Effect of calcium hydroxide on morphology and physicochemical properties of Enterococcus faecalis biofilm

Calcium hydroxide Ca(OH)2 has been used as an intracanal medicament to targets microbial biofilms and avert secondary infection in the root canal system. This study evaluated the effects of this material on the morphology and physicochemical properties of an established in-vitro biofilm of Enterococcus faecalis. A biofilm of E. faecalis was grown in multichannel plates. The chemicals including Ca2+, OH−, and saturated Ca(OH)2 (ie 21.6 mM) were prepared in order to evaluate which component eradicated or amplified biofilm structure. Various biochemical and microscopic methods were used to investigate the properties of the biofilm. Biofilms treated with Ca(OH)2 absorbed more Ca2+ because of the alkaline pH of the environment and the ions affected the physicochemical properties of the E. faecalis biofilm. A denser biofilm with more cavities and a granular surface was observed in the presence of Ca2+ ions. This resulted in a decrease in the surface-to-biofilm ratio with increases in its biomass, thickness, colony size, and volume. Calcium hydroxide did not destroy E. faecalis biofilms but rather contributed to the biofilm structure. This in-vitro study sheds light on a missing link in the formation of E. faecalis biofilm in which the Ca2+ in Ca(OH)2


Scientific Reports
| (2022) 12:7595 | https://doi.org/10.1038/s41598-022-11780-x www.nature.com/scientificreports/ Calcium hydroxide Ca(OH) 2 is widely used as a temporary root canal medicament to hinder the regrowth of bacteria in the root canal between treatment sessions 11 . One of the most important reasons to use it in endodontics is its antimicrobial property. The antimicrobial activity of Ca(OH) 2 relies on the release of hydroxyl ions (OH -) in the presence of water, which results in cytoplasmic membrane distortion, protein denaturation, and DNA damage 12 . However, some studies have reported that calcium hydroxide has limited effectiveness in eliminating bacteria from biofilm 5,13 .
In 2012, van der Waal and van der Sluis hypothesized that calcium plays a key role in the formation of the scaffold and extracellular matrix of a biofilm 14 . They observed that exposure to calcium resulted in distinct changes in the biofilm. They suggested that chelators like Ethylenediaminetetraacetic acid (EDTA) can absorb these calcium ions in the EPS and facilitating the disruption of biofilm. With this as a basis, we investigated the effect of calcium hydroxide ingredients (Ca 2+ and OH -) on the morphology and physicochemical properties of E. faecalis biofilms. This bacterium has been commonly used in endodontic biofilm models because it has been coupled with endodontic treatment failures 13,15 .
The antimicrobial properties of this molecule against planktonic microbial cells have been described but its action against biofilm results to be unclear and controversial. The null hypothesis of this study was that calcium hydroxide does not show antibiofilm activity but rather contributes to the biofilm matrix architecture.

Results
This laboratory study investigated the effect of calcium hydroxide, calcium and hydroxyl ions on 21-day-old E. faecalis biofilms, grown on different artificial surfaces. Evaluation included microscopy (SEM, CLSM, LM, AFM) and determination of calcium and polysaccharide content of the biofilm as well as quantity of viable cells. It was found that the different chemical formulations affected the biofilm morphology and physicochemical properties.
Biofilm surface morphology. Microscopy showed differences of biofilm growth under different solutions.
Scanning electron microscopy (SEM) images revealed that in the Ca(OH) 2 group an increase in cell size was observed (Fig. 1b). Furthermore, the cells had changed from oval to cigarette-shaped and a small number of them were dividing. Empty holes and a large volume of EPS also was observed in this group. In the Ca 2+ group, the cell sizes were normal and some of them were dividing. In the OHgroup, an increase in cell size was observed. These cells also had a smooth surface.
In the Ca(OH) 2 and Ca 2+ groups, biofilm surface was granular due to the huge amount of EPS while in the control and OHgroups, it was smooth (Fig. 1). The granular surface could have been the result of the Ca 2+ ion bonds, which produced different morphologies when compared with other groups. Furthermore, the SEM images ( Fig. 1) showed that in the Ca(OH) 2 group, the biofilm density was increased, while, in the OHgroup, no further biofilm was formed. These observations confirmed the role of Ca 2+ ions in biofilm progression.
3D structure of biofilm. According to the confocal laser scanning microscopy (CLSM) data, the structural properties of biofilm was described in terms of thickness, colony volume, biomass, colony size and surfacebiovolume ratio (Supplementary Table S1 and S2). The CLSM images showed that the presence of Ca 2+ ions induced structural changes and a denser biofilm formation (Fig. 2). In the control group, the biofilm covered a larger surface, but its volume and the thickness were smaller and contained living cells (as expected).
The maximum thickness of the green and red dyed biofilm was considerably different between groups. The Ca(OH) 2 group had the most biofilm thickness followed by Ca 2+ group. The OHand control groups had the least biofilm thicknesses (Fig. 3A, Tables S1 & S2).
The Ca(OH) 2 group had significantly highest average biofilm thickness (p-value < 0.0001) followed by the Ca 2+ , OH -, and control group (Fig. 3B, Tables S1 & S2). Statistical analysis indicated that the average colony volume related to the green and red dyes were significant. The Ca(OH) 2 group was significantly different (p-value < 0.0001) from other groups (Fig. 3C, Tables S1 & S2). This group formed significantly much more biomass than did the other groups (p-value < 0.0001). The difference between the other groups was not significant (Fig. 3D, Tables S1 & S2). The Ca(OH) 2 and Ca 2+ groups had larger colony size; meanwhile, the OHand control groups formed smaller one (Fig. 3E, Tables S1 & S2). The OHgroup had the most surface/volume ratio followed by the control, Ca(OH) 2 , and Ca 2+ groups (Fig. 3F, Tables S1 & S2).
Morphology and roughness of the bacterial biofilm. The atomic force microscopy (AFM) microscopy images revealed the variable morphology and roughness of the bacterial biofilms (Fig. 4). The roughness values of the samples were 52.6, 56.7, 42, and 43.4 for the Ca(OH) 2 , Ca 2+ , OH -, and control groups, respectively. Statistical analysis using the Kruskal-Wallis test showed no significant difference between Ca(OH) 2 and Ca 2+ groups (P = 0.727). Meanwhile, the morphological difference pertinent to Ca(OH) 2 and OHgroups revealed the cooperation of calcium in biofilm progression where the environment was alkaline.
Polysaccharide staining of biofilm. The amount of total polysaccharide increased in the presence of free Ca 2+ ions in the Ca(OH) 2 (Fig. 5b) and Ca 2+ (Fig. 5c) groups. However, the amount of polysaccharide that formed in the OHgroup (Fig. 5d) was very low and similar to that of the control group (Fig. 5a).
Quantification of biofilm polysaccharide. The mean concentration of free polysaccharides was measured for in all groups (Fig. 6). A remarkable difference was observed between groups. Overall, it could be concluded that the concentration of polysaccharides in the Ca(OH) 2 group was significantly different from the other groups. The results showed that the mean Ca 2+ concentration in the control, Ca(OH) 2 , Ca 2+ , and OHgroups were 30.8, 18,555, 10,948 and 2211 ppm, respectively (Fig. 7). The Ca 2+ ions content of the biofilm was at maximum levels in the Ca(OH) 2 group, which was in compliance with previous results and confirmed that Ca 2+ ions played major role in biofilm progression in alkaline environment.

Quantity of viable cells in biofilm.
The number of surviving bacteria in the OHgroup was lowest followed by the Ca +2 , Ca(OH) 2 , and control groups (Fig. 8). This demonstrates the protective nature of the biofilm under a higher concentration of Ca +2 , although the alkaline environment disrupted the viable cells.

Discussion
This study investigated the effect of calcium hydroxide on the morphology and physiochemical properties of E. faecalis biofilms. SEM, CLSM, and AFM methods were used to evaluate the morphologic and structural properties; whereas, viable cells in the biofilm, polysaccharides, Ca 2+ , and EPS components were measured to assess the chemical properties of the biofilms. The results revealed that Ca(OH) 2 components, ie Ca 2+ , and alkaline pH, through a cooperative manner strengthen biofilm. Therefore, Ca(OH) 2 did not eradicate E. faecalis biofilm, but promoting growth of biofilm.   2 and Ca 2+ groups formed a granular surface; while, in the control and OHgroups, the surface was smooth. The granular surface was produced by the Ca 2+ ion bonds, which led to morphological differences when compared to the smooth surfaces in the absence of Ca 2+ ions. These findings are in consistent with those of Safari et al.,who showed that the addition of Ca 2+ to the biofilm produced a granular surface 16 . Moreover, Mangwani et al. observed that the addition of Ca 2+ caused cavities in the biofilm. An increased in cavities in the biofilm indicates the accumulation of large amounts of EPS 17 .
In regard to the qPCR and CLSM results, it can be concluded that most of the green dye displayed in the Ca(OH) 2 and Ca 2+ groups were live cells, while in the OHgroup the number of viable cell dramatically reduced. In the Ca(OH) 2 group, because of alkaline pH of the environment, the uptake of Ca 2+ increased in the biofilm such that number of living bacteria was more than other groups 18 . This finding is consistent with the report pertinent to elevated biomass by adding Ca 2+ to the biofilm 9,16,17,19 .
The results of quantitative analysis of free polysaccharides in the EPS revealed that the lowest concentration of free polysaccharides was for the Ca(OH) 2 group. This can be explained by the mechanisms of the Smith-Gilkerson reaction, in which the OH part of the sixth carbon of the saccharide ring in the polysaccharide structure, used to produce chromogenic substance, was associated with large amounts of Ca 2+ ions and was unable to interact with the Smith-Gilkerson reagent. We concluded that most of the polysaccharides in the biofilm were not free and had not been measured by the Smith-Gilkerson method.
In conclusion, the presence of Ca 2+ ions caused a denser biofilm with more cavities and indicates an increase in EPS. The presence of this ion also created a granular surface in the biofilm. Expansion of the biomass and increases in the thickness, colony size, and volume of the biofilm as well as declining the surface-to-biofilm ratio were the results for the Ca 2+ ions in the biofilm. The alkaline pH of the environment enhanced absorption of Ca 2+ when a biofilm was treated with Ca(OH) 2 ; thus, this ion was able to affect the morphology, structure, and chemical properties of the E. faecalis biofilm as well.
This study has shown that Ca 2+ , as a part of calcium hydroxide, serves a missing link in the biofilm progression of E. faecalis. Calcium hydroxide does not destroy the biofilm of E. faecalis, but actually participates in strengthening the biofilm of this bacterium.

Material and methods
Solution preparation and biofilm assay. In this study, the standard E. faecalis strain (ATCC 29,212) was used in all experiments. Brain heart infusion (BHI) agar (Merck; Germany) and BHI broth culture medium (Merck; Germany) were prepared according to manufacturer instructions. The isolated colonies were cultured on BHI agar plates and incubated under aerobic conditions at 37 °C for 24 h. Thereafter, a bacterial suspension in normal saline was prepared adjusted to the 0.5 McFarland standard and diluted to equal 10 6 CFU/mL. Chamber slides, cover glass, mica, and falcon tubes were used in which form the biofilm for microscopic observations. To prepare the Ca(OH) 2 solution, 1.65 g of powder was dissolved in 1000 mL of deionized water (i.e. 21.6 mM) and stirred for 3 h. After overnight incubation at room temperature, the solution was centrifuged at 8000 RPM for 10 min. The supernatant was used as an 100% Ca(OH) 2 solution. To prepare pure Ca 2+ ions, a part of aforementioned solution was neutralized using glacial acetic acid at pH 7. To produce the OHions, 100% Ca(OH) 2 solution was chelated using ethylenediaminetetraacetic acid (EDTA). Thereafter, the Ca 2+ content was quantified using Eriochrome Black T indicator solution (Sigma-Aldrich; Germany) to ensure all of the Ca 2+ had chelated (https:// chem. libre texts. org/@ go/ page/ 75718). For convenience, the groups were denoted as Ca(OH) 2 , Ca 2+ , and OH -.
After 21 days of incubation and formation of a mature biofilm, it was exposed to one of three solutions for 7 days to simulate clinical conditions. All experiments were carried out in triplicate.

Scanning electron microscopy (SEM).
To assess the surface morphology and composition of the E.
faecalis biofilm under the different solutions, they were examined with scanning electron microscopy (SEM). For www.nature.com/scientificreports/ this purpose, a 1 cm × 1 cm cover glass was embedded in 6-well plates (Guangzhou Jet Bio-Filtration; China), and 5 mL of freshly inoculated E. faecalis BHI media was poured into each well. The cumulative number of inoculated bacteria was adjusted to 10 6 CFU/mL. The biofilm culturing and treatments were the same as for the biofilm assay. Thereafter, the cover glass was placed into 4% glutaraldehyde (Merck; Germany) to fixate the biofilms. After 1 h, the samples were washed with double-distilled water (DDW) and air dried. They then were mounted on a gold-coated SEM stub and photographed using a MIRA3 scanning electron microscope (Tescan; Czech) 22 . The accumulation of bacteria and biofilm formation under different conditions were investigated.

Confocal laser scanning microscopy (CLSM).
CLSM was used to examine the reconstruction of threedimensional structures in the biofilm. The biofilm was grown on a chamber slides (Nunc Lab-Tek; Thermo Fisher Scientific; Denmark) and exposed to the solutions as described in the biofilm assay. The slides were stained with fluorescein diacetate (FDA) green fluorescent dye (Sigma-Aldrich; Germany) for live bacteria and with propidium iodide (PI) red fluorescent dye (Sigma-Aldrich; Germany) for dead bacteria. The PI was not able to cross the membrane of living cells but can stain the DNA of dead bacteria or eDNA in EPS.
After staining, the samples were incubated at room temperature for 20 min in a dark place. Then they were gently rinsed with phosphate buffer saline (PBS) to remove non-adherent bacterial cells. For imaging, an inverted microscope (Leica TCS-SPE system; USA) was used at × 10 magnification. The excitation wavelength used for the FDA and PI were 488 nm and 532 nm, respectively, and the emission wavelengths were 500-550 and 580-700 nm, respectively. Four to six regions were randomly selected for each biofilm in each group. The biofilm parameters of biomass, mean thickness, maximum thickness, average colony size, average colony volume, and surface-tovolume ratio were evaluated. The three-dimensional images were analysed using Comstat software (V. 2.1, www. comst at. dk) 17,21,23 . Atomic force microscopy (AFM). The roughness value of E. faecalis biofilm surface under the solutions were assessed using AFM. After the biofilm was grown on mica and then put into contact with the solutions, each was washed twice with DDW and dried in a desiccator overnight. The imaging of the prepared samples Light microscopy. To ensure that changes in the parameters measured by Comstat in the CLSM experiment were not solely due to the changes in the number of bacteria, the biofilms was stained with Alcian blue (Sigma-Aldrich; Germany). The matrix and acidic extracellular polysaccharides of the biofilm became visible. The biofilm was examined with a light microscope (PH2-RFCA Olympus, Japan) at a × 400 magnification 6 .
Biofilm polysaccharide measurement. The amount of free polysaccharides in the biofilms was quantified using the Smith-Gilkerson method 24 . Biofilm was grown in a 50 mL falcon tube and brought into contact with the solutions, as described previously. The samples were dried in an incubator (3 days; 40 °C), their weight was measured, and the falcon walls were then carefully scraped using a spatula to remove the biofilm. Next, 2.5 mL of DDW was added to each sample. The samples were then sonicated eight times in 30-s cycles at 60% power with the a sonicator (Ultrasound Technology, UP200h; Germany) to release the polymers from the bacterial cells. These samples then were centrifuged at 5000 g for 30 min at 12 °C to separate the supernatants from the bacterial cells. A volume of 1.5 mL of the clear supernatant was removed from the suspension and centrifuged again for 10 min at 12,000 g. Finally, 1 mL of the final clear supernatant was used to determine the polysaccharides concentration. The polysaccharide compounds were hydrolyzed using 0.5 M of hydrochloric acid (Merck; Germany). Then, 3-methyl-2-benzothiazolinone hydrazine (MBTH) (Sigma-Aldrich; Germany), a chromophore, formed a colour complex and the optical density was measured at 650 nm.
Biofilm calcium content. Inductively coupled plasma was used to measure the amount of calcium in the biofilm matrices. In four falcon tubes, E. faecalis was grown to form biofilms and then was put into contact with the solutions as described previously. The samples were dried in a desiccator and the falcons were weighted. Then, 1 mL of nitric acid (Merck, Germany) and 1 mL of hydrogen peroxide (Merck, Germany) were added to each sample. Finally, the volume of each sample was increased to 10 mL with DDW and Ca 2+ ion concentration was measured by inductively coupled plasma-optical emission spectroscopy (OES-ICP; 730-ES; Varian; USA) at a wavelength of 397/847 nm 25 .  for each group and for positive (non-treated) and negative (vancomycin-treated) controls. After 21 days of biofilm formation, the plates were gently rinsed twice with PBS to remove the unattached bacteria. To enumerate the viable cells, the RNA of each well was harvested using TRI reagent (Sigma Aldrich; Germany) following manufacturer protocols with a few modifications. Briefly, 100 µL of lysozyme (20 mg/mL) was added to each well for 10 min to reduce the EPS. Then, RNase free DNase (ThermoFisher Scientific) was poured into each well to digest the eDNA. Finally, 100 µL of TRI reagent was used in each well followed by RNA extraction. The upper phase containing RNA was transferred to a RNeasy column extraction kit (Takara Bio; Japan) to prepare the pure RNA. DNase treatment was performed to eliminate probable DNA contamination. The purity and quantity of RNA were detected using NanoDrop 1000 spectrophotometer (Thermo Scientific, Waltham, MA; USA). The cDNA was constructed using a commercial kit (Takara Bio; Japan). Quantitative real-time polymerase chain (qPCR) reaction was done to determine the absolute number of viable bacteria in the biofilm using SYBR reagent (Takara Bio; Japan) and specific primers 26 on a C1000 Touch™ Thermal Cycler (Bio-Rad;PA; USA). The RNA content was translated into the bacterial content in accordance with a standard curve and was reported as colony forming units (CFU) per mL. The experiment carried out in triplicate. Data analysis. The normality of the continuous variables was assessed using the Shapiro-Wilk test. In order to check the parameters of CLSM images and polysaccharide quantity, one-way ANOVA and Bonferroni posthoc tests were used, respectively. The roughness values in the AFM and the calcium content in the biofilms were evaluated by applying the Kruskal-Wallis and Mann-Whitney tests. All data analyses were performed using Stata version 12.0 (StataCorp., USA), and P < 0.05 indicated statistical significance. The SEM results were descriptively assessed and reported.

Data availability
The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.