Suspension bioprinted whole intervertebral disc analogues enable regional stiffness- and hypoxia-regulated matrix secretion by primary human nucleus pulposus and annulus fibrosus cells.

Intervertebral disc (IVD) degeneration is a leading cause of back pain, and while studies have revealed the roles resident nucleus pulposus (NP) and annulus fibrosus (AF) cells play in degeneration, tissue-engineered IVD models are needed to better investigate the mechanisms underpinning these cell-driven changes. This study therefore integrated suspension baths with bioprinting to create four multi-material, whole IVD analogues and investigated the combined effect of reduced oxygen tension and increased regional matrix stiffness on disc cell phenotype since these factors correlate with IVD degeneration. Primary NP and AF cells were seeded into alginate-collagen hydrogels and bioprinted into biphasic IVD structures. The nascent area, intensity, and integrated density of pro-collagen type I, collagen type VI, aggrecan, and hyaluronic acid were quantified using immunofluorescence staining in each region. Stiffness-mediated collagen and glycosaminoglycan production was observed in the AF, and increased stiffness downregulated collagen type VI in the AF but upregulated it in NP. Oxygen tension impacted proteoglycan production, with hypoxia increasing aggrecan and hyaluronic acid in both regions. This work represents a step towards the automated biofabrication of whole IVD analogues and expands the state-of-the-art in suspension bioprinting using regionally specific matrix cues. The findings provide important insights into two key microenvironmental factors driving IVD degeneration. STATEMENT OF SIGNIFICANCE: This manuscript outlines an original application of suspended layer additive manufacturing to biofabricate novel, biphasic intervertebral disc analogues containing patient-derived primary human cells. Significantly, the bioprinted models demonstrated biological function and were used to assess the effect of stiffness and oxygen concentration on regional matrix production using a range of internationally-recognized phenotypic intervertebral disc cell markers. The study therefore furthers the state-of-the-art in suspended bioprinting using regionally specific matrix cues and paves the way for future bioprinted disc models that can serve as biosimulators capable of generating insights into key mechanisms governing tissue development, homeostasis, and degeneration.