Improved Chondrogenic Differentiation of rAAV SOX9-Modified Human MSCs Seeded in Fibrin-Polyurethane Scaffolds in a Hydrodynamic Environment

Abstract

The repair of focal articular cartilage defects remains a problem. Combining gene therapy with tissue engineering approaches using bone marrow-derived mesenchymal stem cells (MSCs) may allow the development of improved options for cartilage repair. Here, we examined whether a three-dimensional fibrin-polyurethane scaffold provides a favorable environment for the effective chondrogenic differentiation of human MSCs (hMSCs) overexpressing the cartilage-specific SOX9 transcription factor via recombinant adeno-associated virus (rAAV) -mediated gene transfer cultured in a hydrodynamic environment in vitro. Sustained SOX9 expression was noted in the constructs for at least 21 days, the longest time point evaluated. Such spatially defined SOX9 overexpression enhanced proliferative, metabolic, and chondrogenic activities compared with control (reporter lacZ gene transfer) treatment. Of further note, administration of the SOX9 vector was also capable of delaying premature hypertrophic and osteogenic differentiation in the constructs. This enhancement of chondrogenesis by spatially defined overexpression of human SOX9 demonstrate the potential benefits of using rAAV-modified hMSCs seeded in fibrin-polyurethane scaffolds as a promising approach for implantation in focal cartilage lesions to improve cartilage repair.

Keywords: SOX9; bioreactors; cartilage repair; chondrogenesis; fibrin-polyurethane scaffolds; hMSCs; rAAV.

Figure 1

Model system: Human bone marrow-derived mesenchymal stem cells (hMSCs) were isolated from bone marrow aspirates, placed in monolayer culture, released, and hMSC aggregate cultures (2 × 10⁵ cells/pellet) were prepared and next transduced with rAAV as described in Materials and Methods. After 24 h, the transduced cells were placed in a fibrin/thrombin mixture and seeded onto fibrin-polyurethane (PU) scaffolds. The constructs were next transferred to rotating bioreactors and maintained in chondrogenic medium in hydrodynamic culture for 21 days for further evaluations as depicted.

Figure 2

Detection of transgene (SOX9) expression in hydrodynamic cultures of rAAV-transduced hMSCs seeded in fibrin-PU scaffolds. Cells were transduced with rAAV-lacZ or rAAV-FLAG-hsox9 (40 µL each vector), seeded in PU scaffolds, and cultivated in rotating bioreactors for 21 days using chondrogenic medium as described in Figure 1 and in Materials and Methods. Samples were processed after bioreactor cultivation to detect immunoreactivity to SOX9 (magnification ×10; scale bar: 200 µm; representative data). Note the highest presence of immunoreactivity to SOX9 at the surface of the constructs which can be attributed to a diffusion gradient of the nutrients into the center of the constructs at homogenous cell seeding.

Figure 3

Histological analysis in hydrodynamic cultures of rAAV-transduced hMSCs seeded in fibrin-PU scaffolds. Cells were transduced, seeded in PU scaffolds, and placed in rotating bioreactors as described in Figure 1 and Figure 2 and in Materials and Methods. The samples were processed after 21 days for histological staining with toluidine blue and to detect immunoreactivity to aggrecan and to type-II collagen (magnification ×10; scale bar: 200 µm; all representative data). Similar to the immunoreactivity to SOX9, the high signals at the surface of the constructs can be attributed to a diffusion gradient of the nutrients into the center of the constructs at homogenous cell seeding.

Figure 4

Biochemical analyses in hydrodynamic cultures of rAAV-transduced hMSCs seeded in fibrin-PU scaffolds. Cells were transduced, seeded in PU scaffolds, and placed in rotating bioreactors as described in the Figure 1, Figure 2 and Figure 3 and in Materials and Methods. The samples were processed after 21 days to monitor the proteoglycan contents by dimethylmethylene blue dye method, the type-II collagen contents by ELISA, and the DNA contents using Hoechst 33258 in the constructs. * Statistically significant compared with rAAV-lacZ.

Figure 5

Immunohistochemical analysis in hydrodynamic cultures of rAAV-transduced hMSCs seeded in fibrin-PU scaffolds. Cells were transduced, seeded in PU scaffolds, and placed in rotating bioreactors as described in the Figure 1, Figure 2, Figure 3 and Figure 4 and in Materials and Methods. The samples were processed after 21 days to detect immunoreactivity to type-I and -X collagen and for histological staining with alizarin red (magnification ×10; scale bar: 200 µm; all representative data). Note the high signals at the surface of the constructs as a result of a diffusion gradient of the nutrients.

Figure 6

Real-time RT-PCR analysis in hydrodynamic cultures of rAAV-transduced hMSCs seeded in fibrin-PU scaffolds. Cells were transduced, seeded in PU scaffolds, and placed in rotating bioreactors as described in the Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5 and in Materials and Methods. After 21 days of bioreactor cultivation, mRNA was directly isolated from the constructs of rAAV-transduced hMSCs in fibrin-PU scaffolds and the gene expression profiles of SOX9, SOX5, SOX6, aggrecan (ACAN), type-II (COL2A1), type-I (COL1A1), type-X collagen (COL10A1), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2) were monitored, with GAPDH serving as a housekeeping gene and internal control (all primers are listed in Materials and Methods). Ct values were obtained for each target and for GAPDH as a control for normalization, and fold inductions (relative to lacZ-treated samples) were measured by using the 2^−ΔΔCt method. * Statistically significant compared with rAAV-lacZ.