An in vitro evaluation of efficacy of ViaSpan, aloe vera, gatorade solution, and propolis storage media for maintaining the periodontal ligament cell viability.

Background: Replantation is a commonly performed method for avulsed tooth. A vital periodontal membrane (periodontal ligament [PDL]) is significant for the successful healing of replanted teeth. Hence, various storage media are used to preserve the viability of periodontal cells before replantation.
Objectives: The present study was conducted to evaluate the efficacy of ViaSpan, Aloe vera, Gatorade solution, and propolis storage media for maintaining the PDL cell viability. Materials and
Methods: The present study was conducted on 40 recently extracted teeth which were randomly divided into four study storage groups: Group I: ViaSpan, Group II: Aloe vera, Group III: Gatorade solution, and Group IV: Propolis. Later they were subjected to centrifugation, and the cells from supernatant were colored with 0.4% trypan blue for determination of viability. The obtained data were statistically evaluated with SPSS package (21.0 version, Inc.; Chicago, IL, USA) using analysis of variance, Mann-Whitney test, and Post hoc tests.
Results: The mean viable periodontal cell in Group I was 30.2 cumm, in Group II was 24.6 cumm, Group III was 14.5 cumm, and Group IV in 31.4. The difference was significant (P < 0.01). Post hoc test between different groups revealed a significant difference in mean viable periodontal cells (P < 0.001). Propolis, ViaSpan, and Aloe vera had higher pH and osmolality values.
Conclusion: This study found that propolis had higher periodontal cell viability followed by ViaSpan solution and Aloe vera and least in Gatorade solution. Propolis, ViaSpan, and Aloe vera media can be used as a storage media.

INTRODUCTION

Avulsion (tooth loss) injury is one of the most severe forms of dental trauma, which affects the esthetics of the patients.[1] The reported incidence of tooth avulsions ranges from 1% to 16% of all traumatic injuries occurring in permanent dentition.[2] The consequences of these traumas affect neurovascular supply and usually results in loss of pulp vitality.[12]

A vital periodontal membrane (periodontal ligament [PDL]) is significant for the flourishing healing of replanted teeth.[2] Instantaneous reposition of the avulsed tooth may not be possible most of the time. In such cases, avulsed tooth should be stored in an appropriate storage media to avoid dehydration and to preserve the vitality of tooth.[2] The management of tooth avulsion includes instant replantation of tooth in its socket if the periodontal membrane is still vital. Two of the most critical factors affecting the outcome of an avulsed tooth after replantation are extraoral dry time and the storage medium in which the tooth is placed.[3] However, the ability of a storage/transport medium to support cell viability is more important than the extraoral dry time to prevent ankylosis and replacement resorption.[4]

There are different storage mediums available as natural media such as saliva, saline, milk, Aloe vera, pomegranate juice (PJ), propolis, coconut water, and green tea. Synthetic storage media available are contact lens solution, Gatorade, Hank's balanced salt solution (HBSS), Emdogain, ViaSpan, and culture media.[24567] Propolis is a muggy resin that leaks from the buds or bark of trees. It is made up of the resin (rich in flavonoids) (45%–55%), waxes and fatty acids (23%–35%), essential oils (10%), pollen proteins (5%), and additional organic components and minerals. Propolis has antibiotic, antiseptic, antifungal, antiviral, antibacterial, antioxidant, antithrombotic, anticarcinogenic properties. It has tissue regenerative and immunomodulatory properties.[78]

HBSS is an ideal storage media for preserving the viability of PDL cells for up to 24 h at 4°C and at room temperature. The American Association of Endodontists suggested HBSS as a storage medium of preference for the management of avulsed teeth because of its ability to offer long-term maintenance of PDL cell viability.[6] However, it is expensive and not easily available. Hence, other inexpensive and easily available naturally occurring storage media have been tried by many researchers.[24]

ViaSpan is a cold transplant organ storage material. It has an osmolality of 320 mOsm/kg, and its pH is approximately 7.4.[8] It is expensive, short vitality termination, and not easily available. It has been observed that its clonogenic capability is comparable to HBSS after 8 h.[8]

Aloe vera is a cactus-like plant that belongs to the family Liliaceae. The inner gel of Aloe vera consists of more than 75 energetic ingredients.[7] 98–99% of gel is composed of water, and the rest 1%–2% consists of active components such as aloe-emodin, aloesin, aloin, acemannan, aloemannan, naftoquinones, aloeride, flavonoids, methylchromones, sterols, saponin, amino acids, and vitamins. Aloe vera gel has antibacterial, anti-inflammatory, antioxidant, and immune-boosting properties. It has wound healing, cell proliferating potential, and hypoglycemic properties because of its active components.[79]

Gatorade solution is a noncarbonated sports drink frequently used by nonathletes as a snack drink. It has a pH of 3 and 280–360 mOsm/L range of osmolality.[8]

Studies pertaining to natural tooth storage media are scares; hence, the present in vitro study was conducted to evaluate the efficacy of ViaSpan, Aloe vera, Gatorade solution, and propolis storage media for maintaining the PDL cell viability.

MATERIALS AND METHODS

The present study was conducted from April to July 2018 in the Department of restorative dentistry, Al Russ Dental College, Qassim University, Saudi Arabia. The study protocol was approved by the institutional ethics committee IRB. Ref. No. Code#:DRC/009FA/18.

Forty teeth extracted due to orthodontic reason with intact crown and close apices having a healthy PDL were used for the study. Samples size was calculated at 95% confidence interval and 90% power; hence the minimum sample size required was calculated as per group 10 samples and total 40 samples for 4 groups. Inclusion criteria were freshly extracted teeth without cracks, caries, or any pathology, no history of systemic conditions. Exclusion criteria include teeth extraction with periodontal and periapical pathology.

The teeth were randomly separated into four experimental storage groups with 10 in each. Group I had ViaSpan (DuPont Pharmaceuticals, Wilmington), Group II had Aloe vera, Group III had Gatorade solution (GATORADE-Quaker Oats Company, Chicago, Illinois, USA), and Group IV had propolis storage media.

pH level and osmolality determination

pH level of each tested storage media was evaluated using an Orion pH Meter model 720 A (Orion Research, Inc., Boston, MA, USA). Osmolality measurements were determined with a Vapro model 5520 Vapor Pressure Osmometer calibrated from 100 to 500 mosm/kg (Wescor, Inc., Logan, UT, USA).

Commercially available ViaSpan and Gatorade solutions were used for the study, and propolis and Aloe vera were manually prepared as below.

Propolis preparation

Hard propolis was grounded into fine powder using mortar and pestle. Fifty percent propolis was prepared with addition of 50 mg ground propolis powder/250 ml to 0.4% ethanol solution. This solution was mixed thoroughly for 15 min to make a homogenous mix.

Aloe vera extract preparation

The Aloe vera leaf was cleaned carefully and added to 70% ethanol alcohol. Following aseptic procedure, the leaf was split opened, and with the help of scalpel, the viscous gel was scrapped from the inner part of the leaf, followed by homogenizing and filtering by means of a 0.45 μm filter mesh.

Immediately after atraumatic extraction, teeth were washed in sterile saline solution by holding coronal portion of tooth with forceps to remove residual blood. Then, they were held using forceps at the coronal region, and with a curette, coronal 3 mm of PDL was scraped to eliminate damaged cells during extraction procedure. After dry storage for 30 min, these teeth were immersed for 45 min in one of the four experimental media. Teeth were moved to storage solution by grasping only crown part with extraction forceps and by avoiding to bother the viable cells on root surface. Later 2.5 ml of stock solution containing Grade II Dispase and collagenase were added to each tooth and incubated for 30 min at 37°C.[91011] After incubation, 50 μL of fetal bovine serum was added to each tube with the help of micropipette. All tubes were then centrifuged for 4 min at 1000 rpm and supernatant was removed with sterile micropipettes. The cells from supernatant were colored with 0.4% trypan blue for determination of viability. The number of viable PDL cells was counted under light microscope with hemocytometer at ×40 magnification. Uncolored (translucent) cells were counted as viable, and the cells stained in blue were not counted.

In our research, we used trypan blue cell staining method since it is simple and fast to carry out and distinguishes viable cells from nonviable one. Viable cells will have a plain cytoplasm, but a nonviable cell will have a blue cytoplasm. In the present study, PDL cells were treated with collagenase and Dispase Grade II to protect maximum viability and to reduce exposure of cells to active trypan. It has been seen that collagenase and Dispase will help in upholding the utmost cellular integrity.[2]

The obtained data were statistically evaluated with SPSS package (21.0 version, Inc.; Chicago, IL, USA) using analysis of variance, Mann–Whitney test, and Post hoc test. P ≤ 0.5 was statistically significant.

RESULTS

The mean viable periodontal (PDL) cell count in Group I was 30.2 cubic millimeter (cumm), in Group II was 24.6 cumm, Group III was 14.5 cumm, and Group IV was 31.4 cumm [Table 1]. It indicates an expressively higher number of viable PDL cells in propolis (Group IV) compared to others. The difference was significant (P < 0.01). The mean absorbance value at 3 h, 6 h, 12 h, 24 h, 48 h and 72 h in different groups were compared by Mann–Whitney test (P < 0.01). Group IV (31.4 cumm) and Group I (30.2 cumm) had higher mean absorbance value than other groups after 72 h [Table 2]. Post hoc test for comparison between group for periodontal cell viability and time interval revealed a significant difference in mean periodontal cells concentration. There was higher cell viability for propolis and ViaSpan compared to other media (P < 0.001) [Table 3]. Table 4 indicates mean pH level and osmolality. pH value for ViaSpan, Aloe vera, Gatorade solution, and propolis was 7.4, 7.1, 3.2, and 7.6, respectively. Osmolality value for ViaSpan, Aloe vera, Gatorade solution, and propolis was 328, 321, 285, and 345, respectively. Only Gatorade solution has acidic pH with lower osmolality.

Table 1

Comparison of mean viable periodontal cells in each group

Group	Mean (cumm)	SD	P
Group I: Viaspan	30.2	4.09	0.01
Group II: Aloe vera	24.6	3.65
Group III: Gatorade solution	14.5	3.72
Group IV: Propolis	31.4	3.24

Test used - ANOVA significance: P<0.01. SD=Standard deviation

Table 2

Mean absorbance values at different time intervals

Time	Group I	Group II	Group III	Group IV	P
3 h	27.5	24.2	13.5	29.4	0.01
6 h	28.6	24.2	13.7	30.5
24 h	29.4	24.6	14.3	30.7
48 h	30.1	24.4	14.4	31.2
72 h	30.2	24.6	14.5	31.4

Mann–Whitney test P<0.01

Table 3

Comparison between group for periodontal cell viability and time interval

Tukey’s HSD	Significant
Group I versus
Group II	0.004
Group III	0.001
Group IV	0.235
Group II versus
Group III	0.323
Group IV	0.001
Group III versus
Group IV	0.001

Test used - Post hoc test. Significance: P<0.001. HSD=Honestly significant difference

Table 4

Mean pH level and osmolality value of experimented storage media

Storage media	pH level	Osmolality (mosmol)
Viaspan (Group I)	7.4	328
Aloe vera (Group II)	7.1	321
Gatorade solution (Group III)	3.2	285
Propolis (Group IV)	7.6	345

DISCUSSION

Various media have been used for storage of avulsed teeth. The ideal storage media for an avulsed tooth should have a neutral pH, physiological osmolality, low bacterial content, with essential nutrients.[2]

In our study, we observed the highest viable PDL cell count with propolis, followed by ViaSpan, and Aloe vera and least with Gatorade solution.

Babaji et al. assessed the effectiveness of propolis, HBSS, PJ, and Aloe vera in maintaining the viability of PDL cells. They found higher cell viability with propolis compared to Aloe vera and HBSS;[2] this is similar to our results. In contrast to our results, Sanghavi et al. concluded from their study that number of PDL cell viability was greater in coconut water compared to 50% propolis and ORS.[10] Moreover, Osmanovic et al. done a systemic review from 15 selected studies out of 5701 records on tooth storage media to maintain PDL cell viability, and found ViaSpan as a best media, followed by Dulbecco's Modified Eagle Medium (DMEM) and 10% propolis.[6] Hiltz and Trope have evaluated milk, ViaSpan and HBSS storage media for avulsed tooth and found ViaSpan as a suitable media.[12] Several researchers observed ViaSpan as the best storage medium and found 37.6% of living cells after 18 h. They stated that ViaSpan and HBSS solution are equally effective as a storage medium.[1213]

We found the higher mean absorbance value for propolis and ViaSpan after 72 h compared to other groups. There was significantly higher periodontal cell viability and time interval for propolis and ViaSpan compared to Aloe vera and Gatorade solution. These findings are similar to Sanghavi et al. study.[10]

Badakhsh et al. evaluated the efficiency of several concentrations of Aloe vera in contrast to egg white and DMEM. They found Aloe vera was more effective than other media and suggested that Aloe vera at 10%, 30%, and 50% concentrations can be used as a storage media.[9] Abraham et al., evaluated the effectiveness of Aloe vera, milk, and HBSS in preserving the viability of PDL cells. They found the highest viable ligament cells in HBSS, followed by milk and Aloe vera.[14] Sharma et al. evaluated Aloe vera with egg white and milk, and they found Aloe vera better than the other two media.[15]

Anegundi et al. compared the milk, Gatorade, coconut water, saline, tap water, egg white, and contact lens solution for preservation of pdl cell viability, and they found higher cell viability with milk followed by contact lens solution and least with Gatorade.[16] We also found Gatorade as least desirable compared to other tested media.

We observed higher pH and osmolality with propolis, ViaSpan, and Aloe vera, while Gatorade solution showed acidic pH.

Efficacy of the storage media depends mainly on osmolality value and pH level of the medium. The optimal cellular proliferation happens at pH level of 7.2–7.4 and an osmolality in range of 230–400 mosmol/kg. In the present study we have observed, higher cellular viability in propolis, ViaSpan, and Aloe vera groups owing to their high osmolality and pH value and due to their antibacterial, tissue healing capacity. ViaSpan has a capacity to re-establish the vitality of damaged pdl cells.[17] Aloe vera plant is familiar in wound healing supporter, and it consists of necessary nutrients for survival of cells.[9] the current study suggests that Gatorade is not an appropriate storage medium for avulsed teeth because of its low pH and high osmolality, which can result in cell death.[817]

Following are the drawback of the study; sample size was small, this study does not compare the result with HBSS storage media, and it was in vitro study. Further long-term in vivo studies are required to evaluate and compare with other storage media.

CONCLUSION

In our study, we have compared natural media, such as propolis and Aloe vera, with artificial media, such as ViaSpan and Gatorade solution. We have selected natural media as they have higher pH, easily available, and inexpensive compared to artificial media. This article helps to save tooth using natural storage media. The present research found that propolis had a higher periodontal cell viability followed by ViaSpan and Aloe vera and least in Gatorade solution. Therefore, propolis, ViaSpan, and Aloe vera can be used as a storage media for maintaining the viability of the PDL of avulsed teeth.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.