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Table of Contents   
ORIGINAL ARTICLE  
Year : 2020  |  Volume : 23  |  Issue : 1  |  Page : 42-45
Assessment of the viability of human periodontal ligament cells in black tea, lime juice, and passion fruit concentrate – A comparative in vitro study


1 Junior Resident, Department of Conservative Dentistry and Endodontics, Mahatma Gandhi Post Graduate Institute of Dental Sciences, Puducherry, India
2 Professor and Head of Department, Department of Conservative Dentistry and Endodontics, Mahatma Gandhi Post Graduate Institute of Dental Sciences, Puducherry, India
3 Professor, Department of Conservative Dentistry and Endodontics, Mahatma Gandhi Post Graduate Institute of Dental Sciences, Puducherry, India

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Date of Submission01-Jun-2020
Date of Decision07-Jun-2020
Date of Acceptance27-Jul-2020
Date of Web Publication10-Oct-2020
 

   Abstract 

Background: Tooth avulsion is considered as a severe form of dental trauma, causing damage to the periodontium. Hence, the preservation of healthy periodontal ligament (PDL) cells in the storage medium are pivotal for the success of replantation.
Aim and Objective: The aim of this study is to assess the viability of human PDL cells in black tea, lime juice, and passion fruit concentrate.
Methods: Human periodontal cells were cultured and stored in three experimental media – black tea, lime juice, and passion fruit concentrate and subjected to 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay for 1 h and the cell viability was determined. Mean and standard deviation were statistically analyzed using one-way analysis of variance to identify the significant groups.
Results: The human PDL cells showed 100% viability in lime juice and passion fruit concentrate, followed by 98% viability in black tea.
Conclusion: Black tea, lime juice, and passion fruit concentrate can be used effectively as storage media for maintaining PDL cells viability in avulsed teeth, with 100% viability exhibited by lime juice and passion fruit concentrate.

Keywords: Avulsion; black tea; lime juice; passion fruit; storage media; viability

How to cite this article:
Deepthi K, Borthakur BJ, Swathika B, Ganesan S. Assessment of the viability of human periodontal ligament cells in black tea, lime juice, and passion fruit concentrate – A comparative in vitro study. J Conserv Dent 2020;23:42-5

How to cite this URL:
Deepthi K, Borthakur BJ, Swathika B, Ganesan S. Assessment of the viability of human periodontal ligament cells in black tea, lime juice, and passion fruit concentrate – A comparative in vitro study. J Conserv Dent [serial online] 2020 [cited 2020 Oct 26];23:42-5. Available from: https://www.jcd.org.in/text.asp?2020/23/1/42/297670

   Introduction Top


An avulsion is defined as the complete displacement of a tooth from its socket in alveolar bone owing to trauma.[1] Periodontal ligament (PDL) tissues begin to dehydrate the following avulsion, and its vitality plays a pivotal role in the successful healing of replanted teeth,[2],[3] for which various storage media have been suggested.[4],[5],[6] Yet, there is a continuous search for an ideal and accessible storage media.

Hence, the present study aimed to assess the viability of human PDL cells in black tea, lime juice, and passion fruit concentrate as alternatives to already available and previously proposed storage media for the avulsed tooth.


   Methods Top


The viability of human PDL cells were assessed in– vitro, using L929 fibroblast cell line in three different media, namely, Black tea, Lime juice, and Passion fruit concentrate and were compared.

Cell line culture

The cell line (L929 fibroblasts) was propagated in Dulbecco modified minimum essential medium in a humidified atmosphere of 5% CO2 and 95% O2 at 37°C with antibiotic and anti-mycotic solutions, to prevent contamination and were incubated for 48 h and then visualized under inverted microcope.[7]

Sample preparation

The black tea solution was prepared by boiling the tea powder (Brookbond 3 roses, Hindustan Unilever Limited, India) in 50 ml of water at 100°C for 10 min.[8] The solution was allowed to cool and filtered. Lime juice was prepared by squishing five lemons, and the solution was filtered. Passion fruit concentrate was prepared by crushing two fruits in a mixer, and then the solution was filtered. All three test solutions were then transferred to small plastic containers of 5 ml capacity filled in five containers for each sample.

Cell viability analysis

The test samples were divided into the following three groups, which were tested against Control containing only cells in the well with no added test solutions.

  • Group 1 – Black tea solution
  • Group 2 – Lime juice
  • Group 3 – Passion fruit concentrate.


The test solutions were then transferred and inoculated into the 96-well culture plates containing the fibroblasts cells and placed in an incubator of 5% CO2 at 37°C for 24 h and subjected to 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The MTT substrate was then prepared in a physiologically balanced solution, added to cells in culture, at a final concentration of 0.2–0.5 mg/ml, and incubated for 1 h. The quantity of formazan (presumably directly proportional to the number of viable cells) was measured by recording changes in absorbance at 570 nm using a plate reading spectrophotometer.[9] The percentage of viable cells for each test sample was then calculated at the end of 1 h.

The mean and standard deviation were estimated from the triplicate values of results that were statistically analyzed (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY, USA.) using one-way analysis of variance to identify the significant groups at P ≤ 0.05.


   Results Top


The cell viability of human periodontal cells at different concentrations with three experimental transport media is shown in [Table 1]. The difference in the cell viability among the 10% concentration of lime, black tea, passion fruit and control is statistically significant (P < 0.05). However, no statistically significant difference has been found in the cell viability among the other concentrations of lime, black tea, passion fruit, and control.
Table 1: Cell viability of human periodontal cells at different time intervals with three experimental transport media

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The results show that the human PDL cells showed 100% viability in lime juice and passion fruit concentrate, followed by 98% viability in black tea.


   Discussion Top


The most severe type of traumatic tooth injury is avulsion because it causes damage to several structures and also results in the complete displacement of the tooth from its socket in the alveolar bone.[10] Hence, the ideal solution is to replant an ex-articulated tooth immediately after an avulsion, because the extra-oral time is one of the determinant factors for treatment success and for a good prognosis. This statement is in agreement with Lekic et al., who confirmed in their study that thein vitro clonogenic capacity of PDL cells is temporarily associated with the ability of PDL progenitor cells to attach and recolonize the root surface after replantation. The critical period for rapid loss of cell viability begins 15 min after tooth storage notably.[11] Moreover, an extra-oral dry time of 60 min makes the survival of the periodontal cells unlikely, and literature supports moist storage to be a more productive approach to optimize their survival.[12] Furthermore, the choice of storage medium for preserving traumatically avulsed teeth is important for the success of future replantation.[13]

In the case of permanent tooth avulsion, there is a considerable risk for pulp necrosis, root resorption, and ankylosis. Recent evidence suggest that successful replantation is dependent upon many factors, some of which the clinician can manipulate in a manner that favors more successful outcomes. Hence, the two most crucial factors affecting the prognosis of an avulsed tooth are extra-oral dry time and the storage medium, in which tooth is placed or transported before re-implantation. The dry period should be as less as possible, and it would be better if immediate replantation can be achieved. Practically, it is not always possible to replant the tooth immediately, so the need for storage media arises. The delay in replantation can vary from few minutes to hours, as in cases of accidents, emergencies, and unavailability of dentists, so a storage media that can maintain the PDL cell viability for a long duration is needed. Experimental studies have indicated that the storage medium is a more critical prognostic factor than the extra-alveolar period.[14],[15]

For avulsed tooth storage, the American Association of Endodontists recommends Hank's balanced salt solution as the standard storage solution. However, in an effort to find alternate media, several solutions such as milk, egg white, etc., have been tested. These media, apart from maintaining the cell viability, should also be easily available, inexpensive, and simple to use.

The ability of lime, black tea, and passion fruit to preserve the viability of periodontal cells was evaluated in the present study.

The periodontal fibroblasts were tested for cytotoxicity in this study as they are the predominant cell type in the soft connective tissues of the periodontium and consequently play a central role in normal function and in pathologic alterations.[16] L929 lineage of fibroblasts was cultured, as it is one of the most commonly used lineages for endodontic research.

In the present study, the cell viability was evaluated using the MTT assay, which is a quantitative colorimetric assay forin vitro cytotoxicity tests. Viable cells with active mitochondria cause cleavage of MTT dye into water-insoluble dark blue formazan crystals, whereas dead cells remain uncolored.[17]

Tea is the second most consumed beverage in the world, and the ease of its availability made black tea to be tested as one of the samples. Commercially available green tea was tested for its avulsion tooth storage properties and advocated it as an alternate due to superior osmolarity, easier availability, and cost-effectiveness.[9] The PDL cell viability in Black tea might be attributed to various properties shared by both green and black tea. The presence of similar catechins and flavonoids in green and black tea might contribute to the proven anti-oxidant and anti-inflammatory effect. Certain tea infusions are also known to have high fluoride levels, the presence of which is appreciated in this scenario. Chatterjee et al. concluded that both green and black tea leaves possess a marked anti-inflammatory effect againstin vitro denaturation of the protein.[18]

Citrus limon (Lime), a potential source of vitamin C is known for its antibacterial potential, anti-oxidant and anti-cancer activities.[19] Itsin vitro anti-cariogenic potential has also been proved.[20] Lime juice exhibited 98% viability of cells, and this reduction in its viability as compared to the other two samples, might be attributed to the acidic pH and osmolality range of lime. It has been proven that physiologic osmolality and pH, along with appropriate temperature, are required to allow optimal cell growth and survival.[21]

Passion fruit is a good source of ascorbic acid (Vitamin C) and carotenoids (Vitamin A) known for its anti-oxidant and anti-carcinogenic properties.[22] In this study, 100% viability was demonstrated in passion fruit concentrate, which might be due to its composition, acceptable osmolality, pH range, and also the absence of water (concentrated solution). Also, a minimal concentration of 0.625%passion fruit concentrate exhibited highest viability compared to 10% lime juice which showed lowest viability [Figure 1]. It has been documented for the presence of nonnutritive phytochemicals, carotenoids, and polyphenols within the fruit with anti-oxidant properties.[23] Septembre-Malaterre et al. characterized the ability of polyphenol-rich fruit extract of passion fruit to protect cells against free radical damage and high concentration of Vitamin C.[24]
Figure 1: 10% lime juice showed lowest viability and 0.625% passion fruit concentrate showed highest viability

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   Conclusion Top


Under the limitations of this study, it can be concluded that Lime juice, Black tea, and passion fruit concentrate can be used as effective alternatives for avulsed tooth storage, because of their ability to maintain high viability. Furthermore, black tea and lime juice are easily accessible popular beverages around the world. While passion fruit is not very accessible, its desirable anti-oxidant and high viability properties, make it a choice, which can be marketed commercially for usage as a storage medium.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
   References Top

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Dr. K Deepthi
No. 36, Second Floor, Main Street, Shivaji Nagar, Pondicherry - 605 006
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/JCD.JCD_253_20

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