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Purpose: Osteoblasts are capable to produce different compounds directly connected to bone mineralization process.
This study aims to standardize the reverse transcriptase polymerase chain reaction (RT-PCR) for adult osteoblasts to
observe the effect of low level laser therapy on bone mineralization. Methods: Five-millimeter long fragments obtained
from the mead femoral region of male Wistar rats were assigned into group A (n=10, laser) and group B (n=10, no laser),
submitted to mechanic and enzymatic digestion. After 7 days, cultures of group A were irradiated daily on a single spot
with a GaInAs laser, λ=808nm, 200mW/cm2, 2J/cm2, bean diameter of 0,02mm, 5 seconds for 6 days. Group B was manipulated but received no laser irradiation. After 13 days the cells were trypsinized for 15 minute and stabilized with RNA later® for RNA extraction with Trizol®. cDNA synthesis used 10μg of RNA and M-MLV® enzyme. PCR was accomplished using the β-actin gene as a control. Another aliquot was fixed for Hematoxylin-Eosin and Von Kossa staining to visualize bone mineralization areas. Results: Under UV light we observed clearly the amplification of β-actin
gene around 400bp. HE and Von Kossa staining showed osteoblast clusters, a higher number of bone cells and well
defined mineralization areas in group A. Conclusion: The cell culture, RNA extraction and RT-PCR method for adult
osteoblasts was effective, allowing to use these methods for bone mineralization studies. Laser improved bone
mineralization and further studies are needed involving osteogenesis, calcium release mechanisms and calcium related
channels.
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