*3.2. E*ff*ects of rAAV-FLAG-hsox9*/*Polymeric Micelle Delivery on the Anabolic Activities of Human OA Chondrocytes in Inflammatory Conditions*

We next investigated the effects of SOX9 overexpression on the deposition of type-II collagen and proteoglycans following rAAV-FLAG-hsox9 gene transfer via micellar vehicles in human OA chondrocytes monolayer cultures maintained in conditions of inflammation.

Administering of rAAV-FLAG-hsox9 significantly incremented type-II collagen deposition in the cells (up to a 1.3-fold increase with respect to the cell control in the absence of cytokines, *p* = 0.017) (Figure 2A,B). These levels increased over time, chiefly by delivery of the vectors via PF68 micelles (up to a 1.5-fold difference with respect to the negative control in the absence of cytokines on day 10, *p* = 0.040) (Figure 2A,B). Of note, these levels were higher than those achieved with free vector administration (up to a 1.2-fold difference compared with free rAAV-FLAG-hsox9 application on day 10, *p* = 0.011) (Figure 2A,B). Treatment with IL-1β decreased type-II collagen deposition (up to a 1.1-fold difference relative to the negative control in the absence of cytokines on day 1, *p* = 0.300) (Figure 2A,B versus Figure 2C,D). rAAV-FLAG-hsox9 application to IL-1β-treated chondrocytes significantly increased type-II collagen deposition, especially when using micelle-guided vector delivery (up to a 1.5-fold difference with respect to the cell control in the presence of IL-1β on day 10, *p* = 0.011; up to a 1.2-fold difference compared with free vector applying in the presence of IL-1β on day 10, *p* = 0.006) (Figure 2C,D). A similar tendency was noted when applying TNF-α alone or combined as a IL-1β/TNF-α co-treatment, showing modest decreases in type-II collagen deposition compared with cells kept in culture in the absence of cytokines (*p* = 0.290) (Figure 2A,B versus Figure 2E–H). Similarly, overexpression of SOX9 significantly increased type-II collagen deposition over time, particularly when providing rAAV-FLAG-hsox9 in micellar carriers (up to a 1.4-fold difference with respect to the cell control in the presence of TNF-α alone or as an IL-1β/TNF-α combination on day 10, *p* = 0.040) (Figure 2E–H).

**Figure 2.** Remodeling activities in rAAV-FLAG-h*sox9*-transduced human OA chondrocytes using

polymeric micelles. Cells in monolayer culture were directly transduced with the rAAV/polymeric micelles (**A**,**B**) or after pre-incubation for 4 h with IL-1β (10 ng/mL) (**C**,**D**), TNF-α (100 ng/mL) (**E**,**F**), or IL-1β/TNF-α (10/100 ng/mL) (**G**,**H**), as described in Figure 1 and in the Materials and Methods. The cultures were processed after 1 and 10 days to detect type-II collagen deposition by immunocytochemistry (magnification x10, scale bar 200 μm; all representative data) (**A**,**C**,**E**,**G**) with corresponding histomorphometric analyses (**B**,**D**,**F**,**H**), as described in the Materials and Methods. Control conditions included the absence of copolymer or vector treatment (negative control) and the application of free rAAV vector (positive control). \* Statistically significant compared with the negative control at similar time points.

Overexpression of SOX9 in rAAV-FLAG-hsox9-transduced chondrocytes significantly increased the accretion of ECM-proteoglycans compared with untransduced cells (up to an 1.8-fold difference with respect to the negative control in the absence of cytokines on day 10, *p* = 0.030) (Figure 3A,B). Of note, delivery of rAAV-FLAG-hsox9 via micellar systems led to the highest proteoglycan deposition (up to a 1.2-fold increase with respect to free vector administering on day 10, *p* = 0.030) and proliferative index (up to a 1.4-fold increase with respect to the negative control in the absence of cytokines) (Figure 3A,B). Strikingly, treatment with IL-1β significantly decreased the deposition of proteoglycans and the cell proliferation ratio (up to a 1.2-fold difference with respect to the negative control in the absence of cytokines on day 1, *p* = 0.030) (Figure 3A,B versus Figure 3C,D). Additionally, rAAV-FLAG-hsox9-mediated transduction of IL-1β-treated chondrocytes prompted the restoration of proteoglycans, an effect more marked over time (up to a 1.7-fold increase when compared to the control in the presence of IL-1β on day 10, *p* = 0.038), exhibiting higher cell proliferation. Interestingly, providing rAAV-FLAG-hsox9 in micellar carriers led to the highest proteoglycan deposition (up to a 2.1-fold increase relative to the cell control in the presence of IL-1β on day 10, *p* = 0.006), reaching values that were higher than those reached with the free vector administration (up to a 1.3-fold difference with respect to free rAAV-FLAG-hsox9 application in the presence of IL-1β on day 10, *p* = 0.046) (Figure 3C,D). Treatment with TNF-α also decreased the deposition of proteoglycans (up to a 1.2-fold difference with respect to the negative control in the absence of cytokines on day 10, *p* = 0.203) and the cell proliferation index (Figure 3A,B versus Figure 3E,F). Transduction of TNF-α-treated chondrocytes with rAAV-FLAG-hsox9 significantly increased the cell proliferation and proteoglycan deposition, especially when the vectors were delivered via micellar systems (up to a 2-fold difference compared with the cell control in the presence of TNF-α on day 10, *p* = 0.010) (Figure 3E,F). Simultaneous IL-1β/TNF-α administration significantly decreased the deposition of proteoglycans (up to a 1.1-fold difference with respect to the negative control in the absence of cytokines on day 1, *p* = 0.011) and the cell proliferation rates (Figure 3A,B versus Figure 3G,H). Again, SOX9 overexpression increased the deposition of proteoglycans following IL-1β/TNF-α treatment, especially when the vectors were transferred via micellar vehicles (up to 2-fold difference relative to the cell control in the presence of IL-1β/TNF-α on day 10, *p* = 0.043; up to a 1.5-fold difference compared with free vector administration in the presence of IL-1β/TNF-α on day 10, *p* = 0.009) (Figure 3G,H). Likewise, genetic modification of chondrocytes via rAAV-FLAG-hsox9 resulted in an increased proliferation index (up to a 1.8-fold relative to the cell control in the presence of IL-1β/TNF-α on day 10, *p* = 0.001).

*3.3. E*ff*ects of rAAV-FLAG-hsox9*/*Polymeric Micelle Delivery on the Viability Processes in Human OA Chondrocytes in Inflammatory Conditions*

We finally examined the effects of SOX9 overexpression on the cell viability processes following rAAV-FLAG-hsox9 gene transfer via micellar systems in human OA chondrocytes monolayer cultures maintained in conditions of inflammation.

**Figure 3.** Biosynthetic activities in rAAV-FLAG-h*sox9*-transduced human OA chondrocytes using polymeric micelles. Cells in monolayer culture were directly transduced with the rAAV/polymeric micelles (**A**,**B**) or after pre-incubation for 4 h with IL-1β (10 ng/mL) (**C**,**D**), TNF-α (100 ng/mL) (**E**,**F**), or IL-1β/TNF-α (10/100 ng/mL) (**G**,**H**), as described in Figures 1 and 2 and in the Materials and Methods. The cultures were processed at the denoted time points for Alcian blue staining (magnification x10, scale bar 200 μm; all representative data) (**A**,**C**,**E**,**G**) with spectrophotometric evaluations for cell proliferation and proteoglycan deposition following solubilization in 6 M guanidine hydrochloride (**B**,**D**,**F**,**H**), as described in the Materials and Methods. Control conditions included the absence of copolymer or vector treatment (negative control) and the application of free rAAV vector (positive control). \* Statistically significant compared with the negative control at similar time points.

In concordance with our previous observations [23], no cytotoxic effects from none of the gene transfer procedures (polymeric vehicles, free vector supply) were noticed with respect to the control condition (*p* = 0.130) (Figure 4A). A similar tendency was evidenced when providing copolymer solutions in the absence of vector treatment (not shown). Moreover, while separate cytokine treatment resulted only in slight decreases in cell viability (~90%) (Figure 4B,C), concomitant administration of both cytokines led to higher toxicity especially in untransduced cells (~75% cell viability on day 10 in the negative control in the presence of IL-1β/TNF-α) (Figure 4D). Strikingly, overexpression of SOX9 led to higher cell viability indices in the presence of both cytokines (~100% compared with the cell control in the presence of IL-1β/TNF-α on day 10, *p* = 0.045) (Figure 4D).

**Figure 4.** Cell viability in rAAV-FLAG-hsox9 modified human OA chondrocytes using micellar systems. Cell monolayer cultures were directly transduced with the rAAV/polymeric micelles (**A**) or after pre-incubation for 4 h with IL-1β (10 ng/mL) (**B**), TNF-α (100 ng/mL) (**C**), or IL-1β/TNF-α (10/100 ng/mL) (**D**), as described in Figures 1–3 and in the Materials and Methods.
