Abstract
Objectives
Evaluate a new modified quaternary ammonium silane irrigant solution for its antimicrobial, cytotoxic and mechanical properties of dentine substrate.
Methods
Root canal preparation was performed using stainless steel K-files™ and F4 size protaper with irrigation protocols of 6% NaOCl + 2% CHX; 3.5% QIS; 2% QIS and sterile saline. Biofilms were prepared using E. faecalis adjusted and allowed to grow for 3 days, treated with irrigants, and allowed to grow for 7 days. AFM was performed and surface free energy calculated. MC3T3 cells were infected with endo irrigant treated E. faecalis biofilms. Raman spectroscopy of biofilms were performed after bacterial re-growth on root dentine and exposed to different irrigation protocols and collagen fibers analysed collagen fibers using TEM. Antimicrobial potency against E. faecalis biofilms and cytoxicity against 3T3 NIH cells were also. Resin penetration and MitoTracker green were also evaluated for sealer penetration and mitochondrial viability. Data were analysed using One-way ANOVA, principal component analysis and post – hoc Fisher’s least-significant difference.
Results
Elastic moduli were maintained amongst control (5.5 ± 0.9) and 3.5% QIS (4.4 ± 1.1) specimens with surface free energy higher in QIS specimens. MC3T3 cells showed reduced viability in 6%NaOCl+2%CHX specimens compared to QIS specimens. DNA/purine were expressed in increased intensities in control and 6% NaOCl + 2% CHX specimens with bands around 480−490 cm −1 reduced in QIS specimens. 3.5% QIS specimens showed intact collagen fibrillar network and predominantly dead bacterial cells in confocal microscopy. 3.5% QIS irrigant formed a thin crust-type surface layer with cytoplasmic extensions of 3T3NIH spread over root dentine. Experiments confirmed MitoTracker accumulation in 3.5% treated cells.
Significance
Novel QIS root canal irrigant achieved optimum antimicrobial protection inside the root canals facilitating a toxic effect against the Enterococcus faecalis biofilm. Root dentine substrates exhibited optimum mechanical properties and there was viability of fibroblastic mitochondria.
1
Introduction
The main objective of root canal treatment procedures is to promote healing of apical periodontitis induced by the micro-organisms and their toxins [ ]. This is either the source of primary infection or associated with failure of secondary or persistent root canal treatments [ ] Complex biofilms are adhered to the intraradicular dentine [ ] extending within the root canal system including lateral canals, isthmi and recesses. Residual bacteria survive in these areas putting the entire treatment outcome at risk in the presence of a newly found nutrient supply [ ]. The infected root canal can harbor many bacterial species including Treponema, Streptococcus, Fusobacterium, Porphyromonas as well as other cultivable species [ ]. Interactions of these biofilm community members may result in symptomatic endodontic infections [ ] and swelling [ ]. Enterococcus faecalis (E. Faecalis) is prevalent in root canal infections, being one of the most common microorganism in intraradicular infections [ ], that survive in harsh environments of high alkalinity and it is resistant to many antimicrobial agents [ ]. It is difficult to fully eradicate E. faecalis bacterium colonised inside the root canal using antimicrobial agents [ ]. Due to the emergence of antibiotic resistance for many bacterial species, new disinfection protocols are obviously needed.
The purpose of a root canal treatment is to remove pulp tissues, microorganisms and potential substrate for microbial regrowth [ ]. Complete elimination of bacteria and their metabolic by-products have rarely been observed using chemo-mechanical instrumentation and intracanal medications [ ]. Traditional non-specific antimicrobials and irrigants appear to be limited in controlling the infection. Dakin’s solution, an antiseptic consisting of a dilute solution of sodium hypochlorite (NaOCl), is one of the most important types of chlorine-releasing agents that has been used as primary root canal irrigant as early as the 1920s [ ]. In the decomposition reaction, release of chloride ions and oxygen is possible. Because of its prominent antimicrobial properties and ability to dissolve organic tissue remnants [ ], NaOCl still remains the most pivotal irrigating solution employed in the contemporary endodontic practice [ ]. The tissue-dissolving capability is primarily related to its concentration. The recommended concentration of NaOCl ranges from 0.5% to 6% in the past, with no consensus on the ideal concentration [ ]. Apart from its tissue dissolution properties, NaOCl has deleterious effects on the mineralised dentine canal wall, damaging the dentine integrity [ ]. Previous studies have pointed out changes in the composition of dentine with exposure to NaOCl irrigation protocols during root canal therapy [ ] reducing the mechanical properties (strength) of dentine predisposing teeth to fracture [ ]. Moreover, NaOCl irrigant solution is also known for its hypersensitivity or allergic reactions [ ] including palatal tissue necrosis [ ], lip burning and diffuse pain [ ], which have been reported in the literature as complications caused by the inadvertent handling of NaOCl. With the use of NaOCl and chlorhexidine (CHX) as irrigants, a brownish-orange precipitate is formed commonly known as parachloroaniline (PCA). This primarily causes discolouration [ ] and subsequent resistance to free flow of resin material due to blockage of laterals canals causing inadequate sealing of obturating materials [ ]. Hence, there is a constant need to find effective but also safe irrigating solutions which display excellent antimicrobial properties and good biocompatibility with the surrounding tissue.
Silanes are synthetic inorganic–organic hybrid C Si compounds used in numerous dental applications [ ]. A quaternary ammonium silane (QAS; codenamed K21; C92H204Cl4N4O12Si5; CAS number 1566577-36-3) was synthesized using the sol-gel reaction, by allowing tetraethoxysilane to react as the anchoring unit and 3- (triethoxysilyl)-propyldimethyloctadecyl ammonium chloride using a molar ratio of 1:4 [ ]. This promising class of contact-killing antibacterial has a broad spectrum and low toxicity [ ]. The antimicrobial activity is attributed to its long, lipophilic –C18H37 alkyl chain penetrating the bacterial membranes causing autolysis and cell death [ ]. The quaternary ammonium silane is covalently grafted via Si-O-Si linkages due to the presence of silanol groups [ ]. The presence of tetraethoxysilane (TEOS), enables a three-dimensional network comprised of silicate units with condensation of tetra and tri -ethoxysilane species with remaining silanol (-Si−OH) groups [ ]. When applied to acid etched dentine, this silicate network condenses within the dentinal substrate providing a long term antimicrobial effect. In general, such compounds have positively charged nitrogen atoms in their lipophilic chemical structures, making them favourable to be taken by mitochondria inhibiting mitochondrial oxidative phosphorylation at low concentrations [ ]. Mitochondrial dysfunction by inhibition of oxidative phosphorylation via quaternary ammonium toxicity is a known process causing cell apoptosis [ ]. Bacterial infection can enhance apoptosis of osteoblastic cells promote programmed osteoblast death with fate decided between the interaction of pro and anti-apoptotic proteins [ ]. E. faecalis is known to also survive in harsh environments possessing complicated virulence factors such as lipoteichoic acid (LTA) and gelatinase, enabling its strong pathogenicity [ ].
This antimicrobial irrigation system was inspired by our previous work to formulate a new endodontic irrigant, as its function as an irrigant against the traditional NaOCl irrigants was still unknown. One of the most promising novel approaches to overcome endodontic infections was to use QAS as reported by Daood et al. [ ]. Therefore, in this study, as a potential determinant, it was hypothesized that the incorporation of QIS (Quaternary Ammonium Silane Irrigant Solution) as a pure endodontic irrigant, and not as a mixture with NaOCl, can bring significant and safe changes in endodontics. In addition, other effects such as antibacterial potency, cytotoxicity effect of QAS, its potential to reduce apoptosis and mitochondrial effect were evaluated along with an extensive study of E. faecalis biofilms. The null hypothesis tested in the present laboratory study was that there is no difference in antibacterial properties of QIS used as a potential endodontic irrigant when compared to other potent solutions (NaOCl, NaOCl + CHX).
2
Materials and methods
Extracted human mandibular third molars ( n = 120 ) with fused roots (mesiobuccal canal attempted) and extracted non-carious single rooted anterior teeth ( n = 90 ) were collected after attaining patient’s informed consent. The teeth chosen had complete root formation, were sound with no carious lesion. Teeth collection was approved by the Institutional Review Board of International Medical University Kuala Lumpur and stored in 0.5% Chloramine-T solution for 3 weeks and then in distilled water at 4 °C until use. All soft tissues around the teeth were removed by immersing the teeth in 5% hydrogen peroxide and gently removing any remains using spoon shaped excavators before use. Unless stated otherwise, the disinfectant solution is provided by fiteBac Kimmerling Holdings Group, LLC (KHG).
2.1
Synthesis of 3.5% QAS/k21 Endo irrigant QAS/k21 endo irrigant
The k21 silane endodontic dispersions were prepared by appropriately diluting the as received k21 [(QAS(k21); KHG FiteBac ® Technology, Marietta, GA, USA)] organosilane in absolute ethanol (99.6%) to achieve 3.5% concentration (adjusting with absolute ethanol). A 3.5% concentration was selected after prior string of results using different concentrations of QAS/k21 disinfectant on E. faecalis biofilms and observing effect on extracted dental pulp tissue in-vitro (data not shown). Next, 0.3 ml NaOH (M r 39.997 g/mol = 0.025 m/l) and 0.3 ml EDTA (M r 292.24 g/mol = 0.003 mol/l × 3 times) as a disodium salt were blended with 0.75 mg of alexidine ( a bis -biguanide). The pH of the dispersions was adjusted with 1.0 M acetic acid solution or potassium hydroxide in a 100 ml round bottom flask. The mixture above was blended with dispersions for at least 10 min to co-disperse all ingredients to produce an even mix with 3.5% k21 silane under inert environment. The k21 silane dispersion was then added slowly with 0.75 mg DDAB, dimethyldioctadecylammonium (M r 631 g/mol; C 38 H 80 NBr), further immersed in a glass tubings set up at 30 °C. The reaction mixture was further stirred for 2 h. The solution was kept for 2 h in a sealed vial at ambient temperature. The solution was finally filtered with a target pH ranging between 5-6.5 ( details unprovided ). The Raman spectroscopy analysis revealed presence of water, ethanol and all-important silanol groups. Open Si-O-Si cyclic species are identified similar to the QAS disinfectant previously used ( Fig. 1 A–B).
2.2
Tooth preparation
After cleaning teeth using periodontal curettes, and rinsing with sterile saline, the teeth were mounted on an isomet cutting machine (Buehler IsoMet Low Speed, Lake Bluff, IL, USA) under water cooling to remove the occlusal crown completely from just below the cemento-enamel junction to obtain the roots of teeth.
3
The irrigation protocol
Root canal procedures were performed by a trained endodontist (Senior Consultant Faculty) using stainless steel K-files (no. 10-15-20) (Dentsply Sirona, Tulsa Dental, USA) canals for shaping the canals using watch wind motion. Protaper™ rotary file system (Dentsply Maillefer, Ballagues, Switzerland) was used up to file size F4 at a speed of 300 RPM. The working length was kept 1 mm short in the entire procedure in all samples length equal to 11 mm. Crown down technique was performed using #2 and #3 gates glidden drills (Dentsply Maillefer, Ballaigues, Switzerland) with apical portion prepared using #35 file (as master apical file). Irrigation was performed using 2 ml of 6% NaOCl (Calasept, Upplands Väsby, Sweden) after each use. Next 6% NaOCl (2 ml) and 17% (2 ml) EDTA (Pulpdent Corporation, Warwick, UK) were used for 2 min. enabling removal of inorganic smear layer and subsequently rinsed with sterile saline. After autoclaving (121 °C, 15 lbs psi), all teeth were randomly divided to one of the following irrigation protocols of 2.5 ml each ( n = 5 ): Group A: 6% NaOCl; Group B: 6% NaOCl + 2% CHX; Group C: 3.5% QIS; Group D: 2% QIS and Group E: Sterile Saline. (Ultrasonic agitation was performed for all groups (VDW Ultra, VDW GmbH, Munich, Germany; tip set of E8 for 30 s).
3.1
Biofilm preparation
For biofilm formation , E. faecalis (ATCC 29212™) was used in Brain Heart Infusion broth (BHI; Difco Laboratories, Detroit, MI, USA) and adjusted at the 1.5 McFarland turbidity standard. Bacteria were cultured on blood agar plates at 37 °C for 20 h anaerobically. The bacterium was then transferred in brain heart infusion (BHI) broth supplemented with 8% sucrose (pH 7.4) and a minimal amount of xylitol (0–2%) at 37 °C for 48 h. The cells were than centrifuged at 4000 rpm for 15 min and cell pellets washed three times with phosphate buffered solution (PBS, 0.01 M, pH 7.2). The cells were suspended in 100 ml of the growth medium and adjusted to a concentration of McFarland standard no. 3 (109 cells/mL). Using sterilizing syringes, 5 ml of BHI broth and bacterial inoculum were used to fill the canals and allowed to react for 3 days. After 3 days, the irrigation protocol was performed and the bacteria allowed to grow for 7 more days. After 7 days, each tooth was dried with sterile paper points under aseptic conditions. Parallel grooves (2) were made on external surfaces across the mesio-distal direction facilitating a split fracture performed by one operator only. Roots were then separated using a chisel and a hammer and teeth taken for scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM) and Raman analyses.
3.2
AFM instrumentation
A nano wizard II atomic force microscope (JPK Instruments, Berlin, Germany) with CSC38/NO AL (Mikromasch, Tallinn) probe and cantilever B was used: resonance frequency, 10 kHz, cone with a full cone angle of 40°, force constant, 0.03 N/m, and thickness, 1.0 m. The cantilevers were previously washed with sterile MilliQ water as all AFM procedures were performed at room temperature. AFM imaging was performed using the contact mode with a scan rate of 0.1 to 0.5 Hz with a retraction speed of 2 μm/s. The Young’s modulus was used to quantify the stiffness of the sample.
3.3
Surface free energy
With each irrigation protocols, 4 mm dentine blocks were obtained by horizontally sectioning below cemento-enamel junction (1 mm) for each group. After splitting the blocks into semi-cylindrical halves, different irrigant formulations were applied in the form of a drop (2 μL) and profile recorded using contact angle analyzer (Dental Simulation Lab, IMU Laboratory). The contact angle was calculated using t/Image J software fitting within the contour of the droplet after placing on the dentine surface. The following Eq. (1) was used to calculate the free surface energy [ ]:
where L and S subscripts represent different phases, θ and ϒ are surface tension and contact angle of the reference liquid used. D, + and – represent the Lewis and dispersive phases. All images were captured in 5 min after placement on dentine surface.
3.4
MC3T3 cells infected with Endo irrigant treated E. Faecalis biofilms
α-MEM was used with 10% FBS (Invitrogen, Carlsbad, CA, USA) at 37 °C for culturing of mouse osteoblastic MC3T3-E1 cells (ATCC, Manassas, VA, USA) in presence of 5% CO 2 and saturated humidity. E. faecalis (ATCC 29212) was cultured in Tryptone Soya Medium (Hardy Diagnostics, Santa Maria, CA, USA). A single colony was picked and grown at 37 °C in their respective medium to get the microbial suspension for experimental conditions. The microbial suspension’s turbidity was adjusted to 0.5 McFarland standard. About 10 ml of the microbial suspension was taken in a sterile tube for E. faecalis with respective media. The pre-prepared sterilized dentine blocks were aseptically placed into the tubes with the microbial suspension and allowed to grow for 2 days. Fresh microbial broth was replaced on the third day and maintained up to 21 days. All the experiments were performed in an aseptic condition under a BSL II laminar flow hood (Bioair Safemate, Italy).
The mouse osteoblasts cells were grown overnight in 96-well plates at 3.5 × 10 3 /well to investigate the effects of E. faecalis on osteoblasts. The density of osteoblasts was maintained at 8 × 105/well and incubated with E. faecalis at a multiplicity of infection (MOI) of 1,000:1 for 3 h. Once the cells were infected, the cells were treated with 5 mg/mL nisin for 3 h along with 10 μg/mL of ciprofloxacin. The mouse osteoblastic cells were cultivated for 6, 12, 36 and 48 h and cells centrifuged at 2000 rpm for 10 min. The cells were incubated with CCK-8 solution and optical density measured at 450 nm (triplicates taken) using a microplate reader (Tecan, Reading, UK); while blanking was conducted with the culture supernatant which was untreated.
3.5
Raman spectroscopy of biofilms
After removing the dentine discs from culture plates, the specimens were dried for 20 min at 35 °C and placed on x-y-z-axis-Raman positioning stage on a low fluorescent quartz microscopic slide. Horiba Jobin Yvon LabRam HR800 micro spectrometer (Horiba Jobin Yvon, NJ, USA) was used for spectrum acquisition using a 100x objective: 514.5 nm green laser excitation, 785 nm with argon ion (with spectral resolution 1.6 cm −1 ), <500 μW were applied. Dark count correction with noise removal was carried out for all spectra with normalization with spectral peaks calculated with OriginPro 8.5.1 software (Origin Lab, Northampton, MA, USA) and Rayleigh scattering photons blocked with a notch filter that has a spectral range of −120 to 130 rel cm −1 , with an ellipsoid measurement of approximately 1.0 μm. The spatial resolution was maintained with 350 nm and 2.0 μm in the horizontal plane. Raman peaks were centered around 434 cm −1 (stretching vibration of ν2PO 4 ) [ ] and 484 cm −1 (polysaccharides or carbohydrates) [ ] and 1665 cm -1 .
Thirty-six spectra from each spectrum were used for spot-to-spot location for calculation of coefficient of variation. The average spot was calculated location-to-location for the sample. The values were determined by using mean and dividing the standard deviation (SD) to the mean and multiplying it by 100. Also the different disinfectant was successfully discriminated using Principle Component Analysis (PCA).
3.6
Transmission electron microscopy of collagen
Root dentine blocks were exposed to different irrigation protocols to simulate the contact of various irrigation solutions. The specimens ( n = 5 ) were then thoroughly washed with deionized water and split into halves for TEM sections. To evaluate the root dentine collagen structure after irrigation protocols, each specimen was completely demineralized in 0.1 M formic acid/sodium formate (pH 2.5). The specimens were fixed with Karnovsky’s fixative (2.5 wt% glutaraldehyde and 2% paraformaldehyde in 0.1 mol/l cacodylate buffer; pH, 7.3) for 8 h, and post fixed using 1% osmium tetraoxide (OsO 4 ) for 1 h. Next, the specimens were subjected to dehydration using ascending grades of ethanol (75%-100%) with propylene oxide as a transition medium and finally embedded in pure epoxy resin. An ultramichrotome (Leica Microsystems, Inc., Bannockburn, IL, USA) was used to cut 90 nm thick sections of dentine specimens which were examined unstained using 2% uranyl acetate and lead citrate. The images of sections were taken using JEM-1230 TEM (JEOL, Tokyo, Japan) at 110 kV.
3.7
Confocal analysis and CFU log of E. Faecalis biofilms
Live/Dead Baclight bacterial viability (molecular probe #L7012 LIVE/DEAD BacLight stain; Invitrogen) was used to analyse viability of bacteria in single- specie biofilms on dentine was using a confocal light microscope (Fluoview FV 1000, Olympus, Tokyo, Japan). After mixing the stain according to manufacturer’s instructions, the biofilm was imaged between 500 and 550 nm with an excitation wavelength of 488 nm and × 100 objective lens for direct observation. Each specimen was examined using bioimageL software (v.2.0. Malmö, Sweden) showing green-red staining based on colour segmentation algorithms in MATLAB™. Percentages for live and dead bacteria were calculated.
E. faecalis biofilms from the root dentin specimens were collected in 1 mL sterile BHI broth at a pH of 7.4 and further incubated for 24 h at 37 °C. A 100 μL of each broth from different groups was centrifuged (Centrifuge 5430 R; Eppendorf AG, Hamburg Germany) five times in 100 μL of PBS inside eppendorf tubes (plastibrand micro centrifuge Tube/Z334006; Sigma-Aldrich). Once completed, 5 μL of each serial diluted sample was played on selective BHI agar plates for incubation for 24 h. The microbial colonies in colony-forming units [CFU/mL] were counted and further converted to log CFU.
3.8
SEM and resin penetration
Randomly selected dentine root canals ( n = 3 ) were subjected to different irrigant protocols according to the groups. After making two parallel grooves in the mesio-distal direction, a chisel and hammer was used to create a split fracture. The specimens were then transferred in in ascending grades of ethanol (33%, 66%, 85%, 95%, 2 × 100%, for 20 min in each) for dehydration. Later, the specimens were transferred to critical point drying machine (CPD 30, Leica). Two 1 mm slices were cut and polished using 1200 silicon carbide paper for analysis of interface morphology, between resin-based dentine sealer and root dentine. After etching specimens with 35% ortho-phosphoric acid and washed using deionised water for 10 s, specimens were immersed in 5.25% NaOCl solution for 15 min and rinsed under running water for 5 min. Specimens were fixed using osmium tetra oxide and again immersed in ascending grades of ethanol for 20 min. The specimens were air dried, and mounted on stubs for gold sputtering for 120 s under vacuum to analyse in Philips/FEI XL30 FEG at an accelerating voltage of 10 kV.
3.9
Mitotracker
The NIH 3T3 mouse fibroblastic cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Sigma–Aldrich Corp., St. Louis, MO, USA) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (10,000 U/100 g/mL) inside a humidified incubator with 5%CO 2 incubator at 37 °C. After expanding the cell culture to 80% confluence, the cells were utilized at sixth passage. The cells were seeded on the pulpal side of dentine discs in 12-well plates ( n = 10 ) in an incubator with 5% CO 2 and 98% air at 37 °C. The cells received the irrigant treatments using saturated microbrush for 20 s. The cells were then incubated with 200 nM MitoTracker Green (mitochondria-specific fluorescent dye; CN M7514- San Francisco, USA ) for 20 min and analysed using Axiovert microscope (Carl Zeiss) equipped with a Neofluar 100 NA 1.3 objective (488 nm laser, 490 nm excitation wavelength and 516 nm emission wavelength).
4
Statistical analysis
All experiments in this paper were repeated thrice and the results/data were presented as mean ± standard deviation (SD) using SPSS 20.0. The expression p < 0.05 stand for statistically significant, **p < 0.01 for very significant and ***p < 0.001 for extremely significant values, respectively. The microbiological (percentage of live and dead bacteria) and mechanical property (young’s modulus) experiments were analysed using One-way ANOVA followed by the Tukey’s test showing 95–99% significance level. The PCA was used for statistical analysis to identify unique characteristics of each irrigant tested against the biofilms from the original spectral data. Data were also analyzed using the post-hoc Fisher’s least-significant difference analysis for multiple comparisons at p < 0.05 .
5
Results
Table 1 shows the values of elastic modulus of different experimental dentine sections are essentially the same as the bulk tissue value. No significant differences were obtained using One-Way Anova amongst control (5.5 ± 0.9), 3.5% QIS (4.4 ± 1.1) and 6% NaOCl (2.9 ± 1.4) specimens (p < 0.05). Fig. 1 includes the representative data from all experimental groups at locations in each root dentine specimen. The images depict high peaks in control and 6% NaOCl+2% CHX specimens indicating presence of some biofilms. The experimental specimens treated with QIS irrigants demonstrated a great effectiveness in the action against formed biofilm with peaks upto 1.1 μm.
| Groups | Elastic Modulus GPa | Surface Free Energy m/J/m 2 | osteoblastic apoptosis 6 12 36 72 |
||||
|---|---|---|---|---|---|---|---|
| Control | 5.5 ± 0.9 A | 69.4 m/J/m 2 ±22.1 A | 0.07 | 0.08 | 0.38 | 0.69 | α |
| 2%QIS | 3.8 ± 1.8 AB | 107.4 m/J/m 2 ±17.8 B | 0.08 | 0.09 | 0.61 | 0.83 | β |
| 3.5 %QIS | 4.4 ± 1.1 A | 146.7 m/J/m 2 ±9.9 C | 0.09 | 0.09 | 0.58 | 0.71 | ∞ |
| 6%NaOCl + CHX | 2.1 ± 3.1 BC | 87.8 m/J/m 2 ±26.7 D | 0.05 | 0.06 | 0.09 | 0.23 | ɸ |
| 6%NaOCl | 2.9 ± 1.4 B | 57.6 m/J/m 2 ±11.8 E | 0.03 | 0.03 | 0.05 | 0.11 | Ω |
It is clear from the results that the surface free energy of QIS specimens was higher than compared to other specimens. Table 1 shows that the QIS irrigation protocol increased the surface free energy with highest proportion of polar component. It can be seen that surface free energies for all specimens were different with increased values in 2% QIS (107.4 ± 17.8 m/J/m 2 ) and 3.5% QIS (146.7 ± 9.9 m/J/m 2 ) specimens promoting spreading of the irrigant. As compared to control specimens (69.4 m/J/m 2 ± 22.1), the values for 6% NaOCl+2% CHX (87.8 m/J/m 2 ± 26.7) were significantly higher. This was due to differences in the polar components of different specimens. The estimated ϒS D values remained different for all QIS specimens as increased values not just compared to controls, but also when compared to 6% NaOCl+2% CHX specimens.
The MC3T3 cells showed ascending in the line graphs for cell viability as time lapsed. They were observed to have significantly reduced in 6% NaOCl+2% CHX treated specimens compared to the control and QIS treated specimens at 6, 12, 36 and 72 h (p < 0.05). However, the viability of cells in the control group ( Table 1 ) was significantly lower at 36 and 72 h (p < 0.05) when compared with QIS specimens. Moreover, no significant differences were observed between the QIS specimens (0.8–0.9 OD) at any given indicated time point (p < 0.05).
The changes in the chemical compositions of biofilms grown by different irrigation protocols were characterised using Raman spectroscopy ( Fig. 2 ). There were several variations in peak intensities observed after treatment of treated groups where signature spectral peaks at 730 cm −1 , 484 cm −1 , 1580 cm −1 and 1670 cm −1 were observed. The band at 730 cm −1 is a strong band ( Fig. 2 A) attributed to the main components of a peptidoglycan, the C N stretching mode of N-acetyl- d -glucosamine or N-acetylmuramic acid. The intensity decrease at 730 cm −1 in 3.5% QIS specimens is due to disruption of purine bases. This can be correlated to the effect of QIS also seen in the 2% specimens. Different from QIS specimens, it was observed that DNA/purine entities were expressed in increased intensities in control, 6% NaOCl + 2% CHX and 6% NaOCl alone specimens. The bands around 480-490 cm −1 exhibited changes with different irrigation protocols ( Fig. 2 B). The bands were assigned to the ring ‘breathing’ of polysaccharide chains of the polysaccharide linkages. There was a striking reduction in signals in QIS specimens as compared to the NaOCl and control specimens which showed the presence of overlapped maxima with highest intensity signal seen in control specimens (p < 0.05) originating from the COC ring deformations. A similar decrease was seen in QIS specimens in the intensity of the peaks at 1575 cm −1 region, also referring to Amide II region and assigned to the ring-breathing modes of RNA/DNA bases, such as uracil, cytosine, adenine, guanine and thymine appearing at different positions in different specimens ( Fig. 2 C). The identified bands were the proteins as the main discriminant components for the biofilm formed and grown. The Amide I bands of the spectra are related to the molecular polypeptide chains of Type I collagen. The superimpositions of the Raman spectra within this region indicated increased intensities for QIS specimens with concomitant reduction in the intensity in NaOCl and control specimens ( Fig. 2 D). The PCA plots obtained from the biofilms ( Fig. 3 A–B) formed on substrates showed that E. faecalis could be differentiated significantly. The loadings of the PCs show the spectral variabilities at molecular level with loadings observed at PC1 and PC2 in 837 cm −1 , 1662 cm −1 , and 1046 cm −1 on dentine substrates of different groups ( Fig. 3 C), the highest loadings being the 6% NaOCl + 2% CHX and control specimens.
