RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECTS FOR DISSERTATION
1 / NAME OF THE CANDIDATE AND ADDRESS / DR. GANGA. A2/12 5TH CROSS, MARUTHINAGAR
MADIWALA, BANGALORE-68
KARNATAKA
2 / NAME OF THE INSTITUTION / VOKKALIGARA SANGHA DENTAL COLLEGE AND HOSPITAL , BANGALORE
3 / COURSE OF STUDY AND SUBJECT / MASTER OF DENTAL SURGERY – PEDODONTICS AND PREVENTIVE DENTISTRY
4 / DATE OF ADMISSION TO COURSE / 30TH MAY 2010
5 / TITLE OF THE TOPIC / “EFFECT OF TWO DIFFERENT HYDRATING SOLUTIONS ON ANTIBACTERIAL PROPERTY OF WHITE MINERALTRIOXIDE AGREGGATE AGAINST ENTEROCOCUSS FAECALIS - AN INVITRO COMPARITIVE STUDY”
6 .BRIEF RESUME OF THE INTENDED WORK :
6.1 NEED FOR THE STUDY :
Mineral trioxide aggregate (MTA) was developed at the Loma Linda University, California, USA, as a root-end filling material for surgical endodontic treatment. Over the years, further research on the material has resulted in MTA being applied in various clinical situations in addition to its use as a suitable root-end filling material.
The diverse application of MTA in the practice of paediatric dentistry is evident in its use as an apical barrier in immature non-vital teeth and in the coronal fragment of fractured roots, as a pulpotomy medicament in primary and permanent teeth, a pulpcapping agent in young permanent teeth, and as a repair material for perforation and resorptive defects.
Traditionally, calcium hydroxide has been the material of choice for the apexification of immature permanent teeth but MTA holds significant promise as an alternative to multiple treatments with calcium hydroxide. Calcium hydroxide also is an effective antimicrobial agent because of its high alkalinity. MTA has shown better sealing ability and structural stability, but equal or less potent antimicrobial activity compared with that of calcium hydroxide.
Physicochemical analyses have revealed that MTA not only acts as a “calcium hydroxide-releasing” material, but also interacts with phosphate-containing fluids to form apatite precipitates.
Chlorhexidine is a broad spectrum antibacterial agent, irrigation with chlorhexidine gluconate has been suggested based on its antimicrobial effect and substantivity. CHX is incorporated into endodontic materials to improve prognosis by enhancing their antimicrobial properties.
Antimicrobial activity of root canal materials may help to eliminate residual
microorganisms unaffected by the effects of both chemomechanical preparation and intracanal medication and in turn controlling infection. Thus the use of filling materials presenting an antimicrobial capacity represents one of the more important aspects for achieving success in endodontic therapy.
Thus the purpose of this in vitro study is to check the antibacterial activity of white mineral trioxide aggregate mixed with sterile water against E.faecalis and comparing it with white mineral trioxide aggregate mixed with two different hydrating solutions, i.e, 2% chlorhexidine and phosphate buffered saline.
6.2 REVIEW OF LITERATURE :
An vitro antimicrobial activity of MTA was carried out by Caroline Sousa Ribeiro et al in 2010 using E. coli mutant (AB2463-RecA13), triple mutant (BW535), and wild type (AB1157) on Müller-Hinton agar (MHA) by diffusion method. The plates were incubated at 37°C for 48 hours under aerobic and anaerobic conditions. The zones of inhibition were measured. Also the damage caused to the plasmid DNA in the presence of the cement was evaluated through 0.8%agarose gel electrophoresis.
It was possible to observe a zone of inhibition under aerobic conditions promoted by both cements on both of the mutant E. coli but not in the wild-type E. coli. On the other hand, both cements were not able to induce any bacterial inhibition under anaerobic conditions, suggesting that the inhibitory action is a result of ROS production. Both cements promoted damage in the DNA plasmid when compared with distilled water (control).
They concluded that MTA under aerobic conditions provoked antimicrobial activity by induction of ROS. Furthermore, the antimicrobial activity occurred by damage caused directly to the bacterial DNA.1
Stowe et al. determined the effect of the substitution of 0.12% CHX for sterile water as a mixing agent on the antimicrobial activity of white MTA. They found that substituting 0.12% CHX for water enhanced the antimicrobial activity of MTA.2
An experiment, conducted by Dennis M .Holt et al in 2007, on the antimicrobial effect of MTA mixed with 2% chlorhexidine , accomplished by placing freshly mixed MTA samples on agar plates inoculated with Enterococcus faecalis and comparing the zones of inhibition at 24 hours. The antimicrobial effect experiment, showed that the zones of inhibition were significantly larger for samples mixed with 2% chlorhexidine.3
Mahmut Sumer et al conducted a study to histopathologically examine the biocompatibility of the high-copper amalgam, intermediate restorative material (IRM), mineral trioxide aggregate (MTA), and MTA mixed with chlorhexidine (CHX). This study was conducted to observe the rat subcutaneous connective tissue reaction to the implanted tubes filled with amalgam, IRM, MTA, and MTA mixed with CHX. Amalgam, IRM, and MTA mixed with CHX caused a weak inflammatory response on days 15, 30, and 60. MTA provoked an initial severe inflammatory response that subsided at the 30 and 60 day study period. A clear fibrous capsule was observed beginning from the 15 days in all of the groups. Within the limits of this study, amalgam, IRM, MTA, and MTA mixed with CHX materials were surrounded by fibrous connective tissue indicated that they were well tolerated by the tissues, therefore, MTA/CHX seemed to be biocompatible.4
Khalid Al-hezaimi et al.2006 studied the antibacterial effects of gray-colored MTA (GMTA) and white-colored MTA (WMTA) against Enterococcus faecalis and Streptococcus sanguis in vitro using the tube dilution test. A direct correlation was found between GMTA and WMTA concentrations and their antibacterial effect. They concluded that the susceptibility of E. faecalis and S. sanguis to MTA differed and that GMTA requires lower concentrations than WMTA to exert the same antibacterial effect against each of the microorganisms tested.5
Martin RL et al in 2007 conducted study to examine the sealing properties of two MTA apexification procedures using an in vitro apexification model. A Flodec fluid filtration device was used to record fluid flow across filled root canals at 48 hours and after 4 weeks of immersion in phosphate-buffered saline (PBS). Although MTA root fillings exhibited a better seal than MTA apical plugs at 48 hours, seals of these two groups were not significantly different after 4 weeks. They concluded that interaction of MTA with PBS may result in apatite deposition that improves the seal of MTA apical plugs with time.6
A study was conducted by Ahmad M. Al-Kahtani to investigate the in vitro antibacterial activity of white MTA mixed with different vehicles on a mix of S. aureus, using MTA mixed with water, local anesthetic and Chlorhexidine. After 24 hour set test materials were placed on the surface of the inoculated media and incubated in appropriate atmospheres for 48 hours at 37 degrees centigrade. Augmentin suspension was used as a positive control. Dry sterile tin foil disc was used as a negative control. The antibacterial effects of each material were determined by measuring the diameter of the zones of inhibition in millimeters. Chlorhexidine group had significantly bigger zone of inhibition (P< 0.05). Hence he Concluded that substituting 0.2% Chlorhexidinegluconate for water enhanced the antimicrobial activity of tooth-coloredProRoot MTA.7
Jessie F.Reyes-Carmona et al.conducted study using WMTA with artificially created open apices to form 5mm thick plug and used Phosphate-buffered Saline as intracanal medicament. After 2 months, SEM observations Showed that Phosphate-buffered Saline intracanal dressing improves the biomineralization ability of Mineral Trioxide Aggregate Apical Plugs.8
6.3 OBJECTIVES OF THE STUDY :
1. To evaluate antibacterial properties of WMTA against E.faecalis.
2. To compare the antibacterial effect Of WMTA mixed with 2% clorhexidine,
Phosphate Buffered Saline, and sterile water against E.Faecalis.
7. MATERIALS AND METHODS :
7.1 Sources of data :
Enterococcus faecalis will be obtained from the Department Of Microbiology
Kempegowda Institute Of Medical Sciences And Hospital, Bangalore.
PLACE OF STUDY ;
Department Of Pedodontics And Preventive Dentistry
V.S.Dental College and Hospital ,K.R.Road , V.V.Puram, Bangalore.
Laboratory : Department Of Microbiology
Kempegowda Institute Of Medical Sciences and Hospital, Bangalore.
STUDY DESIGN :
Comparative , Experimental study
STUDY DURATION :
One and half years
SAMPLE DESIGN :
Purposive sampling
SAMPLE SIZE :
4 agar plates with 3 wells of 5mm diameter on each plate , placed equidistantly.
PROPOSED STATISTICAL ANALYSIS :
Chi square and Z test
7.2 METHODS OF COLLECTION OF DATA &METHODOLOGY:
Group 1: MTA / Sterile water
Group 2: MTA / 2% chlorhexidine
Group 3: MTA / Phosphate buffered saline
The antimicrobial effect test will be accomplished by placing freshly mixed MTA samples on trypticase soy agar (TSA) plates inoculated with Enterococcus faecalis and comparing the zones of inhibition at 24 h.
n E.Faecalis is rehydrated with trypticase soy broth (TSB) and subcultured to TSA with 5% sheep blood (TSA II) and incubated at 37oc overnight. Colonies are harvested from the TSA II and suspended in TSB.
n Turbidity is adjusted by adding E.faecalis suspended in TSB to equal a 0.5 McFarland turbidity standard.
n A sterile cotton swab is used to inoculate the bacterial suspension on TSA II plate for lawn growth.
n Agar plates are prepared for diffusion test after plating 100 microlitre aliquots of E.fecalis, 3 wells of 5-mm diameter placed at equal distance are created in 4 separate TSA II, plates.
n MTA is mixed using sterile spatula on sterile glass slab according to manufacturer’s instructions by mixing 1g powder with 0.35 ml sterile water. Each group is mixed exactly the same way by substituting the test liquids for sterile water.
n The MTA is placed into wells created by using a new sterile amalgam carrier and gently condensed into place.
n The plates are incubated for 24 h at 37oc and zone of inhibition measured to nearest mm using zone measuring scale and recorded.
7.3 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR OTHER
INTERVENTION TO BE CONDUCTED ON PATIENTS OR HUMANS OR
ANIMALS? IF SO PLEASE DESCRIBE BRIEFLY:
NO
7.4 HAS THE ETHICAL CLEARANCE BEEN OBTAINED FROM YOUR
INSTITUTION?
NOT REQUIRED
8. LIST OF REFERENCES :
1. Caroline Sousa Ribeiro DDS, MiriamF. ZaccaroScelza DDS, MSD, PhD,
Raphael Hirata Júnior PhD and Lidia Maria Buarque de Oliveira PhD. The antimicrobial activity of gray-colored mineral trioxideaggregate (GMTA) and white-colored MTA (WMTA) under aerobic and anaerobic conditions. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontology 2010 Jun;109(6):e109-e112.
2. Ted J. Stowe DDS, Christine M. Sedgley BDS, MDSc, MDS, FRACDS, PhD.
The Effects of ChlorhexidineGluconate (0.12%) on the Antimicrobial Properties of Tooth-ColoredProRoot Mineral Trioxide Aggregate.
Journal of Endodontics. 2004 Jun;30(6): 429-31.
3. Dennis M. Holt, J. Dustin Watts, Thomas J. Beeson, Timothy C. Kirkpatrick, Richard E. Rutledge. The Anti-microbial Effect Against Enterococcus faecalis and the Compressive Strength of Two Types of Mineral Trioxide Aggregate Mixed With Sterile Water or 2% Chlorhexidine Liquid. Journal of Endodontics. 2007 Jul;33(7):844-7.
4. Mahmut Sumer DDS, PhDlow, MehtapMuglali DDS, PhD.EmreBodrumlu DDS, PhD and TolgaGuvencPhD Reactions of Connective Tissue to Amalgam, Intermediate Restorative Material, Mineral Trioxide Aggregate, and Mineral Trioxide Aggregate Mixed With Chlorhexidine. Journal of Endodontics
2006 Nov; 32(11):1094-6.
5. Khalid Al-Hezaimi BDS, Thakib A. Al-Shalan BDS, PhD, JafarNaghshbandi DDS, Samuel Oglesby DDS, James H.S. Simon DDS and IlanRotstein DDS Antibacterial Effect of Two Mineral Trioxide Aggregate (MTA) Preparations Against Enterococcus faecalis and Streptococcus sanguis In Vitro. Journal of Endodontics. 2006 Nov;32(11):1053-6.
6. Martin RL, Monticelli F, Brackett WW, Loushine RJ, Rockman RA, Ferrari M, Pashley DH, Tay FR. Sealing properties of mineral trioxide aggregate orthograde apical plugs and root fillings in an in vitro apexification model.
J Endod. 2007 Mar;33(3):272-5.
7. Ahmad M. Al-Kahtani Antibacterial activity of white MTA mixed with different vehicles. Egyptian dental journal. 2007 Apr;53:1581.
8. Jessie F.Reyes-Carmona DDS, MSD,phD, Mara S. Fellippe DDS, MSD,phD and Wilson T.Felippe DDS,MSD,phD . A Phosphate-buffered Saline Intracanal Dressing Improves the Biomineralization Ability of Mineral Trioxide Aggregate Apical Plugs. Journal of Endodontics. 2010 Oct;36(10):1648-52.
9. C. R. Sipert, R. P. Hussne, C. K. Nishiyama, S. A. Torres. In vitro antimicrobial activity of Fill Canal, Sealapex, Mineral Trioxide Aggregate, Portland cement and EndoRez . International Endodontic Journal. 2005 Aug; 38(8):539–43.
10. Fabiane Piva, Italo Medeiros Faraco junior, Carlos Estrela Antimicrobial activity of different root canal fiiling pastes used in deciduous teeth. Journal of Material Research. 2008;11(2):171-3.
9 / Signature of Candidate
10 / Remarks of the guide / The study is feasible to be conducted.
11 / Name and designation of
11.1 Guide
11.2 Signature
11.3 Co- Guide
11.4 Signature
11.5 Head Of The
Department
11.6 Signature / DR. VENKATESH BABU
PROFESSOR AND HEAD
DEPARTMENT OF PEDODONTICS & PREVENTIVE DENTISTRY
DR. VENKATESH BABU
PROFESSOR AND HEAD
DEPARTMENT OF PEDODONTICS & PREVENTIVE DENTISTRY
12 / 12.1 Remarks of the Chairman and principal
12.2 Signature