Comparative evaluation of the viability of periodontal ligaments cells in green tea and turmeric as storage media – An ex vivo study

1 Introduction

Tooth avulsion is defined as a complete displacement of a tooth from its alveolar socket. The most indicated procedure for this kind of dental trauma is re-implantation [1]. The management of this issue is both time and technique sensitive.

The prognosis of the tooth to be re-implanted is governed by the existence of viable cells in the periodontal ligament that are able to proliferate at the damaged site on the root. This is a complex healing process and relies primarily on time and the storage of the tissue prior to replantation [2]. In order to maintain cell viability, an avulsed tooth needs to be stored in an appropriate media to avoid desiccation. Exposure of an avulsed tooth to air or to dry conditions causes necrosis of PDL cells thus resulting in a severe inflammatory response when replanted [3]. Some of the ideal properties of the transport media are: it should be capable of preserving the viability of the cellular PDL; preserve the majority of the functional capacities of the cells of PDL; maintain the vitality of the remaining PDL; physiologic pH.(7.2–7.4); physiologic osmolality (290–330 mosm/kg.; antimicrobial characteristics; favorable proliferative capacity of cell (Clonogenic & Mitogenic capacity);unreactive with body fluids; not produce any antigen–antibody reaction; reduce the risk of post-reimplantation root resorption or ankylosis; good shelf life; effective in different climates and different conditions; wash off extraneous materials and toxic waste products, aid in reconstitution of depleted cellular metabolites [4].

Although Hank’s Balanced Salt Solution (HBSS) is currently the storage media recommended by the AAE, AAPD, and IADT for an avulsed tooth, the search for accessible and economical storage media is still intense. Pharmacological safety and small cost make natural products attractive agents to investigate further. Extensive research on turmeric has demonstrated a wide spectrum of therapeutic actions such as anti-inflammatory, antibacterial, antiviral, antifungal, anti-diabetic, anticoagulant, hepato-protective, anti-ulcer, hypo-tensive, and hypo-cholesteremic [5–9]. Similarly, Green tea (GT), extracted from Camellia sinensis, is a widely consumed beverage throughout the world. GTE has been reported to have remarkable cytostatic properties for preserving cells, anti-inflammatory, antioxidant, and anti-carcinogenic effects [10–12]. However, literature regarding the use of turmeric and green tea based products as storage solutions for PDL cells is scarce. Hence, the present study was undertaken to comparatively evaluate the efficacy of storage media HBSS, green tea and Curcumin at two hour’s time intervals.

2 Materials and methods

The present study was carried out in Department of Paediatric and Preventive Dentistry at Rural Dental College, Loni. Ethical clearance for the study was obtained from the Institutional Ethical Review Board and Research Committee (Ethical Clearance No. PIMS/RDC/IEC-UG/2017/PG/12 dated 26/09/2017). Sixty single rooted human premolars freshly extracted for orthodontic purpose were collected and used for the study. Factors considered under inclusion criteria were premolars extracted for orthodontic purpose, teeth free from caries, cracks, dilacerations and developmental anomalies. Whereas, teeth with external or internal resorption and developmental anomalies were excluded from study.

2.3 Periodontal ligament cell viability assessment

Sixty premolars indicated for orthodontic extraction with healthy PDL were used for the study. Immediately after the extraction, the tooth under study was immersed in respective storage media to maintain equal baseline standard for viable cells.

Periodontal cells viability was evaluated at time intervals of 2 hours. At this time interval, premolar from the storage media was washed with normal saline and with the help of a sterile BP blade, the root of the premolar was scrapped off to obtain the PDL cells in a Petri dish. 100μl of the cell suspension was obtained with the help of a micropipette to which 100μl of Trypan blue stain was added and mixed well. The collected solution was transferred to Neubauer’s chamber. The viable cells were counted using a Neubauer’s chamber under the light microscope at 40X magnifications (Olympus, Japan) (Fig. 1). The total numbers of cells were calculated using the following formula Cell count = No . of cells counted × dilution factor Volume of the chamber ( Area × depth )

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Fig. 1

Counting of viable cells under the light microscope at 40X magnifications (a) HBSS, (b) green tea, (c) turmeric (Olympus, Japan).

The data were assessed for normality using the unpair t-test and analysis of variance (ANOVA). Level of significance was set at P≤0.05. The observed values for all media were plotted over the 2-hour time period (Fig. 2). The mean number of viable cells at two hours interval were 262020 in HBSS, 214020 in Green tea extract and 209426 in turmeric solution (Table 1). Intergroup comparison between three storage groups showed that viability of cells were more with turmeric in comparison with green tea extract when HBSS was considered as control group. The cell viability was much better in HBSS than Turmeric when Green tea extract was taken as control and HBSS had more cell viability count as compared to Green Tea when Turmeric was taken as the control group (Table 2).

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Fig. 2

Cell viability of storage media after two hours.

Avulsion type of dental injury, one of the most severe forms of trauma, is characterized by complete displacement of the tooth from its alveolar socket. The reported incidence of complete avulsion ranges from 1% to 16% of all traumatic injuries to the permanent dentition and 7% to 21% in primary dentition [13]. In young age group, relatively resilient alveolar bone provides only minimal resistance to extrusive forces and the maxillary central incisors are the most commonly affected teeth.

Ideally, the tooth should be replanted immediately after the avulsion injury in an effort to preserve the viability of the PDL cells, and so as to have adequate healing and minimum root resorption. In a study of 110 human avulsed teeth, evaluated by Andreasen JO and Hjorting-Hansen E, 90% of cases showed no sign of resorption if replanted within 30 minutes, while delayed replantation resulted in significantly more cases with root resorption [14]. However, immediate replantation is not always possible in all the avulsion cases. In some cases, management of concomitant injuries, such as soft tissue laceration, alveolar fractures, and skeletal fractures, may delay replantation of an avulsed tooth, as reported by Petrovic et al. [15].

One of the important prognostic factors for the avulsed teeth is the viability of PDL cells at the time of replantation [16]. In a study by Andreasen et al., it was observed that all the significant factors for healing of the replanted teeth (i.e. root development status, extra-alveolar dry time, immediate replantation, and wet storage time) had the common denominator: viability of the PDL cells [17]. In turn, the viability of PDL cells depends on extra-alveolar dry time [18]. Animal studies and in vitro studies show that after 60 minutes of dry storage, very few PDL cells remain vital, and there may be no viable PDL cells left after 120 minutes of dry storage.

HBSS is regarded as a standard tooth storage medium, and its commercial version is available in the USA as Save-A-Tooth. EMT Tooth saver (Dentosafe) is a commercial tooth storage medium that is widely used more in European countries [19]. Availability of HBSS is still a major concern, so there is a need to find alternate storage media which can be easily made available and will be equally effective. Natural transport media like skimmed milk, coconut water, green tea, pomegranate juice and egg white score over HBSS based on their storage time, ease of availability and economical price [20].

In an attempt to find the alternative agent, the present study was conducted in order to compare green tea, turmeric and HBSS, in terms of their effects on viability of periodontal ligament fibroblast as a function of exposure time.

The storage procedure explained in the previous literature was followed in the current study as it was closely replicating the clinical scenario [21]. At a predetermined time, the teeth were taken out of the medium and PDL was isolated to evaluate the cell viability. Trypan blue exclusion assay quickly and easily differentiates nonviable cells from viable ones. Trypan blue stain used in this study assessed only viability of the cells [22].

In the present study Hank’s Balanced Salt Solution (HBSS) was selected as control as it is recommended by AAE, AAPD and IADT as the standard storage medium for an avulsed tooth [1, 2]. HBSS is sterile and contains essential nutrients [23], including sodium chloride, D-glucose, potassium chloride, sodium bicarbonate, potassium phosphate, calcium chloride, magnesium chloride and magnesium sulphate [24]. Its pH and its osmolality, is suitable for cell growth. In present study HBSS preserved significantly higher number of viable PDL cells than any other sample solutions at determined interval of time. Several studies have shown that HBSS can preserve the viability of PDL cells for an extended period of time [12, 25]; promote proliferation [26] and high mitogenicity [25] of PDL cells; and is beneficial to PDL cells that were initially stored in the dry condition [27, 28] or in saliva [29]. Cells stored in HBSS had better cell integrity than those in milk, saline or saliva [30]. In addition, animal studies have demonstrated that storing extracted teeth in HBSS resulted in high rates of healing upon replantation [31, 32]. The main drawback of HBSS as a tooth storage medium is its limited availability to the public at or near the site of incidences of tooth avulsion [26].

Green tea in comparison to other samples showed more viable cells followed by turmeric. Green tea 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. Catechin, epicatechin, epigallocatechin, epigallocatechin-3-gallate, epicatechingallate, and quercetinglycosides are the most important polyphenols of green tea. 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. Epigallocatechin-3-gallate (EGCG) is a major polyphenol of green tea, having anti oxidative, anti-carcinogenic, anti- mutagenic, anti-inflammatory, anti- microbial and anti-viral activities. In present study green tea has shown number of viable cells similar to HBSS. This finding is in agreement with a study carried out by Hwang et al and Jung et al who reported enthusiastic results with green tea, with the maintenance of 90% of cell viability for up to 24 h, similar to the HBSS control [12, 33]. Furthermore, it has previously been used for allograft with positive results [34].

Turmeric (Curcuma longa) is a common antiseptic belonging to the family Zingiberaceae. The proven properties of Curcumin include anti-inflammatory, antioxidant, antimicrobial, antiseptic, and antimutagenic [21]. Antimicrobial effect of Curcumin inhibits the growth of various microorganisms. It is a natural medicament with a wide spectrum of biologic actions [35, 36]. It has a pH balance of 7.1 with an osmolality of 260mosmol/kg. Mandrol et al. [35] investigated in vitro cytotoxicity of Curcumin against primary dental pulp fibroblasts by MTT assay. No cytotoxicity was detected for Curcumin at any of the concentrations used (25%, 50%, and 100%). The results revealed that the viability of primary dental pulp fibroblasts increased with an increasing concentration of Curcumin. Curcumin promotes cell viability and induces proliferation of dental pulp fibroblasts and thus can be used as a suitable natural storage medium. The result of present study showed that turmeric has less potential than HBSS and green tea to maintain the viability of PDL cells which is in accordance with recently study done by Pranjali et al. in case of turmeric extract medium, there were 81.63% of cells which were viable [37].

According to results of our study, it was found that the maximum percentage of viable cells was found in HBSS, followed by green tea and turmeric. More data and further investigations are required to demonstrate the optimum storage time of different media and the form and manner of use. Hence, it is necessary to conduct further studies so that the features of successful replantation can be assessed.

5 Conclusion

The results of our experiment suggest that HBSS effectively maintains the viability of PDL for two hours. Green tea was equally effective in maintaining the viability of PDL cells at two hours interval. Turmeric was less effective in studied time interval. Within the limitation of this in vitro study, it can be concluded that green tea can be a practical and effective medium for storage of an avulsed tooth and can be a promising choice to HBSS followed by turmeric to store an avulsed tooth for at least two hours.