Propolis, chemicals and reagents
Malaysian propolis was collected from Pahang, Malaysia with the following geographical coordinates: north latitude 3.8126°, east latitude 103.3256° and height of 12 m above sea level. To study the content of Malaysian propolis, reversed phase high performance liquid chromatography (RP-HPLC) analysis was carried out. The flavonoids such as pinocembrin, kaempferol and quercetin were identified to be in the highest concentration in Malaysian propolis.
Preparation of Ethanolic extracts of Malaysian propolis
Propolis was manually cut into small pieces and a 20% (w/v) extract of propolis free from wax was prepared using 80 % ethanol under constant agitation in a rotary shaker (Certomat Model S II, Sartorius, Goettingen, Germany) at 200 rpm, 37oC for 48 hours. This was later centrifuged (Eppendorf Model 5810 R, Hamburg, Germany) at 3000 rpm for 15 mins, filtered through Whatman no.1 filter paper and subjected to distillation under reduced pressure using a rotary evaporator (Buchi Rotavapor R–215, Flawil, Switzerland) at the set pressure 175 mBar, temperature 52 °C and speed 95 rpm to remove the solvent. The ethanolic extract of propolis was then placed in a glass container and left for approximately 3 days for the residual solvent to evaporate; as a result, the extraction yield (Final weight/Initial weight x 100) was determined for extracts of propolis.
Preparation of propolis nanoparticles
Propolis nanoparticles were prepared by the ultrasonication method and compositions of the formulation are shown in Table 1. Propolis and tween 80 (stabilizer) were added to a conical centrifuge tubes containing distilled water. Then, the above mixture was briefly mixed in a vortex mixer for 1 minute followed by sonication for 20 minutes using a probe-type sonicator. To avoid thermal degradation of propolis during the sonication, formulation tubes were kept in an ice bath.
Particle size analysis
The particle size distribution and polydispersity index of the propolis nanoparticles were determined by dynamic light scattering using Zetasizer Nano S90 (Malvern, Worcestershire, UK) at fixed angle of 90°. The formulation was suitably diluted with distilled water and measured at 25°C. Data were collected after 2 minutes of equilibration time and averaged over three measurements (Figure 1).
Microorganism
A single colony of E. faecalis (ATCC 29212) was used in this study. The medium used was tryptic soy broth (TSB) (BD DifcoTM, NJ, USA).
Dentine block specimens
A total of 210 extracted intact human anterior single canalled teeth with complete root formation were selected for this study. The teeth were cleaned and stored in saline during all procedures to avoid dehydration. A middle third of the root (6mm in height) was obtained by sectioning the tooth below the cementoenamel junction and the apical part of the root using a slow speed diamond disc (Bredent®, Wittighausen, Senden, Germany) mounted on a milling machine under water cooling. The root canal was enlarged to an internal diameter of 0.9mm using Pesso Reamer no. 2 (Mani®, Utsunoniya, Tochigi, Japan) in a slow speed hand-piece (Kavo, Charlotte, North Carolina, USA) to standardize the internal diameter. The smear layer was removed using 5.25% NaOCl (Clorox®, Oakland, California, USA) and 17% EDTA (Calasept®, Nordiska Dental, Ängelholm, Skåne Country, Sweden) for two minutes with EndoActivator (Dentsply, Weybridge, Surrey, UK). The dentine blocks were thoroughly rinsed with sterile saline and autoclaved (LTE®, Oldham, Lancashire, UK) for 20 minutes at 121°C. The outer surfaces of the specimens were covered with nail varnish to prevent contact of the E. faecalis or any irrigant with the external surface. The dentin blocks were fixed to the petri dishes with wax but before that, the apical end of dentine blocks was blocked using a thin small square of sterilized parafilm (Parafilm M®, Brand, Wertheim, Baden-Württemberg, Germany) to prevent any softened wax from entering the canals.
Inoculation of dentinal blocks with E. faecalis
E. faecalis were suspended in 20.0 ml of TSB broth. The cell suspension was adjusted to match the turbidity of 1.5 x 108 CFU mL–1 (equivalent to 0.5 McFarland standards). Five hundred microliters of E. faecalis suspension were transferred into the dentine blocks with the use of sterile 5.0mL syringes (Terumo®, Somerset, New Jersey, USA) with 30-gauge needles (Terumo, Somerset, New Jersey, USA) in a sterile laminar flow hood. The coronal part of the dentine blocks was then sealed immediately using parafilm (Parafilm M®, Brand, Wertheim, Baden-Württemberg, Germany). The dentine blocks were incubated at 37°C for 21 days, with renewal of E. faecalis every 3 days.
Disinfection of dentinal blocks
Following the inoculation period, 210 dentine blocks were randomly divided into seven groups (n = 30) according to the different endodontic irrigants: Group I: Saline, Group II: Propolis 100µg/ml (P100), Group III: Propolis 300µg/ml (P300), Group IV: Propolis Nanoparticle 100µg/ml (PN100), Group V: Propolis Nanoparticles 300µg/ml (PN300), Group VI: 6% Sodium Hypochlorite (6% NaOCl) (Calasept, Upplands Väsby, Sweden), Group VII: 2% Chlorhexidine (CHX) (Calasept, Upplands Väsby, Sweden). 5 ml of each endodontic irrigant was placed into the root canal using a 30-gauge side vented needle (Endo-EZE, Ultradent) Each group was further divided into three subgroups based on the time period for 1, 5 and 10 minutes.
Collection of dentinal shavings
At the end of the experimental periods, the dentine blocks were removed from the petri dishes and the canals were dried with sterile paper points. Samples of dentinal shavings were collected after one minute of exposure A1, B1, C1, D1, E1, F1 and G1, after five minutes of exposure for A2, B2, C2, D2, E2, F2, and G2 and after ten minutes of exposure A3, B3, C3, D3, E3, F3 and G3. Dentinal shavings were collected using Pesso Reamer (Mani®, Utsunoniya, Tochigi, Japan) size no. 4 (1.3 mm diameter) followed by size no. 6 (1.7 mm diameter), in a low speed handpiece (Kavo®, Charlotte, North Carolina, USA).
Antimicrobial assessment
The collected dentinal shavings were transferred into a micro-centrifuge tube (Axygen, NY, USA) containing 1ml sterile TSB broth. A sterile micro tip was used to take 0.1ml of broth containing dentinal shavings, transferred to another tube containing 0.9ml sterile TSB broth. The content of each tube was serially diluted from 10–1 until 10–4. 300µl of the diluted shavings was spread evenly using a L-shaped glass rod and triplicated on three occasions. These plates were incubated for 24 hours at 37°C and the colonies were counted and readings were tabulated.
Total numbers of colony forming units were calculated for assessing the remaining vital viable microbial population. The SPSS computer software version 18.0 (SPSS Inc., Chicago, Illinois, USA) was used to perform statistical analysis. The values were analysed using non-parametric Kruskal-Wallis test and Mann Whitney U test to compare the reduction of E. faecalis between all intracanal medicaments. Probability values of P < 0.05 were set as the reference for statistically significant results.
SEM Analysis
Dentinal blocks (n = 3) were prepared as described in the methodology mentioned in materials and methods section. E. faecalis ATCC 29212 was cultured in 10 ml TSB broth supplemented with 8% sucrose (pH 7.4) with a minimal amount of xylitol (0–2%) at 37oC for 48 h and later incubated at 37oC for 24 h. After 4000 rpm centrifugation for 15 minutes, each of the cell pellet was washed three times with sterile phosphate buffered solution (PBS, 0.01 M, pH 7.2), re-suspended (O.D reading of 0.11 at 660 nm) in 100 ml of the respective growth medium and adjusted to a concentration of 0.5 McFarland (108 cells/mL) before use. Five millilitres of TSB broth were mixed with equal weight of bacterial inoculum using sterilized syringes of sufficient volume to fill the root canal during 21 days’ period. After 21 days, endodontic irrigants were placed according to the groups mentioned above. Two parallel grooves were made onto the external surfaces, in a mesiodistal direction, to facilitate a split fracture. Final separation was made using chisel and hammer. All specimens were dehydrated in ascending grades of ethanol (35%, 65%, 85%, 95%, 2x100%, for 20 min in each) and immediately transferred to the pressure chamber of the critical point drying machine (CPD 30; Leica). The specimens were mounted on aluminium stubs with double-sided conductive tape, sputter-coated with 30 nm-thick layer gold/palladium (120 s) and examined using SEM (Philips/FEI XL30 FEG SEM) at an accelerated voltage of 5kV. Different magnifications and images were observed to evaluate the qualitative reduction of E. faecalis. Four-score scale system based on percentage of residual isolated microbial cells was used to assess the microbial coverage on SEM images of the canal walls [26].
CLSM Analysis
After disinfection solution regimen, specimens were rinsed in 0.1wt% fluorescein for 24 h and rinsed with deionised water. Determination of bacterial viability was done using Live/Dead kit (BacLightTM kit L–13152; Molecular Probes, Inc., Eugene) with dyes applied for 5 min at a 1:1 ratio and examined using confocal laser scanning microscope (CLSM; Leica Fluoview FV 1000, Olympus, Tokyo, Japan) equipped with a 60 × /1.4 NA oil immersion lens using 480–500 nm argon/helium and a 633 nm krypton ion laser illumination both in reflection and fluorescence modes. This was adjusted to 473-nm laser emission for SYTO 9 and to 559 nm emission including propidium iodide. Reflected and fluorescence signals were detected with a photomultiplier tube to a depth of 20 μm and then converted to single-projection images for better visualization and qualitative analysis. This was maintained at a resolution of 512 pixels with a zoom factor of 1.0, giving a final pixel resolution of 0.41 mm/pixel. Stacks of fluorescent images obtained of the biofilm were examined using bioimage L software (v.2.0. Malmő, Sweden), which provided information on the structure of the biofilm, including green and red-stained bacteria volume on a two dimensional x-y section based on color segmentation algorithms written in MATLAB.
E. faecalis isolates from patients with failed root canal treatment (Antibacterial effect of PN as an endodontic irrigant in clinical samples)
Patients were selected from those who attended the clinic, with a need for non-surgical endodontic re-treatment. A detailed medical and dental history were obtained from each patient. Patients who have received antibiotic treatment during the last three months or have a systemic disease were excluded from the study. Ten teeth were included; all have been previously root filled and show radiographic evidence of apical periodontitis. Failure of root-canal treatment were determined on the basis of clinical and radiographical examinations. Tooth type, clinical signs and symptoms such as tenderness on percussion presence or absence of a sound coronal restoration, caries, sinus, swelling, periodontal status of the tooth, mobility, status of the root canal in terms of any exudate, timing and radiographic quality of the root-canal filling were recorded. After obtaining the consent the procedure was carried out. An access cavity was made without the use of water spray under manual irrigation with sterile and by using sterile high-speed diamond bur. Root-filling material was removed by rotary instrumentation and K-files (Dentsply-Maillefer, Ballaigues, Switzerland) in a crown-down technique without the use of chemical solvent, accomplished by irrigation with sterile saline. A sterile pyrogen-free paper point (Dentsply-Maillefer, Ballaigues, Switzerland) was then introduced into the full length of the canal and retained in position during 60 seconds for sampling. Culture procedure was done using the selective E. faecalis plates (Slanetz Bartley Agar (m-Enterococcus A.), Liofilchem, Italy) and the CFUs were grown. Thereafter, the grown E. faecalis were suspended in 20.0 ml of TSB. The cell suspension was adjusted to match the turbidity of 1.5 x 108 CFUs mL–1 (equivalent to 0.5 McFarland standards). One ml of E. faecalis suspension was transferred into the Eppendorf tube and 50 microlitre of each irrigant according to these seven groups including group I: saline, group II: P100, group III: P300, group IV: PN100, group V: PN300, group VI: 6% NaOCl, group VII: 2% CHX. 50 microlitre of each endodontic irrigant was placed into 1 ml of E. faecalis suspension into the Eppendorf tube for one minute, five and ten minutes. After this exposure, the content of each tube was serially diluted and this was spread evenly onto the agar plate using a L-shaped glass rod and triplicated on three occasions. These plates were incubated for 24 hours at 37°C and the colonies were counted, and readings were tabulated.