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Table of Contents   
ORIGINAL ARTICLE  
Year : 2015  |  Volume : 18  |  Issue : 1  |  Page : 47-50
In vitro viability of human periodontal ligament cells in green tea extract


1 Department of Pediatrics, Faculty of Dentistry, Babol University of Medical Sciences, Babol, Iran
2 Department of Pharmacology and Physiology, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran
3 Department of Cellular and Molecular Research Centre, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran
4 Department of Biochemistry and Biophysics, Faculty of Medicine, Babol University of Medical Sciences, Babol, Iran

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Date of Submission30-Jun-2014
Date of Decision21-Sep-2014
Date of Acceptance04-Oct-2014
Date of Web Publication8-Jan-2015
 

   Abstract 

Context: Delayed replantation of avulsed teeth may be successful if the majority of periodontal ligament cells (PDL) survive. A proper transport medium is required when immediate replantation is not possible. Green tea extract (GTE) may be effective in preserving the cells because of its special properties.
Aims: This study was done to evaluate the potential of GTE in periodontal ligament cells preservation.
Materials and Methods: Fifty-four extracted human teeth with closed apices were randomly divided into three groups each with 18 teeth as follow: GTE, water (negative control), and Hank's balanced salt solution (HBSS) (positive control). The specimens were immersed in the media for 1, 3, and 15 hours at 4 o C (n = 6) and treated with collagenase 1A for 45 minutes. Cell viability was determined using the trypan blue exclusion technique.
Statistical Analysis: Data were analyzed by one-way analysis of variance (ANOVA), post hoc Tukey and paired t-test at significance level of P < 0.05.
Results: Means (standard deviation, SD) of viable cells in HBSS, water, and GTE were estimated 348.33 ± 88.49, 101 ± 14.18, and 310.56 ± 56.97 at 1 hours; 273.4 ± 44.80, 64.16 ± 16.44, and 310.2 ± 11.21 at 3 hours; and 373.72 ± 67.81, 14.41 ± 2.88 and 315.24 ± 34.48 at 15 hours; respectively. No significant differences were found between HBSS and GTE at all the time intervals. Both these solutions could preserve the cells more than water significantly.
Conclusion: GTE and HBSS were equally effective in preserving the cells and were significantly superior to water.

Keywords: Avulsion; green tea; periodontal ligament cell; storage medium

How to cite this article:
Ghasempour M, Moghadamnia AA, Abedian Z, Amir MP, Feizi F, Gharekhani S. In vitro viability of human periodontal ligament cells in green tea extract. J Conserv Dent 2015;18:47-50

How to cite this URL:
Ghasempour M, Moghadamnia AA, Abedian Z, Amir MP, Feizi F, Gharekhani S. In vitro viability of human periodontal ligament cells in green tea extract. J Conserv Dent [serial online] 2015 [cited 2019 Nov 14];18:47-50. Available from: http://www.jcd.org.in/text.asp?2015/18/1/47/148894

   Introduction Top


In cases of avulsion, in spite of the obscure long-term survival of replanted permanent teeth, immediate replantation is strongly recommended. [1],[2]

The prognosis of replantation is directly dependent upon the viability of periodontal ligament (PDL) cells which is affected by two critical factors; the extra alveolar time and the storage condition. [1],[3],[4],[5]

When immediate replantation is not possible, preserving the tooth in a physiologic solution at an appropriate temperature is advised to allow optimal cell growth and viability. [6]

Green tea extract (GTE) as an easily available physiologic medium has been of interest recently because of its anti-bacterial, anti-oxidative, and anti-inflammatory effects. [4],[7],[8]

The present study aims to evaluate the potential of GTE to preserve the PDL cells of avulsed teeth.


   Materials and Methods Top


The study protocol was approved by the Ethics Committee of Babol University of Medical Sciences. GTE was prepared based on the method previously described by Howang et al. Ten grams of the commercial spring green tea leaves (Refah Lahijan Co, Iran) harvested in the north of Iran, were infused in 100 ml of boiled water for 5 minutes, then filtered and cooled to 4° C. [4] The pH and osmolality were measured twice with a pH meter (Crison pH-meter Basic 20 +, Spin) and an osmometer (Robin, Germany), respectively.

Fifty-four human teeth with closed apices were extracted as atraumatically as possible for orthodontic reasons and were randomly divided into three experimental groups each with 18 teeth as follows: GTE, water (negative control), and Hank's balanced salt solution (HBSS) (positive control) (Baharafshan, Iran). Following the extraction, the specimens were immediately immersed in one of the storage solutions for 1, 3, and 15 hours at 4° C (six teeth at each time interval). After that, 3 mm of cervical PDL tissues were scraped with a sharp scalpel, the teeth gently washed with relevant solutions and incubated at 37° C (5% CO2) for 45 minutes in 15-ml Falcon tubes with 2.5 ml of 0.5 mg ml - collagenase 1A (Sigma-Aldrich, USA) in phosphate-buffered saline. Following the incubation, 50 μl of fetal bovine serum was added to each tube and all the tubes were centrifuged at 1500 r. p. m for 4 minutes. Subsequently, the supernatant was removed with a sterile micropipette and dead cells were labelled with 0.4% trypan blue (Sigma-Aldrich, USA). [9] The number of viable cells was counted using a hemocytometer under a light microscope at × 20 magnifications (Olympus, Japan). Data were analyzed using one-way analysis of variance (ANOVA) and post hoc Tukey tests supplemented by paired t-test using statistical package of social sciences (SPSS) software (version 18). The level of significance was considered at P < 0.05.


   Results Top


Acidity and osmolality of experimental media are shown in [Table 1].
Table 1: The mean values of pH and osmolality of experimental solutions

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Generally, within the study parameters no significant differences were found between data of HBSS and GTE (P = 0.46) while there was a significant difference in viable cell counts of both solutions compared with water (P = 0.00, P = 0.00). The mean (standard deviation, SD) of viable cells in different study solutions are displayed in [Table 2].
Table 2: The mean (standard deviation, SD) of viable cells in each experimental medium based on time

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The average viable cell counts in HBSS exhibited no significant differences between 1 and 3 hours as well as between 3 and 15 hours (P = 0.18, P = 0.07; respectively). The viable cell counts in HBSS exhibited also no significant differences between the first time interval compared with the third experimental time interval (P = 0.30).

No significant differences were found between data of GTE at three time intervals compared with each other (P = 0.98, P = 0.79; P = 0.79; respectively).

Unlike the current solutions, the number of viable cells in water exhibited a significant difference at all the study time intervals compared with each other (P = 0.01, P = 0.00, P = 0.00).

[Figure 1] illustrates the differences of data in all the experimental groups based on time [Figure 1].
Figure 1: The number of viable cells in all experimental groups based on time

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   Discussion Top


The present results confirmed the hypothesis that GTE might be effective in preserving the PDL cells of avulsed teeth. It was revealed that GTE can keep the PDL cells alive for 15 hours, as well as HBSS. This result was consistent with the report made by Hwang and colleagues. They found that most of the PDL cells survive in both GTE and HBSS up to 24 hours. [4]

Interestingly, like HBSS, the capability of GTE for maintaining the cells was not affected by time. The same outcome was also achieved by Hwang and co-workers. [4]

American Association of Endodontists (AAE) recommends HBSS as the standard storage solution. [10] This isotonic salt solution with physiologic pH is a sterile and non-toxic medium commonly used for culturing or transporting cells. [11],[12] However, it is not usually available where traumatic injury is likely to happen and not widely sold on the market. [10],[13]

In an effort to find proper media that would be easily accessible for use, several solutions such as milk, egg white, coconut water, etc. have been introduced. [1],[13]

In the present study, the ability of GTE to preserve the cells was evaluated. This medium was selected because of its special properties such as anti-inflammatory, anti-microbial, and anti-oxidative potentials. It is produced from leaves of Camellia sinensis plant which is considered a major source of polyphenols. [7] Catechin, epicatechin, epigallocatechin, epigallocatechin-3-gallate, epicatechingallate, and quercetin glycosides are the most important polyphenols of green tea. [5],[7] This medium also contains critical elements for cell growth such as calcium, magnesium, selenium, zinc, iron, and fluoride as well as some carbohydrates such as glucose, fructose, sucrose, and vitamins B, C, and E. [8] Furthermore, it is an accessible popular beverage around the world and has previously been used for allografts and cell studies. [14]

It has been demonstrated that physiologic osmolality and pH along with an appropriate temperature are required to allow optimal cell growth and survival. [6],[15] Since almost all the cellular reactions are dependent upon the pH of the environment, little changes may influence the main biological processes. Additionally, the osmolality of medium affects the water absorption of the cells. Both the rise and the reduction of osmolality are critical for the cell viability. [15] The suitable pH and osmolality for cell growth are around 6.6-7.8 and 230-400 mosmol/kg; respectively. [4],[13]

In the current study, unlike the pH, the osmolality of GTE was measured under normal physiologic limits. Although, the pH value of GTE was estimated to be slightly lower than the favorable range, but the protective capacity of GTE was not seriously affected.

Surprisingly, it was revealed that some of the PDL cells survived in water for a longer duration than what was previously shown. [4],[13] It is supposed that extra oral time plays a key role in such cases. In fact, an attempt was made to minimize the adverse effect of dry time storage in the present study, so that freshly extracted teeth were placed in the solutions immediately, while in other studies tooth placement in the storage media were postponed for a couple of minutes. [1],[4],[10] However, consistent with previous studies, the number of viable cells in water was significantly lower than other understudied solutions and rapidly dropped by time. [4],[13] Generally in spite of optimal pH for water, the hypotonicity of this medium results in cell rupture and maintaining the PDL cells in water has not been recommended for more than 20 minutes. [2]

An ideal transport solution should be capable of preserving both viability and mitogenicity of PDL cells. [6] Considering the present results, the authors think that the osmolality of the transport solution is relatively more important than the pH in preserving the viability of cells but not essentially in preserving the mitogenicity and clonogenic capacity of cells.


   Conclusion Top


GTE and HBSS were equally effective in preserving the cell viability and were significantly superior to water. GTE may be considered a potential transport medium for avulsed teeth and it is better to be compared with other media such as milk. Also, studies are recommended on the ability of GTE to maintain the mitogenicity and clonogenic capacity.


   Acknowledgement Top


Special thanks to the surgeons Ataei, Pour Ahmadi, Sajadi and Movahhedi who participated in the sample collection. Also, it is necessary to appreciate the staff of Cellular and Molecular Research Centre of Babol University of Medical Sciences because of scientific helps.

 
   References Top

1.
Gopikrishna V, Thomas T, Kandaswamy D. A quantitive analysis of coconut water: A new storage media for avulsed teeth. Oral Surg Oral Med Oral Pathol Oral Radiol Endod 2008;105:e61-5.  Back to cited text no. 1
    
2.
Andreasen JO, Andreasen FM. Avulsions. In: Andreasen JO, Andreasen FM, Andersson L, editors. Textbook and color atlas of traumatic injuries to the teeth. 4 th ed. Oxford: Backwell Munksgaard; 2007. p. 444-88.  Back to cited text no. 2
    
3.
Goswami M, Chaitra T, Chaudhary S, Manuja N, Sinha A. Strategies for periodontal ligament cell viability: An over view. J Conserv Dent 2011;14:215-20.  Back to cited text no. 3
[PUBMED]  Medknow Journal  
4.
Hwang JY, Choi SC, Park JH, Kang SW. The use of green tea extract as a storage medium for the avulsed tooth. J Endod 2011;37:962-7.  Back to cited text no. 4
    
5.
Gramaza A, Korczak J, Amarowicz R. Tea polyphenols- their antioxidant properties and biological activity: A review. Pol J Food Nutr Sci 2005;14/55:219-35.  Back to cited text no. 5
    
6.
Chamorro MM, Regan JD, Opperman LA, Kramer PR. Effect of storage media on human periodontal ligament cell apoptosis. Dent Traumatol 2008;24:11-6.  Back to cited text no. 6
    
7.
Friedman M. Overview of antibacterial, antitoxin, antiviral, and antifungal activities of tea flavonoids and teas. Mol Nutr Food Res 2007;51:116-34.  Back to cited text no. 7
    
8.
Cabrera C, Artacho R, Gimenez R. Beneficial effects of green tea - A review. J Am Coll Nutr 2006;25:79-99.  Back to cited text no. 8
    
9.
Polverini PJ, Leibovich SJ. Induction of neovascularization in vivo and endothelial proliferation in vitro by tumor-associated macrophages. Lab Invest 1984;51:635-42.  Back to cited text no. 9
[PUBMED]    
10.
Caglar E, Sandalli N, Kuscu OO, Durhan MA, Pisiriciler R, Caliskan EA, et al. Viability of fibroblasts in a novel probiotic storage media. Dent Traumatol 2010;26:383-7.  Back to cited text no. 10
    
11.
Eskandarian T, Badakhsh S, Esmaeilpour T. The effectiveness of oral rehydration solution at various concentrations as a storage media for avulsed teeth. Iran Endod J 2013;8:22-4.  Back to cited text no. 11
    
12.
Nozari A, Esmaeilpour T, Fijan S. Investigation of the capability of the new storage media in keeping the periodontal ligament cells viability. Sadra Med Sci J 2013;1:103-12.  Back to cited text no. 12
    
13.
Khademi AA, Saei S, Mohajeri MR, Mirkheshti N, Ghassami F, Torabi nia N, et al. A new storage medium for an avulsed tooth. J Contemp Dent Pract 2008;9:25-32.  Back to cited text no. 13
    
14.
Cavet ME, Harrington KL, Vollmer TR, Ward KW, Zhang JZ. Anti-inflammatory and anti-oxidative effects of the green tea polyphenol epigallocatechin gallate in human corneal epithelial cells. Mol Vis 2011;17:533-42.  Back to cited text no. 14
    
15.
Gomes MC, Westphalen VP, Westphalen FH, Silva Neto UX, Fariniuk LF, Carneiro E. Study of storage media for avulsed teeth. Braz J Dent Traumatol 2009;1:69-76.  Back to cited text no. 15
    

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Correspondence Address:
Dr. Samane Gharekhani
Department of Pediatric, Faculty of Dentistry, Babol University of Medical Sciences, Babol
Iran
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Source of Support: Babol University of Medical Sciences, Conflict of Interest: None


DOI: 10.4103/0972-0707.148894

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