Table of Contents  
ORIGINAL ARTICLE
Year : 2022  |  Volume : 14  |  Issue : 2  |  Page : 81-87

Comparative Evaluation of Cytotoxicity of Two Novel Herbal Intracanal Medicaments on Human Periodontal Fibroblasts—An In Vitro Study


1 Department of Conservative Dentistry and Endodontics, Maratha Mandal’s Nathaji Rao G. Halgekar Institute of Dental Sciences and Research Centre, Belagavi, India
2 Department of Conservative Dentistry and Endodontics, V.S. Dental College, Banglore, India
3 Department of Microbiology and Immunology, Maratha Mandal’s Nathaji Rao G. Halgekar Institute of Dental Sciences and Research Centre, Belgavi, India
4 Department of Oral Pathology and Microbiology, Maratha Mandal’s Nathji Rao G. Halgekar Institute of Dental Sciences and Research Centre, Belgavi, India
5 Central Research Laboratory, Maratha Mandal’s Nathaji Rao G. Halgekar Institute of Dental Sciences and Research Centre, Belgavi, India

Date of Submission22-Jul-2022
Date of Decision22-Oct-2022
Date of Acceptance25-Oct-2022
Date of Web Publication10-Jan-2023

Correspondence Address:
Dr. Sheetal Basavaraj Ghivari
Professor, Department of Conservative Dentistry and Endodontics, Maratha Mandal’s Nathaji Rao Halgekar Institute of Dental Sciences and Research Centre, Belagavi-590010
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jofs.jofs_187_22

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  Abstract 


Introduction: Intracanal medicaments used during root canal treatment comes in direct contact with cells of pulp and periapical tissues. This study aimed to compare the cytotoxic effect of two novel herbal intracanal medicaments Ganoderma lucidum (GL), Psidium gujava (PG) on human periodontal fibroblasts (HPF) by evaluating cell viability using Methyl Thiazolyl tetrazolium assay (MTT assay). Materials and methods: Temperature modulated in situ gel matrix of G. lucidum (GL, P. guajava L (PG) along with positive control calcium hydroxide (CH) and modified triple antibiotic paste (MTAP) was prepared by mixing carbapol and pluronic F-127. All four medicaments were serially tested at various concentrations from 400 to 12.5 µg/mL for six times on Human periodontal Fibroblasts (HPF) for their viability using MTT assay and evaluation of half maximal inhibitory concentration (IC50) after 48 hours. Results: The novel thermo reversible gel matrix of GL has shown lowest cytotoxicity followed by PG, CH, and MTAP. The cytotoxicity was minimal at lower concentration (12.5 µg/mL) and at increased concentration cytotoxicity was higher (400 µg/mL). The IC50 concentration of GL was highest (1681 µg/mL) followed by PG (1555 µg/mL), CH (1295 µg/mL), and MTAP (665 µg/mL). Conclusion: Novel thermo reversible gel matrix of GL and PG were found to be less toxic than CH and MTAP and also they have shown higher cell viability.

Keywords: Calcium hydroxide, cytotoxicity, herbal intracanal medicament, Ganoderma lucidum, modified triple antibiotic paste, Psidium gujava


How to cite this article:
Ghivari SB, Meena N, Bhat KG, Babji DV, Kumbar VM, Rao PM. Comparative Evaluation of Cytotoxicity of Two Novel Herbal Intracanal Medicaments on Human Periodontal Fibroblasts—An In Vitro Study. J Orofac Sci 2022;14:81-7

How to cite this URL:
Ghivari SB, Meena N, Bhat KG, Babji DV, Kumbar VM, Rao PM. Comparative Evaluation of Cytotoxicity of Two Novel Herbal Intracanal Medicaments on Human Periodontal Fibroblasts—An In Vitro Study. J Orofac Sci [serial online] 2022 [cited 2023 Jan 28];14:81-7. Available from: https://www.jofs.in/text.asp?2022/14/2/81/367442




  Introduction Top


Anatomical complexities of root canal system harbor some residual microorganisms which may survive biomechanical preparation and cause persistent periapical infections. Intracanal medicaments play an important role in eliminating residual microorganisms by endotoxin neutralization, prevention of microorganism’s entry from saliva, reduction of pain, inflammation and by creating a favorable environment for repair of the hard tissues.[1]

Intracanal medicaments used in endodontic treatment comes in direct contact with viable cells of pulpal and periapical tissues which promote healing as well as repair of the periapical tissues.[2] In today’s world of evidence based dentistry, any new medicament with possible clinical utilization must undergo a series of tests to demonstrate its tissue compatibility, tissue viability, and its efficacy in root canal disinfection.[3]

Biocompatibility of any new intra canal medicaments can be evaluated by various criterion such as genotoxicity, mutagenicity, carcinogenicity, and histocompatibility or cytotoxicity.[4],[5] Methyl Thiazolyl tetrazolium assay (MTT) helps in determining cytotoxicity of new medicament after exposing viable cells to test medicaments. It is a colorimetric assay based on ability of mitochondrial succinate dehydrogenases present in viable cells which gets reduced to a yellow tetrazolium dye MTT (3-{4, 5-dimethylthiazol–yl}-2, 5-diphenyltetrazolium bromide) to violet formazan product which can be detected spectrophotometrically at 540 nm. More the purple color higher the number of viable cells.[6]

However calcium hydroxide (CH) is the most commonly used intracanal medicament which exert its antimicrobial activity due to its alkaline pH and can induce tissue mineralization.[1] In an in vivo situations due to high pH it can cause chronic inflammation and cell death. Various studies have proven that CH is abortive in eliminating bacteria from dentinal tubules.[7],[8]

Triple antibiotic paste (TAP) is a combination of Ciprofloxicin, Metronidazole, and Minocycline, effective against most of the endodontic pathogens. Due to presence of Minocycline in TAP may cause tooth discoloration which can be overcome by addition of Cefaclor, which is known as modified triple antibiotic paste (MTAP). The acidic contents present in these antibiotics can induce demineralization in root dentin causing changes in the mineral content which may reduce dentin microhardness.[9] Ruparel et al.,[10] exposed triple and double antibiotic paste to stem cells of apical papilla (SCAP) to assess their effect and found that both are detrimental to SCAP cells.

The toxic side effects of these synthetic drugs and increasing number of antibiotic resistant strains have led the scientist’s to search for herbal alternatives with antimicrobial properties such as phytochemical extract of Ganoderma lucidum (GL) which is popularly known as red mushrooms, and Psidium guajava L (PG) which is a extract of Guava leaf.

GL have been in use for thousands of years due to its varied medicinal properties in traditional Chinese Medicine. It is known to have many biologically active components like triterpenes, polysaccharides, ganoderic acids, etc. which provide antimicrobial, antiviral, immune modulatory, antioxidant, antitumor, and anticancer properties. Recently many studies have proven that GL and other Ganoderma species contain antibacterial components that are responsible to inhibit gram positive and gram positive bacteria.[11]

PG is also known as guava. Its bark, fruit, and leaves are used in folk medicine for the treatment of several diseases such as wounds, ulcers, bowels, and cholera. The leaves of PG contain an essential oil rich in cineol, tannins, triterpenes, and flavonoids.[12],[13] Psidium.Cattleianum (P. cattleianum) ethanolic extract has shown highest antimicrobial activity against Enterococcus Fecalis (E. fecalis) biofilm remarkably reducing the microbial load in 24 hours.[14]

Therefore present study was aimed to evaluate cytotoxic effects of the newer herbal medicaments on the HPF cells using MTT assay.


  Materials and Methods Top


Ethical approval for this study (protocol no. 54, 13/04/2018) provided by Ethical Committee NAC of Rajiv Gandhi University of Health Sciences, Bengaluru on April 13, 2018. HPF cells were obtained from institutional repository of central research laboratory isolated from freshly extracted third molars and teeth extracted for orthodontic correction. GL extract used in present study was obtained from hot aqueous extraction method and PG used was an ethanolic extract both were prepared in the institutional laboratory.

Preparation of test materials

Preparation of Ganoderma extract

Hot aqueous extraction of GL powder was carried out in Soxhlet extraction unit. The GL powder was placed in paper cylinder made from a filter paper and it was placed in the body of the Soxhlet extractor. The water in the flask was heated until boiling which gets converted into vapors. The vapor enters into the condenser falls on column of the drug this process of alteration of filling and emptying leads to the soluble active constituents of the drug remain in the flask where solvent was repeatedly volatilized. The dried extracts were stored in dark bottles at −20 °C until use.[15]

Guava extract preparation

The Guava leaves were washed and dried at 37 °C for 1 week then dried and ground to a fine powder (Maceration). Ethanolic extract was prepared by mixing 100 g of leaf powder with 250 mL of 80% ethanol and shaking vigorously five times a day for 12 days. The ethanolic solution was then filter sterilized with 0.22 µm mixed cellulose ester membranes (EMD Millipore, Billerica, MA, USA). The extract was concentrated under reduced pressure by a rotary evaporator linked to a vacuum pump. The dried extract was stored in dark bottles at −20 °C until use.[16]

Preparation of in situ gels

Briefly, the required amount of Pluronic F-127 (19% w/v) was added slowly to the ice cold Milli-Q water with continuous stirring. The resulting solution was kept in the refrigerator overnight at 4°C to ensure the complete wetting and dissolution of the polymer. The extract powder of GL and PG (2% w/v) was gently mixed into the prepared polymer solution at 4°C followed by the adding of preservatives methyl paraben (0.18% w/v) and propyl paraben (0.02% w/v) dissolved in appropriate amount of polyethylene glycol to the prepared polymer solution with stirring at 4°C to prepare the in situ gel [Figure 1].[17]
Figure 1 Insitu gel preparations

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Preparation of calcium hydroxide and modified triple antibiotic gel

Saturated solution containing CH powder (Merck India Ltd., Mumbai, India) was prepared by mixing (2% w/v) CH with 1 mL of sterile distilled water. The mixture was centrifuged at 3000 rpm for 15 minutes to clarify the solutions, and the aqueous top layers were filter sterilized using 25 mm syringe filter. The in situ gel preparation was done as mentioned above.

Entricoating of the tablets Ciprofloxacin (500 mg)-5% w/w (Cipla, India), Metronidazole (400 mg)-1% w/w ((Flagyl 400 mg) Medi India), Cefaclor (200 mg)-1% w/w (Biocef 250 mg DT, Aristo Pharmaceuticals Ltd.) were removed using BP blade and each tablet was crushed in the mortar using pestle into fine powder. The three crushed tablets were taken in (1:1:1) proportion, 2% w/v of compounded antibiotic powder was mixed in 1 mL of distilled water. The suspension was centrifuged at 3000 rpm for 15 minutes to clarify the solutions, aqueous supernatant was filter sterilized using syringe filter. The in situ gel preparation was done as mentioned above.

Cell culture

Material used

The HPF cells were grown in DMEM medium which was supplemented with 10% heat inactivated foetal calf serum (FBS) and 1% antibiotic − Antimycotic 100× solution. The cells were seeded at a density of approximately 5 × 103 cells/well in a 96-well flat-bottom micro plate and maintained at 37 °C in 95% humidity and 5% CO2 for overnight. Test compounds naming GL, PG extract along with positive control CH, and MTAP compounds [Figure 1] at different concentrations of 100, 200, 300, 400 μg/mL, were placed in the 96 well microtitre plate. The fibroblast cells were treated with medicament gels at the above mentioned concentration and incubated at 37°C for another 48 hours. The cells in the wells were washed twice with phosphate buffer solution, the MTT solution (Sigma Aldrich, St Louis, MO, USA) was added to each well of the plate and incubated for 1 to 4 hours in an incubator. After dissolution of formazan crystals, the optical density (OD) of the solution was read at 450 nm using micro plate reader (BioTek instruments Inc., Winooski, VT, USA). Only HPF cells were tested without use of any medicament as negative control. For reproducibility of results the test was repeated at least six times for each medicament and after 48 hours the viability and proliferation of cells were evaluated. [Figure 2].
Figure 2 (a) Viable cells after treatment with Ganoderma lucidum. (b) Viable cells after treatment with Psidium gujava. (c) Viable cells after treatment with calcium hydroxide. (d) Viable cells after treatment with modified triple antibiotic paste

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The cell viability for four compounds calculated by the following formula:



Using graph Pad Prism Version 5.1, IC50 value of compounds was calculated.

Based on the number of viable cells the test compounds were categorized as severe (<30%), moderate (30–60%), mild (60–90%), non-toxic (>90%).[16]

Determination of IC50 value

Is the concentration of the test material required to inhibit the growth of 50% of cells in dose dependent manner. IC50 value (half maximal inhibitory concentration) of all the test materials was determined.

Statistical analysis

All the tests were carried out six times to minimize errors. The data were analyzed using IBM SPSS statistics for windows (22.0 IBM Corp., Armonk, NY, USA) using descriptive statistics. For inter group comparison among the groups Two way Analysis of Variance (ANOVA) followed by Bonferroni correction was used.


  Results Top


Absorbance of viable cells was calculated in terms of optical density and expressed in numerical values. [Table 1],[Table 2],[Table 3] represent mean cell viability post MTT assay, which was measured through mitochondrial activity where MTT was reduced to metabolically active cell. The test medicaments with higher cell viability showed lower cytotoxicity giving higher optical density and vice versa. According to [Table 2], the results of present study demonstrated a statistically significant difference of P < 0.05 between GL&CH at 400 and 100 μg/mL concentration whereas at 50 μg/mL there was moderately significant difference (P < 0.01) was found. Highly significant results were found between PG and CH at 100, 50, and 25 μg/mL concentrations (P < 0.001), whereas, at 12.5 μg/mL (P < 0.05) it showed significant difference [Table 3] When GL and PG was compared at 400 μg/mL concentration highly significant difference was observed, at other concentrations both the medicaments were equally effective [Table 3].
Table 1 Shows Two way ANOVA

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Table 2 Bonferroni post test shows cytotoxic effect of intergroup comparision of GL, PG, CH and MTAP on human periodontal fibroblasts

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Table 3 Bonferroni post test shows cytotoxic effect of intergroup comparison of GL with CH and MTAP on human periodontal fibroblasts

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IC50 values of all the test medicaments against HPL cells for MTAP (665 μg/mL) showed lowest viable cells followed by CH (1295 μg/mL) and PG (1555 μg/mL). IC50 value of GL was 1681 μg/mL which was high amongst all the medicaments.


  Discussion Top


An ideal requirement of an intracanal medicament is its antibacterial activity which should overreach its cytotoxicity, such agents may eliminate bacteria but can cause damage to periapical tissues.[18] A study done by Triveno et al., medicaments and irrigants having antibacterial activity used in endodontic treatment greatly affect the survival of stem cells in the root canal ecosystem which makes it essential to evaluate their cytotoxicity profile.[19]

Cell type is an important factor in cytotoxicity research,[18] as fibroblasts metabolically resemble the cells of pulp and periapical region. They are quick and easy to grow, in addition they are found abundantly in pulp and periapical tissues which are vulnerable to the effect of intracanal medicaments and their by-products. Fibroblasts plays a key role in wound healing and repair as they produce large amount of collagen.[20],[21]

To provide real ideal condition for growth of cells, low glucose DMEM broth supplemented with 10% bovine serum was used. In order to avoid biased results cells were recultured until sixth passage to get juvenile new cells and get rid of weak senile cells followed by disinfection to prevent programmed cell death.[20],[21],[22]

The herbal medicaments GL and PG in its hot aqueous and ethanolic form were used against various microorganisms has shown maximal antimicrobial efficacy.[23],[12] The medicaments used in the present study were in thermo reversible hydro-gel form which are prepared by incorporation of micro-particulate drug molecules in biodegradable synthetic polymers such as Pluronic F-127. The thermo modulate hydro-gel fills the canal three dimensionally and leads to sustained drug release into difficult/incompletely disinfected root canals.[24] Various studies conducted have shown in situ gels exert minimal cytotoxic, inflammatory, and biocompatibility responses with surrounding tissues.[17],[25]

In the present study, MTT assay showed higher cytotoxicity at higher concentration (400 μg/mL) for all the tested medicaments, at lower concentration (12.5 μg/mL) the cytotoxicity was minimal with higher cell viability. Chuensombat et al., in his study stated that cytotoxic effect of intracanal medicaments depends upon its concentration and combination of drugs used.[26] Both the herbal intracanal medicaments have shown less cytotoxicity at all the concentrations with more cell viability 60% at (400 μg/mL), followed by 75% (200 μg/mL), 80% (100 μg/mL), 82% (50 μg/mL), 90% (25 μg/mL), and 95% (12.5 μg/mL). [Figure 3].
Figure 3 Chart showing HPF cell viability percentage at different IC50 values at different concentrations.

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Comparison of GL and CH showed statistically significant difference at higher concentration 400, 100, and 50 μg/mL (P < 0.05). Between PG and CH a highly statistical difference was found at 100, 50, 25 μg/mL (P < 0.001) and 12.5 μg/mL (P < 0.05). At 400 μg/mL no statistical significance was found. The finding of the present study was consistent with that of Matin et al., who stated that cytotoxicity of CH was concentration dependent, CH at 0.1 mg/mL was slightly toxic but at 1 and 10 mg/mL was severely cytotoxic.[27] Cytotoxicity of CH may be attributed to high PH and released hydroxyl ions which promotes enzymatic denaturaton and destruction of cell membrane leading to cell death.[28] Al-Shaher et al., in 2004 evaluated effect of CH on PDL fibroblasts at different concentrations, according to his study at higher (0.4 mg/mL) concentration less than 25% of cells survived.[29] [Table 2] and [Table 3].

In present study GL and MTAP were compared which showed highly significant difference (P < 0.001) at all the concentrations. PG and MTAP also have shown highly significant difference at all the concentrations except at 12.5 μg/mL. Matin et al., in his study stated that triple antibiotic paste (TAP) is less cytotoxic than CH and its effect is also concentration dependent, at 0.1 and 1 mg/mL concentration had mild cytotoxicity but at 10 mg/mL more cytotoxicity was seen.[27] Yadlapati et al., in his study stated that TAP and double antibiotic paste (DAP) compared for cell viability using XTT multi parametric assay TAP showed highest cytotoxicity with 30% cell viability whereas DAP showed more than 70% cell viability this could be attributed to minocycline present in TAP.[25] In present study MTAP displayed more than 40% cell survival due to replacement of Cefaclor for minocycline. Findings of the present study are analogous to Sipert et al., MTAP at 250 and 1000 μg/mL demonstrated decreased cell viability of 50% at first day, at 3rd day at decreased concentration 15.65 to 1000 μg/mL increased cytotoxicity was observed, and at day 5 and 7 cytotoxicity was seen de novo with 250 and 1000 μg/mL.[29] Chuensombat et al. in his study used original 3 Mix paste for 7 days and showed 50% viability reduction for dental pulp cells and 70% apical papilla cells.[26] [Table 2] and [Table 3].

The comparison between GL and PG showed statistically significant difference only at 400 μg/mL. Fernandes et al., conducted in vitro short term cytotoxicity analysis and evaluated wound healing potential of traumatic ulcers in rats using PG extract along with commonly used corticosteroids. He found cytotoxicity of PG extract showed a reduced cell viability in vitro while accelerated the wound healing in vivo.[30] Guava leaves contain medicinal compounds such as carbohydrates, flavonoidal glycosides, steroids, and tannins as their main constituents, flavanoids are responsible for the antimicrobial activity.[12] Flavanoids are synthesized by plants in response to bacterial infection, their activity is to complex with bacterial cell wall and disrupt. Tannins exert their antimicrobial activity by their ability to inactivate bacterial adhesion, cell envelops, and transport proteins. Gao et al. suggested that potent constituents present in Ganoderma lucidum such as polysaccharides and triterpinoids may inhibit viral replication of HSV, HBV, HIV and other types of viruses by interfering with their absorption, virus hepatocyte fusion and endocytosis, viral integration assembly and release. GL plays an important role in antimicrobial activity by activation of immune effector cells such T cells, macrophages, and natural killer cells and also it caused immune modulating effects by cytokines and radicles facilitating killing of viruses and bacteria. A study on mouse by Zohu et al. indicated that proteoglycan with carbohydrate protein ratio of 11.5:1 isolated from GL stimulated proliferation of spleen lymphocytes which in turn caused increased proliferation of B cells in vivo, producing immunoglobulins which neutralize viral and bacterial endotoxin.[31] [Table 2].

The IC50 value helps in determining the concentration of the drug needed to kill 50% of experimental cells at that moment. GL (1684 μg/mL) and PG (1555 μg/mL) have shown highest IC50 value among all the tested medicaments. There is no notable difference found in IC50 value of viable cells of both the herbal intracanal medicaments could be attributed to the formulation and the phytochemical constituents of the medicaments. The thermomodulated hydrogel formed of pluronic F127 has helped in sustained drug release which lead to decreased cytotoxicity.[17] [Graph 1].

Application of these novel herbal intracanal medicaments need to be evaluated in in vivo clinical situation as the cytotoxic effect of intracanal medicaments depends on the state of byproducts produced by bacteria which has effect on cell activation.[31] Future studies should focus upon detailed cytotoxic evaluation on different cell lines using newer techniques.


  Conclusion Top


Evaluation of cytotoxicity of novel intracanal medicaments plays vital role in establishing security profile before placing any medicament in direct contact with viable tissues. In present study novel polymeric gel matrix of herbal intracanal medicaments GL and PG has similar or better biocompatibility than CH and MTAP. However the properties of novel polymeric gel matrix of GL and PG need to be evaluated as an intracanal medicament in an in vivo situations such as in animal research and human clinical trials.

Author contributions

All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Dr Sheetal Ghivari, Dr N. Meena, and Dr Kishore Bhat. The first draft of the manuscript was written by Dr Deepa Babji, Dr Vijay Kumbar, and Dr Malleshwar Rao. All authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.

Financial support and sponsorship

Nil.

Conflicts of interest

The authors report no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3]



 

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