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High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex-vivo gene therapy to repair articular cartilage defects

https://doi.org/10.1053/joca.2000.0382Get rights and content
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Abstract

Background Primary perichondrial cells and chondrocytes have been used to repair articular cartilage defects in tissue engineering studies involving various animal models. Transfection of these cells with a gene that induces chondrocytic phenotype may form an ideal method to affect tissue engineering of articular cartilage.

Design A protocol for high-efficiency transfection of primary perichondrial and cartilage cells was optimized. Plasmids carrying the marker β-galactosidase (β-gal), PTHrP and TGF-β1 genes driven by a strong mammalian promoter were transfected into primary perichondrial cells and chondrocytes. A three-step method was used to achieve high efficiency of transfection: (1) permeabilization of primary cells using a mild detergent, (2) association of plasmid DNAs with a polycationic (poly-l-lysine) core covalently linked to a receptor ligand (transferrin), (3) introduction of cationic liposomes to form the quaternary complex. For in-vivo assessment, polylactic acid (PLA) scaffolds seeded with β-gal transfected perichondrial cells were implanted into experimentally created osteochondral defects in rabbit knees for 1 week.

Results The efficiency of transfection was determined to be over 70%in vitro. The transformed cells continued to express β-gal, in vivo for the entire test period of 7 days. Furthermore, primary perichondrial cells transfected with TGF-β1 and PTHrP over-expressed their cognate gene products.

Conclusion The ability to transfect autologous primary perichondrial cells and chondrocytes with high efficiency using a non-viral system may form a first step towards tissue engineering with these transformed cells to repair articular cartilage defects.

Keywords

Gene therapy, Tissue engineering, Articular cartilage repair, Transfection, Primary perichondrial cells, Primary chondrocytes, PTHrP, TGF-β1

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Address correspondence to: Randal S. Goomer, PhD, Dept of Orthopedics, 0630, UCSD School of Medicine, 9500 Gilman Dr., La Jolla, CA 92093-0630, U.S.A. E-mail:[email protected]