gms | German Medical Science

Objectives: Controlling the release of gene transfer vectors from biocompatible materials is an attractive strategy to improve the processes of cartilage repair. Here, we examined the ability of the self-assembling peptide hydrogel RAD-16-I in a pure (M0) or combined form using hyaluronic acid (HA) (M5) to encapsulate and release rAAV vectors in a controlled fashion by testing their bioactivity and their effects upon the chondrogenic differentiation of hMSCs.

Methods: rAAV-lacZ carries the E. coli β -galactosidase (lacZ) gene. Bone marrow aspirates were obtained from the distal femurs of donors undergoing total knee arthroplasty for hMSC isolation. Hydrogel spheres were prepared by mixing 0.9% or 1% (v/v) RAD-16-I with rAAV (40:60) for self-assembly in DMEM (M0) or DMEM/HA (M5). rAAV release from the spheres was estimated using an AAV Titration ELISA. Bioactivity studies were performed by incubating rAAV-lacZ-loaded spheres to hMSC monolayer cultures for 21 days with controls of cells cultured with or without a similar amount of vector solution. Cell viability was determined using the Cytotoxity Detection KitPLUS (LDH). hMSC chondrogenesis upon contact with optimized spheres was evaluated in aggregate cultures by histological and immunohistochemical analyses. Each condition was performed in duplicate in three independent experiments. The t-test was employed with P below 0.05 considered statistically significant.

Results and Conclusion: A more sustained release was noted using spheres formed at 0.9% (35% for M0 and 22% for M5) and a more rapid release for those at 1% (73% and 93%, respectively). After 6 days, all the systems released most of the vectors except for M0 at 1% (90% of release after 21 days). Concordantly, the initial higher levels of rAAV-lacZ released from M0 and M5 at 1% during the first days led to the highest levels of lacZ expression with a peak on day 10, while the lowest levels occurred from spheres at 0.9%. At 21 days, lacZ expression using lacZ-loaded spheres significantly decreased compared with free vector treatment. hMSC viability upon contact with rAAV-loaded spheres reached 95-100% in all conditions tested like with unloaded spheres or without sphere (~ 95%). hMSC chondrogenic differentiation was noted in all types of aggregates, independently from the presence or absence of optimized (1%) spheres or of vector treatment as noted by robust toluidine blue staining and type-II collagen deposition while moderate type-X collagen expression and alizarin red staining were reported. Self-assembling peptide-based hydrogels pure (M0) or combined with HA (M5) are suitable compounds for rAAV release, allowing for high and sustained levels of transgene expression without impeding the chondrogenic differentiation of hMSCs. Controlled release of therapeutic rAAV from such hydrogels is a promising tool to improve articular cartilage repair.