RT-PCR standardization and bone mineralization after low-level laser therapy on adult osteoblast cells

RT-PCR standardization and bone mineralization after low-level laser therapy on adult osteoblast cells

Author Costa do Bomfim, Fernando Russo Autor UNIFESP Google Scholar
Gonzalez Sella, Valeria Regina Autor UNIFESP Google Scholar
Zanaga, Jessica Querido Google Scholar
Pereira, Nayara Simionatto Google Scholar
Andree Nouailhetas, Viviane Louise Google Scholar
Plapler, Helio Autor UNIFESP Google Scholar
Choi, B. Google Scholar
Kollias, N. Google Scholar
Zeng, H. Google Scholar
Kang, H. W. Google Scholar
Wong, BJF Google Scholar
Ilgner, J. F. Google Scholar
Tearney, G. J. Google Scholar
Gregory, K. W. Google Scholar
Marcu, L. Google Scholar
Mandelis, A. Google Scholar
Institution Universidade Federal de São Paulo (UNIFESP)
Abstract 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, lambda=808nm, 200mW/cm(2), 2J/cm(2), 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 (R) for RNA extraction with Trizol (R). cDNA synthesis used 10 mu g of RNA and M-MLV (R) enzyme. PCR was accomplished using the beta-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 beta-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.
Keywords Osteoblasts
low-level laser therapy
bone
osteogenesis
Language English
Date 2014-01-01
Published in Photonic Therapeutics and Diagnostics X. Bellingham: Spie-int Soc Optical Engineering, v. 8926, 6 p., 2014.
ISSN 0277-786X (Sherpa/Romeo, impact factor)
Publisher Spie-int Soc Optical Engineering
Extent 6
Origin http://dx.doi.org/10.1117/12.2037340
Access rights Closed access
Type Conference paper
Web of Science ID WOS:000337574500062
URI http://repositorio.unifesp.br/handle/11600/37284

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