Table 1.
Overview of studies focusing on the combination of gene transfer and biomaterials for cartilage tissue engineering.
Table 2.
Primer sequences and product sizes, for quantitative RT-PCR.
Fig 1.
Transgene expression by MSCs in hydrogels during 21 days of culture following adenoviral gene transfer of GFP, SOX9, TGFB1 or BMP2.
Primary MSCs were transduced with Ad.GFP, Ad.SOX9, Ad.TGFB1 or Ad.BMP2 at 5 x 102 vp/cell for each vector, seeded into type I collagen hydrogels and analyzed for the respective transgene expression during 21 days. Expression of the GFP transgene was analyzed by fluorescence microscopy in the hydrogels modified with Ad.GFP (a) or Ad.SOX9/GFP (b). Expression of the TGFB1 (c) and BMP-2 (d) transgenes was measured by ELISA. The data represent mean values ± SD from measurements of supernatants of n = 3 hydrogel aggregates per condition and time point; m = 3 marrow preparations were analyzed. Each experiment was performed in triplicate. Statistically different values from GFP+ control hydrogels are marked by asterisks (p < 0.05).
Fig 2.
Histological appearance of MSC hydrogels after chondrogenic differentiation by adenoviral gene transfer of GFP, SOX9, TGFB1 or BMP2.
MSCs in monolayer cultures were transduced with Ad.GFP (Control; a), Ad.SOX9 (b), Ad.TGFB1 (c) or Ad.BMP2 (d) at 5 x 102 vp/cell for each vector as indicated, seeded into hydrogel aggregates 24 h post infection and cultured in serum-free medium for 21 days. Representative examples after 21 days are shown, that were stained with H & E for assessment of cell morphology and cellularity. (a-d) Panels are shown at low (50x; bar = 200 μm) or high (200x; bar = 50 μm) magnification as indicated.
Fig 3.
Histological appearance of MSC hydrogels after chondrogenic differentiation by adenoviral gene transfer of SOX9, TGFB1 or BMP2.
MSCs in monolayer cultures were infected with Ad.GFP (Control; a), Ad.SOX9 (b), Ad.TGFB1 (c) or Ad.BMP2 (d) at 5 x 102 vp/cell for each vector as indicated, seeded into hydrogel aggregates 24 h post infection and cultured in serum-free medium for 21 days. Representative examples after 21 days are shown, that were stained with alcian blue for evaluation of matrix proteoglycan content. (a-d) Panels are shown at low (50x; bar = 200 μm) or high (200x; bar = 50 μm) magnification as indicated.
Fig 4.
Immunohistochemical analyzes for chondroitin-4-sulfate in MSC hydrogels after chondrogenic differentiation by adenoviral gene transfer of SOX9, TGFB1 or BMP2.
Monolayer cultures of MSCs were infected with Ad.GFP (Control; a), Ad.SOX9 (b), Ad.TGFB1 (c) or Ad.BMP2 (d) at 5 x 102 vp/cell for each vector as indicated, seeded into hydrogel aggregates 24 h after infection and cultured in serum-free medium for 21 days. Immunohistochemical staining for chondroitin-4-sulfate (CS4), one of the proteoglycan matrix components, was performed on representative sections after 21 days. (a-d) Panels are reproduced at low (50x; bar = 200 μm) or high (200x; bar = 50 μm) magnification as indicated, and regions of positive immunostaining appear brown.
Fig 5.
Immunohistochemical analyzes for type II collagen in MSC hydrogels after chondrogenic differentiation by adenoviral gene transfer of SOX9, TGFB1 or BMP2.
MSCs in monolayer cultures were infected with Ad.GFP (Control; a), Ad.SOX9 (b), Ad.TGFB1 (c) or Ad.BMP2 (d) at 5 x 102 vp/cell for each vector as indicated, seeded into hydrogel aggregates 24 h post infection and cultured in serum-free medium for 21 days. Immunohistochemical staining for type II collagen (COL II), the predominant collagen in hyaline cartilage, was performed on representative sections after 21 days. (a-d) Panels are shown at low (50x; bar = 200 μm) or high (200x; bar = 50 μm) magnification as indicated, and regions of positive immunostaining appear brown.
Fig 6.
Immunohistochemical analyzes for type X collagen in MSC hydrogels after chondrogenic differentiation by adenoviral gene transfer of SOX9, TGFB1 or BMP2.
MSCs in monolayer cultures were infected with Ad.GFP (Control; a), Ad.SOX9 (b), Ad.TGFB1 (c) or Ad.BMP2 (d) at 5 x 102 vp/cell for each vector as indicated, seeded into hydrogel aggregates 24 h post infection and cultured in serum-free medium for 21 days. Immunohistochemical staining for type X collagen (COL X) was performed on representative sections after 21 days. (a-d) Panels are shown at low (50x; bar = 200 μm) or high (200x; bar = 50 μm) magnification as indicated; regions of positive immunostaining appear brown.
Fig 7.
Histochemical analyzes for alkaline phosphatase in MSCs hydrogels after chondrogenic differentiation by adenoviral gene transfer of SOX9, TGFB1 or BMP2.
MSCs in monolayer cultures were infected with Ad.GFP (Control; a), Ad.SOX9 (b), Ad.TGFB1 (c) or Ad.BMP2 (d) at 5 x 102 vp/cell for each vector as indicated, seeded into hydrogel aggregates 24 hours post infection and cultured in serum-free medium for 21 days. Histochemical staining for alkaline phosphatase (ALP) was performed on representative sections after 21 days. (a-d) Panels are shown at low (50x; bar = 200 μm) or high (200x; bar = 50 μm) magnification as indicated, and regions of positive staining appear blue.
Fig 8.
Biochemical composition of MSC hydrogels during three weeks of culture following adenoviral gene transfer of SOX9, TGFB1 or BMP2.
Primary MSCs in monolayer cultures were infected with Ad.GFP (Control; a), Ad.SOX9, Ad.TGFB1 or Ad.BMP2 at 5 x 102 vp/cell for each vector as indicated, seeded into hydrogel aggregates 24 h post infection and cultured in serum-free medium for 21 days. (a) Cell proliferation was quantified by ATP assay at days 3, 7, 14 and 21. (b) The concentration of DNA was measured at days 3,7,14 and 21. (c) In addition, the GAG assay was performed for all SOX9, TGFB1 and BMP2 modified hydrogels. (d) As a quantitative parameter for hypertrophy ALP activity was measured and normalized to the DNA content. (a-d) The data represent mean values ± SD from n = 3 aggregates per condition and time point from marrow preparations of m = 3 different patients. Each experiment was performed in quadruplicate. Asterisks indicate values that are statistically different (p < 0.05) from marker gene vector–transduced control cultures.
Fig 9.
Gene expression profiles determined by quantitative real-time PCR in MSC hydrogels after adenoviral gene transfer of SOX9, TGFB1 or BMP2.
The chondrogenic marker genes studied include type II collagen alpha 1 (COL2A1) and SOX9 (panels a), whereas the hypertrophic markers include type X collagen alpha 1 (COL10A1) and ALPL (panels b). Primer sequences and product sizes are presented in Table 2, with elongation factor α (encoded by EFAA1) serving as housekeeping gene and internal control. RNA was extracted from 3 hydrogels for each preparation/patient, treatment group and time point, and n = 3 were analysed depending on group and time point. For quantitative RT-PCR analysis values are mean +/- SD and normalized using the EFAA1 reaction products and the ΔΔ-CT method. Asterisks indicate statistically different values (p < 0.05) from the GFP-transduced control group.