Endurable injectable mesenchymal stem cell therapy for osteoarthritis by encapsulation in alginate constructs

Purpose: Mesenchymal stem cells (MSC) are promising as cell therapy in osteoarthritis (OA) due to their immunomodulatory and trophic capacities. Therapeutic results of intra-articularly injected MSC are however modest and MSC disappear within one to two weeks from the joint. Our hypothesis is that therapeutic efficacy of MSC can be enhanced by prolonging their intra-articular presence, providing a longer interplay between MSC and diseased tissues. We developed an injectable MSC construct for intra-articular use to retain MSC in the desired location while protecting MSC against the host immune system and preserving their immunomodulatory capacity. Methods: Clinical grade high mannuronate (M) (L. Pallida) and high guluronate (G) (L. Hyperborean) alginate in 1.1% concentration were used for encapsulation. Encapsulated MSC (N = 3; 4x106 MSC/ml) survival was measured by DNA analysis and live/dead assay. Immunomodulation capacity of IFNγ and TNFα (50 ng/ml) activated MSC was studied by IL-6 secretion and IDO activity for 14 days in vitro. Protection of the encapsulation of superparamagnetic iron oxide (SPIO)-labeled allogeneic MSC was evaluated by subcutaneous (s.c.) implantation in immune competent rats (N = 3; 18 pockets) for one month and analyzed by magnetic resonance imaging (MRI) and histology. Alginate solution viscosity was studied by rheometry, while construct integrity was studied by gadolinium (Gd) crosslinking and MRI in rats (N = 5). For intra-articular use, microbeads were produced with the Büchi B-395 Pro encapsulator, Gd crosslinked and injected in rat knees (N = 8) ex vivo, and imaged by MRI. Results: After 14 days, approximately 50–70% of the MSC survived (Fig 1A,B) and retained their immunomodulatory capacities (Fig 1C,D). No differences were observed in these biological aspects between high G and high M alginate. S.c. implanted SPIO-MSC constructs remained clearly visible on MR images over 30 days (Fig 2A,B). Histology showed a homogenous distribution of SPIO labelled cells in alginate constructs with a viable aspect and a negative staining for CD3 and CD68 immunohistochemistry, indicating that these cells were not macrophages or lymphocytes, but SPIO-MSC (Fig 2C). In rat tissues surrounding the constructs, very limited host immune reaction was observed without cell infiltration (Fig 2D). No differences were observed between high M and high G alginate. Physical aspects of high M and high G alginate solutions were different before polymerization with a viscosity of 1565 vs 297 mPa●s respectively. For construct integrity, MRI did not show any difference between the alginates up to 30 days in vivo (Fig 3A,B). Low solution viscosity is desirable for microbead production. Therefore, high G alginate is used for standardized microbead production. A homogenous microbead population was producible (mean diameter of 250 μm) and these beads could be injected intra-articularly in a rat knee ex vivo and could be visualized individually by MRI (Fig 3C). Conclusions: We present a reproducible encapsulation method to prolong the local presence of injected MSC at the diseased location. MSC retain their immunomodulatory properties for at least two weeks after encapsulation in vitro. Viable MSC were observed in constructs after s.c. implantation with very little host immune reaction. By using a specific alginate type and encapsulation protocol we produced homogenous bead populations which could be injected intra-articularly in rat knees and visualized by MR imaging. These results offer a possibility to enhance the already promising effects of cell therapy with MSC as an injectable long term disease modifying anti-osteoarthritic drug.