title: High-efficiency non-viral transfection of primary chondrocytes and perichondrial cells for ex-vivo gene therapy to repair articular cartilage defects.
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.
  
  
  METHODS
  A protocol for high-efficiency transfection of primary perichondrial and cartilage cells was optimized. Plasmids carrying the marker beta-galactosidase (beta-gal), PTHrP and TGF-beta1 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 beta-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 beta-gal, in vivo for the entire test period of 7 days. Furthermore, primary perichondrial cells transfected with TGF-beta1 and PTHrP over-expressed their cognate gene products.
  
  
  CONCLUSIONS
  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.
authors: Department of Orthopedics, University of California, San Diego School of Medicine, 9500 Gilman Dr., La Jolla, California, 92093-0630, USA. rgoomer@ucsd.edu
authors: R S
authors: RS
authors: Goomer
authors: L J
authors: LJ
authors: Deftos
authors: R
authors: R
authors: Terkeltaub
authors: T
authors: T
authors: Maris
authors: M C
authors: MC
authors: Lee
authors: F L
authors: FL
authors: Harwood
authors: D
authors: D
authors: Amiel
mesh_terms: D000818:Animals; D002357:Cartilage Diseases / Q000628:therapy*; D002358:Cartilage, Articular; D019902:Chondrocytes; D005818:Genetic Engineering / Q000379:methods*; D015316:Genetic Therapy / Q000379:methods*; D006614:Hindlimb; D007596:Joints; D044162:Parathyroid Hormone-Related Protein; D011506:Proteins / Q000235:genetics; D011817:Rabbits; D014162:Transfection / Q000379:methods*; D016212:Transforming Growth Factor beta / Q000235:genetics; D053773:Transforming Growth Factor beta1; D016896:Treatment Outcome; D001616:beta-Galactosidase / Q000235:genetics
chemical_list: D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D001616:beta-Galactosidase