Assessment of Kangen Water as a Storage Media for Maintaining Viability of Periodontal Ligament Cells of Avulsed Teeth: An Ex Vivo Study

Abstract

Aim and background

Avulsion is the most complex traumatic dental injury (TDI) encountered. Treatment modality is immediate replantation or prompt storage of tooth. Storage is the most common modality. Hanks’ balanced salt solution (HBSS) and normal saline are some investigated and efficient storage media to maintain periodontal ligament cell viability. Kangen water is alkalinized water enriched with minerals; thus, it can be used as a storage media. Therefore, the present study aimed to assess the efficacy of Kangen water as a storage media.

Material and methods

Teeth extracted for orthodontic purposes were included and disinfected. Extracted tissues were transferred to a Petri dish consisting of the respective storage media. Normal saline (pH 5.5): group A and HBSS (pH 7.2): group B, Kangen water (pH 8.5): group C, (pH 9.0): group D, and (pH 9.5): group E were study groups. Samples were stained using trypan blue exclusion staining, and cell viability was assessed at 0.5, 1, 2, 6, 24, and 48 hours using a hemocytometer. Data was analyzed using analysis of variance (ANOVA) followed by post hoc analysis.

Results

Hence, it can be suggested that group B was most efficient in maintaining cell viability at all study time intervals and highly significant on statistical analysis with a p-value <0.05, followed by groups C, D, E, and A.

Conclusion

Kangen water can be used as a storage media/interim transport media. It will be commonly used for domestic purposes. Thus, it will become feasible and easily available.

Clinical significance

Kangen water has properties such as alkaline pH, capable of maintaining cell viability, easily available, and cost-effective. Hence, it can be recommended as a storage media and can be made easily available in areas with a higher incidence of trauma, such as schools, ground, sports complexes, and home.

How to cite this article

Desai A, Kariya PB. Assessment of Kangen Water as a Storage Media for Maintaining Viability of Periodontal Ligament Cells of Avulsed Teeth: An Ex Vivo Study. Int J Clin Pediatr Dent 2025;18(5):539–543.

Introduction

Traumatic dental injuries (TDIs) occur frequently in every individual's life, which mostly affect prominent portions of the face, that is, the nose, teeth, and chin. The worldwide prevalence of TDI in primary dentition is 22.7% and in permanent dentition is 15.2%.¹ Prevalence of TDI children of Indian population is 15% in <6 years old and 13% in >6 years old.²

Among all TDIs, avulsion is the most complex type of injury, which has an impact on a child's general health, well-being, and psychology.³ The periradicular tissue and periodontal ligament cells of the root get damaged and undergo necrosis due to avulsion.⁴ Immediate replantation of the tooth in its socket is the treatment of choice, which preserves periodontal ligament cell viability and has a better prognosis.^5–9 When extraoral dry time increases, the role of storage media comes into play.¹⁰

An increase in extraoral dry time results in dehydration and necrosis of viable cells of the periodontal ligament, making the tooth incompetent for replantation. Extraoral dry time can be reduced by immersing the avulsed teeth in storage media/interim transport media.¹⁰

The objective of storage media is to maintain the viability of periodontal ligament cells, which is achieved by virtue of its characteristics. Ideal properties of storage media should be neutral pH closer to the oral cavity; should not cause any antigen-antibody reaction; should be easily available; should reduce inflammatory reaction and chances of ankylosis after replantation; should have antioxidant properties.¹¹

Among various available storage media such as HBSS, milk, saliva, and normal saline, Hanks’ balanced salt solution (HBSS) is considered to be an ideal storage media. In 1975, Hank discovered this solution and used it for the preservation of tissue culture. The International Association of Dental Traumatology (IADT) has recommended the use of HBSS as storage media for avulsed teeth.¹² But, HBSS is not readily available in the market, and is also not cost-effective. The most readily available and cost-effective storage media is normal saline, but it is not as effective as HBSS.¹⁰

Kangen water is alkalinized water enriched with various minerals, especially hydrogen. It is available in pH values of 7.5, 8.5, 9.0, 9.5, and 11.5. The pH values of 8.5, 9.0, and 9.5 can be assessed as storage media as their pH is closest to the oral environment. It acts as an antioxidant, facilitates healing, and has an antibacterial effect.^13,14 It is supplied from a unit that can be installed for domestic use.¹⁵ Owing to all these properties, Kangen water seems fit to be used as storage media for avulsed teeth. Therefore, the present study aimed to assess Kangen water as a storage media for maintaining the viability of periodontal ligament cells.

Material and Methods

Study Design and Ethical Aspects

The ex vivo study was conducted after receiving approval from the Institutional Ethics Committee (SVIEC/ON/DENT/BNPG20/D21081). Informed written consent with a detailed patient information sheet was obtained from patients whose extracted teeth were procured for the study.

Sample Size

Twenty-five freshly extracted premolars were used to procure the periodontal ligament tissue. The procured tissue was placed in five Petri dishes per group.

Groups

Twenty-five teeth were randomly divided into five groups using the lottery method, that is, five teeth per group. Tissue was procured and transferred to Petri dishes consisting of respective storage media, that is, normal saline (0.9%) (acuLIFE^® Healthcare Pvt Ltd, Ahmedabad, India) (group A) and HBSS (1X) (HiMedia Laboratories Pvt Ltd, Mumbai, Maharashtra, India) (group B); and three different pH of Kangen water (Enagic^® India Kangen Water Pvt Ltd, Bengaluru, Karnataka, India), pH 8.5 (group C), pH 9.0 (group D), and pH 9.5 (group E).

Inclusion and Exclusion Criteria

Atraumatically, freshly extracted premolar teeth for orthodontic purposes were included in the study. Teeth showing signs of caries, periodontitis, periapical lesions, root resorption, cyst or granuloma, and fractured roots were excluded from the study.

Sample Collection and Disinfection

Twenty-five freshly extracted premolar teeth fulfilling inclusion criteria were included. Samples were disinfected by immersing them in povidone Iodine (10%) and saline solution (1:3) immediately after extraction, followed by storage in a sterile Falcon tube (50 mL) consisting of 10 mL phosphate-buffered solution (PBS) during transportation to the laboratory. Teeth were removed from the tube using sterile tweezers, by holding them at the cementoenamel junction (CEJ), and were divided into study groups. BP blade #15 was used to extract attached tissue (i.e., periodontal ligament) from the mesial and distal surfaces of the roots.

Procedure

Tissue procured from the root surface of included teeth were transferred to labeled five Petri dishes per group, comprising 3 mL of storage media groups of the present study for the entire duration of the study. 1 mL of solution from each Petri dish was pipetted and dispensed in the respective labeled Eppendorf tubes. Further, these were placed in a centrifugation machine (Thermo Fisher Scientific Inc., Waltham, USA) and spun at 3,000 rpm for 3 minutes. The supernatant fluid present in the cell suspension was removed.

A 0.4% trypan blue stain was prepared by dissolving 0.4 gm of trypan blue in 80 mL of PBS and was boiled on a slow flame. This solution was cooled down until it reached room temperature. Additional PBS was incorporated to produce a final volume of 100 mL.

A 0.1 mL of solution from the respective Eppendorf tube was pipetted, and stained in another Eppendorf tube utilizing 0.4% trypan blue. A sample from each group was placed in the Improved double ruling Neubauer's chamber one by one. Microscopically, the cells with stained outer membrane were intact and viable, and cells with complete staining were nonviable. Cell counting was carried out by counting viable and nonviable cells in four outer large boxes of the chamber, excluding cells observed adjoining the outer periphery of boxes. Computing was performed with the aid of a light microscope (Labomed Microscope, Ahmedabad, Gujarat, India) at 40× magnification. The same process was followed for the assessment of cell viability in the remaining Petri dishes. Cell viability was assessed at 0.5, 1, 2, 6, 24, and 48 hours. Petri dishes were stored in refrigeration at 4°C between study time intervals.

The total cell count was estimated using a hemocytometer and the cell count was calculated using the formula given below:

Total cell count = No. of cells counted × dilution factor × 10⁴/volume of the chamber (area × depth)

The percentage of viable cells was formulated using the following formula:

Viable cells (%) = Total number of viable cells per mL of aliquot × 100/total number of cells per mL of aliquot.

Statistical Analysis

Data obtained was recorded and tabulated in Microsoft Excel 2021, and subjected to statistical analysis using IBM SPSS Statistics 20.0 (IBM Corporation, Armonk, NY, USA). Analysis of variance (ANOVA) was executed to find the significance of study parameters between the groups, followed by post hoc analysis, to confirm the significance of values obtained from ANOVA. Results on continuous measurements were presented as mean ± SD. The level of significance was fixed at p = 0.05, and any value less than or equal to 0.05 was considered to be statistically significant. Descriptive statistics were computed using Excel statistical operations, and inferential statistics were carried out using SPSS version 21.

Results

The total cell count in study groups at time intervals of 0.5, 1, 2, 6, 24, and 48 hours is mentioned in Table 1.

Table 1.

: Total cell count of periodontal ligament cells in all study groups

Total cell count at different time intervals
Group	A		B		C		D		E		F value	p-value
Duration	Mean	Std. deviation	Mean	Std. deviation	Mean	Std. deviation	Mean	Std. deviation	Mean	Std. deviation
1/2 hour	123.68	1.84	123.24	2.54	106.04	3.41	142.12	2.66	151.46	2.58	223.847	<0.001*
1 hour	115.98	1.87	135.66	2.89	97	1.12	104	3.51	127.78	2.13	213.801	<0.001*
2 hours	119.68	2.67	126.3	3.59	109.6	2.66	135.66	2.96	105	4.35	70.074	<0.001*
6 hours	83.68	2.05	98.7	7.16	118.28	2.6	117.92	2.72	87.24	3.34	83.336	<0.001*
24 hours	89.96	3.09	94.9	2.52	68.26	2.58	77.24	1.85	75.24	2.94	86.907	<0.001*
48 hours	113.1	1.8	119.46	3.07	84.92	2.47	92.86	2.06	65.36	2.67	394.136	<0.001*

^*Highly significant p < 0.001

At half an hour, the highest percentage of viable cells was observed in, group B (74.18 ± 3.31), followed by group A (66.33 ± 1.72), group D (63.25 ± 1.88), group E (62.94 ± 2.14), and group C (53.22 ± 2.18), which was highly significant on statistical analysis (p < 0.001) (Table 2).

Table 2:

Percent of cell viability of periodontal ligament cells in all study groups

Percentage of viable cells
Group	A		B		C		D		E		F value	p-value
Duration	Mean	Std. deviation	Mean	Std. deviation	Mean	Std. deviation	Mean	Std. deviation	Mean	Std. deviation
1/2 hour	66.33	1.72	74.18	3.31	53.22	2.18	63.25	1.88	62.94	2.14	52.802	<0.001*
1 hour	54.98	2.63	63.25	1.9	56.29	2.09	61.31	1.92	55.85	2.32	14.206	<0.001*
2 hours	43.55	2.21	54.98	2.9	52.64	1.45	54.41	2.85	54.51	2.62	18.974	<0.001*
6 hours	40.82	2.2	51.64	2.45	47.36	1.5	44.64	1.78	45.69	2.39	17.693	<0.001*
24 hours	33.4	1.59	49.58	2.42	30.74	1.55	35.28	2.78	35.13	3.03	48.731	<0.001*
48 hours	33.41	2.31	45.64	3.79	35.38	3.48	25.9	2.4	34.1	2.11	29.608	<0.001*

^*Highly significant p < 0.001

At a time duration of 1 hour, more percentage of viable cells was observed in group B (63.25 ± 1.90), followed by group D (61.31 ± 1.92), group C (56.29 ± 2.09), group E (55.85 ± 2.32), and group A (54.98 ± 2.63), which was highly statistically significant (p < 0.001) (Table 2).

At 2 hours, a higher percentage of viable cells was observed in group B (54.98 ± 2.90), followed by group E (54.51 ± 2.62), group D (54.41 ± 2.85), group C (52.64 ± 1.45), and group A (43.55 ± 2.21), which was found to be highly significant on statistical analysis (p < 0.001) (Table 2).

At the time duration of 6 hours, a higher percentage of viable cells were observed in group B (51.64 ± 2.45), followed by group C (47.36 ± 1.50), group E (45.69 ± 2.39), group D (44.64 ± 1.78), and group A (40.82 ± 2.20), which was highly statistically significant (p < 0.001) (Table 2).

At 24 hours, higher viable cells were observed in group B (49.58 ± 2.42), followed by group D (35.28 ± 2.78), group E (35.13 ± 3.03), group A (33.40 ± 1.59), and group C (30.74 ± 1.55), which was statistically significant (p < 0.001) (Table 2).

At 48 hours, more viable cells were observed in group B (45.64 ± 3.79), followed by group C (35.38 ± 3.48), group E (34.1 ± 2.11), group A (33.41 ± 2.31), and group D (25.90 ± 2.40), which was significant on statistical analysis (p < 0.001) (Table 2).

On comparing the mean value and standard deviation of the percentage of viable cells at all time intervals to total cell count, it suggests that HBSS is the most efficient, followed by Kangen water (pH 9.0, 8.5, and 9.5) and normal saline (Fig. 1).

Fig. 1:

Comparison of percent of cell viability of periodontal ligament cells in all study groups

According to the results of all groups, group B (HBSS) was most efficient, followed by group D (Kangen water 9.0 pH), group C (Kangen water 8.5 pH), group E (Kangen water 9.5 pH), and group A (normal saline).

Discussion

The present study assessed the efficacy of Kangen water as a storage media for maintaining periodontal ligament cell viability.

Cell viability can be evaluated by various techniques such as MTT assay, ATP reduction, and hemocytometer. Among these, a hemocytometer is commonly employed. Morten et al.¹⁶ suggested that all methods can be utilized for evaluating cell viability and are sensitive, but a hemocytometer quantifies cell count in the form of cells/mL.

Trypan blue exclusion staining is a technique of labeling cells in which both nonviable and viable cells are differentiated. Kerschbaum et al.¹⁷ observed that trypan blue exclusion is an accurate method for nonviable and viable cells and is a simple technique.

Normal saline (group A) could maintain cell viability at 0.5 and 24 hours. Singh et al.,¹⁸ D'Costa et al.,¹⁹ and Pileggi et al.²⁰ concluded that saline has less capacity to maintain cell viability, similar to the result of the present study. It has demerits, such as it has limited action as storage media, because of a lack of nutrients such as calcium, magnesium, and glucose. Therefore, it has limited application in preserving the periodontal ligament viability of avulsed tooth/teeth.

Hanks' balanced salt solution (group B) was observed to have the most viable cells at 0.5, 1, 2, 6, 24, and 48 hours. Navit et al.,²¹ Saini et al.,²² and Fulzele et al.²³ evaluated HBSS with other storage media and found that HBSS had the most viable cells among other compared storage media. This is in accordance with results of the present study. On the contrary, Babaji et al.²⁴ concluded that maximum cell viability was in propolis, followed by HBSS and aloe vera. Though HBSS is one of the most recommended storage media for avulsed tooth/teeth, its high cost and scarce availability in areas with higher incidence of TDI, its actual application is infrequent.²¹

Kangen water at pH 8.5 (group C) could maintain cell viability at 1, 2, 6, and 48 hours; pH 9.0 (group D) maintained cell viability at 0.5, 1, 2, 6, 24, and 48 hours; and pH 9.5 (group E) maintained cell viability at 0.5, 1, 2, 6, 24, and 48 hours. Results of the present study convey that among groups C, D, and E, group D had a better efficacy in maintaining cell viability.

Kangen water has several beneficial properties, such as acts as an antioxidant, has an antibacterial effect, a few variants have pH values closer to the oral environment, and is composed of ions.^20,21 Therefore, it can be considered as a potent storage media. It has an abundant amount of active hydrogen and oxygen molecules, which are potent in neutralizing free radicals. Because of its alkaline nature, an ample amount of bicarbonates is obtained, which also serves in neutralizing acid produced in the body.²⁵ Kusumakar et al.²⁶ evaluated the effect of Kangen water and reverse osmosis water on dental plaque, salivary pH, and salivary Streptococcus mutans counts. They concluded that alkaline Kangen water, especially with pH 9 effectively reduces microbial count in dental plaque.

There are a few limitations in the present study; more advanced cell analysis techniques should be considered to improve the credibility of study outcomes. More number of cells should be evaluated for accurate results. Single-cell suspension and an automated analyzer can be utilized for accuracy in cell counting.

Conclusion

Hanks' balanced salt solution is considered as a gold standard storage media for preserving the viability of periodontal ligament cells, and the same was seen in the result of present study, but it is not easily available and as well as not cost-effective. Kangen water with pH 8.5, 9.0, and 9.5 showed a significant result in maintaining cell viability. It can be concluded that alkalinized Kangen water can be used as a potent storage media as it is capable of maintaining periodontal ligament cell viability, easy availability, and cost-effectiveness. Further research should be executed employing advanced cell analysis techniques.

Clinical Significance

Kangen water has properties such as alkaline pH, capable of maintaining cell viability, easily available, and cost-effective. Hence, it can be recommended as a storage media and can be made easily available in areas with a higher incidence of trauma, such as schools, ground, sports complexes, and home.

Orcid

Arohi Desai https://orcid.org/0000-0003-4206-649X

Pratik B Kariya https://orcid.org/0000-0001-8240-3142