Abstract
Objectives: The main objective of this research was to assess the formation of toxic volatile organic compounds (VOCs) and disinfection by-products (DBPs) following the interaction of sodium hypochlorite (NaOCl) with a model system of different sources of natural organic matter (NOM) present in infected root canals, including dentine powder, planktonic multi-microbial suspensions, bovine serum albumin and their combination. A novel tooth model was further developed to study the apical extrusion of VOCs and DBPs as well as their release as effluents, during instrumentation and irrigation of artificially infected root canal specimens, with 2.5% NaOCl and 17% ethylenediaminetetraacetic acid (EDTA). Finally, this research aimed to examine alternative concepts of root canal disinfection, following the preparation of silver nanoparticles (AgNPs) synthesized on an aqueous graphene oxide (GO) matrix (Ag-GO), with different irrigant delivery methods to enhance the disinfection regimen, using a novel ex vivo infected tooth model.Materials and methods: Dual proof of concept experiments were conducted to study the short-term oxidising effect of NaOCl at different concentrations and volumes on the structural integrity of mineralised dentine powder (MDP), at different masses, with the aid of attenuated total reflection Fourier Transform Infrared Spectrometer (ATR/FTIR). To examine the formation of chlorinated DBPs, the aliquots obtained from samples of MDP mixed with NaOCl or ultrapure water (UPW) were stirred for different time internals, from 30s to 90min. The analysis of the compounds was performed with gas chromatography coupled to a mass spectrometer equipped with electron impact ionisation source (GC-EI-MS). The use of selected ion flow tube mass spectrometry (SIFT-MS) was found appropriate to analyse the chemical derivatives present in aliquots, obtained from the real time interaction of 2.5% NaOCl with combined sources of NOM present in infected root canals. Samples were stirred at 370C in aerobic and anaerobic conditions for 30min to approximate a clinically realistic time. Single-rooted human teeth were decoronated to obtain 15mm-long root specimens and working length was determined 1mm short of root apex. All specimens were initially preflared to create a standardised conical space for the inoculation of the root canals with 5 selected endodontic pathogens and the development of a nutrient-stressed multispecies biofilm. Then they were randomly assigned into three groups. In group1, no endodontic intervention was performed (control). In group2, root specimens were instrumented with rotary files and irrigated with sterile saline. In group3, root specimens were instrumented with rotary files and irrigated with 2.5% NaOCl and 17% EDTA. A customised experimental model apparatus was fabricated for each specimen, with the apical root third inserted in a glass vial filled with sterile ultrapure water, to simulate high-compliance periradicular space. A portable suction was used to aspirate the effluent during irrigation procedures. The reaction products of the aliquots obtained from the glass vials and the collected effluents were analysed in real time, by selected ion flow tube mass spectrometry (SIFT-MS) in triplicates. The aliquots obtained from group3 were further treated with silver-impregnated activated carbon (Ag-AC) to examine the effect of Ag-AC on the residual chlorine availability as well as the reduction of forming VOCs and DPBs. For the development of Ag-GO, AgNPs were prepared by reducing AgNO3 with 0.01M NaBH4 in presence of GO. Elemental analysis was performed with scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS) and scanning transmission electron microscopy (STEM) was used for size and morphology analysis of GO and AgNPs. Nutrient stressed, multi-species biofilms were grown in prepared root canals of single-rooted teeth. The tested irrigants were sterile saline, 1% and 2.5% sodium hypochlorite (NaOCl), 2% chlorhexidine gluconate (CHX), 17% EDTA and an aqueous suspension of 0.25% Ag-GO. The antimicrobial efficacy of root canal irrigants was performed with paper point sampling and measurement of quantitative microbial counts. The biofilm disruption capacity on dentine surfaces was analysed with confocal laser scanning microscopy (CLSM).
Results: With regard to the proof of concept studies, NaOCl concentration, MDP mass, irrigant volume and their interaction had a significant effect on the values of Amide I intensity peaks (P<0.05). The use of GC-EI-MS disclosed the presence of chloroform (CHCl3) and carbon tetrachloride (CCl4) within blank NaOCl solutions, as well as following the incubation with MDP, at varying concentrations, at all exposure times. SIFT-MS analysis showed the formation of several types of VOCs and DPBs, including chlorinated hydrocarbons, particularly chloroform, together with unexpected higher levels of some nitrogenous compounds, especially acetonitrile. No difference was observed between aerobic and anaerobic conditions. The chemomechanical preparation of root canals in groups 2 and 3 resulted in the apical extrusion of VOCs and DBPs. In group3, the aliquots obtained from periradicular space and the collected effluent released high concentrations of methanol, propanol, ammonia, chloroform, together with unexpected higher levels of formaldehyde, which were statistically significant compared to group2 (P<0.05). In group1, VOCs and DBPs were not detected. The use of Ag-AC efficiently reduced the concentration levels of chloroform, but did not affect the presence of ammonia and formaldehyde.
SEM/EDS analysis confirmed impregnation of Ag within the GO matrix. TEM images showed edge-shaped GO sheets and spherical AgNPs of diameter 20-50nm, forming a network on the surface of GO sheets. The microbial killing efficacy of 2.5% NaOCl was superior compared to the experimental groups. The use of ultrasonic activation enhanced the efficacy of Ag-GO compared to 1% NaOCl, 2% CHX, 17% EDTA and sterile saline (P<0.05). The maximum biofilm disruption, in dentine tubules, was achieved by 2.5% NaOCl. Ag-GO caused a significant reduction of total biovolumes compared to the rest experimental groups (P<0.05%).
Conclusions: The chemical interaction of NaOCl with NOM resulted in the formation of toxic chlorinated hydrocarbons. SIFT-MS analysis proved to be an effective analytical method. The mechanical preparation and irrigation of artificially infected root canals with rotary NiTi files, 2.5% NaOCl and 17% EDTA resulted in the formation of VOCs and DBPs in a water-closed periradicular space and aspirated effluent aliquots. The risks from the rise of toxic compounds require further consideration in dentistry. The biofilm killing and disruption capacity of Ag-GO was successfully documented in a novel ex vivo infected tooth model. Ultrasonic activation selectively improved the antimicrobial efficacy of Ag-GO.
| Date of Award | 1 Mar 2020 |
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| Original language | English |
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| Supervisor | Francesco Mannocci (Supervisor) & Sanjukta Deb (Supervisor) |