Ethanol-mediated compaction and cross-linking enhance mechanical properties and degradation resistance while maintaining cytocompatibility of a nucleus pulposus scaffold.

Intervertebral disc degeneration is a complex, cell-mediated process originating in the nucleus pulposus (NP) and is associated with extracellular matrix catabolism leading to disc height loss and impaired spine kinematics. Previously, we developed an acellular bovine NP (ABNP) for NP replacement that emulated human NP matrix composition and supported cell seeding; however, its mechanical properties were lower than those reported for human NP. To address this, we investigated ethanol-mediated compaction and cross-linking to enhance the ABNP's dynamic mechanical properties and degradation resistance while maintaining its cytocompatibility. First, volumetric and mechanical effects of compaction only were confirmed by evaluating scaffolds after various immersion times in buffered 28% ethanol. It was found that compaction reached equilibrium at ~30% compaction after 45 min, and dynamic mechanical properties significantly increased 2-6× after 120 min of submersion. This was incorporated into a cross-linking treatment, through which scaffolds were subjected to 120 min precompaction in buffered 28% ethanol prior to carbodiimide cross-linking. Their dynamic mechanical properties were evaluated before and after accelerated degradation by ADAMTS-5 or MMP-13. Cytocompatibility was determined by seeding stem cells onto scaffolds and evaluating viability through metabolic activity and fluorescent staining. Compacted and cross-linked scaffolds showed significant increases in DMA properties without detrimentally altering their cytocompatibility, and these mechanical gains were maintained following enzymatic exposure.