In vitro viability, mitogenicity and clonogenic capacities of periodontal ligament fibroblasts after storage in four media supplemented with growth factors.

Abstract

The choice of storage medium for preserving traumatically avulsed teeth is important for the success of future replantation. The objective of this study was to evaluate the effectiveness of growth factors (IGF-1 and PDGF-BB) when added to storage media in preserving the functional abilities of cultured periodontal ligament fibroblasts (PDLF). The evaluated storage media were: ViaSpan, Hanks' balanced salt solution (HBSS), alpha minimal essential medium (alpha MEM), and alpha MEM supplemented with FCS and antibiotic (alpha MEM-S). PDLF were obtained from explants of human healthy extracted teeth. Plates with confluent PDLF were soaked in the various media supplemented with IGF-1 (10 ng/ml) and PDGF-BB (4 ng/ml) for 2, 8 and 24 h at room temperature (24 degrees C). The control group was incubated with the examined storage media without growth factors at 24 degrees C. An additional control group was incubated with culture medium at 37 degrees C without growth factors. After incubation, the viability of the cells was determined by Trypan blue exclusion test. Viable cells were then analyzed for mitogenic (with thymidine) and clonogenic (by culturing one cell/well) capacities. Storage of PDLF with growth factors (GF) for 2, 8 and 24 h decreased their vitality by only 3% (not statistically significant). The mitogenicity of PDLF stored for 2, 8 and 24 h in various media with GF was statistically comparable to that of the control group. Generally, the highest mitogenic capacity of PDLF stored with or without GF was found after 8 h of storage. Increasing the storage period to 24 h decreased the mitogenic capacity of the cells stored with GF by only 10-40% compared to the control group. In contrast, the clonogenic capacity of PDLF stored with GF increased with increasing storage periods by 100-300%, and the highest clonogenic capacity was found in most storage media after 24 h of storage with GF. The highest clonogenic and mitogenic capacities were found in cells stored in HBSS followed by alpha MEM-S. The mitogenic and clonogenic capacities of PDLF stored in various media supplemented with GF for 2-8 h were generally lower than without GF supplementation. The mitogenic and clonogenic effects of GF-supplementation was observed only after 24 h of storage. After 24 h of storage with GF, the clonogenic capacity increased by 8-224% and the mitogenicity by 20-37%, except in cells stored in alpha MEM (-1%). However, these differences were generally not statistically significant. In conclusion, the mitogenic and clonogenic effects of GF were observed only after 24 h of storage at room temperature. HBSS and alpha MEM-S supplemented with GF were the most effective media for preserving the viability, mitogenicity and clonogenic capacity of PDLF stored for 24 h at room temperature. For short periods of storage (2 and 8 h), HBSS and alpha MEM-S without GF were preferable.