Encapsulation in alginate beads prolongs mesenchymal stem cell longevity in vivo but does not enchance their therapeutic efficacy in a murine model for osteoarthritis

Purpose: Mesenchymal stem cells (MSCs) are promising as a biological therapeutic for osteoarthritis (OA). Clinical results of intra-articular injected MSCs are modest and the MSCs disappear from the joint within a few weeks. We hypothesize that efficacy of MSCs can be enhanced by prolonging their intra-articular longevity. Here we examined if encapsulation in alginate was suitable to prolong the longevity and efficacy of MSCs in vivo in a monoiodoacetate (MIA) induced rat OA model. Methods: First we performed in vitro experiments in which, two alginate types, high mannuronic (High M) and high guluronic (High G), were tested as possible candidates for MSC encapsulation. After encapsulation of human MSCs in these two alginate types, viability was evaluated using both a DNA and Live/Dead assay. The immunomodulatory function after in vitro stimulation with TNFα and INFγ, was tested by IDO activity. Next, in the first in vivo experiment, alginate construct integrity and safety for both alginates were assessed subcutaneously in immune competent male Wistar rats (n=4/group). Based on these results and alginate type specific properties, one type of alginate was chosen for the creation of MSC-alginate beads suitable for intra-articular injection, with the Buchi Encapsulater device. These alginate beads were used in the next in vivo experiments, in which OA was induced by 0.3mg MIA injection in, 16 weeks old, male Wistar rats (Week 0). Treatments were given one week hereafter (Week 1). Here we evaluated the effect of encapsulation on the presence of MSCs in an OA joint. We compared injection of MSC-alginate beads versus freely injected MSCs (n=8/group). Firefly luciferase-expressing allogeneic rat MSCs were used for longitudinal tracking of the MSC viability on bioluminescent imaging (BLI). By crosslinking alginate with Gadolinium, in vivo tracking of bead integrity was made possible. The animals were scanned weekly with Magnetic Resonance Imaging (MRI) and BLI. Finally, we evaluated the therapeutic efficacy of MSCs encapsulated in alginate. Human MSC-alginate beads were compared to freely injected human MSCs and saline control (n=14-16 knees/group). Outcome parameters were weekly pain measurement by von Frey filaments (Pre-OA till week 8), synovial inflammation (Week 4 and 8) and cartilage damage (Week 8) scored on histology. Results: Two weeks after encapsulation no differences were seen between High G and High M alginates on MSC survival and IDO activity in vitro. In addition, subcutaneous implantation of MSC alginate constructs showed no differences on construct integrity and lead to a mild foreign body reaction by forming a very thin capsule, for both alginate types. The lower viscosity of High G alginate lead to smaller and more homogeneous bead production, with an average bead diameter of 284 ±28μm, thus suitable for intra-articular injection in a rat knee. Therefore High G alginate was selected to use in future experiments. Longitudinal imaging demonstrated that the number of MSC-alginate beads decreased from 73 ±36 per knee at baseline to 46 ±34 per knee at week 4 (p=0.028), and remained stable afterwards till week 8 (37±20). In accordance, the fluorescence on BLI in the MSCs-alginate group decreased significantly from week 2 to week 3 (p =0.028), but remained stable hereafter and significantly stronger than in the free MSC group (p< 0.04, for week 3 till 8). Next we proceeded to test the therapeutic efficacy of alginate encapsulated MSCs. One week after the treatment, the animals in the saline control group showed an additional significant decrease in withdrawal threshold (p=0.001), whereas animal injected with either free or encapsulated MSC did not, indicating an early pain inhibiting effect of MSCs. No additional treatment effects on pain, cartilage or synovial inflammation were observed. There was a trend though (p=0.058), to a thicker synovium in the MSC-alginate group (218±22μm) versus saline control (162±12 μm). Conclusions: Here, we demonstrate the ability to produce MSC-alginate constructs, suitable for intra-articular injection. Longitudinal construct integrity tracking on MRI showed detectable alginate beads till the end of experiment. Encapsulation in alginate beads prolonged the presence of metabolically active allogenic MSCs in the knee joint of immunocompetent rats for at least 7 weeks. This could, however, not improve the efficacy of human MSCs as a biological therapeutic for OA, possibly due to the foreign body reaction to the alginate.