It is a randomized controlled in-vitro study to compare between the efficacy of the application of a chlorohexidine diacetate varnish (cervitec plus) and that of a selenium containing sealant ( DenteShieldTM)in the prevention of biofilm formation on teeth.
The study was conducted following the approval of the Institutional review board of the Faculty of Dentistry, Alexandria University. (IRB: 00010556-IORG: 0008839). All methods were carried out in accordance with the CRIS guidelines and Regulations.
The study was conducted at the Orthodontic and Biomaterials Departments at the Faculty of Dentistry, Alexandria University as well as the Microbiology department of the Medical Research Institute, Alexandria University.
Sample Preparation:
The sample size estimate was 30 premolars with at least 10 premolars per group. This was calculated using GPower software version 3.1.9.2 using an error of 0.05 and a study power of 80%16. To cater for any damage or loss during the study, 45 premolars that had been extracted for orthodontic purposes were used and divided equally into three groups using simple randomization. Informed consent was sought from each individual or their guardians before taking their teeth. Only Premolars with an intact enamel surface without caries, restorations or cracks were included in the study.
Immediately after extraction, the teeth were thoroughly cleaned under running water. All calculus and soft tissue remnants were removed using a hand scaler. The teeth were then stored in distilled water at room temperature to avoid dehydration.
Using simple randomization, the teeth were divided into three groups I, II, III of 15 teeth each: I -Control. II - Chlorohexidine varnish. III- Oganoselenium sealant.
The apical third of the root was sectioned off. A hole was drilled near the apical part by means of a rose-head bur on a high speed hand piece. A ligature wire was passed through this hole to allow handling of the teeth during the experimental period and to attach tags that carried unique numbers to the teeth. The apical part of the tooth was sealed with composite.
At the onset of the study, the enamel surface was cleaned and polished with a rubber prophylaxis cup at low speed using fluoride-free pumice and water. The teeth were then thoroughly rinsed, Placed in a sterile bag and autoclaved. Each tooth was assigned a number from 1 to 15 for identification purposes and to facilitate re-examination in their respective groups. The experiment proceeded as shown in the flow chart fig. 2.
Isolation of Streptococcus mutans
A sample from dental plaque was collected on a tip of sterile wooden toothpick from myself. Plaque was dispersed in 1ml of sterile phosphate buffer saline and vortexed to obtain a homogenous suspension. The suspension was then plated on modified MSB agar (Himedia, India) fig.3 and incubated anaerobically at 37°C for 48 hours. BD Phoenix™ automated identification and susceptibility testing system (fig.4) was used for identification of the colonies. The identified S. mutans Isolate was stored at -800C in LB-glycerol culture till needed17. It was to be used for initiating biofilm formation during the experiments and to prepare the standard curve for real-time PCR.
Standard suspensions of each isolate containing 107 S.mutans /mL were prepared
A 10 μL aliquot of each thawed and homogenized sample, that was stored previously, was streaked with a loop onto Petri dishes containing the MSB (Himedia, India) agar. The aliquots were seeded onto brain heart infusion (BHI) agar and incubated for 48 h at 37°C in a candle jar. After incubation, the microorganisms were cultured on blood agar (Oxoid, USA) for at least 18 h at 37°C in a candle jar. To confirm the identity of S. mutans, BD Phoenix™ automated identification and susceptibility testing system was used. The bacterial cultures were prepared using Mueller-Hinton broth (Oxoid, USA). Briefly 5ml of sterile Mueller-Hinton broth inoculated with colonies of S. mutans to a concentration
of 0.5McFarland standard as read by the BD PhoenixSpec™ nephelometer(fig.5) to make the standard S. mutans suspension18. This concentration was later diluted 1 to 10 to get 107 cells /mL.
Induction of biofilm formation by S.mutans.
The biofilms were grown on specimens of human teeth. The specimens were placed in sterile glass test tubes, suspended in each test tube by means of a ligature wire, with 1.8 mL sterile broth and were inoculated with 0.2 mL S. mutans standard suspensions. The ligature supports were made to suspend the specimens, with the crowns submerged to the cementoenamel junction, to simulate the oral cavity. They were then placed in candle jar and left in the incubator at 37°C for 24 hours.
To induce biofilm formation each tooth was placed alone in a glass test tube with 2ml Mueller Hinton broth inoculated with 107 S.mutans cells / ml. Test tubes were incubated in a candle jar at 37°C for 24 hours.
Thermal and Mechanical aging
Teeth were brushed for 3000 strokes using brushing machine with soft bristled brushes with a constant force applied to each brush simulating the oral cavity for 5 months19.
Teeth were further subjected to a thermocycling protocol (SD Mechatronik, Feldkirchen-Westerham, Germany) and were cycled in water between 10˚C and 50˚C with a dwell time of 30s and a transfer time of 10secs for 500 cycles.20
Assessment of the number of S. mutans attached on to the surface of the teeth:
Real time PCR were used. The investigator carrying out the PCR reactions was blinded to which samples belonged to which group. At each stage, the specimens were washed by dipping in physiologic saline, placed in tubes with 10 mL sterile physiologic solution (0.85% NaCl) and vortexed for 3 minutes.
Extraction of chromosomal DNA
The extraction of DNA was as described by Oho, Yamashita, Shimazaki, Kushiyama, and Koga (2000)21. Briefly 5ml of bacterial suspension in saline were centrifuged at 13000 rpm for 1 minute to obtain bacterial pellet. Pellet was resuspended in 200 µl of Tris-EDTA buffer and incubated in boiling water bath for 10minutes. After centrifugation at 13000rpm for 10minutes, the supernatant was used as a source for bacterial DNA.
Real Time PCR
The PCR Technique was as described by Yano, Kaneko, Ida, Yamaguchi and Hanada (2002)22. PCR was used to quantify S.mutans in biofilm molecularly. Primer Pairs were selected targeting a conserved region of S.mutans. The sequence of the forward gtfB primer, Smut3368-F, was: 5′-GCCTACAGCTCAGAGATGCTATTCT-3′. The sequence of the reverse gtfB primer, Smut3481-R, was: 5′- GCCATACACCACTCATGAATTGA-3′. The PCR was performed in 15µL final reaction volume containing 7.5µL sybr green master mix, (Thermoscientific, Lithuana), 0.5µL of each primer (10pmol/µL) and 1µL of DNA extract, 5.5µL of distilled water with 0.07 Rox dye. A standard curve was instituted using a series of 10 fold serial dilutions liquid culture. PCR was conducted using Strategene mx 3000P a real time PCR detection system with the following conditions: 95°C for 15minutes for enzyme activation followed by 40 cycles of two temperature PCR with denaturation at 95°C for 15s followed by annealing and extension at 60°C for 40s.