STORAGE MEDIUM FOR AVULSED TEETH: A LITERATURE REVIEW

Avulsion injury is one of the most severe types of traumatic dental injuries. Following avulsion, periodontal ligament tissues are injured and the vessels and nerves of the pulp rupture at the apical foramen which causes pulp necrosis. In studies it was reported that the key to retention of the knocked-out teeth was to maintain the viability of the periodontal ligament. Storage media plays an important role in preserving the viability of PDL cells during extra alveolar time. This article highlights the different storage medias available for avulsed teeth, along with their merits and demerits.

Avulsion injury is one of the most severe types of traumatic dental injuries. Following avulsion, periodontal ligament tissues are injured and the vessels and nerves of the pulp rupture at the apical foramen which causes pulp necrosis. In studies it was reported that the key to retention of the knocked-out teeth was to maintain the viability of the periodontal ligament. Storage media plays an important role in preserving the viability of PDL cells during extra alveolar time. This article highlights the different storage medias available for avulsed teeth, along with their merits and demerits.

…………………………………………………………………………………………………….... Introduction:-
Dental trauma refers to injury to the teeth and periodontium (gums, periodontal ligament,alveolar bone), and nearby soft tissues such as the lips, tongue, etc .Dental trauma is accounting for a major part of dental problems in children and adolescents.Falling, fight, sports, accidents, and hittingobjects or people are among common etiologic factors. Traumatic dental injuries are neglected oral condition despite its relatively high prevalence. Among various types of dental injuries-Dental avulsion is an emergency in which prompt management (within 20-40 minutes of injury) results in favourable prognosis of the tooth. 1 Tooth avulsion (exarticulation) implies total displacement of the tooth out of its socket. This kind of dental trauma causes the periodontal ligaments to be severed with or without fracture of the alveolus. Reported incidence of dental avulsion is 0.5-3%of all dental injuries. The age group of 7-14 years appears to be most affected. 2 At this age, the loosely structured periodontal ligament and low mineralized bone surrounding erupting teeth provide only minimal resistance to an extrusive force. When a tooth is avulsed, attachment damage and pulp necrosis occurs. 2 Since most avulsions occur before the patient's facial growth is complete it is critical to maintain the integrity of the surrounding bone.The ideal treatment of avulsion injury is immediate replantation, but this can rarely be achieved. 3 In such conditions the avulsed tooth should be stored in appropriate storage media. Storage medium keeps the attached periodontal ligament cells in hydrated state and maintain their viability, allows healing with regenerated periodontal ligament cells when replanted. However, if excessive drying occurs before replantation , the damaged periodontal ligament cells will elicit an inflammatory response over a diffuse area on the root surface which will result in replacement resorption (ankylosis). 2 This literature review attempts to discuss various storage media in depth, including their advantages and disadvantages for maintaining viability of attached PDL cells and allows favourable prognosis following Replantation.

Storage Medium
The first line of treatment for an avulsed tooth is immediate replantation, however, most of the times it is not possible,therefore in situations when immediate replantation cannot be performed storage of avulsed teeth in an appropriate storage media is recommended.

Definition
A storage medium may be defined as a physiological solution that closely replicates the oral environment to help preserve the viability of PDL cells following avulsion.

A) Naturally available i)Tap water
Most of the time the available adult or parents at the accident site place avulsed teeth in tap water because it is most readily available .But it is not an ideal storage media because several studies have shown that cells stored in water did not maintain their morphology, with visible destruction and rapid cell death. 5 Thus tap water should only be used to avoid tooth dehydration only if there is no other appropriate media available ,as water is inadequate for preservation of PDL cells attached to the avulsed teeth.(figure:1)

Advantages
It is the most readily available medium at the site of accident VARIOUS STORAGE MEDIA AVAILABLE FOR AVULSED TEETH Naturally available Commercially available

Human saliva (buccal vestibule)
Saliva can be collected in a cup and tooth can be dropped into it or the tooth can be either placed in the patient mouth under the tongue or in the vestibule. But it is used as a storing medium for a short period of time as it can damage the cells of the periodontal ligament if used for longer than 1 hour. 6 (figure:2)

Advantage
It is the most immediately available storage medium at all accident sites.

Disadvantages
1. Osmolality is much lower than the physiologic (60-70 mOsm/kg). 7 2. Has lesser amount of nutrients like glucose and essential growth factors to maintain PDL cell viability.

Milk
The American association of endodontics indicates milk as a storage medium for avulsed teeth for maintaining the viability of PDL cells. Milk is an excellent storage medium for upto 6 hours after which it loses its effectiveness. 8 877 Not all types of milk are equally effective as storage medium. Avulsed teeth stored in chilled milk upto 6 hour can maintain sufficient number of viable PDL cells to support replantation of tooth and possibility of PDL healing. 9 Milk with lower fat content is considered to be more appropriate at maintaining cell viability than milk with higher fat content. 10 (figure:3) Advantages 1. Isotonic liquidwith physiologic osmolality(275 mOsm /kg) 2. Neutral ph (6.5-6.8) 11 3. Low or no bacterial content because of pasteurization 4. Contains growth factors and essential nutrients for cells 5. Milk contains epithelial growth factor which stimulates the proliferation and regeneration of epithelial cell rests of Malassez

Disadvantage
As regular pasteurized milk has a short shelf life and requires refrigeration which makes it less readily available at the trauma site.  Sousa et al. (2008) evaluating human PDL adhered to extracted tooth roots and maintained in this storage medium observed that the egg white provided cell viability and histologicalcharacteristics similar to those of milk. 12 Khademi et al (2008) compared milk and egg white as solutions for storing avulsed teeth, and found that teeth stored in egg white for 6 to 10 h had a better incidence of repair and lower surface resorption than those stored in milk for the same amount of time.

Coconut water
Several studies have been performed to use this substance as a storage medium for avulsed teeth, but the results are contradictory.  found greater efficacy of coconut water over HBSS and milk for maintaining the viability of PDL cells. 13 Thomas et al. (2008) found that 15 to 120 min storage in coconut water is as efficient as storage in HBBS. On the other hand, Pearson et al. (2003) and  observed that inflammatory resorption was more frequent when the tooth was maintained in coconut water compared with milk. (figure:5)

Advantages
1. Biologically pure,sterile product 2. Rich in amino acids,proteins,vitamins and minerals. 3. Aids in replenishing fluids, electrolytes and sugars lost from the body

Disadvantages
The acidic ph (4.13) is a concern because of deleterious effect on cell metabolism. 14 In view of this, the use of green tea extract and its compounds may be an alternative for the preservation of avulsed teeth and its beneficial effect is enhanced by higher extract concentrations.

Disadvantages
Very difficult to procure at the accident site.

Morusrubra(red mulberry)
It is a natural product usually availablein Southern Europe, Middle East,Northern Africa and Indian subcontinent which contains flavonoids, alkaloidsand polysaccharides. Ozan et al. (2008) reported that when teeth were stored in red mulberry for up to 12 h, its capacity to maintain the viability of PDL cells was better than that of HBSS; however, if a longer storage time is required, it is advisable to employ higher concentrations of the fruit juice. 17 There are very few studies evaluating the use of red mulberry juice as a transport medium for avulsed teeth and its biological properties have not been established yet. Further research is necessary before its use can be recommended.(figure:8)

Advantages
Contain essential nutrients important for cell preservation.

Disadvantage
Poor availability at the site of accident.

Ricetral:
Dehydration, as in diarrhea cases, is treated with oral rehydration solutions like Ricetral. These solutions are available in drugstores at low cost and their components are able to maintain the body hydrated by replenishment of liquids lost in the intestine. Rajendran etal. (2011) evaluated the PDL cell viability of extracted human teeth by the Tripan blue exclusion method and the results of the study showed that the effectiveness of Ricetral was similar to the HBSS control and both were superior to milk as storage medium. In an another study by Subramanian P et al(2014)the viability of PDL cells in Ricetral was maintained for 45 minutes, when it was used as storage media whereas in HBSS the viability of cells was maintained for 24 hours. 18 Thus HBSS was found to be more effectiveas compared to Ricetral in terms of storage time when both were used as storage medium for avulsed teeth.(figure:10) Advantages 1. It contains essential nutrients like glucose and vital salts in concentrations considered adequate for the cell metabolism. 2. Low cost 3. Readily available at drug stores.

Disadvantage
Efficacy is not good as that of HBSS as it can be used as storage medium for only 45 minutes. 19

Gatorade
It is a sports drinks used for rehydration and is available at sport's equipment stores. Sigalas et al. (2004) evaluated Gatorade efficacy in maintaining the viability of PDL cell culture by the Tripan blue exclusion method and the results showed that at 37 0 C it was toxic to the cells; but this may be an alternate medium to HBSS and milk when used as ice cold and for a short time.(figure:12).

Advantages
It is readily available at the centre of sports activities.

Disadvantages
1. It has osmolality of 360 mOsm/kg, which may cause damage to the cells due to hypertonicity (Goswami M et al.2011). 7 2. Has low ph of 3.

Hank's Balanced Salt Solution (HBSS)
Hank's balanced salt solution has been commercially prepared storage medium for avulsed teeth.It contains active ingredients like 8 g/L sodium chloride, 0.4 g/L D-glucose, 0.4 g/L potassium chloride, 0.14 g/L calcium chloride, 0.35 g/L sodium bicarbonate, 0.09 g/L monobasic sodium phosphate, 0.1 g/L anhydrous magnesium sulphate and water as a vehicle and has the ideal osmolality of 270-290 mOsm/kgand pH of 7.2. 7 HBSS is available at Sigma Aldrich Pharmaceuticals, Bangalore, India .
Hwang et al. reported 94% cell viability after storage of cultured human PDL cells for 24 h in this medium, which is considered as an excellent result, and Souza et al. had also reported similar results with HBSS. Moreover it has also beenshown to replenish metabolites which have been depleted from PDL cells. Hence, it has been recommended to place avulsed teeth in HBSS for 30 minutes before replantation in order to replenish the PDL cells, even if the avulsed teeth had been stored in an appropriate storage medium(figure:13). 11 In some countries ,HBSS is available in emergency kits [Save-A-Tooth, PA, USA]. This kit comprises a small basket to hold the avulsed tooth while it is submerged in HBSS. Each tooth that goes into Save-A-Tooth gets into its own protective slot that prevents any serious damage while transporting the avulsed tooth and the design of the slots allows the dentist to remove the tooth without accidentally damaging the root.( figure:14) Advantages 1. It has physiologic ph of 7.2 and osmolality of 270-290 mOsm/kg. 7 2. The presence of magnesium, glucose,calcium and other essential nutrients can maintain the PDL cell viability. 3. Replenishes the metabolites that had been depleted from the PDL cells 4. It has a long shelf life of 2 years and does not require refrigeration.

Disadvantages
1. It is not readily available at an accident site, which makes it difficult to be used as a storage medium. 2. High cost 3. It has been recommended to use HBSS at 37 0 C in a controlled incubator. 21

Culture media
Culture media such as Eagle's medium, alpha-Minimum Essential Media (MEM)and αMEM-S (supplemented with foetal calf serum and antibiotic) have been shown to maintain the viability and proliferative activity of PDL cells for an extended period of time (48-53 hours) with a reduced rate of inflammatory resorption as reported by ( Andreasen JO et al.1978,Pohl Y et al. 1999,Ashkenazi M et al.2000,Sigalas E 2004. In India it can be bought from Sigma Aldrich Pharmaceuticals, Banglore.

Advantages
1. The culture medium contains all the required essential nutrients for the growth and proliferation of PDL cells.
2. Supplementation and the addition of growth factors (platelet derived growth factor, insulin-like growth factor, epidermal growth factor, etc) in a culture medium has also been shown to increase the mitogenic and clonogenic capacity of PDL cells for as long as 24 hours (Ashkenazi M et al.2000). 22 3. Act as a potent biologic mediator and proposed to aid in regeneration of PDL. 4. The addition of antioxidant, catalase supplementation to a medium have shown to produce reduced rate of surface resorption.

Disadvantages
Poor availabilty at the accident site.

Viaspan
It is a widely used solution for the storage and transportation of organs to be transplanted. In Dentistry it can be used as a storage medium for avulsed teeth.(figure:16). It can be bought online at a site www.medindia.net.

Advantages
It has 320mOsm/kg osmolality and 7.4 pH that helps to maintain the PDL cells viability. 7

Disadvantages
The limited access to it, especially at the site of the accident, makes itdifficult to use .