Prolonged intra-articular retention of mesenchymal stem cells by advanced microencapsulation

Abstract

Purpose: Intra-articular injection of mesenchymal stem cells (MSCs) has shown promising results as a treatment for osteoarthritis (OA). Rodent models show a reduction of inflammation and limited cartilage regeneration. However, the treatment efficacy is limited most likely by the rapid clearance of the injected MSCs from the joint. These findings are supported by phase one clinical trials which suggest that this therapy can decrease pain, increase motion range, and reduce cartilage damage. We hypothesize that the encapsulation of MSCs in micrometer sized hydrogel matrices can prolong the intra-articular retention of MSCs, and thus increase their therapeutic potential. Therefore we have pioneered a novel method to prevent the MSCs’ rapid clearance: microfluidic encapsulation of MSCs in micrometer sized hydrogel matrices (microgels). These microgels were designed to associate with minimal therapeutic cell escape and host cell infiltration while simultaneously supporting the function, viability, and metabolism of encapsulated cells. The goal of this study is to examine the effect of microencapsulation of MSCs in microgels on the duration of intra-articular retention of MSCs. Methods: MSCs were harvested from 12 week old Wistar rats (Charles-River), and labelled with a near infrared (NIR) label (DIR, Invitrogen). Enzymatically cross linkable, biocompatible, polysaccharide hydrogels (tyramine-conjugated dextran) and a chip-based microfluidic droplet generator were used to encapsulate MSCs in microgels. In vitro viability and metabolic activity was assessed using live/dead staining and presto blue assay. Microgels were intra-articularly injected in knee joints of 12 week old Wistar rats (n=6). Non-encapsulated MSCs were used as a control. For four months, long term non-invasive quantitation of intra-articular NIR signal was performed using whole animal NIR-imaging (Pearl). Rats were sacrificed after 8 and 16 weeks, and had the inside of their knees scraped with a paper tissue, to retrieve microgels and determine their presence and morphology. The presence of NIR signal from MSCs in the retrieved microgels was confirmed using confocal microscopy, and the viability of retrieved MSCs was assessed using live/dead staining (Invitrogen). The animal study was approved by the Utrecht University Medical Ethical Committee for animal studies. Results: Microfluidic encapsulation allowed for the formation of homogenous, monodisperse microgels (diameter 100μm, coefficient of variation 5%), which contained on average 13 cells/gel. In vitro experiments revealed that these MSCs maintained <60% of their initial metabolic activity and stayed alive for at least four weeks. MSC microencapsulation increased the intra-articular retention of MSCs from four weeks to four months (figure 1 A and B). After one month, the normalized signal of the encapsulated cells was as high as 63%±24 while non-encapsulated MSCs had already decreased to 13%±4. Impressively, it took encapsulated MSCs four months to reach a similarly low level (11%±2). Additionally, microgels retrieved from the knee joint contained viable, NIR-positive MSCs, confirming that the NIR signal originated from the injected cells (figure 1 C and D). Conclusions: Our study showed that micro-encapsulation of MSCs in microgels is suited for increasing the intra-articular retention of MSCs to at least four months. Additionally, the microgels protected MSCs against the harsh environment within the joint by sustaining their viability and function for this entire period, suggesting a cytoprotective role of the microgels. Our micro-encapsulation approach allows for a single intra-articular injection with a significantly extended retention of therapeutic cells within the intra-articular joint cavity. Currently we are investigating the effect of the prolonged retention of MSCs in a therapeutic OA model. [Figure presented]