Evaluation of effect of dentin powder on antibacterial properties of Mineral Trioxide Aggregate - An in vitro study

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1 ENDODONTOLOGY Volume: 26 Issue 1 June 2014 Original Research Evaluation of effect of dentin powder on antibacterial properties of Mineral Trioxide Aggregate - An in vitro study Anuraag Gurtu # * Sumit Mohan # * Anurag Singhal # * Chandrawati Guha # * ABSTRACT Aim : To compare the additive effect of Dentin powder on antibacterial properties of Mineral Trioxide Aggregate against Enterococcus faecalis. Materials and Methods : The antimicrobial effectiveness of Mineral Trioxide Aggregate against a standard strain and an isolated strain of Enterococcus faecalis was evaluated in vitro. Fresh MTA powder, powder from crushed set materials and pieces of uncrushed set cements were suspended in autoclaved water at different concentrations for direct exposure testing. The antimicrobial effect of the cements mixed with equal amounts of human dentin powder and liquid in 1.5:1.5:1 was also tested. Results and Observations : The addition of equal amounts of dentin powder to the suspension of MTA powder (both fresh and set) resulted in faster elimination of the bacteria (p < 0.05) in standard strain. Similar inhibitory potential was observed in an isolated strain from a case of apical periodontitis which are expected to be more resistant than standard strain when higher concentration of mix was used. Conclusion : Addition of dentin powder to mineral trioxide aggregate significantly increased its antibacterial efficacy against Enterococcus faecalis. Key Words : Antimicrobial effect, Enterococcus faecalis, Mineral Trioxide Aggregate (MTA), Dentin powder. Introduction Throughout the dental history, a wide variety of materials have been used for retrograde fillings and perforation repair. 1,2 An ideal root-end filling material should produce a complete apical seal, be nontoxic, well tolerated by the periradicular tissues, non-resorbable, dimensionally stable, easy to manipulate, and radiopaque. In addition, it should be bactericidal or bacteriostatic. Although numerous materials have been recommended as root-end filling materials, none has so far been found to be totally ideal. Most endodontic failures are attributable to inadequate cleansing of the root canal and egress of bacteria and other antigens into the periradicular tissues. Therefore, in addition to sealing ability and biocompatibility, root-end filling materials should ideally have some antibacterial activity to prevent bacterial growth. 3 Success of any endodontic material also depends on its antimicrobial activity. It is evident that an infected root canal system is a unique niche for selective species of microorganisms. 4 Enterococcus faecalis is The star survivor in the root canal and is the most frequently recovered microorganism from refractory periapical periodontitis.. Enterococcus faecalis has been # Department of Conservative Dentistry & Endodontics, * Institute of Dental Sciences, Bareilly

2 EVALUATION OF EFFECT OF DENTIN POWDER ON ANTIBACTERIAL PROPERTIES OF MINERAL TRIOXIDE AGGREGATE - AN IN VITRO STUDY shown to invade dentinal tubules and is the most commomly isolated bacterial from endodontic retreatment cases. It can colonize root canal and survive without the support of other bacteria. The prevalence of different Enterococcal species appears to vary according to host and is also influenced by age, diet and other factors that may be related to changes in physiologic conditions such as demographic factors, underlying diseases and antimicrobial thyerapy. 5 Over the years many studies have been conducted to assess the antibacterial efficacy of MTA against E. faecalis which concluded that MTA has limited inhibitory action against this resistant microorganism. 6-8 Many additives have been tested for their effect on various properties of MTA. 1 Dentin powder is a biocompatible material with excellent inactivation potential. 9 It has been known to possess optimal inhibitory properties against E. faecalis when it was added to Bioactive Glass. 10 Previous study by Hui Zang et al 11 indicated that dentin enhances the antibacterial effect of Mineral Trioxide Aggregate and Bioaggregate. Hence this in vitro study was planned to evaluate the effect of dentin powder as an additive on antibacterial efficacy of MTA against a different strain of E. faecalis commonly found in Indian population. Many materials have been used clinically as root-end fillings including amalgam, glass ionomer cement, composite resin, intermediate restorative material, Super EBA, and mineral trioxide aggregate (MTA) The antimicrobial activity of MTA has been tested in several previous studies The purpose of this in vitro study was to evaluate the antibacterial effect of MTA against Enterococcus faecalis and to compare it with the usage of dentin powder as an additive to MTA; this combination has shown to possess shorter setting time as delayed setting is a common problem with MTA. The effect of dentin powder on the antibacterial efficacy of MTA against a standard strain of bacteria and one isolated from a case of apical periodontitis was measured, as the result found against a standard strain of E. faecalis would indicate towards the antibacterial effect of MTA and its combination with dentin and the result against an isolated strain from oral cavity would indicate towards the clinical utility of MTA and its combination with dentin in retreatment cases. Materials and methods The samples were divided into three groups:- Group I: In this group the antibacterial effectiveness of only MTA samples were checked against Enterococcus faecalis. This group was further subdivided as: Group I (a)- Fresh Set MTA Group I (b)- Crushed Set MTA Group I (c)- Uncrushed Set MTA Group II: Dentin powder was added to MTA and sterile liquid in 1.5:1.5:1and its antibacterial properties were evaluated against a standard strain of E.Faecalis. This group was further subdivided as: Group II (a)- Fresh Set MTA + Dentin Powder Group II (b)- Crushed Set MTA + Dentin Powder Group II (c)- Uncrushed Set MTA + Dentin Powder Group II (d )- Dentin Powder (Control Group) Group III: Dentin powder was mixed with MTA and sterile liquid in ratio 1.5:1.5:1and was introduced to prepared bacterial suspensions. The test organism used was the one isolated from apical periodontitis. This group was further subdivided as:

3 ANURAAG GURTU, SUMIT MOHAN, ANURAG SINGHAL, CHANDRAWATI GUHA Group III (a)- Fresh Set MTA + Dentin Powder Group III (b)- Crushed Set MTA + Dentin Powder Group III (c)- Uncrushed Set MTA + Dentin Powder Group III (d )- Dentin Powder (Control Group) Preparation of dentin powder Extracted human teeth were preserved in 0.5% sodium hypochlorite to remove soft tissue and prevent bacterial growth. They were cleaned of debris and stains using ultra sonic scaler. The teeth were rinsed with hydrogen peroxide followed by 0.9% normal saline and autoclaved at C for 15 minutes. The crowns of the teeth were removed with a diamond disc and the roots were crushed. The crushed dentin was then ground in a mortar using a pestle to obtain fine dentin powder. 9 Cements The mineral-based endodontic cement used in this study was ProRoot MTA (Dentsply/Tulsa Dental, Tulsa, OK). Suspensions of fresh powder of MTA in sterile water were prepared at concentrations of 25, 50, and 100 mg/ml. To prepare suspensions of crushed set cements, MTA was mixed with sterile water according to the manufacturers instructions and allowed to set in 100% humidity for 7 days. After complete setting, some of the blocks were crushed into powder with a particle size similar to that of fresh, non-set material. Crushing of the set cement to powder was finished with alumina mortar and pestle. E.Faecalis used in study Zones of Inhibition of Group II Mineral trioxide aggregate Zones of Inhibition of Group III Dentin Powder

4 EVALUATION OF EFFECT OF DENTIN POWDER ON ANTIBACTERIAL PROPERTIES OF MINERAL TRIOXIDE AGGREGATE - AN IN VITRO STUDY Bacteria E. faecalis isolated from a case of apical periodontitis and a standard strain were used as test organisms. They were grown at 37 C in air overnight on tryptic soy agar plates for the experiments. After checking for purity, E. faecalis was suspended in sterile water and adjusted spectrophotometrically to the density of 3 x10 7 colony-forming units (CFU)/mL. Antibacteriall Efficacy of MTA Antibacterial efficacy of the Groups (I, II and III) were evaluated against E.faecalis. After incubation at room temperature for 1, 6, 15, 60 minutes and 4 hours, the survival of the bacteria in the solutions was assessed by 10-fold serial dilutions and culture on TSA plates. After incubation for hours at 37 C, colonies on the plates were counted, and CFU/mL was calculated. All experiments were performed in triplicate. Results Group Ia (Fresh MTA powder) and Group Ib and Ic (MTA powder from set materials) all killed >99% of the bacterial cells in 1 minute. It was observed that after 6 minutes of exposure, 50 mg/ ml of MTA had killed all bacteria. Powder obtained by crushing the set cements (Group Ib) showed quicker killing of E. faecalis at 1 minute and 6 minutes than the fresh powder (p <0.05). Set uncrushed cement (Group Ic) killed all bacteria within 1 hour (p >0.05). Group IIa and IIb IIc Dentin powder increased the antibacterial activity of fresh powder and powder from crushed set cement of MTA (p <0.05). Dentin powder (Group IId) alone did not cause any reduction in the number of viable bacteria during the 24-hour experiment. It was also observed that though MTA has an effective antibacterial activity against E. faecalis but addition of dentin powder significantly increased the antimicrobial action against a standard strain of bacteria for all groups of MTA used. In Group III MTA and dentin were assessed for their antibacterial effectiveness against E. faecalis which was isolated from apical periodontitis, MTA inhibited E. faecalis effectively after a short time of exposure of 1 minute to 4 hours. Limited inhibitory action was observed by Groups III a and III b. Group III c increased inhibition was observed at 100 mg/ ml. No inhibitory action was observed in Group III where dentin powder was used alone. Discussion MTA is biocompatible cement with several clinical applications in endodontics. 1, 23 The major components of MTA are tricalcium silicate, dicalcium silicate, gypsum, and tricalcium aluminate. When mixed with water, tricalcium silicate and dicalcium silicate hydrate to form alkaline calcium silicate hydrate gel, which sets in a few hours Calcium hydroxide in silicate matrix accounts for the high alkalinity 25,26 and biocompatibility. 23 Enterococcus faecalis has been only occasionally found in cases of primary endodontic infections but frequently isolated or detected in cases in which the endodontic therapy has failed which usually form the tougher cases and cases where MTA is usually indicated i.e periapical surgery, perforation repair etc. It is resistant to the antimicrobial effects of calcium hydroxide, partly due to an effective proton pump mechanism which maintains optimal cytoplasmic ph levels. The rapid emergence of antimicrobial resistance among Enterococci helps to shift the microbial flora in favor of Enterococcus faecalis

5 ANURAAG GURTU, SUMIT MOHAN, ANURAG SINGHAL, CHANDRAWATI GUHA The present study evaluated the effect of dentin powder on antibacterial efficacy of MTA. In our study MTA powder (Group I) was compared in its antibacterial efficacy against MTA mixed with dentin (Group II and III). These samples were evaluated for their antibacterial properties against a standard strain of Enterococcus faecalis and one which was isolated from apical periodontitis. It was observed that addition of dentin powder to mineral trioxide aggregate significantly increased its antibacterial properties in group II where standard strain of bacteria was used. This result was in agreement with the one inferred by Hui Zhang et al. 11 Limited inhibitory potential was observed in group III where samples were tested against an isolated strain of E. faecalis. This can be attributed to the fact that isolated bacteria especially those isolated from apical periodontitis are more resistant than standard species. 5 But the uncrushed set sample at 100 mg/ml inhibited bacteria significantly. Moreover representatives of group III show more clinical relevance as human micro flora does not have a standard strain but a more resistant strain. The rationale behind using an isolated strain from apical periodontitis was based on the fact that they would make the study clinically relevant. A direct exposure test can be used to assess the antibacterial activity of water-insoluble root-end filling materials before and after set setting to obtain quantitative and reproducible results. In the present study suspensions of MTA and at low concentrations were used to investigate their antibacterial effect against E. faecalis. MTA showed comparable bacterial killing and completely eradicated the bacteria within 1 hour, which was consistent with the results with MTA by McHugh et al. 20. Al-Hezaimi et al. 18 and Holt et al. 21 reported that grey MTA showed greater E. faecalis growth inhibition than white MTA. Another study by Miyagak et al. 8 reported no antimicrobial activity of MTA against E. faecalis in agar diffusion test. Sipert et al. 19 reported that MTA demonstrated antimicrobial activity against E. faecalis, Micrococcus luteus, Staphylococcus aureus, S. epidermidis, Pseudomonas aeruginosa, and Candida albicans but not against Escherichia coli with double-layered diffusion method. However, it is not meaningful to make comparisons among studies with different methodologies. Agar diffusion studies might reflect more the type of chemical interaction of the agar medium and the material to be tested than the antibacterial effect of the material. The advantage of direct exposure test is that the number and effect of possible confounding factors can be minimized. In an empty root canal after finishing the chemomechanical instrumentation, the main remaining elements are in fact dentin, the filling material, moisture, and possibly some surviving microbes, reflecting the design of the present model. However, the model is less likely to be suitable in predicting the antimicrobial efficacy and other biologic effects of these materials in contact with periapical tissues where a number of other factors including tissue fluid and inflammatory exudate modify and complicate the interaction between the materials and the microbes. E. faecalis,the most frequently recovered microorganism from refractory periapical periodontitis is highly resistance to certain intracanal medicaments and ability to survive conventional root canal therapy. 27 Calcium hydroxide is produced by MTA by a hydration reaction. MTA is capable of releasing its soluble fraction continuously to an

6 EVALUATION OF EFFECT OF DENTIN POWDER ON ANTIBACTERIAL PROPERTIES OF MINERAL TRIOXIDE AGGREGATE - AN IN VITRO STUDY aqueous environment. It is possible that the increased ph resulting from the dissociation of calcium hydroxide into calcium and hydroxyl ions is responsible for their antimicrobial efficacy. 24 Calcium hydroxide has been shown to be ineffective in killing E. faecalis when used in the root canal, because the buffering capacity of dentin makes it difficult to maintain ph above 11.5 in the dentinal tubules with current calcium hydroxide utilization techniques. 20,28-30 The proton pump of E. faecalis is probably the key factor in its resistance to alkali and survival during intracanal calcium hydroxide treatment. Zehnder et al. 31 reported that dentin potentiated the antimicrobial effect of bioactive glass (BAG) against E. faecalis. 32 BAG and dentin preincubated up to 24 hours showed significantly increased bacterial killing as compared with BAG alone, and the increased killing was detected from 1 hour to 5 hours. In the study by Zehnder et al. and in the present study, similar concentrations of bacteria, cements, and dentin were used. Even without preincubation of the cements with dentin, mixing dentin with MTA resulted in complete killing of E. faecalis in 1-6 minutes, indicating a greater antibacterial potential for MTA than what has been reported for BAG. Zehnder et al. showed that dentin powder triggered BAG dissolution, causing elevated local ph and silica levels. Gubler et al. 33 suggested that a mechanism of bacterial killing by BAG that is not directly linked to ph is dependent on ion release from the BAG material. Although not shown in the present study, it is possible that increased killing by MTA of E. faecalis in the presence of dentin might be triggered by a mechanism similar to that suggested for BAG. Another reason for improved antibacterial efficacy of MTA dentin combination against E. faecalis might be due to the buffering capacity of dentin that inhibits its proton pump. The reason for MTA to possess excellent antibacterial efficacy can also be attributed to its components. GMTA consists of dicalcium and tricalcium silicate and bismuth oxide whereas WMTA is composed of tricalcium silicate and bismuth oxide. On mixing MTA with liquid calcium hydrate and calcium silicate hydrate are formed. It has been suggested that placing MTA in an acidic environment such as inflammatory tissue might result in the release of bismuth oxide. Bismuth oxide does not encourage cell proliferation in cell culture. 1 Conclusion The results of the present study showed that MTA has strong antimicrobial activity against E. faecalis. The presence of dentin powder further enhances killing of E. faecalis by MTA. MTA dentin mix inhibited standard strain more significantly. Enhanced antibacterial properties of dentin MTA combination was observed at 100 mg/ml against isolated strain of E.Faecalis which makes this study clinically relevant. Conflicting results from antibacterial investigation on MTA might be attributed to various species of microorganisms, the source of preparing material, as well as the type and concentration of MTA used. 34 Altering the ratio of dentin and MTA powder and powder to liquid ratio might affect the antibacterial properties of MTA. Further long term studies pertaining to E. faecalis obtained from different dental locations & patients along with the sealing ability and biocompatibility of this mix are suggested before recommending this combination for clinical usage

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