Abstract
Background
This study aimed to investigate the effect of different sodium hypochlorite (NaOCl) concentrations on the efficacy of tissue dissolution with PIPS activation in artificial root canal models.
Methods
Artificial root canal models were divided into eight groups (n = 30/group), based on irrigation method (conventional needle irrigation or PIPS) and irrigant concentration (saline, 1% NaOCl, 2.5% NaOCl, and 5.25% NaOCl). Irrigation was performed using standardized protocols. Soft tissue samples were obtained from porcine palatal mucosa and placed at coronal 1/3, middle 1/3 and apical 1/3 of the root canal. Tissue dissolution was quantified by measuring weight loss of each tissue sample. Free available chlorine (FAC), pH and temperature of irrigant were also assessed. For normally distributed data, factorial repeated measures analysis of variance (ANOVA) and Least-Significant Difference (LSD) comparison were used. Paired t-test and Wilcoxon signed-rank test were used to assess tissue dissolution after irrigation.
Results
PIPS activation contributed to significantly higher mass reduction percentage of tissue samples (p <.05). 5.25% NaOCl exhibited the highest mass reduction percentage of all irrigants under PIPS activation. The highest dissolution occurred with 5.25% NaOCl under PIPS, achieving near-complete tissue dissolution in the apical third. Decrease in pH and increase in temperature of NaOCl after irrigation were also observed.
Conclusions
NaOCl showed greater tissue dissolving efficacy under PIPS activation than CNI. The tissue dissolving ability of NaOCl increased as the concentration increased. PIPS demonstrates superior apical irrigation efficiency compared to CNI, highlighting its clinical potential.
Keywords: Photon-initiated photoacoustic streaming (PIPS), Sodium hypochlorite, Tissue dissolution
Background
Root canal treatment is the principal method of treating endodontic and periapical pathologies, focusing on the removal of necrotic pulp tissue, debris and microbial biofilms such as Enterococcus faecalis [1]. Complete root canal treatment is achieved by thorough preparation of the root canal, both instrumentally and chemically [2]. Given that more than 35% of the root canal surface remains uninstrumented regardless of the technique used, chemical preparation with disinfecting solutions is necessary for irrigating the root canal system [3].
Sodium hypochlorite (NaOCl) is the most commonly used irrigant for chemical preparation of root canals. One of the important functions of NaOCl is to dissolve residual pulpal tissues in the root canal system in order to avoid secondary infections that may lead to failure of root canal treatment. Conventional chemical preparation has proven to be inadequate for the complete elimination of infection due to the complexity of the root canal system [4–6]. Additionally, it has been proved that there are significant differences in irrigation effect between different positions of root canal [7–9]. For this reason, several techniques have been developed to ensure sufficient disinfection and debridement of the root canal system.
Laser-activated irrigation (LAI) utilizes lasers to activate the irrigant during root canal treatment to remove biofilm, debris and smear layer [10]. In comparison to traditional methods such as sonic irrigation and conventional needle irrigation (CNI), LAI can achieve superior debridement results [11–17]. This is due to the fact that LAI is capable of generating a larger area and higher flow rate of fluid movement within the root canal, which effectively activates the root canal irrigant, enhancing its antimicrobial efficacy [18]. Photon-initiated photoacoustic streaming (PIPS) is an improved system of LAI, which entails the generation of acoustic streaming and shockwaves within the root canal by Er: YAG laser irradiation at a low energy setting [19]. Compared with CNI, PIPS can better eliminate residual debris in the root canal [20].
Recent research has shown that LAI can be applied with lower concentrations of NaOCl while maintaining its antimicrobial effect during the activation of NaOCl irrigant [21, 22]. It has also been demonstrated that the tissue dissolution ability of NaOCl could also be significantly enhanced by PIPS when compared to passive irrigation [23]. However, as this experiment is unable to simulate the fluid dynamics during root canal irrigation, the results cannot be directly extrapolated to clinical practice. Additionally, the research only tested changes in the tissue dissolving capacity of 5.25% NaOCl under PIPS activation. The objective of this study was to investigate the effect of different NaOCl concentrations on the efficacy of tissue dissolution with PIPS activation in artificial root canal models.
Methods
Tissue sample preparation
Tissue was obtained from the porcine palatal mucosa after the pigs were slaughtered for food production. Subsequently, the epithelial tissue and connective tissue of the porcine palatal mucosa were isolated using a scalpel consisting of a #3 handle and a #11 blade. The resulting fragments (mean initial weight ± SD: 1.45 ± 0.28 mg) were stored in normal saline until use. To ensure comparability between experimental groups, these fragments were randomly distributed into groups using a computer-generated random number sequence. The surface area of samples located in the same section of the root canal that received irrigation was almost the same. The process of tissue samples preparation is shown in Fig. 1A.
Fig. 1.
A schematic representation of tissue sample preparation and root canal preparation: (A) Splitting palatal mucosa after isolation of connective tissue into blocks. (B) The “coronal 1/3, middle 1/3 and apical 1/3” mentioned above refers to the upper edge of the tissue being 2 mm, 6 mm and 10 mm from root canal orifice respectively. (C) The needle used in CNI was inserted to 1 mm above the sample. (D) The laser tip was placed at the canal entrance with a 22G syringe injecting the irrigants on the side of the tip
Root canal model preparation
Artificial root canal models were fabricated from resin with a straight root canal and a coronal reservoir resembling a pulp chamber (Endo Training Block; Dentsply Maillefer, Ballaigues, Switzerland). The length of root canal was 12 mm. The root canal was prepared using M3 file (UnitedDental, Shanghai, China) to #30/0.04.
Groups and irrigation
Sample size calculation was performed via a priori power analysis using the G*Power software (Heinrich Heine University, Düsseldorf, Germany), with effect size derived from pilot data, achieving 80% power at α = 0.05. Tissue samples obtained from the same palatal mucosa (n = 240) were randomly distributed into two groups (n = 120/group): CNI or PIPS irrigation, each containing four subgroups (n = 30/subgroup) using saline, 1% NaOCl, 2.5% NaOCl and 5.25% NaOCl as irrigants respectively. To find out the differences of tissue dissolution in different root canal positions, tissue in every subgroup was placed in coronal 1/3 (n = 10/subgroup), middle 1/3 (n = 10/subgroup) and apical 1/3 (n = 10/subgroup) of the artificial root canal model.
Prior to the start of the experiment, the tissue samples were removed from saline, drained of surface moisture and weighed, after which they were positioned into the appropriate position by periodontal probe. The “coronal 1/3, middle 1/3 and apical 1/3” mentioned above refers to the upper edge of the tissue being 2 mm, 6 mm and 10 mm from the root canal orifice, respectively (Fig. 1B). All placement procedures were performed by a single trained operator to ensure consistency. Tissues were gently guided (not folded or compressed) to the predetermined position. After irrigation, the location of the sample was recorded to calculate its displacement. Finally the sample was taken out using a barbed broach and weighed again to determine the tissue dissolution percentage. To prevent measurement bias, the operator performing weight measurements was blinded to group allocation throughout the study. Samples were coded numerically prior to measurement, with group identities revealed only after data collection was complete. This blinding protocol was implemented to prevent any potential measurement bias.
For CNI, a 22 G open-ended needle (Transcodent) was inserted to 1 mm above the sample (Fig. 1C). Each sample was irrigated at a flow rate of approximately 0.17 mL/s, with a total volume of 5 mL delivered over 30 s in three cycles. For PIPS irrigation, a 2,940-nm Er: YAG laser (AT Fidelis; Fotona, Ljubljana, Slovenia) with a handpiece (R14-PIPS, Fotona) holding a 400-µm-diameter quartz tip (XPulse 400/14, Fotona) was used to activate the irrigants during the irrigation procedure. The tip was applied according to the manufacturer’s guidelines at 0.3 W, 15 Hz, and 20 mJ per pulse without water/air spray. The laser tip was placed at the canal entrance with a 22G syringe injecting the irrigants on the side of the tip (Fig. 1D). The irrigants in the root canals were activated for 30 s with 5 ml of different irrigants in a cycle. Three cycles were performed on each sample. The tip was left stationary and the irrigant was activated at predetermined settings with constant irrigation to the pulp chamber, allowing passive filling of the canal and pulp chamber with irrigant throughout the irrigation period.
Displacement
Tissue displacement was determined by two independent observers using visual inspection. The reference point was defined as the apex of the specimen. The distance between the reference point and the canal orifice was measured before and after irrigation using a digital caliper (accuracy: ±0.1 mm). Displacement values were calculated as:
 |
Displacement > 0 refers to moving towards apical direction, < 0 refers to moving towards coronal direction. Inter-observer agreement was assessed using a two-way random-effects intraclass correlation coefficient (ICC). The final displacement value for each sample represents the average of two observers’ measurements to minimize bias.
Free available chlorine, pH and temperature
Experiments were conducted at 24℃ and the initial temperature of all the irrigants used in this experiment was also 24℃. All the irrigants were collected after irrigation. The pH and temperature of the irrigants were measured using a pH meter (HI 2211 pH-ORP Meter; HANNA Instruments, Woonsocket, RI) before and after the experiment. Before each measurement, calibration is performed using standard samples provided by the manufacturer. The amount of free available chlorine (FAC) was tested using an iodine/thiosulfate titration method [24]. We investigated FAC of the four irrigants before irrigation. For groups using PIPS, we took the FAC tested after irrigation without tissue in the root canal model as the initial FAC. When measuring temperature, pH value, and effective chlorine, we repeated the measurement three times and recorded the average value.
Statistical analysis
Percentage of tissue dissolution was calculated using the formula: [(initial weight-final weight)/initial weight] *100. Statistical Package for the Social Sciences (SPSS) software Version 17.0 (IBM SPSS Inc, Chicago, IL) was used to perform statistical analysis.
For data confirming the assumption of normal distribution of data, a factorial repeated measures analysis of variance (ANOVA) and Least-Significant Difference (LSD) comparison were used to detect whether there is a difference between groups. For data not satisfying normal distribution, Kruskal–Wallis test and Dunn’s comparison procedures were used. Additionally, paired t-test and Wilcoxon signed-rank test were used to compare whether significant changes occur after irrigation.
Results
Tissue dissolution
Comparisons of mass reduction percentage of tissue using CNI or using PIPS are shown in Fig. 2. PIPS activation contributed to significantly higher mass reduction percentage of tissue samples (p <.05).
Fig. 2.
(A1-C1) Comparisons of percent weight loss of tissue using CNI or photon-initiated photoacoustic streaming (PIPS) in coronal 1/3, middle 1/3 and apical 1/3, respectively. Data are presented as mean ± SD (n = 10 per group). * indicate significant differences between the CNI and PIPS irrigation groups for each irrigant concentration. (A2-C2) Comparisons of percent weight loss between different irrigants in coronal 1/3, middle 1/3 and apical 1/3 respectively. Data are presented as mean ± SD (n = 10 per group). * denote significant differences between different groups within the same irrigation protocol. *p <.05, **p <.01, ***p <.001, ****p <.0001
Comparisons of mass reduction percentage between different irrigants in coronal 1/3, middle 1/3 and apical 1/3 are shown in Fig. 2. All concentrations of NaOCl showed significantly higher mass reduction percentage of tissue samples than saline (p <.05). 5.25% NaOCl exhibited the highest mass reduction percentage of all the irrigants, achieving nearly total dissolution under PIPS activation. Mass reduction percentage of tissue situated at different positions in root canal is shown in Table 1. Tissue in apical 1/3 was almost totally dissolved by NaOCl under PIPS activation.
Table 1.
Percent weight loss (%) of tissue situated at different positions in root Canal (mean ± SD)
| Irrigant | Position | CNI groups | PIPS groups |
|---|---|---|---|
| Saline | coronal 1/3 | 0.56 ± 1.76a | 0.56 ± 1.76a |
| middle 1/3 | 0.00 ± 0.00a | 0.67 ± 2.11a | |
| apical 1/3 | 2.50 ± 5.63a | 0.00 ± 0.00a | |
| 1%NaOCl | coronal 1/3 | 10.40 ± 4.17a | 51.06 ± 7.53a |
| middle 1/3 | 10.63 ± 3.91a | 58.80 ± 8.67a | |
| apical 1/3 | 19.14 ± 12.95a | 98.18 ± 5.75b | |
| 2.5%NaOCl | coronal 1/3 | 31.06 ± 6.25a | 59.97 ± 8.67a |
| middle 1/3 | 39.12 ± 6.91b | 71.95 ± 8.35b | |
| apical 1/3 | 56.93 ± 7.94c | 100.00 ± 0.00c | |
| 5.25%NaOCl | coronal 1/3 | 82.19 ± 6.81a | 98.97 ± 2.16a |
| middle 1/3 | 87.04 ± 8.25a | 100.00 ± 0.00a | |
| apical 1/3 | 96.59 ± 5.90b | 100.00 ± 0.00a |
Lower-case letters in a columns are for the comparison of percent weight loss between different positions using the same irrigation protocol. Same lower-case letters follow means that do not differ statistically (p >.05)
Abbreviations: CNI conventional needle irrigation, PIPS photon-initiated photoacoustic streaming, NaOCl sodium hypochlorite
Displacement
Displacement of tissue in all groups is shown in Table 2. ICC analysis showed excellent reliability (ICC = 0.997 (95% CI: 0.996–0.998.996.998)). It can be found that PIPS caused more displacement of sample than CNI. There is no significant difference between groups using CNI (p >.05). With PIPS activation, 1% NaOCl caused the greatest displacement of tissue in apical 1/3. 5.25% NaOCl caused apical displacement while other irrigants caused coronal displacement.
Table 2.
Displacement (mm) of tissue (median ± quartiles) in all groups
| Irrigant | Position | CNI groups | PIPS groups | p-value |
|---|---|---|---|---|
| Saline | coronal 1/3 | −0.00 ± 0.03a | −0.65 ± 0.80a | < 0.05 |
| middle 1/3 | 0.00 ± 0.00a | −1.45 ± 0.95b | < 0.05 | |
| apical 1/3 | 0.00 ± 0.00a | −1.90 ± 0.72c | < 0.05 | |
| 1%NaOCl | coronal 1/3 | −0.00 ± 0.03a | −0.80 ± 0.98a | < 0.05 |
| middle 1/3 | 0.00 ± 0.00a | −1.80 ± 0.98b | < 0.05 | |
| apical 1/3 | 0.00 ± 0.00a | −2.80 ± 0.97c | < 0.05 | |
| 2.5%NaOCl | coronal 1/3 | 0.00 ± 0.00a | −0.85 ± 0.48a | < 0.05 |
| middle 1/3 | 0.00 ± 0.00a | −1.40 ± 0.53b | < 0.05 | |
| apical 1/3 | 0.00 ± 0.00a | ND | ND | |
| 5.25%NaOCl | coronal 1/3 | 0.10 ± 0.43a | 0.00 ± 0.55 | > 0.05 |
| middle 1/3 | 0.00 ± 0.38a | ND | ND | |
| apical 1/3 | 0.00 ± 1.53a | ND | ND |
Lower-case letters in a columns are for the comparison of displacement between different positions using the same irrigation protocol. Same lower-case letters follow means that do not differ statistically (p >.05). Displacement >0 refers to moving towards apical direction, <0 refers to moving towards coronal direction. ND denotes that displacement could not be measured as the tissue sample was completely dissolved
Abbreviations: CNI conventional needle irrigation, PIPS photon-initiated photoacoustic streaming, NaOCl sodium hypochlorite, ND not detected
Free available chlorine, pH and temperature
Comparisons of FAC between different subgroups are shown in Fig. 3. FAC of NaOCl was significantly increased by activation of PIPS. 5.25% NaOCl showed the highest content of FAC. pH and temperature of all subgroups before and after irrigation are shown in Table 3. Decrease in pH and temperature elevation can be observed in most of the groups.
Fig. 3.
Comparisons of FAC between groups using different irrigation protocols. * indicate significant differences. Data are presented as mean ± SD (n = 30 per group). *p <.05, **p <.01, ***p <.001, ****p <.0001
Table 3.
pH and temperature (℃) of irrigant(mean ± SD) in all groups
| Groups | pH | Temperature(℃) | ||
|---|---|---|---|---|
| Initial | Final(mean ± SD) | Initial | Final(mean ± SD) | |
| Saline + CNI | 6.98 | 7.02 ± 0.03a | 24.00 | 23.97 ± 0.20a |
| Saline + PIPS | 6.98 | 6.99 ± 0.04a | 24.00 | 24.72 ± 0.29b |
| 1%NaOCl + CNI | 12.51 | 12.27 ± 0.07a | 24.00 | 24.17 ± 0.27a |
| 1%NaOCl + PIPS | 12.51 | 12.56 ± 0.12b | 24.00 | 25.46 ± 0.90b |
| 2.5%NaOCl + CNI | 12.72 | 12.51 ± 0.10a | 24.00 | 24.19 ± 0.19a |
| 2.5%NaOCl + PIPS | 12.72 | 12.65 ± 0.03b | 24.00 | 27.12 ± 0.23b |
| 5.25%NaOCl + CNI | 12.86 | 12.75 ± 0.05a | 24.00 | 25.18 ± 0.42a |
| 5.25%NaOCl + PIPS | 12.86 | 12.76 ± 0.05a | 24.00 | 25.19 ± 0.32a |
Lower-case letters in a column are for the comparison of percent weight loss between different positions using the same irrigation protocol. Same lower-case letters follow means that do not differ statistically (p >.05)
Abbreviations: CNI conventional needle irrigation, PIPS photon-initiated photoacoustic streaming, NaOCl sodium hypochlorite
Discussion
The current study investigated the effect of different concentrations of NaOCl on the tissue dissolving efficacy with PIPS activation. Previous studies often involve immersing standardized tissue samples in irrigants and measuring their dissolution rates [24–27], but these results cannot be directly extrapolated to clinical practice. This research simulated the flow of irrigants in the root canal system, which is of clinical relevance in research concerning PIPS.
Mass reduction percentage is used to compare tissue dissolving efficacy of different irrigation protocols directly, while FAC is used to represent it indirectly. 5.25% NaOCl in conjunction with PIPS can reach a satisfying irrigation result that almost 100% dissolved tissue samples, suggesting that high concentrations of NaOCl still have irreplaceable ability for chemical preparation to those at low concentrations, especially when activated by PIPS. Notwithstanding, the tissue dissolving capacity of 5.25% NaOCl may still be overestimated because hypertonic irrigants will draw water out of the samples and reduce their weight [28].
A visualization study shows that during PIPS activation, the maximal velocity of both 2.5% NaOCl and 5.25% NaOCl in the apical region of the root canal is higher than that observed in the middle region [29], showing potential of PIPS to dissolve apical tissue. In this research, tissue in apical 1/3 is almost totally dissolved by NaOCl under PIPS activation, indicating that tissue located at the apical area can also be dissolved thoroughly under activation of PIPS. However, previous studies have shown that tissue in the apical area does not dissolve as well as that in the coronal area during root canal irrigation [7–9]. The differences in weight of tissue placed at different positions may lead to this result. Owing to the larger space in coronal 1/3, tissue placed here was heavier and bigger than that placed at apical 1/3, thus more difficult to dissolve.
Basically, subgroups with higher mass reduction percentage of tissue also have higher initial FAC of irrigants. In other words, there is more chlorine reserve in high concentration of NaOCl, which is crucial in tissue dissolution and disinfection [30]. Besides concentration of NaOCl, activation techniques like PIPS are also related to FAC, which is validated in the current research.
The displacements of tissue samples in this study were calculated from the distances between the initial and final positions of the tissue specimens. 5.25% NaOCl caused displacement in the opposite direction from other irrigants. This is due to the fact that after the experiment, the tissue samples all underwent significant dissolution, resulting in a decrease in volume. After the irrigation was completed, the tissues would slide towards the apical region due to gravity. The coronal displacement was caused by PIPS activation through the cavitation effect [31]. In the present study, PIPS caused coronal displacement while CNI did not, which is meaningful. Extrusion of irrigants into the periapical region can happen with any irrigation protocol because the open apex provides a larger contact area [32]. The application of PIPS may reduce the risk of apical extrusion.
The relative ratio of OCl- and HOCl in NaOCl irrigants is significantly influenced by pH value. When pH is decreased, HOCl, a strong antiseptic, becomes more prevalent, resulting in greater antibacterial efficacy of the irrigants, while its tissue-dissolving capacity is diminished [33]. A finding needed to be explored more in the present study is pH of 1% NaOCl with PIPS activation after irrigation is even higher than that of before irrigation, which means there may still a dissolving potential of it. NaOCl’s reaction rate could be enhanced by heating the irrigants, namely raising the temperature [34]. Heating NaOCl while preserving its FAC enhances tissue dissolution, bacteria elimination, and smear layer removal [35]. We didn’t assess the contribution of temperature change to tissue dissolution. Since it is widely recognised that temperature increases occur during high-concentration sodium hypochlorite irrigation or LAI, and that this promotes tissue dissolution, no dedicated control group was set up in this experiment. This remains to be explored further in the future. It is worth noting that the temperature increase after irrigation did not exceed 4℃ at most in this experiment, which means that PIPS activation won’t lead to overheating of the irrigants and burning of the oral soft tissue.
Artificial resin-made root canal model was used in many studies to explore tissue dissolution efficacy of different irrigation technique [36, 37]. There are some limitations like it can’t simulate dentin that will chemically interact with irrigants [38]. What’s more, the design of straight root canal structure is simple without considering areas that are difficult for irrigants to access like isthmus and lateral canal. But anyway, standardization of the extracted teeth may not be as accurate as the root canal model. Additionally, resin teeth offer absence of biological variability and reproducibility—key factors for controlled in vitro studies. Considering that we also aim to investigate the efficacy of tissue dissolution in different areas of root canal, we finally chose resin artificial teeth as our experimental model. The present study still makes great progress from laboratory results to clinical practice. As far as the current results are concerned, 5.25% NaOCl still shows the greatest applicability in root canal preparation.
Conclusions
Within the limitations of this laboratory study, the tissue dissolving ability of NaOCl increased as the concentration increased. NaOCl showed greater tissue dissolving efficacy under PIPS activation than CNI. PIPS can achieve satisfying irrigation effect in apical region of root canal.
Acknowledgements
The authors acknowledge all the support and contributions of participators.
Abbreviations
- PIPS
Photon-initiated Photoacoustic Streaming
- NaOCl
Sodium Hypochlorite
- CNI
Conventional Needle Irrigation
- FAC
Free Available Chlorine
- LAI
Laser-activated Irrigation
Authors’ contributions
Q.Z.J. and Y.T.H. supervised the experiment and manuscript drafting. R.J.H. and C.C. contributed to the design of the study and performed most of the experiments. Y.C., L.X.G and H.X.D. participated in the experiment. R.J.H. carried out data analysis. R.J.H and Y.T.H. participated in drafting the manuscript and performing critical revision of the draft. All authors have read and approved the final version of the manuscript. R.J.H and Y.T.H contributed equally and are shared first authors of this manuscript.
Funding
This work was supported by GuangDong Basic and Applied Basic Research Foundation Project (2022A1515110601) and Guangzhou Municipal Science and Technology Bureau, Basic and Applied Basic Special Research Project (Youth Doctoral “Voyage” Project, Grant No. 2024A04J3387).
Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Runjie Hu and Yuting Huang contributed equally and are shared first authors of this manuscript.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.