Scaffold-guided gene transfer offers strong systems to develop non-invasive, convenient therapeutic options for the treatment of articular cartilage defects, especially when targeting bone marrow aspirates from patients containing chondroregenerative mesenchymal stromal cells in a native microenvironment. In the present study, we examined the feasibility of delivering reporter (RFP, lacZ) rAAV vectors over time to such samples via biocompatible, mechanically stable poly(caprolactone) (PCL) films grafted with poly(sodium styrene sulfonate) (pNaSS) for improved biological responses as clinically adapted tools for cartilage repair. Effective transgene expression (RFP, lacZ) was noted over time in human bone marrow aspirates using pNaSS-grafted films (up to 90% efficiency for at least 21 days) versus control conditions (ungrafted films, absence of vector coating on the films, free or no vector treatment), without displaying cytotoxic nor detrimental effects on the osteochondrogenic or hypertrophic potential of the samples. These findings demonstrate the potential of directly modifying therapeutic bone marrow from patients by controlled delivery of rAAV using biomaterial-guided procedures as a future, noninvasive strategy for clinical cartilage repair.

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Background: The delivery of therapeutic genes in sites of articular cartilage lesions using non-invasive, scaffold-guided gene therapy procedures is a promising approach to stimulate cartilage repair while protecting the cargos from detrimental immune responses, particularly when targeting chondroreparative bone marrow-derived mesenchymal stromal cells in a natural microenvironment like marrow aspirates. Methods: Here, we evaluated the benefits of providing a sequence for the cartilage-specific sex-determining region Y-type high-mobility group box 9 (SOX9) transcription factor to human marrow aspirates via recombinant adeno-associated virus (rAAV) vectors delivered by poly(ε-caprolactone) (PCL) films functionalized via grafting with poly(sodium styrene sulfonate) (pNaSS) to enhance the marrow chondrogenic potential over time. Results: Effective sox9 overexpression was observed in aspirates treated with pNaSS-grafted or ungrafted PCL films coated with the candidate rAAV-FLAG-hsox9 (FLAG-tagged rAAV vector carrying a human sox9 gene sequence) vector for at least 21 days relative to other conditions (pNaSS-grafted and ungrafted PCL films without vector coating). Overexpression of sox9 via rAAV sox9/pNaSS-grafted or ungrafted PCL films led to increased biological and chondrogenic differentiation activities (matrix deposition) in the aspirates while containing premature osteogenesis and hypertrophy without impacting cell proliferation, with more potent effects noted when using pNaSS-grafted films. Conclusions: These findings show the benefits of targeting patients’ bone marrow via PCL film-guided therapeutic rAAV (sox9) delivery as an off-the-shelf system for future strategies to enhance cartilage repair in translational applications.

Polycaprolactone (PCL) is a widely used biodegradable polyester for tissue engineering applications when long-term degradation is preferred. In this article, we focused on the analysis of the hydrolytic degradation of virgin and bioactive poly(sodium styrene sulfonate) (pNaSS) functionalized PCL surfaces under simulated physiological conditions (phosphate buffer saline at 25°C and 37°C) for up to 120 weeks with the aim of applying bioactive PCL for ligament tissue engineering. Techniques used to characterize the bulk and surface degradation indicated that PCL was hydrolyzed by a bulk degradation mode with an accelerated degradationthree times increased rate constant -for pNaSS grafted PCL at 37°C when compared to virgin PCL at 25°C.The observed degradation mechanism is due to the pNaSS grafting process (oxidation, radical polymerization) which accelerated the degradation until 48 weeks, when a steady state is reached. The PCL surface was altered by the pNaSS grafting, introducing hydrophilic sulfonate groups that increase the swelling and smoothing of the surface, which facilitated the degradation. After 48 weeks, pNaSS was largely removed from surface and the degradation of virgin and pNaSS grafted surfaces were similar. The cell response of primary fibroblast cells from sheep ligament were consistent with the surface analysis results: a better initial spreading of cells on pNaSS surfaces when compared to virgin surfaces and a tendency to become similar with degradation time.It is worthy to note that during the extended degradation process the surfaces were able to continue inducing better cell spreading plus preserve their cell phenotype as shown by collagen genes expressions.