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ORIGINAL ARTICLE
Year : 2022  |  Volume : 40  |  Issue : 2  |  Page : 195-200
 

An in vitro evaluation of ice apple water, Aloe vera, and propolis as a storage medium to preserve viability of human periodontal ligament fibroblasts


1 Department of Pediatric and Preventive Dentistry, JSS Dental College and Hospital, JSS Academy of Higher Education and Research, Mysore, Karnataka, India
2 Department of Public Health Dentistry, JSS Dental College and Hospital, JSS Academy of Higher Education and Research, Mysore, Karnataka, India

Date of Submission28-May-2021
Date of Decision05-Apr-2022
Date of Acceptance25-Apr-2022
Date of Web Publication15-Jul-2022

Correspondence Address:
Dr. Raghavendra Shanbhog
Department of Pediatric and Preventive Dentistry, JSS Dental College and Hospital, JSS Academy of Higher Education and Research, Mysore, Karnataka
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jisppd.jisppd_193_21

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   Abstract 


Background: A number of media that create the best possible conditions to maintain periodontal ligament (PDL) cell viability after dental avulsion have been reported. Aim: The aim of this study is to evaluate ice apple water (IAW), Aloe vera, and propolis as a storage medium to preserve the viability of human PDL fibroblasts. Methods: An in vitro comparative type of study was performed on a PDL cell culture model. PDL fibroblasts obtained from the roots of healthy premolars were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and treated with ice apple water (IAW), 7% propolis extract (PE), 30% Aloe vera extract (AVE), positive control DMEM supplemented with fetal bovine serum, negative control (NC) without any agent, and incubated at 37°C for 1 h, 3 h, and 24 h. Cell viability was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay after every test period. Optical density was measured at a wavelength of 490 nm. Statistical Analysis Used: The effects of the test storage media were evaluated by one-way analysis of variance test, followed by post hoc Tukey's multiple comparison test (P < 0.05). Results: Seven percent PE demonstrated the highest capacity of maintaining PDL cell viability at 1 h and 24 h. IAW showed a statistically significantly lower percentage of viable cells at all three test periods as compared to 7% PE. After 3 h, 30% AVE demonstrated maximum viable cells. Conclusions: Within the limitations of this study, propolis at a concentration of 7% was the most effective medium for maintaining PDL cell viability.


Keywords: Aloe vera, avulsion, periodontal ligament cells, propolis, storage media


How to cite this article:
Bijlani S, Shanbhog R, Godhi BS, Talwade P, Tippeswamy H M. An in vitro evaluation of ice apple water, Aloe vera, and propolis as a storage medium to preserve viability of human periodontal ligament fibroblasts. J Indian Soc Pedod Prev Dent 2022;40:195-200

How to cite this URL:
Bijlani S, Shanbhog R, Godhi BS, Talwade P, Tippeswamy H M. An in vitro evaluation of ice apple water, Aloe vera, and propolis as a storage medium to preserve viability of human periodontal ligament fibroblasts. J Indian Soc Pedod Prev Dent [serial online] 2022 [cited 2022 Oct 5];40:195-200. Available from: http://www.jisppd.com/text.asp?2022/40/2/195/351045





   Introduction Top


Dental trauma can at times overtake dental caries with a prevalence rate between 6.1% and 58.6%, of which avulsion, the total dislodgment of an intact permanent tooth from its socket, accounts for approximately 0.5%–3%.[1] Trauma has a multitude of consequences for the traumatized individual, family members, and society. The impact is not only physical but also psychosocial and economical.[2] Management protocol for avulsion includes the management of the pulp and the periodontal ligament (PDL) cells, with the latter being more important to improve the long-term survival and prognosis of the tooth.[3] The factors that play a role in healing of the PDL after reimplantation of an avulsed tooth are primarily the amount of physical damage to the root surface, the type of medium in which the avulsed tooth is stored, and the extraoral dry time.[4]

Maintaining the tooth in an adequate medium that can preserve, as long as possible, the vitality of the PDL cells that remain on the root surface is the key to success of reimplantation. Recent research[5] has led to the development of storage media that produce conditions that closely resemble the original socket environment, with adequate osmolality (cell pressure), pH, nutritional metabolites, and glucose and thus create the best possible conditions for storage.

A recent study reported that even if there is a delay in replantation, the buccal and palatal alveolar bone dimensions are maintained for future implant prosthesis. Tooth replantation is essential to provide esthetic appearance for a young adolescent in the interim phase of growth to boost his/her self-esteem.[6]

Until now, there is not a single product or solution that possesses all the characteristics required to be indicated as the ideal storage medium for avulsed teeth. Such a medium must be capable of preserving the vitality of the PDL and pulp cells, while presenting compatible physiological pHand osmolality, antioxidant property, no or minimal microbial contamination and also high availability, ready accessibility at accident sites, homes, schools, hospitals, and low cost. For this reason, natural storage media are preferred over synthetic.[5] The different natural products that have been evaluated as storage media for avulsed teeth include milk, coconut water, propolis, egg white, green tea, Aloe vera, neem, turmeric, pomegranate, saliva, soya milk, probiotic solution, royal jelly, dragon's blood sap, red mulberry, and rice water.[1],[5],[6],[7],[8]

Storage media such as propolis and green tea need to be processed; other natural products such as coconut water and Aloe vera are readily available, and they can be used in their unprocessed form.[6] Coconut water has been proven to be a promising medium for avulsed teeth and is superior to Hank's balanced salt solution (HBSS), milk, or propolis; besides being sterile, there is no need of processing it, and is available easily at different sites. Botanically ice apple (Borassus flabellifer), a locally available fleshy juicy fruit in coastal regions of India, has a similar composition as coconut water, which is a positive factor in the nourishment and viability of the cells. Hence, it could be evaluated as a storage medium in this study.

A number of studies that evaluated Aloe vera as a storage medium for avulsed teeth[9],[10],[11] concluded that it had a higher capacity to maintain PDL cell viability at 10%, 30%, and 50% concentrations of the extract.

Propolis is a yellowish to brownish resinous product of honeybees, which has antibacterial and anti-inflammatory properties and a number of biologically active compounds which benefit to preserve the viability of PDL cells. The properties and composition of propolis vary from region to region. In this study, propolis was obtained from Coorg, Karnataka honey farms, and evaluated as a storage medium for an avulsed tooth.

In the present study, propolis extract (PE), Aloe vera extract (AVE), and ice apple water (IAW) have been evaluated as storage media for avulsed teeth. The null hypothesis for this study was that the potential for cell viability and hence cell proliferation is same across the different categories of products tested.

Methods

Study design and location

This was in vitro comparative study. The procedure protocol was reviewed and approved by the Institutional Research Ethics Committee before the commencement of the study (JSSDCH IEC Research Protocol no: 14/2018).

Collection of ice apple water

The ice apple fruit was acquired from a local farm. It is borne in 2–4 cluster seeds and covered with a black husk. The top portion of the fruit has to be cutoff to uncover the sweet translucent pale-white jelly seed sockets. The jelly fruit comprises a watery fluid inside and is covered with a thin, yellowish-brown skin. An 18-gauge needle was inserted into the jelly part of the fruit through the yellowish-brown skin, and the fluid was aspirated in a sterile syringe and then filtered using syringe filters (0.45 μm).

Propolis extract

Ten gram of the raw propolis was collected from honey farms in Coorg district, Karnataka. It was first dissolved in 70% ethanol, and this mixture was placed in a rotary shaker for 6 days at 50 rpm at room temperature. After 6 days, refluxing was done for 2 h at 40°C. The ethanol extract was then filtered with no. 4 Whatman filter paper. Rotary evaporator was used to remove the excess of ethanol. The extracts were stored at 4°C. At the time of the experiment, the sterile extracts were diluted in Dulbecco's Modified Eagle's Medium (DMEM) to yield 7% PE.

Aloe vera extract

Aloe vera leaves were freshly cut from the plant, washed, and disinfected with 70% ethanol. Under a sterile hood and using a sterile knife, the inner gelatinous portion was carefully removed by separating it from the outer leaf rind. This inner gel was then triturated using a mortar and pestle to make it a homogenous mix and later filtered using a 0.45-mm filter paper to obtain AVE. About 60 ml of AVE was diluted with 140 microliters of DMEM solution to obtain 30% of AVE.

Cell culture

The PDL cell culture experimental model was used in this study. Cells were procured from healthy human teeth extracted for clinical necessity. The coronal portion of the tooth was held using forceps and with the use of a #15 scalpel blade, the PDL was scraped. This tissue was then transferred to sterile  Petri dish More Detailses and washed with DMEM supplemented with 10% fetal bovine serum (FBS), penicillin (100 units/ml), streptomycin (100 units/ml), and fungizone (5 μg/ml) in a humidified atmosphere containing 5% CO2 at 37°C. Then, it was split into smaller pieces before transferring into tissue culture flasks. These pieces of tissue were maintained in the supplemented DMEM and allowed to stick to the surface of the flask in an atmosphere with 5% CO2 at 37°C. Renewal of the culture medium was done two times in a week till the time the cells reached confluence. Then, using 0.25% trypsin and 0.02% ethylenediaminetetraacetic acid for 3–5 min, the cells were detached from the culture flasks and subcultivated. A master bank and a working bank of cells were prepared using the first and second passages of cells, respectively. Further, these cells were suspended in a cryopreservation medium which contained 20% FBS and 10% dimethyl sulfoxide. The culture flasks were stored frozen in liquid nitrogen. Before starting the experiment, cells of the third and fourth passages were thawed at 37°C and seeded into the culture plates. The cell seeding into the 96-well plate was done at a density of 104 cells/well, and incubated for 2 h in a CO2 incubator at 37°C. On the day of the experiment, this culture medium was removed from each of the wells, and the cells were subjected to 200 microliters of the test storage solutions:

  1. IAW
  2. 7% PE
  3. 30% AVE
  4. Negative Control (NC) – without any agent
  5. Supplemented DMEM – DMEM + FBS.


The 96-well plates were incubated for 1 h, 3 h, and 24 h in a CO2 incubator at 37°C.

Determination of cell viability

To evaluate the viability of PDL cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was performed. In vitro viability and growth proliferation of cells were assessed by spectrophotometric determination of conversion of MTT into “formazan blue” by living cells. This is a colorimetric assay which measures the reduction of yellow MTT by mitochondrial succinate dehydrogenase. This MTT penetrates the mitochondria in the cells and is reduced to an insoluble, dark purple formazan crystal. After incubation for 3 h, the cells are solubilized with an organic solvent. Thus, the released, solubilized formazan product is measured spectrophotometrically. The viability of cells is measured by the level of activity of the cells since the reduction of MTT occurs only in metabolically active cells. After every test period, the test storage media was removed from the wells. Sixty microliters of MTT solution were added to each of the wells and incubated at 37°C for 3 h. The supernatant was carefully aspirated, and the precipitated crystals of “formazan blue” were solubilized by adding DMSO (100 μl).

The optical density was measured at a wavelength of 490 nm using iMark™ Microplate Reader. The result recorded is the mean of three readings. Each experiment was repeated three times.

Formula:



Statistical analysis

Statistical analysis of the recorded data was carried out using Kolmogorov–Smirnov one-sample test for normality and Levene's test for homoscedasticity (same variances). Results displayed the normal distribution of the data, and homogenous variances were observed (P < 0.05). The effects of storage solutions at each time point were evaluated by one-way analysis of variance test. This was followed by post hoc Tukey's multiple comparison tests. Type I error rate of 0.05 was used as statistical significance threshold.


   Results Top


The pH of storage media was assessed using a Mettler Toledo benchtop pH meter [Table 1].
Table 1: pH values of test storage media

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[Table 2] represents the mean percentage of viable cells for the different media tested at different time periods. At 1 h, the maximum mean percentage of viable cells was seen with 7% PE, i.e., 152%. This was followed by IAW with 129.67% viable cells at 1 hr. On comparing all these test media, a statistically significant difference was observed (P = 0.001). After 3 h, 30% AVE showed the highest mean percentage of viable cells, i.e., 135% with a statistically significant difference when compared to the other two test media (P = 0.001). The results after 24 h suggested that 7% PE showed the maximum mean percentage of viable cells. IAW and 30% AVE showed a statistically significantly lesser mean percentage of viable cells. By the end of 24 h, only 30% of mean viable cells were seen in the NC group. [Table 3] represents the multiple comparison analysis of the percentage of viable cells of the different test groups at 1 hr. According to this, there was a statistically significant difference observed across all the test groups. In [Table 4], multiple comparison analysis of the percentage of viable cells at 3 h is displayed. Here, the mean percentage of viable cells displayed by 7% PE and IAW did not show any statistically significant difference. When comparing DMEM + FBS with 7% PE, no statistically significant difference was noted either. [Table 5] represents the multiple comparison analysis of the mean percentage of viable cells after 24 h. A statistically significant difference was observed when 7% PE was compared with IAW and 30% AVE.
Table 2: Mean absorbance values of test storage media at different time periods

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Table 3: Multiple comparison analysis of different storage media at 1 h

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Table 4: Multiple comparison analysis of different storage media at 3 h

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Table 5: Multiple comparison analysis of different storage media at 24 h

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   Discussion Top


Avulsion is characterized by severance of the PDL leading to loss of nutrient supply to the remaining viable cells on the root cementum. It is necessary to prevent dehydration and provide the required nutrition for maintaining the viability of these cells by supplying vital metabolites. The treatment plan is directed to limit the infection and inflammation of pulpal and periradicular tissues, thus, leading the balance toward cemental healing rather than unfavorable osseous replacement or inflammatory resorption.[12] To avoid complications, avulsed teeth should be replanted immediately. However, immediate replantation rarely occurs due to several factors, such as lack of knowledge of proper storage medium and the need to access it as early as possible.[13] For this purpose, it is best that the avulsed tooth is temporarily stored in a capable medium which has the potential properties of preserving PDL cell viability.[14] In this in vitro experiment, all three test storage media showed better cell viability compared to negative and positive controls at all the different time intervals. Seven percent PE after 1 h of in vitro storage displayed the highest capacity to maintain cell viability, followed by IAW. Since this is the first study that has been conducted to evaluate the efficacy of ice apple, there are no previous comparative studies for the same. The maintenance of viability of PDL cells by IAW could possibly be due to the presence of numerous phytochemicals with antioxidant and anti-inflammatory properties of ice apple fruit. The vitamin and mineral content of IAW being similar to that of intracellular fluid may be a positive factor in providing nutrition and maintaining the viability of PDL cells.[15],[16]

To evaluate the proliferative capacity of Aloe vera on human keratinocytes, Tudose et al.[17] affirmed that a higher concentration of the extract was required for better cellular proliferation. Thirty percent AVE was chosen in the study as the results obtained by Badakhsh et al.[9] showed that Aloe vera is capable of maintaining PDL cell viability at concentrations ranging from 10% to 50%. In yet another study, 30% concentration of AVE tested at different time periods showed promising results with up to 98.76% viable cells.[10] The antiviral, antioxidant, antigenotoxic, anti-inflammatory, and such other properties of Aloe vera could be the contributing factors to the maintenance of the viability of PDL cells.[11] Our results in the study after 3 h with 30% AVE are in agreement with the previous studies.[9],[10],[11],[17],[18]

However, in the present study, at 24 h also, 7% PE showed a maximum capacity to maintain the viability of PDL cells. Propolis obtained from the Coorg district was evaluated as a storage medium. Previously, it has been shown that minor differences in relation to the qualities and quantities of the active compounds in PE can occur due to climatic variances, several plant sources from diverse geographical locations, and also different species of honeybees.[19] Hence, it is reasonable to say that this material obtained from a different region is effective in maintaining PDL cell viability even up to 24 h. Previous studies have shown that the lower the concentration of propolis, higher are the viability results. The results of this study are in agreement with previous literature where propolis has been reported to be a suitable storage medium for avulsed teeth.[20],[21],[22],[23] However, further evaluation of different concentrations of propolis would be required for more accurate results.

Most of the avulsed teeth are not replanted immediately and need to be stored in a suitable medium until replantation. In this experimental cell culture in vitro model, to mimic the natural circumstances of replantation of teeth, three different time periods were chosen. A number of in vitro studies have been conducted with varying time periods ranging from short-term storage of 30 min[24] to long-term storage of up to 120 h.[25] In the present study, storage time of 1 h, 3 h, and 24 h was chosen to make it more clinically relevant.

This study demonstrated that all three media tested were effective in maintaining PDL cell viability. The limitation in using ice apple as a test media was that over the course of 24 h the solution underwent oxidation and appeared darker in color. This color change could have affected the optical density value of these test media.

However, 7% PE was most effective in maintaining PDL cell viability up to 24 h among the three tested media. In a comparative study conducted by Babaji et al.,[26] it was concluded that propolis showed more viable PDL cells as compared to HBSS, Aloe vera, and pomegranate juice. A recent network meta-analysis also concluded that propolis may be the preferred storage media for storing avulsed teeth for the purpose of preserving the viability of PDL cells before replantation when it is available to actual settings.[27]


   Conclusions Top


Within the limitations of this study, it can be concluded that IAW, 30% AVE, and 7% PE were found to be nontoxic to human PDL cells, and among them, 7% propolis was the most effective medium for maintaining the viability of PDL cells up to 24 h.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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