Basso Fernanda Gonçalves, Pansani Taisa Nogueira, de Oliveira Camila Fávero, Turrioni Ana Paula Silveira, Soares Diana Gabriela, Hebling Josimeri, Costa Carlos Alberto de Souza
UNICAMP - University of Campinas, Piracicaba School of Dentistry, PiracicabaSP, Brazil.
UNESP - Univ Estadual Paulista, Araraquara School of Dentistry, AraraquaraSP, Brazil.
Braz Dent J. 2013 Nov-Dec;24(6):551-8. doi: 10.1590/0103-6440201302229.
Bisphosphonate-induced osteonecrosis has been related to the cytotoxicity of these drugs on oral mucosa cells. A previous study showed that 5 µM of zoledronic acid (ZA), a nitrogen-containing bisphosphonate, is the highest concentration of this drug found in the oral cavity of patients under treatment. Therefore, in order to simulate an osteonecrosis clinical condition, the aim of this study was to evaluate the highest concentration of ZA applied on human epithelial cells (HaCaT) and gingival fibroblasts. For this purpose, cells (3 × 10(4) cells/cm2) were seeded in wells for 48 h using complete culture medium (cDMEM). After 48 h incubation, the cDMEM was replaced by fresh serum-free culture medium (DMEM-FBS) in which the cells were maintained for additional 24 h. Then, 5 µM ZA were added to the DMEM-FBS and the cells incubated in contact with the drug for 48 h. After this period, the number of viable cells (trypan blue), cell viability (MTT assay), total protein (TP) production and cell morphology (SEM analysis) were assessed. Data were analyzed statistically by Mann-Whitney, ANOVA and Tukey's test (α=0.05). ZA caused a significant reduction in the number of viable cells and decreased the metabolic activity of both cell lines. However, decrease of TP production occurred only in the epithelial cell cultures. Morphological alterations were observed in both cell types treated with ZA. In conclusion, ZA (5 µM) was cytotoxic to human epithelial cells and gingival fibroblast cultures, which could be associated, clinically, with the development of bisphosphonate-induced osteonecrosis.
双膦酸盐诱导的骨坏死与这些药物对口腔黏膜细胞的细胞毒性有关。先前的一项研究表明，5微摩尔的唑来膦酸（ZA），一种含氮双膦酸盐，是在接受治疗患者口腔中发现的该药物的最高浓度。因此，为了模拟骨坏死临床状况，本研究的目的是评估应用于人类上皮细胞（HaCaT）和牙龈成纤维细胞的ZA最高浓度。为此，使用完全培养基（cDMEM）将细胞（3×10⁴个细胞/cm²）接种于孔中48小时。孵育48小时后，将cDMEM替换为新鲜的无血清培养基（DMEM - FBS），细胞在其中再维持24小时。然后，向DMEM - FBS中加入5微摩尔的ZA，细胞与药物接触孵育48小时。在此之后，评估活细胞数量（台盼蓝法）、细胞活力（MTT法）、总蛋白（TP）产量和细胞形态（扫描电子显微镜分析）。数据采用曼 - 惠特尼检验、方差分析和图基检验进行统计学分析（α = 0.05）。ZA导致活细胞数量显著减少，并降低了两种细胞系的代谢活性。然而，TP产量的降低仅发生在上皮细胞培养物中。在用ZA处理的两种细胞类型中均观察到形态学改变。总之，5微摩尔的ZA对人类上皮细胞和牙龈成纤维细胞培养物具有细胞毒性，这在临床上可能与双膦酸盐诱导的骨坏死的发生有关。
