Cytocompatibility of calcium hydroxide medicament combined with cationic peptide – An in vitro study on L929 fibroblast cells

Abstract

Background:

Intracanal medication is recommended after the debridement of the canal to eliminate bacteria to prevent apical periodontitis. Combining calcium hydroxide (CH) with DJK-5 peptide will improve the penetration of medicament into root canal dentin.

Aim:

This study was performed to evaluate the cytotoxicity of a combination of DJK-5 peptide with CH on murine fibroblast cells.

Settings and Design:

This is an in vitro experimental study performed using the L929 fibroblast cells.

Materials and Methods:

L929 fibroblast cells were grown in Dulbecco’s Modified Eagle’s Medium and administered with DJK-5 peptide at a concentration of 10 µg/mL, CH+ saline, CH+ 2% chlorhexidine (CHX), 2% CHX, and CH+ DJK-5 for 24 h and 48 h. Cytotoxicity assessment of the treatments was carried out using the methyl thiazolyl tetrazolium assay and Live/Dead assay by acridine orange and ethidium bromide dual-fluorescence staining.

Statistical Analysis:

One-way analysis of variance was used for group-wise comparison of cytotoxicity of the test compounds. Pairwise comparison between groups was analyzed by Tukey’s test.

Results:

The DJK-5-treated group exhibited no cytotoxicity at 24 h and 48 h and exposure time points. Highest cytotoxicity was exerted by the 2% CHX-treated group, which increased when combined with CH, and the cytotoxicity increased with time. Combining the DJK-5 with CH reduced the cytotoxicity when compared to CH or CHX alone.

Conclusions:

At its used 2% concentration, CHX was found to exhibit high cytotoxicity. The least cytotoxicity was found in DJK-5-treated cells, and it also significantly alleviated the cytotoxicity rendered by CH treatment.

INTRODUCTION

Apical periodontitis occurs due to inflammation by polymicrobial infection of the root canal system.[1] Bacteria organize themselves in a biofilm. Intracanal medication is highly recommended after the debridement of the canal to eliminate as many remaining bacteria as possible.[2]

Calcium hydroxide (CH) has been widely employed for the intracanal treatment, possessing high alkalinity (pH ~12.5), tissue dissolution, and endotoxin neutralization properties. Bacteria like Enterococcus or Streptococcus can survive the CH medication since they can tolerate high pH or are intrinsically resistant to the medication.[1] Chlorhexidine (CHX) is the most commonly used intracanal medicament due to its broad-spectrum antimicrobial nature. Some studies have also stated improved antimicrobial properties when CH is combined with CHX.[1] However, several studies have reported dose- and time-dependent cytotoxic effects of CHX on periapical tissues and fibroblasts.[1]

DJK-5, a synthetic peptide, exhibits antibiofilm and antimicrobial action against endodontic pathogens.[3] Combining the CH with DJK-5 as a vehicle will improve the action and enhance medicament penetration into the dentinal tubules of root canal dentin.

There is a need to establish the cytocompatibility of the dental materials for their clinical acceptance.[4] In the present study, the cytotoxic effects of the combination of DJK-5 peptide with CH were evaluated.

MATERIALS AND METHODS

Ethical clearance

Ethical approval was not applicable as the present study was an in vitro study involving the cell line. However, ethical exemption was obtained from the institutional ethics committee (Protocol No. YEC2/2024/061).

Study location

This study was conducted at Yenepoya Dental College and Yenepoya Research Centre, Yenepoya (Deemed to be University).

Stock solution preparation

The DJK-5 peptide was commercially procured from GaloreTx Pharmaceuticals Private Limited (Bengaluru, Karnataka, India), and its stock solution at a concentration of 100 µg/mL was made by dissolving the peptide powder in autoclaved deionized water. Initially, 1 mg of the peptide was dissolved in 1 mL of sterile water to make the stock. From this, 100 µL of peptide solution was taken and mixed with 900 µL of autoclaved deionized water to obtain a final concentration of 100 µg/mL.

Cells and culture conditions

L929 murine fibroblast cells were provided by the National Centre for Cell Science, Pune, India. Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum and 1% antibiotic–antimycotic solution was used for culturing the fibroblasts. After attaining 70% confluence, cells were subjected to trypsinization for subculturing. Cells were maintained in a humidified setup in an incubator at 37°C and a 5% CO₂ atmosphere. After three consecutive passages, cells were used for the experiments.

Treatment protocol

Cells were randomly assigned to six experimental groups according to the treatment administered. Group 1 served as the untreated control, receiving no material. Cells in Group 2 were treated with DJK-5 at a final concentration of 10 µg/mL. Cells in Group 3 were treated with CH at a concentration of 0.425 mg/mL, prepared by mixing CH with saline to achieve the desired final concentration. Group 4 cells received a combination treatment of CH (0.425 mg/mL) and 2% CHX. Cells in Group 5 were treated with 2% CHX alone. Cells in Group 6 were treated with a combination of CH (0.425 mg/mL) and DJK-5 (10 µg/mL). All treatments were administered for 24 h and 48 h before subsequent analyses.

Assessment of cytotoxicity using methyl thiazolyl tetrazolium assay

The methyl thiazolyl tetrazolium (MTT) assay was utilized to assess the cytotoxicity of the test compounds on the L929 fibroblasts. In this method, cell proliferation and viability are evaluated based on the quantification of mitochondrial succinate dehydrogenase activity. The 96-well microtiter plates were seeded with fibroblast cells (5000 cells/well) and incubated in a humidified atmosphere for 24 h at a temperature of 37°C under 5% CO₂. Various treatments, DJK-5, CH+ saline, CHX, and their combinations, at specified concentrations, were administered to the cells and further incubated for 24 h and 48 h. The cells were further incubated for 4 h after the addition of MTT reagent (1 mg/mL, 100 µL). Dimethyl sulfoxide was employed for dissolution of the formazan crystals, which was followed by recording of the absorbance with a multimode microplate reader (FLUOstar Omega, BMG Labtech), at 570 nm.

The formula used to calculate the cell viability percentage was as follows:

Cell viability percentage = Absorbance of treated cells at 570 nm/Absorbance of control cells at 570 nm × 100

Detection of apoptosis in fibroblasts using acridine orange–ethidium bromide dual-fluorescence staining

L929 fibroblast cells were plated into a 24-well surface-treated plate (10,000 cells per well) and incubated at 37ºC and 5% CO₂, in complete DMEM. After adherence, the cells were subjected to the treatments of test compounds and their combinations, as outlined under the section treatment protocol. Postexposure to the test compounds, acridine orange (AO) and ethidium bromide (EB) mixture (1:1, 2 µg/mL) was used for staining the cells for 20 min in the dark at 37ºC. Excess stain was removed in phosphate-buffered saline (PBS) bath, and 1X PBS was used to overlay the cells. Photomicrographs were recorded at ×20, under a fluorescence imager (ZOE, BioRad) in green and red channels.

Statistical analysis

For comparing the cytotoxicity of different treatment groups, one-way analysis of variance followed by Tukey’s test was used. GraphPad Prism version 9.5.1 (GraphPad Software, LLC, Boston, Massachusetts, United States) was used for data analysis.

RESULTS

Cytotoxicity assessment using methyl thiazolyl tetrazolium assay

The results of cytotoxicity assessment indicated that cytotoxicity was observed in all tested groups, with a highly significant intergroup difference. It was observed that at both 24 h and 48 h time points, the cells exposed to pristine DJK-5 peptide exhibited no cytotoxicity. The highest cytotoxicity was exerted by 2% CHX, which was found to increase when combined with CH. The increase in cytotoxicity percentage was observed to be time-dependent. Combining the DJK-5 with CH reduced the cytotoxicity when compared to CH or CHX alone.

At 24 h, CH+ CHX exhibited moderate cytotoxicity, which was statistically highly significant from other groups. The CH alone and CHX alone groups were slightly cytotoxic, which was highly significant between each other and among other groups. DJK-5 was noncytotoxic when compared to other groups, which was highly significant among all the groups. Combination treatment of DJK-5 with CH alleviated the cytotoxicity of CH, which was highly significant (P < 0.0001) from other tested groups.

At 48 h, the cytotoxicity of the test groups was found to be increased with time. The CH+ CHX group exhibited the most cytotoxicity when compared with other groups. The cytotoxicity of CHX alone was noted to be increased over time than CH when compared with CH at 24 h. The combination CH and DJK-5 also exhibited an increase in cytotoxicity with time. All the results were statistically highly significant (P < 0.0001) between all other groups [Figure 1]. The quantitative results of the MTT assay along with statistical analyses are summarized in Tables 1 and 2 for the 24 h and 48 h time points, respectively.

Figure 1.

Cytotoxicity of the test compounds on L929 fibroblasts, as evaluated using methyl thiazolyl tetrazolium assay. Results are represented as mean ± standard deviation (n = 3). ****P < 0.0001. CH: Calcium hydroxide, CHX: Chlorhexidine

Table 1.

Cytotoxicity of test compounds on L929 fibroblasts at 24 h (methyl thiazolyl tetrazolium assay)

Group	Treatment	Dead cells (%), mean±SD	Statistical significance*
I	Control	0.00±0.00	a
II	DJK-5 (10 μg/mL)	1.15±0.90	a
III	CH	37.24±2.60	b
IV	CH+ CHX	62.00±1.02	c
V	CHX	32.87±1.04	d
VI	CH+ DJK-5	17.85±0.76	e

*Different letters indicate statistically significant differences between the groups (Tukey’s post hoc test, P<0.05). One-way ANOVA: P<0.0001. SD: Standard deviation, CH: Calcium hydroxide, CHX: Chlorhexidine

Table 2.

Cytotoxicity of test compounds on L929 fibroblasts at 48 h (methyl thiazolyl tetrazolium assay)

Group	Treatment	Dead cells (%), mean±SD	Statistical significance*
I	Control	0.00±0.00	a
II	DJK-5 (10 μg/mL)	1.50±0.57	a
III	CH	64.06±0.85	b
IV	CH+ CHX	83.01±0.46	c
V	CHX	73.68±0.80	d
VI	CH+ DJK-5	38.52±1.25	e

*Different letters indicate statistically significant differences between the groups (Tukey’s post hoc test,P< 0.05). One-way ANOVA: P<0.0001. CH: Calcium hydroxide, SD: Standard deviation, CHX: Chlorhexidine

Apoptosis detection using acridine orange–ethidium bromide dual-fluorescence staining

AO stains both live and apoptotic cells, while EB selectively stains the cells undergoing apoptosis and displays red-fluorescing nuclei. Results of the investigation indicated that viable cells were present in the untreated control and cells treated with DJK-5 alone groups, as evidenced by green fluorescing nuclei. Cells treated with CH and CHX showed yellow-green nuclei, indicating the induction of early apoptosis. Cells treated with a combination of DJK-5 with CH displayed a reduction in the number of nuclei with yellow-green fluorescence, further confirming the protective action of DJK-5 peptide on the cytotoxicity rendered by CH. Photomicrographs of L929 cells stained with the dual fluorochromes, 48 h posttreatment with the test compounds, are depicted in Figure 2.

Figure 2.

Photomicrographs of L929 cells treated with the test compounds for 48 h and stained using acridine orange–ethidium bromide (×20). CH: Calcium hydroxide, CHX: Chlorhexidine

DISCUSSION

The cytotoxicity of intracanal medicament CH when combined with CHX and DJK-5 peptide on murine fibroblast cells (L929) was evaluated in the present investigation using MTT assay and dual-fluorescence staining. It is essential to assess the cytocompatibility of materials that come in contact with tissues to confirm their clinical acceptance.[5] Cytotoxicity testing is considered to be a critical aspect of the biological evaluation of medical devices, as per the ISO 10993-5 requirements.[6] L929 fibroblast established cell line is a widely used cell model that enables quantifiable as well as reproducible results, when used in a controlled laboratory setting, and is commonly used in MTT assay.[7]

CH (pH ~12.5) is widely used as an intracanal medicament. It acts by generating free radicals and provides a physical barrier. CH can dissolve the residual tissues.[1] However, not all bacteria are efficiently killed by CH; therefore, proper disinfection cannot be achieved by its use.[8] The clinical application of the CH is affected by the vehicle to which CH is mixed.[8] The CH concentration used in the present study was 0.425 mg/mL, which was observed to be noncytotoxic on human gingival fibroblast cells (HGF -1 Cells) by Ehlinger et al.[9]

CHX has been widely employed as a cationic bisbiguanide with substantive residual activity; however, necrotic tissue remnants could not be dissolved effectively with its use.[10] CHX is considered to possess a significant antibacterial effect, attributable to its biguanide action.

DJK-5 is an enantiomeric peptide that exhibits broad-spectrum antibiofilm activity.[11] The peptide DJK-5 efficiently prevented the growth of biofilms in the case of oral biofilms.[11] Further, DJK-5 is resistant to the action of proteases, thereby confirming its enhanced efficacy against biofilms.[5] The concentration of the peptide DJK-5 was determined to be 10 µg/mL based on the earlier studies by Wang et al.[5]

In the current study, results showed that the highest viability percentage of living cells with the least cytotoxicity was found in the peptide DJK-5 alone group, followed by the CH+ DJK-5 group. CH and CHX groups showed an increase in cytotoxicity with time. The combination of CH and CHX showed the lowest viability percentage of living cells with the highest cytotoxicity at both time points and was found to increase with time. This cytotoxic effect may be attributed to the additive or synergistic effects of the combination. CHX treatment has been associated with dose- and time-dependent cytotoxic effects on mammalian cells through compromising the cell membrane integrity, oxidative stress, and disruption of mitochondrial activity. When combined with CH, the high alkalinity may facilitate sustained release of CHX, leading to cumulative toxicity over time. Further, chemical interactions between CH and CHX may cause ionic imbalance and cellular stress, further contributing to reduced cell viability.

The cytotoxic effects of CH were reduced when combined with the peptide DJK-5. This is in line with a study conducted by combining DJK-5 with endodontic irrigants, where cytotoxicity of ethylenediaminetetraacetic acid (EDTA) and CHX was reduced in combination with DJK-5.[12] Thus, DJK-5, with its antibiofilm and antimicrobial properties, along with its cytoprotective effect, might have the potential to be used with CH as a vehicle to reduce the microbial load within the root canal and periapical region.

As confirmed by our results, the cationic peptide DJK-5 possesses good biocompatibility. It is further proved that combining CH with DJK-5 reduces the cytotoxicity of CH. However, these results represent their compatibility only under in vitro conditions, hence demanding careful interpretation. In the in vivo environment, the lymphatic and vascular systems are involved in the neutralization of the test materials, which makes them less harmful in a clinical scenario compared to their equivalent concentrations in in vitro studies.[13] These results were further supported by the images of AO-EB-stained cells, wherein DJK-5 treatment rendered the protective effect on the cytotoxicity induced by CH. DJK-5 peptide and CH combination showed better cytocompatibility, which may be attributed to their distinct antimicrobial mechanisms. CH predominantly exerts its effect through sustained alkalinity exerted on the host cells. DJK-5, a synthetic antimicrobial peptide, works by disrupting the bacterial intracellular pathways with no significant damage to mammalian cell membranes, thereby exhibiting selective antimicrobial activity with no cytotoxicity on the host cells. In addition, the use of peptides may enhance antimicrobial efficacy without compromising the physicochemical stability of CH, making such combinations promising for intracanal applications sustaining both efficacy and biocompatibility.

The study results need further validation using both in vivo assessments and clinical trials. Studies must be conducted to evaluate the antimicrobial efficacy of the CH and DJK-5 combination against various endodontic pathogens and its effect in regenerative procedures as well. Future studies involving animal models and long-term evaluations will be required.[14,15]

CONCLUSIONS

In the current study, the combined use of CH with DJK-5 was interpreted to be cytocompatible. The CH and CHX combination group exhibited the highest cytotoxicity among all the test groups, which was also found to be time-dependent. The peptide exhibited a cytoprotective effect alone and while combining with CH. Thus, peptide DJK-5 has the potential to be used along with CH as an intracanal medicament during endodontic treatment.

Conflicts of interest

There are no conflicts of interest.

Funding Statement

Nil.