Development of nanocellulose-hyaluronic acid bioinks for 3D bioprinting facial cartilages

Nanocellulose (NC) possesses desirable biological and mechanical properties for 3D bioprinting. This study aimed to derive a novel NC-hyaluronic acid (HA) bioink for 3D bioprinting facial cartilages and assess its printability, biocompatibility and chondrogenicity. Pulp-derived NC blend was combined with a HA hydrogel to make composite NCHA bioinks ranging from 100% HA to 20% HA. Human nasoseptal chondrocytes were cultured in the bioinks for 21 days to assess chondrogenicity through gene expression analysis, quantitative protein assays and histology. Successful crosslinking using hydrogen peroxide solution (5 μM) occurred within 5 minutes with no detriment to cell survival. All NCHA mixtures demonstrated increases in aggrecan, collagen and SOX9 expression over 21 days with bioinks comprising 20-40% HA being the most chondrogenic (p=0.0001). Printability was assessed using rheology and printability assays, demonstrating appropriate mechanical properties for 3D printing and shape retention as auricular cartilages. Biocompatibility with Live-Dead, lactate dehydrogenase and AlamarBlue assays demonstrated cell viability and proliferation sustained over 21 days. We conclude that NCHA bioinks promote chondrogenicity, possess favourable mechanical properties and have excellent biocompatibility for cartilage tissue engineering.