
==== Front
BMC Oral Health
BMC Oral Health
BMC Oral Health
1472-6831
BioMed Central London

4852
10.1186/s12903-024-04852-z
Research
Evaluation of pulp tissue dissolving efficiency of sodium and calcium hypochlorite solutions activated by ultrasonics and laser: an in vitro study
http://orcid.org/0000-0001-5866-3155
Ozturk Oznur
http://orcid.org/0000-0002-3864-9310
Genc Sen Ozgur ozgurgencsen@gmail.com

https://ror.org/041jyzp61 grid.411703.0 0000 0001 2164 6335 Dentistry Faculty, Department of Endodontics, Van Yuzuncu Yil University, Zeve Campus, 65080 Tusba, Van Turkey
6 9 2024
6 9 2024
2024
24 104630 4 2024
2 9 2024
© The Author(s) 2024
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by-nc-nd/4.0/.
Objectives

This study aimed to compare the tissue dissolving capacities of sodium and calcium hypochlorite [NaOCl and Ca(OCl)2] solutions applied without activation or with two irrigant activation methods.

Materials and Methods

One hundred and eight dentin cavities were prepared. Preweighed tissue pieces were placed in these dentin cavities, and samples were divided into nine groups of twelve. In groups 1, 2, and 3, NaOCl was used with non-activation (NA), passive ultrasonic (PUA), and laser activation (LA). With the same techniques, Ca(OCl)2 was used in groups 4, 5, and 6, and distilled water was used in groups 7, 8, and 9. The weight loss of tissue samples was calculated and analyzed using Two-way ANOVA and Duncan tests.

Results

Distilled water groups showed no tissue dissolution in any conditions. NaOCI and Ca(OCI)2 showed statistically similar dissolving effectiveness when used with the same technique. Activated groups dissolved significantly greater tissue than non-activated ones, the highest in LA.

Conclusions

Ca(OCI)2 can be an alternative to NaOCl; for both, the first choice of activation may be the LA.

Keywords

Calcium Hypochlorite
Laser
Irrigant
Sodium Hypochlorite
Tissue dissolution
Ultrasonics
issue-copyright-statement© BioMed Central Ltd., part of Springer Nature 2024
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pmcBackground

The removal of organic and inorganic residues, microorganisms, and toxic products in the root canal system plays an essential role in the success of root canal treatment. Pulp tissue residues left in the system may create an environment in which microorganisms can live and multiply, causing postoperative pain and treatment failure [1].

For this reason, using irrigation solutions with high tissue-dissolving ability is of great importance in cleaning both the main canal and physically hard-to-reach areas during root canal instrumentation.

Sodium hypochlorite (NaOCl), a widely used irrigation solution in endodontics, can efficiently eliminate pulp tissue, necrotic debris, and microbial biofilms from the root canal system [2]. Although its excellent tissue-dissolving capacity is one of the main reasons for using this solution, it performs best at high concentrations, which, unfortunately, require careful use, as there is a risk of toxicity [3]. NaOCl also has a chemically unstable structure, and some external factors (such as temperature, light, and storage conditions) can suppress the solution’s tissue-dissolving ability by affecting the number of chloride ions it contains [2]. For the above reasons, the search for an alternative irrigation solution for root canal treatment continues.

Since sodium hypochlorite efficiently dissolves vital and necrotic pulp tissue, it has long been used in biopulpectomy and necropulpectomy cases. The solution’s dissolving efficacy is directly proportional to its chlorine content [4]. Chlorine, an active oxidizer, also allows the irreversible inhibition of essential bacterial enzymes [5]. Studies have reported that calcium hypochlorite [Ca(OCl)2] had a higher percentage of usable chlorine ions than NaOCl [6, 7] and demonstrated a considerable dissolving effect on organic tissue [8]. Furthermore, cytotoxicity studies showed that Ca(OCl)2 provided more positive results for cell viability and induced far lower inflammatory response than NaOCl [9]. Based on these findings, Ca(OCl)2 could be a promising root canal irrigant alternative to NaOCl.

Although conventional needle irrigation has been the most widely used method for intracanal irrigation, it is known that this method is not effective enough for the irrigation solution to be delivered to and penetrate the complex three-dimensional microstructure of the root canal system, especially to the apical third of the canal [10]. Therefore, irrigant activation methods are recommended to increase the efficiency of irrigation solutions in the root canal system and ensure their adequate dispersion in root canal irregularities [11].

Passive ultrasonic activation (PUA) stands out as an irrigation technique whose effectiveness has been demonstrated by many studies. In this technique, a non-cutting file or flat wire is attached to an ultrasonic device to vibrate the solution. It has been shown that PUA removes pulp tissue residues, dentine debris, and planktonic bacteria more effectively than the conventional technique [12]. In addition, laser applications of different wavelengths have been suggested as adjuvants or alternatives to cleaning procedures in root canal treatment [13]. Erbium lasers have been successfully used for irrigant activation in root canals, and several studies have reported that these lasers increase the pulp tissue-dissolving efficiency of NaOCl [14, 15].

NaOCl's superior tissue dissolving effect is well reported; nevertheless, it is also known that it may lead to severe injuries when it is extruded into the periapical area. The risk of accidental extrusion of solutions from the root end is a possibility that will not entirely be eliminated, and it increases significantly in teeth with apical resorption or a wide foramen [16–18]. For this reason, research should be conducted to increase the effectiveness of more biocompatible irrigants by supporting them with activation, and clinicians should be offered a safer alternative than NaOCl. Although various studies have investigated the tissue-dissolving abilities of Ca(OCl)2 and NaOCl, to the authors’ knowledge, no comparative evaluation has yet been carried out of these two solutions’ tissue-dissolving capacities when activated. Therefore, this study aimed to compare the pulp tissue-dissolving efficacies of NaOCl and Ca(OCl)2 solutions when applied with non-activation (NA), PU, and Er, Cr: YSGG laser activation. The study adopted the following null hypotheses: (i) there would be no statistically significant difference in the pulp tissue dissolution amounts after the use of NaOCl and Ca(OCl)2 irrigants, and (ii) the type of irrigation technique would not lead to a statistically significant difference in terms of pulp tissue dissolution.

Methods

The manuscript of this laboratory study has been written according to Preferred Reporting Items for Laboratory studies in Endodontology (PRILE) 2021 guidelines [19] (Fig. 1).Fig. 1 Preferred Reporting Items for Laboratory studies in Endodontology (PRILE 2021) flow chart

Sample size calculation

The sample size calculation based on data obtained from a pilot study indicated that 12 teeth would be sufficient per group (type-1 alpha error = 5%, effect size = 0.8, power = 80%).

Provision of bovine pulp tissue

This study used freshly extracted mandibular incisors from commercially slaughtered bovine. Therefore, this study had no influence on the animals’ fate. Teeth crowns were cut from the enamel cement junctions with a diamond fissure bur (Diatech AG, Switzerland) and separated from the roots. The pulp tissue was carefully removed (Fig. 2a) via hemostatic forceps and washed with distilled water to remove excess blood and debris. After the teeth were dried on blotting paper, they were placed in Eppendorf tubes and stored at -40 °C until the experiments were carried out.Fig. 2 a Bovine tooth and extirpated pulp tissue, b Dentine cavity prepared in the inverted bovine tooth crown, c Tube-placed dentine cavity

Preparation of the dentine cavity models

One hundred and eight anterior teeth of similar sizes that had been extracted from commercially slaughtered bovine were selected. Dental crowns were cut from cement–dentine junctions with a diamond fissure bur and separated from their roots. The crowns were reversed, and standard dentine cavities were prepared from the cervical to the incisal direction. Dentine cavities with standard dimensions (length: 5 mm; width: 4 mm; depth: 5 mm) were prepared (Fig. 2b), and these samples were randomly assigned to nine groups (n = 12) using a computer algorithm (http://www.random.org).

Preparation of the irrigation solutions

To prepare the 2.5% (w/v) Ca(OCl)2 solution, a tablet form of Ca(OCl)2 (Jianghan Salt & Chemical Complex, Qianjiang, Hubei, China) was crushed into granules. Distilled water was added to the preparate while it was mixed with a magnetic stirrer until a 2.5% solution was obtained.

The solution of 2.5% NaOCl was obtained by diluting 5% NaOCl preparate (Mikrovem AF, Istanbul, Turkey) sold for dental use with distilled water.

The solutions’ pH values were measured using a digital pH meter (WTW, Wellheim, Germany). Each measurement was repeated three times, and averages were recorded.

Preparation of the pulp tissue samples

Before the irrigation applications were started, the frozen pulp tissues were left to thaw at room temperature for approximately 1 h. The tissues were cut into smaller particles with a #15 surgical blade (Swann Morton Surgical Blades, Sheffield, UK) and mixed to create a random tissue mass. The pulp tissue pieces were taken from this mass and weighed three times using a precision balance. The average weight of each sample was calculated and equalized to 8 ± 0.5 mg. These weights were recorded as initial weights (W0).

Irrigation and activation processes

Before the applications were begun, 3-mm-thick pieces were cut from the transparent saliva ejector tube and fixed with cyanoacrylate adhesive around the dentine cavities (Fig. 2c) to prevent the irrigation solution from spilling out of the cavity.

Group 1 (NaOCl – Non-Activation, n = 12): Preweighed pulp tissue samples were placed in the dentine cavities, and the cavities were filled with 1 mL of 2.5% NaOCl solution using a dental syringe. After 90 s, the solution was aspirated using a dental syringe, and 1 mL of fresh 2.5% NaOCl was injected into the cavity. In this way, four periods of 90 s were applied.

Group 2 (NaOCl – PUA, n = 12): Pre-weighed pulp tissue samples were placed in the dentine cavities, and the cavities were filled with 1 mL of 2.5% NaOCl solution using a dental syringe. The #25 IRRI-S tip (Satelec Acteon Group, Merignac, France) driven by an ultrasonic device (VDW Ultra, VDW, Munich, Germany) was placed in a stationary position 3 mm deep into the solution in the cavity, and activation was performed at 30% power. Activation for 30 s was followed by 60 s of waiting time. This procedure was repeated four times and took a total of 6 min. Between each period, the irrigant solution in the dentine cavity was removed via aspiration with a dental injector, and 1 mL of fresh 2.5% NaOCl solution was injected into the cavity.

Group 3 (NaOCl – LA, n = 12): Pre-weighed pulp tissue pieces were placed in the dentine cavities, and the cavities were filled with 1 mL of 2.5% NaOCl solution using a dental syringe. The Er, Cr: YSGG laser device (Waterlase MD, Biolase, Irvine, CA, USA) was set to 0.75 W and 20 Hz. The 21-mm Biolase RFT-2 (Biolase, Irvine, CA, USA) tip of the laser device was positioned 3 mm deep into the solution in the dentine cavity, and the procedure was performed with 30 s of stationary activation of the irrigant and 60 s of waiting. This procedure was repeated four times and took 6 min. Between each period, the irrigant in the cavity was aspirated using a dental syringe, and 1 mL of fresh 2.5% NaOCl solution was injected into the cavity.

Group 4 (Ca(OCl)2 – Non-activation, n = 12): The Group 1 procedure was performed using Ca(OCl)2 solution as the irrigant instead.

Group 5 (Ca(OCl)2 – PUA, n = 12): The Group 2 procedure was performed using Ca(OCl)2 solution as the irrigant instead.

Group 6 (Ca(OCl)2 – LA, n = 12): The Group 3 procedure was performed using Ca(OCl)2 solution as the irrigant instead.

Group 7 (Distilled water – Non-activation, n = 12): The Group 1 procedure was performed using distilled water as the irrigant instead.

Group 8 (Distilled water – PUA, n = 12): The Group 2 procedure was performed using distilled water as the irrigant instead.

Group 9 (Distilled water – LA, n = 12): The Group 3 procedure was performed using distilled water as the irrigant instead.

Following the completion of each irrigation procedure, pulp tissue samples were removed, rinsed with 2 mL distilled water, blotted dry on absorbent papers, and weighed three times on the same precision balance. The averages were calculated and recorded as the final weights (W1). The weight loss of each pulp tissue sample was calculated by subtracting W1 from W0. These results were subjected to statistical analysis.

Statistical analyses

Descriptive statistics regarding the dissolution amounts of pulp tissue are given as mean, standard deviation, minimum, and maximum values. Normality was tested and confirmed by the Kolmogorov–Smirnov test. A two-way analysis of variance (ANOVA) was performed to determine whether there was a difference between the groups, and the Duncan test was applied for multiple comparisons. Since the interaction was significant, multiple comparisons were made at the subgroup level. The IBM SPSS (ver: 21) statistical package program was used for calculations, and the statistical significance level was set at 5%.

Results

Table 1 shows the amount of weight loss (in mg) in pulp tissue samples after different applications. Table 1 Means ± standard deviations, minimum and maximum values of weight loss (in mg) in pulp tissue samples after using different irrigants and activation procedures

	NaOCl	Ca(OCl)2	Distilled Wate	
Mean ± SD	Min	Max	Mean ± SD	Min	Max	Mean ± SD	Min	Max	
NA	2.2 ± 0.47cA	1.4	3.2	2.18 ± 0.41cA	1.6	3	0.27 ± 0.14aB	0.0	0.5	
PUA	3.5 ± 0.44bA	2.7	4.3	3.57 ± 0.47bA	2.8	4.4	0.3 ± 0.11aB	0.1	0.5	
LA	4.87 ± 0.42aA	4.2	5.6	4.82 ± 0.43aA	4.1	5.6	0.3 ± 0.1aB	0.2	0.5	
a, b, c: ↓ Different lowercases in the same column represent the significant difference between activation methods (p < 0.05)

A, B: → Different capitals on the same line represent the significant difference between groups receiving different irrigation solutions (p < 0.05)

When different applications using 2.5% NaOCl were compared, a significantly higher amount of pulp tissue dissolution was observed in the groups where irrigation activation systems (LA, PUA) were applied than in the non-activated group (p < 0.05). There was a statistically significant difference between the LA and PUA groups (p < 0.05), and the LA group exhibited significantly more pulp tissue dissolution than the PUA group.

When different applications using 2.5% Ca(OCl)2 were compared, a significantly higher amount of pulp tissue dissolution was observed in the activated groups (LA, PUA) than in the non-activated group (p < 0.05). The LA group exhibited significantly greater pulp tissue dissolution than the PUI group (p < 0.05).

In distilled water groups, there was no significant difference between application methods in terms of tissue dissolution (p > 0.05).

There was no statistically significant difference between NaOCl and Ca(OCl)2 solutions when the same activation method was used. NaOCl and Ca(OCl)2 solutions dissolved significantly more tissue than distilled water in the activated and non-activated groups (p < 0.05). The distilled water group exhibited the least tissue dissolution regardless of the application method.

The solutions’ pH values were found to be high and approximately the same [pH Ca(OCl)2 = 11.8; pH NaOCl = 12].

Discussion

Necrotic or vital pulp tissue residues remaining in the system after root canal treatment may ruin the healing process and cause infection. Therefore, the complete removal and dissolution of the organic tissue plays an essential role in the success of root canal treatments. Although NaOCl is commonly used because of its strong dissolving effect on pulp tissue, it has been demonstrated that Ca(OCl)2 is a more stable chemical containing more available free chlorine ions than NaOCl [9]. In addition, cytotoxicity studies have shown that Ca(OCl)2 demonstrated favorable results for cell viability in vitro and induced less inflammatory response in tissues than NaOCI [9]. Therefore, this study examined the dissolving effects of sodium and calcium hypochlorite solutions on pulp tissue when applied with different irrigation techniques.

It has been reported that dentine has a significant buffering effect against acid and alkaline solutions and reduces the tissue-dissolving capacity of NaOCl [20, 21]. Arslan et al. [22] applied NaOCI to pulp tissue in dentine models for 1 h and achieved less dissolution than the 30-min application by Almeida et al. [21] to pulp tissue in a plastic tube. According to the researchers, the buffering effect of dentine could explain this result [22]. Therefore, we opted for the dentine cavity model recommended by Slutzky‐Goldberg [23] in our study to account for dentine’s buffering effect and simulate clinical conditions as much as possible.

This study used bovine pulp tissue, which is highly similar to human dental pulp. The pulp tissue samples were placed in dentine cavities instead of plastic tubes during the applications due to the dentine’s possible buffering effect on the solutions. Naturally, the structural, dimensional, and geometrical differences between a dentine cavity and a root canal may be considered a limitation of this study and may have changed the pulp tissue–irrigant interaction. On the other hand, it is reported that the dissolution effect of the irrigant may not be observed if a small amount of irrigating solution is used relative to the pulp tissue’s quantity or an insufficient reservoir of solution is used [24]. In this respect, a root canal model may prevent the irrigant from accessing some areas of the canal space, limiting the solution’s contact with the pulp tissue in the root canal. The current study aimed at comparatively evaluating only the tissue dissolving efficiency of the two solutions; for this reason, like some previous researchers [22, 23], we preferred a dentine cavity model to provide an experimental standardization and a precise examination.

As the tissue-dissolving capacity of irrigation solutions may vary depending on concentration [25], the current study used the same concentrations (namely, 2.5%) of Ca(OCl)2 and NaOCl solutions. The solutions demonstrated statistically similar dissolution effects on pulp tissue; thus, our null hypothesis (i) was accepted. De Paula et al. [26] compared the tissue-dissolving capacities of Ca(OCl)2 and NaOCl solutions at 0.5%, 1.0%, 2.5%, and 5.25% concentrations. Similar to our findings, they found no statistically significant difference between solutions at the same concentration. In contrast, Dutta and Saunders [6] reported that the amount of tissue dissolved in 5 min by NaOCl was significantly higher than that dissolved in Ca(OCl)2 when the solutions were of similar concentrations (4.65% and 5%). However, in contrast to the current study, Dutta and Saunders [6] employed bovine muscle tissue placed in test tubes. We assume that the discrepancy between their results and ours may be due to the different dissolution resistances of pulp and muscle tissue and dentine’s buffering effect on the solutions.

The positive impact of passive ultrasonic activation on the tissue-dissolving ability of hypochlorite solutions has been demonstrated in many studies [27, 28]. In agreement with these results, in the current study, PUA enhanced the tissue-dissolving ability of NaOCl and Ca(OCl)2, resulting in a significantly greater pulp dissolution than the NA group. This study used the intermittent flush technique, as it is easier to control the ultrasonic tip’s depth and the irrigant’s volume during the application. In the literature, various durations and cycle counts for PUA have been suggested [29]. Although some authors [11] have claimed that the continuous flush technique, which consists of a single uninterrupted cycle with a continuous irrigant supply, provides more effective results and reduces the time needed for irrigation, it has also been reported that the intermittent method can be equally or more efficacious, depending on the irrigation time applied [30]. It has been shown that a longer agitation time (30 s) in PUA results in increased tissue dissolution [31] and that intermittent flushing creates a cumulative effect after the third cycle in removing the pulp tissue [30, 32]. Therefore, to both allow sufficient time for activation and benefit from the cumulative effect, in the current study, PUA was applied with 30 s of activation and 60 s of waiting time, and these processes were repeated four times using fresh solutions.

Erbium lasers are long known to be promising tools for irrigation effectiveness; however, they have disadvantages, such as the high prices of the devices and the need for expensive disposable tips, so their usage among clinicians spreads slower than that of the other systems. Irrigant activation by these lasers has been reported to positively influence the degree of tissue dissolution by enhancing NaOCl’s reaction rate and fluid movement [14]. In our study, the dissolving effect of both hypochlorite solutions on tissue was highest when laser activation was used, resulting in a significant difference from both PUA and non-activated applications. Thus, our second (ii) null hypothesis was rejected. Consistent with our results, Kuhn et al. [14] demonstrated that NaOCl revealed highly effective pulp tissue dissolution when activated with an Er:YAG laser. An in vitro study by Srinivasan et al. [15] compared pulp tissue dissolution rates when 5.25% NaOCl was used with Er,Cr:YSGG laser activation, with PUA, and without activation. Similar to our results, it was observed that the highest pulp tissue dissolution occurred in the Er, Cr: YSGG laser group followed by the PUA group. Efficacy may have been highest in the laser group because of the shock waves and rapid streaming caused by laser-induced vapor and bubbles within the irrigants [33].

Our findings show that the Er: Cr, YSGG laser had no dissolving effect on pulp tissue since no tissue dissolution was detected in distilled water activated with the laser. This finding emphasizes that laser activation only has a supportive effect on irrigation solutions that can dissolve tissue, consistent with previous research results from Guneser et al. [34].

Conclusions

Based on the findings of this study, the pulp tissue-dissolving effectiveness of 2.5% NaOCl and 2.5% Ca(OCl)2 solutions was similar when the same application method was used, and the LA method showed the best performance on tissue dissolvement with both irrigants. Therefore, Ca(OCl)2 could be an alternative to NaOCl in root canal irrigation since it is less toxic to periapical tissues. Additionally, to enhance the dissolution ability of these hypochlorite solutions, laser activation by Er,Cr:YSGG may be an appropriate choice if the cost is ignored. Future studies can be conducted to examine whether these irrigants’ lower concentrations provide the same advantages when activated with different laser settings.

Abbreviations

NaOCl Sodium hypochlorite

Ca(OCl)2 Calcium hypochlorite

NA Non-activation

PUA Passive ultrasonic activation

LA Laser activation

Acknowledgements

This article was derived from Oznur Ozturk’s specialization thesis.

Authors gratefully acknowledge SiddikKeskin for performing the statistical analyses.

Authors’ contributions

Conception and Design of the Work: OGS Performing the Experiments: OO The First Draft of the Manuscript written by: OGS, OO Revising the Work Critically for Intellectual Content: OGS Final Approval of the Version to be Published: OGS, OO.

Funding

No funding was received for this study.

Availability of data and materials

The datasets used and/or analysed during the current study are available from the corresponding author on a reasonable request.

Data Availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent for participate

Van Yuzuncu Yıl University’s Animal Experiments Local Ethics Committee (Van YUHADYEK) evaluated and approved that the study protocol does not require ethical approval (decision date: 30/09/2021; number: 2021/09-11).

Consent for publication

Not applicable.

Competing interest

The authors declare no competing interests.

Publisher’s Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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