Evaluation of Cell Viability in Histidine−Tryptophan−Ketoglutarate Solution: A Storage Media for Avulsed Teeth

ABSTRACT

Objective

To evaluate the effectiveness of histidine−tryptophan−ketoglutarate (HTK) solution or Custodiol HTK solution as a storage media for avulsed teeth by testing the viability of periodontal ligament (PDL) cells.

Materials and methods

Ten freshly extracted noncarious premolars from patients undergoing orthodontic treatment were taken as tooth samples, which were immersed in HTK solution immediately after extraction for 30 minutes. Then the apical two-thirds of the root portion was scraped into a Petri dish to collect the PDL cells and treated with type 1 collagenase and phosphate buffer. The samples were centrifuged and stained with tryptan blue, loading it onto Neubauer's chamber, and the percentage of viable cells from the total number of cells was calculated.

Results

Teeth stored in HTK solution demonstrated significantly 58% of total viable cells. All statistical tests were performed at 95% confidence intervals, keeping p-value < 0.05 as statistically significant.

Conclusion

The PDL cells retained their viability significantly when stored in HTK solution; hence, HTK solution can be used as a storage media for avulsed teeth.

How to cite this article

Tamhankar SS, Sahu A, Patil RU, et al. Evaluation of Cell Viability in Histidine−Tryptophan−Ketoglutarate Solution: A Storage Media for Avulsed Teeth. Int J Clin Pediatr Dent 2025;18(6):617–620.

INTRODUCTION

Teeth avulsion is quite common in children, which is the complete displacement of a tooth from the socket. It is a stressful situation in young children and requires both expedient and expert management by a dental practitioner.¹ In such cases, the primary aim should be to preserve the viability of the periodontal ligament (PDL) cells, which are attached to the root, until appropriate management can be done, which may bring about reattachment to the socket.² Viability of the PDL cells depends on various factors like extra-alveolar conditions of the root, storage media, and the time between the injury and reimplantation of the tooth. All these factors determine the outcome.^3,4 However, immediate reattachment may not be possible in many situations. In these conditions, the tooth should be kept in a suitable medium until replantation. An ideal storage medium should maintain or improve the life of PDL cells during the out-of-socket period by preventing desiccation of the cells.³ The medium used is also a determining factor for extending the life of the tooth.⁴

A storage medium is a natural liquid that closely replicates oral conditions to help preserve the viability of PDL cells. Various storage media have been studied, such as Hank's Balanced Salt Solution (HBSS); organ storage solutions like Viaspan, Custodiol, and Euro-Collins; saline; culture media like Minimum Essential Medium (MEM); and commonly available products like saliva, milk, water, propolis, green tea, Morus rubra (red mulberry), egg white, and coconut water. Rehydrating solutions like Ricetral, Gatorade, and contact lens solutions have also been tested.⁵ Histidine−Tryptophan−Ketoglutarate (HTK) solution was first prepared by Bretschneider as a cardioplegia solution in the 1970s.⁴ HTK/Custodiol HTK solution, a high-flow, low-potassium preservation solution, is commonly used in the medical field for organ transplantations. HTK content is similar to that of extracellular fluid that occurs naturally in the body. Histidine aids in buffering; mannitol and tryptophan help improve membrane stability; and ketoglutarate helps in ATP generation during cell reperfusion.⁶ The objective of this study was to check the effectiveness of HTK solution in storing avulsed teeth by testing the viability of periodontal cells.

MATERIALS AND METHODS

Ten extracted noncarious premolars from patients undergoing orthodontic procedures were taken as tooth samples (Fig. 1). The extractions were atraumatic and performed using forceps only, so as not to harm the apical PDL tissue. The sample teeth were immersed in HTK solution (Courtesy: Sandor Medicaids Pvt. Ltd) after extraction for 30 minutes (Fig. 2). Then the coronal two-thirds of the crown portion and one-third of the root portion were scraped to remove debris and periodontium damaged due to the use of forceps for extraction. The apical two-thirds of the root portion was scraped into a Petri dish to collect the PDL cells (Fig. 3). The Petri dish was loaded with 0.5 mg of type 1 collagenase and 2.5 mL of phosphate buffer^7,8 (Fig. 4). The Petri dish was then incubated for 30 minutes in an incubator, and the mixture was transferred to a test tube followed by centrifuging at 1000 rpm for 5 minutes. The supernatant was discarded, and to the residue, equal amounts of 0.4% tryptan blue were added. The resulting mixture was then loaded into the Neubauer's chamber with the help of a micropipette. Following the staining, viable cells appeared pink/colorless and nonviable cells appeared blue when viewed under an optical microscope.

Fig. 1:

Atraumatically extracted premolar used as tooth sample

Fig. 2:

Histidine−tryptophan−ketoglutarate/Custodiol solution

Fig. 3:

Scraping of apical two-thirds root portion with scalpel

Fig. 4:

Petri dish loaded with scrapings, type 1 collagenase, and 2.5 mL phosphate buffer

The determination of the cells was done at ×40 magnification of an optical microscope, and cell counting was performed in a 4 × 1 mm² area of the counting chamber as viable cells (Fig. 5) and nonviable cells (Fig. 6). After collecting the number of viable and nonviable cells from all ten samples, the percentage of viable cells in each sample was evaluated.

Fig. 5:

Viable cells at 40× magnification

Fig. 6:

Nonviable cells at 40× magnification

RESULTS

The amount of viable cells/nonviable cells was measured by the formula: [viable cells / total number of cells stained] × 100. The mean/standard deviation of the number of viable cells was calculated, and statistical analysis was performed. The Chi-squared test of proportion was done to evaluate the p-value.

The teeth stored in HTK solution demonstrated significantly 58% of total viable cells (Fig. 7). The mean of viable cells was observed to be 6.2, nonviable cells 4.7, and the mean of total number of cells was 10.9 (Fig. 8).

Fig. 7:

Percentage of viable cells

Fig. 8:

Mean viable, nonviable and total cells

The data obtained were statistically analyzed using Statistical Package for the Social Sciences (SPSS) software (v. 21.0). Descriptive statistics were performed for viable cells, nonviable cells, total cells, and percentage of viable cells (Table 1). The statistical analysis gave the mean value of viable cells as 6.20 and standard deviation as 3.76, significantly, with the Chi-square p-value as 0.03. The descriptive statistics showed that the mean of percentage of viable cells was significantly 58 and the standard deviation was 8.97, with the Chi-square p-value 0.001 when p-value was kept <0.05. Mean of total cells, which included both viable as well as nonviable cells, showed 10.90 and standard deviation was 6.99, with Chi-square p-value as 0.01. Chi-squared test for proportion was done in order to determine significant differences within each of the viable cells, nonviable cells, total cells, and percentage of viable cells. All statistical tests were done at 95% confidence intervals, maintaining p-value of <0.05 (as statistically significant).

Table 1:

Descriptive statistics of viable, nonviable, and total cells and percentage of viable cells

	N	Minimum	Maximum	Mean	Std. deviation	Chi-square p-value
Viable cells	10	1.00	13.00	6.20	3.76	0.03*
Nonviable cells	10	1.00	11.00	4.70	3.46	0.066*
Total cells	10	2.00	24.00	10.90	6.99	0.01*
Percentage of viable cells (%)	10	45.40	69.20	58.00	8.97	0.001*

*p-value < 0.05 statistically significant. Interpretation—Chi-squared test of proportion showed statistically significant differences among the viable cells and total cells (p-value < 0.05)

DISCUSSION

The worst effect of dental trauma is PDL cell loss, which can cause poor long-term prognosis, even after replantation.² After tooth avulsion, successful healing of reimplantation can be achieved when viable PDL cells are present on the root surface. An ideal medium for avulsed teeth must closely resemble the intraoral environment. So, osmolality, pH, and nutrients required for the metabolism are taken into account when estimating storage media. Also, ideal storage media should possess antimicrobial properties, maintain the viability of PDL cells, be easy to use, and be easily available.⁴

The most important factor for the success of a reimplanted tooth is the preservation of PDL viability during the extra-alveolar period by using an appropriate storage medium. Inappropriately storing can cause PDL cell death, which may lead to ankylosis or root replacement resorption.⁵ Viability of PDL cells that remain on the root surface after trauma is dependent on the availability of cell metabolites. If, within 60 minutes, these metabolites are not supplied to the cell, there would be cell death. To avoid such a situation, it is important for the cells to receive the proper metabolites required for their survival. These metabolites are present in the right storage media, which help in the survival of the periodontal cell when kept within 60 minutes of the dental trauma. The survival may be due to prevention of protein synthesis in bacterial cells, enhancement of the action of fibroblasts, and the healing of connective tissue may bring recovery of the PDL.⁹

A recent study in 2012¹⁰ suggested that a suitable medium is very necessary for a successful outcome. ViaSpan, HBSS, and Eagle's medium have better ability to maintain PDL cells in a viable state, but considering practicality and public availability, they are far from ideal. The outcome of dental reimplantation is directly related to the viability of PDL cells, and its success is dependent on the appropriate use of storage media that is available in the market and used within 30 minutes of dental trauma.¹¹

The differential gene expression profiling of PDL cells after storage in 50% HTK for 2–3 hours showed cell participation in various biological processes, like angiogenesis, cell proliferation, and inflammatory response.⁴ Angiogenesis plays a vital role in both the maintenance of periodontal cells' health and inflammatory development. When the PDL cells were stored in 50% HTK solution, the gene encoding fibroblast growth factor 9 was upregulated 4-fold.¹² However, further elaborated research can be carried out on specific genes involved in maintaining PDL cell viability.

HTK has high flow and contains less potassium. It is used for preserving donor organs, is easier to handle due to in situ perfusion into organs, and results in good postoperative function as well, even with kidneys of nonheart-beating donors.¹³

Viability of the PDL cells in any storage medium can also be checked at different intervals of time. In a review by Udoye et al. similar study used different storage media and compared their effectiveness at different intervals of time and the capacity of different aqueous solutions to maintain PDL cell life in ice and at room temperature. They recommended that the amount of viable cells in all groups exposed to ice was quite less during the initial time, as compared to groups exposed to room temperature. In conclusion, they reported that teeth stored on ice gave better results than at room temperature.¹⁰ In a similar study, aloe vera gel showed satisfactory results when used as a storage medium, when availability of other storage media is an issue.¹⁴ Easy availability of media plays a crucial role, as well as the cost of the storage media. Looking at this, various studies are carried out on natural products that are easily available in the market or in day-to-day life, which have shown better results in in vitro studies, but more experiments are required to estimate their potential in storing under a dynamic oral environment.¹⁵

HTK solution is highly workable, easy to store at 4–8°C in the refrigerator, has a shelf life of 1 year, is cost-effective, and is most commonly and regularly used for organ storage and transportation, but it is rarely used in dental practices. Since children are naturally prone to trauma due to their playful activity,^16,17 in the future, considering the cost-benefit and availability of the material, further studies can be carried out comparing HTK solution with other storage media.

CONCLUSION

In the present study, within limits, the PDL cells retained their viability when stored in the HTK solution. It can be effectively used as a storage medium after dental trauma to reimplant the tooth.