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ORIGINAL ARTICLE
Year : 2019  |  Volume : 37  |  Issue : 2  |  Page : 140-145
 

Evaluation of the efficacy of neem (Azadirachta indica) and turmeric (Curcuma longa) as storage media in maintaining periodontal ligament cell viability: An in vitro study


Department of Pedodontics and Preventive Dentistry, Hitkarini Dental College and Hospital, Jabalpur, Madhya Pradesh, India

Date of Web Publication26-Jun-2019

Correspondence Address:
Dr. Pranjali Dhimole
Department of Paedodontics and Preventive Dentistry, Hitkarini Dental College and Hospital, Hitkarini Hills, Dumna Road, Jabalpur - 482 005, Madhya Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1319-2442.261356

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   Abstract 


Aims: The aim of the study is to evaluate the efficacy of neem and turmeric as storage media in maintaining periodontal ligament (PDL) cell viability. Materials and Methods: Ninety premolar extracted teeth were randomly selected and divided into three groups, namely milk as a control group and neem and turmeric as experimental groups. The teeth were placed in one of the three storage media for 30 min. Scrapped PDL fibers were collected in Falcon tubes, incubated, and centrifuged for 5 min at 800 rpm. Obtained PDL cells were stained with trypan blue, observed, and counted in a hemocytometer under microscope, which was followed by the calculation of percentages of viable cells. One-way ANOVA was applied for comparison between different groups, and Tukey's test was applied for pair-wise comparison. Results: Mean percentage of viable cells in milk was 89.98 ± 4.11%, whereas in neem and turmeric extracts, it was 88.00 ± 5.85% and 81.63 ± 7.12%, respectively. There was a significant difference between all the three storage media for the viable PDL cells (P = 0.001). Intergroup comparison of the different storage media showed that there was a statistically highly significant difference between milk and turmeric (P ≤ 0.001). There was a statistically significant difference in between turmeric and neem (P ≤ 0.531) for the viable cell percentage, with neem being better storage medium than the turmeric. Conclusion: Within the limitations of the current study, it can be concluded that neem is as efficient as milk in maintaining the PDL cell viability. Turmeric, though is an efficient storage medium, was not as efficient as milk and neem.


Keywords: Periodontal ligament, storage media, tooth avulsion, viability


How to cite this article:
Dhimole P, Bhayya DP, Gupta S, Kumar P, Tiwari S, Pandey S. Evaluation of the efficacy of neem (Azadirachta indica) and turmeric (Curcuma longa) as storage media in maintaining periodontal ligament cell viability: An in vitro study. J Indian Soc Pedod Prev Dent 2019;37:140-5

How to cite this URL:
Dhimole P, Bhayya DP, Gupta S, Kumar P, Tiwari S, Pandey S. Evaluation of the efficacy of neem (Azadirachta indica) and turmeric (Curcuma longa) as storage media in maintaining periodontal ligament cell viability: An in vitro study. J Indian Soc Pedod Prev Dent [serial online] 2019 [cited 2019 Jul 19];37:140-5. Available from: http://www.jisppd.com/text.asp?2019/37/2/140/261356





   Introduction Top


Traumatic injuries to the permanent anterior teeth are most common in the age group of 7–10 years. Out of these traumatic injuries, 0.5%–16% results in tooth avulsion.[1] The World Health Organization has defined “exarticulation or avulsion” as the complete displacement of a tooth from its alveolar socket due to traumatic injury along with damage to the periodontal ligament (PDL), cementum, alveolar bone, and gingival and pulp tissues.[2],[3],[4]

At the time of accident, avulsed tooth should be immediately replanted at the site of the accident to prevent further damage to the PDL cells from desiccation.[5],[6] Under certain conditions, immediate repositioning of teeth is not always possible. In such cases, the storage medium is used to preserve PDL cell viability. The prognosis of an avulsed tooth after replantation depends on two critical factors: extraoral dry time and storage medium in which the tooth is placed before treatment is rendered.[7]

The ideal requirements for storage medium being antimicrobial, maintaining the viability of periodontal fibers, favoring proliferative capacity of cells as well as the extraoral time to prevent ankylosis and replacement resorption.[7] A suitable medium for storage of exarticulated teeth from the time of trauma to the arrival at the dental clinic has to fulfill two requirements. First, it should provide the best possible conditions for the cells in the PDL to survive. Second, it has to be available at the time of need.[8] The American Association of Endodontists has recommended Hank's Balanced Salt Solution (HBSS) as a storage medium of choice for avulsed teeth which was proven to maintain PDL viability, but it is not readily available, hence an alternative medium is requires that is not only comparable performance to HBSS, but is also readily available and inexpensive.

One of the alternate media which are frequently been studied is milk, and it is the most practical transport medium for the short-term storage of avulsed teeth because of its ready availability in almost all situations. It contains amino acids and vitamins and is capable of inactivating enzymes harmful to the PDL cells. It has a pH of 6.5–7.2 and osmolality of 270 mosmol/kg, which is similar to the extracellular fluid.[4]

The usage of herbal products is not new to dentistry, and they have been used for various purposes from pain-relieving capabilities to the implant coating in dental implantology. There are various herbs which have been used successfully for the benefit of human health, and among them, neem and turmeric are very popular.

Neem (Azadirachta indica) is an extremely healthy plant which has attracted worldwide attention. Neem leaf and its constituents have been verified to exhibit immunomodulatory, anti-inflammatory, antihyperglycemic, antifungal, antiviral, antioxidant, antimutagenic, and anticarcinogenic properties. It has several active constituents such as nimbidin, nimbin, nimbolide, azadirachtin, mahmoodin, and cyclic trisulfide which are responsible for its antibacterial action.[9],[10] It is biocompatible with pH balanced at 7–7.5 and has an osmolality of 270 mosmol/kg.

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. Antimicrobial effect of curcumin inhibits the growth of various microorganisms. It is a natural medicament with a wide spectrum of biologic actions.[10],[11] It has a pH balance of 7.1 with an osmolality of 260 mosmol/kg.

There is a wide literature gap in considering the possibilities of using the herbal extract as a storage medium for the avulsed tooth. Hence, the current study was undertaken with the hypothesis that the herbal products such as neem and turmeric are as efficient as the milk in preserving the vitality of the PDL of the avulsed tooth. Neem and turmeric were selected because of their beneficial characteristics, availability, feasibility, efficacy, and accessibility.


   Materials and Methods Top


The presentin vitro study was carried out in the Department of Paedodontics and Preventive Dentistry in collaboration with the Department of Oral and Maxillofacial Surgery and Excellent Bio Research Solutions Pvt. Ltd. (Daksh Laboratories). The study was assessed and approved by the Institutional Ethical Committee (HDCH/IEC/2016/521).

A sample of 90 noncarious single-rooted human premolar teeth with closed apices and healthy PDL that were extracted for the orthodontic purpose was utilized in this study. The age of the patients ranged from 13 to 28 years. Inclusion criteria were teeth undergoing extraction for orthodontic therapeutic purposes, noncarious human premolar teeth, and teeth with normal periodontium and closed apices. Teeth with periodontal problems, extensive root and crown caries, fractured teeth involving root, aberrant anatomy, and teeth with developmental anomalies were excluded in the present study.

The teeth were extracted as atraumatically as possible with elevator and forceps. After extraction, the teeth were held by the anatomical crown and were rinsed with water for 10 s to remove any blood and/or saliva from the gingival and apical tissues. The coronal 3 mm of PDL was scrapped with a sharp curette to remove cells that may have been damaged by the forceps during the extraction procedure. After curettage, all the teeth were dried for 15 min, followed by 30 min immersion, and then randomly divided to be placed into storage solution groups, namely

  • Group I (n = 30): Teeth specimens stored in milk (control)
  • Group II (n = 30): Teeth specimens stored in neem extract (A. indica) (experimental)
  • Group III (n = 30): Teeth specimens stored in turmeric extract (C. longa) (experimental).


Neem extract was prepared according to the procedure documented in the earlier study.[9] Fresh neem leaves were washed in sterilized water and were then added to the 50 ml ethanol solution. This is then thoroughly mixed for 1–2 min and filtered. The alcohol part of the extract is separated to obtain 25 ml of extract and then diluted with phosphate-buffered saline solution to get it in an alkaline form. An optimum concentration of 10% was taken for the storage purpose.

Turmeric extract was prepared according to the guideline presented in the previous literature.[12] Dried turmeric rhizomes were crushed in a ceramic mortar and were grounded into powder and are extracted with 500 ml ethanol in a Soxhlet extractor for 16 h. This extract is then transferred to a conical flask for drying in hot air oven (60°C ± 20°C) overnight. The extract is dissolved in dimethyl sulfoxide (DMSO). DMSO acts as a solvent without changing any property of the ethanolic extract. Again, a concentration 10% of solution was considered as an optimal one.



After 30 min immersion in respective experimental storage media, irrigation of the root surface was done twice with sterile isotonic saline to remove the residual storage media. The apical two-third of the root surfaces were scraped in a  Petri dish More Details (ETO 90 mm Sterile, Healthcare Pvt. Ltd. Delhi, India) to obtain the PDL cells. These PDL scrapings were collected in Falcon tubes (Remi, Mumbai, India) containing 2.5 ml of phosphate-buffered solution (100 ml), 10X solution, and in this, 0.5 mg of type I collagenase (100 mg) (HiMedia Labs, Mumbai, India) was added and this has been incubated for 30 min and centrifuged for 5 min at 800 rpm. In collected residue, an equal volume of 0.4% trypan blue stain was added and mixed. In trypan blue stains (HiMedia Labs, Mumbai, India), nonviable cells appear blue and viable cells appear pink. The cells were then observed and counted with the help of hemocytometer (Neubauer's counting chambers) under optical microscope (Labomed Vision 2000).

The cell count was done using the following formula:[2]



The collected data were entered in Microsoft Excel 2016 for Windows. Mean, standard deviation, and minimum and maximum values of total cells, viable cells, nonviable cells, and percentage viable cells in different storage medium groups were calculated. One-way ANOVA was applied for comparison between different groups. When ANOVA showed a significant difference between the groups, Tukey's test was applied for pair-wise comparison. All data analyses were performed using version 21.0 of the Statistical Package for Social Sciences (SPSS version 21.0: IBM Corporation, Armonk, New York, USA). P < 0.05 was accepted as statistically significant.


   Results Top


Mean percentage of viable cells in milk, neem, and turmeric is represented in [Table 1]. The mean percentage of viable cells in milk (89.98 ± 4.11) and neem (88.00 ± 5.85) is significantly higher than turmeric (81.63 ± 7.12) when used as storage media. There is no significant difference between milk and neem.
Table 1: Mean percentage of viable cells in milk, neem, and turmeric

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[Table 2] depicts the intergroup comparison using one-way ANOVA complemented by the Tukey's test. There existed a statistically highly significant difference (P = 0.001) between the milk and turmeric groups, with milk (89.98 ± 4.11) being superior in maintaining the viability of PDL cells than the turmeric (81.63 ± 7.12). Whereas, a statistically significant difference (P = 0.003) was noted between the neem and turmeric groups, with neem being superior to turmeric in maintaining the vitality of the PDL cells. However, no significant statistical difference was noted between the milk and neem extracts.
Table 2: Intergroup comparison using one-way ANOVA complemented by the Tukey's test

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   Discussion Top


The prognosis of dental reimplantation is directly related to the viability of PDL cells that remain on the root surface of the avulsed tooth, or the success of dental reimplantation depends on the existence of viable cells on the PDL, which is able to proliferate on the denuded areas of the root surface.[3] For successful replantation of an avulsed tooth, preservation of viability, mitogenicity, and clonogenic capacity of the injured PDL fibroblasts and their progenitors is necessary.[5]

The replantation of teeth beyond 5 min of avulsion has been defined by Andreasen et al. as delayed replantation.[13] In cases of delayed replantation, the avulsed tooth should be stored in appropriate media, which can prevent desiccation and subsequent loss of vitality of PDL.[14],[15] The reason why it is necessary to put the tooth in some storage media is for the maintenance of the viability of PDL cells on the root surface for an extended time, and in some cases, it even stimulates its proliferation and it is available at the moment of dental avulsion. An ideal storage medium should be capable of preserving the viability of cellular PDL so that the cells could go through mitosis and form clones of the PDL fibroblasts and its generating cells. This is essential for the surface of the root to be repopulated by fibroblasts, thus avoiding the adherence of osteoclasts in this area.[16],[17],[18]

To avoid root resorption and maintain PDL cell viability, various transport media have been proposed. Various studies have found HBSS, Viaspan, and Eagle's medium to be suitable for transportation of avulsed teeth. However, easy access to this solution for the first aid purposes is a major drawback as in most places where avulsion occurs such as at sports fields or schools, where it might be most needed. Cost is also a major prohibitive factor which makes it impracticable as a storage medium.[19] Therefore, there should be an effort to find a storage medium that is easily available and cheap. Therefore, milk, neem, and turmeric were taken since they are easily available and economic. Hence, the present study was carried out to compare the efficacy of three most commonly available and cost-effective storage media, i.e. milk, neem, and turmeric.

A method suggested by Ragnarsson et al.[20] and Sharma et al.[2] has been quoted in the literature to evaluate the efficacy of different storage media in preserving the viability of dental fibroblasts. They removed fibroblasts from the root surfaces and cultured them after adding them to a storage medium. The viability of cells was evaluated at different time intervals. However, Doyle et al.[21] placed the extracted teeth directly in a storage medium. At a predetermined time, the teeth were taken out of the medium and PDL was isolated to evaluate the cell viability. The storage procedure explained in the previous literature was followed in the current study as it was closely replicating the clinical scenario.[2],[21] To quantitate the number of viable PDL cells in the current study and to preserve the maximum cell viability, the root surfaces were treated with type I collagenase for rapid cell retrieval and cellular integrity.[22],[23] 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.

Milk as a storage medium is the most practical transport medium for the short-term storage of avulsed teeth because of its ready availability in almost all situations.[19],[24] The American Academy of Endodontics indicates milk as a storage medium for avulsed teeth to maintain the periodontal cell viability.[25]

It contains amino acids and vitamins and is capable of inactivating enzymes harmful to the PDL cells.[26] Due to these advantages, in the present study, milk was chosen as a control medium. In the present study, milk maintained the highest number of viable cells (89.98%) among neem extract and turmeric extract which could be attributed to its composition. The pasteurization of milk is responsible for diminishing the number of bacteria and bacteriostatic substances[27],[28] also for the inactive presence of enzymes, which could be potentially harmful to the fibroblasts of the PDL. Blomlöf et al. (1983)[29] also recommend milk as an excellent storing solution for 6 h and gold standard for transporting avulsed teeth. Huang et al.[30] found that milk maintains 85% viability to cells which are approximating the results of the present study also. Patel et al.[31] using fluorescein showed that no significant difference was found in the number of viable cells on the root surfaces after 24 h storage in either milk or saliva, whereas Blomlöf (1981)[27] showed that 50% of the cells were viable after 12-h storage in milk and no cells were viable after 3 h storage in saliva. Milk maintains the viability of PDL cells at a clinically significant level for up to 1 h of extraoral time.[32]

A previous study suggested that propolis, as a storage medium, maintained higher viability of PDL cells due to its antibacterial and anti-inflammatory properties.[13],[33],[34],[35] Natural products such as neem and turmeric possess these properties and they are not yet explored as storage media for exarticulated teeth. These herbal products being readily accessible at the trauma site might prove as a boon for effective storage capacity and maintenance of cell viability. Keeping in mind the medicinal effects of these herbal medicaments, they were chosen as experimental media.

The percentage of viable cells was found to be 88.00%. The results of the current study showed that around 88% of cells were viable in case of the neem extract storage medium. turmeric. The results of neem (A. indica) storage medium in the present study is attributed to its several active constituents such as nimbidin, nimbin, nimbolide, azadirachtin, and cyclic trisulfide which are responsible for its antibacterial action.[9] Phenolics exhibit antioxidant activity by inactivating lipid-free radicals or preventing decomposition of hydroperoxides into free radicals. Due to the importance of these compounds, it is necessary in maintaining the optimal antioxidant activity of polyphenols. It is time that neem extracts are incorporated in the present day oral and dental care and can be used in maintaining the viability of PDL cells.

In case of turmeric extract medium, there 81.63% of cells which were viable. The results can be attributed due to the turmeric's anti-inflammatory, antioxidant, anti-carcinogenic, antimutagenic, antibacterial, and antifungal activities. Chemical constituents of turmeric include volatile oil (6%), curcumin, and α- and β-turmerone.[36] Mandrol et al.[11] investigated thein 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. According to the results of the present study, there was no significant difference between milk, neem, and turmeric for percentage viable cells. However, there was a highly significant difference between neem and turmeric for the presence of viable cells with neem storage medium showing increased rate maintaining the viability of cells (P ≤ 0.001).

Fibroblast function is known to be affected by age, trauma, and inflammation. Therefore, noncarious mature human premolar teeth undergoing extraction for orthodontic therapeutic purposes were selected. For the same reason, teeth from young healthy individuals without periodontal disease were included in the study.[37] On average, it takes about 10–15 min for the victim and the person caring for him/her to recover from the traumatic event and act appropriately. According to Pohl et al.,[38] PDL cells remain in a noncompromised state with up to 15 min of dry time. In the present study, it was decided to keep the extraoral dry time for 15 min to simulate avulsion injury and to ensure that sufficient viable PDL cells are available for assessment. Following a 15-min dry time, the teeth were placed in different storage media for 30 min. This period was very important because the PDL cells were most susceptible to damage during this time. Hence, the preservation of teeth in storage media might reduce the damage.[22] According to results of our study, it was found that the maximum percentage of viable cells was found in milk (89.98%), followed by neem (88.00%) and turmeric (81.63%). 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 conductin vivo studies so that the features of successful replantation can be assessed.


   Conclusion Top


Based on the results of the present study, the following conclusions are drawn:

  1. Percentage of viable cells in milk was significantly higher than turmeric extract
  2. Percentage of viable cells in neem was significantly higher than turmeric extract
  3. There is no significant difference between milk and neem extracts.


Within the limitations of this study, it appears that neem maintains the PDL cell viability as comparable as milk but better than turmeric.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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