Cell Culture

Cell line hFOB 1.19 (ATCC® CRL-11372^TM) of human osteoblast was acquired from the American Type Culture Collection (ATCC), provided by the renal biology laboratory of São Paulo University (USP-SP) and cultured according to Harris et al.²¹ Cells were grown in a six-well plate with a growth area of 9.07 cm², and 10⁴ cells were seeded per well with 2 mL of Ham’s F12 Dulbecco’s modified medium and Eagle’s medium in a 1:1 mixture, without phenol red, plus 2.5 mM L-glutamine, fetal bovine serum (10% - Gibco), geneticin - G418 (50 mg/mL - Sigma Chemical Co., St. Louis, MO, USA) and fungizone (250 μg/mL - Sigma Chemical Co, St. Louis, MO, USA) at 34 °C and 5% CO₂, with the culture medium changed every 3 days. When 70% confluence was reached in each well of each plate, the oven temperature was adjusted to 39 ºC for differentiation of the cell line according to ATCC instructions. After 4 days at 39 ºC, the osteoblasts acquired an adult phenotype and the laser group (L) was irradiated. During the experiment, the control group (C) followed an identical procedure with the device in the off position. The groups were as follows: C (n = 78; n = 6 per day) without low-level laser application and L (n = 78; n = 6 per day) with low-level laser application.