Micro-CT determination of the porosity of two tricalcium silicate sealers applied using three obturation techniques

Abstract

Purpose: Using X-ray micro-computed tomography (micro-CT), the aim of this study was to measure the porosity of two tricalcium silicate sealers (EndoSequence BC and NeoSealer Flo) applied using three obturation techniques (single-cone, warm-vertical, and cold-lateral) to six single-rooted human teeth.

Methods: Six extracted, single-rooted human teeth were shaped with ProTaper Next rotary files and obturated with EndoSequence BC or NeoSealer Flo sealers and gutta-percha (GP) using one of the three techniques above. Micro-CT was used to map the full length of the canals. Deep learning cross-sectional segmentation was used to analyze image slices of the apical (0-2 mm) and coronal (14-16 mm from the apex) regions (n = 230-261 per tooth) for the areas of GP and sealer, as well as porosity. Median (%) with interquartile range of porosity were calculated , and the results were statistically analyzed with the Kruskal-Wallis test.

Results: In the apical region, EndoSequence BC had significantly fewer pores than NeoSealer Flo with the single-cone obturation (% median-interquartile range, IQR: 0.00-1.62) and warm-vertical condensation (5.57-10.32) techniques, whereas in the coronal region, NeoSealer Flo had significantly fewer pores than EndoSequence BC with these two techniques (0.39-5.02) and (0.10-0.19), respectively. There was no significant difference in porosity between the two sealers for the cold-lateral condensation technique in both the apical and coronal regions.

Conclusion: For optimal obturation, the choice of technique and sealer is critical.

Introduction

Non-surgical root canal treatment (NSRCT) has two primary objectives: disinfection of the canal system and establishment of a hermetic seal to thwart any resurgence of microbial activity [1]. The foremost cause of NSRCT treatment failure is infiltration of microorganisms through gaps between or through the endodontic sealer and canal walls, particularly at the apical or coronal regions. Hence, reduction of such gaps is paramount for ensuring the long-term success of endodontic treatment [2]. The capacity of a sealer and obturation technique to achieve effective filling of a root canal system is a critical determinant of successful sealing [2,3].

A variety of endodontic sealers with different chemical formulas have been developed, including zinc oxide-eugenol, salicylate, fatty acid, glass ionomer, silicone, epoxy resin, methacrylate resin sealer, and tricalcium silicate sealer [4]. Tricalcium silicate sealers are gaining popularity because of their exceptional sealing efficacy, biocompatibility, and antimicrobial characteristics [5,6,7]. Tricalcium silicate sealers set with water and form calcium hydroxide within a hydrated inorganic cement matrix, which imbues these sealers with the capacity to precipitate a biomimetic apatite layer on their surface [8]. This surface reaction of calcium hydroxide with interstitial fluid contributes to excellent sealing and antibacterial properties. Moreover, these sealers have less cytotoxicity and greater antibacterial properties because of their alkaline pH and absence of resins [6]. However, despite the promising properties of tricalcium silicate sealers, comprehensive studies of their properties and ability to obturate without gaps have been limited [6].

EndoSequence BC sealer (Brasseler, Savannah, GA, USA) was introduced in 2009, and a substantial body of research has since been conducted to assess its efficacy [9,10,11]. EndoSequence BC sealer was originally indicated for the single-cone obturation technique, and not the warm-vertical condensation technique, which had been the preferred method for epoxy resin sealers [12]. NeoSealer Flo (Avalon Biomed Division of NuSmile Ltd., Houston, TX, USA) was introduced in 2020, and instructions for use included its suitability for both warm and cold obturation techniques. The porosity associated with these different obturation techniques has not yet been reported for NeoSealer Flo sealer.

Many methods have been used to study microleakage, including fluid filtration, dye penetration, bacterial leakage models, protein leakage models, and scanning electron microscopy. However, researchers have questioned the reliability of the results obtained [13,14,15]. X-ray micro-computed tomography (micro-CT) is a newer, non-destructive imaging method for visualizing the microanatomy of roots and measuring the areas of sealer, gutta-percha (GP), and voids with an accuracy greater than that achievable with other techniques [3,16].

This study measured and compared the porosity of obturated root canals using micro-CT and deep learning cross-sectional segmentation analysis [17]. Two tricalcium silicate sealers were used for three obturation techniques (single-cone, warm-vertical, and cold-lateral), and the results were compared.

Materials and Methods

Preparation of extracted human teeth

Fully erupted, human, permanent maxillary anterior teeth devoid of caries or defects were collected from local oral surgeons. The specimens were immersed in 10% formalin solution for disinfection (IRB: Not Human Subject Research #19.11.12-006, University of New England). Six teeth were selected based on their radiographs. The selected teeth had single canals and were devoid of cracks, fractures, resorption, caries, immature apices, or root curvature exceeding ten degrees [18]. The six teeth were decoronated at the cementoenamel junction using a high-speed diamond bur, and the roots were meticulously standardized to 16 mm using precision digital calipers.

Root canal preparation

This preparation procedure was conducted by a single operator. The pulp canal was accessed, and a 15 mm working length was established (1 mm short of the apical foramen) using a size 10 K-file (Dentsply Sirona, Johnson City, TN, USA). Canal patency was checked by extending a #10 K-file 1 mm past the anatomical apex. Canals were cleaned and shaped using ProTaper Next NiTi rotary files with a ProMark torque-limited electric motor and instrument sizes X1 to X5 (#50/variable taper) in succession. Canals were irrigated at each instrument change using 2 mL of 2.5% sodium hypochlorite (NaOCl) with a 27-G needle (Ultradent Inc., South Jordan, UT, USA). Final irrigation was performed with 5 mL of 17% EDTA for 1 min, followed by 5 mL of 2.5% NaOCl. The canals were dried with ProTaper Next absorbent points (Dentsply Sirona).

Root canal filling with a GP point and sealer using three different obturation techniques

The instrumented teeth were fitted with a ProTaper NEXT variable-taper X5 gutta percha point (Dentsply Sirona) that matched the last instrument used, i.e., a ProTaper NEXT NiTi rotary file size X5. The six teeth were divided into two groups (n = 3) for obturation with either EndoSequence BC or NeoSealer Flo sealer and GP. Table 1 summarizes the physico-chemical properties and compositions of the two sealers including setting times, radiopacity, and flow [19,20,21].

Each tooth was obturated using one of the three obturation techniques. The sealer was introduced into a cleaned and shaped canal using an injection needle. The GP point or points were “buttered” with one of the sealers, then inserted into the canal to the working length using the single-cone, cold-lateral, or warm-vertical technique.

For the single-cone technique, only one GP point was “buttered” with one of the sealers. For cold-lateral condensation, in addition to using one GP point “buttered” with one of the sealers, accessory points were used to fill the remaining canal spaces. For the warm-vertical technique, teeth were obturated using the same method as that for single-cone obturation, and the GP was removed until only the apical 5 mm remained. The rest of the canal space was backfilled with additional sealer, then the GP was thermoplasticized using a Calamus Pack heat carrier system (Dentsply Sirona). Excess GP was cut off and removed with a heated Calamus Pack heat carrier system, and the remainder was vertically condensed with an endodontic plugger. Coronal orifices were restored with glass ionomer cement (GC Fuji Triage, Tokyo, Japan); the apical orifices had no external covering. The obturated roots were placed in phosphate-buffered saline (PBS, VWR, Radnor, PA, USA) for up to 57 days until they were scanned using micro-CT.

Table 1 Physicochemical properties of the two tricalcium silicate sealers

Product name (manufacturer, country)	Lot number	Composition	Setting time (h)	Radiopacity (mm Al)	Flow (mm)	Solubility (%)
NeoSealer Flo (Avalon Biomed Division of NuSmile Ltd., Houston, TX, USA)	2021050702	tricalcium silicate, dicalcium silicate, calcium aluminate, tantalite, organic liquid	8.0	5.5	19.0	<3.0
EndoSequence BC (Brasseler USA, Savannah, GA, USA)	21002SP	tricalcium silicate, dicalcium silicate, zirconium oxide, calcium hydroxide, fillers, thickening agents, organic liquid	7.5	6.6	23.1	2.9

NeoSealer Flo data are from company’ IFU and Ref. 19; EndoSequence BC sealer data are from Ref. 20 and 21

 

Micro-CT scanning and image analysis

Each tooth was scanned and reconstructed with an X-ray micro-CT scanner (CT Lab HX, Rigaku Americas Corp., The Woodlands, TX, USA) with a 1 mm aluminum filter at 130 kV and 61 mA, and a voxel size of 7.25 mm. The detector was calibrated before the scans to minimize artifacts. All datasets were exported in tiff file format. The areas of GP, sealer, and voids were analyzed using Dragonfly software (Object Research Systems, Montreal, Canada). Deep learning cross-sectional segmentation was used to quantify the areas of GP and sealer, as well as porosity. Each cross-sectional transverse slice was spaced at 7.7-8.8 µm, and 230-261 slices per tooth were analyzed. Figure 1 shows sagittal section images of a tooth, overlaid with micro-CT images.

Fig. 1 Representative micro-CT images and cross-sectional segmentation results after deep learning. *Scale bar: 1 mm

A: Sagittal section of the tooth obturated with NeoSealer Flo using the cold-lateral condensation technique. The red dotted line indicates the location of the coronal section shown in panel B. The segmentation results, juxtaposed with micro-CT images, magnify the components in the root canal system: GP (pink), sealer (orange), and voids (light blue). The arrow indicates voids. The top right panel of A shows the magnified apical (0-2 mm) region; the bottom right panel of A shows the coronal (14-16 mm) region. B: A coronal section of the tooth obturated with NeoSealer Flo using the cold-lateral condensation technique at the level corresponding to the dotted line in panel A.

Statistical analysis

Descriptive statistics were computed for voids in both the apical and coronal regions. The Shapiro-Wilk test was conducted to assess data distribution, revealing that the data did not have a normal distribution. Therefore, the Kruskal-Wallis test was conducted to determine statistical significance in different groups employing the three obturation techniques and two tricalcium sealers. Subsequent pairwise comparisons were conducted using the Mann-Whitney test to identify statistically significant differences between specific pairs of groups. Microsoft Excel, equipped with QI macros, was utilized to analyze the data with non-parametric tests, setting the significance level at α = 0.05.

Results

Porosity analysis of coronal sections

Tables 2 and 3 summarize the average area of the GP and sealer, total area, and pore area in mm² in the apical and coronal regions. The total areas among the six groups did not differ significantly among the two sealers and three techniques, suggesting that the shaped canal areas were similar across the six teeth. There were no statistically significant differences in the areas occupied by GP and sealer. These findings confirmed the consistency of the GP and sealer distribution among the six teeth, each constituting one group.

The percentages of porosity in the apical region (0-2 mm from the apex) and the coronal region (14-16 mm from the apex) are included in Tables 2 and 3. NeoSealer Flo showed the lowest porosity at the apex with the cold-lateral technique, followed by the single-cone and warm-vertical techniques, being 0.28% (1.14), 3.10% (8.87), and 11.68% (9.79) (median-interquartile range, IQR), respectively. EndoSequence BC sealer had the lowest porosity at the apex when obturated using the single-cone technique, followed by the cold-lateral technique and warm-vertical technique: 0.00% (1.62), 0.70% (1.06), and 5.57% (10.32) (median-IQR), respectively.

In the apical region, the teeth obturated with NeoSealer Flo and EndoSequence BC showed similar porosity levels for the cold-lateral technique. However, the apical region obturated with NeoSealer Flo had significantly more voids than EndoSequence BC for the single-cone and warm-vertical techniques.

In the coronal region of the canals (Table 3) obturated with NeoSealer Flo, the percentage (median-IQR) porosity was 0.39% (5.02), 0.10% (0.19), and 0.75% (0.56) for the single-cone, warm-vertical, and cold-lateral techniques, respectively. For canals obturated with EndoSequence BC sealer, the percentages (median-IQR) were 1.25% (1.76), 0.76% (0.89), 0.55% (2.32), respectively. Canals obturated with NeoSealer Flo had less porosity than EndoSequence BC for the single-cone and warm-vertical techniques in the coronal region. NeoSealer Flo showed the least porosity with the warm-vertical technique, followed by the single-cone and cold-lateral techniques. EndoSequence BC sealer showed the least porosity with the cold-lateral technique, followed by the warm-vertical and single-cone techniques.

Figure 2 illustrates the distribution of voids along the entire root, providing insight into the porosity. EndoSequence BC sealer showed porosity peaks within the 0-2 mm and 13 mm regions when the single-cone technique was used (Fig. 2A). For NeoSealer Flo sealer with the single-cone technique (Fig. 2D), pores were more numerous in the 0-2 mm and 11 mm regions. Both EndoSequence BC and NeoSealer Flo sealers (Fig. 2B, E) applied using the warm-vertical technique showed prominent peaks of porosity within regions 0-2 mm and 7 mm from the apex. However, with the cold-lateral technique, the two sealers (Fig. 2C, F) had multiple areas of slightly higher porosity 0-8 mm from the apex.

Table 2 Pore area and percentage, apical region

Sealer	Technique	Pore area (mm2)	GP area and sealer area (mm2)	Total area (mm2)	Percentage of porosity (%) in area	Statistical significance of % porosity		Number of slices (n)
		Median-IQR				obturation technique	sealer
NeoSealer Flo	single-cone	0.01 (0.02)	0.25 (0.18)	0.27 (0.17)	3.10 (8.87)	a	c	250
	warm-vertical	0.02 (0.02)	0.16 (0.13)	0.19 (0.14)	11.68 (9.79)	a	d	232
	cold-lateral	0.00 (0.00)	0.20 (0.14)	0.21 (0.14)	0.28 (1.14)	a	ns	232
EndoSequence BC	single-cone	0.00 (0.00)	0.16 (0.09)	0.16 (0.08)	0.00 (1.62)	b	c	235
	warm-vertical	0.01 (0.01)	0.13 (0.17)	0.13 (0.16)	5.57 (10.32)	b	d	261
	cold-lateral	0.00 (0.00)	0.32 (0.11)	0.33 (0.12)	0.70 (1.06)	b	ns	227

(a) Statistically significant difference of techniques in NeoSealer Flo sealer groups; (b) Statistically significant difference of techniques in EndoSequence BC sealer groups; (c) Statistically significant difference between NeoSealer Flo and EndoSequence BC sealer for single-cone obturation; (d) Statistically significant difference between NeoSealer Flo and EndoSequence BC sealer for warm-vertical condensation; ns: no statistical significance

Table 3 Pore area and percentage, coronal region

Sealer	Technique	Pore area (mm2)	GP area and sealer area (mm2)	Total area (mm2)	Percentage of porosity (%) in area	Statistical significance of % porosity		Number of slices (n)
		Median-IQR				obturation technique	sealer
NeoSealer Flo	single-cone	0.01 (0.06)	1.44 (0.29)	1.45 (0.22)	0.39 (5.02)	a	d	250
	warm-vertical	0.00 (0.00)	2.07 (0.43)	2.07 (0.43)	0.10 (0.19)	a,b	e	232
	cold-lateral	0.01 (0.01)	1.85 (0.49)	1.87 (0.50)	0.75 (0.56)	b	ns	232
EndoSequence BC	single-cone	0.02 (0.03)	1.73 (0.31)	1.75 (0.28)	1.25 (1.76)	c	d	235
	warm-vertical	0.01 (0.01)	1.31 (0.45)	1.36 (0.45)	0.76 (0.89)	c	e	261
	cold-lateral	0.01 (0.04)	1.55 (0.23)	1.61 (0.24)	0.55 (2.32)	c	ns	227

(a) Statistically significant difference between single-cone and warm-vertical techniques in NeoSealer Flo group; (b) Statistically significant difference between cold-lateral and warm-vertical techniques in NeoSealer Flo group; (c) Statistically significant difference of techniques in EndoSequence BC sealer groups; (d) Statistically significant difference between NeoSealer Flo and EndoSequence BC sealer for single-cone obturation; (e) Statistically significant difference between NeoSealer Flo and EndoSequence BC sealer for warm-vertical condensation; ns: no statistical significance

 

Fig. 2 Percentage of porosity from the apical (0 mm) to the coronal (16 mm) region

A, B, and C show the relationships between the percentage of porosity and the distance from the apex to the coronal region when using EndoSequence BC sealer (BC) in conjunction with single-cone (SC), warm-vertical (WV), and cold-lateral (CL) techniques, respectively. D, E, and F show the relationship between the percentage of porosity and the distance from the apex to the coronal region when using NeoSealer Flo sealer (Neo) with SC, WV, and CL techniques, respectively.

Discussion

This study compared the porosity of two tricalcium silicate sealers applied to six teeth using three obturation techniques. The differences in measured porosity were found to depend on the obturation technique employed.

In the apical region (0-2 mm from the apex), the cold-lateral technique created fewer voids for both NeoSealer Flo and EndoSequence BC. The single-cone and warm-vertical techniques created significantly higher apical porosity than the cold-lateral technique. The initial step of the warm-vertical technique and single-cone obturation is to insert a GP into a cleaned and shaped canal. For warm-vertical technique, a heated tip is used to remove GP except for the apical 4-5 mm. The warm-vertical technique adds more apical pressure with the condensation, but creates more voids.

In the coronal region (14-16 mm from the apex), the highest porosity was observed for teeth obturated using the single-cone technique for EndoSequence BC and using the cold-lateral condensation technique for NeoSealer Flo. Conversely, NeoSealer Flo and the warm-vertical technique resulted in the least porosity in the coronal region.

EndoSequence BC sealer has been widely investigated since its introduction for single-cone or cold-lateral techniques [9,10,22,23], particularly in terms of its porosity with single-cone obturation, except in a few studies [24,25]. The present study is the first to have investigated the porosity resulting from three obturation techniques using NeoSealer Flo. In previous studies, the void volumes for the single-cone obturation technique examined by micro-CT ranged from 3.8% to 13.1% [26,27,28]. The present study demonstrated less void volume than previous studies, i.e., about 1.8%. This lower void volume may have been due to operator variation and the use of single-rooted maxillary anterior teeth with circular canal shapes, unlike the molar or premolar teeth employed in previous studies with different canal shapes and larger volumes. The present results were derived from cross-sectional segmentation analysis involving 231 to 260 slices of each tooth and utilizing a smaller voxel size for detailed analysis. These factors may have contributed to the lower observed porosity.

Celikten et al. used micro-CT to measure porosity variations in EndoSequence BC sealer among single-cone, cold-lateral, and Thermafil obturation techniques [9]. Porosity was higher for the single-cone technique and lower for the Thermafil technique across all root regions [9]. The present study demonstrated higher porosity in the coronal region with the single-cone technique relative to cold-lateral or warm-vertical condensation.

Using micro-CT, Penha da Silva et al. found no significant differences in void volume within the full canal between cold-lateral condensation (8.44% ± 10.21%) and single-cone obturation (13.11% ± 10.75%) when EndoSequence BC sealer was used [23]. This observation differs from the present study, which found less porosity for the cold-lateral technique.

The porosity of NeoSealer Flo and EndoSequence BC sealer varied depending on the obturated regions and the obturation technique employed. EndoSequence BC sealer had fewer pores when applied using single-cone obturation in the apical region and when applied using cold-lateral condensation in the coronal region. NeoSealer Flo showed fewer pores with cold-lateral condensation in the apical region and with warm-vertical condensation in the coronal region. Vertical compaction with NeoSealer Flo reduced the extent of pores.

Both NeoSealer Flo and EndoSequence BC are tricalcium silicate-based sealers that employ different radiopacifiers and liquids. These differences in composition, which affect flow and film thickness, may be responsible for the differences in porosity. NeoSealer Flo contains tantalum oxide, while EndoSequence BC contains zirconium oxide [8]. Differences in particle size distribution affect their physico-chemical behavior, including flow and film thickness [29]. Although the organic liquid contents of these paste sealers are undisclosed, they certainly differ in their thermal stability, viscosity, and molecular weight. Flow is affected by many factors, including liquid content, particle size, and powder-to-liquid ratio [20]. The flowability of NeoSealer Flo is reported to be 19 mm [20], while that of EndoSequence BC is 23.1 ± 0.7 mm [19,21,25].

EndoSequence BC HiFlow sealer purportedly has a lower viscosity, higher flow, and higher radiopacity than EndoSequence BC sealer [25]. However, previous studies have shown that iRoot SP/EndoSequence BC sealer sets faster and has less flow after heating, indicating that some liquid has evaporated [25,30]. In warm-vertical technique, heat is applied with a carrier at 200°C while the temperature inside the canal is around 56-100°C [31]. Well-controlled studies using the validated ISO 6876:2012 and ANSI/ADA 57:2021 methods are important for comparing the behavior of sealers [8].

In this study, pores were observed predominantly at interfaces between the sealer and dentin or the sealer and GP, rather than within the sealer (Fig. 1). This differs from the micro-CT study by Radwanski et al., who observed porosity for tricalcium sealers (Total Fill BC and BioRoot RCS sealers) [32]. This difference may have been due to variations in the prepared canal shape, space, and the fit of the GP among the respective studies.

Radwanski et al. studied oval-shaped canals prepared with ProTaper Next files up to X4 and obturated them with ProTaper Next X4 GP, resulting in an irregular canal space that required more sealer [32]. In contrast, the present study used round-shaped canals prepared with ProTaper Next files up to X5 and obturated them with ProTaper X5 GP. The prepared canals were well fitted with a GP point, minimizing any sealer spaces. Furthermore, micro-CT scans were conducted on the prepared teeth 57 days after preparation. Radwanski et al. observed closed pores that might transition to open pores over time when samples were compared between 7 days and 60 days. Further investigation of the nature of these pores would be an intriguing avenue of future research.

A previous investigation using the single-cone obturation technique reported greater dye penetration for EndoSequence BC sealer in the coronal region relative to NeoSealer Flo, although not at the apex [18]. Likewise, in the present study, the coronal porosity of NeoSealer Flo, which exhibited less dye penetration, was significantly lower than that of EndoSequence BC sealer. Interrelationships among dye penetration, porosity, and microleakage warrant further comparison [18].

This study measured the porosity of two tricalcium silicate sealers (EndoSequence BC and NeoSealer Flo) applied using three techniques (single-cone, warm-vertical, and cold-lateral) to provide clinical insights into the results of obturation. EndoSequence BC sealer had notable porosity around the apex and coronally (13 mm) for the single-cone technique (Fig. 2A). NeoSealer Flo sealer had more porosity at 0-2 mm and 11 mm from the apex. Cold-lateral condentation resulted in much less apical porosity; lateral condensation pressure reduced the porosity in two of the teeth.

When EndoSequence BC and NeoSealer Flo sealers were applied using the warm-vertical technique (Fig. 2B, 2E), two peaks of porosity were seen at 0-2 mm and 7 mm from the apex. The peak around 2 mm was similar to that seen with the single-cone technique, which began using the same procedure. The peak around 7 mm is where thermoplastic GP was injected for the warm-vertical technique.

When EndoSequence BC sealer and NeoSealer Flo sealer were applied using the cold-lateral technique, several areas of small porosity peaks were noted at 0-8 mm from the apex. The coronal region achieved better sealing than the apical region without the accessory GP points and lateral condensation. Higher porosity at the coronal end (16 mm) in Fig. 2C and 2D is represented by a peak, which can be explained by deeper removal of GP at the coronal end in these experimental groups relative to the other groups.

On the basis of the above data, clinical implications would include removal of the coronal few millimeters of GP, especially when using the single-cone technique, to allow space for an orifice barrier. By doing so, a robust coronal seal would be obtained in situations involving delayed post-core placement and full coverage restoration; this seal would limit microbes and microbial products resulting from apical and coronal leakage, which are the main reasons for pain and infection after NSRCT [1]. A meta-analysis has reported that tricalcium silicate-based sealers showed the least microleakage among various root canal sealers [4]. Both of the present sealers have a porosity spike about 7 mm from the apex when the warm-vertical technique is used, which could be concerning. The application of small-diameter endodontic pluggers under an endodontic microscope is recommended for precise clinical operation to ensure adequate compaction at 7 mm from the apex, especially when a post and core build-up is planned. A follow-up study with a larger sample size and more tricalcium silicate sealers will be needed to measure the porosity and its dependence on the chosen technique. Variations in canal anatomy and their effects on porosity are also important.

Within the limitations of this study, NeoSealer Flo showed superior performance in achieving lower porosity in the coronal region with the single-cone and warm-vertical condensation techniques. Conversely, EndoSequence BC sealer yielded better results in the apical region with the single-cone and warm-vertical condensation techniques. Teeth obturated with NeoSealer Flo and EndoSequence BC showed similar levels of porosity for the cold-lateral condensation technique in both the apical and coronal regions. For successful endodontic treatment, it is critical to select the optimal obturation technique and sealer.

Abbreviations

GP: gutta-percha; IQR: interquartile range; micro-CT: micro-computed tomography; NaOCl: sodium hypochlorite; NSRCT: non-surgical root canal treatment; PBS: phosphate-buffered saline

Ethical Statements

Ethical approval for the use of human teeth was obtained from the institutional review board of the University of New England (#19.11.12-006).

Conflicts of Interest

Carolyn Primus is the inventor of the NeoSealer Flo material. She did not participate in the data gathering or statistical analysis.

Funding

This study was supported by NuSmile/Avalon Biomed, the University of New England College of Dental Medicine, and the University of North Carolina at Chapel Hill Adams School of Dentistry.

Author Contributions

JK: investigation, data acquisition, data curation, visualization, software, and original draft preparation; KV: investigation, data acquisition, visualization, and software; GSD: writing, validation, review, and editing; AT: conceptualization, methodology, software, review, and editing; CP: writing, methodology, review, and editing; TK: conceptualization, methodology, data curation, writing, review, editing, and supervision. All authors reviewed and approved the final version of the manuscript.

ORCID iD

¹⁾JK: [e-mail], http://orcid.org/0000-0002-2196-8737

¹⁾KV: [e-mail], NA

²⁾GSD: [e-mail], NA

³⁾AT: [e-mail], NA

⁴⁾CP: [e-mail], http://orcid.org/0000-0003-3884-5191

²⁾TK*: [e-mail], http://orcid.org/0000-0002-6364-8440

Acknowledgments

The authors would like to thank Ms. Cindy Stewart for reference support, Dr. Miki Furusho for table/figure consultation, and Ms. Lori Rand for writing consultation and editing.

Data Availability Statements

Data are available upon reasonable request to the corresponding author. All data generated or analyzed during this study have been included in the published article.

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