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ORIGINAL ARTICLE
Year : 2018  |  Volume : 8  |  Issue : 1  |  Page : 19-24

Evaluating the effectiveness of rehydrating solutions in preserving periodontal ligament cells vitality: An in vitro study


1 Department of Pedodontics and Preventive Dentistry, Panineeya Institute of Dental Science and Hospital, Hyderabad, Telangana, India
2 Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh, India
3 Department of Pedodontics and Preventive Dentistry, SNDC, Vijayawada, Andhra Pradesh, India

Date of Web Publication10-Jan-2018

Correspondence Address:
Dr. A J Sai Sankar
Department of Pedodontics and Preventive Dentistry, Sibar Institute of Dental Sciences, Guntur, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/sej.sej_37_17

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  Abstract 

Background and Purpose of the Study: Two of the most critical factors affecting the prognosis of an avulsed tooth after replantation are extraoral dry time and the storage medium in which the tooth was placed before treatment could be rendered. The ability of a storage medium to support cell viability of the periodontal ligament (PDL) can be an important factor in the prognosis, and thus, the aim of this study was to evaluate the efficacy of commonly available storage media.
Materials and Methods: Cultured PDL cells of sixty freshly extracted human teeth were randomly divided into four groups of 15 each, Group I: Electral solution, Group II: Ringer's lactate, Group III: Oral rehydration salt liquid (ORS-L), and Group IV: Coconut water. Samples in each group were further divided into three subgroups depending on the time duration they were placed in the storage medium, i.e., 1, 4, and 8 h. Further these samples were subjected to Collagenase assay, the cells were stained with trypan blue and viable PDL cells were counted under light microscope.
Results: Statistical analysis showed that Group II demonstrated significantly (P < 0.001) more viable PDL cells in than Groups IV and I, least viable PDL cells are seen with Group III samples.
Conclusion: Ringer's lactate maintained highest PDL cell viability followed by coconut water, electoral solution, and ORS-L at various time intervals. More studies with large samples are required to prove the efficacy of Ringer's lactate as viable storage media.

Keywords: Avulsed teeth, coconut water, oral rehydration solutions, periodontal ligament fibroblasts, Ringer's lactate, storage media


How to cite this article:
Mahesh C, Sai Sankar A J, Srideevi E, Charishma B, Manoj Kumar M G, Radhika M. Evaluating the effectiveness of rehydrating solutions in preserving periodontal ligament cells vitality: An in vitro study. Saudi Endod J 2018;8:19-24

How to cite this URL:
Mahesh C, Sai Sankar A J, Srideevi E, Charishma B, Manoj Kumar M G, Radhika M. Evaluating the effectiveness of rehydrating solutions in preserving periodontal ligament cells vitality: An in vitro study. Saudi Endod J [serial online] 2018 [cited 2018 Oct 23];8:19-24. Available from: http://www.saudiendodj.com/text.asp?2018/8/1/19/222758


  Introduction Top


Reimplantation of an avulsed tooth is the most desirable physiologic and also carries an inherent psychological benefit to the patient when compared to the replacement of lost tooth with prosthesis. Even at the instances of successful reimplantation the longevity of outcome cannot be guaranteed. Many variables found to influence the long-term success of such reimplanted teeth which include amount of physical damage occurred to the root surface at the incidence of trauma, type of medium in which exarticulated tooth is stored and the time lapse between exarticulation and reimplantation. All the above aspects directly or indirectly influence the number of viable periodontal ligament (PDL) cells at the time of reimplantation,[1],[2],[3] which is a key factor that directly determine the ultimate success.[3],[4]

Number of natural and synthetic solutions have been endeavored for preserving PDL cells and reported fluctuating success, as a part, these studies have evidenced that the vitality of thus preserved PDL cells is determined by the composition of storage medium, its temperature, and duration of storage. The recent research has shown that along with the composition other dynamics which got tremendous influence on the capability of storage media would be the pH and osmolality.[5],[6]

Although Hank's balanced salt solution (HBSS) and Viaspan have been recommended by many authors as an ideal storage media for avulsed teeth, lack of their ready availability becomes an obstacle in selecting them.[7],[8] Thus, the search for readily available solutions which could act as storage media for avulsed teeth is progress. The rehydrating solutions which contain solutes to preserve the cell metabolism at the instances of dehydration may help in preserving periodontal cell viability at the instances of tooth avulsion. The factors taken into consideration were composition, pH, osmolality, and easy availability.

The Ringer's lactate which is an efficient intravenously administered rehydrating solution at the instances of burns and trauma was selected as storage solution. For comparison purpose, two commercially available oral rehydrating solutions - electral and oral rehydration salt liquid (ORS-L), and one natural rehydrating agent such as coconut water has been selected for the study. The aim of this study was to evaluate and compare the PDL cell vitality of extracted human teeth in different solutions at various time intervals.


  Materials and Methods Top


Sixty intact (n = 60) freshly extracted human premolars with closed apices that were extracted for orthodontic purpose were selected for the study. Extractions were performed as atraumatically as possible. After extraction, the teeth were held with forceps at the coronal region, and the coronal 3 mm of PDL tissue was scraped with a curette.

Selected teeth were then randomly divided into four groups of 15 (n = 15) each depending on experimental storage solution used. Group I: Electral powder (FDC Ltd., India), Group II: Ringer's lactate solution (Claris Life Sciences Ltd., India), Group III: ORS-L (Jagdale Health Care, Health Care Division of Jagdale Industries Ltd., Bengaluru, India), and Group IV: Tender coconut water.

These four groups were further divided into three subgroups (A, B, and C) of 5 teeth in each depending on the duration of storage (1, 4, and 8 h, respectively). To prepare electral solution, 1 teaspoon of powder was dissolved in 200 ml (one glass) of distilled water just before tooth immersion.[9] Ringer's lactate, ORS-L, and coconut water were freshly opened as and when required.

Five samples from each group were kept in one of the designated storage media for prescribed period after a dry period of 30 min. Then, stored teeth were transferred to a sterile test tube containing freshly prepared 2.5 ml solution of 0.2 mg/ml of type II collagenase (High Media Laboratories, LBS Marg, Mumbai, India) in phosphate-buffered saline (High Media Laboratories, LBS Marg, Mumbai, India). These test tubes were placed in an incubator for 30 min to simulate room temperature. After declared 30 min of incubation, 50 μL of fetal bovine serum (High Media Laboratories, LBS Marg, Mumbai, India) was pipetted into each test tube to halt the enzymatic activity. The solution containing the cellular suspension was transferred to another test tube and centrifuged (Centrifuge REMI R-4C) for 4 min at 1000 rpm. The supernatant formed at the top of the test tube was drawn off with a sterile micropipettes and precipitate collected at bottom was utilized. One drop of sediment was pipetted onto a glass slide, to which a drop of 0.4% trypan blue dye solution (High media Laboratories, LBS Marg, Mumbai, India) was added, mixed and then loaded onto the Neubauer chamber (Marienfeld, Germany).

The number of viable cells in a grid of Neubauer chamber was counted under a light microscope at ×40 magnification. The live cells possess intact cell membrane that excludes penetration of certain dyes; on the contrary, dead cells take up the stain [Figure 1]. The whole experimental procedure including counting of vital cells was performed by a single operator. However, to avoid bias in the results, cell counts of few samples were randomly reevaluated by another examiner who was unaware of the earlier values. As the inter-examiner variability was <0.05 (not significant), the values derived by the first investigator were only considered and the values thus obtained were tabulated and subjected to statistical analysis. The mean and standard deviations were calculated by descriptive statistics, and the groups were compared by using ANOVA with post hoc test. The P value was taken as significant when it was <0.05.
Figure 1: Photo micrograph showing viable (red arrow) and nonviable (yellow arrow) periodontal ligament cells

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  Results Top


[Table 1] exhibited viable PDL cell counts at different time intervals in various experimental solutions along with their mean and standard deviations.
Table 1: Intra-group comparison of viable periodontal ligament cells at various time intervals

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Intra-group comparison revealed statistically significant difference in the mean PDL cell viability with respect to duration of storage (<0.001). ANOVA with post hoc test analysis revealed that mean cell viability at 1 h was significantly higher than at 4 and 8 h intervals. Similarly, cell viability at 4 h was significantly higher than 8 h.

The inter-group comparison among tested solutions revealed that at all the tested time periods (1, 4, and 8 h), Group II demonstrated significantly higher mean viable cell counts than Groups I, III, and IV. Similarly, Group IV had significantly higher mean viable cells than Groups I and III. Likewise, Group I had significantly higher mean viable cells than Group III [Table 2].
Table 2: Inter-group comparison of viable periodontal ligament cells at various time intervals

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  Discussion Top


The complications and outcomes of tooth avulsion and its re-implantation do not require much of illustration as it is a well-known fact for the dentists worldwide. To achieve reasonable success at these instances, many attempts were made to tackle the critical stages after avulsion and before reimplantation. The critical being extraoral time and the most precarious aspects are how the tooth being handled at this crucial period. The role of storage media at these vital hours is indispensable.[10]

At the instances of tooth avulsion, some amount of damage to the PDL is inevitable. However, if the vitality of the cells in undamaged portion is maintained, the mitogenic and clonogenic potential of these cells assist in proper healing of PDL apparatus, and thus, it is ideal to reimplant the tooth immediately. The scene at the site of trauma, lack of awareness, and many more reasons often hamper immediate reimplantation.[3],[11] This delay in reimplantation causes drying of PDL cells which elicits a diffuse area of severe inflammatory response ultimately leading to reimplantation failure.[10],[12]

Thus, an ideal physiological storage medium should play an influential role by supplying essential nutrients to the vital cells and maintain their metabolism. On the other hand, the medium should wash out all damaged necrotic and dying cells and their inflammatory stimulators.[13] To accomplish these sanctified properties, the storage media should possess few essentialities. The composition of media should be able to provide required nutrients to the cells so that the metabolism of the cells is maintained, and also, the optimal pH and osmolality of storage media are important for maintaining physiologic and metabolic health of live cells.[10]

In the literature, many natural and synthetic solutions have been tested as storage media and few more have been exclusively developed for this purpose and reported varying degrees of success rates. Although HBSS (recommended by the American Academy of Endodontics), Viaspan, and Eagle's medium have greater potential to maintain the PDL cell viability, lack of their ready availability to the general public make them less ideal.[5],[10],[14]

Ringer's solution was first introduced by the physiologist Sidney Ringer in 1882 for preserving the frog's heart.[15],[16] Since then, it has been used to restore circulating blood volume in victims of burns and trauma. Later studies have demonstrated that lactate was as effective as glucose in preserving retinal vascular endothelial cell viability and then Hartmann introduced lactated Ringer's solution.[17],[18] In the present study, Ringer's lactate was selected because of its few noticed similarities in composition with HBSS,[19] reported tissue friendly properties of its pH (6.2–7.5), osmolality (274 m OSM/L), and its incredible property of rehydration.[20] For comparison purpose, other natural and synthetic rehydration solutions with considerable osmolality and pH such as coconut water and ORS solutions have been chosen.

The PDL fibroblasts behave differently in cell cultures in terms of growth rate, levels of alkaline phosphatase, protein synthesis, and collagen production than human fibroblasts obtained from other tissues;[21] hence, the PDL cells from extracted teeth were selected. After extraction, coronal 3 mm of PDL was scrapped to remove the cells that might have been damaged during the process of extraction.[2],[4],[22],[23] Many researchers evaluated the critical extraoral dry times; Andreasen and Hjorting-Hansen [24] reported that teeth replanted within 30 min had a better success rate. A study by Söder et al.[25] has shown that severe cell damage occurs at a dry period of 60 min, and at 2 h, it would not be possible to demonstrate any cell viability. In the absence of major injuries, 30 min would be a reasonable time for an injured patient to seek dental attention.

Cell viability can be determined by various methods which include Trypan blue dye exclusion test, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and neutral red uptake assay. In the present study, trypan blue dye exclusion assay was followed as it is most sensitive, quick, easily performed and distinctively differentiates nonviable cells.[26] To assess the relationship between cell viability and time period, the samples were tested at various time intervals (1, 4, and 8 h).

The intra-group comparison yielded highest mean viable cell counts at 1 h when compared to 4 and 8 h interval for all the tested solutions, and this time dependent decrease in mean viable cell counts was statistically significant (P < 0.001). Thus, the results of the study has reconfirmed the fact that irrespective of storage media, the cell destruction is directly proportional to the duration of extraoral time.[24],[25]

On inter-group comparison, the highest number of mean viable PDL cells was obtained with Ringer's lactate solution (Group II) at all the tested time periods. The mean viable cell count obtained in the present study with Ringer's lactate solution at 1 h duration was 906.40 ± 60.50. The existing studies in the literature pertaining to HBSS have reported the mean viable cell counts in the range of 1387–250.[2],[4],[9],[27],[28] Accordingly, in the respective individual studies, their positive controls had shown the mean viable cell counts almost double to the derived experimental values. The wide variation in the cell counts among different studies can be attributed to the different study protocols. The viable cell counts obtained in the present study with Ringer's lactate solution at 1 h duration was comparable to the upper range of reported values with reference to HBSS. These findings are in accordance with the studies of Buffa et al.[18] and Bharath et al.[29] The attributable reason for this could be the presence of components such as sodium chloride, calcium chloride, and sodium bicarbonate in similar concentrations both in the Ringer's lactate solution and that occur in body fluids. In addition, it is isotonic and the osmolality of 278 mOsm/L and pH of 7.2 makes it more physiologic.

Next to Ringer's lactate solution, coconut water (Group IV) has proven to be effective among the tested solutions. The coconut water being liquid endosperm of the coconut, extensively been studied recently due to its emphasized nutritive, antioxidant, and growth promoting characteristics. These characteristics have been attributed to its physiological nature (with optimum osmolality of 372 mOsm/L) and composition which include substances such as sugars, proteins, vitamins, minerals, and growth factors, i.e., essential amino acids such as lysine, cystine, phenylalanine, histidine, and tryptophan along with antioxidants and cytokines. It is readily accepted by the human body and is a safe means of rehydration particularly in patients suffering from potassium deficiency. These characteristics may explain its ability to preserve the vitality of the cells.[7],[30],[31] Studies conducted by Mojumdar (1951),[32] Edirweera [33] indicate that it is sterile when injected intravenously, it did not cause any hemolysis thus supporting its physiological acceptability. The mean viable cell count obtained in the present study at 1 h storage in coconut water was 555 ± 36.49. In two different studies conducted by Gopikrishna et al.,[27],[30] the mean viable cell counts reported were 525.00 ± 9.51 and 532.8 ± 9.7, and thus, the values obtained in the present study were comparable with these studies. Accordingly, Sanghavi et al.[31] have compared coconut water with propolis and ORS and shown that the coconut water preserved more number of cells compared to propolis and ORS. However, contrasting results were reported by Moreira-Neto et al.,[34] Souza et al.[35] by comparing coconut water with milk and HBSS and shown that coconut water is inferior to HBSS and milk in maintaining cell vitality. The variation in the results can be attributed to different methodologies and solutions that were selected for comparison. Despite many desirable qualities of coconut water, its comparative less success rate can be attributed to its acidic pH (4.7).

Electral powder and ORS-L are electrolytes containing the ORSs which were strongly recommend by WHO for children suffering from dehydration. Its composition includes sodium 75 mmol (1725 mg), glucose 75 mmol (13.5 g, 1.35%), potassium 20 mmol (782 mg), chloride 65 mmol (2801 mg), and base (citrate) 10 mmol and a sweetener, with osmolality around 245 mOsm/L. Studies by Mousavi et al.[36] and Rajendran et al.[22] reported the ability of oral rehydration solutions to retain PDL cell vitality, similar to HBSS and better than milk.

The mean number of viable cells obtained with electral and ORS-L in the present study was 496 ± 31.62 and 385 ± 31.32, respectively. In two different studies on ORS-L by Subramaniam et al. reported a mean viable cell count of 513.5 ± 19.2 and 343.5 ± 11.66.[4] Thus, when it is viewed in terms of mean viable cell count, the obtained values in the present study were comparable with that counts reported in the literature. However, the mentioned studies reported ORS-L to be more or less effective as HBSS in preserving the PDL cell vitality.[22],[36] However, the results of the present study were contradictory, where the mean viable cell counts in ORS-L were significantly less compared to other experimental solutions.

Among the four tested solutions, the Ringer's lactate and coconut water can be considered as most suitable solutions in terms of preserving PDL cell vitality as viable cell counts obtained with Ringer's lactate solution were almost comparable to HBSS. Although the coconut water preserved less number of cells compared to Ringer's lactate considering its easy availability and familiarity to the general public, its use at the instances of tooth avulsion can be promoted while providing dental education to the public. Although the study has proven the effectiveness of Ringer's lactate, it is only relative, even in the study design, positive control has not been incorporated probably making less number of the cells available for the experiment; also with the procedures employed in the present study, we could only differentiate viable and nonviable cells but their mitogenic and clonogenic potential which are the determining factors in reestablishment of vital PDL apertures is not known.


  Conclusion Top


The present study definitely evidenced the effectiveness of Ringer's lactate as storage solution when compared with other solutions. Whatever is the storage medium used, the duration of extraoral time has a definite negative influence on mean viable cell counts.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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