In vitro evaluation of the effects of low level laser therapy on cultured osteoblasts, fibroblasts and keratonicytes submitted to zoledronic acid therapy

Abstract

Oral osteonecrosis has been associated to bisphosphonates therapy. It has been demonstrated that these drugs may cause cytopathic effects to cells of bone tissue and to adjacent tissues, such as fibroblasts and keratinocytes. Some clinical studies demonstrated excellent results when the patients with osteonecrosis were submitted to low-level laser therapy. Therefore, the aims of this in vitro study are to evaluate the effects: 1) of aminobisphosphonate Zoledronate (Zometa® - Novartis, Brasil) on cultured of osteoblasts, gingival fibroblasts, and keratinocytes; 2) low-level laser therapy on these cells previously treated with Zometa® For the first experiment, the cells will be seeded (30,000 cells/cm2) in wells of 24-well plates using complete culture medium (DMEM) and incubated for 24 hours. Then, the DMEM will be replaced by a solution of Zometa® (5μM/mL) diluted in a new DMEM with no concentrations of fetal bovine serum (FBS). After 48-hour incubation, the cells will be evaluated for cell metabolism, total protein dosage, alkaline phosphatase activity and morphology will be evaluated. For the second part, the cells treated with Zometa® will be irradiated three times with LLL in specific parameters (780±3 nm, 40 mW, 3 J/cm2) every 24 hours, giving rise to the following experimental groups: G1- DMEM (control); G2- DMEM+Zometa®; G3-DMEM+Lasertherapy; G4- DMEM+Zometa®+Lasertherapy. Then, all groups will be evaluated by the same analyses described before and also by gene expression for VEGF, bFGF, Col-1, RANK, OPG, KGF e EGF (RT-PCR). The data will be analyzed by the normality, and if this is respected the chosen tests will be ANOVA, complemented by the Tukey. If normality could not be achieved, the test chosen will be Kruskal-Wallis, complemented with Mann-Whitney at significance level of 5%. (AU)