"Introduction. Intervertebral disc (IVD) degeneration is one of the main cause of low back pain (LBP), affecting over 600 million individuals worldwide. Mechanisms associated with the degeneration remain poorly characterized, in part due to the lack of adequate IVD models. Reproducing in vitro the complex structure of an IVD, with its gelatinous core Nucleus Pulposus (NP) surrounded by the anisotropic lamellae of the Annulus Fibrosus (AF), represents a challenge for tissue engineers. To develop a bioprinted IVD model, we selected collagen type I (Col) and tyramine-conjugated Hyaluronic Acid (THA) with different ratios, according to the native composition of the IVD, and we evaluated the interactions of ovine NP and AF cells with these bioinks.
Methodology. Col 0.4%-THA 0.6% (ratio 1:1.5) and Col 0.4%-THA 0.1% (ratio 1:0.25) hydrogels were selected to mimic the NP and AF tissues, respectively. To determine the effect of THA concentration on the stiffness of the hydrogels, Young's modulI were measured with a MicroTester equipment (CellScale). NP and AF cells were harvested from healthy discs of lambs (age 6 months), in collaboration with the Nantes ONIRIS veterinary school. We encapsulated 1 to 6 million of these cells per mL (Col-THA 1:1.5 for NP cells and Col-THA 1:0.25 for AF cells). To characterize the interaction between the cells and the bioinks, cell viability was assessed with a Live/Dead assay and cell morphology was visualized by cytoskeletal actin labeling with fluorescent phalloidin- Alexa Fluor 647 (up to 28 days). Cell proliferation was assessed by a DNA assay (PicoGreen assay) and by a nucleoside incorporation assay during the cell cycle (EdU assay).
Results. The two hydrogels exhibited different mechanical properties with a Young's modulus of 2.0 kPa and 0.6 kPa for the 1:1.5 and 1:0.25 ratios, respectively. In addition, an increase in THA concentration increased the stiffness of the bio-inks. NP and AF cells encapsulated in both formulations remain alive for at least 28 days, confirming that the bioinks are not cytotoxic. While actin labeling revealed a rounded morphology of AF and NP cells in Col-THA after 1 day of culture, the cells showed an elongated morphology afterwards, suggesting cell adhesion to the polymer network. AF and NP cells proliferated in 2D culture and also when seeded on a Col-THA gel, however there was absence of cell proliferation in the two bioinks, as shown by PicoGreen and EdU assays. Interestingly, we also evidenced a significant contraction of the bioink over time, as a function of the initial cell density (from 0 to 6 million cells/mL), suggesting cell-mediated remodeling of the bioink.
Conclusion. Col-THA bioinks are not cytotoxic for ovine NP and AF cells and promote cell adhesion. Interestingly, cell seeding induces a bioink contraction upon culture, allowing to control the final dimensions of the bioprinted IVD construct."