INTRODUCTION:

Traumatic dental injuries are the most common reason for emergency appointments in dental practice. 18% of injuries in preschool children are due to oral trauma out of which 1%–16% are avulsion¹. Avulsion commonly seen between the age group of 7 and 10 years². Avulsion is characterized by the complete displacement of the tooth from its alveolar socket. The ideal procedure for an avulsed tooth is immediate replantation at the site of trauma to maintain PDL cell viability. However, factors such as the patient's conscious state, and lack of first aid knowledge at an accident site make it difficult to immediately replant³. The length of extra-alveolar time, the preservation of the root, and the storage medium in which the tooth is stored all affect the viability of PDL cells, which significantly impacts the likelihood of successful replantation¹.

A storage medium is a physiological solution that closely mimics the oral environment to help PDL cells maintain viability. When periodontal ligament cells are removed from their alveolar socket, these treatments prolong their survival.⁴Immediate replantation of avulsed tooth positively impacts the viability of PDL cells which results in 85% of PDL healing⁵. Reimplantation of the tooth must be within 5 minutes ensuring the normal functioning of PDL cells The ability of cells to develop into fibroblasts is lost after exceeding fifteen minutes of dry time. Almost all PDL cells still present on the root of the tooth will begin to become necrotic within 30 minutes of dry storage⁶.

EFFECT OF STORAGE MEDIA ON PERIODONTAL HEALING:⁶

When a tooth is knocked out and before it is replanted, it goes through a period of drying out. To ensure that the cells responsible for supporting the tooth are still alive, it's important to reimplant the tooth as soon as possible. To prevent the tooth from drying out, it should be stored in appropriate storage media. Numerous clinical trials stated that PDL and pulpal healing are vastly dependent on storage media and period. If a tooth is avulsed, it should be immediately kept in an appropriate storage media which keeps the attached PDL cells in a hydrated state and maintains their viability. This allows healing with the regeneration of PDL cells when replanted. If excessive dryness occurs in PDL cells it will often lead to inflammatory resorption or replacement resorption (ankylosis)⁷. If immediate replantation of a tooth is not possible, using the right storage medium can help maintain the vitality of the periodontal ligament (PDL) cells. In order for a storage medium to be suitable for avulsed teeth in a clinical setting, it must have specific properties. This improves the chances of successful replantation^6.

IDEAL REQUIREMENTS OF A STORAGE MEDIUM FOR AVULSED TEETH⁵:

Ideal storage medium should:

· Should possess antimicrobial characteristics.

· Able to preserve the feasibility of cellular PDL.

· Should be able to maintain the viability of periodontal fibers for at least 24 hours.

· Should favor the proliferative capacity of cells and should have the same osmolality as that of body fluids.

· Should not react with body fluids.

· Should not produce any antigen-antibody reactions.

· Reduce the risk of post-reimplantation root resorption or ankylosis.

· Should possess a good shelf life.

· It must be effective in different climates and different conditions.

· Wash away foreign matter and toxic waste.

· Should aid in the reconstitution of depleted cellular metabolites

CLASSIFICATION OF TRANSPORT/STORAGE MEDIUM ²

Different types of storage media for avulsed teeth are classified under two broad categories: Natural and Synthetic (Flow chart 1)

Natural	Synthetic
Tap water	Saline solution
Saliva	Hank’s balanced salt solution
Milk	Gatorade
Egg white	Ricetral
Propolis	ViaSpan
Coconut water	Contact lens solution
Green tea extract	Eagle’s minimal essential medium
Soy milk	Culture medium
Morus rubra fruit	Probiotic solution
Emdogain	Custodial
Dentosafe	Growth factors
Pomegranate	Casein phosphopeptides
Aloe vera	Catalase supplementation
Rice water-conditioned media
Patient’s own serum	Euro Collins
Dragon’s blood sap	Ascorbic acid
Cranberry	Levodopa
Bakul	Cryoprotective agent
Plum	Castor oil
Guava
Basil (Tulsi)
Saliva Officinalis extract

· Tap water:

The pH and osmolality of tap water is 7.4–7.79 and 30 mOsm Kg. The use of tap water to preserve avulsed teeth is not advised since it is more bacterially contaminated, is non-physiological, and incompatible with PDL cells due to its hypotonic osmolarity, which results in cell lysis and replacement resorption of avulsed teeth⁸.

· Saliva:

Saliva is more effective than tap water, but it is a potential source of bacterial contamination for PDL cells. The osmolality of saliva is 60-70 mOsm/kg. Cells are swollen and membranes get damaged when stored in saliva. Ease of availability is the only advantage. Avulsed tooth stored in the saliva of the buccal vestibule is always better than transporting the tooth under dry conditions⁹.

· Milk:

As milk is readily available it can be used as a storage/transport medium for the short-term storage of avulsed teeth. Its pH is 6.5 to 7.2 and its osmolality of 270 mOsm kg, which is similar to extracellular fluid. It can maintain PDL cell viability for up to 2 hours. Using chilled milk with an ice pack or refrigerator reduces cell swelling, increases cell viability, improves recovery, and can be kept for a longer duration¹⁰.

· Honey milk:

Recently honey milk has been studied as a storage medium due to its long shelf life. Honey milk consists of 8% non-fat solid milk, 3 grams protein, 11gram carbohydrate, 0.1 grams of calcium, 0.6 grams of minerals 0.12 grams of phosphorous and natural honey 5%. It has extended storage capability up to 6 months without refrigeration. Compared to HBSS, honey milk can be used as an appropriate storage medium, better than fresh milk¹¹.

· Egg white:

The pH of egg white is 8.6–9.3 and its osmolality is 258 mOsm kg-1. There is no significant difference between HBSS and egg white at storage times of 1, 2, 4, 8, and 12 h and egg white was more suitable than water and milk¹⁰. de Sousa et al.¹²conducted a microscopic study and found that there was no difference between egg white, milk, and artificial saliva.

· Coconut water:

Coconut water is biologically pure, sterile, and rich in amino acids, proteins, vitamins, and minerals. Natural isotonic fluid with a pH of 4.1, is naturally available directly from the coconut, having long-shelf life. Coconut water has a greater capacity to maintain PDL cell vitality when compared to propolis, HBSS, and milk¹³.

· Rice water:

Due to its great nutritional value, rice water was a popular energy drink in the past. It contains high levels of potassium, niacin, thiamin, iron, and zinc while being low in sodium. It assists with collagen formation and prevents acute inflammation. Studies using various storage mediums revealed that rice water kept PDL cells viable even after 30 minutes².

· Patient’s serum:

During the time between avulsion and replantation, the tooth stored in the patient's serum was found to have viable PDL fibers. Histological examination of the avulsed tooth stored in serum showed that the tissues were well stained and indicated viable PDL cells².

· Green tea extract:

Green tea is commercially and easily available at the site of the accident. It is prepared by boiling 10 grams of green tea leaves in 100 ml of distilled water and boiling for 5 minutes and then the extract is filtered. The osmolality and pH of green tea extract also are not ideal, but experiments showed the best ability for storage of avulsed teeth amongst HBSS, tap water, milk, commercial green tea, and green tea extracts¹⁴.

· Emdogain:

Emdogain is available commercially as an Enamel Matrix Derivative (EMD), which is derived from the porcine enamel of developing embryos and contains several matrix proteins. It can affect PDL cells' ability to migrate, adhere, proliferate, and synthesize substances. Along with topical glucocorticoids and systemic doxycycline, it has also been utilized in anti-resorptive regenerative therapy. When applied to the avulsed tooth's root surface and/or placed directly into the alveolar socket before implantation, Erdogan has been demonstrated to increase the incidence of repaired PDL. It seemed to help prevent or delay ankylosis and root resorption¹¹.

· Pomegranate juice:

Pomegranate is regarded as "a pharmacy unto itself" in Ayurvedic medicine. It has strong anti-inflammatory, antioxidant, and anti-carcinogenic qualities. Pomegranate affects the growth of fibroblast cells¹⁵. At lower concentrations (1% and 2.5%) the proliferative effect is found for 1 hour but at higher concentrations (5% and 7.5%) a general proliferative effect is seen. There’s a peak increase in cell viability up to 6 hours. It also enhances cell attachment. Pomegranate juice and HBSS can preserve the spindle-like morphology of periodontal fibers for up to 24 hours after storage. As a result, it could be a useful storage medium¹⁶.

· Aloe vera:

Aloe Vera is a medicinal cactus plant. It consists of 98–99% water and 1–2% active ingredients (aloecin, aloin, aloe-emodin, aloemannan, acemannan, aloeride, methylchromone, flavonoids, naphthoquinones, saponins, sterols, amino acids, vitamins, etc.). It is anti-inflammatory, antioxidant, antibacterial, antifungal, and anticarcinogenic, and its antioxidant properties help to maintain the viability of PDL cell fibers^2,14. Badaksh et al.¹⁷studied aloe vera at concentrations of 10%, 30%, and 50% and found it comparable to cultures supplemented for up to 9 h and superior to egg whites. Accordingly, aloe vera can be used as a preservative.

· Propolis:

Propolis is a natural product that bees use to build and maintain their hives. It possesses anti-inflammatory, antibacterial, anti-oxidant, anti-fungal, and tissue-regenerating properties18. It can inhibit the late stages of osteoclast maturation and thus may be useful as an intracanal medicament to reduce the resorption of traumatized teeth. According to recent literature, propolis can be used to treat avulsed teeth, preferably stored for 6 6-hour to 60-minute storage period. The major disadvantage is that it is not easily available and thus has little value as a storage medium for avulsed teeth¹⁰.

· Dentosafe:

The brand name for Dentosafe is a tooth rescue box and includes a unique cell culture medium. It has vitamins, carbohydrates, and amino acids. If unopened, the shelf life is three years. According to Pohl et al. when avulsed teeth are placed in a Dentosafe solution, functional healing is observed. It is strongly advised that Dentosafe be added to the emergency kit at any locations where avulsion incidents are frequent, such as schools, sporting venues, and ambulances^19. Dentosafe is simple to use and understand for the average person. Despite being an extremely efficient media, it is still not widely accessible in many nations².

SYNTHETIC STORAGE MEDIA:

· Saline:

The solution in normal saline contains 0.90% w/v sodium chloride and has an osmolality of 280 mOsm/kg. Despite being compatible with PDL cells, it lacks vital minerals including magnesium, calcium, and glucose that are necessary for PDL cells to function normally metabolically. After 4 hours of storage²⁰, Moreira N et al.²¹ reported that 55% of the grown cells were still alive, and Pileggi et al.¹⁶ discovered that only 20% of the PDL cells were dead after 45 minutes of being kept in this medium.

· Hank’s balanced salt solution:

John H. Hancock developed the HBSS solution for tissue culture preservation in 1975. It is a widely used standard solution for the growth of cells in biomedical research. The pH and osmolality of HBSS are 7.2 and 320 mOsm/kg, making it non-toxic and biocompatible with periodontal ligament cells. 8 grammes of sodium chloride, 0.4 grammes of D-glucose, 0.4 grammes of potassium chloride, 0.35 grammes of sodium bicarbonate, 0.09 grammes of sodium phosphate, 0.14 grammes of potassium phosphate, 0.14 grammes of calcium chloride, 0.1 grammes of magnesium chloride, and 0.1 grammes of magnesium sulphate make up HBSS. After 8 and 24 hours of storage, Ashkenazi et al. found that HBSS had the highest mitogenicity of PDL cells. In order to preserve the viability of PDL cells over the long term, the American Association of Endodontists recommends HBSS as the recommended storage medium for avulsed tooth therapy. It is advised that the avulsed teeth be placed in HBSS for 30 minutes prior to reimplantation to replace the PDL cells, even if they have been kept in an acceptable storage medium⁷.

· Viaspan:

ViaSpan is an organ transplant cold storage solution. The pH and osmolality are 7.4 and 320 mm/kg, respectively, and it is a strong antioxidant. According to Ashkenazi et al., cells kept in ViaSpan for 8 hours had a high clonogenic capacity that was comparable to HBSS and superior to Milk. Viaspan's three main drawbacks are its limited shelf life, requirement that it be refrigerated, and high price⁷.

· Contact lens solution:

Contact lens solution fatty acid monoester with a cationic antimicrobial component. Due to the presence of preservatives in it which are harmful to the PDL cells is worse than saline solution, milk, and HBSS and is not recommended widely¹.

· Ascorbic acid:

When ascorbic acid is added to osteoblastic cell lines stimulates type I collagen production, which is followed by the expression of a specific marker associated with osteoblastic phenotypes, such as alkaline phosphatase (ALP) and osteocalcin. Ascorbic acid stimulates ALP activity, which is required for PDL cell binding to type I collagen via 2 beta 1 integrin, the expression of which is also stimulated by ascorbic acid. Type I collagen production is thought to be the first step in the differentiation of PDL cells and it could be used as a storage medium²³.

· Eagle’s minimal essential medium:

Eagle's Minimal Essential Medium contains L-glutamine; penicillin; streptomycin; nystatin; and calf serum. According to Ashkenazi et al., when this medium is stored for 8 hours at 4 C, it shows relatively high viability and mitogenic and clonogenic capacities. When stored for 24 hrs it is less effective than milk and HBSS. Studies have established that the use of special cell culture media (SCCM) as a storage medium for avulsed teeth is more effective at retaining PDL mobile viability than HBS for terms longer than 24 hours. Another variant of Eagle's Modified Crucial Medium (EME) is Dubelco's Modified Eagle's Medium (DME), which contains more nutrients and amino acids in addition to glucose compared to EME. Since it is more effective in a culture medium, EMEM is not readily available⁷.

· Euro-collins solution:

Euro Collins solution is a hypothermal medium that is developed for preserving organs to be transplanted and can also be used as a storage medium for avulsed teeth. Its pH and osmolality are 7.4 and 340 mOsm/kg⁶.

· Growth factor:

Polypeptide growth factors have been suggested for use in promoting PDL regeneration because they function as a potent biological intermediary regulating a number of wound-healing processes ²³. According to Lynch et al.²⁴, the periodontal attachment apparatus can form more rapidly during the earliest stages of wound healing when platelet-derived growth factor and insulin-like growth factor are combined for a short period of time.

· Levodopa:

Levodopa induces GH secretion from the pituitary gland. Levodopa speeds up the recovery process. According to Partovi et al.,²⁵ when employed as a storage medium, levodopa has a local effect on human PDL cells, assists in cell proliferation, and can affect avulsed teeth².

· Cryoprotective agents:

To protect biological tissue from freezing harm, cryoprotective compounds are utilized. The PDL cells of the reimplanted teeth can be preserved to varying degrees depending on the cryoprotective used and the controlled freezing rate at -196°C²⁶, according to research by Schwartz and Andreasen on the effects of 5%, 10%, and 10% glycerol and 5% and 10% DSMO on PDL⁷.

· Cling film:

It is the newest form of alternate transport that can serve as a storage medium for up to 6 hours. SOS ZahnboxR, UHT milk (4 °C), saline solution, water (tap water), and cling film were all compared in a study done by Anja Zeissler et al. In comparison to a tooth rescue box, they found that within two hours²⁶, there had been an 86% increase in cell proliferation. Up until six hours, it stayed the same, but at 24 hours, it had decreased to about half (43%)³.

· Gatorade:

Athletes use it as a non-carbonated sports drink which replaces lost electrolytes. It contains the glucose, salt, calcium, and potassium needed for cell metabolism²⁷. Despite being easily accessible during sporting events where injuries frequently occur, its high osmolarity makes it unsuitable as a medium for long-term storage. According to studies, Gatorade is a more effective storage medium than tap water⁷.

CONCLUSION:

Despite numerous studies and examinations of different mediums, it hasn't been proven that there is a perfect storage medium for an avulsed tooth that satisfies the ideal criteria. The lack of availability and high cost of several storage mediums are their major disadvantages⁷. Clinical studies have demonstrated that all storage mediums deteriorate with time²⁷. Because of qualities like pH and osmolality Saliva, Gatorade, saltwater, and tap water are considered non-physiological and should not be used⁵.Due to a lack of availability and less knowledge, HBSS and tooth rescue boxes are less than ideal even though they have high potential for preserving PDL cells after avulsion. Natural remedies are more effective at preserving PDL cell viability as compared to synthetic treatments²⁹. By reducing the temperature or cooling the storage media, the preservation property may be enhanced¹². The effectiveness of several storage media in preserving the survival of PDL cells can be summed up as follows: Eagle's culture medium = Viaspan = Euro collins = Custodiol = HBSS > Milk > Propolis > Green tea > Egg > coconut water > Ricetral. According to a review of the literature, milk and patient's serum seem to be the most practical transport medium for an avulsed tooth in terms of affordability and public accessibility³⁰. Both the International Association of Dental Traumatology and the American Academy of Paediatric Dentistry recommend using these two materials, and they can also be used as a useful temporary storage medium for avulsed teeth¹.

CONFLICT OF INTEREST:

no conflict of interest

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Received on 10.10.2023 Modified on 18.01.2024

Research J. Pharm. and Tech 2024; 17(7):3527-3532.

DOI: 10.52711/0974-360X.2024.00551

· Levodopa:

Levodopa induces GH secretion from the pituitary gland. Levodopa speeds up the recovery process. According to Partovi et al.,25 when employed as a storage medium, levodopa has a local effect on human PDL cells, assists in cell proliferation, and can affect avulsed teeth2.

· Cryoprotective agents:

Levodopa induces GH secretion from the pituitary gland. Levodopa speeds up the recovery process. According to Partovi et al.,²⁵ when employed as a storage medium, levodopa has a local effect on human PDL cells, assists in cell proliferation, and can affect avulsed teeth².