2.1

In vitro experiment

2.1.1

Tooth selection and preparation

Forty-four intact, caries-free premolars were used in the in vitro part of the study. The teeth were collected based on a protocol approved by the Human Assurance Committee of the Augusta University, with informed consent obtained from the donating subjects with respect to the use of human tissues. The extracted teeth are stored in 0.9% (w/v) NaCl containing 0.02% sodium azide at 4 °C before use. The crown of each tooth was decoronated to achieve a 15 mm long standard root length. After access to the pulp cavity, a size 10 K-file (Dentsply Sirona, York, PA, USA) was used to achieve apical patency and establish working length 1 mm short of the apical foramen. The root canals were instrumented with ProTaper Gold nickel titanium rotary instruments (Dentsply Sirona) using a crown-down technique. Each instrumented canal was irrigated with 2.5% NaOCl after each file until F4 apical size was achieved. The rationale for using reduced NaOCl concentration for the in vivo experiment was to enable microbial loads to be discerned more accurately after the use of the apical negative pressure delivery protocol for different time periods (to be described later). Final irrigation was performed using 2 mL of 17% ethylenediamine tetra-acetic acid (EDTA). The root apex was filled with light-cured block-out resin and the surface were varnished with nail polish twice to produce a closed canal system. All specimen were autoclaved at 121 °C for 30 min.

2.1.2

Bacterial culture

Enterococcus faecalis (ATCC 29212, American Type Culture Collection, Manassas, VA, USA) was propagated in sterilized brain and heart infusion broth (BHI, Thermo Scientific, Waltham, MA, USA) at 37 °C in an aerobic chamber for 24 h. The optical density of bacteria was adjusted to 1 at 600 nm (≈1*10 ⁸ cells/mL) [ ]. Twenty microliter of bacterial inoculum was added to each canal. A sterile size 10 K-file was gently inserted into canal to the apical terminus and retrieved to uniformly coat the canal wall with the inoculum. All teeth were incubated at 37 °C in the aerobic chamber for 21 days to generate mature, single-species biofilms on the canal walls. Fresh inoculum was added to each canal every alternative day. At the end of every week, a sterile size 15 K-file was used to collect bacterial sample from one randomly chosen canal; gram staining was performed to affirm bacteria contamination of the canal walls.

2.1.3

Root canal irrigation

After 21 days of incubation, the teeth were divided into 5 groups (N = 8) based on the time employed for irrigation with the EndoVac MicroCannula. The remaining 4 teeth were not irrigated and used as the positive control group (0 s Group). All procedures were performed by the same researcher. Irrigation with EndoVac system was performed using 2.5% NaOCl as the irrigant. According to the manufacturer’s instruction, irrigation was initially performed using the EndoVac MacroCannula in up and down motion for 10 s (0.5 mL 2.5% NaOCl was delivered). This was followed by the use of the MicroCannula, inserted to full working length to ensure irrigant replacement thought the entire root canals.

The 2.5% NaOCl was delivered in the following manner:

0s group : positive control. No irrigation was conducted.

15s group : irrigation with the MacroCannula for 10 s (0.5 mL NaOCl) and subsequently with the MicroCannula for 15 s (0.75 mL NaOCl was delivered).

30s group : irrigation with the MacroCannula for 10 s and subsequently with the MicroCannula for 30 s (1.5 mL NaOCl).

45s group : irrigation with the MacroCannula for 10 s and subsequently with the MicroCannula for 45 s (2.25 mL NaOCl).

60s group : irrigation with the MacroCannula for 10 s and subsequently with the MicroCannula for 60 s (3 mL NaOCl).

90s group : irrigation with the MacroCannula for 10 s and subsequently with the MicroCannula for 90 s (4.5 mL NaOCl).

2.1.4

Bacterial sample collection

After irrigation, the root canals were rinsed with 2 mL of 1% sodium thiosulfate to inactivate the NaOCl. Sterile size 30 Hedstrom file, size15 K-file and medium-sized paper point were placed into each canal for 1 min per file/point to collect bacterial sample. The collected sample was transferred into tubes containing 3 mL of BHI broth. The tubes were gently sonicated for 1 min. After incubating the tubes at 37 °C in an aerobic chamber for 4 h, ten-fold serial dilutions were performed and 100 μL of each dilution was plated on BHI agar and incubated at 37 °C for 24 h. Bacteria load was quantified as colony forming units (CFU)/mL using an automatic colony counter (Flash & Go, IUL, S.A., Barcelona, Spain). The original tubes were remained in aerobic chamber for 1 week for further microbiological evaluation. The samples that remained clear after 1 week of incubation were recorded as culture-negative, while samples that were turbid at the end of 1 week were recorded as culture-positive.

2.2

In vivo experiment

2.2.1

Patient selection

Twenty patients attending the Department of Endodontics, The Dental college of Georgia, Augusta University were recruited for the study. All the selected teeth were diagnosis as pulpal necrosis and apical periodontitis using clinical diagnostic tests and radiography. All the teeth were single-root and had intact pulp cavity without detected crown or root fracture. Teeth with irreversible pulpitis were excluded from the study. Consent was received from each patient for root canal treatment. Because the bacterial sampling procedures were performed using materials that were destined to be discarded after use, without alteration of the clinical procedures, no additional consent was received for those procedures.

2.2.2

Treatment protocol

Bacterial sampling from those teeth followed strict aseptic principles. After rubber dam was placed on the designated specific tooth, Hibiclens (Mölnlycke Health Care, Georgia, USA), which contains 4% chlorhexidine gluconate, was used to clean the operation area, followed by 2.5% NaOCl. After access with a sterile bur, the same disinfection procedures were performed with Hibiclens and 2.5% NaOCl.

Root canal samples taken immediately after access into the root canals and prior to further instrumentation (S0 s) were used as the baseline. Sampling was performed with a sterile size 10 K-file, which was inserted to 1 mm short of the radiographic apex and left in the canal for 1 min. Each root canal was sampled with one file. After sampling, the file was transferred to a tube containing 2 mL of normal saline that contained 1% sodium thiosulfate. The samples were left inside an anaerobic chamber for at least 24 h for sufficient gas exchange. Root canal instrumentation was subsequently performed with ProTaper Gold nickel titanium rotary instruments. A chelating solution, 17% EDTA, was used as irrigant for removing the inorganic component of the smear layer and loose debris between each file.

A second bacterial sample was taken after canal instrumentation (S1). The S1 sample was collected using a sterile size 30 K-file and left in the canal for 1 min. The sample was transferred to a new tube containing 2 mL normal saline containing 1% sodium thiosulfate. The EndoVac apical negative pressure system was subsequently employed for irrigant delivery. Based on the result of in vitro experiment, the irrigation time was set at 10 s (0.5 mL 8.25% NaOCl) with the MacroCannula and 60 s (3 mL 8.25% NaOCl) with the MicroCannula. A sterile size15 K file was used to sample microorganisms from the canal wall (S2). After sampling, the file was transferred into 2 mL normal saline containing 1% sodium thiosulfate.

All the samples were immediately transferred to a microbiological laboratory after collection and agitated for 60 s. Ten-fold serial dilutions were made to achieve optical densities in the range of 10 ⁻³ for the S0 sample, 10 ⁻² for the S1 sample and 10 ⁻¹ for S2 sample. One hundred microliter of each dilution was inoculated onto Brucella agar plates that were supplemented with 5% sheep blood, 5 mg/L hemin and 10 mg/L vitamin K1 (Becton Dickinson and Company, Franklin Lakes, NJ, USA). The agar plates were incubated in an anaerobic chamber for 7 days. After incubation, CFUs were counted using the automatic colony counter and actual CFUs were calculated according to the dilution ratio.

2.2.3

Statistical analysis

Data collected from the in vitro experiment were transformed to log ₁₀ CFU/mL and analyzed with SPSS Statistics 22.0 (IBM Corporation, Armonk, NY, USA). Because the normality (Shapiro-Wilk test) and equal variance (modified Levene’s test) assumptions of the data sets did not appear to be violated, one way analysis of variance was performed to examine the effect of NaOCl delivery time via the MicroCannula on elimination of intracanal bacterial load. Post hoc pairwise comparisons were conducted using the Holm-Šidák statistic. For in vivo part, data were analyzed non-parametrically using the Friedman test, to detect differences in bacterial loading across multiple sampling attempts. For all analyses, statistical significance was pre-set at α = 0.05.