Ethanol-Mediated Compaction and Crosslinking Enhance Mechanical Properties and Degradation Resistance While Maintaining Cytocompatibility of a Nucleus Pulposus Scaffold

Abstract

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 crosslinking 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 2-6x after 120 min of submersion. This was incorporated into a crosslinking treatment, through which scaffolds were subjected to 120 min pre-compaction in buffered 28% ethanol prior to carbodiimide crosslinking. 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 crosslinked scaffolds showed significant increases in DMA properties without detrimentally altering their cytocompatibility, and these mechanical gains were maintained following enzymatic exposure.Low back pain (LBP) is the leading cause of Years Lived with Disability in all developed countries, 1 with a lifetime prevalence of 70-84%. 2 LBP has severe economic ramifications on the individual and societal levels, leading to premature workforce departure, short/long-term fiscal losses, 3 and cumulative yearly costs exceeding $100 billion in the US. 4 The most common diagnosis for patients experiencing LBP is intervertebral disc (IVD) degeneration (IDD). 5IVDs are avascular, fibrocartilaginous structures connecting adjacent vertebrae in the spinal column. Each IVD comprises an aggrecan-rich core known as the nucleus pulposus (NP), 15-25 concentric sheets of aligned collagen type I encircling the NP known as the annulus fibrosus (AF), and thin layers of hyaline cartilage covering the cranial and caudal surfaces of the IVD known as the cartilage endplates (CEP). 6 Adjacent vertebrae interface with the IVD through the CEP, which sequesters the NP within the disc while allowing for nutrient exchange with vertebral blood vessels. 7 The encapsulation of the NP and its anionic, dense matrix impart triphasic timedependence to its mechanical properties, allowing it to dissipate loads via fluid exudation and prevent harmful stress concentrations in AF and CEP through hydraulic pressurization. 6,8,9 IDD is a multi-factorial, cell-mediated process originating in the NP in which normal tissue turnover becomes imbalanced, favori...