Cytotoxic Evaluation of Effective Ecoproduce (EEP) as a Potential Root Canal Irrigant: A Preliminary In Vitro Study

Featured Application

Through this initial in vitro study, the most effective and safe concentration of EEP can be established, which will give a useful insight into the biocompatibility and safe concentration of EEP in root canal treatment. Further, the findings from this study will lead to further exploration on the antibacterial properties of the solution against other oral bacteria and the antibiofilm effect against multi-species biofilm, which is more clinically relevant. This will later help in the establishment of the solution as a potential alternative endodontic irrigation solution.

Abstract

Concerns have been raised about the usage of sodium hypochlorite (NaOCl) in endodontics following its toxic effects. Effective ecoproduce (EEP), an organic solution produced through the fermentation of fruit peels, exhibits antibacterial and antibiofilm action, suggesting its potential as an endodontic irrigant. However, studies on its cytotoxicity are limited. This in vitro study aimed to evaluate the cytotoxic effects of EEP at different concentrations and fermentation periods against the MC3T3-E1 cell. EEP derived from orange and pineapple peel waste and fermented for 3 and 6 months was prepared from 100% to 0.78% concentration. Briefly, 2.5% NaOCl was used as the comparison group. Cell viability was analysed using Alamar Blue and Live and Dead Cell assay. A transmission electron microscope (TEM) was used to evaluate ultrastructural changes to the cells. Data analysis was performed using a two-way mixed Analysis of Variance (ANOVA). EEP exhibited concentration-dependent cytotoxicity regardless of the fermentation period (p > 0.05). A concentration below 6.25% was non-cytotoxic and comparable to the negative control (p > 0.05). Live and Dead Cell assay and TEM analysis complement the findings. The mean cell viability of EEP at all concentrations for both fermentation periods was significantly higher than that of 2.5% NaOCl (p < 0.05). Conclusively, 6.25% EEP fermented for 3 and 6 months are non-cytotoxic and can serve as an alternative endodontic irrigants.

1. Introduction

Untreated dental caries can have a massive impact and consequences towards a patient’s oral health and quality of life. Following carious enamel breakdown, the bacteria can travel through the dentinal tubules and reach the pulp chamber [1], leading to pulpal inflammation and, later, pulp necrosis and periapical infection [2]. Irritable pain, sleep disturbance, localised intra-oral or facial swelling and school or work absences are among the outcomes that have been reported in the literature [3,4]. A life-threatening event caused by airway obstruction following cellulitis of dental origin has been reported [5], suggesting the importance of treating the infection promptly.

Root canal treatment has been the treatment of choice in managing teeth with infected and necrotic pulp tissue, provided that the tooth is still restorable and not indicated for extraction. The aims of root canal treatment are to reduce intracanal bacteria populations through the removal of the pulp tissue, optimise root canal disinfection via the delivery of antimicrobial agents and prevent reinfection of the canal post-treatment [6,7]. Removal of the biofilm and adequate antimicrobial action are of paramount importance to eradicate pulpal and periapical infections [1,8].

Mechanical instrumentation of the canal space has been the main step in eradicating the necrotic tissues, microbes, biofilm and debris from the root canal space. However, proper canal disinfection through mechanical instrumentation alone is hampered by the complex anatomy of the root canal systems, where access to the lateral canals, isthmuses and apical delta is limited by the rigidity of the endodontic instrument [9,10]. The presence of this complex anatomy indirectly provides shelter for the bacteria, acting as a niche for their accumulation and growth [1,9].

Irrigation of the root canal system with antibacterial solutions has been proposed as one of the strategies to overcome the shortcoming of mechanical instrumentation alone [9,11]. Canal shaping achieved through mechanical instrumentation facilitates the irrigation process that allows the antimicrobial solutions to penetrate and disinfect the areas which cannot be efficiently accessed by mechanical instrumentation. The procedure also helps in flushing out the debris from the root canal space. These combinations, also referred to as chemo-mechanical root canal preparation, create a sterile environment within the root canal system prior to final obturation and restoration [1,9].

For almost a century, sodium hypochlorite (NaOCl) has been the irrigant of choice in disinfecting root canals [12] and predominates other types of solutions available on the market for this purpose. NaOCl has been regarded as the gold standard irrigation solution for root canal treatment, mainly due to its capability to dissolve the pulpal tissue, good antimicrobial activity and ability to remove the biofilm and other organic components following mechanical instrumentation [13]. A concentration between 2 and 2.5% has been advocated, citing its antimicrobial efficacy against Streptococcus sp., Enterococcus faecalis and Actinomyces israelii as well as eliminating Gram-negative bacteria endotoxins [6,14].

Despite their excellent antimicrobial activity, clinicians have voiced their concern regarding the usage of NaOCl as a root canal irrigant, especially at high concentrations. This is owing to the fact that NaOCl itself is a bleaching agent, which can cause corrosive damage to the endodontic instruments and soft tissue ulceration and necrosis [15]. In addition, several case reports have described severe outcomes following NaOCl accidents during root canal treatment, which include pain, facial oedema and swelling, oro-pharynx ulceration, dyspnoea and respiratory distress [16,17,18]. Further, alternating irrigation between NaOCl and chlorhexidine gluconate, another antimicrobial solution can lead to the formation of a brown precipitate which may contain para-chloroaniline, a byproduct with mutagenicity potential [19].

Following the controversy surrounding the use of NaOCl in endodontics, researchers have been exploring other potential alternative irrigation solutions. They have turned their attention to developing irrigants based on the extracts of natural products since these extracts are considered cheap, readily available and, most importantly, safe [20]. Additionally, the use of natural products provides a plenteous source of bioactive molecules that can be beneficial in endodontic treatment. Extracts of neem, aloe vera, garlic, miswak and propolis are among those that have been investigated with comparable antibacterial action to those exhibited by NaOCl [21,22]. Another natural-based product that has caught our attention is effective ecoproduce (EEP).

EEP, an organic solution produced following the fermentation of fruit and vegetable organic waste, was first introduced by Dr Rusukon Poompanvong in Thailand [23,24]. It is also commonly known as garbage enzyme [25] or eco enzyme [26], although the word ‘enzyme’ might be inappropriate since the solution also contains organic acids and other secondary metabolites as well [27]. EEP has been used both in the agricultural and waste-management industry, mainly as a pesticide, insecticide, fertiliser, water treatment and sludge management [24,28,29]. Further, it also has been used as an alternative to bleaching agents for household disinfection and cleaning solutions [30].

In dentistry, in vitro studies have shown that EEP fermented for 3 and 6 months exhibits good antibacterial and antibiofilm action against Enterococcus faecalis, a resistant bacterial strain which is usually associated with root canal reinfection [25,26,27]. Additionally, Ng has reported a comparable antibiofilm action of EEP fermented for 6 months with NaOCl at a 6.25% concentration [31], highlighting the potential usage of EEP as an alternative irrigation solution for root canal treatment. Despite this promising application, the biocompatibility of EEP, particularly in endodontic practice, has not been thoroughly investigated yet. This missing link should be addressed accordingly in order to produce a safe, biocompatible and highly effective root canal irrigant. Thus, we have conducted this in vitro study with the aim of evaluating the cytotoxic effects of EEP at different concentrations and fermentation periods towards pre-osteoblast cells.

2. Materials and Methods

2.1. Ethical Approval

This study received ethical approval from the institutional research ethical committee (UKM PPI/111/8/JEP-2021-673).

2.2. Preparation of Test Materials and Control Solutions

EEP extracts were prepared based on the method outlined by Ng et al. [25] and Arun and Sivashanmugam [32]. The study used the same variety of Josephine pineapple (Ananas comosus L. var. comosus cv. Josephine) and Navel orange (Citrus sinesis), both obtained from the same vendor. A mixture of fruit peels was prepared, consisting of 30 g of orange peels and 45 g of pineapple peels mixed in a 6:4 ratio. Then, EEP extract was prepared in triplicate by combining 75 g of fruit peels, 25 g of molasses and 250 mL of water in a 3:1:10 ratio, kept in airtight containers. The containers were stored in the same location at room temperature. The containers were opened for 10 s every day to release the trapped gas. The fermentation was carried out for 3 and 6 months. Each sample from different fermentation periods was prepared three times on different days to create three biological triplicates.

At 3 months of fermentation, 3 months of EEP (3 M EEP) extract was sterile-filtered using a 0.22 µm pore-size syringe and further diluted to a concentration of 100%, 50%, 25%, 12.5%, 6.25%, 3.13%, 1.57% and 0.78% with culture medium using two-fold serial dilution method. The same dilution method was repeated for EEP fermented for 6 months (6 M EEP). For the cytotoxicity test, different concentrations of EEP were freshly prepared just before conducting the test.

The comparison group used in this experiment was 2.5% NaOCl, which is the gold standard for endodontic irrigants. It was prepared by diluting 5.25% NaOCl with distilled water to achieve the final concentration of 2.5% NaOCl. Different positive control was used for different cytotoxic tests conducted in this study. Briefly, 100% reduced Alamar Blue reagent and 10% dimethyl sulfoxide (DMSO) were used for the Alamar Blue and Live and Dead Cell assay, respectively. Culture media containing cells was used as an untreated negative control for both cytotoxic assays.

2.3. Cell Culture

The mouse pre-osteoblast cell lines (MC3T3-E1) used in this study were acquired from the institution’s bank cell. The cell subculturing procedure was performed in accordance with American Type Culture Collection (ATCC) animal cell culture guide [33,34] and Freshney [35]. The cells were cultured in tissue culture flask with Alpha Minimum Essential Medium (α-MEM) (Thermo Fisher Scientific, Waltham, MA, USA) and supplemented with 10% fetal bovine serum (Thermo Fisher Scientific, Waltham, MA, USA), 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and 1 mM sodium pyruvate (Thermo Fisher Scientific, Waltham, MA, USA) under standard cell-culture conditions at 37 °C in a humidified atmosphere of 95% air and 5% CO₂. At 70–80% confluency, the cells were trypsinised with 0.25% trypsin/EDTA (Thermo Fisher Scientific, Waltham, MA, USA) and split at a subcultivation ratio of 1:6 [33] for further passaging. The cells were passaged every 3 days and checked regularly for the absence of mycoplasma contamination. Cells with passage numbers four to six were employed in this study.

For both cytotoxicity assays, MC3T3-E1 cells were cultured in flat-bottom 96-well plates (Jetbiofil, Guangzhou, China) at a density of 5000 cells/well in 100 µL of culture media [36,37]. The cells were allowed to attach to the base of the plates for 24 h, incubated in a humidified atmosphere at 37 °C, 95% air and 5% CO_2. Upon completion of the incubation period, the culture medium was discarded from the well plates. Then, 3 M and 6 M EEP in eight concentrations, ranging from 100% to 0.78%; 2.5% NaOCl solutions; and positive and negative controls were added into 96-well plates in triplicates according to their respective group. This method was performed according to ISO 10993-5 [38].

Following the addition of test materials and control solutions, the well plates were incubated at 37 °C with 5% CO₂ for 24 h before the commencement of cytotoxicity tests.

2.4. Alamar Blue Cytotoxic Assay

After 24 h of incubation, the test solutions or culture media from each well were removed and then rinsed with 200 µL of phosphate-buffered solution. This step was taken to prevent any potential interaction between test solutions and the Alamar Blue reagent. Then, 100 µL of fresh complete culture media was added to each well. Blank wells were filled with culture media without cells. Then, 10 µL of Alamar Blue reagent (Thermo Fisher Scientific, Waltham, MA, USA) was added to the wells and left incubated at 37 °C with 5% CO₂ for 4 h. Later, the Alamar Blue reagent reduction was measured by measuring the fluorescence (reduced fluorescence unit, RFU) at an emission wavelength of 590 nm and excitation wavelength of 560 nm using the microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The test was conducted in triplicate and repeated at three independent times, n = 9. The percentage of reduction was calculated to determine the gain adjustment of fluorescence for each well against the well with fully reduced Alamar Blue reagent, in line with the manufacturer’s protocol [39] and Zachari et al. [40]. The percentage of Alamar Blue reagent reduction serves as a quantitative indicator of metabolic activity and reducing capacity of live cells. It measures the ability of viable cells to convert the non-fluorescent oxidised resazurin into the fluorescent reduced resorufin [41]. A higher percentage of reduction indicates greater cell viability [42].

$$\mathrm{\%} \mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{t}\mathrm{i}\mathrm{o}\mathrm{n} \mathrm{o}\mathrm{f} \mathrm{a}\mathrm{l}\mathrm{a}\mathrm{m}\mathrm{a}\mathrm{r} \mathrm{b}\mathrm{l}\mathrm{u}\mathrm{e} \mathrm{r}\mathrm{e}\mathrm{a}\mathrm{g}\mathrm{e}\mathrm{n}\mathrm{t}=\frac{\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{R}\mathrm{F}\mathrm{U} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}-\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{R}\mathrm{F}\mathrm{U} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}}{100\mathrm{\%} \mathrm{r}\mathrm{e}\mathrm{d}\mathrm{u}\mathrm{c}\mathrm{e}\mathrm{d} \mathrm{p}\mathrm{o}\mathrm{s}\mathrm{i}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{o}\mathrm{n}\mathrm{t}\mathrm{r}\mathrm{o}\mathrm{l} \mathrm{R}\mathrm{F}\mathrm{U} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}-\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{R}\mathrm{F}\mathrm{U} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}}\times 100$$

(1)

Additionally, the relative cell viability expressed as a percentage of untreated control was calculated in accordance with ISO 10993-5 [37].

$$\mathrm{R}\mathrm{e}\mathrm{l}\mathrm{a}\mathrm{t}\mathrm{i}\mathrm{v}\mathrm{e} \mathrm{c}\mathrm{e}\mathrm{l}\mathrm{l} \mathrm{v}\mathrm{i}\mathrm{a}\mathrm{b}\mathrm{i}\mathrm{l}\mathrm{i}\mathrm{t}\mathrm{y}=\frac{\mathrm{e}\mathrm{x}\mathrm{p}\mathrm{e}\mathrm{r}\mathrm{i}\mathrm{m}\mathrm{e}\mathrm{n}\mathrm{t}\mathrm{a}\mathrm{l} \mathrm{R}\mathrm{F}\mathrm{U} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}-\mathrm{b}\mathrm{l}\mathrm{a}\mathrm{n}\mathrm{k} \mathrm{R}\mathrm{F}\mathrm{U} \mathrm{v}\mathrm{a}\mathrm{l}\mathrm{u}\mathrm{e}}{\begin{array}{c}\mathrm{untreated} \mathrm{neative} \mathrm{control} \mathrm{RFU} \mathrm{value}-\mathrm{blank} \mathrm{RFU} \mathrm{value}\end{array}}\times 100$$

(2)

2.5. Live and Dead Cell Assay

The Live and Dead dye solution (Abcam, Boston, MA, USA) was prepared following the instructions provided by the manufacturer. To prepare a 5× Live and Dead dye solution, a 5 µL volume of the concentrated 1000× Live and Dead dye was diluted with 1 mL of phosphate-buffered saline (Thermo Fisher Scientific, Waltham, MA, USA) to a final concentration of 5× Live and Dead dye through 200-fold dilution. Following 24 h of incubation, the test solutions or control solutions in the 96-well plate were carefully aspirated. Subsequently, 200 µL of a 5× Live and Dead dye solution was introduced into each well and left for 10 min in a dark room prior to examination under the fluorescence microscope (Olympus, Tokyo, Japan) to analyse the cell viability and staining patterns.

2.6. TEM Analysis

TEM analyses were conducted for a detailed examination of possible ultrastructural changes in the cells after being treated with different concentrations of EEP and control solutions. After determining the cytotoxic concentration of EEP extract, the following samples were evaluated using TEM:

  i.

Cells exposed to culture medium only (untreated negative control);

 ii.

Cells exposed to 3 M 12.5% EEP (lowest concentration that was cytotoxic);

iii.

Cells exposed to 3 M 6.25% EEP (highest concentration that was non-cytotoxic);

 iv.

Cells exposed to 6 M 12.5% EEP (lowest concentration that was cytotoxic);

  v.

Cells exposed to 6 M 6.25% EEP (highest concentration that was non-cytotoxic).

Transmission electron microscopy was not performed on the 2.5% NaOCl group due to profound cell damage and lysis, which resulted in the absence of a cell pellet for analysis. The sample preparation for TEM in this study was performed in accordance with the protocol by Tinari et al. [43]. The M3CT3-E1 cells were cultured in a tissue culture flask (Thermo Fisher Scientific, Waltham, MA, USA) in a humidified atmosphere at 37 °C, 95% air and 5% CO₂. After incubation with 3 M 12.5% EEP, 3 M 6.25% EEP, 6 M 12.5% EEP, 6 M 6.25% EEP and control solutions for 24 h, a single cell suspension for each treatment group was collected via trypsinisation with 0.25% trypsin/EDTA (Thermo Fisher Scientific, Waltham, MA, USA). The cell suspension was then centrifuged at 125× g for 10 min. The cell pellet collected was fixed in 4% glutaraldehyde (EMS, Hatfield, PA, USA) in 0.1 M PBS (Thermo Fisher Scientific, Waltham, MA, USA) for 12 h at 4 °C. Post-fixation was performed with 1% osmium tetroxide (EMS, Hatfield, PA, USA) for 2 h at 4 °C. After that, the samples were dehydrated in ascending order of acetone (HmbG, Petaling Jaya, Malaysia) and embedded in epoxy resin (Agar Scientific, Stansted, UK). The ultrathin sections (60–100 nm) were prepared with an ultramicrotome and treated with uranyl acetate (Ted Pella, Inc., Redding, CA, USA) and lead citrate (BDH, Dubai, UAE) stains before being analysed under TEM (Carl Zeiss, Oberkochen, Germany).

2.7. Data Analysis

The data are presented as the mean and standard error of the mean values of percentage of reduction and relative cell viability, based on the data collected from triplicates in three independent experiments (n = 9). The relative cell viability and half-maximal inhibitory concentration (IC₅₀) were calculated in accordance with the ISO guide for in vitro cytotoxicity tests [38]. The IC₅₀ concentration of EEP was determined using GraphPad Prism version 9.4.0. Eight different concentrations of EEP were transformed to Log10 and plotted on the X-axis, while the relative cell viability was plotted on the Y-axis in a dose–response graph. The software calculated the IC₅₀ value, which was then presented along with the plotted graph.Top of Form.

The effect of different treatment groups and two fermentation periods on the percentage of reduction were analysed using two-way mixed Analysis of Variance (ANOVA), by means of Statistical Package for the Social Sciences (SPSS) version 26 (IBM, Armonk, NY, USA). The normality of data distribution was assessed using Shapiro–Wilk’s test. Homogeneity of variance and covariances were assessed using Levene’s test of homogeneity of variances and Box’s test of equality of covariance matrices, respectively. A further post hoc Turkey test was carried out to determine where the difference between groups lies. The test involved pairwise comparisons of various concentrations of EEP at different fermentation periods with both 2.5% NaOCl and the untreated negative control. Statistical significance was determined by considering p-values less than 0.05.

3. Results

3.1. Alamar Blue Assay

An Alamar Blue assay was conducted to evaluate the cytotoxicity of EEP. The test material was considered non-cytotoxic if the relative cell viability of the sample extract was more than 70% of the control group, in line with ISO 10993-5 [38]. There was an inverse relationship between the relative cell viability and concentration of EEP at both fermentation periods. EEP at a concentration of 6.25% and below for both fermentation periods were non-cytotoxic with a relative cell viability more than 70%, as shown in

Table 1. Relative cell viability of different concentrations of EEP at 3 and 6 months of fermentation.

Concentrations	Relative Cell Viability (% of Control)
	3 Months of Fermentation (Mean ± SE)	6 Months of Fermentation (Mean ± SE)
100% EEP	8.25 ±0.25	8.28 ± 0.28
50% EEP	9.65 ± 0.39	9.03 ± 0.27
25% EEP	11.28 ± 0.39	10.14 ± 0.36
12.5% EEP	24.97 ± 0.33	25.24 ± 0.26
6.25% EEP	96.20 ± 0.21 *	96.06 ± 0.30 *
3.13% EEP	99.35 ± 0.23 *	99.02 ± 0.32 *
1.57% EEP	100.49 ± 0.24 *	100.03 ± 0.31 *
0.78% EEP	101.13 ± 0.23 *	100.82 ± 0.24 *

SE: standard error. * Cell is regarded as viable when the percentage of relative cell viability is more than 70% [38].

. On the other hand, for both fermentation periods, the percentage of relative cell viability was less than 25% for EEP at a concentration above 12.5%, reflecting its toxicity towards the cell.

The IC₅₀ concentration of EEP, which represents the point where 50% inhibition of cell viability occurs, was calculated following the dose–response graph recommended by ISO 10993-5 [38]. At 3 months of fermentation, the IC₅₀ value for EEP was determined to be 10.38% (Figure 1a), while at 6 months of fermentation, it was found to be 10.39% (Figure 1b). In simpler terms, when the concentration of EEP reached approximately 10.38% and 10.39% for 3 and 6 months of fermentation, respectively, approximately half of the treated cells experienced cell death or cytotoxic effects.

3.2. The Effects of Different Concentrations and Fermentation Periods on the Cytotoxicity of EEP Extracts

The effect of different treatment groups and fermentation periods on the cytotoxicity of EEP extract was analysed with two-way mixed ANOVA. The data were normally distributed, as assessed with Shapiro–Wilk’s test (p > 0.05) and a normal Q-Q plot. There was homogeneity of variance (p > 0.05) and covariances (p = 0.435), as assessed with Levene’s test of homogeneity of variances and Box’s test of equality of covariance matrices, respectively.

Cell viability of MC3T3-E1 cells was expressed as the percentage of reduction. Higher percentages indicated greater Alamar Blue reduction and higher cell viability, and vice versa. Two-way mixed ANOVA revealed that the interaction effects of different treatment groups and fermentation periods on the percentage of reduction were not statistically significant, as shown in

Table 2. Results of two-way mixed ANOVA and the subsequent main effect of fermentation periods and different treatment groups.

Interaction between Different Treatment Groups and Fermentation Periods
Two-Way Mixed ANOVA	F = 0.724, p = 0.713
Main Effect	Different Treatment Groups F = 38,354.39, p < 0.001 *	Fermentation Periods F = 3.408, p = 0.068

* p is statistically significant at α = 0.05.

. There was no significant difference in the percentage of reduction of EEP at 3 and 6 months of fermentation periods (p > 0.05).

On the other hand, two-way mixed ANOVA showed that the effect of treatment groups was significant. Further post hoc Turkey tests and pairwise comparisons with the negative control and 2.5% NaOCl were run where the mean, mean difference, standard error and p-value were reported, as shown in

Table 3. Pairwise comparison of the percentage of reduction for different concentrations of EEP with negative control at 3 and 6 months of fermentation.

3 Months of Fermentation				6 Months of Fermentation
Source	Percentage of Reduction (Mean ± SE)	Pairwise Comparison with Negative Control		Percentage of Reduction (Mean ± SE)	Pairwise Comparison with Negative Control
		Mean Difference (Mean ± SE)	p-Value		Mean Difference (Mean ± SE)	p-Value
100% EEP	7.89 ± 0.35	87.68 ± 0.45	0.001 *	7.91 ± 0.27	87.62 ± 0.44	<0.001 *
50% EEP	9.22 ± 0.38	86.34 ± 0.45	<0.001 *	8.62 ± 0.26	86.91 ± 0.44	<0.001 *
25% EEP	10.78 ± 0.37	84.78 ± 0.45	<0.001 *	9.69 ± 0.35	85.84 ± 0.44	<0.001 *
12.5% EEP	23.86 ± 0.31	71.70 ± 0.45	<0.001 *	24.11 ± 0.25	71.42 ± 0.44	<0.001 *
6.25% EEP	91.93 ± 0.20	3.64 ± 0.45	<0.001 *	91.73 ± 0.29	3.76 ± 0.44	<0.001 *
3.13% EEP	94.94 ± 0.22	0.62 ± 0.45	0.993	94.60 ± 0.31	0.94 ± 0.44	0.597
1.57% EEP	96.03 ± 0.25	−0.47 ± 0.45	0.996	95.56 ± 0.30	−0.03 ± 0.44	1.00
0.78% EEP	96.65 ± 0.22	−1.08 ± 0.45	0.400	96.31 ± 0.23	−0.78 ± 0.44	0.82
NaOCl	95.56 ± 0.38	89.31 ± 0.45	<0.001 *	95.53 ± 0.50	89.28 ± 0.44	<0.001 *
Positive control	100.00 ± 0.34	−4.44 ± 0.45	<0.001 *	100.00 ± 0.27	−4.47 ± 0.44	<0.001 *

SE: standard error. EEP: effective ecoproduce. NaOCl: sodium hypochlorite. Post hoc Turkey test for comparison of different concentrations of EEP and NaOCl with negative control, p is statistically significant at α = 0.05, indicated by *.

and

Table 4. Pairwise comparison of the percentage of reduction for different concentrations of EEP with NaOCl at 3 and 6 months of fermentation.

3 Months of Fermentation				6 Months of Fermentation
Source	Percentage of Reduction (Mean ± SE)	Pairwise Comparison with NaOCl		Percentage of Reduction (Mean ± SE)	Pairwise Comparison with NaOCl
		Mean Difference (Mean ± SE)	p-Value		Mean Difference (Mean ± SE)	p-Value
100% EEP	7.89 ± 0.35	−1.63 ± 0.45	<0.021 *	7.91 ± 0.27	−1.67 ± 0.44	0.013 *
50% EEP	9.22 ± 0.38	−2.97 ± 0.45	<0.001 *	8.62 ± 0.26	−2.37 ± 0.44	<0.001 *
25% EEP	10.78 ± 0.37	−4.52 ± 0.45	<0.001 *	9.69 ± 0.35	−3.43 ± 0.44	<0.001 *
12.5% EEP	23.86 ± 0.31	−17.61 ± 0.45	<0.001 *	24.11 ± 0.25	−17.86 ± 0.44	<0.001 *
6.25% EEP	91.93 ± 0.20	−85.67 ± 0.45	<0.001 *	91.73 ± 0.29	−85.52 ± 0.44	<0.001 *
3.13% EEP	94.94 ± 0.22	−88.68 ± 0.45	<0.001 *	94.60 ± 0.31	−88.34 ± 0.44	<0.001 *
1.57% EEP	96.03 ± 0.25	−89.78 ± 0.45	<0.001 *	95.56 ± 0.30	−89.31 ± 0.44	<0.001 *
0.78% EEP	96.65± 0.22	−90.39 ± 0.45	<0.001 *	96.31 ± 0.23	−90.06 ± 0.44	<0.001 *
Negative control	95.56 ± 0.38	−89.31 ± 0.45	<0.001 *	95.53 ± 0.50	−89.28 ± 0.44	<0.001 *
Positive control	100.00 ± 0.34	−93.74 ± 0.45	<0.001 *	100.00 ± 0.27	−93.75 ± 0.44	<0.001 *

SE: standard error. EEP: effective ecoproduce. NaOCl: sodium hypochlorite. Post hoc Turkey test for comparison of different concentrations of EEP and NaOCl with negative control, p is statistically significant at α = 0.05, indicated by *.

. EEP negatively affected cell viability in a concentration-dependent manner (Table 3). The four highest dilutions of EEP resulted in a significant decrease in the percentage of reduction compared with the negative control (p < 0.001). Conversely, the lower dilutions showed biocompatibility. A noteworthy observation was that when the EEP concentration was 6.25% or lower, regardless of the fermentation periods, there was a remarkable increase in the mean percentage of reduction, surpassing 90%. For EEP at concentrations of 3.13%, 1.57% and 0.78% at both fermentation periods, the percentage of reduction was comparable to the negative control, with no significant difference (p > 0.05), as indicated by the symbol “#” in Figure 2. Similar results were observed when visually analysing the Alamar Blue assay, where the colour shift to pink indicated the presence of viable cells at the four lower concentrations of EEP during both fermentation periods, as depicted in Figure 3. The group treated with 2.5% NaOCl showed a significantly lower percentage of cell viability reduction compared with the negative control (p < 0.001).

Furthermore, when comparing different concentrations of EEP with 2.5% NaOCl, all concentrations of EEP exhibited significantly higher percentages of reduction at both fermentation periods (p < 0.05), as illustrated by “*” in Figure 2 and Table 4. Notably, the percentage of reduction observed in the 2.5% NaOCl group was relatively low (less than 7%).

3.3. Live and Dead Cell Assay

The Live and Dead Cell assay was conducted to evaluate the viability of MC3T3-E1 cells following exposure to different treatment groups, including different concentrations of EEP at both fermentation periods, 2.5% NaOCl and control solutions. This assay utilises dual-colour fluorescence staining, where viable cells are stained with green fluorescence and dead cells are stained with red fluorescence. The positive control group (Figure 4a), treated with 10% DMSO, displayed an abundance of cells with red fluorescence, indicating a higher number of dead cells with increased intercellular space and less cell density. Conversely, the untreated cells in the negative control group (Figure 4b) showed increased cell density and an abundance of cells with green fluorescence, indicating viable cells.

The qualitative assessment from the Live and Dead Cell assay illustrated the cytotoxicity of EEP in a concentration-dependent manner for both fermentation periods. For the cells exposed to 100% and 50% EEP at both fermentation periods (Figure 4d–g), a notable number of intensely bright red fluorescent cells were observed, indicating increased cell death. When MC3T3-E1 cells were treated with 25% and 12.5% EEP at both fermentation periods (Figure 5a–d), there was a mixture of green and red fluorescence, with red fluorescence predominating, suggesting a combination of viable and dead cells. At an EEP concentration of 6.25% (Figure 5e,f), there was a noticeable increase in the density of cells with green fluorescence, and red fluorescent cells were barely seen, indicating a higher proportion of viable cells. Further reduction in the concentration of EEP to below 3.13% (Figure 6a–f) resulted in a significant increase in the density of cells with green fluorescence, similar to the negative control (Figure 4b), indicating comparable viable cell numbers. This trend was observed for both fermentation periods. On the other hand, cells exposed to 2.5% NaOCl (Figure 4c) exhibited red fluorescence with a fragmented pattern, indicating the presence of dead cells with fragmented nuclei.

3.4. TEM Analysis

The untreated negative control cells (Figure 7a,b) showed characteristics similar to the cells exposed to 6.25% EEP at 3 and 6 months of fermentation respectively (Figure 8a–d). The cells exhibited an intact cell membrane and a smoothly outlined nucleus with an intact nucleus membrane, containing nucleoli (Nu) and heterochromatin (HChr) deposited along the nuclear membrane. The cytoplasm showed intact organelles, including mitochondria (indicated by a white arrow) with preserved cristae, endoplasmic reticulum (indicated by a yellow arrow) and minimal cytoplasmic vacuolisation (V).

On the other hand, cells exposed to EEP 12.5% at both 3 and 6 months of fermentation showed evidence of cell death, as depicted in Figure 9a–d. The cells demonstrated cell surface changes with plasma membrane blebs and the formation of numerous apoptotic bodies (ApBs) that were seen detaching from the plasma membrane (Figure 9a,c). At the nuclear level, the nucleus lost its regular shape and membrane integrity. The chromatin (Chr) became densely condensed with the disappearance of the nucleolus. Additionally, there were numerous nucleus aggregates (yellow arrows) and cytoplasmic aggregates (white arrows), as observed in Figure 9a–d. These aggregates were small, round or oval-shaped fragments evident in both the nucleus and cytoplasm.

Furthermore, noticeable morphological alterations were observed in the cytoplasm and organelles. The cytoplasm exhibited an increased number of fragmented organelles (red arrow) and autophagic vacuoles (V) containing cell debris. In addition, the mitochondria (M) lost their regular shape, and the internal cristae were collapsed and not discernible.

4. Discussion

The present study was our preliminary work on the development of a novel natural-based root canal irrigant by utilising the clinical benefits of EEP. Cytotoxic evaluation of EEP and the establishment of a safe and effective concentration is of paramount importance since the existing synthetic chemical irrigants present with many potential hazardous side effects. A combination of different cytotoxic assays was used in the present study to assess the cytotoxic effects of EEP towards the cells. Alamar Blue assay was used as a quantitative measurement of cell viability, calculated as the percentage of reduction of the non-fluorescence blue resazurin to the fluorescent pink resorufin through the oxidation-reduction reaction provided by the mitochondrial redox enzyme [44]. The Live and Dead Cell assay measures the cellular membrane integrity and the activity of intracellular esterase enzymes. The assay provides a qualitative assessment of the cell viability, which can be visualised as green and red fluorescence under a microscope, representing the live and dead cells, respectively [45]. The use of both quantitative and qualitative assessment methods provides a comprehensive evaluation of the cytotoxic effects of EEP from different points of view. This enables the cross-validation of results, which enhances the reliability and validity of the study and ensures greater confidence in the conclusions drawn [46]. We did not conduct any MTT/XTT cytotoxic assays since both assays measure similar mitochondrial enzymatic activity as the Alamar Blue assay, but at a lesser sensitivity. Furthermore, the MTT cytotoxic assay requires an organic solvent to dissolve the crystals prior to the absorbance measurement, which may itself cause cytotoxicity towards the cells [45].

The results from our study showed that the cytotoxicity of EEP is concentration-dependent, regardless of the fermentation period. EEP at a concentration below 6.25% were not toxic to the cells, with cell viability of more than 70% [38] as depicted by the Alamar Blue cytotoxic assay. Further, there was no statistically significant difference in terms of toxicity between the EEP at 3.125% and the negative control. These findings are consistent with the qualitative assessment of the cells done through the Live and Dead Cell assay. Cells exposed to EEP at a concentration below 6.25% were densely packed, with green fluorescence cells predominating, while the red-stained cells were barely seen. The intensity of the green fluorescence increased as the concentration of EEP decreased, suggesting the concentration-dependent toxicity of the solution. Further, at similar concentrations, TEM analysis of the cells showed intact cell and nucleolar membranes and preservation of the organelle structure comparable to the untreated negative control. In contrast, the presence of autophagic vacuolisation and organelle derangement, a feature that is associated with cell apoptosis [47] and autophagy [48], were detected in cells exposed to EEP at a 12.5% concentration. This explains and validates the gradual reduction in cell viability following exposure to EEP at a concentration higher than 6.25%, as shown by the Alamar Blue cytotoxic assay.

We also compared the toxicity of EEP with NaOCl, the gold standard irrigation solution in root canal treatment. Briefly, 2.5% NaOCl is toxic to the cells, with less than 7% of cell viability as measured through the Alamar Blue assay. Interestingly, EEP at all concentrations for both fermentation periods showed a statistically significant difference as compared with 2.5% NaOCl, showing higher superiority of the solution in terms of cell viability over NaOCl. Our findings were consistent with the outcome reported by Teixeira et al. [49]. In their study, the authors demonstrated the cytotoxicity of 2.5% NaOCl towards the human fibroblast cell lines using an MTT assay. Further, NaOCl has been found to be cytotoxic even at a lower concentrations, as described by Alkahtani et al. [50] and Bohle et al. [51], using NaOCl at 0.005% and 0.08%, respectively. Both of the authors were in agreement and added that the cytotoxic effects exerted by NaOCl are both concentration- and time-dependent.

Our observation through the Live and Dead Cell assay supports the previously discussed cytotoxic outcome of NaOCl, where exposure to 2.5% NaOCl showed an abundance amount of red fluorescent cells, indicating the non-viability of the cells. One of the glaring comparisons was the presentation and appearance of the cells in samples exposed to 2.5% NaOCl and 100% EEP. Although both were cytotoxic, the latter showed red cells with more intense fluorescence, bigger cell sizes and more compact cell distribution, in contrast to the former, which presented with multiple loosely-bound small-size cells in an ununiform cell orientation, suggestive of cellular fragmentation, and the formation of cell corpses. The presence of cellular fragmentation and cell corpses is classified as type III cell death or necrosis, the most severe form of cellular damage [48,52]. The pH of NaOCl is thought to be responsible for these findings, as it promotes tissue penetration and interrupts the cellular membrane integrity, causing damage and fragmentation to the cells’ organelles and DNA [50,53].

In addition, we were unable to perform TEM analysis for the cells exposed to 2.5% NaOCl due to excessive cell damage and fragmentation, which resulted in the absence of a cell pellet available for analysis. This is highly suggestive that NaOCl is more toxic to the cells as compared with EEP. In a study by Alkahtani et al. [50], the authors reported extreme cell lysis following exposure to 0.5 mg/mL NaOCl for 2 h, leaving almost no cell structures as observed through a scanning electron microscope. We believe that the extent of cell lysis in the current study was more profound since the concentrations of NaOCl used were higher and the duration of exposure was longer.

We postulated that the presence of the active component within EEP is the main reason for the cytotoxic effects exerted by the solution at a concentration beyond 6.25%. Even though the main constituents of EEP used in the present study are still under investigation, various studies in the literature have identified and described the active component of EEP. Organic acids, mainly acetic acids, have been consistently reported as the main constituents of EEP by Arun et al. [28], Vikas et al. [54] and Vama et al. [55], regardless of the type of fruit used. In addition, the presence of naringenin and naringin, a flavonoid compound, has been reported in citrus fruit peel-based EEP [27,56,57].

Acetic acid can induce cell death via two mechanisms: the disruption of cellular homeostasis and the induction of oxidative stress in cells, as proposed by Chaves et al. [58]. The former action is induced as the acid penetrates the cell membrane through simple diffusion and dissociates into acetate and hydrogen ions. The pKa of acetic acid is 4.76, which is lower than the intracellular pH value. At high concentrations, the accumulation of hydrogen ions creates an acidic cellular environment and a drop in the intracellular pH value. This leads to the disruption of normal cellular protein folding and other enzymatic activity, contributing to cell death. In our previous study, Ng [31] reported an initial pH of EEP at 4.14 and raised up to 7.38 as the concentration of EEP diluted to 6.25% and below. This observed pH alteration following dilution could potentially explain the concentration-dependent cytotoxicity of EEP.

In addition, both acetic acid and naringenin have been shown to cause the accumulation of and increases in reactive oxygen species (ROS) within the cell in a dose-dependent manner. Upon exposure to high concentrations of these substances, the level of ROS starts to build up within the cell, exceeding the natural antioxidant threshold for the reduction of ROS. This subsequently activates the signalling pathways, which leads to a significant alteration in the cellular morphology and, later, cell apoptosis [58,59,60,61]. Chaves et al. [58] and Rego et al. [61] suggested that the mitochondria could be the first organelle affected by acetic acid, triggering the release of cytochrome c and further activating the cell apoptosis process. These effects were evident and correlated well with the TEM analysis in our study, where the presence of the apoptotic body and degraded mitochondria were observed when the MC3T3-E1 cells were exposed to higher concentrations of EEP beyond 6.25% for both fermentation periods. Additionally, the presence of cell debris within the vacuolated cytoplasm, chromatin condensation and plasma membrane blebbing, as visualised through the TEM analysis, is associated with the activation of the autophagy process [62], a critical step in cell death. This further strengthens the role of ROS-induced cell death exerted by acetic acid and naringenin. The cytotoxic effects exerted by naringenin [63] and acetic acid [61] in a concentration-dependent manner also explained the increasing toxicity of EEP as observed in our study.

Based on the two cytotoxic assays and TEM findings from the present study, we proposed the safe concentration of EEP at 6.25% with a 6-month fermentation period. EEP at this concentration was significantly less cytotoxic than the 2.5% NaOCl, the gold standard irrigant used in root canal treatment. Since NaOCl has been used widely as an irrigation solution for root canal treatment with a good success rate, EEP offers huge potential which can be explored further as an alternative irrigant to NaOCl.

Furthermore, we proposed the abovementioned concentration at 6 months of fermentation based on the findings of our previous study [31]. In the study, using an antibiofilm assay, EEP at a lower concentration below 6.25% showed a positive percentage of Enterococcus faecalis biofilm removal. The effects were comparable to those depicted by 2.5% NaOCl, suggesting its high antibiofilm potential. On the other hand, EEP at higher concentrations did not show antibiofilm activity; instead, an increase in the biomass of the biofilm was detected through crystal violet staining, leading to a negative percentage of biofilm removal. In addition, six-month-old EEP showed better antibiofilm effects than three-month-old EEP, where more biofilm was removed in the former fermentation period as compared with the latter.

The assessment of the antibacterial activity of an endodontic irrigant through its antibiofilm removal efficacy is more clinically relevant since bacteria such as Enterococcus faecalis and Streptococcus mutans reside within the root canal system in the form of biofilm rather than in isolation [64]. These bacterial species have the ability to produce a sessile biofilm consisting of the exopolymeric matrix, which is attached to the dentinal surface [65]. The slow-growing bacteria within its exopolymeric matrix make it more resistant to host-defence mechanisms and other antimicrobial agents [66,67]. Thus, it is not surprising that these bacteria are recognised as the primary etiological factor that is responsible for the development of pulpal necrosis, periapical infection and post-root-canal treatment reinfection [64,68]. Ghivari et al. [68] have shown that 5.25% NaOCl is effective at removing the Enterococcus faecalis biofilm following 10 s of application.

Nevertheless, the present study has some limitations. The study is still in the preliminary stage, where the analysis of the active compound of EEP is currently under investigation using the High-Performance Liquid Chromatography (HPLC) method. Based on the previous study conducted on the EEP produced through the fermentation of fruit peels, acetic acid and naringenin have been identified as the main components, which we believe will also be detected in our sample. Additionally, other active components and secondary metabolites may be present as well, and the analysis will give a better insight and strengthen our understanding of the exact mechanism of EEP towards the cell. These findings will add more value to the development of EEP-based irrigants for root canal treatment.

The EEP in this study was prepared through the conventional fermentation process as described previously by Arun et al. [32] and Ng et al. [25]. This fermentation method does not provide a stringent controlled environment for fermentation, thereby introducing potential batch-to-batch variation. The use of a bioreactor can be considered, as this device provides a stringent environment for the fermentation process, allowing precise regulations of the pH and temperature together with air filtration and other sterilisation capabilities, which will prevent the introduction of unwanted contamination during fermentation.

In addition, the present study was conducted in a well-controlled environment and did not replicate the actual clinical scenario of an oral environment. The cytotoxic effects measured were solely based on the cell’s response, without considering the involvement of the inflammatory reaction provided by the host defence mechanism [50]. However, the biocompatibility profile of EEP obtained from this study will ethically justify the use of a similar concentration for in vivo studies on animals [69]. Future in vivo studies can be conducted, either by injecting the material into the subcutaneous tissue of the animal to elucidate the inflammatory reaction towards EEP or by replicating the actual root canal treatment protocols in laboratory animals, followed by clinical assessment, radiographic imaging and histopathological evaluation of the periapical tissue reaction [70].

Clinically, since EEP is a fruit-based product, this irrigant might not be suitable to be used on patients with citrus allergies. Although the prevalence is rare [71], precautionary measures should be taken, which may limit its application in clinical settings. Exploration of the antibiofilm activity of EEP at 6.25% against the multi-species bacteria that cause endodontic infection should be investigated in the future. Further, the penetrative ability of EEP and its effect on the microhardness, tensile strength and modulus of elasticity of the dentine should be assessed, either in vitro or in vivo. Additionally, its potential interactions with restorative and obturation materials are yet to be explored. Addressing these questions requires further research efforts in the future.

5. Conclusions

Within the limitation of this study, the cytotoxicity of EEP is concentration-dependent. EEP at a 6.25% concentration was biocompatible and non-cytotoxic towards MC3T3-E1 cell lines regardless of the fermentation period, as observed through the in vitro cytotoxic tests. EEP at all concentrations for both fermentation periods achieved significantly higher cell viability and exhibited less cellular toxicity as compared with 2.5% NaOCl, which is the gold standard root canal irrigation solution. The 6 M EEP at a 6.25% concentration was proposed as a potential root canal irrigant as it provides comparable antibiofilm activities to those exerted by NaOCl. Future research is needed to clinically explore the effects and interaction of 6 M EEP at 6.25%.