ABSTRACT Introduction: Management of tooth avulsion, to most dental practitioners is a challenge.

Although success can be achieved by immediate reimplantation, if the tooth cannot be immediately reimplanted, the tooth has to be preserved in a suitable transport medium to maintain viability of the PDL cells. Aim: To Evaluate PDL Cells Viability in Three Different Storage Media: Aloevera, Rice Water and Egg White. Methods: Forty mature premolar teeth were randomly and equally distributed within 4 study groups and stored in three storage media with milk as control group. The scrapings of the periodontal ligament of these extracted teeth were collected in falcon tubes having collagenase in 2.5 ml of phosphate buffer saline and were incubated for 30 minutes and centrifuged for 5 minutes at 800 rpm. Then the PDL cells were stained with Trypan Blue and were observed under optical microscope for their viability following which the cell count was performed. Results: Highest percentage of viable cells were seen in Aloevera (88.33.1) followed by Rice water (83.0 4.1) and Egg white (75.6 2.6) while least in Milk (73.3 2.5). Conclusion:Aloevera and rice water have the potential to be used as a practical transport media for an avulsed tooth.

MANUSCRIPT

INTRODUCTION & LITERATURE REVIEW: According to the World Health Organization (WHO) classification for traumatized teeth, avulsion or exarticulation is the complete displacement of a tooth from its alveolar socket due to traumatic injury. 1 The main etiologic factors of avulsion in permanent dentition are fight and sports injuries.2 Following avulsion, periodontal ligament cells are injured along with severance of the neurovascular bundle of the dental pulp at the apical foramen resulting in pulp necrosis. In these conditions the tooth should be maintained in a suitable medium until it can be promptly reimplanted by a dentist. 3 Research has shown that an avulsed tooth can be reimplanted without any complications if the tooth is re-inserted into the socket immediately or stored in a suitable storage media for 30 minutes to 1 hour after avulsion. When the tooth is dry for more than 20 minutes, its periodontal ligament cells begin to necrose and on replantation, inflammatory resorption sets in which is proportional to the extra-oral dry time.4 A variety of storage media have been proposed till date which include saliva, milk, Hanks Balanced Salt Solution (HBSS), Minimum Essential Medium (MEM), Viaspan, Propolis and many more. 5The American Associations of Endodontists (AAE) recommends the use of HBSS as the storage medium of choice for treatment of avulsed teeth because of its ability to provide long-term preservation of PDL cell viability. However, HBSS is not an easily obtainable over the counter product. Hence need arises to identify a media that will be readily obtainable and yet effective. Blomlof et al considered milk as a gold standard for transporting avulsed tooth to the laboratory. It has the physiologic osmolality similar to that of serum and contains fewer bacteria as compared to the saliva.23 Aleovera is remarkable because it is one of the most durable plants known to mankind. The plant has a miraculous ability to self-seal and heal, and contains all the nutrients within its leaf in order to survive. In recent studies human kidney cells were utilized to determine the effectiveness of Aleovera on cellular longevity. The cellular death rate was found to be reduced when cultured with the Aloe gel.5

Rice is an extremely healthy food for a number of reasons. Rice has low sodium content and contains useful quantities of potassium, Vitamin B, thiamin and niacin. In the Medicinal Book of Malayan Medicine, it is prescribed that boiled rice "greens" can be used as an eye lotion and for use with acute inflammation of the inner body tissues. Rice water is prescribed by the Pharmacopoeia of India as an ointment to counteract inflamed surface.6 Egg white can be a suitable storage medium for an avulsed tooth because it has a pH of 9.3 and is more likely to be available at the site of a traumatic event.7 The aim of the present study is to evaluate the effectiveness of Aloevera, Rice water and Egg white as storage media for avulsed tooth in maintaining the periodontal cell viability in comparison with milk as control. MATERIALS AND METHODS Forty non-carious human mature premolar teeth with apparently normal periodontium undergoing extraction for orthodontic therapeutic purposes were randomly and equally distributed within 4 study groups. Informed consent was obtained from the patients prior to the extraction. Following extractions, the teeth were held with forceps by the coronal region, and the coronal 3mm of PDL were scraped with a curette to remove cells that may have been damaged during extraction.7Immediately after, the teeth were left in mud for 15 minutes to simulate avulsion injury.8 Then the teeth were placed in four different storage media for 30 minutes. (Figure 1) Group I: Teeth stored in Milk (G1M, Control group) Group II: Teeth stored in Aloevera gel (G2A, Experimental group) Group III: Teeth stored in Rice water (G3R, Experimental group) Group IV: Teeth stored in Egg White (G4E, Experimental group) Method for obtaining the PDL cells Following 30 minutes of storage in the respective media, the teeth were held by the crown portion and the root surface was cleaned by irrigating twice with sterile isotonic saline to remove the storage media residues.

The cleaned teeth were then placed in a petridish and the apical two third of the root surfaces (as measured from epithelial attachment) were scraped with the No.15 scalpel blade, to obtain the periodontal ligament cells. (Figure 2) The obtained scrapings were added to the 2.5ml of phosphate buffer saline into a falcon tube. To the above mixture, 0.5mg of collagenase enzyme (0.2mg for every ml of phosphate buffer saline was weighed using a digital weighing machine) was added and then the mixture was incubated for 30 minutes. Following incubation, the falcon tubes containing the above mixture were centrifuged for 5 minutes at 800 rpm.7The obtained supernatant liquid was discarded and the centrifuged residue was collected. To this equal volume of 0.4% Trypan blue stain was added and mixed thoroughly. Following staining, the cells were observed with the help of haemocytometer under optical microscope. Preparation of Aloevera gel: Firstly, the Aloevera leaves were collected and washed. Then, a kitchen knife was used to cut off the outer spiked edges of Aloevera leaf. The skin of the one side of the leaf was removed with a vegetable peeler and the inner pulp was scooped out with a spoon and was placed into a blender. The contents were blended thoroughly and filtered through a piece of muslin cloth and was placed into a glass jar with a tight fitting lid.9 Preparation of Rice Water: Six cups of water was taken into a vessel and placed over medium heat until boiling. One cup of rice was added into boiling water and stirred frequently. After rice was cooked, the liquid was separated from the cooked rice using a sieve and then the liquid was cooled.10 Preparation of Egg white: The egg was cracked on the edge of a bowl then the egg was turned upright and the shell was opened into two halves and the yolk was kept into lower half. Over the bowl, the content was poured from one half of the broken shell into the other, letting the egg white into the bowl, but the yolk was kept intact

in the egg shell halves. The process was repeated until all the white had fallen into the bowl and only the yolk was left in the shell.11 Determination of the number of cells (total and viable): The cells were viewed under microscope at 100x magnification. The number of cells (total and non-viable) was counted overlying four x 1 mm2 areas of the counting chamber. The cell count was done as Total cells - Stained cells x100 Total cells The number of viable cells in percentage was obtained.8

STATISTICAL ANALYSIS: All the results were recorded and tabulated. To evaluate and compare the efficacy of the test groups, the records were statistically analyzed by One-Way Analysis of Variance (ANOVA) complemented by the Tukeys test. RESULTS: The results of the present study were evaluated under the following headings; Table 1: Denotes the comparison of number of viable and % viable cells in study groups and with control group. Table 2: Mean Values of Viable, Nonviable and %Viable PDL cells count in study groups and comparison with control group. Table 3: Contains intergroup comparison of viable cells and % viable cells in study media and control medium. It also contains comparison of different media in difference between groups by p value. The teeth stored in Aloevera demonstrated the highest number of viable PDL cells followed in rank order by rice water, egg white and milk. There was no significant difference between Aloevera and rice water. There was also no significant difference in the number of viable PDL cells between egg white and

milk. The results of the present study showed that the number of viable and % viable cells were highest for G2A and least for G1M.In contrast nonviable cells were highest for G1M and least for G2A. DISCUSSION: Recent clinical studies have shown that avulsed teeth replanted within 5 minutes have the best prognosis, resulting in much higher reattachment success.12If the tooth cannot be immediately replanted, the viability of PDL cells on the surface of the root can be maintained in a suitable transport medium which will lead to a decreased incidence of root resorption. The fundamental philosophy for the storage of avulsed tooth is that the teeth should be stored in an environment that most closely replicates the oral environment. The normal, morphologic, physiologic and metabolic conditions of the teeth should be simulated in the storage medium as closely as possible to the oral cavity. Of utmost importance is the prevention of drying, which causes loss of normal physiologic metabolism and morphology of the periodontal ligament cells.13,14 Numerous studies have tried to assess the optimal storage media for PDL cell viability and preservation in different conditions. Hanks Balanced Salt Solution, Viaspan and Eagles medium allow cell reconstitution, thus they could be ideal storage media. However, the disadvantages with these media are their high cost and unavailability. It is fundamental to have a storage medium that is easily available, so that more avulsed teeth can be reimplanted with better prognosis. Hence the present study was conducted to evaluate and compare the effectiveness of Aloevera, Rice water, Egg white as alternate transporting media for avulsed teeth. Fibroblast function is known to be affected by age, trauma and inflammation. Therefore, non-carious mature human premolar teeth undergoing extraction for orthodontic therapeutic purposes were selected. For the same reason, teeth from young healthy individuals without periodontal disease were included in the study.15

In this study, avulsion injury was simulated by extraction of tooth and leaving it on mud for at least 15 minutes. This is the time the victim and the attender take to recover from the traumatic event and act appropriately.8In the current investigation, a 15 minute dry time was chosen, as this seems to be a critical time at which damage has been done to many PDL cells, yet some cells remain for assessment. Pohl et al stated that 15 minute dry time is the time when PDL cells remain in non compromised state.16Other investigators have shown that at 2 hours dry time, no vital PDL cells remain.17 Following the 15 minute dry time, the teeth were placed in different storage media for 30 minutes. This period is very important because the periodontal cells are damaged during this time. Hence preservation of teeth in the storage media might reduce the damage.18 In the present study, Aloevera gel was chosen as a transport medium because it contains 99% water and over 75 nutrients which include 20 minerals, 19 amino acids, and 12 vitamins. Aloevera contains all eight essential amino acids, and 11 out of 14 secondary amino acids that the human body requires. It promotes healing and can be used in any surgical wound. Aloevera can also be used around dental implants to control inflammation. Recently, the cellular death rate of human kidney cell was found to be reduced 2/3rd when cultured with the aloe gel.19,20 Rice water was chosen as a storage medium because rice is easily available. Rice is an extremely healthy food which has low sodium content and contains useful quantities of potassium, Vitamin B, thiamine and niacin. In Malayan Medicine, it is prescribed that boiled rice "greens" can be used as an eye lotion and for use with acute inflammation of the inner body tissues.21 The Egg white was chosen due to its nutritive constituents. It was seen that Egg white from a single egg contains 4.7grams of 40 different proteins, 0.3 grams of carbohydrate, 62 milligrams of sodium and the remaining being water.22 Blomlof et al considered milk as a gold standard for transporting an avulsed tooth. It has the physiologic osmolality similar to that of serum and contains fewer bacteria as compared to that of

saliva. It is one of the most frequently used transport medium. Due to all the above advantages, in present study, milk was chosen as the positive control.23 To evaluate the efficacy of the different storage media in preserving the viability of dental fibroblasts two methods have been quoted in the literature, Ragnarssons and Doyles method. In Ragnarssons method (1985)24 the fibroblasts were first removed from the root surfaces and added to the storage media for culturing. The viability of cells were evaluated at different time interval and counted. In another method Doyle et al.(1998)25placed the extracted tooth directly in the storage medium. After a predetermined time, the teeth were taken out of the medium and PDL cells were isolated to evaluate cell viability. In the present study Doyle,smethod was followed because it more closely replicates the actual clinical scenario.25 To quantitate the number of viable PDL cells in the current study, to preserve maximum cell viability, the root surfaces were treated with collagenase type1 as was performed by Pillegi et al. 18 This procedure allowed rapid cell retrieval and maintained maximum cellular integrity.26 In this study, trypan blue exclusion staining technique was used as it is quick, easily performed and distinctively differentiates nonviable cells from viable cells. The reactivity of Trypan blue is based on the fact that the chromopore present on the cell membrane is negatively charged and does not take up the stain unless the membrane is damaged. Therefore, all the cells which exclude the dye are viable. 13Trypan Blue stain used in the study would assess only viability of the cells and not the actual physiologic health and metabolic capabilities of the cells.27 According to the results, maximum percent of viable cells were in Aloevera (88.3) followed by Rice water (83.0) and Egg white (75.6) while least in Milk (73.3). Aloevera maintained highest viable cells, could be attributed to composition. It contains a glycoprotein with cell proliferating- promoting activity.28 According to another study, topical application of the Aloevera-derived Allantoin gel stimulated fibroblast activity and collagen proliferation.29 Rice water which is another medium, showed 83% of cell viability. It also performed better than egg white and milk. This can be explained by the fact that it contains low sodium content, useful quantities of

potassium, Vitamin B, thiamine and niacin. It also has anti-inflammatory properties.21The iron and zinc in its composition help in synthesis of collagen. Although not statistically significant, egg white maintained superior cell viability than milk. This may be attributed to the high pH (9.38), and also to the large amount of proteins in egg white that might act as a foreign body. This is in agreement with Sousa et al (2008) who microscopically analysed the human periodontal ligament attached to the extracted tooth after one hour of extra alveolar time, compared to milk, egg white and artificial saliva. The results of teeth stored in milk and egg white were similar concerning the organization of collagen fibres and the number of cells.30 On the contrary, Khademi et al found statistically significant difference between the viability of PDL cells in egg white as a storage medium in comparison with milk and water.31 The opinion is that there can be some quantitative and qualitative factors which may interfere with the preservation of the vitality of periodontal ligament cells. In the present study, milk has shown least number of viable cells (75.3%). On comparison of milk with egg white, number of viable cells was higher for egg white but it was statistically not significant. On comparison of milk with Aloevera and rice water, number of viable cells was least for milk and results were statistically significant. This can be due to various enzymes present in the milk, which could be potentially harmful to the fibroblasts of the periodontal ligament. According to Gamsen et al (1992) milk does not have ability to reconstitute depleted cell metabolites and restore viability of periodontal ligament cells and cells stored in the milk lack the cell energy and ions to permit the repopulation of the periodontal ligament.32Blomlof et al (1981) found that milk was a compatible storage medium only when it was cold and fresh.33 Further research, including in vivo studies are needed to investigate whether Aloe vera or rice water at extended extra oral dry time can maintain viability of PDL cells. CONCLUSION:

Aloevera, Rice water and Egg white have potential to be used as a transporting

media for avulsed tooth. and milk. Aloevera and rice water maintain PDL cell viability are better than Egg white

REFFERENCE:

1.

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http://www.ehow.com/how-2154587-make-aloevera-gel.html. 10. Howtomake ricewater[online][Cited2010March12]Availablefrom:URL:http://www.ehow.com/how-4503461make-rice-water.html. 11. Howtoseparate eggwhites[Online][Cited2010March12] Available from:

URL:http://www.ehow.com/how-2640-separate-egg-whites.html. 12. Santos CLV, Sonoda CK, Poi WR, Parzarini SR, Sundefeld MLMM, Negri MR. Delayed replantation of rat teeth after use of reconstituted powdered milkas a storage medium. Dent Traumatol 2009;25:51-7. 13. Soder PO, Otteskog P, Andreasen, Modeer. Effect of drying on viability of periodontal membrane. Scand J Dent Res.1977;85:164-8. 14. Andreasen JO, Kristersson L. The effect of limited drying or removal of the periodontal ligament: Periodontal healing after replantation of mature permanent incisors in monkeys. Acta Odontol Scand 1981;39:1 15. Khademi AA, Saei S, Mohajeri MR, Mirkeshti N, Ghassami F, Torabi N, et al. A new storage medium for an avulsed tooth. J Contemp Dent Pract 2008;9:1-5. 16. Pohl Y, Filippi A, Kirschner H. Results after replantation of avulsed permanent teeth. II. Periodontal healing and the role of physiologic storage and antiresorptive-regenerative therapy. Dent Traumatol 2005;21:93-101. 17. Andreasen J. Effect of extra oral period and storage media upon periodontal and pulpal healing after replantation of mature permanent incisors in monkeys. Int .J. Oral Surg.1981;10(1): 43-53.

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en.wikipedia.org/wiki/ eggwhite 23. Blomlof, Lindskog, Andersson, Hedstrom. Storage of experimentally avulsed teeth in milk prior to replantation Journal of Dent Res.1983;62(8):912-16. 24. Ragnarsson B, Carr G, Daniel JC. Basic biological sciences isolation and growth of human periodontal ligament cells in vitro. J Den Res 1985;64:1026-30. 25. Doyle DL, Dumsha TC, Sydiskis. Effect of the soaking in Hanks balanced salt solution or milk on PDL cell viability of dry stored human teeth. Endod Dent Traumatol. 1998;14:221-4. 26. Sheher Abdul, Wallace James,Aggarwal S, Bretz W, Baugh D. Effect of Propolis on human fibroblasts from pulp and periodontal ligament. J Endod 2004;30:5,359-61. 27. Martin MP, Pileggi R.A quantitative analysis of Propolis: a promising new storage media following avulsion. Dent Traumatol 2004;20:85-9. 28. Yagi A, Egusa T, Arase M, Tanabe M, Tsuji H. Isolation and characterization of the glycoprotein fraction with a proliferation-promoting activity on human and hamster cells in vitro from aloe vera gel. Planta Medica 1997;63:18-21. 29. Davis RH, Leitner MG, Russo JM, Byrne ME. Anti-inflammatory activity of against a spectrum of irritants. J Am Podiat Med Assocg 1989;79:263-76. aloe vera

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FIGURE LEGENDS: Figure1- Four storage Medias

Figure 2- Removing PLD scraping from apical 2/3rd

Table 1: Comparison of viable and %viable cell count in study groups and with control group Milk Aloe vera Ricewater Egg white Milk Aloe vera Ricewater Egg white

viable

Viable

viable

Viable

%viable

%viable

%viable

%viable

103

117

116

106

76.3

86.7

82.9

74.6

99

108

102

102

73.3

85.0

81.0

74.5

105

115

114

109

70.5

89.8

83.8

72.7

98

113

110

106

74.8

90.4

75.3

77.9

117

130

125

112

70.9

92.2

86.2

72.3

105

123

120

108

69.1

91.8

88.2

72.0

101

113

103

106

75.4

85.6

79.2

76.3

98

107

122

101

74.8

84.3

88.4

78.3

97

109

104

97

71.9

85.8

80.6

75.2

90

103

105

92

75.6

91.2

84.0

79.3

Table 2: Mean Values of Viable, Nonviable and %Viable PDL cells count in study groups and control group

GROUPS

Ligament cells ( Mean SD)

Total

Viable

Non viable

%Viable

Milk (G1M)

138.6 7.8 (119 165)

101.3 7.1 ( 90-112)

37.3 6.7 ( 29 48)

73.3 2.5 ( 69.1 76.3)

Aloe vera (G2A)

128.9 7.8 (113-141)

113.8 8.0 ( 103-130)

15.1 4.0 ( 10-20)

88.3 3.1 (84.3- 92.2)

Rice water (G3R)

135.1 7.4 (125-146)

112.1 8.5 (102-125)

23.0 5.8 (16-36)

83.0 4.1 (75.3-88.4)

Egg white (G4E)

138.3 11.8 (116-155)

103.9 6.0 (92-112)

34.3 6.3 (24-43)

75.3 2.6 (72.0- 79.3)

Table 3: Inter group comparison using One-Way Analysis of Variance (ANOVA) complemented by the Tukeys test. GROUPS Viable cells % Viable cells

Mean SD I. II. III. IV. Milk (control) Aloe vera Rice water Egg white F* P 101.3 7.1 113.8 8.0 112.1 8.5 103.9 6.0 6.70 < 0.01, S Mean difference Difference between I-III groups
**

Median 100 113 112 106

MeanSD 73.3 2.5 88.3 3.1 83.0 4.1 75.3 2.6 49.06 < 0.01, S

Median 74.1 88.3 83.3 74.9

ANOVA,

Range

Mean difference

Range

I-II

12.5 10.8 2.6 1.7 9.9 8.2

< 0.01, S < 0.05, S 0.86, NS 0.95, NS < 0.05, S 0.08, NS

15 9.7 2.0 4.7 13 7.7

< 0.01, S < 0.01, S 0.47, NS < 0.01, S < 0.01, S < 0.01, S

I-IV ( P-values) II-III II-IV III-IV

*One way ANOVA, ** Post- hoc Tukey,s Test

Fig 1.

Fig 2

FIGURE LEGENDS Figure1- four storage Medias

Figure 2- removing PDL scraping from apical 2/3rd

Comments: ( Reviewer 1. Dr.Venaktesh Babu, Prof & Hod, Dept.of Pedodontics, V.S. Dental College, Bangalore)
1. 2. 3. 4.