Effect of Nicotine and Porphyromonas gingivalis on the Differentiation Properties of Periodontal Ligament Fibroblasts

Abstract

Abstract Objectives This study aimed to evaluate the effect of Porphyromonas gingivalis and nicotine on the in vitro osteogenic differentiation of periodontal ligament (PDL) fibroblasts. Materials and Methods PDLs were cultured in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum at 37°C under 5% CO2 and 100% humidified atmosphere. Cells were incubated with various concentrations of nicotine and P. gingivalis extracts, and cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. To study cell differentiation, PDLs (5 × 104cells) were treated with the osteogenic differentiation medium containing 10 mM β-glycerophosphate, 10 nM dexamethasone, 50 mg/mL ascorbic acid, 1 μM nicotine, and 50 µg/mL P. gingivalis lysate. mRNA samples were collected at 0, 7, and 14 days. Odontogenic-related gene expression, namely, Runt-related transcription factor 2 (Runx2), collagen type I (COL1A1), and alkaline phosphatase (ALP) was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). Calcified nodule formation was determined on day 28 using Alizarin Red S. Analysis of variance and Tukey’s test were used to compare the difference among groups at significant level of p < 0.05. Results It showed that 50 µg/mL of P. gingivalis lysate and 1 µM of nicotine showed no toxicity to PDLs. Runx2, COL1A1, and ALP expression were found to decrease significantly after 7 days of treatment, while osteocalcin expression was found to decrease after 14 days. The nodule formation in the control group was much greater in both number and size of nodules than in experimental groups, which implied a positive sign of calcium deposition in controls. Conclusion The results indicated that nicotine and P. gingivalis showed adverse effect on osteogenic differentiation properties of PDLs.