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

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 engineeri ng of articular cartilage. Design: A protocol for high-efficiency transfection of primary perichondria l and cartilage cells was optimized. Plasmids carrying the marker beta -gal actosidase (beta -gal), PTHrP and TGF-beta1 genes driven by a strong mammal ian promoter were transfected into primary perichondrial cells and chondroc ytes. A three-step method was used to achieve high efficiency of transfecti on: (1) permeabilization of primary cells using a mild detergent, (2) assoc iation of plasmid DNAs with a polycationic (poly-l-lysine) core covalently linked to a receptor ligand (transferrin), (3) introduction of cationic lip osomes to form the quaternary complex. For in-vivo assessment, polylactic a cid (PLA) scaffolds seeded with beta -gal transfected perichondrial cells w ere implanted into experimentally created osteochondral defects in rabbit k nees for 1 week. Results: The efficiency of transfection was determined to be over 70% in vi tro. The transformed cells continued to express beta -gal, in vivo for the entire test period of 7 days. Furthermore, primary perichondrial cells tran sfected with TGF-beta1 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 repai r articular cartilage defects. (C) 2001 OsteoAtthritis Research Society Int ernational.