Cytotoxicity of oregano essential oil and calcium hydroxide on L929 fibroblast cell: A molecular level study.

Introduction: The purpose of antimicrobial agents is to eliminate the microorganisms without causing toxicity to host cells. This study aimed to assess the cytotoxic effect of oregano essential oil on L929 fibroblast cells. Materials and
Methods: L929 fibroblast cells were exposed to four different concentrations of oregano essential oil (25-200 μg/ml) and calcium hydroxide (1 mg/ml). Dose-response curve was evaluated using 3-(4,5 dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. The toxicity of L929 fibroblast cells was determined by lactate dehydrogenase activity (LDH). The results were analyzed using one-way analysis of variance and turkey post hoc test. P <0.05 was considered statistically significant.
Results: Oregano essential oil showed a higher percentage of cell viability than the calcium hydroxide group. At 50 μg/ml, fibroblast cells showed arbitrarily 80% of cell viability compared to calcium hydroxide. There was a statistically significant difference with P < 0.05 on evaluating the effect of oregano essential oil on cytotoxicity measurement by LDH release of L929 fibroblast cells.
Conclusion: Within the limitation of the study, Oregano essential oil at 50 μg/ml reported to show reduced cytotoxicity compared to calcium hydroxide at 1 mg/ml. Therefore, perhaps after evaluating other properties, it might be considered an intracanal medicament. Copyright:

INTRODUCTION

Successful endodontic regeneration aims at creation of a bacteria-free biological environment inside the root canal space through the use of intracanal antibacterial medicaments. It must be used at concentrations that are bactericidal while having minimal effects on cell viability in periapical tissues.[1] The choice of this agent depends on biologic characteristics; such medicaments should be nonirritant, able to control the intensity and duration of inflammatory processes and infection, and have healing potential.[2]

Calcium hydroxide is the most commonly used intracanal medicament. It has the ability to neutralize the bacterial by product. Because of its high pH, calcium hydroxide can lead to chronic inflammation and cell necrosis in vivo.[3]

Owing to the disadvantage essential oil from aromatic and medicinal plants has known to possess antimicrobial and antioxidant properties.[45] Origanum vulgare (Lamiaceae family), is an endemic plant found in India, southern part of Iran and Mediterranean region, that has been used traditionally used for antiseptic purpose. Oreganum essential oil is edible plant oil used in food products. Reports have shown oregano oil possess antimicrobial property and reported to be an effective antimicrobial agent against Enterococcus faecalis.[6] Hence, the present agent can be used as an intracanal medicament in endodontic infections.

MATERIALS AND METHODS

Preparation of oregano essential oil

The fresh leaves of O. vulgare were collected from southern region of India (Tamil Nadu). Oreganum leaves were dried in hot air oven at 60°C till a constant weight was obtained. The dried leaves were ground and placed inside a soxhlet apparatus. Petroleum ether was used as a solvent for extraction of oil. Soxhlet apparatus was run at 60°C for 8 h. After which the solvent (40°C–60°C) was evaporated in a rotary evaporator to isolate the essential oil.

Cell culture

L929 cells were procured, placed in 25 cm2 culture flasks, and cultured in Roswell Park Memorial Institute medium 1640 culture medium, with 10% fetal bovine serum (FBS), L–glutamine, 1% penicillin (100 U/ml), and streptomycin (100 μg/ml) at 37°C in a humidified CO2 (5%) chamber and 95% air. The cells were detached using 0.25% ethylenediaminetetraacetic acid trypsin. Neutralization of the trypsin was achieved using Dulbecco's Modified Eagle's Medium (DMEM) containing 10% FBS and 1% antibiotic and cells were mechanically separated using a pipette. There were 96-well plastic culture plates filled with 200 μl of medium containing in each well. The plates were then incubated at 37°C in a humidified atmosphere containing 5% CO2 and 95% air for 24 h to permit attachment of the cells to the plates.

Reagent preparation

Stock solutions of 10 mg/ml of oregano oil were freshly diluted to working concentrations in 10% DMSO. From this stock solution, different lower dilutions (25–200 μg/ml) were prepared. There were three experimental groups.

Study groups

Group 1: Control group without any treatment

Group 2: Treatment with oregano oil

Group 3: Treatment with standard calcium hydroxide (1 mg/ml).

L929 cells were cultured in 96-well plates (1 × 103 cells/ml) and treated with various concentrations (25–200 μg/ml) of oregano oil, respectively.

3-(4, 5dimethythiazol-2-yl)-2, 5-Diphenyl tetrazolium bromide assay

3-(4, 5dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay is a colorimetric assay that measures the reduction of yellow MTT by mitochondrial succinate dehydrogenase. The L929 cells were seeded at the density of (1 × 103 cells/ml) were plated on into well plates and treated with oil for 24 h. The cells were permitted to adhere for 24 h, and the growth medium (MEM) removed using micropipette and the monolayer of cells washed twice with MEM without FBS to remove dead cells and excess FBS. Cell culture medium (DMEM) was used as negative control for assessment of cell viability. 1 ml of medium (without FBS) containing different dilution of drugs were added in respective wells; 200 μl of MTT (5 mg/ml in PBS) were added to each well, and the cells incubated for a further 67 h in 5% CO2 incubator. After removal of the medium, 1 ml of DMSO was added to each well and the calcium hydroxide (5 mg/ml) was tested.

The effect of oregano oil on cell growth inhibition was assessed as percent cell viability, where vehicle-treated cells were taken as 100% viable. The supernatant was removed and 50 μl of propanol was added and the plates were gently shaken to solubilize the formed formazan. The MTT enters the cells and passes into the mitochondria where it is reduced to an insoluble, colored (dark purple) formazan product. Since the reduction of MTT can only occur in metabolically active cells the level of activity is a measure of the viability of the cells. The plates were placed on a shaker for 15 min and the absorbance was read on an enzyme-linked immunosorbent assay reader at 570 nm.[7]

From the values obtained, the percentage cytotoxicity (half maximal inhibitory concentration [IC50] value) was calculated. Each experiment was carried out in triplicate and the IC50 of the oil as the percentage survival of the cells was calculated according to the formula:

The optical density (OD) was measured with a microplate reader at 570 nm filter and calculated the cell viability. Cell viability (%) = (Mean test OD/Control OD) ×100

The percentage of cell viability over 90% indicated nontoxicity, while the amount between 60% and 90%, 30%–60% and <30% indicated mild, moderate, and severe toxicity respectively.

Cell membrane integrity (lactate dehydrogenase release assay)

Percentage release of lactate dehydrogenase (LDH) was measured by the Promega 96 CytoTox assay kit (Madison). In brief, L929 cells were seeded into 24-well plates and left to grow to 60% confluence, achieved typically within 48 h from seeding at 40%. After 48 h the cells were treated either with different concentrations of oil (25–200 μg/ml). L929 cells without any serum-free DMEM were used as a negative control. After 3 days of incubations, the medium was measured for LDH release, whereas in a duplicate well cells were lysed by the addition of Triton X-100 (to a final of 1%) to measure total LDH. After mixing the samples with the substrate and incubated at room temperature for 30 min, OD was measured using a micro plate reader (Biorad) with filter of 492 nm.[8] The percentage of LDH release was calculated from the medium and total values corrected for background.

Calculation of cytotoxicity of test compounds as follows:

Statistical analysis

For statistical analysis of data, multiple comparisons were performed using one-way analysis of variance followed by the Least Significant Difference test for post hoc analysis. Statistical significance was accepted at a level of P < 0.05. Data were analyzed using SPSS software package, version13.0 (IBM SPSS predictive analytics community, Armonk, New York).

RESULTS

3-(4, 5dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay

Graph plotted from the percentage of cell viability calculated using the absorbance of test sample and control sample [Table 1]. Figure 1 shows cell viability using microtiter plate assay. Microscopic Observations of L929 Cell lines subjected to calcium hydroxide and different concentration of oregano oil was visualized using Phase contrast Microscope at 10x magnification [Figure 2]. Figure 3 depicts a higher percentage of cell viability with oregano essential oil than calcium hydroxide group. The negative control showed arbitrarily 100% cell viability. Fibroblast exposed to 25 μg/ml showed 90% cell viability. At 50 μg/ml oregano essential oil the cell viability was found to be arbitrarily 80%. At 100 and 200 μg/ml, the presence of viable cells was found to be lower than 80%. Although the percentage of cell viability of oregano essential oil was reduced compared to negative control. There was a statistically significant difference with P < 0.05 between the control calcium hydroxide and oregano essential oil.

Table 1

3-(4, 5dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay

	Negative control	Oregano oil (concentration in µg)				Ca (OH)2
		25	50	100	200
Percentage of cell viability	100	90.1	80.6	71.3	60.5	30.6

Figure 1

Cell viability –Microtitre plate assay. Negative control-1; oregano oil (25 μg)-2; oregano oil (50 μg)-3; oregano oil (100 μg)-4; oregano oil (200 μg)-5; calcium hydroxide (1 mg/ml)-6

Figure 2

3-(4, 5dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay – Microscopic observations of image of L929 cell lines by phase contrast microscope at ×10. (a) Negative control; (b) oregano oil (100 μg); (c) oregano oil (200 μg); (d) Ca (OH)2

Figure 3

Results of 3-(4, 5dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay. Figure depicts cytotoxicity result was determined by 3-(4, 5dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide assay and results expressed as mean ± standard error of the mean. *P< 0.05 significant as compared with negative control

Graph plotted from the absorbance reading of test sample and control sample [Table 2]. Microscopic Observations of L929 Cell lines subjected to calcium hydroxide and different concentration of oregano oil was visualized using Phase contrast Microscope at 10x magnification [Figure 4]. Figure 5 depicts the effect of oregano essential oil on cytotoxicity measurement by LDH release. Fibroblast exposed to 50 μg/ml showed insignificant amount of LDH release. At 100 and 200 μg/ml, the amount of LDH release was significant when compared to negative control. Release of LDH assay was found to be related to the integrity of the cell wall. More the release of LDH greater the loss of cell wall integrity.

Table 2

Lactate dehydrogenase activity cytotoxicity assay

	Negative control	Oregano oil (concentration in µg)				Ca (OH)2
		25	50	100	200
Absorbance reading	0.132	0.175	0.394	0.416	0.502	0.632

Figure 4

Lactate dehydrogenase assay – Microscopic observations of image of L929 cell lines by phase contrast microscope at ×10. (a) Negative control; (b) oregano oil (100 μg); (c) oregano oil (200 μg); (d) Ca (OH)2

Figure 5

Lactate dehydrogenase assay. Figure represents effect of oregano oil on cytotoxicity by lactate dehydrogenase release of L929 cells. Results are represented as mean ± standard error of the mean. *P< 0.05 statically different as compared with as compared with positive control

DISCUSSION

Modern endodontics deals regeneration of techniques with minimal or no instrumentation. Therefore, in such cases, intracanal medicament plays a vital role. Reports have shown necrotic immature teeth may contain some vital pulp cells that can play significant roles in endodontic regeneration.[91011] Recent studies have shown that intracanal medications at concentrations even lower than that currently used in endodontic regeneration have negative effects on DPCs and stem cells of the apical papilla.[112] Moreover, antibacterial activity of intracanal medicament must exceed cytotoxicity. The evaluation of cytotoxicity of these agents beside antibacterial activity seems to be necessary as these agents have enough potential to eliminate bacteria that may damage periapical tissues.[1314]

Previous studies have assessed the cytotoxicity of carvacrol compound and oregano extracts using various assays which were majorly concentrated on medical-related diseases.[1516171819] Most of the studies related to the endodontic literature have assessed on the murine fibroblastic cell lines.[2021] Various primary cell cultures were also used to assess the in vitro cytotoxicity.[2223] Cell lines such as human fibroblastic cell lines,[24] Murine macrophage like RAW cell lines,[25] Human dental pulp stem cell lines[26] were used in assessing the in vitro cytotoxicity of the agents tested as root canal irrigants or sealers. Previous reports have tested the efficacy of L929 fibroblastic cell lines as appropriate to assess the root canal sealers.[2728]

As the present study was mainly assessing the cytotoxicity of the agent, to be used as an intracanal medicament we thought it would be appropriate to the select the L929 fibroblastic cell lines which simulate the periapical fibroblasts. Upon the discussion of the type of cells used for the assessment of cytotoxicity, fibroblasts cells are commonly preferred. This type of cells is said to produce collagen tissues and aid in tissue repair process.[29] Moreover, these types of cells were found to be available in the periapical tissues that are susceptible to intracanal medicament.[30]

Although the culture mediums vary the ideal conditions and the agents used for maintaining the cell lines and protocol of the incubation of cell lines correlated with the previous literature.[7825] Hence, the protocol of cell line collection is standardized and reliable.[3132]

The MTT assay was introduced for cell viability analysis. This method assesses the ability of viable cell in changing the water-soluble tetrazolium salts to the insoluble formazan crystals through the activity of mitochondrial dehydrogenase enzymes. This method assesses the cytotoxicity of dental materials based on the changes in the number of viable cells, cell metabolism, and cell morphology. This method is simple and reproducible and does not require radioisotopes. In this method, only cell death, in the apoptotic phase, is detected when cellular metabolism decreases.[3334]

The protocol of MTT assay for screening of cytotoxicity was similar to the previous studies.[78] A recent systematic review has compared MTT assay versus other cell assays to evaluate the biocompatibility of the root canal filling materials.[35] The results concluded that MTT assay do not over or underestimate the cell viability during the cytotoxic screening and can be considered as reliable for endodontic assessments. The recent development of protocols on assessing the cytotoxicity is based on the 3D-tissue-like cultures, aiming to improve their predictability in the clinical scenario collected.[35]

LDH assay, this method indirectly reflects the compromised cell membrane integrity, which is associated with necrosis.[36] Necrosis is another form of cell death that will provoke an inflammatory response of surrounding cells through the leakage of intracellular contents. The cell membrane damaged by the cytotoxic agent allowed the release of intracellular LDH molecules into the culture medium.[37] The protocol used for LDH assay in the present study was standardized according to the currently proposed guidelines.[38]

Single cytotoxicity assays are no longer considered sufficient to evaluate or predict all aspects of a material's cytotoxicity.[39] In the current study, both MTT assay and LDH assay were performed. Hence, the presently used protocols and the assays were reliable and standardized with the current standards of assessments to prove their reliability.[3132]

Calcium hydroxide has been introduced as an effective intracanal medicament because of its alkaline PH and antibacterial effect. However, because of high PH, calcium hydroxide is potentially toxic and tends to dissolve soft tissues.[3] In addition, evidence indicates that a several microorganisms such as E. faecalis[40] are resistant to calcium hydroxide. Due to these disadvantages, the new and natural materials with minimum side effects and high antibacterial activity have attracted the attention of endodontists in endodontic therapy. One of these materials is oregano essential oil.

Essential oil is mainly composed of oxygenated monoterpenes and monoterpene hydrocarbon. The major constituent of the essential oil was carvacrol (41%). Oxygenated monoterpenes carvacrol causes inhibition of ATPase activity and increasing the nonselective permeability of bacterial cell membrane. It inhibits the microbial colonization and also makes the microbes more sensitive to antibacterial agents.[41] Carvacrol is known to possess strong antioxidant properties and also exhibit antibacterial activity against several bacteria.[4243444546]

This study intended to examine the effect of oregano essential oil on the fibroblasts of dental pulp as the first step in its possible use as an alternative intracanal medication. Previous studies were done by the same agent and assessed the efficacy on endodontic pathogens and proved its efficacy on E. faecalis.[47] In addition, we investigated various compounds through Gas chromatography-mass spectrometry analysis and analyzed the active compounds responsible for the activity.[47] We also did a preliminary animal study, where we assessed the oregano extract in comparison with other intracanal medicaments. Our colleagues have been constantly exploring the compound and its activity especially for usage of an endodontic irrigant and intracanal medicament.[4748] In the present study, we planned to explore a step ahead to assess the cytotoxicity using some fibroblasts and using a phase contrast microscopy.

The previous study which was performed on rat fibroblast has reported calcium hydroxide to be cytotoxic, but it is still known to be biocompatible.[49] Guigand et al. demonstrated that calcium hydroxide constituents were as toxic as the calcium hydroxide itself.[50] Hirschman et al. conducted a study on skin fibroblasts to evaluate the percentage of viable cells for the assessment of the cytotoxicity of calcium hydroxide and reported it to be 37%.[51] The results of the present study were in corroboration with the previous studies with the reduction in cell viability was observed with calcium hydroxide.

In the present study, MTT assay revealed a higher percentage of viability cells with oregano essential oil when compared to calcium hydroxide. LDH assay also revealed that lesser percentage of cytotoxicity. Because of antimicrobial properties and the ability to enhance immune response[3] and lower cytotoxicity, oregano essential oil can be considered an intracanal medicament.

Future implication

However, further studies should be performed to investigate other properties of this substance.

CONCLUSION

Within the limitation of the study, Oregano essential oil at 50 μg/ml was reported to show reduced cytotoxicity compared to calcium hydroxide at 1 mg/ml. Therefore, perhaps after evaluating other properties, it might be considered an intracanal medicament.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.