In vitro viability of human periodontal ligament cells in green tea extract.

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°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.

INTRODUCTION

In cases of avulsion, in spite of the obscure long-term survival of replanted permanent teeth, immediate replantation is strongly recommended.[12]

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.[1345]

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.[478]

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

MATERIALS AND METHODS

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

Acidity and osmolality of experimental media are shown in Table 1.

Table 1

The mean values of pH and osmolality of experimental solutions

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

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

DISCUSSION

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.[1112] However, it is not usually available where traumatic injury is likely to happen and not widely sold on the market.[1013]

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.[113]

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.[57] 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.[615] 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.[413]

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.[413] 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.[1410] However, consistent with previous studies, the number of viable cells in water was significantly lower than other understudied solutions and rapidly dropped by time.[413] 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

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.