Fig. 3

Evaluation of hypertrophic and terminal differentiation processes in rAAV-transduced human bone marrow aspirates in static versus dynamic culture conditions. Aspirates (150 μl) were prepared and transduced with rAAV-luc or rAAV-FLAG-hsox9 as described in Fig. 1 and 2 or let untransduced for maintenance in dynamic or static culture conditions for up to 28 days (n = 4 independent samples tested in triplicate per vector treatment and culture condition in three independent experiments) as described in the Methods. The samples were processed (a) for alizarin red staining and to detect the expression of type-X collagen after 7, 21, and 28 days as described in the Methods (magnification x10, scale bars: 200 μm, all representative data) and (b) to evaluate the gene expression profiles (COL10A1, ALP, MMP13, RUNX2, and β-catenin, with GAPDH serving as a housekeeping gene and internal control) after 21 days by real-time RT-PCR amplification as described in the Methods and in Fig. 2. Ct values were obtained for each target and GAPDH as a control for normalization, and fold inductions (relative to untreated aggregates) were measured by using the 2^-ΔΔCt method. Statistically significant relative to ^ano vector treatment and ^brAAV-luc application