Regenerative Capacity of Human Dental Pulp and Apical Papilla Cells after Treatment with a 3-Antibiotic Mixture

Abstract

IntroductionA 3-antibiotic combination (3Mix) has been widely used in regenerative endodontics. Recent studies recommend that a safe concentration of 3Mix is in the range of 0.39 μg/mL and 1 mg/mL because higher concentrations may limit tissue regeneration. The aim of this study was to determine the regenerative capacity of isolated human dental pulp cells (DPCs) and apical papilla cells (APCs) after a 7-day treatment with selected doses of 3Mix. MethodsPrimary human DPCs/APCs from the third passage were divided into control and experimental groups. In the control group, cells were cultured in regular complete media. In the experimental group, cells were cultured in complete media containing 0.39 μg/mL or 1 mg/mL of 3Mix for 7 days. After the treatment period, the media were changed, and the cells were further tested for proliferation and differentiation potential. For cell proliferation, a colorimetric qualification of 3-[4, 5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide was used on days 1, 3, 5, and 7. For differentiation potential, a dentinogenic differentiation medium was added into treated cells and cultured for 7, 14, and 21 days. Results were analyzed using quantitative alizarin red S staining and real-time reverse-transcription polymerase chain reaction. ResultsAfter 7 days of treatment, 100% cell death was discovered in the 1-mg/mL 3Mix group. The proliferative capacity of 0.39 μg/mL 3Mix-treated DPCs and APCs was significantly lower than that of untreated cells at all time points (P < .05). Mineralized nodule formation was found both in the 3Mix-treated and control groups, but it was significantly less in the 3Mix-treated groups at 7, 14, and 21 days (P < .01). Quantitative reverse-transcription polymerase chain reaction showed no statistically significant difference (95% confidence interval) in bone sialoprotein, alkaline phosphatase, and dentin matrix protein 1 gene expression in either 3Mix-treated DPCs or APCs compared with control groups. ConclusionsOne milligram per milliliter of 3Mix had strong toxicity to DPCs/APCs when applied for 7 days, whereas 0.39 μg/mL 3Mix showed no toxicity but still affected cell proliferation and mineralization potential. However, no differences in dentinogenic gene expressions were observed between the 3Mix-treated and untreated groups.