*3.4. E*ff*ects of rAAV-Mediated SOX9 and TGF-*β *Overexpression on the Biological Activities in hMSCs upon Vector Delivery via Carbon Dots*

The ability of the delivery systems to trigger the biological activities (cell proliferation, matrix deposition) in hMSCs over time (21 days) relative to control conditions (-/CD-2, rAAV-*lacZ*/CD-2) was then examined with the two formulations rAAV-FLAG-h*sox9*/CD-2 and rAAV-hTGF-β/CD-2 by evaluating cell viability using the Cell Proliferation Reagent WST-1 and matrix deposition via spectrophotometric detection of alcian blue staining (glycosaminoglycans) and via immunocytochemical detection of type-II, -I, and -X collagen expression.

Administration of rAAV-hTGF-β in hMSCs via CD-2 led to significantly higher levels of cell proliferation relative to all other conditions after 21 days (1.3-, 1.3-, and 1.2-fold difference using rAAV-hTGF-β/CD-2 versus -/CD-2, rAAV-*lacZ*/CD-2, and rAAV-FLAG-h*sox9*/CD-2, respectively; always *P* ≤ 0.001), while no difference was seen with rAAV-FLAG-h*sox9*/CD-2 (*P* ≥ 0.065 versus -/CD-2 or rAAV-*lacZ*/CD-2) (Figure 6A). Delivery of either rAAV-FLAG-h*sox9* or rAAV-hTGF-β in hMSCs via CD-2 led to significantly higher levels of glycosaminoglycans relative to all other conditions after 21 days (1.3- and 1.2-fold difference using rAAV-FLAG-h*sox9*/CD-2 versus -/CD-2 and rAAV-*lacZ*/CD-2, respectively, always *P* ≤ 0.002; 1.8- and 1.7-fold difference using rAAV-hTGF-β/CD-2 versus -/CD-2 and rAAV-*lacZ*/CD-2, respectively, always *P* ≤ 0.001), with a stronger effect of TGF-β relative to SOX9 (1.4-fold difference; *P* ≤ 0.001) (Figure 6B). Delivery of either rAAV-FLAG-h*sox9* or rAAV-hTGF-β to hMSCs via CD-2 led to significantly higher levels of type-II collagen expression relative to all other conditions after 21 days (17.8- and 15.4-fold difference using rAAV-FLAG-h*sox9*/CD-2 versus -/CD-2 and rAAV-*lacZ*/CD-2, respectively, always *P* ≤ 0.001; 14.4- and 12.5-fold difference using rAAV-hTGF-β/CD-2 versus -/CD-2 and rAAV-*lacZ*/CD-2, respectively, always *P* ≤ 0.001), with a stronger effect of SOX9 relative to TGF-β (1.2-fold difference; *P* ≤ 0.002) (Figure 6C and Table 2).

Interestingly, administration of rAAV-FLAG-h*sox9* or of rAAV-hTGF-β in hMSCs via CD-2 led to significantly lower levels of type-I collagen expression relative to all other conditions after 21 days (20.1- and 20.2-fold difference using rAAV-FLAG-h*sox9*/CD-2 versus -/CD-2 and rAAV-*lacZ*/CD-2, respectively, always *P* ≤ 0.001; 22.7- and 22.9-fold difference using rAAV-hTGF-β/CD-2 versus -/CD-2 and rAAV-*lacZ*/CD-2, respectively, always *P* ≤ 0.001), without difference between SOX9 and TGF-β (*P* = 0.319) (Figure 6D and Table 2). Similar results were noted when analyzing type-X collagen expression (6.2- and 6.1-fold difference using rAAV-FLAG-h*sox9*/CD-2 versus -/CD-2 and rAAV-*lacZ*/CD-2, respectively, always *P* ≤ 0.001; 5.7-fold difference using rAAV-hTGF-β/CD-2 versus -/CD-2 or rAAV-*lacZ*/CD-2, respectively, always *P* ≤ 0.001), again without difference between SOX9 and TGF-β (*P* = 0.257) (Figure 6E and Table 2).

**Figure 6.** Biological activities in hMSCs transduced with the rAAV/CD-2 system. The rAAV-FLAG-h*sox9*, rAAV-hTGF-β, and rAAV-*lacZ* vectors (40 <sup>μ</sup>L each vector, i.e., 8 <sup>×</sup> <sup>10</sup><sup>5</sup> transgene copies) were formulated with CD-2 (40 μL), and the resulting rAAV/CD systems (80 μL) were incubated with hMSCs (10,000 cells in 48-well plates; MOI = 80) for up to 21 days. Cell proliferation was examined using the Cell Proliferation Reagent WST-1 (**A**), glycosaminoglycans using alcian blue staining (light microscopy; magnification ×4; scale bars: 200 μm; all representative data) with spectrophotometric analysis after solubilization (histograms) (**B**), and the deposition of type-II collagen (**C**), type-I collagen (**D**), and type-X collagen (**E**) using immunocytochemistry (magnification ×20; scale bars: 50 μm; all representative data). rAAV-*lacZ*/CD-2 and CD-2 lacking rAAV were used as controls. Statistically significant relative to <sup>a</sup> -/CD-2, <sup>b</sup> rAAV-*lacZ*/CD-2, and <sup>c</sup> rAAV-FLAG-h*sox9*.
