title: Delivery of plasmid DNA to articular chondrocytes via novel collagen-glycosaminoglycan matrices.
abstract: Our primary objective was to fabricate a porous gene-supplemented collagen-glycosaminoglycan (GSCG) matrix for sustained delivery (over a period of several weeks) of plasmid DNA to articular chondrocytes when implanted into cartilage lesions. The specific aims of this in vitro study were to determine the release kinetics profiles of plasmid DNA from the GSCG matrices, and to determine the ability of the released plasmid DNA to transfect adult canine articular chondrocytes. In particular, we evaluated the effects of two variables, cross-linking treatment and the pH at which the DNA was incorporated into the matrices, on the amount of the plasmid DNA that remained bound to the GSCG matrices after passive (nonenzymatic) leaching and on the expression of a reporter gene in articular chondrocytes grown in the GSCG matrices. Collagen-glycosaminoglycan matrices were synthesized without cross-linking, and by three cross-linking treatments: dehydrothermal (DHT) treatment, 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) treatment, and exposure to ultraviolet (UV) radiation. The plasmid DNA was incorporated into the collagen-glycosaminoglycan matrices in solutions at pH 2.5 or 7.5. Transmission electron microscopy studies revealed plasmid DNA bound to the walls of the porous GSCG matrices. In general, the GSCG matrices fabricated at pH 2.5 retained a larger fraction of the initial DNA load after 28 days of incubation in Tris-EDTA buffer. The passive, solvent-mediated release of the plasmid DNA from the GSCG matrices showed a biphasic pattern consisting of a faster, early release rate over the initial 8 hr of leaching followed by a slower, late release rate that was relatively constant over the subsequent 28 days of leaching. Electrophoretic analyses revealed that the plasmid DNA released from the GSCG matrices fabricated at pH 2.5 had been linearized and/or degraded; whereas the plasmid DNA leached from the GSCG matrices prepared with a DNA solution at pH 7.5 was primarily supercoiled and linear. Plasmid DNA released from all GSCG matrix formulations was able to generate luciferase reporter gene expression in monolayer-cultured chondrocytes transfected with the aid of a commercial lipid reagent, and in chondrocytes cultured in the GSCG matrices without the aid of a supplemental transfection reagent. Luciferase expression in chondrocyte-seeded GSCG constructs was evident throughout the culture period (28 days), with the EDC and UV cross-linked matrices prepared at pH 7.5 providing the highest transgene expression levels. We conclude that released plasmid DNA continually transfected canine articular chondrocytes seeded into GSCG matrices in vitro for a 4-week period as evidenced by luciferase reporter gene expression. Thus, GSCG matrices can be fabricated to provide sustained release of plasmid DNA carrying a potential therapeutic gene.
authors.affiliation: Department of Orthopedic Surgery, Brigham and Women's Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115, USA.
authors.forename: R E
authors.initials: RE
authors.lastname: Samuel
authors.forename: C R
authors.initials: CR
authors.lastname: Lee
authors.forename: S C
authors.initials: SC
authors.lastname: Ghivizzani
authors.forename: C H
authors.initials: CH
authors.lastname: Evans
authors.forename: I V
authors.initials: IV
authors.lastname: Yannas
authors.forename: B R
authors.initials: BR
authors.lastname: Olsen
authors.forename: M
authors.initials: M
authors.lastname: Spector
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D019902:Chondrocytes / Q000378:metabolism; D003094:Collagen / Q000378:metabolism*; D004247:DNA / Q000378:metabolism; D004285:Dogs; D004587:Electrophoresis, Agar Gel; D018014:Gene Transfer Techniques; D015316:Genetic Therapy / Q000379:methods; D006025:Glycosaminoglycans / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008156:Luciferases / Q000378:metabolism; D008854:Microscopy, Electron; D008855:Microscopy, Electron, Scanning; D010957:Plasmids / Q000378:metabolism*; D011485:Protein Binding; D013997:Time Factors; D014162:Transfection; D014466:Ultraviolet Rays
chemical_list: D006025:Glycosaminoglycans; D003094:Collagen; D004247:DNA; D008156:Luciferases