria in the long term but have less effect on the acidity of S.mutans biofilm

(Friedl et al., 1997;Van Loveren et al., 1991;van der Hoeven and Franken, 1984), which was evident from our findings.

5.3 Comparison of the surface roughness between the test materials polished with three different polishing systems after 30 days S.mutans biofilm culture.

The second objective was to compare the surface roughness (Ra) of three test materials polished with three different polishing systems after 30 days S.mutans biofilm culture.

Results showed that there was no significant difference in the surface roughness between nanofilled glass-ionomer and nanofilled composite when polished with Enhance/Pogo and Astropol. However, the surface roughness of nanofilled glass-ionomer was less than nanofilled composite when polished with Sof-Lex disks, Table 4.2.

Previous studies which are conducted only on nanofilled composite showed that Sof-Lex polishing disks produced the smoothest surface compared to Enhance/Pogo and

CompoSystem based on the fact that the surface roughness is related to the surface morphology and to the average filler size (Choi et al., 2005;Uctasli et al., 2007;Janus et al., 2010).

However in this study, the surface roughness of nanofilled glass-ionomer was less than nanofilled composite polished with Sof-Lex disks. Since both have the same filler size (5-

25 nm), the difference in the surface roughness can be due to the nanofilled glass- ionomer fluoride releasing property which minimizes the reaction between S.mutans biofilm and the surface of the material.

However, the surface roughness of nanofilled glass-ionomer was significantly less compared with the surface roughness of microfilled glass-ionomer when polished with all three polishing techniques. This was due to the larger filler size of microfilled glass- ionomer compared to nanofilled glass-ionomer which overrides the fluoride effect.

This study also compared the polishing systems within each test material in order to find

the best polishing system for each material. Mylar strip formed the smoothest surface in all test materials as also shown in many previous studies (Chung, 1994;Hondrum and

Fernandez, 1997;Roeder et al., 2000;Ozgunaltay et al., 2003). However, clinically, it is

difficult to perform a restoration without the need for removal of excess material (Yap et

al., 1998), which in turn will disturb the surface finished with Mylar strip.

The results of this study showed that multiple step polishing system (Sof-Lex) was more

effective than one step polishing system (Enhance/Pogo and Astropol) in both nanofilled

and microfilled glass-ionomer. Enhance/Pogo recorded the roughest surface on nanofilled

glass-ionomer compared to Sof-Lex and Astropol.

However, Astropol produced the smoothest surface with nanofilled composite compared

to Sof-Lex disks and Enhance/Pogo. However the comparison between Astropol and

Enhance/Pogo was not statistically significant. The finding is in conflict with previous

studies which concluded that flexible aluminum oxide disks (Sof-Lex) are the best

instruments for providing low roughness on composite surfaces (Scheibe et al.,

2009;Venturini et al., 2006;Lu et al., 2003). However, the result of this study is in

agreement with several previous studies which concluded that current one-step systems

appear to be as effective as multiple-step systems for polishing dental composites (St-

Georges et al., 2005;Yap et al., 2004). Other studies also concluded that Enhance/Pogo

generally produced the smoothest surfaces in all composites (Da Costa et al.,

2007;Turkun and Turkun, 2004). These previous studies have used various types of

composites including micro-hybrid (Scheibe et al., 2009), (Lu et al., 2003), hybrid (Filtek

Z250) (Venturini et al., 2006), micro-hybrid composites and hybrid composite (Filtek

Z250) (St-Georges et al., 2005) and microfilled composite (Z100) (Yap et al., 2004).

Several polishing systems have been used over the years, ranging from multiple-step

systems using fine and superfine diamond burs, abrasive disks, diamond and silicon

impregnated soft rubber cups, to one-step polishing systems containing diamond impregnated cups and silicon carbide brushes. The factors that might have influenced the different results of the surface roughness studies may possibly be due to different filler particle size and type of abrasives used in the polishing system, the time used for each polishing procedure, the way which the particles are bound within the matrix, as well as the composition of the matrix which is different for each of system, thus, affecting their polishing efficiency. If the matrix wears at a more similar rate as the polishing particle, it is likely that the particle would be less likely to extrude significantly from the matrix; therefore, it would have less of a gouging effect on the materials at the end product surface roughness (Yap et al., 1997).

5.4 Evaluation and comparison of the S.mutans biofilm thickness cultured for 14 days and 30 days between the test materials polished with three different polishing systems.

The S.mutans biofilm thickness was measured after 14 days of culture on nanofilled glass-ionomer, microfilled glass-ionomer and nanofilled composite polished with

Enhance/Pogo, Astropol and Sof-Lex, in order to evaluate the interaction between

S.mutans biofilm and three types of materials, Table 4.3. These materials have different consistency, filler size and fluoride level. In this study, a standard pattern for the behavior of S.mutans biofilm cannot be confirmed. The biofilm thickness increase and decrease with the change of the test material and polishing techniques. The basic fact is that physico-chemical surface properties play an important role in the formation of biofilm

(Quirynen and Bollen, 1995). In addition, the fillers and matrices of resin dental materials

have a big influence on the growth of bacterial biofilm. However, the amount of biofilm

accumulation varies according to the particle size of fillers and monomer components of

the resin matrix (Imazato et al., 2003;Takahashi et al., 2004).

In this study, after 14 days of culture, the S.mutans biofilm thickness on the surface of nanofilled composite was slightly less compared to the nanofilled and microfilled glass-

ionomer. This finding is in agreement with previous studies, in which nanofilled

composite have less surface roughness than glass-ionomers (Fucio et al., 2008). The

difference in matrix composition between composite and glass-ionomer may contribute to the findings. The difference in S.mutans biofilm thickness between the three test materials was not significant. This can be consoled to the short period of culturing.

With regards to the polishing techniques, the specimens cured against Mylar strip and the

ones polished with Enhance/Pogo and Astropol have almost the same biofilm thickness.

On the other hand, specimens polished with Sof-Lex disks acquired less S.mutans biofilm

thickness in 14 days. It was previously proven in this study that Sof-Lex produced the

smoothest surface in nanofilled and microfilled glass-ionomer which would explain the

least biofilm thickness compared to Mylar strip, Enhance/Pogo and Astropol.

Surface roughness influences the adhesion of the biofilm, probably because of the greater

surface area provided and the provision of protected sites for colonization. Polishing the

surfaces is another important factor. The polishing process should entail the consideration

of resisting biofilm adherence and growth. Selection of polishing technique is another

factor influencing biofilm adherence. Without neglecting the fact that polishing did not

cause all dental materials to be similarly resistant to biofilm formation (Ono et al., 2007).

It has been proven that materials with lower surface roughness had less bacterial adhesion

(Ikeda et al., 2007).

S.mutans biofilm thickness after 30 days of culture was significantly different from that of 14 days. This may be due to many factors such as fluoride concentration, pH and surface roughness since all of which have direct effect on the biofilm thickness.

After 30 days of culture, regardless of the polishing technique, S.mutans biofilm thickness was less on microfilled glass-ionomer specimens compared to nanofilled glass- ionomer and nanofilled composite, Table 4.4.

The only explanation for that is the low pH and high fluoride concentration of microfilled glass-ionomer (Nakajo et al., 2009;Markovic et al., 2008;Marczuk-Kolada et al., 2006).

Generally, antibacterial activity is attributed to fluoride release and low pH during setting of microfilled glass-ionomer (Konishi et al., 2003). Low concentrations of fluoride can modify the bacterial metabolism with a concomitant decrease in acid production (van

Loveren et al., 1990), in addition to the low initial pH of the material during setting

(Barkhordar et al., 1989), polyalkenoic acids, zinc oxide (Scherer et al., 1989) and

HEMA (Benderli et al., 1997).

Fluoride release from a glass-ionomer may occur by diffusion from within the material or dissolution at the surface (Yap et al., 1999). In addition to that, the concentration of fluoride is maximized at the surface of the material (Hadley et al., 2001). Under acidic conditions, the dissolution process is expected to be more dominant and this probably explains the greater fluoride release for each material under these conditions (Forss and

Seppa, 1990). Fluoride releasing materials significantly inhibited bacterial metabolism, resulting in lower pH drop. It is well-known that fluoride affects bacterial growth and metabolism (Hamilton, 1990).

The difference in the behavior of the various materials may be related to the ratio of resin to salt in the material matrix. Hence, materials which are predominantly salt matrix (GIC and RMGIC) are more sensitive to changes in pH than products which have predominantly a resin matrix (Al-Naimi et al., 2008).

The fluoride concentration of microfilled glass-ionomer (Fuji II™ LC) is higher compared to nanofilled glass-ionomer (Ketac™N100) which is 4.6 and 2.5 wt (%), respectively. Voids, cracks and microporosities seen at the disk surface of microfilled glass-ionomer explain the high fluoride concentration compared to nanofilled glass- ionomer. Fluoride release is related to glass-ionomer degradation, destruction of the

material surface is followed by extensive fluoride release (Markovic et al., 2008). This supports our finding of S.mutans biofilm thickness after 30 days on microfilled glass- ionomer was less than S.mutans biofilm thickness on nanofilled glass-ionomer and

nanofilled composite.

With regards to the polishing systems, Sof-Lex disks also produced less biofilm thickness

in all test materials after 30 days of culture compared to Mylar strip, Enhance/Pogo and

Astropol.

With regards to the comparison between the means of S.mutans biofilm thickness after 14

days and 30 days of culture on nanofilled glass-ionomer, microfilled glass-ionomer and

nanofilled composite polished with Enhance/Pogo, Astropol, Sof-Lex and Mylar strip,

the results show that S.mutans biofilm thickness for 14 days was significantly less than

30 days S.mutans biofilm thickness, Table 4.5.

The result of this study are in agreement with a study conducted by Sissons et al.(1995) which showed that plaque weight of biofilm increased for more than 18 days, although it is clear that bacterial growth rate decreased sharply during that period.

The characteristics of biofilm vary, depending on dynamic changes in the micro

environment. Thickness is considered a primary factor in determining the properties of

the biofilm. Diffusion of nutrients and gases across the biofilm is the crucial determinant

in the physiology of the adhered bacteria (Bowden and Li, 1997). Low diffusion co-

efficiency will result in stress conditions within the biofilm thus decreasing bacterial

growth.

In this study, the results showed that S.mutans biofilm thickness was around 110 µm after

14 days of culture compared to around 180 µm after 30 days of culture. This increase in

biomass suggests that extracellular enzymes such as GTF and FTF are enzymatically

active in the biofilm and they synthesize exocellular matrix as glucans and fructans from

sucrose, thus contributing to the increasing thickness of the biofilm (Schilling and

Bowen, 1992).

Viability and growth of bacteria within the biofilm is influenced by numerous

environmental conditions, such as nutrition supply, outflux of metabolites, pH gradient

and oxygen tension (Marsh and Bradshaw, 1997).

In addition to the physiological parameters, cell density and quorum sensing which is the

process by which many bacteria coordinate gene expression according to the local density

of bacteria producing signaling molecules are implicated in the growth rate of biofilm

bacteria (Costerton et al., 1999). Whereby, lack of nutrition and accumulation of byproducts may account for a decline in the growth of bacteria in suspension and in biofilm.

One of the major growth rate controlling factors in a biofilm is the diffusion rate of

ingredients and gases across the biofilm (Dibdin, 1997). This fact may explain the slight

decrease in the biofilm growth rate from day 14 to day 30 of S.mutans biofilm culture.

6 CONCLUSIONS AND RECOMMENDATIONS

6.1 Conclusions

1. Surface roughness of microfilled glass-ionomer cured against Mylar strip

increased significantly after 30 days of S.mutans biofilm culturing, however there

were no significant increase in the surface roughness after 30 days of S.mutans

culture both on nanofilled glass-ionomer and nanofilled composite.

2. Nanofilled glass-ionomer and microfilled glass-ionomer polished with Sof-Lex

disks showed the least surface roughness, while nanofilled composite showed the

least surface roughness when polished with Astropol after 30 days of S.mutans

culture.

3. After 14 days S.mutans biofilm culturing, nanofilled composite have slightly less

biofilm thickness compared to the nanofilled and microfilled glass-ionomer.

However, after 30 days S.mutans biofilm culturing, microfilled glass-ionomer

displayed less S.mutans biofilm thickness compared to nanofilled glass-ionomer

and nanofilled composite. Sof-Lex disks produced the least S.mutans biofilm

thickness in both periods, but not all results were statistically significant within

the comparative groups. S.mutans biofilm thickness after 14 days of culture was

less than in 30 days of culture in all test materials regardless of the polishing

systems.

6.2 Limitations of the study

This study was carried out to compare the surface roughness of the test materials using

three different polishing systems, which were subjected to 30 days S.mutans culture and

also to evaluate and compare the S.mutans biofilm thickness after 14 and 30 days of

culture. It was impossible to carry out the correlation between the surface roughness and

the biofilm thickness of the test materials throughout the test period of 30 days as it was

technically impossible to do so. Bacterial growth on the sample will be interrupted when

the samples were subjected to AFM for surface roughness measurement. Therefore

measurement of surface roughness and the biofilm thickness cannot be carried out on the

same sample, hence the inability to correlate between the surface roughness and the

biofilm thickness.

6.3 Recommendations

6.3.1 Clinical recommendations

Nanofilled glass-ionomer has all the good properties of the mirofilled glass-ionomer, and also some of the good properties of the nanofilled composite. This includes chemical

bonding to tooth structure which produces minimal marginal microleakage, and making it

less technique sensitive compared to nanofilled composite. It also has the good feature of

being aesthetically comparable to nanofilled composite. However it is not as strong as

nanofilled composite and its fluoride release is lower than the microfilled glass-ionomer.

Therefore the nanofilled glass-ionomer is most beneficial to be used in Class V cavity where aesthetic is of concern, and in patients with low caries risk.

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It can also be used in patients with root caries or patients with external cervical

inflammatory resorption (ECIR) where aesthetic is important. Isolation is difficult in

these cases, and the risk of marginal microleakage is high if composite material is used

on its own. Normally, in these cases clinicians would use sandwich technique where

missing dentine is replaced with glass ionomer, which minimizes marginal microleakage,

and composite is used in the outer layer to achieve better aesthetic. However, if

nanofilled glass-ionomer used, the clinicians can avoid using sandwich technique as the

nanofiled glass-ionomer can minimize marginal microleakage, and at the same time is

aesthetic.

Nanofilled glass-ionomer is less technique sensitive compared to composite material.

Therefore, nanofilled glass-ionomer is recommended when the tooth isolation is difficult,

for example in children and special need patients with low caries risk.

6.3.2 Recommendations for future research

Although this study focused on the surface roughness of the nanofilled glass-ionomer, wear resistance is one of the material properties which determined by the filler size, wear resistance of nanofilled glass-ionomer is an important factor should be taken into considerations in future research. Discoloration of the nanofilled glass-ionomer is another factor which has not been evaluated; In vitro study can be carried out to estimate discoloration, wear resistance and hardness. In vivo study should also be conducted to evaluate the longevity of the nanofilled glass-ionomer materials.

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APPENDICES

1. Raw data

Test Polishing Surface Biofilm Biofilm Surface Surface materials systems roughness thickness thickness roughness roughness Ra after after 14 after 30 at day 0 at day 30 30 days days days S.mutans S.mutans S.mutans S.mutans S.mutans culture culture culture culture culture (nm) (nm) (nm) (µm) (µm) 1 1 1 45.0 113.0 176.0 27.0 43.0 2 1 1 36.0 115.0 188.0 25.0 39.0 3 1 1 32.0 115.0 197.0 23.0 31.0 4 1 1 43.0 117.0 194.0 26.0 45.0 5 1 1 30.0 118.0 191.0 23.0 29.0 6 1 1 42.0 118.0 194.0 25.0 43.0 7 1 1 37.0 134.0 188.0 24.0 37.0 8 1 2 144.0 108.0 222.0 9 1 2 108.0 110.0 207.0 10 1 2 117.0 110.0 218.0 11 1 2 105.0 111.0 213.0 12 1 2 100.0 111.0 217.0 13 1 2 119.0 112.0 220.0 14 1 2 109.0 115.0 209.0 15 1 3 84.0 111.0 103.0 16 1 3 75.0 110.0 202.0 17 1 3 72.0 112.0 192.0 18 1 3 80.0 113.0 198.0 19 1 3 85.0 115.0 200.0 20 1 3 93.0 119.0 198.0 21 1 3 92.0 123.0 194.0 22 1 4 121.0 102.0 169.0 23 1 4 258.0 105.0 165.0 24 1 4 134.0 105.0 167.0 25 1 4 266.0 107.0 172.0 26 1 4 125.0 108.0 178.0 27 1 4 129.0 107.0 164.0 28 1 4 199.0 109.0 174.0 29 2 1 44.0 124.0 196.0 32.0 46.0 30 2 1 45.0 128.0 202.0 33.0 43.0 31 2 1 30.0 129.0 197.0 29.0 28.0

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32 2 1 44.0 130.0 195.0 34.0 46.0 33 2 1 53.0 130.0 196.0 37.0 52.0 34 2 1 47.0 131.0 200.0 31.0 48.0 35 2 1 43.0 133.0 199.0 31.0 44.0 36 2 2 201.0 132.0 194.0 37 2 2 173.0 125.0 216.0 38 2 2 249.0 126.0 210.0 39 2 2 130.0 127.0 199.0 40 2 2 146.0 129.0 203.0 41 2 2 210.0 133.0 207.0 42 2 2 113.0 137.0 191.0 43 2 3 78.0 110.0 209.0 44 2 3 50.0 110.0 215.0 45 2 3 71.0 119.0 212.0 46 2 3 79.0 134.0 208.0 47 2 3 90.0 134.0 218.0 48 2 3 68.0 141.0 209.0 49 2 3 71.0 144.0 211.0 50 2 4 65.0 112.0 191.0 51 2 4 55.0 112.0 199.0 52 2 4 52.0 115.0 194.0 53 2 4 77.0 117.0 198.0 54 2 4 56.0 118.0 198.0 55 2 4 73.0 120.0 193.0 56 2 4 72.0 126.0 193.0 57 3 1 117.0 122.0 177.0 43.0 109.0 58 3 1 74.0 122.0 185.0 40.0 90.0 59 3 1 113.0 113.0 188.0 45.0 126.0 60 3 1 161.0 124.0 180.0 47.0 145.0 61 3 1 133.0 124.0 186.0 46.0 134.0 62 3 1 138.0 125.0 188.0 49.0 132.0 63 3 1 109.0 128.0 181.0 41.0 114.0 64 3 2 550.0 119.0 181.0 65 3 2 749.0 121.0 172.0 66 3 2 368.0 121.0 186.0 67 3 2 180.0 123.0 180.0 68 3 2 789.0 124.0 178.0 69 3 2 705.0 124.0 184.0 70 3 2 489.0 126.0 176.0 71 3 3 305.0 120.0 174.0 72 3 3 678.0 120.0 170.0 73 3 3 518.0 122.0 175.0 74 3 3 403.0 123.0 171.0 75 3 3 684.0 125.0 177.0 76 3 3 758.0 125.0 179.0

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77 3 3 579.0 127.0 178.0 78 3 4 187.0 119.0 171.0 79 3 4 306.0 120.0 179.0 80 3 4 213.0 121.0 173.0 81 3 4 307.0 122.0 176.0 82 3 4 249.0 124.0 169.0 83 3 4 338.0 122.0 176.0 84 3 4 244.0 126.0 172.0 materials 1= nanofilled composite (Z350) 2= nanofilled glass-ionomer (Ketac N100) 3= microfilled glass-ionomer (Fuji II LC) polishing 1= Mylar strip 2= Enhance/Pogo 3= Astropol 4= Sof-Lex

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2. Publication

Archives of BioCeramics Researches Volume 10, 2010. Proceeding of The 10th Asian BioCeramics Symposium (ABC 2010).

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3. Presentation and publication.

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