INTRODUCTION: Cell therapies have potential to slow the progression of painful intervertebral disc (IVD) degeneration,¹ which costs $134.5 billion in domestic healthcare spending.² To support cell-mediated healing, injectable cell delivery biomaterials may be used as a platform to retain cells in the IVD.³ Engineering annulus fibrosus (AF) cell delivery biomaterials is challenging because of the need to balance biomechanical and biological performance.⁴ To overcome this challenge, we developed a novel composite biomaterial for AF repair. It uses cell-laden, degradable oxidized alginate (OxAlg) microbeads (MBs) to deliver AF cells within high-modulus genipin-crosslinked fibrin (FibGen) hydrogels (FibGen+MB composites). This study evaluates the long-term biomechanical and biological performance of FibGen+MB composites in an ex vivo bovine organ culture model. 

METHODS: Intact, Injury and Repair IVDs [N=5-6 per group] were for cultured for 42 days using a custom Loading system for Disc Organ Culture (LODOC),⁵ which simulates physiologic loading (Fig. 1A). Herniation risk [Gross morphology & Histology], disc height changes [X-rays & LODOC] and stiffness [LODOC] were measured to assess biomechanical repair. Biological repair was assessed using culture viability [Nitric oxide], cell tracking [PKH67] and extracellular matrix (ECM) synthesis [Alcian blue, Picrosirius red and Collagen I]. Two-way ANOVA with Bonferroni [Biomechanics & Nitric Oxide] and one-way ANOVA with Tukey’s post-hoc [Disc Height Change] tests found significant differences (p<0.05).

RESULTS: Morphological and histological assessments showed that composites remained within defects at culture day (D) 42 with high adhesion, suggesting FibGen+MB composites have very low herniation risk under cyclic compressive loading (Fig. 1B). Repair IVDs had significantly less disc height loss when compared to Injury IVDs, and no differences between Intact and Repair IVDs were detected (Fig. 1C). Repair also prevented injury-induced IVD stiffening at D7, but by D42 there were no significant differences between groups (Fig. 1D). This is likely due to mechanical compaction in all groups from approximately 96,000 loading cycles over the 42-day culture. All IVDs remained viable with constant, low nitric oxide levels throughout culture (Fig. 1E). Delivered AF cells were present within the degraded OxAlg MB space of composites at D42, suggesting successful cell delivery and retention (Fig. 1F). Delivered AF cells synthesized ECM as OxAlg MBs degraded within FibGen+MB composites, as shown by positive staining for Alcian blue, picrosirius red and collagen I in the interstitial space between cells (Fig. 1G).

DISCUSSION: FibGen+MB composites restored IVD height and improved the biomechanical properties of injured IVDs, while delivering AF cells capable of ECM synthesis. FibGen+MB composites did not herniate after approximately 96,000 cycles of compression in situ, which is a very rigorous challenge for an experimental IVD repair biomaterial.⁶ Importantly, composites also restored disc height loss to intact levels, which is one of the most sensitive measurements of IVD repair.⁷ Lastly, delivered AF cells synthesized ECM within composites, suggesting this composite strategy shows promise for long-term tissue synthesis and IVD repair. Overall, we demonstrated a proof-of-concept for this composite repair strategy as a next-generation AF repair method that balances biomechanical and biological repair.