Pomegranate Juice (Punica Granatum): A New Storage Medium for Avulsed Teeth

Headnote

Abstract

Objective: There is evidence indicating that pomegranate juice contains many of the essential properties necessary to retain cell viability and cell proliferation. These properties indicate that pomegranate juice is a suitable storage medium for avulsed teeth. However, this idea has not yet been tested. In this study, the capacity of pomegranate juice (PJ) as a storage medium for retaining avulsed teeth was evaluated.

Materials and Methods: PDL fibroblasts were obtained from healthy human premolars and cultured in Dulbecco's Modified Eagle's Medium (DMEM). Cultured cells were subjected to different concentrations of pomegranate juice (PJ), 1% Hank`s balanced salt solution (HBSS) and tap water for 1, 3, 6 and 24 hours. PDL cell viability was assessed by the neutral red uptake assay.

Results: The results indicated that 7.5% PJ was the most effective solution for maintaining PDL cell viability amongst all the experimental solutions and time intervals (P<0.05). The results also showed that 1% PJ was as effective as HBSS for maintaining PDL cell viability. The amount of cell viability increased with increasing concentration of PJ at all time intervals (P<0.001). This effect is suggestive of the proliferative potential of PJ solution.

Conclusion: In conclusion, PJ can be recommended as a suitable transport medium for avulsed teeth.

Key words: Punica Granatum; Tooth Avulsion; Periodontal Ligament; Neutral Red; Cell Culture

Journal of Dentistry, Tehran University of Medical Sciences, Tehran, Iran (2014; Vol. 11, No. 2)

INTRODUCTION

The reported incidence of tooth avulsion ranges from 1 to 16% of all traumatic injuries to the permanent dentition [1]. Studies have shown that a dry period of 2 hours results in necrosis of almost all of the periodontal liga- ment cells [2]. After avulsion, immediate rep- lantation is the treatment of choice to prevent further damage to the PDL cells from desicca- tion. However, clinical experience has shown that most avulsed teeth are replanted only after an extended extra-alveolar time, while being dry or stored in inadequately moist conditions [3]. If the tooth cannot be replanted for any reason, the extraoral dry time must be mini- mized by placing the tooth in a suitable sto- rage medium [4]. The optimum storage me- dium should have the ability to preserve the viability, mitogenicity and clonogenic capacity of the injured PDL cells and their progenitors [3]. While HBSS, Viaspan®, milk, normal sa- line, saliva and tap water [5, 6] are some sug- gested storage media for an avulsed tooth, re- cent studies have verified the application of some other storage media with natural sources such as propolis [7], egg white [8], Morus ru- bra [9], coconut water [10] and green tea ex- tract [11]. Previous studies have indicated that inflammatory resorption and ankylosis are fre- quent sequelae after tooth replantation [12]. For increasing the replantation success, using a suitable medium with anti-inflammatory, antioxidant and antibacterial properties would be of great advantage [9].

Pomegranate is the fruit of Punica granatum (Punicaceae) that has been extensively used in the folk medicine of many cultures. In tradi- tional medicine, pomegranate fruit has been used to treat acidosis, dysentery, microbial infections, diarrhea, hemorrhage, respiratory pathologies, and hypertension [13]. Besides, pomegranate is a rich source of polyphenolic flavonoids; which possess direct antioxidant properties, such as radical scavenging ability, and indirect antioxidant properties such as in- duction of endogenous antioxidant enzymes [14, 15]. Pomegranate flavonoids also have anti-inflammatory properties such as restric- tion of low stimuli activation of inflammation [16]. Moreover, in-vitro studies of pomegra- nate flavonoids have shown anti-bacterial properties; which relate to gingivitis [17]. Pre- vious studies have indicated that rinsing the mouth with pomegranate extract had the ca- pacity to remove dental plaque bacteria from the teeth [18]. Furthermore, it has been de- clared that the effects of pomegranate fruit and peel extracts on proliferation and differentia- tion of osteoblasts significantly improved the pocket depth, level of attached gingiva and bleeding on probing in gingival pockets [19, 20]. To date, PJ extract has never been tested for PDL cell viability. Based on these proper- ties of PJ, which offer the essential features of a good storage medium, this study was de- signed to evaluate the capability of PJ for this purpose.

MATERIALS AND METHODS

Preparation of Pomegranate Juice

Fresh fruits of pomegranate (scientifically au- thenticated as Punica granatum) containing red pulps/red peels were collected from Iran (Shi- raz) at its fruit season. The amount required for this study was prepared and the grains were separated carefully in order to extract the juice. PJ was prepared by squeezing the grains and filtering the juice. Pure juice was placed in Rotary Flash evaporator until the optimal con- centration was obtained.

It was then filtered again by filtering paper to be suitable for passing through the 0.2 µm fil- ter. The prepared PJ was kept in the dark at +4°C until tested.

Primary Culture of Human PDL Cells

PDL cells were obtained from clinically healthy premolar teeth that were extracted for orthodontic purposes from patients under 18 years of age. The teeth were extracted as atraumatically as possible and then immersed in Dulbecco's Modified Eagle's Medium (Gibco, UK) that was supplemented with 10% fetal calf serum and 1% 1x penicil- lin/streptomycin (Gibco, UK). The samples were washed with saline solution and rinsed twice in phosphate-buffered saline (PBS) to eliminate residual blood.

The PDL tissues were removed from the root area with a #15 scalpel using an aseptic tech- nique. The detached tissues were then im- mersed in 25 cm2 culture dishes (Nunc, Roskilde, Denmark); which contained 5ml of culture medium.

All tissue manipulations were carried out un- der sterile laminar airflow. The dishes were then incubated at 37°C, 5% CO2 for an aver- age of 2 weeks in order to observe the HPLF cells (passage zero).

The culture medium was renewed twice per week until the cells reached confluence. The cells were then sub-cultured using 0.25% Trypsin/EDTA (Gibco, UK). Passages number 3-6 were used in this study.

Exposure of PDL Cultures to Different Solu- tions

HPLF cells from passage 3-6 were cultured in 24-well plates at a density of 4x104 cells /well. After 24 hours, the cells were treated with 1%, 2.5%, 5% and 7.5% PJ solutions; tap water was the negative control and 1% HBSS was the positive control.

A time course study including 1hr, 3hr, 6hr and 24hr time periods was designed and cell viability was assessed at each time point for all tested concentrations using neutral red as- say. Cell morphology was visualized with phase contrast microscopy (Leitz, Germany).

Determination of Cell Viability by Neutral Red Assay

The media in each well were aspirated and cells were washed twice with phosphate- buffered saline (PBS); 300 microliters of neu- tral red was added to each well and plates were incubated at 37° C, 95% O2/5% CO2 conditions for 90 min. The dye was removed and cells were exposed to 300 microliters of solubilizer [Absolute ethanol: 0.1 M Citrate buffer, pH 4.2 (21.01 g citric acid + 200 ml of 1M NaOH per litter [A]); 60 ml of [A] +40 ml 0.1 M HCL mixed 1:1 v/v] after washing. Fol- lowing 20 minutes of shaking at room temper- ature, absorbance was measured at 540 nm using ELISA reader. Each experiment was re- peated 3 times. The percentage of viable cells was determined by using the following equa- tion: % of cell viability = (mean absorbance of experimental wells/mean absorbance of HBSS wells at 1hour) × 100%.

Statistical Analysis

Differences in cell viability among different tested media during all time intervals were analyzed using -one way repeated measures ANOVA, and multiple comparison was car- ried out using Tukey's post-hoc test. The level of significance was 5% (P<0.05).

RESULTS

Microscopic Examination

Results of microscopic assessment showed that compared to tap water, both HBSS and PJ preserved spindle-like cell morphology even after 24 hours, and PJ was more successful in retaining cell viability and natural cell mor- phology (Figure 1).

Cell Viability Test by Neutral Red Assay

The mean absorbance, standard deviation and mean percentage values of cell viability in dif- ferent mediums at various periods are summa- rized in Table 1. Results revealed statistically significant differences between different tested media and within various time intervals (P<0.001).

There was a significant interaction between the tested media and time intervals (p<0.001, Figure 2).

The results showed that during all four time intervals, 1% PJ solution performed similarly to HBSS, and the differences were insignifi- cant (P>0.05). Across all time intervals, 7.5% PJ solution was found to have the most signif- icant effect on maintaining cell viability com- pared to HBSS, 1% and 2.5% PJ solutions (P<0.05, Table 2).

For comparison of the four PJ experimental groups, repeated measures ANOVA revealed a significant interaction between the time and experimental group factors (P<0.001); al- though after eliminating the one-hour time point from the time intervals, this interaction was no longer significant (P=0.351).

The results of Tukey's test revealed that at all time intervals, the viability of cells increased with increasing concentration of PJ solution (P<0.001); therefore, 7.5% PJ solution main- tained the highest number of vital cells, and this ability decreased with decreasing concen- tration of PJ solution. At the 6 hr time interval, all four experimental groups had the ability to maintain the highest number of vital PDL cells. Although amongst all concentrations of PJ, the number of vital PDL cells reduced at the 24 hr time interval, this was still signifi- cantly higher in 2.5%, 5% and 7.5% concen- trations of PJ compared to HBSS (P<0.001).

DISCUSSION

Avulsion is the worst traumatic injury to teeth; which leads to detachment of periodontal li- gament from the alveolar socket. The treat- ment of choice for avulsion is immediate rep- lantation of the tooth. However, due to the lack of knowledge, stress and different condi- tions at the time of accident, immediate rep- lantation rarely occurs [21, 22].

In this situation, maintenance of PDL vitality until provision of dental treatment is of high importance for a good prognosis.

The prognosis depends on two factors: the in- terim transport medium and the extra-alveolar time. The capacity of a storage medium to pre- serve cell vitality is considered more critical than the extra-alveolar time in prevention of inflammation and replacement root resorption [23]. To date, several studies have suggested various substances as storage media [5, 6].

Tap water is not a suitable medium due to its hypotonicity leading to rapid cell lysis. Saliva as a storage medium is more effective than tap water, although it is not widely accepted due to its potential for bacterial contamination [24]; furthermore, saliva is a hypotonic solu- tion which causes cell swelling.

Thus, it may be used as a short-term storage medium, while not being suitable for long- term use [25].

In several studies, milk was identified as a suitable short-term storage medium [8] for up to 48h [26, 21].

HBSS is a widely used standard solution rec- ommended by the International Association of Dental Traumatology as a suitable storage medium for avulsed teeth [1].

Although HBSS has the ability to provide long-term preservation of PDL fibroblasts, a major obstacle in using synthetic storage me- dia is their difficult accessibility during an ac- cident.

This indicates the need to identify other ac- ceptable storage media; which provide a suita- ble environment for maintenance of PDL cell vitality [27].

Up to now, pomegranate juice has not been tested for preserving PDL cell viability.

In this study, different concentrations of PJ were studied and compared with HBSS and tap water. The results showed a significant difference between HBSS and 7.5% PJ solu- tions at 1 hr time interval. There was also a significant difference between 2.5%, 5% and 7.5% PJ solutions and HBSS at 6 and 24hr time intervals. This means that the PDL cell viability increases by increasing time and con- centration of PJ solution.

According to Figure 2, it can be observed that PJ increased cell proliferation. This indicates the effects of PJ on fibroblast cell prolifera- tion; which resulted in three times more cell viability in 7.5% PJ compared to HBSS after 24 hours. Also, it was observed that cell via- bility decreased at 1% and 2.5% concentra- tions of PJ at the 1hr time interval; although a general proliferative effect was observed at 5% and 7.5% concentrations of PJ. This may result from the initial cytotoxicity of PJ; which is followed by proliferative effects at higher doses. Furthermore, cell viability increased in all concentrations of PJ at various time inter- vals, with the peak effect being observed at the 6hr time point. Buttke and Trope suggested that the storage of avulsed teeth in a medium containing one or more antioxidant compo- nents may improve the prognosis of replanta- tion [28]. Nowadays, pomegranate as an an- cient fruit is attracting tremendous attention due to its strong antioxidant properties. The potent antioxidant activity of PJ is attributed to its polyphenols including punicalagin, the ma- jor fruit ellagitannin and ellagic acid (EA). Punicalagin is the major antioxidant polyphe- nol ingredient in PJ [29]. Thus, it is assumed to be effective for maintaining PDL cell via- bility. A previous study suggested that propo- lis as a storage medium maintained higher viability of PDL cells due to its antibacterial and anti-inflammatory properties [7].

It is known that pomegranate flavonoids have anti-inflammatory and antibacterial properties, while pomegranate polyphenols have antioxi- dant and antiviral properties which may result in higher viability of PDL cells [13].

An interesting phenomenon observed in our pilot study was that PJ promotes an extremely strong cell attachment for a long duration of time. Although initially trypan blue dye exclu- sion test was used for determining cell viabili- ty, the cells treated with PJ did not detach by trypsin nor collagenase (even with various concentrations and exposure times of the en- zymes). Therefore, neutral red assay was pre- ferred. According to a study by Kwak et al, pomegranate contains components such as el- lagic acid and punicalagin that are specific in- hibitors of beta-secretase (BACE1) and have an inhibitory effect on alpha-secretase (TACE) and other serine proteases such as chymotryp- sin, trypsin, and elastase [30]. Moreover, in another study that evaluated anti-collagenase, anti-elastase and anti-oxidant activities of ex- tracts from 21 plants, it was reported that po- megranate showed 15% anti-elastase and 11% anti-collagenase activity [31]. Strong attach- ment of cells may occur as a result of serine protease inhibitor components of PJ; which deactivate enzymes such as trypsin and colla- genase.

CONCLUSION

In conclusion, the results of this study suggest that pomegranate juice promotes cell viability and induces proliferation of PDL cells.

Therefore, it is recommended to use PJ as a suitable storage medium for avulsed teeth, al- though further in vitro/ in vivo studies are re- quired in this field.

ACKNOWLEDGMENTS

This research was supported by a grant (No. 9729) from the Research Deputy of Tehran University of Medical Sciences and a grant from Shahed Medical University. The authors wish to thank these universities for financially supporting this research project.

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