*2.5. Gene Transfer in Inflammatory Conditions via rAAV-FLAG-hsox9*/*Polymeric Micelles*

Human OA chondrocytes (3000 cells/well or 40,000 cells/well for Alcian blue staining) were seeded in 96-well plates and maintained for 12 h at 37 ◦C under 5% CO2 as previously described [22,23]. Monolayer cultures of OA chondrocytes were directly transduced with the rAAV-FLAG-h*sox9*/polymeric micelles (2 <sup>×</sup> 108 transgene copies, micellar concentration 2%) or after pre-incubation for 4 h with IL-1β (10 ng/mL) [32] or TNF-α (100 ng/mL) only [33], or concomitantly with IL-1β and TNF-α (10 and 100 ng/mL, respectively). Control conditions included cells cultured without vector treatment or copolymer solution (negative control) and cells transduced with free rAAV vector (positive control). Cultures were maintained for 10 days with 3 weekly medium changes.

Expression of SOX9 was monitored by immunocytochemistry using and anti-SOX9 specific primary antibody, a biotinylated secondary antibody, with the ABC method with diaminobenzidine (DAB) as previously described [23,29]. To control for secondary immunoglobulins, OA chondrocytes in monolayers cultures were assayed with exclusion of the primary antibody. All cultures were inspected under light microscopy (Olympus CKX41).

### *2.6. Histological and Immunocytochemical Analyses*

Chondrocytes in monolayer cultures were harvested after 1 and 10 days and fixed in 4% formalin [21–23] prior to the immunocytochemical analyses. Expression of SOX9 and type-II collagen was detected using specific primary and biotinylated secondary antibodies, and the ABC method with DAB chromogen, with examination under light microscopy (Olympus CKX41) [21,22]. Alcian blue staining was involved to detect matrix proteoglycans [21,22,34,35]. Briefly, fixed monolayer cultures were stained with Alcian blue (1% in HCl 1 N) and excess stain was washed with double distilled

water. The staining was solubilized by overnight incubation in 6 M guanidine hydrochloride and the absorbance at 595 nm was quantified with a GENios spectrophotometer (Tecan Crailsheim, Germany).
