In osteochondral defect cultures, a higher deposition of type-II collagen and reduced hypertrophic events were noted

In osteochondral defect cultures, a higher deposition of type-II collagen and reduced hypertrophic events were noted. and poly (propylene oxide) (PPO) (poloxamer and poloxamine) polymeric micelles as a means to overexpress the therapeutic factor transforming growth factor-beta (TGF-) in human OA chondrocytes and in experimental human osteochondral defects. Application of rAAV-human transforming growth factor-beta using such micelles increased the levels of TGF- transgene expression compared with free vector treatment. Overexpression of TGF- with these systems resulted in higher proteoglycan deposition and increased cell numbers in OA chondrocytes. In osteochondral defect cultures, a higher deposition of type-II collagen and reduced hypertrophic events were noted. Delivery of therapeutic rAAV vectors via PEO-PPO-PEO micelles may provide potential tools to remodel human OA cartilage. in human OA chondrocytes in vitro and in experimental osteochondral defects without detrimental effects on the biological activities of the cells nor on their phenotype, also affording protection when anti-AAV capsid neutralizing antibodies were present.37 In light of such promising findings, the purpose of the present study was to test whether PF68 and T908 polymeric micelles can deliver a candidate rAAV TGF- vector to human OA chondrocytes and to human osteochondral defects in order to overexpress the growth factor as a Rabbit Polyclonal to NCoR1 potent therapeutic approach for the future treatment of articular cartilage injuries. Materials and methods Materials Pluronic? F68 and Tetronic? 908 were kindly provided by BASF (Ludwigshafen, Germany). The anti-TGF- (V) was from Santa Cruz Biotechnology (Heidelberg, Germany). The anti-type-II collagen (II-II6B3) antibody was from DSHB (Iowa City, IA, USA) and the anti-type-X collagen (COL-10) antibody from Sigma (Munich, Germany). Biotinylated secondary antibodies and the ABC reagent were from Vector Laboratories (Alexis Deutschland GmbH, 6-Methyl-5-azacytidine Grnberg, Germany). The Cy3 Ab Labeling Kit was from Amersham/GE Healthcare (Munich, Germany). The cell proliferation reagent WST-1 and the Cytotoxicity Detection KitPLUS (LDH) were from Roche Applied Science (Mannheim, Germany). The TGF- enzyme-linked immunosorbent assay (ELISA) (hTGF- Quantikine ELISA) was from R&D Systems (Wiesbaden, Germany). Other reagents were from Sigma (Munich, Germany). Cells and osteochondral defect model Human OA cartilage (Mankin score of 7C9) was from total knee arthroplasty samples (n=7) from patients who previously signed informed consent.32 The study was approved by the Ethics Committee of the Saarland Physicians Council (Approval Ha67/12) and all procedures were in accordance with the Helsinki Declaration. Human OA chondrocytes were isolated as previously described32 and used not later than passage 3. Cells were incubated at the denoted cell densities in DMEM, 10% fetal bovine serum, 100 U/mL penicillin G, 100 L/mL streptomycin (growth medium) for 12 h 6-Methyl-5-azacytidine at 37C under 5% CO2 prior to addition of the rAAV/copolymer systems or free rAAV preparations (see below for concentrations) for up to 10 days for 6-Methyl-5-azacytidine consistency with our previous study with reporter vectors.37 Osteochondral defects were created in human OA cartilage biopsies (n=7) using a 1-mm drill 6-Methyl-5-azacytidine needle in standardized cylindrical (6-mm diameter) as previously described37 and incubated in growth medium prior to addition of the rAAV/copolymer systems or free rAAV preparations at the concentrations indicated thereafter for 10 days. Plasmids and rAAV vectors The constructs were derived from pSSV9, an AAV-2 genomic clone.38,39 rAAV-hTGF- carries a 1.2-kb human transforming growth factor-beta 1 (hTGF-) cDNA fragment under the control of the cytomegalovirus immediate-early promoter.32,36,40 The vectors were packaged as conventional (not self-complementary) vectors using a helper-free, 2-plasmid transfection system in 293 cells 6-Methyl-5-azacytidine with the packaging plasmid pXX2 and the adenovirus helper plasmid pXX6.32 The vector preparations were purified by extensive dialysis and titrated by real-time polymerase chain reaction,32,36,40 averaging 1010 transgene copies/mL (~1/500 functional recombinant viral particles). Cy3 labeling rAAV vectors were labeled using the Cy3 Ab Labeling Kit as previously described.41 Briefly, rAAV (1 mL) was dispersed in sodium carbonate/sodium bicarbonate buffer (pH 9.3), kept.