RESOURCES

Abstract
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.

Abstract
The intervertebral disc (IVD) primarily comprises an outer ring of collagen fibers (annulus fibrosus, AF), which encases a soft, gelatinous core (nucleus pulposus, NP). Existing in vitro models have failed to integrate these two tissues effectively or accurately replicate their intricate organization. By combining two biofabrication techniques, we developed a novel 3D in vitro model that closely mimics the organization of an ovine IVD. Our approach employs a polycaprolactone (PCL) frame produced via melt electrowriting to recreate the multilamellar architecture of the annulus fibrosus. Ovine primary cells, encapsulated in a photocrosslinkable alginate hydrogel, were precisely extruded within the multilamellar structure, thereby mimicking the native shape and size of an ovine disc. The bioink containing the NP cells was deposited at the center of the construct, while the bioink with the AF cells was strategically layered in between the lamellae of the PCL frame. Photocrosslinking was optimized to match the native stiffness of the disc. The constructs were maintained in culture for 28 days, during which we thoroughly assessed reproducibility, stability, and cell viability and phenotype. The results unequivocally demonstrated that the PCL frame effectively guided the alignment and proliferation of AF cells, while the alginate hydrogel preserved NP cell phenotype. This model successfully replicates the organization of the IVD, providing a promising platform for advancing our understanding of disc biology and driving the development of novel therapeutic strategies.

Abstract
Intervertebral disc (IVD) function is achieved through integration of its two component regions: the nucleus pulposus (NP) and the annulus fibrosus (AF). The NP is soft (0.3–5 kPa), gelatinous and populated by spherical NP cells in a polysaccharide-rich extracellular matrix (ECM). The AF is much stiffer (∼100 kPa) and contains layers of elongated AF cells in an aligned, fibrous ECM. Degeneration of the disc is a common problem with age being a major risk factor. Progression of IVD degeneration leads to chronic pain and can result in permanent disability. The development of therapeutic solutions for IVD degeneration is impaired by a lack of in vitro models of the disc that are capable of replicating the fundamental structure and biology of the tissue. This study aims to investigate if a newly developed suspended hydrogel bioprinting system (termed SLAM) could be employed to fabricate IVD analogues with integrated structural and compositional features similar to native tissue. Bioprinted IVD analogues were fabricated to recapitulate structural, morphological and biological components present in the native tissue. The constructs replicated key structural components of native tissue with the presence of a central, polysaccharide-rich NP surrounded by organised, aligned collagen fibres in the AF. Cell tracking, actin and matrix staining demonstrated that embedded NP and AF cells exhibited morphologies and phenotypes analogous to what is observed in vivo with elongated, aligned AF cells and spherical NP cells that deposited HA into the surrounding environment. Critically, it was also observed that the NP and AF regions contained a defined cellular and material interface and segregated regions of the two cell types, thus mimicking the highly regulated structure of the IVD.