gms | German Medical Science

Objectives: Human bone marrow-derived mesenchymal stromal cells (hMSCs) modified by recombinant adeno-associated virus gene vectors (rAAV) are a potent source of implantable cells in articular cartilage defects, especially when rAAV are provided via scaffolds to control their release [1]. The objective was to test whether rAAV can be formulated in poly (sodium styrene sulfonate)-grafted poly (ethylene glycol diacrylate) (pNaSS-PEGDA) and pNaSS-PEGDA/tricalcium phosphate (pNaSS-PEGDA/TCP) hydrogels to target hMSCs for cartilage repair.

Methods: rAAV-RFP carrying the reporter Discosoma sp. red fluorescent protein (RFP) gene and rAAV-hTGF-β the transforming growth factor beta 1 cDNA (both under the CMV-IE promoter) were routinely prepared and labeled with Cy3 [2], [3]. Bone marrow aspirates from two donors were used to isolate hMSCs as described [3] and placed in DMEM, 10% fetal bovine serum, 100 U/ml penicillin, and 100 μ l/ml streptomycin (5x103 cells/well in 48-well plates). The pNaSS-PEGDA and pNaSS-PEGDA/TCP hydrogels were formulated using pNaSS, PEGDA, TCP, rAAV (40–80 µl), and 0.002% poly-L-lysine and added to the cells for up to 21 days (duplicate experiments) [2]. Cy3 and RFP signals were monitored by live fluorescence and cell viability using the WST-1 assay [2], [3]. The t-test was employed with P≤0.05 considered statistically significant.

Results: The pNaSS-PEGDA and pNaSS-PEGDA/TCP hydrogels could successfully formulate rAAV as seen by the strong Cy3 signal when using Cy3-labeled vectors (Figure 1 A, B [Fig. 1]) and effectively deliver them in hMSCs in a controlled, dose-dependent manner as noted by the strong RFP signal when applying rAAV-RFP (Figure 1 C,D [Fig. 1]), for up to 21 days versus control conditions (unlabeled vectors, absence of vector) (always P≤0.04). The use of both hydrogels in hMSCs was safe for up to 21 days as assessed with the WST-1 assay versus the control condition (no hydrogel) (P≤0.05) (Figure 1 E [Fig. 1]).

Conclusion: Delivering rAAV via pNaSS-PEGDA and pNaSS-PEGDA/TCP hydrogels can durably and safely target hMSCs as a novel therapeutic cell transplantation strategy for cartilage repair.