Ex vivo microbial leakage analysis of polytetrafluoroethylene tape and cotton pellet as endodontic access cavity spacers.

BACKGROUND: The endodontic spacers are placed between the endodontic appointments or after completion of the endodontic therapy, and until the placement of a definitive restoration. AIMS: The aim of this study was to evaluate the sealing ability of polytetrafluoroethylene (PTFE) access spacer against microbial leakage and to compare it with that of a cotton pellet.
MATERIALS AND METHODS: Fifty-two extracted human single-rooted premolars were divided into two experimental groups (n = 20) according to the endodontic spacer; cotton pellet or PTFE tape, and two control groups (n = 6). Following standardized access cavity, cleaning, and shaping procedures, the access cavities received a standardized thickness of the spacer material followed by a Cavit restoration in all the teeth except for the positive controls, which were left empty. Negative controls had the root surfaces completely sealed with nail polish. A dual-chamber microbial leakage model was used with Enterococcus faecalis as the test strain. At days 7 and 30, samples of the lower chambers' solution were obtained and subjected to the quantitative real-time polymerase chain reaction (qPCR) analysis to quantify bacterial levels. Furthermore, broth turbidity in the lower chambers was recorded weekly. The Mann-Whitney U test and Wilcoxon test were used to compare E. faecalis counts between and within groups, respectively.
RESULTS: At days 7 and 14, the experimental groups leaked similarly as determined by broth turbidity. However, at days 21 and 30, a significantly higher number of cotton pellet samples exhibited microbial leakage. Analysis by qPCR revealed higher levels of E. faecalis counts in cotton pellet samples compared with PTFE samples. This difference was statistically significant at day 7, but not at day 30.
CONCLUSIONS: PTFE spacer showed improved sealing ability compared with the commonly used cotton pellet and may serve as an alternative endodontic access cavity spacer. Copyright:

INTRODUCTION

It has been well established that the main etiological factors of pulpal and periradicular diseases are related to a microbial origin.[12] Therefore, the aim of the root canal treatment is to eliminate all bacteria from the root canal system by mechanical and chemical debridement and to maintain it in this disinfected state by preventing a further influx of bacteria during or after endodontic treatment.[34]

In cases of vital noninfected teeth, endodontic treatment can be completed in a single visit, eliminating the need for temporization.[5] However, in many situations, a one-visit treatment may not be feasible because of time limitation, case complexity, persistent bacteria,[6] and/or patient-related factors. Moreover, despite the large body of evidence indicating no difference in the outcome of endodontic treatment performed in either single or multiple visits,[7] many endodontists still prefer the multiple-visit approach to ensure the absence of pain or complications and for dressing infected canals with antibacterial intracanal medicaments.[7] This requires the placement of a temporary restoration to seal the endodontic access cavities between appointments and to prevent microbial recontamination from oral fluids.[5]

It has been a common practice to place a spacer material onto canal orifices and beneath the provisional restorations. These spacers are placed between the endodontic appointments or after completion of the endodontic therapy, and until the placement of a definitive restoration.[8] The placement of a spacer has many objectives, with the main ones being to allow easier removal of the temporary fillings and for readily identifying canal orifices.[5] The ideal material for this purpose would be easy to place and remove, minimize bacterial leakage, and not promote bacterial growth.[9] Additional desirable characteristics include being inexpensive, inorganic, easily accessible, autoclavable, and consisting of minimal volume.[10]

Until today, the cotton pellet has been the most commonly used endodontic spacer.[91011] However, its use has been associated with some serious complications that can negatively affect the intended seal. One of the material drawbacks has been related to the fibrous and organic nature of the cotton, which may provide it with the potential to enhance microbial leakage and promote bacterial growth.[58910]

Recently, polytetrafluoroethylene (PTFE) tape has gained some popularity as an endodontic access spacer.[8910] It is a polymer material that is used in versatile technologies and in various medical and dental applications.[912] It shares the advantages of the cotton pellet as an acceptable spacer material, but also has some other benefits. Because it is inert, nonbiodegradable,[13] and nonfibrous,[10] it has the potential to overcome the limitations of the cotton pellet. Laboratory and clinical studies have evaluated PTFE tape as a potential alternative spacer to the conventional cotton pellet and reported promising results.[8910] However, these studies were very limited, and the quantitative microbial leakage data, if obtained, were based on culturing techniques. Therefore, the aim of this ex vivo study was to evaluate the sealing ability of PTFE tape against bacterial microleakage, when used as an endodontic access spacer between endodontic appointments, in comparison with that of the cotton pellet and to analyze microbial leakage quantitatively using a more sensitive molecular-based method, which is the quantitative real-time polymerase chain reaction (qPCR). The null hypothesis tested was that there would be no differences between the cotton pellet and PTFE spacers in terms of sealing ability against microbial leakage.

MATERIALS AND METHODS

Tooth selection and specimen preparation

The present study was approved by the Institutional Ethics Committee of the College of Dentistry Research Center (PR0060). Fifty-two single-rooted human extracted premolars were collected. Only intact or minimally carious teeth with straight or minimally curved canals, complete apical development, and no resorption or detectable cracks were included in the study. The integrity of the teeth and the configuration of the single canal were confirmed by high magnification and buccolingual and mesiodistal digital radiographs. The crowns were sectioned with a diamond bur to produce flat occlusal surfaces. The access cavities were prepared with a tapered diamond bur (Kerr Dental, Orange, CA, USA). The shape of the access opening was generally oval and approximately 2.5 mm in width, 3 mm in length, and 5.5 mm in depth. The crowns were adjusted to a standardized working length of 18 mm that would be 1 mm short of the point at which a #10 K-file exited the apical foramen. The canals were instrumented using rotary files (K3, Kerr Dental, Orange, CA, USA) in a crown-down technique up to an apical size of 40/04 and irrigated with 2 ml of 5.25% sodium hypochlorite (NaOCl) between each instrument. An apical stop was confirmed by placing a #40 K-file at the working length. Teeth not possessing this stop were excluded from the study. Final flushing of the canals was achieved using 2 ml of 17% ethylenediaminetetraacetic acid (EDTA) (SmearClear, SybronEndo, Orange, CA, USA) for 2 min followed by 2 ml of 5.25% NaOCl and 5 ml of distilled water. The teeth were then steam autoclaved at 121°C for 20 min, and after that, all procedures were performed using sterilized materials and instruments. All experiments were done by one operator; an endodontist and blinding was not applicable.

Preparation of polytetrafluoroethylene pellets

Pieces of approximately 2 cm of PTFE tape (PTFE thread seal tape, ACE, Illinois, USA) were cut and made into pellets by utilizing a resin mold, designed in the laboratory, of a size-four cotton pellet (Richmond Dental, USA). Before use, the PTFE pellets were steam autoclaved at 121°C for 20 min.

Sample size and temporization procedure

Sample size calculation revealed that at a significance level of α = 0.05, with a standard deviation = 1.5 × 104, a power of 0.9, and a maximum difference of 2%, the sample size for each experimental group should be at least 18 teeth. Therefore, the teeth were divided into two experimental groups (n = 20) according to the endodontic spacer; cotton pellet or PTFE tape, and two control groups (n = 6). For the cotton pellet group, a size-four cotton pellet was used as the access cavity spacer. The sterilized cotton pellet was placed over the canal orifices with a standardized thickness of approximately 2 mm, followed by 3.5 mm of the provisional restoration (Cavit) (3M ESPE, Seefeld, Germany). For the PTFE group, the temporization procedures were conducted similarly, except for using the PTFE pellets as the access spacer [Figure 1a–d]. All the teeth surfaces in the experimental and positive control groups were coated with two layers of nail polish, except for the access openings and the apical 2 mm of the roots. Root canals of the positive control group were left completely patent, and the access cavities were left empty. The teeth in the negative control group received an access spacer (either cotton pellet or PTFE) and were temporized similar to the experimental groups. The root apices of this group were sealed with composite, and all the teeth surfaces were then covered completely with two layers of nail polish.

Figure 1

(a) Occlusal view of the cotton pellet spacer. (b) Occlusal view of polytetrafluoroethylene spacer. (c) Lateral view of the cotton pellet spacer. (d) Lateral view of polytetrafluoroethylene spacer. (e) The microleakage model. (f) Turbid samples (right) and nonturbid samples (left)

Microbial leakage model

A modification of the dual-chamber microbial leakage model that was described previously was used [Figure 1e].[14] Briefly, Eppendorf centrifuge tubes with sizes of 1.5 ml (Eppendorf Tubes® 3810X, Eppendorf, Hamburg, Germany) and 15 ml (Falcon™, NY, USA) were used to construct the upper and lower chambers, respectively. The tapered end of the 1.5-ml tube was cut, and the tooth was inserted, and hence, the root was protruding through the cut end. The tube-root junction was sealed with composite resin (MultiCore® Flow, Ivoclar-Vivadent, Schaan, Liechtenstein) and cyanoacrylate glue. The tube-root assemblies were placed into the 15-ml tube that contained 10 ml of the sterile brain–heart infusion (BHI) covering the apical 2–3 mm of the root. All procedures were conducted under sterile conditions in an ultraviolet hood (PCR UV3 HEPA, Analytik Jena, Überlingen, Germany).

Preparation of Enterococcus faecalis suspension

Enterococcus faecalis (ATCC 29121) was cultured overnight on agar plates. It was then grown in BHI broth at 37°C for 24 h. After centrifugation of the inculcated broth, E. faecalis cells were harvested and resuspended in phosphate-buffered saline solution. The bacterial suspension was adjusted to a concentration of 1 × 108 cells/mL (optical density of 0.1 at 600 nm). E. faecalis suspension (0.5 ml) was used to inoculate the upper chamber of the leakage model.

Evaluation of bacterial contamination

Microbial leakage was evaluated by two methods. The occurrence of turbidity in the lower chambers [Figure 1f] was monitored on a weekly basis until the end of the experimental period (day 30). Furthermore, qPCR analysis of bacterial counts was conducted on liquid samples obtained from the lower chambers at two time points (day 7 and day 30). During the sampling procedure, 5 ml was obtained from the lower chambers' broth using a 10-ml serological pipet. The samples were centrifuged, and the bacterial pellets were stored at −80°C until further processing for qPCR analysis. At day 7, to replace the drawn solution, 5 ml of a new sterile BHI medium was added to the lower chambers. In addition, the bacterial suspension in the upper chambers was replenished with a fresh one.

DNA extraction and quantitative real-time polymerase chain reaction conditions

DNA from the bacterial samples was extracted using the HiGene™ Genomic DNA Prep Kit (BIOFACT, Daejeon, Korea). Levels of E. faecalis were quantified using 16S ribosomal RNA gene-targeted qPCR with EvaGreen® qPCR Supermix (Solis BioDyne, Tartu, Estonia) run on an ABI 7500 real-time PCR instrument (Applied Biosystems, Foster City, CA, USA) in a total reaction volume of 20 μL. E. faecalis-specific primers were used according to a previous publication[15] in a concentration of 0.2 μmol/L each.

Cycling conditions for qPCR reactions were 95°C for 12 min, followed by 40 cycles of 95°C for 15 s, 60°C for 20 s, and 72°C for 20 s. Other qPCR conditions, standard curve construction, controls, and data analysis were as previously reported.[16] A pure sample of E. faecalis DNA was subjected to a 10-fold serial dilution from 108 to 101 in Tris-EDTA buffer and used for standard curve generation. All measurements were performed in triplicate for samples, controls, and standards.

Statistical analysis

Data were analyzed using the SPSS version 21.0 (IBM Inc., Chicago, IL, USA). Pearson's Chi-square and Fisher's exact tests were used to analyze qualitative data. The Mann–Whitney U test and Wilcoxon test were used to compare E. faecalis counts between and within groups, respectively. The significance level was set at α = 0.05.

RESULTS

Microbial leakage assessment by broth turbidity

Microbial leakage data obtained by recording broth turbidity are presented in Table 1. All positive controls exhibited turbidity in the lower chamber within the first 48 h. The negative controls were leakage-free until day 30. During the sampling procedure at day 7, one sample of the PTFE group got lost due to operator error, and thus was excluded from further analysis.

Table 1

Frequency and percentage of leaked samples as determined by broth turbidity at different evaluation periods

	Positive control, n (%)	Negative control, n (%)	Cotton pellet, n (%)	PTFE, n (%)	P value
Day 7	6 (100)	0	5 (25)	1 (5)	0.182
Day 14	6 (100)	0	11 (55)	6 (31.6)	0.140
Day 21	6 (100)	0	14 (70)	7 (36.8)	0.038*
Day 30	6 (100)	0	14 (70)	7 (36.8)	0.038*

*Statistically significant difference for cotton pellet versus PTFE groups (Pearson’s Chi-square test). PTFE: Polytetrafluoroethylene

Statistical analysis showed no significant difference in the number of leaked samples between the two experimental groups at days 7 and 14 (P > 0.05). However, at days 21 and 30, the number of leaked samples was significantly higher for the cotton pellet group (P < 0.05).

Gram staining of randomly selected samples confirmed the presence of microorganisms with Gram-positive cocci morphology in the turbid lower chambers (data are not shown).

Microbial leakage assessment by quantitative real-time polymerase chain reaction

Quantitative data of microbial leakage obtained by qPCR are presented in Table 2. At day 7, significantly higher levels of E. faecalis counts were observed for cotton pellet samples compared with PTFE samples.

Table 2

Quantity of leaked bacterial cells by quantitative real-time polymerase chain reaction for the two experimental groups at day 7 and day 30

	Cotton pellet	PTFE	P
Day 7
Mean	1.68×107	6.7×106	0.046*
Median	4.85×102	0
Range	0-1.09×108	0-1.34×108
n	20	20
Day 30
Mean	4.52×107	2×107	0.053
Median	2.89×107	5.05×102
Range	0-1.32×108	0-1.06×108
n	20	19
P	0.003**	0.008**

*Statistically significant difference between cotton pellet versus PTFE groups (Mann–Whitney U-test), **Statistically significant difference between day 7 versus day 30 within each group (Wilcoxon test). PTFE: Polytetrafluoroethylene

At day 30, higher levels of E. faecalis counts were also observed for cotton pellet samples; however, the difference was not significant between the two groups.

Comparison of microbial leakage between the two evaluation periods revealed a significant increase in the number of leaked bacterial cells between day 7 and day 30 within each experimental group [Table 2].

DISCUSSION

Endodontic temporization procedures commonly include the placement of a spacer onto the canal orifices and beneath the temporary restoration. These spacers are mainly used to allow for easier removal of the temporary filling and relocating the root canal orifices.[5] Ideally, endodontic spacers should possess favorable physical and biological properties and contribute to, or at least not interfere with, the sealing ability of the temporary restorations.[5910] Therefore, this study evaluated the sealing ability of two endodontic spacers: the conventional cotton pellet and a more recently introduced spacer, the PTFE.

By observing broth turbidity in the lower chambers of the microbial leakage model, microbial leakage was less frequent in PTFE samples compared with cotton pellet samples at all evaluation periods, with significant differences after day 21. Observing for broth turbidity to evaluate microbial leakage is an approach with relatively reduced sensitivity. Thus, it can be expected that in the earlier evaluation periods, the overall number of turbid samples might not be large enough to show statistical differences.

The quantitative qPCR microbial analysis revealed higher levels of leaked E. faecalis count in cotton pellet samples compared with PTFE samples at both the 1-week and 1-month evaluation periods. The difference was significant at day 7, and not significant, although close to being, at day 30.

Overall, the microbial leakage data obtained by recorded broth turbidity and qPCR indicated an improved sealing ability of PTFE spacer compared with the cotton pellet. There could be a number of potential explanations for these findings. First, the organic nature of the cotton pellet might enhance microbial growth.[10] Second, the cotton pellet fibers could act as a wick[58910] that might have promoted liquid absorption from the upper chamber and subsequently led to microbial leakage. In addition, cotton pellet fibers might have interfered with the proper adaptation of the Cavit restoration to the walls of the access cavity.[58] Third, the cotton pellet is of a porous structure, which could further enhance both fluid and microbial leakage.

On the other hand, PTFE spacer is biologically inert and does not promote microbial growth.[1013] It is hydrophobic with low or no tendency for liquid absorption.[913] It also lacks the fibers that enhance bacterial uptake by the wicking action.[8910] Moreover, the absence of these fibers could improve the adaptation of the filling material to the walls of the access cavity and to the surface of the underlying spacer.[58] In addition, it is more solid and nonporous and can be better compacted compared with the cotton pellet.[910] These factors could have contributed to the better sealing ability of PTFE spacer compared with the cotton pellet in the present study.

Previous studies have evaluated PTFE as an endodontic spacer material.[8910] Due to significant differences in methodological designs, direct comparisons may not be possible between the current study and the previous ones. However, it can be stated that all cited studies have arrived at nearly the same conclusion, which was improved sealing ability of PTFE spacer compared with the cotton pellet. This is generally in agreement with the findings of the present study.

In the present study, the 7th day was chosen as an additional time point to obtain samples for qPCR analysis within the 30-day experimental period. Seven days is the recommended interval for effective placement of intracanal medicaments.[17]

CONCLUSION

Within the limitations of this study, it can be concluded that the PTFE spacer showed improved sealing ability compared with the commonly used cotton pellet and may serve as an alternative endodontic access cavity spacer.

Financial support and sponsorship

This work was funded by King Abdulaziz city for science and technology (KACST 1-17-03-001-0032).

Conflicts of interest

There are no conflicts of interest.