Gene and protein expression markers of bone formation under the influence of low-level laser irradiation.

Abstract

Bone tissue has mechanical, mineral deposit and hematopoietic functions being composed of three cell types, osteoblasts, osteocytes and osteoclasts. Several substances are secreted by osteoblasts like protein and calcium ion, which plays a fundamental role in cellular homeostasis and in production of a mineralized bone tissue and whose flow S100A6 genes are involved, osteocalcin and PMCA1b linked to the transport and mineralization process. Many resources can be used to change cell permeability and anticipate the mineralization process like low-level laser therapy. Laser is characterized by a monochromatic light with specific wavelength that has the ability to modulate cell, increasing cellular respiration, DNA synthesis and protein translation. The aim of this study is to evaluate the low-level laser irradiation effects related to calcium signaling and bone mineralization, in genes and proteins S100A6, PMCA1b and osteocalcin expression in adult osteoblastic cells. Cell line of human osteoblasts cells hFOB 1:19 (ATCC® CRL-11372 ") will be assigned into two groups, A (n=12, without laser) and B (n=12, laser). Cells will be re-suspended and cultured in Ham's F12 and Eagle (1: 1) medium, added L-glutamine, fetal bovine serum (10%), gentamicin (10 mg / ml) and Fungizone (250 / mL). From day 1, group B will be irradiated with low-level laser with a Gallium-Arsenide-Aluminium device with »=808nm, 250mW nominal power, power density of 200mW/cm2, power density of 2000mJ/cm2, energy dose of 2J/cm2, beam diameter of 0.02mm, 5s time in one application point until the 13th day. The cells will be cultured for 13 days with daily collections of cells to perform the real time PCR techniques and Western blotting for osteocalcin, S100A6 and PMCA1b and addition to patch clamp. The RNA will be extracted, quantified, submitted to cDNA synthesis and quantification performed by the 2-””Ct comparative technique. Protein extractions will be performed by the M-PER and N-PER kit, proteins will be submitted to SDS-PAGE electrophoresis and subsequent transfer to nitrocellulose membranes and incubation with primary and secondary antibodies revelation for further analysis. Additionally, the patch clamp technique will be performed to evaluate ion channels, specifically calcium channels. It is expected that after the low-level laser irradiation will promote an increase in S100A6, PMCA1b and osteocalcin genes expression and S100A6, osteocalcin and PMCA1b protein expression and intracellular calcium flux. (AU)