Cytotoxicity and Antimicrobial Activity of BioAkt^TM and Phytic Acid: A Laboratory-Based Study

Abstract

(1) Background: To improve endodontic treatments, it is necessary to find good irrigant solutions that balance potential toxic effects with optimal antimicrobial capacity. The aim of this laboratory study was to assess the cytotoxic and antimicrobial capacity of phytic acid (IP6) and BioAkt^TM in a laboratory setting to determine their suitability prior to endodontic evaluation. (2) Methods: The antimicrobial and cytotoxic effects of IP6 and BioAkt^TM were compared with those of 1.5% NaOCl. Cytotoxicity was evaluated via crystal violet assay and CellTiter-Glo^® 2.0 assay, while antimicrobial capacity was tested via disk diffusion and the effect of a 1-h treatment on Enterococcus faecalis. (3) Results: A crystal violet test and CellTiter-Glo^® 2.0 assay showed statistically significant differences in cell viability and cell activity after 4 and 24 h for all tested solutions (p < 0.05). The antimicrobial activity of BioAkt^TM was similar to that of 1.5% NaOCl, while phytic acid showed the lowest antimicrobial activity compared to BioAkt^TM (p < 0.05) and 1.5% NaOCl (p < 0.05). (4) Conclusion: BioAkt^TM showed an optimal balance between antimicrobial activity and cytotoxicity.

1. Introduction

Bacteria infect more than 70% of dentinal tubules in the presence of endodontic infections, and about 65% of these tubules remain infected even after root canal instrumentation and disinfection [1]. Although root canal irrigation procedures are needed for bacterial load reduction [2], an optimal irrigant with robust antibacterial features and marginal cytotoxic effects on the host tissue has not been identified. Sodium hypochlorite (NaOCl), used at various concentrations (0.5 to 6%), combined with ethylenediaminetetraacetic acid (EDTA) remains the gold standard for irrigation procedures thanks to their combined capability in exerting antimicrobial and antifungal activity, dissolving necrotic tissue, acting on endodontic biofilm, and eradicating smear layers [3]. The use of NaOCl is broadly accepted and recommended by the American Association of Endodontists (AAE) (1.5% concentration) and the European Society of Endodontology (ESE) (1.5 to 3% concentration) [4]. Nevertheless, it remains controversial given some drawbacks due to a high risk of weakening of instruments’ structure [5], allergic reaction [6,7], sodium hypochlorite accident [8], peritubular or intertubular erosion via proteolytic degradation, this last enhanced if NaOCl is used with EDTA [9], and finally, damage to clothing [10] or eyes [11]. Similar issues have been reported about EDTA, which is used as a chelating agent in conventional root canal treatment for smear layer removal. EDTA shows some cytotoxic effects at various concentrations [12,13] and, when combined with NaOCl, can reduce the pH of NaOCl in a time-dependent manner thus affecting free chlorine in solution and resulting in increase in chlorine gas and hypochlorous acid. In this way, EDTA reduces the hypochlorite ion [14], reducing NaOCl effectiveness in removing the necrotic tissue and exerting its antibacterial activity [15]. Moreover, given the high surface tension of NaOCl and EDTA, these solutions do not deeply penetrate dentinal tubules or other anatomical irregularities [16].

According to a recent study, the evaluation of biocompatible materials should consider as decisive the adequacy of the host reaction, i.e., the extent of the different biological responses from cytotoxicity and adverse reaction to the biostimulatory effects on tissues, and not the complete non-toxicity of the tested material [17]. In research on the balance between irrigant therapeutic effects against their potential cytotoxicity effects [18], some new irrigants have been introduced, such as BioAkt^TM and phytic acid.

BioAkt^TM is a water solution composed mainly of silver ion (0.003%) and citric acid (4.846%), whose active ingredient is silver citrate dihydrate. This substrate is weakly bound with silver ions and has the function of reversibly combining with another molecule. As a result, bacteria, which identify citric acid as a metabolic nutrient, are first attracted to the compound and then blocked by the compound itself, causing the microorganism’s death. For this reason, BioAkt^TM is also defined as a metabolic antimicrobial substrate. Previous studies showed that acidic solutions such as BioAkt^TM can more efficiently reduce the smear layer [19] and improve antimicrobial activity in the root canal system [20] compared to EDTA-based solutions. However, some doubts have recently arisen about its cytotoxicity when used at higher than 0.5% concentration [21], which can limit its safe clinical use [22]. Although there are no direct comparisons between BioAkt^TM and NaOCl tissue response, studies conducted so far have compared silver nanoparticles, whose action is broadly similar to that of silver ions, and NaOCl showed a mild-to-moderate inflammation reaction in the case of silver nanoparticles and a higher inflammatory response in the case of NaOCl [23].

Phytic acid, i.e., inositol hexakisphosphate (IP6), is a six-fold dihydrogen phosphate ester of inositol, which is a part of human beings’ daily diet and is also present in mammalian cells [24] with a shallow or null cytotoxic profile. It is also the principal storage form of phosphorus in many plant tissues such as bran and seeds. Moreover, it can be easily extracted from rice bran, and thanks to the many phosphorus groups (negative charges) that can interact with calcium, magnesium, or iron ions (cations), it has a solid chelating capability [25]. In addition, phytic acid has an antimicrobial ability due to its damaging membrane activity, a consequence of its detergent characteristics due to different kinds of phenolics and flavonoids [26]. Compared to NaOCl, it showed a similar antimicrobial capacity with significantly higher biocompatibility with osteoblast-like cells [25].

Given the different and, in many aspects, opposite cytotoxic activities of these two compounds and the need to identify new irrigants able to balance toxic effects with an optimal antimicrobial capacity, this study aimed to investigate separately the cytotoxic and antimicrobial capacity of IP6 and BioAkt^TM in a laboratory setting so as to determine their appropriateness before any endodontic evaluation.

2. Material and Methods

2.1. Solutions

This laboratory-based study investigated 1% IP6, 3% IP6, 5% IP6, and 0.5% BioAkt^TM, comparing antimicrobial and cytotoxic effects with those observed for 1.5% NaOCl (Vista Apex, Racine, WI, USA) diluted proportionally from a concentration of 3%. The NaOCl concentration was chosen according to the ESE and AAE guidelines for endodontic regeneration [27,28].

A control group, treated with saline solution, was also introduced. Cytotoxicity was evaluated through two cell viability tests, crystal violet assay and CellTiter-Glo^® 2.0 assay, while antimicrobial capacity was tested through disk diffusion and the effect of 1 h treatment on Enterococcus faecalis. All tests were carried out in duplicates.

2.2. Cell Viability Tests

The J-774 A.1 murine macrophages-like cell line was used (American Type Culture Collection, Rockville, MD, USA). They were maintained overnight in humidified air (5% CO₂) at 37 °C in Dulbecco’s modified Eagle medium (DMEM) containing 10% FCS and 1% penicillin-streptomycin-nystatin solution, pyruvate, and glutamate, respectively.

2.2.1. Crystal Violet Assay

This test is based on the staining of cells attached to cell culture plates so that the detached dead cells are washed off, while the live cells are stained with crystal violet dye. Then, the crystal violet dye is added to a solution and measured via absorbance at 570 nm. Next, 5 × 10⁴ cells were added to each well of 96-well culture plates and incubated overnight in a 5% CO₂ incubator at 37 °C. After 24 h, the medium was changed, and three 3 wells were provided for each test solution. Then, 100 μL of each test solution was added to each well and incubated in a 5% CO₂ incubator at 37 °C for 1, 4, and 24 h. A cell culture placed in fresh medium without test solution was used as a control. After incubation, the culture medium was discarded, cells were washed three times with 100 μL phosphate buffered saline (PBS) to avoid any interaction between the test solutions and subsequent attachments [29]. Then, 50 μL of a 0.5% crystal violet staining solution was added to each well and incubated for 20 min at room temperature on a bench rocker at a frequency of 20 oscillations per minute. After washing the plate four times in a stream of tap water, it was inverted on filter paper and gently tapped out to remove residual liquid. The plate was then air dried for 2 h without a lid at room temperature. After adding 200 μL of methanol to each well, the plate was incubated with the lid on for 20 min at room temperature on a bench rocker at a frequency of 20 oscillations per minute. The optical density in each well was measured at 570 nm (OD 570) using a plate reader.

2.2.2. CellTiter-Glo^® 2.0 Assay

The CellTiter-Glo^® 2.0 assay quantifies ATP to calculate the number of viable cells in the culture and therefore the luminescence, directly proportional to the number of viable cells. In each well of the 96-well culture plates, 5 × 10⁴ cells were plated and incubated overnight in a 5% CO₂ incubator at 37 °C to allow attachment. After 24 h, the medium was changed, and three wells were provided for each test solution. An aliquot of 100 μL of each test solution was added to each well and incubated in a 5% CO₂ incubator at 37 °C for 1, 4, and 24 h. A cell culture in fresh medium without test solution was used as a control. After incubation, 100 μL of CellTiter-Glo^® 2.0 reagent was added to each well and mixed for 10 min to initiate cell lysis. The plate was then incubated at room temperature for 10 min to stabilize the luminescence signal. Luminescence was then calculated.

2.3. Antimicrobial Test

Enterococcus faecalis HH2, taken from a −80 °C stock, was thawed, inoculated into brain heart infusion with supplement (BHIS), and cultured in a shaker at 200 rpm under aerobic conditions at 37 °C for 24 h. Three agar plates (BHIS, Hemin, and Vitamin K) were inoculated with the bacterial solution, and 100 µL of the 24-h BHIS solution was picked with a sterile pipette and transferred to the agar plate. Then, 6 mm sterile paper disks were soaked with the test solution and placed on the corresponding plates. Each plate received a test agent at a different concentration and a disk of sterile PBS as a control. All colonized plates were incubated overnight at 37 °C. The resulting inhibition zone was measured using a millimeter scale [30]. After incubation, the inhibition zones of microbial growth around the cylinder containing the experimented compounds were estimated. The inhibition zone was determined as the shortest distance between the external edge of the cylinder and the starting point of microbial growth.

2.4. Effect of 1 h Treatment

After 1 h, a serial dilution of each solution was prepared in a 96-well plate. For each treatment, one row (12 wells) of the 96-well plate was used: from 1 to 10 for the serial dilution, and the last two were used as controls. First, 270 µL of phosphate buffered saline (PBS) was added to each well from number 2 to number 10, and then, 300 µL of each solution was added to well number 1. Then, 30 µL of the preparation was added to well number 2; after mixing well number 2, 30 µL of well number 2 was added to well number 3. This was repeated until well number 10 was reached. Finally, 30 µL of tube number 10 was discarded. This resulted in a 1:10 dilution of each reagent concentration. The positive control wells received no reagents, and three wells that received only BHIS served as blanks. Then, 200 µL of the bacterial suspension was added to the wells of another 96-well plate with a U-bottom. Bacteria absorbance was determined at 600 nm to determine the number of bacteria at the starting point. The plate was then centrifuged (4000 rpm for 5 min), and the medium was discarded. Then, 200 µL of each solution was taken with a multichannel from the plate prepared with the dilution series reagents and added to the plate containing the bacteria. Only BHIS was added to the control wells. The plate was incubated at 37 °C for 1 h. After incubation, the plate was centrifuged (4000 rpm for 5 min), and the treatments were discarded. Each well was washed three times with PBS (centrifugation was performed between each wash), and then, 200 µL of the new medium was added to the wells (including controls). The kinetics of bacterial growth was monitored by measuring the absorbance (wavelength = 600 nm) over 4 h. The kinetics were used to confirm the minimum inhibitory concentration (MIC) of the previous tests and determine the difference between the number of bacteria at baseline and the first reading after treatment to understand the effect of the 1-h treatment on the bacteria.

2.5. Statistical Analysis

Variables are reported as mean and standard deviation, given the normal distribution of the variables determined through the Shapiro–Wilk test. Comparisons were performed through the Kruskal–Wallis test along with Dunn’s procedure. Statistical significance was fixed at 5% (p < 0.05). Statistical analyses were performed using STATA17 (StataCorp, College Station, TX, USA).

3. Results

Statistical tests showed a statistically significant difference in cell viability between 4 h and 24 h for 1% IP6 (4 h: 0.485 ± 0.058, 24 h: 0.074 ± 0.009; p = 0.031), 3% IP6 (4 h: 0.398 ± 0.063, 24 h: 0.069 ± 0.009; p = 0.031), 5% IP6 (4 h: 0.331 ± 0.058, 24 h: 0.071 ± 0.004; p = 0.031), and BioAkt (4 h: 0.250 ± 0.016, 24 h: 0.013 ± 0.002; p = 0.031) (Figure 1).

Statistically significant different cell activity emerged between 4 and 24 h for 1% IP6 (4 h: 55,207 ± 3973.63, 24 h: 30,431.2 ± 25,493.4; p = 0.031), 3% IP6 (4 h: 8043.33 ± 1381.21, 24 h: 5495.8 ± 350.96; p = 0.031), and BioAkt^TM (4 h: 4389.83 ± 440.84, 24 h: 0 ± 0; p = 0.031).

After 4 h, cell viability was statistically significantly lower in BioAkt^TM (0.233 ± 0.250) than in 1% IP6 (0.508 ± 0.532) (p = 0.002). After 24 h, BioAkt^TM (0.013 ± 0.002) reported lower cell viability compared to 1% IP6 (0.074 ± 0.009, p = 0.002), 5% IP5 (0.071 ± 0.004, p = 0.024). After 24 h, BioAkt^TM reported a statistically significant difference in cell viability compared to 5% IP6 (0.071 ± 0.004, p = 0.024). Overall, 1% IP6 gave the compound with the lowest reduction in cell viability. After 24 h, only BioAkt^TM reached the same reduction in cell viability as 1.5% NaOCl in both tests.

Cell activity in BioAkt^TM was lower after 4 h (4389.83 ± 440.84) and 24 h (0 ± 0) compared to 1% IP6 (4 h: 55,207.33 ± 3973.63, p = 0.014; 24 h: 30431.2 ± 3320.86, p < 0.0001). Statistically significant differences also emerged on comparing BioAkt^TM and 3% IP6 (5495.80 ± 350.96, p = 0.006) (Figure 2).

For antimicrobial activity, the diffusion test showed no statistically significant differences among the four solutions (Figure 3). In contrast, after 1 h of treatment, 1% IP6 reported the lowest antimicrobial activity compared both to BioAkt^TM (p < 0.05) and 1.5% NaOCl (p < 0.05), while BioAkt^TM showed similar antimicrobial activity as that demonstrated by 1.5% NaOCl (Figure 4).

4. Discussion

BioAkt^TM and phytic acid are considered two novel endodontic solutions that can have wide applications both as chelating agents and irrigant solutions [25,31]. Although NaOCl and EDTA continue to be considered the most effective endodontic combination due to their capacity to destroy necrotic pulp and remove the smear layer, new irrigants which appear to have a broad-spectrum antimicrobial effect with minimal adverse effects on the root canal system have not been identified [32]. For some years, increased attention has been paid to phytic acid, a natural compound, and BioAkt^TM, a silver ions solution in citric acid as possible solutions for endodontic solutions for dental adhesive [29,33] and endodontic irrigants [34,35]. Compared to NaOCl, BioAkt^TM and phytic acid might be possible alternatives to reduce the negative consequences related to accidental NaOCl extrusions, such as vascular damage or other irreversible damages [36] and to limit the side effects of NaOCl, such as changes in dentin collagen [37].

From our findings, the antimicrobial capacity of IP6 reported slightly different results between the two experiments. While IP6 did not show statistically significant differences among the three tested concentrations from the disk diffusion test, results showed IP6 was more effective at a concentration of 5% than at 3% or 1% from the 1-h treatment test on E. faecalis. Furthermore, regardless of concentration, IP6 gave results similar to the control group. Our results did not confirm previous studies about the antibacterial activity of IP6, and on the contrary, showed biostatic and biocide activities of IP6 against various microbial strains [34]. In contrast to Nassar et al., 2021, the increasing concentration of IP6 did not increase its antimicrobial activity, and membrane disruption considered at the base of the bacterial mechanism of action seemed not to act in our study. The relevant difference in antimicrobial activity between NaOCl and IP6 does not allow us to suggest IP6 as a reliable alternative to NaOCl or EDTA.

Different findings emerged instead for the antimicrobial activity of BioAkt^TM, which was more effective than IP6 with antimicrobial capacity similar to that observed with 1.5% NaOCl. This result confirms the optimal antimicrobial activity of BioAkt^TM that emerged in previous works [21,35]. As Tonini et al., 2020, hypothesized, BioAkt^TM presents nanometric precipitation and silver-containing crystallites, resulting from the high activity and interaction rates of silver ions. In the presence of E. faecalis, silver ions increase their activity to interact with and penetrate the cell membrane of Gram-positive and Gram-negative bacteria, resulting in deoxyribonucleic acid damage and bacterial death [35].

Some doubts have been raised about BioAkt^TM such as the potential cytotoxicity of silver ions and nanoparticles in endodontics due to a high surface-to-volume ratio and non-specific oxidative injures [38,39]. However, from our findings, all experimented solutions significantly decreased cell viability and ATP production compared to controls, although cell viability appeared less affected than cell activity. Therefore, we hypothesize that the different trends of the two cell viability tests could be explained as a decrease in ATP production in the cell due to the stress caused by each solution, which reduces the cell activity without reducing their viability. Specifically, 1% IP6 was the most significantly biocompatible compound after 4 h, a condition that was not maintained after 24 h where all the concentrations of IP6 reported very similar results. However, cell viability is significantly reduced compared to the control (85% less than control). On the contrary, after 4 h, cell activity is reduced considerably, increasing the phytic acid concentration from 1% to 5%. BioAkt^TM was more cytotoxic than IP6 and the control by significantly reducing cell viability after 4 h and 24 h. After 24 h, BioAkt^TM and NaOCl reported similar cellular ATP production. These findings contrast with Generali et al., 2020, who showed that BioAkt^TM at 0.5% significantly increased cell viability [21].

5. Conclusions

Within the limitations of the methodology used, mainly due to the in vitro design and the difference in environment between wells of the lab plate and the root canal system and the additional penetration of the solutions in the dentinal tubules at different canal depths, our work confirmed the antimicrobial capacity of BioAkt^TM with an optimal balance between antimicrobial effect and cytotoxicity. However, current studies on BioAkt^TM and phytic acid are exclusively focused on evaluating the antibacterial efficacy and cytotoxicity level of these two solutions and no case reports are present about possible negative side effects or consequences of their accidental exclusion. The biocompatibility of BioAkt^TM and its concrete implementation in a real context should be evaluated. Further research should also be conducted into the applications of phytic acid for which the lowest antimicrobial activity was reported with a low cytotoxic level.