Comparative Evaluation of Corneal Storage Medias Used as Tooth Avulsion Medias in Maintaining the Viability of Periodontal Ligament Cells Using the Cell Counting Kit-8 Assay

Abstract

Purpose The prime factor in determining the success of reimplantation of an avulsed tooth is the maintenance of the viability of periodontal ligament fibroblast cells (PDFC). This study aims to evaluate and compare Mc Carey Kaufman media (MK), Cornisol, Dulbecco’s Modified Eagles Medium (DMEM), Hanks Balanced Salt Solution (HBSS) and distilled water in preserving the viability of the PDFC using the Cell Counting Kit-8 assay (CCK-8). Methods Cryopreserved PDFC were suspended in DMEM and incubated in CO2 incubator at 370C with 95% humidity and 5% CO2 for attachment. Once cells attained 80% confluence, they were trypsinised and passed into T-25 culture flasks to expand the culture population. Cells from passage 5 were pooled for experimentation. Trypan blue exclusion test was performed before each experiment to measure cell viability and batches showing more than 95% viability were used in the experiment. The viable PDFC with 1×105 were seeded in 96 well plates and incubated in CO2 incubator at 370C, 95% humidity and 5% CO2 for 24 hours to allow cell attachment. A 100µL of the experimental media were added in the wells and the cells were exposed for 1, 24 and 48 hours respectively. The viability was determined using the CCK-8. Experiment was performed in triplicates and data was subjected to statistical analysis. Results Statistical analysis was performed using repeated measure ANOVA, ANOVA, and post-hoc Bonferroni test with the significance level p<0.05. The values are as follows: MK (1.3146 ±0.0588, 1.9012±0.0511, 2.0723±0.1211) > Cornisol (1.2399±0.0548, 1.9596±0.0652, 1.9592±0.1361) >DMEM (1.1914±0.0691, 1.8479±0.0116, 2.0718±0.0795) > HBSS (0.3665±0.0814, 0.0184±0.0010, 0.0248±0.0042) >distilled water (0.0122±0.0033, 0.0225±0.0085, 0.0104±0.0008) at 1 hour, 24 hours and 48 hours respectively. MK >Cornisol>DMEM>HBSS>distilled water. Conclusion It can be concluded that the corneal preservation solutions showed promising results in preserving periodontal ligament cell viability for extended time periods.