Mesenchymal Stromal Cell Spheroid-Loaded Microscaffolds for Intervertebral Disc Repair: Evaluation in a Comprehensive Ex Vivo Degeneration Model

Mesenchymal stromal cell (MSC) injection is a promising approach to mitigate intervertebral disc (IVD) degeneration (IVDD). However, the harsh mechanical and biochemical disc environment often hinders MSC survival, and leakage of injected cells is a risk. Here, we propose forming MSC spheroid-loaded microscaffolds as a strategy to improve cell retention, viability, and eventually therapeutic efficacy. To study intradiscal injections, we employed a bovine ex vivo IVD model, where degeneration was induced through a novel combination of papain and TNFα to replicate a combination of structural damage and the inflammatory and catabolic features of IVDD. Bovine coccygeal discs were kept in culture and loaded physiologically (dynamic compression: 0.02–0.2MPa, 0.2Hz, 2hrs/d) for 5 consecutive days. Intradiscal injections (26G, 50µL) of papain (3.2U/mL) and TNFα (250ng) were performed after the loadings on day 1 and 3 respectively. IVDD was characterized thoroughly for n=5 discs per group (Fig.1A). On day 5, 500 cellularized microscaffolds/disc were injected (26G). Prior to injection, cellularized microscaffolds were formed for 3 days in vitro by seeding human bone marrow-derived MSCs onto microscaffolds (2,000 cells/scaffold; Fig.1B). The highly porous microscaffolds (⌀200µm) were produced using PCL-based resin (DEGRADINX, BIO INX, Belgium), utilizing a custom-built two-photon polymerization system based on a resonant scanner (see contribution submitted by Balasubramanian et al .). Spheroid viability after injection (26G needle) was tested in vitro using a live/dead staining. To localize microscaffolds post injection, while evaluating the extent of disc degeneration, the disc was consecutively sectioned and stained for Safranin-O/Fast Green. Discs treated with papain and TNFα showed an upregulation of degenerative markers from both individual interventions. A loss of glycosaminoglycans in the center of the disc and a void was observed in the papain and TNFα-injected group compared to the healthy control. MSC spheroids were successfully seeded into microscaffolds and maintained a high viability after injection (Post injection cellularized microscaffolds could be found in the center (nucleus pulposus) of the discs (Fig. 1E). Combined papain and TNFα treatment created a degeneration model that combines key features of the separately used inducers papain and TNFα, enabling more comprehensive testing of multi-modal cell therapies. Cellularized microscaffold were injectable and locatable in the discs, albeit at reduced numbers compared to the total number of injected scaffolds. As next step injection procedure will be optimized to efficiently deliver the cellularized microscaffolds. For any figures or tables, please contact the authors directly.