Evaluation of antimicrobial properties of bioactive glass used in regenerative periodontal therapy.

CONTEXT: Bone grafting materials which have an inherent anti-microbial property against initial colonizers of plaque bacteria would be useful in regenerative periodontal surgical procedures. AIMS: This study was performed to analyze the antibacterial property of a Perioglas™ against a common oral commensal Streptococcus salivarius (early colonizer). SETTINGS AND
DESIGN: In vitro observational study.
MATERIALS AND METHODS: Perioglas™ (in various concentrations) was assessed for its antibacterial property against the ATCC 13419 strain of S. salivarius. The anti-microbial activity was analyzed in terms of reduction in colony-forming units in culture plates and smear following a 24 h incubation at 37°C. STATISTICAL ANALYSIS USED: Observational study - No statistical analysis applicable.
RESULTS: The bioactive glass (BAG) exerted an antibacterial effect against the S. salivarius in the suspending media and smear. The antibacterial activity of BAG increased in proportion with its concentration.
CONCLUSIONS: Perioglas™ demonstrated a considerable antibacterial effect against S. salivarius at 50 mg/mL concentration.

INTRODUCTION

The oral cavity is the gateway of the body to the external environment and represents one of the most biologically complex sites in the human body. It provides a habitat, which is favorable for the microorganisms to thrive and colonize. The tooth surface provides a unique, hard non-shedding surface, which enables large masses of microorganisms to accumulate as a biofilm. These microorganisms evoke an inflammatory response in the periodontal tissues thereby resulting in defects in the tooth supporting bone.[1]

Treatment of periodontal disease involves restoration of lost bone and periodontal attachment apparatus with an aim of improving tooth support and its prognosis. Regeneration is defined as reproduction or reconstitution of a lost or injured part. Regeneration of periodontal defects involves reconstruction of alveolar bone, periodontal ligament, and cementum to their original levels. Bone replacement grafts have been used clinically in attempts to regenerate lost tooth support. Alloplasts are bone replacement substitutes which are of synthetic nature and have a similar chemical composition as natural bone [Table 1].

Table 1

Various bone grafts used in periodontal regeneration

Hench and Paschall[2] discovered the bioactive glass (BAG) and popularized its use in prosthetic applications and in repair of bone defects. It is composed of 45% SiO2, 24.5% CaO, 24.5% Na2O, and 6% P2O5. In vivo the BAG undergoes surface alterations leading to the formation of hydroxycarbonate apatite layer,[3] in addition the release of soluble silica contributes to osteoblast attachment and proliferation on the BAG particle surface.[4]

The wound healing following regenerative therapy can be influenced by many factors, one of the factors being constant exposure of surgical site to the oral microbiota. Streptococcus spp. is one of the oral commensals, which can potentially hamper the outcome of periodontal therapy.

The objective of this study was to analyze the antibacterial property of a commercially available BAG (Perioglas™) against Streptococcus salivarius (early colonizer).

MATERIALS AND METHODS

This in vitro study was conducted to determine the antimicrobial efficacy of Perioglas™ on ATCC strain of S. salivarius.

Bacterial cultivation

The lyophilized specimen of S. salivarius (ATCC strain 13419) [Figure 1] was procured from HIMEDIA laboratories. Manufacturer's instructions were followed to revive the lyophilized specimen. The revived strain was inoculated on 5% sheep blood agar plates and incubated in a 5% CO2 incubator at 37°C overnight. The microbial cultures were harvested and suspended in brain-heart infusion (BHI) broth at a concentration of 105 colony-forming units per milliliter (CFU/mL) (1.0 McFarland standard).

Figure 1

Lyophilized specimen of Streptococcus salivarius (ATCC, 13419)

Sample preparation

Perioglas™ (45S5, US Biomaterials Corp., Alachua, FL, USA) was obtained from a commercial source. Particulates of Perioglas™ with grain size ranges of 90–710 μm diameter was suspended in sterile water at 37°C under constant stirring for 10 min. 4 samples of different solid-to-liquid concentrations were prepared as given in Table 2. Supernatants of the suspensions were obtained by centrifugation at 13,000 ×g for 10 min (Eppendorf, Hanau, Germany).

Table 2

Initial and final concentrations of the test samples containing BAG

Broth dilution method

The maximum inhibitory concentration value of the Perioglas™ was determined by using a broth dilution assay. A serial two-fold dilution of prepared samples of BAG was done by diluting in 0.5 ml of BHI to achieve final dilutions ranging from 1:0.5, 1:5, 1:25, and 1:50 [Table 2]. Each Eppendorf tube® contained 1 mL (0.5 mL of BHI broth + 0.5 mL of sample solution of BAG). One drop of the bacterial species suspension, adjusted to yield a cell concentration of 105 CFU/mL (1 McFarland standard) was inoculated into the Eppendorf tubes® containing serial dilutions of BAG. Tubes containing plain BHI broth with bacterial sample added served as positive control and tubes containing BAG sample solution alone served as negative control. The Eppendorf tubes® were incubated at 37°C for 24 h. Following this the corresponding samples were subcultured onto sheep blood agar plates as well as smeared for further confirmation. The plates were incubated overnight at 37°C in a 5% CO2 incubator. Plate readings were taken after 6 h and after 24 h. The presence or the absence of the microbial colonies was assessed in the primary, secondary, and tertiary streaks, and the results were interpreted in a qualitative manner (presence or absence of growth indicating bacterial viability).[5] During plate readings, the presence of bacterial colonies in the primary streak, secondary streak, and tertiary streak corresponds to 103, 104, and 105 CFUs, respectively.[5] Smears from each Eppendorf tube® were also examined under a light microscope ×100 magnification using oil immersion.

RESULTS

Evaluation of smear

Following gram staining the smears were evaluated under a light microscope. The positive control showed a confluent growth of S. salivarius in chains. As the concentration of BAG increases, the counts of the Streptococcus in the smear reduced by more than 99% [Figure 2].

Figure 2

Reduction in bacterial number as examined in a smear under, ×100

Evaluation of culture plates

The BAG exerted an anti-microbial activity against the Streptococcus species in the suspending media. The anti-microbial activity of BAG increased proportionately with the concentration. The BAG had a definitive growth inhibitory effect at a concentration of 50 mg/mL [Figures 3 and 4]. The CFUs were calculated based on McFarland standard and a reduction to 102 and 103 CFU was observed at 50 mg/mL concentration after 6 h and 24 h of incubation [Figure 5].

Figure 3

Reduction in colony counts of cultured in 5% sheep blood agar with different concentrations of bioactive glass (0.5:1 and 5:1) following 24 h incubation

Figure 4

Reduction in colony counts of cultured in 5% sheep blood agar with different concentrations of bioactive glass (25:1 and 50:1) following 24 h incubation

Figure 5

Bar graph showing the evaluation of colony forming units of cultured in 5% sheep blood agar with different concentrations of bioactive glass following 6 and 24 h incubation

DISCUSSION

Healing of the periodontal tissue takes place in an open system and under a significant bacterial load, which is due to the fact that the oral environment is never sterile. Various bone replacement grafts are being used in regenerative periodontal therapy. Following surgical therapy, anti-microbial agents are prescribed prophylactically in order to control bacterial colonization. However, when a bone replacement graft itself has an inherent antibacterial effect, this would enhance the predictability of the regenerative therapy. Stoor et al.[6] assessed the antibacterial efficacy of BAG paste on oral microorganisms such as Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Actinomyces naeslundii, Streptococcus mutans, and Streptococcus sanguis. The authors determined that among all the periodontal microorganisms examined in the study S. sanguis was the only microbe that had viable cells left even after 60 min following incubation in suspension of BAG (S53P4).

Based on this observation Tai et al.,[7] performed a 6 weeks clinical study wherein the authors evaluated the antigingivitis and anti-plaque effects of a dentifrice containing BAG (Novamin) as compared with a placebo dentifrice. The authors observed a significant reduction in gingival bleeding and supragingival plaque in the Novamin group as compared to the placebo.

The observations of the above-mentioned studies allow us to conclude that BAG has an anti-microbial activity against early colonizers. This effect may be advantageous for a predictable regenerative periodontal therapy as bacterial recolonization can hamper the therapeutic success.

We examined the antibacterial effects of commercially available particulate BAG (Perioglas™) at four different concentrations against S. salivarius. The antibacterial effects were found to vary with concentration and increased proportionately with the concentration of BAG. The results of our study are in agreement with the observations of Allan et al.[8] wherein the authors demonstrated an anti-microbial effect of BAG against Streptococcus and Actinomyces spp. Waltimo et al.[9] assessed the antimicrobial effect of nanometric BAG 45S5 against clinical isolates of enterococci from persisting root canal infections. The authors observed a 10-fold increase in silica release and pH elevation by more than 3 units following the use of nanometric glass particles.

The anti-microbial activity of BAG supernates can be attributed to a pH-related phenomenon. Stoor et al.,[6] reported that the BAG increased the pH to around 7.75 which was responsible for its anti-microbial activity. The particles have high surface area and when in the aqueous state release a large amount of ionic components.[10] The alkaline nature of the BAG not only contributes to antimicrobial activity, it might also be an important determining factor for periodontal regeneration. Han et al.,[11] reported the change in pH induced by BAG, contributed to a reduction in inflammation at the periodontal defect site and this could be an important determinant for the bone regenerative effect of BAG.

The limitations of our study are: (a) Simulated body fluid with ionic concentration similar to blood was not used (b) efficacy of BAG against Gram-negative anaerobic organism was not determined. Further studies in this direction can contribute for the development of antibacterial surface coatings for implantable devices.