|Year : 2019 | Volume
| Issue : 3 | Page : 251-257
In vitro evaluation of cytotoxicity of Emblica officinalis (amla) on cultured human primary dental pulp fibroblasts
Aditi Madhukar Bulbule1, Praveenkumar S Mandroli1, Kishore G Bhat2, Chetna M Bogar2
1 Department of Pedodontics and Preventive Dentistry, Maratha Mandal's NGH Institute of Dental Sciences and Research Center, Belagavi, Karnataka, India
2 Department of Microbiology and Molecular Biology, Maratha Mandal's NGH Institute of Dental Sciences and Research Center, Belagavi, Karnataka, India
|Date of Web Publication||30-Sep-2019|
Dr. Praveenkumar S Mandroli
Department of Pedodontics and Preventive Dentistry,
Maratha Mandal's NGH Institute of Dental Sciences and Research Center, #47 A/2 Near KSRP Ground, Bauxite Road, Belagavi, Karnataka
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Context: The dental pulp tissue is capable of healing after surgical amputation of infected/inflamed tissue during vital pulp therapy, when in contact with a suitable medicament. Emblica officinalis (amla), a traditional medicine, is one such medicament which has never been evaluated for its healing potential in pulp therapy. Aims: The aim of the study was to evaluate the cytotoxicity of E. officinalis (amla) against human primary dental pulp fibroblasts. Settings and Design: This was in vitro study. Subjects and Methods: Human dental pulp fibroblasts were obtained from dental pulp tissue of extracted over-retained primary incisors. The primary cells were cultured using the Dulbecco's Modified Eagle's Medium and used for the study after the fourth passage. The test medicament was E. officinalis with mineral trioxide aggregate (MTA) (100%) and untreated cells as positive and negative controls, respectively. Methyl-thiazol-diphenyl-tetrazolium (MTT) cytotoxicity assay was performed, and the cell survival was observed and analyzed at intervals of 24, 48, and 72 h. Statistical Analysis Used: Cell survival within groups was compared with Wilcoxon matched-paired t-test and in between groups at each point interval was analyzed with the Kruskal–Wallis ANOVA test. The level of significance was set at 0.05. Results: Within the groups, across the time periods of evaluation, there was a decline in cell survival in both the groups but was statistically significant in the MTA group. On interval-wise comparison, the decline in cell survival was statistically significant between the three groups at 72 h (P = 0.001). Conclusions: E. officinalis preserved the vitality of the human primary dental pulp fibroblasts and has the potential to be developed into vital pulp therapy medicament.
Keywords: Emblica officinalis, human primary dental pulp, mineral trioxide aggregate, pulpotomy
|How to cite this article:|
Bulbule AM, Mandroli PS, Bhat KG, Bogar CM. In vitro evaluation of cytotoxicity of Emblica officinalis (amla) on cultured human primary dental pulp fibroblasts. J Indian Soc Pedod Prev Dent 2019;37:251-7
|How to cite this URL:|
Bulbule AM, Mandroli PS, Bhat KG, Bogar CM. In vitro evaluation of cytotoxicity of Emblica officinalis (amla) on cultured human primary dental pulp fibroblasts. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2020 Jan 21];37:251-7. Available from: http://www.jisppd.com/text.asp?2019/37/3/251/268189
| Introduction|| |
Pediatric pulp therapy aims to maintain the integrity and health of the teeth and their supporting tissues. The selection of the treatment depends on the clinical and radiographic diagnosis. Vital pulp therapy (VPT) for primary teeth is the treatment of choice when the tooth is diagnosed with a normal pulp or reversible pulpitis.
VPT is defined as a treatment which aims to preserve and maintain pulp tissue that has been compromised but not destroyed by caries, trauma, or restorative procedures in a healthy state. The procedures included under VPT can be addressed as protective liners, indirect pulp therapy, direct pulp therapy, pulpotomy, and apexogenesis. The dental pulp is a unique, specialized connective tissue mainly containing interstitial fibroblasts in the cell-rich zone in the center of the pulp with the odontoblasts aligning themselves in the periphery. The progenitor cells are embedded among the interstitial fibroblasts mainly adjacent to the blood vessels.
The success or failure of any procedure depends on the reaction of these dental pulp cells to the material which is in direct contact with the tissue. There are several pulp dressing medicaments that have been proposed to maintain radicular pulp vitality. Mineral trioxide aggregate (MTA) has been currently considered as a gold standard for VPT procedures. The biologically favorable properties of MTA include osteoconduction, osteoinduction, and modulation of cytokine production along with antibacterial property due to its alkaline pH. However, the drawback of MTA is the high cost of this material.
Traditional medicine is known to be a fertile ground for modern medicine. Medicinal plants contain many chemical substances which can be a principle source of therapeutic medication for human ailments. Indian gooseberry or amla, also known as Phyllanthus emblica Linn. (Syn. Emblica officinalis Gaertn.), which belongs to the family Euphorbiaceae, is one of the important medicinal herbs in Ayurveda. It has been scientifically used widely in the Ayurveda system of medicine. According to various ongoing researches, E. officinalis is known to exert anti-inflammatory and wound healing action through upregulation of collagen production on dermal wound healing. These properties play an important role in the healing of injured dental pulp. However, the use in the field of dentistry is still under research.
The ISO10993:2009 standardization for the biological evaluation of medical devices used in dentistry states cytotoxicity testing in vitro as a primary test.
Although there are many studies published regarding the effect of E. officinalis in Ayurveda, there are no reported studies to evaluate the effect of this medicine on human primary dental pulp. Hence, the purpose of this study was to evaluate the cytotoxicity of E. officinalis on human primary dental pulp fibroblasts. The null hypothesis to be tested was that E. officinalis and MTA are cytotoxic to the human primary dental pulp fibroblasts.
| Subjects and Methods|| |
The present in vitro study was conducted at the Department of Pedodontics and Preventive Dentistry, in association with the Department of Microbiology and Molecular Biology of the institute after obtaining the clearance from the Ethical Committee of the institute. The test and control medicaments are shown in [Figure 1] and [Table 1].
|Figure 1: (a) Positive control medicament – ProRoot mineral trioxide aggregate (white) Dentsply, Tulsa Dent, USA, (b) Test medicament – Pure powdered extract of Emblica officinalis|
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Method of collection of data
Source of explants
Human primary incisor was obtained from healthy patients presented to the Department of Pedodontics and Preventive Dentistry of the institute for orthodontic extractions, after obtaining informed written consent of the parents and assent of the child. The inclusion and exclusion criteria were as follows:
- Inclusion criteria: Primary incisor teeth with more than half root resorbed [Figure 2]
- Exclusion criteria: Primary incisor teeth presenting with caries, restorations, and/or periodontal disease.
After extraction of teeth, pulp tissue was carefully extirpated from the canal with a sterile broach and a sharp spoon excavator and was transported to a laboratory in the Dulbecco's Modified Eagle's Medium (DMEM) (HiMedia Laboratories, Mumbai, Maharashtra, India), for fibroblast culture.
Fibroblast isolation and harvesting from dental pulp
Pulp tissue was placed in a sterile Petri dish More Details containing solution of 3 mg/ml collagenase and 4 mg/ml dispase for 1 h, till it underwent enzymatic dissociation. Small pieces of pulp tissues were removed using a micropipette, resuspended in 5–10 ml phosphate-buffered saline, and centrifuged at 1800 rpm for 5 min to obtain a pellet containing cells. The supernatant was discarded and the pellets were resuspended in 5 ml of DMEM. Single-cell suspensions of dental pulp were cultured in 24-well microtiter plate with DMEM and then incubated at 37°C in 5% CO2. The culture medium was changed every 3 days until the cell confluence was achieved. Pulp tissue was minced into 1–2 mm fragments, and each piece was placed in 24-well microtiter plate with DMEM (2–3 ml per well) and then incubated at 37°C in 5% CO2. The medium was changed after outgrowth was observed. The outgrown cells at confluence were subcultured at ratio of 1:4. Cells were used for cytotoxicity tests after the fourth passage [Figure 3].
One thousand milligrams of E. officinalis and MTA, respectively, was mixed with 1000 μL dimethyl sulfoxide (HiMedia Laboratories, Mumbai, Maharashtra, India) to prepare 100% concentrations of each material [Figure 1]. One hundred microliters of this concentration of the test agent and control, respectively, was added in triplicate into 96-well microtiter plates containing 2 × 105 cells/well of the fibroblast culture using Eppendorf tubes. Cells without treatment served as a negative control group [Figure 4].
|Figure 4: 96-well microtiter plates containing 2 × 105 cells/well of the fibroblast culture using Eppendorf tubes|
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The number of viable cells after 24-, 48-, and 72-h incubation at 37°C in a humidified atmosphere of 5% CO2 and 95% air was determined by the methyl-thiazol-diphenyl-tetrazolium (MTT) assay.
Methyl-thiazol-diphenyl-tetrazolium assay after 24 h
For this, 20 μL of 5 mg/ml MTT was added to nine wells (three wells of 100 μL of 100% MTA, three wells of 100 μL of 100% E. officinalis, and three wells of cells without treatment) in 96-well microtiter plate and incubated at 37° C, 5% CO2 with 98% humidity for 4 h [Figure 5].
|Figure 5: Addition of the medicaments into the 96-well microtiter plate containing fibroblast culture using Eppendorf tube|
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At the end of the incubation period, the medium with MTT was removed, and 100 μl dimethyl sulfoxide was added to each well. The plate was shaken on the microplate shaker to dissolve the purple MTT formazan. The relative viability of dental pulp fibroblasts was expressed as a color intensity of the number in the experimental wells relative to that of control [Figure 6]. The same process was carried out three times in a triplicate to affirm the readings. Absorbance was recorded at 492 nm on a microplate reader with background subtraction at 620 nm (Lisaplus, Aspen Diagnostics Pvt. Ltd., Mumbai, Maharashtra, India). Cell viability was calculated using the formula:
|Figure 6: 96-well microtiter plate after incubation for 48 and 72 h postaddition of the test medicament (Emblica officinalis) and positive control (mineral trioxide aggregate ProRoot) and the methyl-thiazol-diphenyl-tetrazolium reagent|
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Cell survival = (optical density [OD] values of experimental wells/OD values of control wells) × 100.
The same procedure was repeated for 48- and 72-h evaluation.
The numerical mean and the standard deviation values of optical density, at the end of each time interval, were presented in a tabular form. Within each group, pairwise comparison (i. e., 24 h-48 h, 24 h-72h, 48-72 h) of percentage of surviving cells was done by Wilcoxon matched pair test. In between the groups, the cell survival was evaluated at 24-h, 48-h and 72-h time point interval by the Kruskal–Wallis ANOVA test. P < 0.05 was considered as statistically significant for all the comparisons. The data were analyzed using SPSS for Windows, version 10.0 (SPSS Inc., Chicago, IL, USA).
| Results|| |
The cell survival at 24-, 48-, and 72-h time point interval is represented as the percentage value of the control in [Table 2].
|Table 2: Percentage survival of cultured cells in individual groups at the end of 24, 48, and 72 h by Wilcoxon matched-paired test|
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Comparison within the group at different time intervals
Within the group treated with E.officinalis, comparison at the tested time intervals revealed a statistically significant (P = 0.03) decline in the cell survival at 24–72 h. However, the decline in the cell survival at 24–48 h (P = 0.18) and 48–72 h (P = 0.12) was not statistically significant [Table 2] and [Graph 1].
In the group treated with MTA, the decline in cell survival was not statistically significant (P = 0.18) at 24–48 h, while a statistically significant (P = 0.03) decline in the cell survival was observed at 24–72 h and 48–72 h indicative of cytotoxicity of the medicament.
In the group with no medicament, the cell survival rate was considered to be 100% and served as a negative control for baseline comparison.
Comparison between the groups at different time intervals
On comparison between the groups, the cell survival rate at the end of 24 h was observed to be slightly greater in the groups treated with both the medicaments as compared to the cells without any treatment, indicative of mild cell proliferation on treatment with both E. officinalis and MTA at the end of 24 h [Table 3] and [Graph 2].
|Table 3: Comparison of three groups with respect to percentage of survival cells at 24, 48, and 72-h time points by Kruskal-Wallis ANOVA|
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The intergroup comparison at the end of 48 h showed a slight decline in the cell survival rate in the groups treated with E. officinalis and MTA [Table 3] and [Graph 2].
The intergroup comparison at the end of 72 h showed a statistically significant (P = 0.001) decline in the cell survival rate in the group treated with MTA as compared to the group treated with E. officinalis. This indicated a lower cytotoxicity of E. officinalis as compared to MTA.
| Discussion|| |
In the past decades, the concept for VPT has changed from preserving the vitality of the remaining dental pulp to attempting the regeneration of the tissue. MTA, currently the best medicament available for primary teeth VPT, is considered the gold standard for these procedures as it has shown a high level of clinical and radiographic successes.
E. officinalis has been traditionally suggested for enhancing the production of red blood cells and strengthen the teeth, hair, nails, regulating the blood sugar levels, in bleeding, hemorrhoids, anemia, diabetes, hyperacidity, gout, psoriasis, infections, and a number of different ailments. The applications of E. officinalis are based on its pharmacological activities which include  antioxidant activity, anticancer activity, immunomodulatory effect, antimicrobial activity ( Escherichia More Details coli, Klebsiella ozaenae, Klebsiella pneumonia, Proteus mirabilis, Pseudomonas aeruginosa, Salmonella More Details paratyphi A, S. paratyphi B, and Serratia marcescens), treatment of ophthalmic disorders (conjunctivitis), and promoting absorption of calcium (development of healthier bones, teeth, nails, and hair). However, its application in the field of dentistry is under research.
The ISO10993:2009 standardization  for biological evaluation of medical devices used in dentistry states the different test procedures specific to dental materials, which includes primary cytotoxicity testing in vitro, followed by animal testing of the dental material.
As per the ISO requirements, the selection of the cell culture in the assay should be based on the properties and method of application of the material. In most VPT procedures, since the medicament is in direct contact of the dental pulp tissue, the evaluation of the effect of these on primary dental pulp fibroblasts is of utmost importance.
The present study was thus conducted with the null hypothesis assumed that E. officinalis and MTA are cytotoxic to the human primary dental pulp fibroblasts.
In a study conducted to evaluate the wound healing action of E. officinalis by upregulation of collagen and extracellular-regulated kinases (EKR ½), the observations of the study provided a firm evidence to support the topical application of E. officinalis as a feasible and productive approach to support dermal wound healing.
According to a study conducted to evaluate the anti-inflammatory potential of phenolic compounds from E. officinalis in rats, it was observed that E. officinalis was capable of attenuating the acute and chronic inflammatory responses through antioxidant activity mainly due to the phenolic compounds present in the extracts of the fruit pulp.
Similarly, in a study evaluating the possible anti-inflammatory effect of E. officinalis in a rodent model, it was observed that E. officinalis possessed strong anti-inflammatory activity in both acute and chronic carrageenan-induced rat paw edema models on topical application of the hydroalcoholic extracts of E. officinalis.
In an in vitro study conducted to evaluate the cytotoxicity of E. officinalis on human cancer cell lines, namely of lungs (A-549), liver (Hep-2), colon (S02713 HT-29) and neuroblastoma (IMR-32); the authors concluded that the plant extracts showed selective in vitro cytotoxicity, active against some human cancer cell lines, and others not showed activity.
Another study evaluating the antimicrobial and cytotoxic activity of alkaloid activity of E. officinalis using brine shrimp lethality bioassay concluded that the extracts exhibited cytotoxicity and was biologically active. At present, there are limited or no documented studies evaluating the cytotoxicity of E. officinalis on healthy cell lines in vitro or in vivo.
MTA is derived from ordinary Portland cement with a minor difference in the composition. It is a bioactive material due to its composition and been shown to induce hard tissue formation on topical application. Literature states that placement of MTA on human dental pulp tissue causes proliferation, migration, and differentiation of odontoblast-like cells that produce a collagen matrix, which is then mineralized to produce osteodentin initially, followed by a tertiary dentinal bridge over a few months of application.
In different types of cell cultures, MTA has shown to be least cytotoxic and mutagenic.
In a study conducted to evaluate the cytotoxicity of accelerated white MTA and Malaysian Portland cement on stem cells from human exfoliated deciduous teeth in vitro using MTT assay, it was observed that at the manufacturer recommended concentration, the cytotoxicity of ProRoot white MTA was significantly higher than the Malaysian Portland cement.
A study conducted to evaluate the cytotoxic effect of three materials, namely MTA, calcium-enriched mixture, and new cement on human pulp stem cells using MTT assay, observed a similar cytotoxicity with all the medicaments. These findings were in accordance to the results obtained regarding the possible cytotoxic effect of MTA in the present study.
Any biomaterial to be clinically successful needs to have a balance between the desirable pharmacological actions and safety on the cells of eventual clinical significance. MTA will remain the gold standard for VPT because of its strong biological properties. However, the cost of MTA remains a drawback for its practical use in all clinical situations.
The effect of both the medicaments was tested in an in vitro controlled environment where there was no inflammation induced in the cells treated. There are a number of confounding factors in the oral environment; hence, the results obtained in the present study cannot be completely generalized to the clinical conditions. The present study evaluated the cytotoxicity of the medicaments using MTT assay which is a primary screening test. Furthermore, some chemicals or phytochemicals may change the activity of succinate dehydrogenase or interact with MTT directly with a resultant false-positive or false-negative interpretation.
Based on the available evidence regarding the biological actions and the safety against human dental pulp fibroblasts, it seems reasonable to conclude that the effect of E. officinalis appears to be favorable when used for the topical application on human primary dental pulp and has the potential to be developed as a cost-effective pulp therapy medicament.
| Conclusions|| |
Based on the present study results, we can conclude that E. officinalis preserved the vitality of the human primary dental pulp fibroblasts better than MTA and has the potential to be developed into VPT medicament. Most of the properties of E. officinalis are established for their medicinal use in Ayurveda; however, these properties have not been explored for its potential dental application. Hence, further animal studies and human clinical trials are required to determine the reliability of this medicament in specific clinical situations for dental application.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]
[Table 1], [Table 2], [Table 3]