Year : 2010 | Volume
: 21 | Issue : 1 | Page : 30--34
Antibacterial effect of bioactive glass in combination with powdered enamel and dentin
AR Prabhakar, Santhosh CH Kumar
Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka, India
A R Prabhakar
Department of Pedodontics and Preventive Dentistry, Bapuji Dental College and Hospital, Davangere, Karnataka
Background and Objectives: In endodontics, various intracanal medications have been advocated to eliminate bacteria after root canal instrumentation. A recent study has revealed that addition of powdered dentin to bioactive glass (BAG) led to increased glass dissolution, and an increased antibacterial efficacy. Therefore, the present study was undertaken to compare the effect of enamel and dentin powder on the antibacterial efficacy of a commercially available BAG.
Materials and Methods: Dentin blocks (dbs) were prepared from single rooted human teeth. These dbs were infected with Enterococcus faecalis for two weeks in Tryptic Soy Broth (TSB), while negative controls were kept in sterile TSB. In group I, the infected dbs were filled with BAG, in group II with BAG + Enamel powder and group III with BAG + Dentin powder. Dentin samples were harvested from the dbs and cultured.
Statistical Analysis: Kruskal-Wallis ANOVA was used for multiple group comparison followed by Scheffe«SQ»s post hoc test for pair-wise comparisons.
Results: All the combinations of BAG evaluated significantly reduced the bacterial counts compared to the control group. However, at the end of 24 hours, three days, and five days BAG + Dentin powder showed significant reduction ( P < 0.01) in bacterial counts compared to the other experimental groups.
Conclusion: Among the various materials evaluated, it appeared that though BAG exhibits antimicrobial efficacy, the addition of powdered enamel and dentin in aqueous suspension definitely enhanced this property. However, the addition of enamel powder BAG did not significantly alter its antimicrobial efficacy compared to BAG + dentin powder.
|How to cite this article:|
Prabhakar A R, Kumar SC. Antibacterial effect of bioactive glass in combination with powdered enamel and dentin.Indian J Dent Res 2010;21:30-34
|How to cite this URL:|
Prabhakar A R, Kumar SC. Antibacterial effect of bioactive glass in combination with powdered enamel and dentin. Indian J Dent Res [serial online] 2010 [cited 2021 Feb 25 ];21:30-34
Available from: https://www.ijdr.in/text.asp?2010/21/1/30/62807
Root canal treatment can be performed either in single or multiple visits.  Irrespective of the preferred approach, it should be realized that disinfection of a root canal system takes time, even if fast-acting biocides, such as sodium hypochlorite solutions, are used to irrigate the endodontium.  Consequently, treatment of teeth with apical periodontitis in two visits using an antiseptic root canal interim dressing remains standard. One possible disadvantage of this approach, however, is that the root canal system can be contaminated by resident or transient oral microbiota during the intervisit period through leaking temporary fillings.  Enteric bacteria, especially Enterococcus faecalis strains, have been known to contaminate unsealed root canals.  These bacteria can survive as monoinfectants in pulpless teeth and are hard to eliminate once present. 
Among the intervisit root canal dressings, aqueous calcium hydroxide suspensions appear to be the most efficient disinfectants.  However, they have two disadvantages. First, calcium hydroxide appears to be relatively inefficient against alkali-resistant microbiota such as E. faecalis.  Second, the nonspecific proteolytic action of the hydroxide ions may reduce the flexural strength of dentin, possibly rendering teeth more prone to fracture. 
Preliminary experiments have recently identified a bioactive glass (BAG) of the SiO 2 -Na 2 O-CaO- P 2 O 5 system as a possible alternative to calcium hydroxide.  BAG can induce dentin mineralization and therefore may not negatively affect the mechanical properties of teeth when used as an intracanal dressing. In aqueous suspension, BAG particles exert an antimicrobial effect. The release of Na+ and Ca++ ions from and the incorporation of H 3 O+ protons into the corroding glass results in a high-pH environment, which interferes with microbial viability.  Furthermore, the release of silica, Ca++, and P ions appear to exert direct and indirect effects on the bacteria. 
A recent study  has revealed that addition of powdered dentin to BAG led to increased glass dissolution, leading to an increased antibacterial efficacy of the liquid phase. It was believed that this was due to the calcium/phosphate precipitations, which interfered with bacterial cell integrity.
Based on these promising results, the present study was undertaken to evaluate and compare the antibacterial efficacy of a commercially available BAG (PerioGlas; , NovaBoneProducts, LLC- Alachua USA) following the addition of dentin or enamel powder in an aqueous suspension.
Materials and Methods
The BAG powder used in the study was 45S5 (PerioGlas® , NovaBoneProducts, LLC- Alachua USA). It is composed of 53% SiO 2 (w/w), 23% Na 2 O, 20% CaO and 4% P 2 O 5 with a particle size ranging from 20-63 ΅.
Preparation of human enamel and dentin powder to be mixed with bioactive glass
Third molars extracted for therapeutic reasons were kept at -25°C in an individual tight containers. Before instrumentation, the teeth were thawed at room temperature and immersed in 5% sodium hypochlorite for 10 min to dissolve organic tissue remnants from the root surfaces. The crowns and roots of the teeth were separated with a diamond disc under constant water cooling. The crowns were then sectioned mesio-distally. From each sectioned crown portion, dentin was removed with a diamond bur (ISO 018).The resulting enamel shells were crushed with the help of two clean metal blocks and the grossly crushed enamel fragments (particles 1-4 mm) were separated and then ground to a fine powder in a mortar and pestle. The powder was then passed through a sieve (No. 120) and stored in sterile air tight containers. The root portions were cleared of their cemental layer and the resulting root fragments were similarly crushed to get dentin powder.
Preparation of dentin blocks
Permanent maxillary non carious single rooted teeth extracted for therapeutic reasons were selected and kept at -258C in air tight containers.
All the specimens were disinfected with 5% Sodium Hypochlorite for 15 minutes, and then cleaned with a rubber cup and prophy brush and pumice. The mesio-distal and bucco-lingual diameters of the specimens were determined at the crown limit of the root using a dial caliper. The mean values obtained were 6.02 mm for M-D and 6.64 mm for B-L dimensions. Samples presenting a difference of 20% from the mean were discarded leaving a total of 66 single rooted teeth. 
A rotary diamond disk was used to decoronate the teeth 5 mm below the cemento enamel junction. Cementum was removed from the root surface and root canals were enlarged to a standard diameter of 1.6 mm, using an ISO 016 bur. Organic and inorganic debris, including smear layer were removed in an ultrasonic bath of 17% EDTA for 10 minutes. All the 66 dbs were rinsed with 2 mL tryptic soy broth (TSB, HiMedia Lab, Mumbai) using a 28-gauge needle. Subsequently, the dbs were placed in an individual glass tubes containing 5 ml TSB and autoclaved for 15 mins at 121°C. The tubes were then sonicated in a water bath for 5 mins at room temperature.
An overnight culture of E. faecalis ATCC 35550 was prepared on TSB separately. Subsequently, 60 out of the 66 TSB containing dentin blocks (dbs) were seeded with E. faecalis. The unused six dentin blocks served as negative control. The blocks were then kept in TSB at 37 o C for two weeks during which the broth was regularly changed at two to three day intervals. After two weeks the infected dbs were taken and washed in sterile saline to remove any remnants of the incubation broth.
The 60 dbs were then divided into three experimental groups of 18 blocks each, and a positive control group having six dbs.
The experimental groups were medicated as follows:
Group I: All the 18 dbs were filled with BAG mixed with distilled water in the ratio 1:0.6 wt/vol. 
Group II: Two freshly prepared suspensions, BAG mixed with distilled water (1:0.6 wt/vol) and sterile enamel powder mixed with sterile saline (1:1.5 wt/vol) thoroughly blended together in a ratio of 1:1 vol/vol. This mixture was then placed in each of the 18 dbs.
Group III: Two freshly prepared suspensions, BAG mixed with distilled water (1:0.6 wt/vol) and sterile dentin powder mixed with sterile saline (1:1.5 wt/vol) thoroughly blended together in a ratio of 1:1 vol/vol. This mixture was then placed in each of the 18 dbs.
The control groups were as follows:
Group IV (Positive control): All the six infected dbs were not filled with any medication.
Group V (Negative control): All the six uninfected dbs were not filled with any medication.
Following medication of the dbs, the canal lumina on the top and bottom surfaces of the individual blocks were sealed with paraffin wax and incubated at 37°C in a sterile air tight container. At the end of 24 hours, six samples each from the three experimental groups were randomly chosen. Paraffin wax was removed and subsequently the medication was carefully rinsed from the dbs using sterile saline solution. Dentin samples from each block were harvested by drilling inside the canal lumen of each db with a round bur (ISO 018) and dropping the shavings on a plate containing TSB. The culture plates were incubated up to three days to detect any bacterial growth and the number of bacterial colonies formed (cfus) counted. Purity of the culture was checked by subsequent observation of colony morphology as well as cellular characteristics after Gram staining.
The same procedure was repeated for the three experimental groups at the end of three days and all the groups (Group I, II, III, IV and V) at the end of five days.
Results were expressed as mean ± standard deviation (SD) for continuous data and numbers for scoring pattern. Measurements were in scores and non-parametric analysis was performed. Kruskal- wallis ANOVA was used for multiple group comparison followed by Scheffe's post hoc test for pair-wise comparisons.
Dbs infected with E. faecalis for 2 weeks and then dressed with saline solution for five days (negative control) showed vigorous growth in all the samples. Samples collected showed a statistically significant reduction (P  that becomes activated when contacted by tissue fluids and induces an alkaline pH (>9.5) in a range similar to the newer calcium hydroxide agents. Bioglass is composed of minerals, such as silica, calcium, sodium, oxygen, hydrogen, and phosphorous, which occur naturally in the body.
A recent study  has reported that S53P4 BAG powder showed antiseptic effect, which was stronger than the one observed from calcium hydroxide, and which apparently was not exclusively pH related. The presence of dentin powder in suspension increased the efficacy of S53P4 against a multiplicity of oral microorganisms.
Enamel is the hardest tissue found in the human body. Compared to dentin, it has a higher inorganic content being predominantly hydroxyl apatite, which is rich in calcium. These compositional differences of enamel were believed to influence the antimicrobial efficacy of Bioglass.
Several simplifications and extrapolations were necessary for the present ex vivo model to be functional and reproducible.
The dentin block assay is commonly used to test endodontic antiseptic irrigants and medications.  Organic and inorganic debris including smear layer were removed from the dbs according to the recommendations of Zehnder M, Soderling D, Salonen J, and Waltimo.  The specimens were then sterilized in Tryptone soy broth to allow the optimal penetration of nutrient broth into the dentinal tubules.
E. faecalis was chosen as the test organism primarily because it is among the few facultative organisms associated with persistent apical periodontitis. 
The broth was changed according to the standard protocol previously used in bovine dbs infected with E. faecalis,  resulting in predictably high numbers of viable enterococci.
The antimicrobial potential of bioactive SiO 2 - Na 2 O-CaO-P 2 O 5 glasses in a simple system in vitro is largely a function of their ability to raise the pH in aqueous suspension. These high pH levels are not well tolerated by either bacteria or host cells. In the present study, BAG was an effective medicament against E. faecalis compared to the controls.
The current ex vivo study revealed a superior disinfecting capacity of BAG + dentin powder compared with a conventional BAG suspension in human teeth. In a previous study conducted by Zehnder M, Soderling E, Salonen J, Waltimo T,  they reported that addition of powdered dentin enhanced the antibacterial efficacy of BAG. They speculated that dentin served as a source of Ca++ and P ions, which allowed the BAG to "mineralize" the bacteria i.e. from Ca/P precipitates on their surface, thereby destroying their cellular integrity.
A more recent study,  however, has reported that the combined effect of dentin powder and BAG was not a matter of osmolarity, neither was it linked to binding of bacteria to dentin powder in the presence of BAG nor cellular agglutination. They proved that only combinations of solid BAG and dentin powder and not their supernatants in suspension had an additive effect. According to their study dentin powder with its complex surface acted as a recipient for ions in solution, and thus acted as a catalyst for the dissolution of the glass in aqueous suspension. And it was this ionic flow between the glass and dentin powder appeared to interfere with bacterial viability.
Regardless of the ambiguities about the exact mechanism of action of a combination of BAG and dentin powder, this combination was most effective against E. faecalis at the end of 24 hours and three days. These observations were similar to previous studies. ,
The findings of our study revealed that the addition of powdered enamel to BAG did not significantly affect its antibacterial efficacy at the end of 24 hours and three days. However, at the end of five days, BAG + enamel combination exhibited a marked increase in antimicrobial activity. A recent study conducted by Waltimo T, Zehnder M, and Soderling E,  reported that the addition of bone powder enhanced the antibacterial efficacy of BAG against E. faecalis, but no antimicrobial boosting effect was observed when decalcified bone or hydroxyl apatite was added.
This ex vivo model was a modest attempt to evaluate the antibacterial efficacy of BAG against E. faecalis and also to compare the enhancing antimicrobial effects of addition of enamel or dentin powder. It must be remembered that the antibacterial effect of BAGs is related to their particle size.  Perioglass is commonly used as a bone substitute and thus has a relatively large particle size (20-63μ) and a low antimicrobial effect. The lesser effect of the BAG suspension, despite the fact that its initial pH is comparable to that of the Ca(OH) 2 counterpart, can be explained by the lesser alkaline capacity of the glass suspension compared to Ca(OH) 2 . This factor needs to be considered before extrapolating the findings of our study to an in vivo situation.
The following conclusions were drawn from this study:
In an in vitro model, BAG was relatively effective against E. faecalis.The addition of enamel powder to BAG did not significantly boosts its antimicrobial efficacy at the end of 24 hours and three days. However, at the end of five days, this combination exhibited marked increase in antimicrobial activity.The mixture of BAG plus dentin powder increased the antimicrobial efficacy of BAG at the end of 24 hours and three days, which was statistically significant; although it tapered at the end of five days.Within the limitations of this study and among the various materials evaluated it appeared that though BAG exhibits antimicrobial efficacy, the addition of powdered dentin/enamel in aqueous suspension definitely enhances this property. Before extrapolating the results of this study into clinical practice, it must be reiterated that the results of this study were obtained in an ideal environment in vitro. It is, therefore, premature to draw any conclusions regarding the antimicrobial action of a combination of BAG and dentin/enamel powder in situ. Blood, tissue remnants, occluded dentin tubules, and many other confounding factors may affect the effectiveness of this combination in the root canal system. We recommend further controlled studies on in vivo models to confirm our observations and ascertain the true clinical effects of this combination.
The authors would like to acknowledge Dr. Sadashiva Shetty, Principal of Bapuji Dental College, for his support in making this study a success.
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