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 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.
Goomer
R S
RS
Department of Orthopedics, University of California, San Diego School of Medicine, 9500 Gilman Dr., La Jolla, California, 92093-0630, USA. rgoomer@ucsd.edu
Deftos
L J
LJ
Terkeltaub
R
R
Maris
T
T
Lee
M C
MC
Harwood
F L
FL
Amiel
D
D
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
D044162:Parathyroid Hormone-Related Protein; D011506:Proteins; D016212:Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; D001616:beta-Galactosidase