Deleterious effect of triple antibiotic paste on human periodontal ligament fibroblasts

Abstract

Aim: To evaluate the cytotoxic effects of triple antibiotic paste (TAP), double antibiotic paste (DAP), minocycline and calcium hydroxide and their influence on cytokine mRNA expression levels on human periodontal ligament (HPDL) fibroblasts.

Methodology: Triple antibiotic paste, DAP and Ca(OH)2 test samples were immersed in culture medium and incubated at 37 °C for 24 and 48 h. HPDL cells were seeded at a density of 2 × 10(4) cells and exposed to either culture media (negative control), 0.1% SDS (positive control), 24- or 48-h elutes of each test material and incubated for 24 h. A multiparametric cytotoxicity assay kit (XTT, NR and CVDE) was used to evaluate the cytotoxic effects of each test material. Results were analysed using an ELISA plate reader and light absorbances of 450 and 530 nm as references. Cytokine mRNA expression levels in HPDL cells treated with the materials were also investigated using real-time PCR. Expression levels were calculated using the comparative 2(-ΔΔCt) method. Statistical analyses included anova followed by Bonferroni correction.

Results: Triple antibiotic paste and minocycline were the most cytotoxic materials when compared with DAP and Ca(OH)2 in all three (XTT, NR and CVDE) assays (P < 0.0001). No significant differences (P > 0.05) were found for cytokine gene expression levels after exposure to 24- or 48-h elutes of any of the materials except for IL6, which had significantly higher mRNA levels with the 24-h TAP elute (P < 0.01).

Conclusion: Ca(OH)2 had a minimal effect on cell viability and cytokine production. The TAP showed deleterious effects on HPDL viability and increased expression of the pro-inflammatory cytokine IL6.

Keywords: calcium hydroxide; cytotoxicity; double antibiotic paste; human fibroblasts; minocycline; triple antibiotic paste.