ACKNOWLEGMENT

I take this opportunity of expressing my sincere gratitude to my revered

and esteemed teacher and guide, DR RUPAL J. SHAH, MDS.

Professor and Head, Department of Prosthetic Dentistry, without

whose guidance, constructive criticism and valuable suggestions, this

study would not have been completed. It’s really a proud privilege to

work under her patronage and observation. I wish to place on record,

through this acknowledgement, my deepest sense of indebtness to her

and look forward to her continued support and encouragement in the

future.

I express my sincere thanks to DR. GIRISH PARMAR, MDS., Dean,

Government Dental College and Hospital, Ahmedabad, for providing all

the necessary facilities.

I express my gratitude towards Ex-Assistant Professor and Guide,

DR. SHRUTI P. MEHTA, MDS. whose guidance and motivation helped

in initiation of this work.

I express my sincere gratitude to all the staff members of the

department of Prosthetic Dentistry for helping me whenever I needed.

I am also thankful to library staff MR. R.S. KHETRA and Mrs. V.R.

CHAYA, for their help in providing the necessary academic material.

I am grateful to Mrs. CHANDRALEKHA, Professor and Head,

Department of Microbiology, B.J. Medical College, Ahmedabad for

permitting me to carry out dissertation work at their department. I also

take the opportunity of expressing sincere thanks to Mrs. MONIKA,

Laboratory Technician, who helped me to carry out the necessary tests

in the laboratory.

I am grateful to Mr. VIJAY SHARMA, Biostatistician, N.I.O.H.,

Ahmedabad for carrying out statistical analysis as per the requirements

of my work. My sincere thanks to my colleagues Dr. Vishal, Dr.

Kuldeep, Dr. Alaap, Dr. Gautam, Dr. Prashant, my seniors and my

junior friends, who have provided support throughout my work.

I wish to place on record that it is the encouragement, love and

warmth given by my parents, my brother, my in-laws and my beloved

husband DR. RITESH GUPTA, who helped me make this venture

successful.

DR. SHUCHI GUPTA

CONTENTS

PAGE NO

1. INTRODUCTION 1

2. REVIEW OF LITERATURE 4

3. MATERIALS AND METHOD 21

4. OBSERVATION AND RESULTS 36

5. DISCUSSION 51

6. SUMMARY AND CONCLUSION 62

7. BIBLIOGRAPHY 63

1

INTRODUCTION

The therapeutic procedures used in the treatment of causes do not

always eliminate all the micro-organisms in the residual tissues. The

persisting bacterial presence, together with the lack of a thoroughly

hermetic seal between the prepared tooth walls and final finished fixed

prosthesis walls allow bacterial leakage and thus may be involved in

the development of recurrent caries. One possible solution for this

service problem is to use dental luting materials with a bacteriostatic

capacity.1

Microleakage is a common, unavoidable, clinical phenomenon by

which oral fluids, ions, molecules’, and bacteria gain access to dentinal

tubuli and pulp through the junction between a cemented prosthesis

and the tooth.2

Recurrent human dental caries has been associated with the

dissolution of dental luting agents and the inherent roughness of the

prepared tooth margins or prosthesis. Breakdown is marginal. Areas

between tooth prepared and the prosthesis cemented by any of the

luting cement can provide potential pathways for reinfection.

Carcinogenic micro-organisms present in normal human flora could

easily penetrate underlying dentin through such defects. A well

designed preparation with a smooth and even margin reduces or

preferably prevents such marginal breakdown reducing the chances of

recurrent carries.3

Several species of bacteria may be isolated from plaque associated

with carious lesions and pulpal inflammation. Streptococcus mutans is

one of the bacteria most frequently implicated in dental caries.4

2

When teeth are being prepared for fixed prosthesis, infected and

softened dentin is often found extending into the deeper layers.

Preparations for abutments, which include removal of such carious

Lesions, can often result in the creation of larger cavities.

Consequently, the mechanical strength and retention force of

abutments may be weakened. Human carious lesions can be

thoroughly sterilized by the topical application of certain antibacterial

drug combinations. These sterilized carious lesions can be left

untreated and softened because the dentin recalcifies with time. Thus

this procedure can minimize the amount of infected dentin that must be

removed from the prepared abutments.5

Failure of fixed partial denture is most frequently caused by caries. It

was proposed that carious lesions of the prepared abutments may be

sterilized by covering them with a provisional restoration and

antibacterial provisional cement containing a mixture of antibacterial

drugs.6

Permanent and provisional restorations cemented with temporary

cement need to provide biological and mechanical protection for the

abutments. A temporary cement must be strong enough to retain the

restoration but weak enough to enable removal of the restoration

without damage to the abutment. However, provisional crown luted with

temporary cements are susceptible to cement washout, marginal

leakage, Bacterial infiltration and caries, especially when placed for a

periods longer than a few weeks.7

Antibacterial activity, during and after setting, assumes clinical

relevance because this property may help in the elimination or

reduction of bacteria that have remained viable in the cavity walls or

3

bacteria that may gain access to the cavity through micro leakage

channels.4

The physically superior cement materials can play an important role

in caries prevention. Furthermore, it would be advantageous if such

materials possessed effective antibacterial properties.3

It is therefore, the objective of this study “ANTIBACTERIAL

PROPERTIES OF AGED DENTAL CEMENTS” to access and compare

the incessant antibacterial activity of three luting cements.

1) Glass-Ionomer Cement (Ketac- Cem)

2) Zing Phosphate Cement (Harvard)

3) Polycarboxylate (Poly F)

in 2 powder: liquid ratios (wt/wt)

1) Ketak-Cem 3.8:1

3.5:1

2) Harvard 1.8:1

1.6:1

3) Poly F 2.8:1

1.8:1

by undertaking the following tests:

1) Direct contact test

2) Agar diffusion test.6

4

REVIEW OF LITERATURE

Watts T.L.P., Combe E.C. in 1980 reported that the adhesion to

enamel is an important aspect of periodontal dressing retention. Details

of tensile and stress bond strengths were presented for three dressing

materials i.e. Coe Pak, Peripac, Peripac improved and a positive

polycarboxylate Control (Duralon) and the data was discussed with

regard to optimum levels of retention. The dr4essing materials were

tested 1hr, 1day and 1week after placement, in groups of five

specimens for each test, tensile and shear.

Most shear tests yielded higher results than the corresponding tensile

tests and a level of 2MN/m2 is suggested for shear bond strength and

1MN/m2 for tensile bond strtength.8

Schwartzman, Caputo A.A., Schein B. in 1980 evaluated the

antimicrobial action of various dental cements, against common micro-

organisms frequently based within the components of the normal

microbial oral flora. The cements included in the study were

1) zinc oxide eugenol

2) polycarboxylate

3) zinc phosphate

4) silicate

5) silico-phosphate

6) composite resin.

The antimicrobial power were against the following micro-organisms:

1) Streptococcus mutans

2) Escherichia coli

3) Streptococcus viridians

4) Lactobacillus acidophilus

5

5) Streptococcus pyogenus

The cements were listed in decreasing order of effectiveness:

1) Zinc oxide eugenol

2) Silico-phosphate

3) Zinc phosphate

4) Silicate

The two cements, polycarboxylate and composite resin, exhibited no

measurable antibacterial action.

Walton J.N., Gardener F.M., Agar J.R. in 1986 examined the

parameters and expected that advances in the state of fixed

prosthodontic materials and methods would mean changes in cause of

failures and length of service of fixed restorations.

1) The mean length of service of all fixed restorations observed was

8.3 years.

2) Caries was the most common cause of failure, affecting 22.0%f

the units and leading to the necessity for placement of 24.3% of all

units observed.

3) Mechanical problems accounted for 69.5% of the failed units as

opposed to 28.5% for oral disease.

4) Resin veneer metal crowns provided the longest service of all

crowns types observed (13.9 years) and failed most frequently

because of worn or lost veneers.

5) The resin veneer metal crown also provided the longest service

as a retainer, with a mean length of service of 14.7 years.

6

6) No apparent relationship was found between the span of

prosthesis and its length of service.10

Scherer Warren, Lippman Nita, Kaim James in 1987 compared the

antibacterial properties of 14 different restorative materials, nine of

which were glass-ionomer cements. The materials were mixed

according to manufacturer’s specifications and exposed to four types of

bacteria (Streptococcus mutans, Streptococcus salivarius,

Actinomyces viscosus and Lactobacillus salivarious) commonly found

in caries and plaque. Zones of bacterial inhibition were measured for all

materials in millimeters. They concluded that:

1) Glass- ionomer cement materials used as liners/based and

restorative materials produced zones of inhibnition with the four

bacteria used in this study.

2) The glass- ionomer cement liner/base materials which contain

zinc oxide produced zones of inhibition larger than did those not

containing zinc oxide.

3) The composite material, FluorEver, is capable of producing

zones of microbial inhibition.

4) Dispersalloy amalgam produced zones of inhibition with all

bacteria used in this study.11

Meryon S.D., Johnson S.G. in 1989 assessed the antibacterial

properties of dental restorative materials with ratios of test

material/culture medium volume aiming to simulate conditions around a

restoration. Antibacterial activity is determined by the reduction in

7

optical density of the test culture relative to controls. The method was

used for assessment of the antibacterial activity of five dental materials

of different composition against five oral bacteria. Release of zinc and

fluoride from these materials was also measured and correlated with

antibacterial activity. There was a general trend toward greater

antibacterial activity with increased zinc release, while fluoride release

had a significant effect on only one organism. While all the materials,

when freshly mixed, were strongly toxic to three out of the five bacteria

studied, much of this activity was lost after the material had set.12

Barkhordar R.A., Kempler D., Pelzner R.R.B., Stark M.M in 1989

evaluated the antibacterial activity of six glass-ionomer cements on S.

sanguis and S.mutans. These cements were Glassic, Shofu lining

cement, GC lining cement, Ever Bond, Gingiva Seal and Ketac Bond,

the empty walls swerved as control. They concluded that:

1) GC lining and Ever Bond had significantly a greater overall

inhibition of microbial growth than did other tested liners.

2) Glassic and Ketac Bond had significantly more inhibitory effect

on S. sanguis than on S. Mutans.13

Forss H., Jokinen J., Spets- Happonen, et al in 1991 compared the

levels of fluoride and mutans streptococci in plaque grown on glass-

ionomer and composite restorations in vivo. From tunnels left under the

brackets bonded either with glass-ionomer or composite, 14-day-old

plaque samples were collected 14,28 and 42 days after bonding. For

glass-ionomer the mean counts of mutans streptococci in plaque were

0.5x103 x 6.7x103 and 8.8x103 CFU at the first, second and third

8

collection, whereas for composite restorations the corresponding

values were 32.1x103,14.6x103 and 120.6x103 CFU. For glass-ionomer

the mean concentrations of fluoride were 19,985, 5788 and 5019 ppm

at first, second and third collections of 14-day-old plaque samples,

respectively, whereas for composite restorations the mean

concentrations of fluoride were about 200 ppm throughout the study.

The results show that the fluoride level in plaque growing on glass

ionomer is much higher than that on composite restorations which

seems to affect the level of mutans streptococci in dental plaque.14

Palenik C.J., Behnen M.J., Setcos J.C, Miller C.H. in 1992

measured the in vitro inhibition of growth and adherence of five oral

bacteria by glass-ionomer materials. Disks were prepared from two

cavity liners and four restorative class materials, by use of Teflon

plates with circular walls, five mm wide and two mm deep. The

bacterial species included: A. viscosus, S. mitis, S. mutans, L.casei &

S. sanguis. An ion exchange electrode was used measure fluoride

release over a 7 day period for all six glass ionomers. The 2 cavity

liners and two of the restorative materials produced the largest growth

inhibition zones by direct contact. No growth inhibition occurred when

the specimens were allowed to come into contact with the agars prior

to inoculation. All four restorative materials reduced bacterial

accumulations on enamel surfaces by over 80%. Elevations in short-

term fluoride release levels were positively corr4elated with growth

inhibition.

9

Seppa L., Torppa-Saarinen E., Luoma H. in 1992 studied the effects

of different glass ionomers on the metabolism of Streptococcus

mutans, test slabs of freshly mixed conventional glass-ionomer, silver

glass-ionomer, composite and 2-week old glass-ionomer were fitted

into the bottom of a test tube. A plaque-like layer of S. mutans strain

Ingbritt was centrifuged onto the test slabs, and the samples were

incubated for 20 hours in 1.7% (w/v) sucrosed solution.

Incubation with glass-ionomeer materials led to increase in the

cellular concentration of fluoride in bacteria, but intracellular fluoride did

not correlate with the fall in pH. The lowest pH was associated with the

lowest cellular magnesium content. Ketac-Silver released large

amounts of calcium in the fluid phase, and the cellular calcium content

was doubled in this group. The results show that freshly mixed glass-

ionomers affect acid production and electrolyte metabolism of S.

mutans in vitro. The effect of conventional glass-ionomer, however,

seems to disappear after a few weeks. The effects of calcium and

silver released by cermet glass ionomer deserve further study.16

Eli., Cooper Y., Ben-Amar A and Weiss E. In 1995 assessed the

antibacterial activity of the following three dental liners; Vitrebond,

Dycal and Life. The test was based on a modification of the agar

diffusion test in which samples were placed on agar plates previously

inoculated with Streptococcus mutans and were removed after

predetermined time periods. The materials effect on bacterial growth

was evaluated.

10

Results showed that Vitrebnond had a strong antibacterial effect

that was evident after 1 min of direct contact with the inoculated

bacteria. It was significantly more effective that Dycal or Life. 17

Chong B.S., PittFord T.R., Kariyawasam S.P. in 1997 compared

the short-term tissue responses to two potential root-end filling

materials, light-cured glass ionomer cement and a reinforced zinc oxide

eugenol cement with that to amalgam.

In the 24 premolar teeth of beagle dogs, a collection of endodontic

pathogenic bacteria was first inoculated into the root canals to induce

periradicular lesions. On each root, an apicectomy was performed and

root-end cavities prepared to receive fillings of each material. The teeth

and surrounding were removed after 2 weeks (23 roots) and I week (24

roots); they were then prepared for histological examination.

Apart from amalgam in which healing was marked by the presence of

a localized focus of inflammation adjacent to the root canal filling,

response to Vitrebond and Kalzinol. Both Vitrebond and Kalzinol have

potential as root-end filling materials, as the tissue response was

considerably more favorable than that of amalgam even in the short-

term.18

Van Dijken J.W.V., Kalfas S., et al in 1997 compared the fluoride

concentrations in plaque on 1-year old resin-modified GIC, compomer

and resin composite restorations intra-individually and related to the

occurrence of caries- associated bacteria. Plaque from class III

restorations of the three restorative materials and from a proximal

enamel surface in 18 individuals was analyzed. Low fluoride levels

11

were detected in all the samples, while the resin-modified GIC samples

showed significantly higher amounts. The distribution of oral

streptococci surfaces and did not correlate to the fluoride levels in the

samples. A good correlation was found between the counts of mutans

streptococci in salvia and their proportions in the plaque.

The results indicate that the fluoride concentrations released in vivo

from 1-year-old restoratives are not high enough to affect the plaque

levels of the caries- associated bacteria mutans streptococci and

lactobacilli.

Hori R., Kohno S. and Hoshino E. in 1997 observed the antibacterial

potential of polycarboxylate temporary cement containing a mixture of

metronidazole, ciprofloxacin and cefaclor on carious lesions of

prepared abutments that were designed to leave caries on the

abutments.

Antibacterial efficacy was estimated in vitro and in vivo by measuring

bacterial recovery from the lesions. They concluded:

1) Bacteria counts ranged from 104 x 107 both in vitro and in vivo in

time–zero samples, just before application of the antibacterial

cement.

2) When the lesions were covered by the antibacterial temporary

cement from 1 to 4 days, no bacteria were recovered in most

cases. This finding indicated that the carious lesions were

sterilized by the antibacterial temporary cement. Although a

small number of bacteria were recovered from some samples,

all the bacteria recovered were sensitive to the drug mixture.

3) From the lesions covered by the temporary cement without the

mixture of drugs, more than 103 CFU/mg were recovered,

12

4) indicating that the temporary cement that contained tannin was

not a potent disinfectant.

5) In experiments in vivo, no bacteria were recovered from the

carious lesions of prepared abutments that were covered by

temporary restorations with the antibacterial temporary cement

within 2 weeks.

6) One in vivo case suggested that care should be taken for

marginal leakage and that temporary restoration should not be

left for a long time, even if antibacterial cement is used.20

Herrera M., Castillo A. and et al in 1999 studied the antibacterial

activity of the Glass-Ionomer restorative cements Ketac-Fil, Ketac-

Silver, Fuji II LC and Vitremer in vitro, in conjunction with a total of 32

strains of five bacterial general that may be associated with dental

caries: Streptococcus spp, Lactobacillus spp, Actinomyces spp,

Porphyromnas spp and Clostridium spp. Agar plate diffusion was the

method used for the bacterial cultures, which included a chlorhexidine

control.

All the four glass-ionomer cements were found to inhibit bacterial

growth, though with noteworthy differences in their spheres of action.

Vitremer was the cement determined to have the greatest antibacterial

effects, whereas Ketac-Silver presented the least inhibitory action.

In light of reported results, the use of glass-ionomer cements may be

indicated in the restorative treatment of root surface caries. These

could even prove beneficial for patients with periodontal disease

suffering from root caries.21

Banerjee A., Watson T.F. and Kidd E.A.M. in 2000 reviewed and

discussed some of the techniques available to excavate demineralised

dentine clinically. These methods can be classified as mechanical and

13

non-mechanical, rotary and non-rotary and include: dental hand

pieces/burs, manual excavators, air-abrasion, air-polishing,

ultrasonication, sono-abrasion, chemo-mechanical methods, laser and

enzymes.

All the techniques removed carious denture with differing levels of

efficiency but more importantly it was still unknown if these techniques

would discriminate between the soft, outer, necrotic, highly infected

zone that needs to be excavated and the inner, reversibly damaged,

less infected zone which could be retained. If this discrimination did not

take place, this could lead to over preparation of cavities with little

control over the quality and quantity of tissue removed by individual

operators. There is, therefore, an important need to assess the effects

of these techniques for their efficiency and extent of removal of carious

dentine.22

Herrera M., Castillo A. and et al in 2000 studied the antibacterial

action of different dental resin adhesive materials (Glumma 2000,

Syntac, Prisma Universal Bond 3, Scotchbond Multi-Purpose and

Prima & Bond 2.0) glass- ionomer cements (Ketac-Cem, Ketac-Bond,

Ketac-Silver, Ketac-Fil) resin-modified galss-ionomer cements (Fuji II

LC, Vitremer and Vitrebond) and a Compomer (Dyract). The agar plate

diffusion method was used for the microbial cultures and a

chlorhexidine control.

The growth of the caries-producing microorganisms was effectively

inhibited by the Vitremer and ViteBond cements, and to a lesser extent

by the Scotchbond Multi-Purpose adhesive system.23

Karanika- Kouma A., Dionysopoulos P. and et al in 2001 examined

the antibacterial activities of the bonding systems-Syntec, EBS and

Scotchbond I, the polyacid-modified composite resins-Hytac and

14

Compoglass, and the composite resins-Tetric, Z100 and Scalp-it. They

were evaluated using the cariogenic bacteria Streptococcus mutans,

Lactobacillus salivarius, Streptococcus sorbinus and Actinomyces

viscosus in vitro with a modified cylinder drop plate agar diffusion

assay.

All adhesives of the dentin bonding systems and the polyacid-

modified composite resins exhibited various degree of antibacterial

activity against all of the test bacteria. On the contrary, composite resin

did not affect bacterial growth. The data suggest that the use of these

adhesives and polyacid-modified composite resins may reduce the

consequences of microleakage owing to their antibacterial properties.24

De C. Luz M.A.A,De Lima A.C.P. and et al in 2001 analyzed the long-

term clinical behavior of two dental materials applied as filling under

silver amalgam restorations: glass-ionomer cement (GIC) and

composite resin with adhesive system (CR). In this study, 117 posterior

teeth (29 premolars and 88 molars) were selected with carious lesions

which resulted in great loss of dentin and cusps with unsupported

enamel. After caries removal, cavities were prepared and totally filled

with GIC with. In a following visit, new cavities were prepared, leaving

the employed filling material as a base and support for the enamel,

which were then restored with silver amalgam. Restorations were

evaluated periodically after 6 months and up to 5 years. Both fracture

and pulpal involvements rates were low.

There was a significant association between kind of tooth and long-

term survival of the restorations; and between degree of unsupported

enamel and the same long-term survival. The results confirmed that the

technique in which GIC or CR are used as filling material under silver

amalgam restorations is clinically acceptable.

15

Naou H.J, Chandler N.P. in 2002 compared the methods and

techniques used for short and long term restoration during immediately

after endodontic treatment, and to make clinical recommendations.

Proper clinical assessments could only be obtained from well

designed studies that reflect the superiority of some of the available

materials more accurately in the actual clinical environment and in

more complex cavities.

Because of the nature of these materials, they should be used for as

short a period as possible during the course of endodontic treatment.26

Hauman C.H.J and Love R.M. in 2003 reviewed on the methodology

involved in biocompatibility testing following by a discussion on

biocompatibility of contemporary intracanal drugs and substances used

in endodontics. Research showed that the intracanal drugs and

substances can have deleterious effects on vital tissues. Although

these substances are meant to contact non vital dentine during use,

they often come into contact with the peripical tissue. It is thus

important to consider biocompatibility when choosing and endodontic

irrigant or intracanal medicament.27

Lewinstein I, Further N. and et al in 2003 investigated the:

1) retention and microleakage of provisional crowns cemented with

temporary cements to which to which stannous fluoride

(SnF2)was added

2) solubility of these cements.

Provisional crowns were constructed of acrylic resin with shoulder

preparations for 12molars. The crowns were luted with Tempbond,

Tempbond NE and Freegenol temporary cements, and also with SnF2

added to these cements. Specimens were thermocycled 100 times,

stored for 6days, and immersed in 0.5% basic fuschin. 7days after

16

cementation, crown removal (retention) tests were concluded. Marginal

leakage and solubility in water of cements with and without SnF2was

assessed.

The results confirmed the Freegenol to be more retentive than the

other cements. The incorporation of SnF2significantly increased the

retention capacity of Freegenol and Tempbond NE but had no effect on

Tempbond. Tempbond showed significantly higher dye penetration

than the Freegenol. The addition of SnF2 did not alter the dye

penetration of the cements. There were no significant differences in the

solubility of the cements. However, the incorporation of SnF2increased

the solubility of Freegenol and Tempbond NE P<0.001) and Tempbond

(P<0.01).28

Larger A, Thornqvist E and Ericson D in 2003 analyzed the amount

of viable bacteria after excavation using conventional rose-bur on the

chemo-mechanical Carsilov method, a total of 22 lesions in this open,

control and randomized study. Two samples per lesion were taken

under aseptic conditions using a rose-bur, on superficially in the

carious lesion and one after complete excavation. The samples were

incubated on blood agar, Rogosa agar and mitis salivarius agar.

For blood agar (aerobic) both methods resulted in a significant

decrease in CFU, for blood agar (anaerobic) and MS agar only the

Carisolv method resulted in a significant decrease in CFU and for SL

agar neither method resulted in a significant decrease in CFU.

Comparing the excavation methods, there were no significant

differences, except in the case of blood agar (aerobic), which showed

that Carisolv excavation was more effective in reducing CFU.29

Eick S, Glockmann E and et al in 2004 evaluated the adhesion of

Streptococcus mutans ATCC 25175 to filling materials (Ariston, Tetric,

17

Dyract, Compoglass, Vitremer, Aqua Ionofil, Ketac Fil, amalgam,

Galloy and ceramics as controls). Streptococcus mutans was added to

saliva- coated test specimens, and a nutrient broth permanently

supplied over a time period of 48hrs and then the weight of plaque, the

number and viability of the bacteria adhering to the materials were

determined.

The weights of artificial plaque on all filling materials tested were

higher than those on ceramics, the highest values were measured on

the glass-ionomers. The amount of plaque correlates with the surface

roughness, whereas there was no correlation of the surface roughness

with the number of colony forming units (CFU) of S. mutans. The

plaque of Ketac Fil contained a high number of viable bacteria. The

fluorides of glass-ionomers do not efficiently prevent the attachment

and the viability of S.mutans.30

Ohashi S, Saku S. and Yamamoto K. in 2004 attempted to determine

whether it exerts an antibacterial effect on human saliva bacteria, and

to determine whether it can be used in dental materials. CFUs in 1ml

stimulated human saliva were examined using blood agar and mitis

salivarius agar after immersion, with or without YDA filler. The

antibacterial effect was compared with that of Ketac-Silver.

Human saliva bacteria and mutans streptococci showed reduced

viability following exposure to YDA filler after 12hrs. Two tested strains

showed reduced viability following exposure to dental materials

containing YDA filler. Thus YDA filler may help in the development of

antibacterial dental materials, such as composite resin, glass-inomer or

temporary cement.31

A1-Hebshi N.N, Nielsen O. and Skaug N. in 2005 studied the effect

of crude khat extract on streptococcus mutans growth and sucrose-

18

dependent colonization, and on its glucosyltransferase (GTF) activity

and production. Lyophilised crude aqueous khat extracts were applied

to the different assays at concentrations of 0-1% (w/v). Sucrose-

dependent colonization was assessed as the ability of Streptococcus

mutans UA159 to form adherent biofilm in glass culture tubes. Colony

forming units (CFUs) in the planktonic phase served as a measure of

bacterial growth, while CFUs in the biofilm phase were used to quantify

viability in the biofilms. GTFs production was assayed by comparing

intgensities of GTF bands in Western blots of extract from control and

khat-containing cultures.

The khat extracts effectively inhibited biofilm formation. The extract

also inhibited synthesis of both glucan types, particularly insoluble

glucans, with significant differences among cultivars. However, khat

increased bacterial growth and at sub-MCIC also viability within

biofilms; there were no inter-cultivar differences. It is shown that

Contain water-soluble constituents that inhibit some cariogenic

properties of S. mutans in vitro.32

Vermeersch G, Leloupb G, Delmee M. and Vrfeven J. in 2005

evaluated the antibacterial activity of six products (one conventional

glass-ionomer cement (GIC), two light-activated glass-ionomers, two

polyacid-modified resin composites and one resin composite) on

Streptococcus mutans. The relationship between product acidity and

antibacterial activity was evaluated.

All the GICs demonstrated antibacterial properties in contrast to the

polyacid-modified resin composites and resin composite which did not

show any antibacterial effects. Vitrebond GIC exhibited higher

antibacterial action, probably because of a cytotoxic photo-initiator

19

diphenyliodoniumchloride. A direct relationship between material

acidity and growth inhibition of S. mutans was observed.33

Lewinstein I, Matalon S and et al in 2005 evaluated the antibacterial

properties of 3 dental cements using the direct-contact test and agar

diffusion test. For the direct contract test, wells of microtitre plate were

coated with the tested cements (Harvard cement, Duralon, and Ketac

cement) while Streptococcus mutans suspension was placed directly

on the cements. Bacterial growth was evaluated by a temperature-

controlled microplate spectrophotometer. For the agar diffusion test,

triplicate specimens of freshly mixed cements were poured into uniform

wells punched in the agar plates inoculated with Streptococcus

mutans.

Compared with the control group, Duralon and Harvard cement

demonstrated antibacterial properties even after 3months will the

direct-contract test, while Ketac-Cem exhibited no antibacterial

properties. In the agar diffusion test, no antibacterial property was

observed for any of the tested cements. The different powder/liquid

ratios had a negligible effect on the antibacterial properties of the

tested cedments.34

Takahashi Y, Imazato S. and et in 2006 evaluated the antibacterial,

physical, and bonding properties of glass-ionomer cements (GIC)

containing chlorhexidine (CHX), and to determine optimal

concentrations for incorporation of agents to obtain antibacterial GICs

for use with the ART approach.

All experimental GICs exhibited inhibition of Streptococcus mutans,

Lactobacillus casei and Actinomyces naeslundii, but sizes of the

inhibition zones and concentrations of CHX released were not

dependent upon CHX content. Incorporation of CHX diacetate at 2% or

20

greater, significantly decreased compressive strength, and bond

strength to dentin was adversely affected by addition of CHX diacetate

at 2% or more, although setting time was extended a little by addition

of any concentrations of CHX.35

The present results demonstrate that incorporation of 1% CHX

diacetate is optimal to give appropriate physical and bonding

properties.35

Hengtrakool C., Pearson G.J. and Wilson M. in 2006 investigated

the interaction of Streptococcus sanguis with two glass- ionomer

formulations (GIC: A containing fluoride and GIC: B without fluoride)

with particular reference to bacterial growth and changes in hardness

of the cement with respect to time. Hydroxyapatite discs (HA) were

used as controls.

1) The viable counts of S. sanguis per mm2 on GIC: A were significantly

less than those on HA and GIC:B during the first 5days.

2) The viable counts bacteria on the surface of GIC: B were lower during

the initial 5 days when compared to HA.

3) Exposure of GIC: A and GIC: B to different medium produced softening

to the surface of the cement.

4) It is apartment that the effects of the biofilms are significantly greater

than storage in water but similar to storage in lactic acid pH5.36

21

MATERIAL AND METHODS

The purpose of this study was to evaluate the antibacterial properties of

3 dental cements using the Direct-Contract test and Agar Diffusion

Test.

MATERIALS

For excavation of caries:

1) Spoon excavator ( Fig 1)

2) Phosphate buffered saline (Fig 2)

3) Distilled water

4) Rubber dam (Fig 3)

For direct contact test:

1) The test cements ( Fig 4)

2) Autoclaved BHI Broth with added Bacitacin (Fig 5)

3) Microtitre plate (96-well, flat bottom Nunclon) (Fig 6)

4) 10-µL micro-pipette, small (Fig 6)

5) 235-µL micro- pipette, large (Fig 6)

For agar diffusion test :

1) The test cements (Fig 4)

2) Autoclaved BHI Agar with added Bacitacin (Fig 5)

ARMENTARIUM

1) Electronic weighing machine (Fig 7)2) Clean, sterile glass slab (Fig 4)3) Dental spatula (Fig 4)4) Vortex mixer (Fig 8)5) Auto- mixer (Shaker) (Fig 9)6) Incubator at 370C (Fig 10)7) Temperature- controlled spectrophotometer (THERMOMAX) (Fig 11)

22

23

24

25

26

27

28

METHOD

The cements tested under this study were Harvard (Zinc-Phosphate

Cement), Poly-F (Polycarboxylate cement) and Ketac-Cem (Glass-

lonomer cement). All the cements were exposed to Streptococcus

mutans under similar conditions.

PROCEDURE

Isolation of Streptococcus mutans from caries:

All consecutive young patients making a group of 25 patients, in the

age group of 9-12 years (Streptococcus mutans considered to be the

primary etiological factor), appearing for regular dental examination

participated in this study. Inclusion criteria were vital teeth without

symptoms, either molars with primary caries that included

approximately half the dentine thickness as judged by bitewing

radiographs, or primary buccal caries lesions extending into the dentine

in the incisors, canines or molars. The consistency of the caries lesions

was medium hard and the colour yellow to light brown, No teeth with

pulpal involvement were included.

Rubber dam was applied and surface debris including the outermost

layer of carious dentine was removed using sterile hand instruments.

The cavity was thoroughly rinsed with sterile saline, then dried with a

cotton pellet. The caries were excavated with spoon excavator and the

sample was transferred to a sterile bottle containing phosphate

buffered saline (mixed in a proportion of 9.95gms in 990ml) and

transferred to the laboratory (Fig 1), where it was shaken for 30sec in a

29

vortex mixer to dislodge the dentin sample. 23,39 A clinical isolate of

mutans streptococci (ATCC 27351) that is naturally resistant to

bacitracin was grown aerobically from frozen-stock cultures in brain-

heart infusion(BHI) broth containing 8ug/ml bacitracin (Fig) for 48 hours

at 37 before applying it to the specimens according to the experimental

design.34

Preparation of samples of test cements:

The test cements (Harvard, Poly F and Ketac-Cem) were measured

with an electronic weighing machine in a prescribed proportion and

mixed in2 powder/liquid (P/L) ratios. P/L 1 ratio was in accordance with

the manufacturer’s instructions and P/L 2 was more diluted (P/L ratio

wt/wt 1 and 2).

Tested cements with 2 powder/liquid (P/L) ratios for each cement

BRAND CEMENT TYPE P:L RATIO (wt/wt)

Harvard Zinc Phosphate

cement

1) 1.8:1

2) 1.6:1

Ketac-Cem Glass- lonomer

cement

1) 3.8:1

2) 3.5:1

Poly F Polycarboxylate

cement

1) 2.0:1

2) 1.8:1

These cements represent 3 types of luting agents, which are

commonly used in fixed prosthodontics. The more diluted P/L ratio

30

simulates situations in which the clinician does not follow the

manufacturers’ instructions exactly and prepares a thin mix of cement.

The antibacterial properties of the materials were examined within 60

minutes after mixing (designated hereafter as “fresh preparations”).

Similar experiments were performed in which the test materials were

allowed to age in a phosphate buffered saline (PBS) for 24 hours,

1week, 1mont, and 3months before assaying.

TESTING OF SPECIMENS:-

DIRECT- CONTACT TEST (DCT)

The direct-contact test originally described by Weiss et al is based on

the turbidometeric determination of bacterial growth in 96-well

microtitre plates. The kinetics of the outgrowth in each well was

recorded continuously at 650nm at 370C every 30minutes, using a

temperature- controlled spectrophotometer (THERMOmax). Auto

mixing prior to each reading ensured a homogenous bacterial cell

suspension.

A microtitre plate (96-well, flat-bottom Nunclon) was held vertically

with the plate’s surface perpendicular to the floor, and the sidewall of 4

wells was coated evenly with a measured amount of the test material

(designated as Group A). A thin coat was achieved by using a small

flat-ended dental instrument, such as a dental spatula. The material

was allowed to set in compliance with the manufacturer’s

recommendation. Special care was taken to avoid the materials’ flow to

the bottom of the well, which could interfere with the light path through

the microtitre well and result in false readings.

31

A 10-µL bacterial suspension (approximately 106 bacteria) was

placed on the test material, while the plate remained in a vertical

position. Wells were inserted for evaporation of the suspensions’ liquid,

ensuring direct contact between bacteria and the test materials. This

usually occurred within 1 hour at 370 C. BHI broth supplemented with

bacitracin (235µL) was added to each of the Group A wells and gently

mixed for 2 minutes; 15uL were then transferred from Group A wells,

respectively, to an adjacent set of 4 wells containing 205µL fresh

medium (designated as Group B) (Fig 13). This resulted in 2 sets of 4

wells for each test material containing an equal volume of liquid

medium (220µL), so that bacterial outgrowth could be monitored and

compared, both in the presence and absence of the test material. Two

sets of 4 uncoated wells in the same microtiter plate served as the

positive control. Identical bacterial inoculum was placed on the sidewall

of the uncoated wells and processed as in the experimental Group A

and B wells. The growth curves from both experimental groups were

compared with the control outgrowth A and B, respectively. The

negative control consisted of a set of 4 wells coated with the test

materials, as in experimental Group A, containing an equal volume

uninoculated fresh medium. (Fig 14).

The kinetics of the outgrowth in each well were monitored at 650 Nm

at 370C and recorded every 30minutes, using the same temperature-

controlled microplate spectrophotometer (THERMOmax). Auto-mixing

prior to each reading ensured a homogenous bacterial cell suspension.

Data were recorded in optical density (OD) units. The values of the

negative control wells were considered as the baseline, and were then

subtracted from the respective experimental sets and plotted as growth

32

curves. The first set of data was recorded approximately 1 hour after

incubation. Additional experiments were performed on set test

materials that were allowed to age in the presence of 280µL of PBS for

24 hours, 1 week, 1montgh and 3months, respectively, before being

assayed by the DCT. During the aging period, PBS was replaced twice

a week. The data were subjected to 1-way ANOVA and the Post-Hoc

multiple comparisons test. Parallel to the experimental design,

calibration experiments were performed to establish bacterial

outgrowth under experimental conditions in a quantitative and

reproducible manner. In a typical experiment, 10µL of bacterial

suspension (approximately 106 cells) was placed on each sidewall of 4

wells in a 96-well microtiter plate, as in experimental design. Fresh

medium, 275µL, was added and the plate gently mixed for 2 minutes.

From each well, 55µL were transferred to an adjacent set of wells that

each contained 220µL fresh medium. The dilution transfer was

repeated 6 consecutive times. The microtiter plate was incubated in the

spectrophotometer at 370C, and the bacterial growth was followed for

16 hours. To overcome contamination throughout the experiment,

25mg/ml of bacitracin was included in the BHI medium and an

antibiotic resistant strain was used.

33

34

AGAR DIFFUSION TEST (ADT)

For the ADT, 200µL of bacterial suspension (approximately 5x107

cells) were spread on BHI agar plates (Fig 15). Freshly mixed

specimens (in triplicate) of each test material were placed in holes

5mm in diameter that were punched in the agar using the blunt end of

a sterile Pasteur pipette23 (Fig 16). After incubation at 370C for 24hours,

the agar plates were examined for bacterial lawn was measured (mm)

in 2 perpendicular locations for each specimen. The first set of data

was recorded after 24hours. Additional experiments were performed on

aged specimens for which test materials had been placed in bottles

containing PBS and bacitracin for 1 week, 1month, and 3 months

before assessment by the ADT. During the aging period, PBS was

replaced twice a week. The data were subjected to 1-way ANOVA and

the Post-Hoc multiple comparisons test.34

35

36

OBSERVATION AND RESULTS

The caliberation experiments showed that bacterial outgrowth in

microtiter wells could be monitored in a quantitative and reproducible

manner.

The gradual decrease in viable bacteria, due to serial dilutions at

time zero, had virtually no effect on the bacterial growth rate or final

density of bacteria at the stationery phase in the system.

The result of DCT for freshly mixed and aged cements in different P:L

ratios are shown.

The abbreviations used in the results:

KC: Ketac Cem (GIC Cement)

KC1: I P: L ratio of Ketac Cem

KC2: II P: L ratio of Ketac Cem

H: Harvard (Zinc Phosphate Cement)

H1: I P: L ratio of Harvard

H2: II P: L ratio of Harvard

PF: Poly F (Polycarboxylate Cement)

PF1: I P: L ratio of Poly F

PF2: II P: L ratio of Poly F

37

38

39

40

41

42

43

44

45

46

47

48

49

50

51

DISCUSSION

Success of restoration is dependent on accuracy of each and every

step from preparation to final seating and even upon duration or

longevity of restoration. Generally the preferred margin of the

preparation is supragingival. Subgingival margins of cemented

restorations are major etiologic factor in periodontal disease,

particularly when they encroach on the epithelial attachment.

The junction between a cemented restoration and the tooth is always

a potential site for recurrent caries because of dissolution of the luting

agent and inherent roughness. The more accurately the restoration is

adapted to the tooth, the lesser the changes of failure. For acceptable

margin adaptation, castings should fit within 10um and a porcelain

margin with 50µm. A well designed preparation has a smooth and even

margin as compared to rough, irregular or “stepped” junctions which

increase the margin length and substantially reduces the adaptation

accuracy of the restoration.37

This is consolidated by Eick S. et al (2004) who evaluated the

adhesion of Streptococci mutans ATCC 25175 to filing materials

(Ariston, Tetric, Dyract, Compoglass, Vitremer, Aque Ionfil, Ketac fil,

Amalgam, Galloy and ceramics as control). They found that the

amount of plaque co-relates with surface roughness, whereas

there was no correlation of the surface roughness with the colony

forming units of Streptococcus mutans. The plaque on KETAQC-FIL

contained a higher number of viable bacteria. The fluoride content of GI

do not efficiently prevent the attachment and viability of Streptococcus

mutans.30

52

The cross-section configuration of the margin has been proposed to be

of various types:

1) Feather edge:

Advantage: Conservation of tooth structure

Disadvantage: Does not provide sufficient bulk

Indication: Not recommended;

2. Chisel edge:

Advantage: Conservation of tooth structure

Disadvantage: Location of margin difficult to control

Indication: on titled teeth.

3. Bevel:

Advantage: Removes unsupported enamel

Allows finishing of the metal

Disadvantage: Extends preparation into sulcus if used on apical

margins

Indication: Facial margin of maxillary partial-coverage restorations

and inlay/onlay margins

4. Chamfer:

Advantage: Distinct margin, adequate bulk and easier to control.

Disadvantage: Care needed to avoid unsupported lip of enamel

Indication: Cast metal restoration

Lingual margins of metal ceramic crown

5. Shoulder:

Advantage: Bulk of restorative material

Disadvantage: Less conservative of tooth structure

Indication: Facial margin of metal ceramic crown

Complete ceramic crowns

53

6. Sloped Shoulder:

Advantage: Bulk of restorative material, advantages of bevel

Disadvantage: Less conservative of tooth structure

Indication: Facial margin of metal ceramic crowns.

7. Shoulder with Bevel:

Advantage: Bulk of restorative material advantages of bevel

Disadvantage: Less conservative of tooth structure

Extends preparation apically

Indication: Facial margin of posterior metal-ceramic crown with

supragingival margins.37

Thus if not properly prepared, restoration margins can provide a

potential pathway to leakage of cariogenic micro-organisms present in

normal human flora. Failure of fixed partial dentures is most frequently

caused by caries. Therefore, physically superior cement materials play

an important role in caries prevention and longevity of cemented

restoration. Furthermore, it would be advantageous such materials

possessed antibacterial properties.34

The antibacterial activity of dental cements has been intensively

tested. The direct-contact test (DTC) was developed by Weiss et al to

evaluate antibacterial activity of non- soluble materials. This test is

based on measuring the effect of physical direct contract between test

bacteria and the tested material on the kinetics of bacterial outgrowth.

The DCT, was used to study the antibacterial properties of endodontic

sealers and pit and fissure sealants and the luting cements used to lute

fixed prosthesis to the prepared tooth. The purpose of the present

study was to assess and compare the incessant antibacterial activity of

3 luting cements mixed in 2 powder/liquid ratios using DCT and ADT.

54

Table I depicts the Bacterial growth rate using DCT as

demonstrated by the slope of the linear portion of the growth curve.

Each number is average (x10-2) ±SD (x10-3) of slope of bacterial growth

measured in 8 separate wells in same microtiter plate at different time

intervals.

Two different P:L ratios (designated as 1 and 2 ) for each cement i.e.

Ketac-Cem, Harvard and Poly F, have been depicted in immediate,

24hours,1 week, 1 month and 3 months duration of time.

The gradual decrease in viable bacteria, due to serial dilutions at

time zero (0), had virtually no effect on the bacterial growth rate or final

density of bacteria at the stationary phase in the system according to

Lewinstein I, Matalon et al34 which indicates that the different P: L

ratios had a negligible effect on the antibacterial properties of the

tested cements. This is the reason that the readings of different

dilutions for the same cement could be clubbed for the result purposes

without any deviation in the final results.

The results obtained above are in agreement with the studies of

Ritsuko Hori, Kohno Shoji and Hoshino Etsuro (1997) observed the

antibacterial potential of polycarboxylate temporary cement containing

in mixture of Metronidazole, Ciprofloxacin, and Cefaclor on carious

lesions of prepared abutments that were designed to leave caries on

the abutments.20

Herrara et al who compared the antibacterial activity of various

restorative study materials with cariogenic bacteria using the ADT. The

authors found a wide range of antibacterial activity of different cement

while on 2% of Ketac cement specimens showed positive antibacterial

properties. The antibacterial activity of cement could not be attributed

55

to fluoride contents because not all of the fluoride containing cements

and antibacterial activity. 21

To support above study, Van Dijken, J.W.V. et al (1997) who

compared the fluoride concentrations in plaque on 1 year resin

modified GIC, compomer and resin composite restoration intra

individually and related to the occurrence of caries-associated bacteria.

Low fluoride levels were detected in all the cements, while the resin-

modified GIC cements showed significantly higher amounts. The

results indicated that the fluoride concentration released in vivo from 1-

year old restoratives are not high enough to effect the plaque levels of

the caries associated bacteria, Mutans Streptococci and Lactobacilli.19

In the Table II, the samples (10) of the two P:L ratios of each test

cement specimen were added forming sample size of 20 for each

material (eg. KC1 +KC2=KC). It was illustrated that the mean value of

optical density was 0.0021674 for immediate group-, 0.0005776 for

24hours group, 0.00088937 for 1 week group, 0.0022723 for 1 month

group and 0.0011266 for the 3 months group.

As shown in Table III by applying the ONE WAY ANOVA test, it was

seen that there is statistically significant difference between the optical

densities of Ketac Cem, Harvard and Poly F for each given time group.

This is illustrated by the fact that the value of ‘p’ being <0.05 which

states that the result is significant.

Table IV analyzes the differences in the mean values of different test

cements from other test cements for each time group. The values are

found to be different with a wide range among themselves as is also

depicted in table III by the ‘p’ factor <0.05.

The results are confirmed by Loyola et al who examined the factors

involved in the antibacterial activity of various GICs on Streptococcus

56

mutans. The authors found that the KETAC-CEM cement exhibited low

antibacterial properties which could be attributed to low fluoride release

of the cement.

Another explanation could be the lack of zinc oxide powder in its

composition. It has been suggested that GICs containing zinc oxide

powder are more effective in microbial inhibit due to direct effect of zinc

oxide powder and cationic effect of zinc (Zn2+).

Also, Meryon S.D (1989) assessed the antibacterial properties of

dental restorative materials. Release of zinc and fluoride from these

materials was also measured and correlated with the antibacterial

activity. They found that there was a general trend towards greater

antibacterial activity with zinc release, while fluoride release had a

significant effect on only one organism.12

However, certain authors such as Barkhordar R.A, Kepler D.,

Pezner R.R.B and Stark M.M (1989) disagree and concluded from

their studies that all glass-ionomer cements showed significantly

greater overall inhibition of microbial growth and were more toxic

towards Streptococcus sanguis and Streptococcus mutans.13 In other

study, Palenik C.J., Behnen M.J, et al (1992) measured the inhibition

of growth and adherence of five oral bacteria by glass- ionomer

materials. The two cavity liners and two of the restorative produced the

largest growth inhibition areas by Direct Contact. No growth inhibition

occurred when the specimens were allowed to come in contact with the

agar prior to inoculation. All four restorative materials reduced bacterial

accumulation on enamel surfaces by over 80%. Elevations in short-

term release levels were positively correlated with growth inhibition.15

And Forss H, Jokinsen J, et al (19991) compared the levels of

fluoride and mutans Streptococci in plaque grown on glass-ionomer

57

and composite restoration in vivo and concluded from his study that the

fluoride level in the plaque growing on Glass-io9nomer is much higher

than that on composite restorations which seems to affect the level of

mutans Streptococci in dental plaque.14

Table V depicts the Agar Diffusion Test in which the diameter of the

halo formed in the bacterial lawn was measure(mm) in 2 perpendicular

locations for each specimen for each given time intervals.

Two different P:L ratios (designated as 1 and 2) for each cement i.e.

Ketac-Cem, Harvard and Poly F, have been depicted in immediate, 24

hours, 1week, 1 month and 3 months duration of time.

Dahl studied the short acting antibacterial effect of polycarboxylate

cement (PCC) and zinc phosphate cement (ZPC) and found that in the

agar diffusion test (ADT), ZPC exhibited the strongest antibacterial

properties, while PCC appeared to be the strongest against

Streptococcus mutans in vivo. In another study, the antibacterial

activity of several glass ionomer cements (GICs), dentin bonding

systems, and luting agents were investigated using the ADT and

growth-inhibition tests. Marked antibacterial activity was shown with the

GIC, while amalgam, composites, luting agents, and dentinal bonding

systems did not affect the bacterial growth. However, the antibacterial

activity of GIC ranged from nonactive to very effective (or high

activity).34

In the Table VI, the samples (6) of the two P:L ratios of each test

cement specimen were clubbed forming sample size of 12 for each

material (eg. KC1 +KC2=KC). It was illustrated that the mean value of

the diameter of the bacterial inhibition zone was 5.5536 for immediate

group, 5.4181 for 24 hrs group, 5.3164 for 1 week group, 5.1903 for 1

month group and 5.0775 for the 3 months group. As shown in Table VII

58

by applying the ONE WAY ANOVA test, it was seen that there is not

any statistically significant difference between the bacterial inhibition

zones of Ketac Cem, Harvard and Poly F for each given time group.

This is illustrated by the fact that the value of ‘p’ being>0.05 which

states that the result is not significant.

The lack of evidence for antibacterial activity for all the test cements

using ADT in the present study may be attributed to the characteristics

of this test. ADT is a popular test for antibacterial properties of

restorative material. However, it has several disadvantages. One

primary disadvantage is that it depends on the solubility and diffusion

properties of both the test material and the media. It is possible that the

antibacterial activity of the tested cements, which were not amply

soluble, could not be detected by ADT Schwartzman et al found low

antibacterial activity for Polycarboxylate and Zinc Phosphate, whereas

Dahl found high activity for these cements, For restorative materials

and cements, which are expected to have low solubility and are less

likely to diffuse, the ADT might not detect antibacterial properties.

Furthermore, the results demonstrated that DCT, which relies on direct

and close contact between the test micro-organism and the test

material, independent of the diffusion properties, may be more suitable

for testing restorative materials and cements than ADT. With respect to

caries prevention at the margins of cemented restorations, the DCT

simulates the clinical situation in which the cariogenic micro-organisms

are in contact with the cement better than the ADT. However, it must

be noted that the DCT experimental design employed is still far from

the clinical situation in terms of oral environment, margin location, and

surface area of cement exposed at restoration margins,. Furthermore,

the antibacterial effect in the present study was observed with respect

59

to Streptococcus mutans, which is the most common caries-related

micro-organism. Since restoration margins may be subgingivally

located, it would be of interest to investigate the antibacterial effect of

these cements on the subgingival flora. The prolonged antibacterial

activity found in this study and the initial low pH of Polycarboxylate

cement and Zinc Phosphate cement could indicate that these cements

have the potential for bacterial eradication in prepared abutments and

carious dentin.34

Table VIII analyzes the differences in the mean values of different test

cements from other test cements for each time group. The Values are

not found to be significantly different among themselves as is also

depicted in table VII by the ‘p’ factor >0.05.

The results obtained are in accordance with Vermeersch G., Leloup

G, et al (2005) who used Agar Diffusion test to evaluate the

antibacterial activity of six products (one conventional glass-ionomer

cement(GIC), two light activated glass-ionomer cements, two poly acid-

modified resin composites and one resin composite) on Streptococci

mutans. All The GICs demonstrated antibacterial properties in contrast

to the polyacid-modified resin composite and resin composites which

did not show any antibacterial effects.33

However, Scherer W., Lippman N, Kaim J. disagreed by comparing

antibacterial properties of 14 different restorative materials, nine of

which were GICs. The materials were mixed according to

manufacturer’s specifications and exposed to four types of bacteria

commonly found in caries and plaque. Zones of bacterial inhibition

were measured for all materials in millimeters and they found that GIC

materials containing zinc oxide and amalgam produced measurable

zone of inhinbition.

60

The 3-D bar graph justifies the results obtained in the tables. As is

clearly seen, the optical density of Poly F is remains zero meaning that

it has the highest antibacterial property throughout the given time

durations. The Harvard has zero optical density for immediate, 24hrs

and 1 week duration depicting to be possessing antibacterial property

for that duration after which the antibacterial property declines. The

Ketac-Cem did not show any antibacterial activity for any of the time

groups.

The results can be depicted also by the line graphs as in line

graphs as in line graphs I to VI the bacterial outgrowth in microtiter

wells could be monitored in a quantitative and reproducible manner.

Each point on growth curve is an average of the optical densities

measured in 4 wells at a given same time. The results of DCT for

freshly mixed and aged cements in different P:L ratios are shown. The

immediate curves demonstrate antibacterial activity of Poly F and

Harvard cement with (graph I immediate A) and without (graph I

immediate B) the presence of the cement specimens. Bacterial growth

was significantly reduced when compared with the control (P=0.002).

Ketac Cem did not show antibacterial activity for any of the test groups.

Prominent antibacterial activity was evident for the aged Poly F and

Harvard cements along the test periods (graph II-V). However,

Harvard cement became less antibacterial than the Poly F after 3

months. The different P: L ratios had a negligible effect on the

antibacterial properties of the tested cements. The ADT showed no

antibacterial activity of the te3sted cements for any of the test groups.

In the present study, according to Lewinstein I, Matalon S et al

POLY F and HARVARD cements maintained antibacterial properties

even after three months of aging in Phospated Buffered Saline

61

(PBS).The prolonged antibacterial activity was measurable only in

Direct Contact between bacteria and the cements, whereas in agar

plates, no diffusion of antibacterial components from the cements was

observed. This may confirm that the cement specimens are stable with

low solubility in an aqueous environment and do not excrete sufficient

antibacterial substances to the surrounding media to effect the

bacterial growth.

The limitations of this study include the two P:L ratios of the test

cements may not be so accurately measured thus leading to not exact

proportions as mentioned in the study. The temperature of the

operatory may be an environmental factor affecting the growth of the

bacteria thus affecting some of the results of cements. Not proper

manipulative or technical procedures may inculate inaccuracies in

study affecting the result significantly.

Many studies have tested the antibacterial activity of dental cements

and demonstrated that GIC is the most active (antibacterial cement),

followed by Zinc Phosphate Cement and PolyCarboxylate Cement. On

the contrary, in the present study using the DCT, POLY F was the most

active followed by the HARVARD cement whereas KETAC CEM

showed no antibacterial activity even after 24 hours. In addition to that

the dilution of cements did not seem to affect the antibacterial

properties of the cements.34

62

SUMMARY AND CONCLUSION

Since failure of fixed partial dentures is most frequently caused by

caries, it would be advantageous if cement possessed antibacterial

properties.

For the direct-contact test, wells (n=4) of microtiter plates were coated

with the tested cements (Harvard cement, Poly F, and Ketac-Cem)

while Streptococcus mutans suspension was placed directly on the

cements. Bacterial growth was evaluated by a temperature-controlled

microplate spectrophotometer. Eight wells of bacterial without the

tested cements served as the positive control. Six wells of the tested

cement without bacteria served as the negative control.

For the agar diffusion test, triplicate specimens of freshly mixed

cements were poured into uniform wells (5 mm in diameter) punched in

the agar plates inoculated with Streptococcus mutans. After incubation

at 370C for 24 hours, the agar plates were examined for bacterial

growth and the diameter of the halo formed in the bacterial lawn was

measured.

In both tests, each cement was mixed in 2 different powder/ liquid

ratios. For the direct-contact test, data were initially recorded after 1

hour of incubation. Additional experiments were performed on

specimens that were aged for 24 hours, 1 week, 1 month, and 3

months before assessment by either direct-contact test or agar

diffusion test. The data were subjected 1-way ANOVA with the POST

HOC test (α=.05).

63

CONCLUSIONS

Compared with the control group and within the limitations of this study,

the following conclusions were drawn:

1) No correlation was found between the DCT and the ADT.

2) DCT was more effective in detecting antibacterial properties of

definitive dental cements than ADT.

3) Using DCT, aged Poly F and Harvard cements demonstrated

antibacterial properties even after 3 months.

4) Antibacterial properties of the tested cements were not detected by

ADT.

5) The different P/L ratios had a negligible effect on the antibacterial

properties of the tested cements.

6) Ketac-Cem exhibited no antibacterial activity for any of the test groups.

LIST OF REFERENCES

1) Herrrera M, Castillo A, Baca P, Carrion: Antibacterial activity of Glass-

Ionomer restorative cements exposed to cavity- producing

microorganisms. Operative Dentistry 1999; 24:286-291.

2) Eli I, Cooper Y, Ben-Amar A, Weiss E: Antibacterial activity of three

dental liners. J Of Prosthodontics 1995;4:178-182.

3) Palenik CJ, Behnen MJ, Setcos JC,Miller CH: Inhibition of microbial

adherence and growth by various glass-ionomers ikn vitro. Dent mater

1992;8: 16-20.

4) Vermeersch G. Leloup G, Delmee M. and Vreven J: Antibacterial

activity of glass-ionomer cements, compomers and resin composites:

relationship between acidity and material setting phase. J of Oral

Rehabilitation 2005;32:368-374.

5) Hori R, Kohno S, Hoshino E: Bactericidal eradication from carious

lesions of prepared abutments by an antibacterial temporary cement. J

Prosthet Dent 1997;77:348-352.

6) Lewinstein I, Matalon S, Slutzkey S, Weiss E: Antibacterial properties

of aged dental cements evaluated by direct-contact and agar diffusion

tests. J Prosthet Dent 2005;93 364-371.

7) Lewinstein I, Furher N, Gelfand K, Cardash H, Pilo R: RTetention,

marginal leakage, and cement solubility of provisional crowns

cemented with temporary cement containing stannous fluoride.

International J of Prosthodontics 2003; 16:189-193.

8) Watts TLP, Combe EC: Adhesion of periodontal dressings to enamel in

vitro. J of Clinical Periodontology 1980;7:62-66.

9) Schwartzman, Caputo A A, Schein B: Antimicrobial action of dental

cements. J Prosthet Dent 1980;43:309-312.

10) Walton JN, Gardener FM, Agar JR: A survey of crown and fixed partial

denture failures: length of service and reasons for replacement. J

Prosthet Dent 1986;56:416-421.

11) Scherer W, Lippman N, Kaim J: Antimicrobial properties of glass-

ionomer cements and other restorative materials. Operative Dentistry

1987;14:77-81.

12) Meryon SD, Johnson SG: The modified model cavity method for

assessing antibacterial properties of dental restorative materials. J

Dent Reds 1989;68:835-839.

13) Barkhordar RA Kempler D, Pelzner RRB, Stark MM: Antimicrobial

action of glass-ionomer lining cement on S. sanguis and S. mutans.

Dent Materials 1989;281-282.

14) Forss H, Jokinen J, Spets-Happonen, Seppa, L, Luoma H: Fluoride

and mutans streptococci in plaque grown on glass-ionomer and

composite. Caries Reds 1991; 25:454-458.

15) Palenik CJ, Behnen MJ, Setcos JC, Miller CH: Inhibition of microbial

adherence and growth by various glass-ionmers in vitro. Dent Material

1992;8:16-20.

16) Seppa L, Torppa-Saarinen E, Luoma H: Effect of different glass-

ionomers on the acid production and electrolyte metabolism of

Streptococcus mutans ingbritt. Caries Reds 1992;26:434-438.

17) Eli I, Cooper Y, Ben-Amar A and Weiss E: Antibacterial activity of three

dental liners. J of Prosthodontics 1995;4:178-182.

18) Chong BS PittFord TR, Kariyawasam SP: Short-term tissue response

to potential root-end filling materials in infected root canals.

International Endodontic J 1997;30:240-249.

19) Van Dijken JWV, Kalfas S, Litra V, Oliveby A: Fluoride and mutans

streptococci levels in plaque on aged restorations of resin modified

glass-ionomer cement, compoer and resin composite. Caries Res

1997;31:379-383.

20) Hori R, Kohno S, Hoshino E: Bactericidal eradication from carious

lesions of prepared abutments by an antibacterial temporary cement. J

Prosthet Dent 1997;77:348-352.

21) Herrrera M, Castillo A, Baca P, Carrion: Antibacterial activity of Glass-

Ionomer restorative cements exposed to cavity- producing

microorganisms. Operative Dentistry 1999;24:286-291.

22) Banerjee A, Watson TF, Kidd EAM: Dentine caries excavation: A

review of current clinical techniques. British Dental Journal

2000;188:476-482.

23) Herrera M, Castillo A, Bravo M, Liehnba J, Carrion P: Antibacterial

activity of resin adhesives, glass-ionomer and resin modified glass-

ionomer cements and a compomer in contact with dentin caries

samples. Operative Dentistry 2000;25:265-269.

24) Karanika-Kouma A, Dionysopoulos P, Kolokotronis: Antibacterial

properties of dentin bonding systems, polyacid-modified composite

resins and composite resins. J of Oral Rehabilitation 2001;28:157-160.

25) De C. Luz MAA, De Lima ACP, Netto NG: Long-term clinical evaluation

of fracture and pulp injury following glass-ionomer cement or composite

resin applied as a base filling in teeth restored with amalgam. J of Oral

Rehabilitation 2001;28:634-639,

26) Naoum HJ, Chandler NP: Temporization for endodontics. International

Endontic J 2002; 35:964-978.

27) Human CHJ and Love RM: Biocompatibility of dental materials used in

contemporary endodontic therapy: intracoronal drugs and substances.

International Endontic J 2003:36:75-85.

28) Lewinstein I, Furher N, Gelfand K, Cardash H, Pilo R: Retention,

marginal leakage, and cement solubility of provisional crowns

cemented with temporary cement containing stannous fluoride.

International J of Prosthodontics 2003;16: 189-193.

29) Lager A, Thornqvist E, Ericson D: Cultivable bacteria in dentine after

caries excavation using rose-bur or carisolv. Caries Res 2003;37: 206-

211.

30) Eick S, Glockmann E, Brandl B, Pfister W: Adherence of streptococcus

mutans to various restorative materials in a continuous flow system. J

of Oral Rehabilitation 2004; 31: 278-285.

31) Ohashi S, Saku Sk, Yamamoto K: Antibacterial activity of silver

inorganic agent YDA filler. J of Oral Rehabilitation 2004; 31: 2364-367.

32) Al-Hebshi NN, Nielsen O, Skaug N: In vitro effects of crude khat

extracts on thed growth, colonization and glucosyltransferases of

Streptococcus mutans. J of Oral Microbiology 2004; 244-250.

33) Vermeersch G, Leloup G, Delmee M. and Vreven J: Antibacterial

activity of glass-ionomer cements, compomers and resin composites:

relationship between acidity and material setting phase. J of Oral

Rehabilitation 2005; 32: 368-374.

34) Lewinstein I, Matalon S, Slutzkey S, Weiss E: Antibacterial properties

of aged dental cements efvaluated by direct-contact and agar diffusion

tests. J Prosthet Dent 2005;93; 364-371.

35) Takahashi Y, Imazato S, Frencken JE, Tay FR: Antibacterial effects

and physical properties of glass-ionomer cements containing

chlorhexidine for the ART approach. Dental Materials 2006; 22: 647-

652.

36) Hengtrakool C, Pearson GJ and Wilson M: Interaction between GIC

and S. sanguis biofilms: Antibacterial properties and changes of

surface hardness. J of Dentistry 2006;34: 588-597.

37) Rosenteil SF, Land MF, Fujimoto J: Contemporary fixed prosthodontics

(ed 4). St. Lousis, Mosby, 2007,209-257.