There is increasing interest in biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz) to investigate the effect of the electromagnetic stimulation on SAOS-2 human gene expression. In comparison with static conditions (without electromagnetic stimulation) RT -PCR analysis revealed the electromagnetically upregulated expression of many and specific genes of bone. Results show that the bioreactor increase cell colonization of polyurethane scaffolds. We think that a better result could be obtained using bone marrow stromal osteoblasts instead of SAOS-2 cells and cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair. Materials and Methods. Cells: Human osteosarcoma cell line SAOS-2 was obtained from the American Type Culture Collection (ATCC, HTB85, Rockville, MD, USA). Cells were cultured at 37°C with 5% C02, routinely trypsinized, counted, and seeded onto the porous polyurethane scaffolds. Electromagnetic bioreactor: It consists of a calTying structure custom¬machined in a tube of polymethylmethacrylate; the windowed tube carried a well-plate and two solenoids with parallel planes. Assay for gene expression: At the end of the culture period, the total RNA was extracted from the cultured scaffolds using the RNeasy system according to manufacturer's protocol (Qiagen, Inc.). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed in order to evaluate the gene expression for decorin, fibronectin, osteocalcin, osteopontin, TGF-B, type-I collagen, type-III collagen, and the housekeeping gene expression for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reverse transcriptase reaction was performed from 500 ng of the total RNA using the ThermoScript RT-PCR System (Invitrogen Corporation). The primers (Primm s.r.l., Milan, Italy) were designed according to the published gene sequences, and the polymerase chain reactions were performed by the GeneAmp PCR System 9700 (Applied Biosystems, Inc.). In conclusion, calcified matrix coated scaffolds could be used as products for the bone repair, References: (1) Lohmann et al. J. Orthop. Res. 21, 326,2003. (2) Aaron et al. Clin. Orthop. Relat Res. 30, 2004. (3) Anderson et al. J. Bone Miner. Metab 20, 73, 2002. (4) Bodamyali et aI. Biochem. Biophys. Res. Commun. 250, 458, 1998. (5) Fitzsinunons et aI. J. Bone Miner. Res. 10,812, 1995.

Electromagnetic fields influence the gene expression of SAOS-2 cell line

MALIARDI, VALENTINA;BENEDETTI, LAURA;BORATTO, RENATA;TAZZI, ANTONIO;FASSINA, LORENZO;MICHELETTI, PIERO;SAMPAOLESI, MAURILIO;CUSELLA DE ANGELIS, MARIA GABRIELLA
2005

Abstract

There is increasing interest in biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz) to investigate the effect of the electromagnetic stimulation on SAOS-2 human gene expression. In comparison with static conditions (without electromagnetic stimulation) RT -PCR analysis revealed the electromagnetically upregulated expression of many and specific genes of bone. Results show that the bioreactor increase cell colonization of polyurethane scaffolds. We think that a better result could be obtained using bone marrow stromal osteoblasts instead of SAOS-2 cells and cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair. Materials and Methods. Cells: Human osteosarcoma cell line SAOS-2 was obtained from the American Type Culture Collection (ATCC, HTB85, Rockville, MD, USA). Cells were cultured at 37°C with 5% C02, routinely trypsinized, counted, and seeded onto the porous polyurethane scaffolds. Electromagnetic bioreactor: It consists of a calTying structure custom¬machined in a tube of polymethylmethacrylate; the windowed tube carried a well-plate and two solenoids with parallel planes. Assay for gene expression: At the end of the culture period, the total RNA was extracted from the cultured scaffolds using the RNeasy system according to manufacturer's protocol (Qiagen, Inc.). Reverse transcriptase-polymerase chain reaction (RT-PCR) was performed in order to evaluate the gene expression for decorin, fibronectin, osteocalcin, osteopontin, TGF-B, type-I collagen, type-III collagen, and the housekeeping gene expression for glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The reverse transcriptase reaction was performed from 500 ng of the total RNA using the ThermoScript RT-PCR System (Invitrogen Corporation). The primers (Primm s.r.l., Milan, Italy) were designed according to the published gene sequences, and the polymerase chain reactions were performed by the GeneAmp PCR System 9700 (Applied Biosystems, Inc.). In conclusion, calcified matrix coated scaffolds could be used as products for the bone repair, References: (1) Lohmann et al. J. Orthop. Res. 21, 326,2003. (2) Aaron et al. Clin. Orthop. Relat Res. 30, 2004. (3) Anderson et al. J. Bone Miner. Metab 20, 73, 2002. (4) Bodamyali et aI. Biochem. Biophys. Res. Commun. 250, 458, 1998. (5) Fitzsinunons et aI. J. Bone Miner. Res. 10,812, 1995.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11571/270715
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