Objectives: We examined the effects of storage in Hank\'s balanced salt solution (HBSS), low-fat milk, soy milk, and aloe vera extract on the clonogenic capacity of human periodontal ligament (PDL) cells. The Dulbecco\'s modified Eagle\'s medium (DMEM) was used as control.
Materials and methods: Human teeth that are extracted for the orthodontic purpose were collected and the periodontal ligament tissue was cultured. Cultured experimental PDL cells were exposed to different experimental solutions for 24 hours. To evaluate the clonogenic capacity of the stored cells, cells were inoculated into 96-well plates at a concentration of one viable cell/well. For each tested group, 96 replicates were plated and grown for 3 weeks in a culture medium at 37°C in humidified air containing 7% CO2. Under these conditions, discrete colonies arising from a single cell that covered 70–100% of the well were counted under light microscopy (×200). The percentage of cells with clonogenic capacity was calculated as the number of colonies formed/number of cells seeded × 100. Thus, the clonogenic capacity reflected the likelihood that each colony arose from a single cell. The experiments for 2 and 8 hours were repeated three times (three 96-well plates), and for 24 hours, five times (five 96-well plates).
Results: The highest clonogenic capacities were found in cells stored in HBSS (15.3 ± 1.15) and aloe vera extract (12.3 ± 2.5) followed by low-fat milk (10.3 ± 1.5). Soy milk showed least clonogenic capacity (3.6 ± 1.5). The Dulbecco\'s modified Eagle\'s medium, HBSS, and aloe vera showed similar statistical significance (p = 0.092).
Conclusion: Highest clonogenic capacity was found in cells stored in HBSS, aloe vera extract, and low-fat milk. Soy milk showed least clonogenic capacity.
Application of the international classification of disease and stomatology. Geneva: World Health Organization; 1992.
American academy of pediatric dentistry. Guideline on management of acute dental trauma. Reference Manual 2011;34:230–238.
Andreasen FM, Daugaard-Jensen J. Treatment of traumatic dental injuries in children. Curr Opin Dent 1991;1:535–550.
Trope M. Avulsion of permanent teeth: theory to practice. Dent Traumatol 2011;27(4):281–294. DOI: 10.1111/j.1600-9657.2011.01003.x.
Andreasen JO. Effect of extra-alveolar 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. DOI: 10.1016/S0300-9785(81)80007-5.
Blomlöf L, Andersson L, Lindskog S, et al. Periodontal healing of replanted monkey teeth prevented from drying. Acta Odontol Scand 1983;41(2):117–123. DOI: 10.3109/00016358309162311.
Kotumachagi Sangappa S, Pravin Kumar A, Shruti, et al. Extra-alveolar storage media for teeth: a literature review. Int J Adv Res 2014;2:963–972.
Poornima P, Kotari S, Sasalawad SS, et al. Save cells before tooth replantation: a review. Int J Contemp Dent Med Rev 2015; 1–7. DOI: 10.15713/ins.ijcdmr.61.
Soder P-O, Otteskog P, Andreasen J, et al. Effect of drying on viability of periodontal membrane. Scand J Dent Res 1977;85(3):164–168. DOI: 10.1111/j.1600-0722.1977.tb00549.x.
Oikarinen KS, Seppa ST. Effect of preservation media on proliferation and collagen biosynthesis of periodontal ligament fibroblasts. Endod and Dent Traumatol 1987;7(3):95–99. DOI: 10.1111/j.1600-9657.1987.tb00609.x.
Ashkenazi M, Sarnat H, Keila S. In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in six different media. Endod Dent Traumatol 1999;15:149–156. DOI: 10.1111/j.1600-9657.1999.tb00793.x.
Lekic P, Kenny D, Moe HK, et al. Relationship of clonogenic capacity to plating efficiency and vital dye staining of human periodontal ligament cells: implication for tooth replantation. J Periodontal Res 1996;31(4):294–300. DOI: 10.1111/j.1600-0765.1996.tb00496.x.
Hiltz J, Trope M. Vitality of human lip fibroblasts in milk, Hank's balanced salt solution and ViaSpan storage media. Endod Dent Traumatol 1991;7(2):69–72. DOI: 10.1111/j.1600-9657.1991. tb00187.x.
Trope M, Friedman S. Periodontal healing of replanted dog teeth stored in ViaSpan, milk and Hank's balanced salt solution. Endod Dent Traumatol 1992;8(5):183–188. DOI: 10.1111/j.1600-9657.1992.tb00240.x.
Hupp JG, Trope M, Mesaros SV, et al. Tritiated thymidine uptake in periodontal ligament cells of dogs’ teeth stored in various media for extended periods. Endod Dent Traumatol 1997;13(5):223–227. DOI: 10.1111/j.1600-9657.1997.tb00044.x.
Ashkenazi M, Sarnat H, Keila S. In vitro viability, mitogenicity and clonogenic capacity of periodontal ligament cells after storage in 4 different media supplemented with growth factors. Dent Traumatol 2001;17(1):27–35. DOI: 10.1034/j.1600-9657.2001.170106.x.
Lekic PC, Kenny DJ, Barrett EJ. The influence of storage conditions on the clonogenic capacity of periodontal ligament cells: implications for tooth replantation. Int Endod J 1998;31(2):137–140. DOI: 10.1046/j.1365-2591.1998.00138.x.
Tudose A, Celia C, Cardamone F, et al. Regenerative properties of aloe vera juice on human keratocyte cell culture. Farmacia 2009;57:590–597.
Sharma M, Sharma S, Reddy YG, et al. Evaluation of Periodontal ligament cell viability in three different storage media: an in vitro study. J Dent (Tehran) 2015;12:524–531.
Blomlof L, Lindskog S, Hedstrom K-G, et al. Vitality of periodontal ligament cells after storage of monkey teeth in milk or saliva. Scand J Dent Res 1980;88(5):441–445. DOI: 10.1111/j.1600-0722.1980. tb01251.x.
Moura CC, Soares PB, de Paula Reis MV, et al. Potential of coconut water and soy milk for use as storage media to preserve the viability of periodontal ligament cells: an in vitro study. Dent Traumatol 2014;30(1):22–26. DOI: 10.1111/edt.12042.
Silva EJ, Rollemberg CB, de Souza Coutinho-Filho T, et al. A multiparametric assay to compare the cytotoxicity of soy milk with different storage media. Dent Traumatol 2013;29(4):319–322. DOI: 10.1111/j.1600-9657.2012.01175.x.
Moazami F, Mirhadi H, Geramizadeh B, et al. Comparison of soymilk, powdered milk, hank's balanced salt solution and tap water on periodontal ligament cell survival. Dental Traumatology 2012;28(2):132–135.