Development of a Novel Composite Bioink for Cartilage Tissue Engineering

Abstract

Summary Introduction and aims: Nanocellulose bioinks have promising biological and mechanical properties for 3D bioprinting. The aim of this thesis was to develop a novel natural composite biomaterial derived of nanocellulose to bioprint with cartilage derived cells for ear reconstruction. Methods: Cartilage derived cells were extracted from nasoseptal cartilage and chondroprogenitor cells were isolated using fibronectin adhesion assay. Different combinations of progenitor cells and chondrocytes were created to determine the most chondrogenic combination for 3D bioprinting cartilage. Nanocellulose blend, crystal and fibrils were blended with alginate and compared for printability and chondrogenicity profiles. The most chondrogenic and printable formulation was then mixed with varying proportions of hyaluronic acid to produce composite bioinks. Human nasoseptal chondrocytes from at least 3 separate patients were cultured in the composite biomaterial, and crosslinking with low dose hydrogen peroxide was optimised. Chondrogenicity was determined with PCR, quantitative protein assays and histology. Printability was assessed using rheology and printing assays. Mechanical properties were examined using atomic force microscopy and compression testing. Biocompatibility was demonstrated with Live-Dead, lactate dehydrogenase and alamarBlue assays. Conclusions: There were no benefits to combining cells isolated with the fibronectin assay compared to using native cartilage cell populations, indicating an inadequacy with the validity of this assay in nasoseptal cartilage. All nanocellulose materials demonstrated superior printability and bicompatibilty to alginate, with crystals and blend varieties offering superior biological properties. Instant crosslinking of the nanocellulose-hyaluronic acid bioinks were achievable with no detriment to cell survival. Nanocellulosehyaluronic acid bioink was more chondrogenic than nanocellulose-alginate and hyaluronic acid alone. Cell viability and proliferation was sustained over 21 days. Nanocellulose-hyaluronic acid bioinks demonstrate superior chondrogenicity, favourable mechanical properties and excellent biocompatibility for bioprinting cartilage for reconstructive surgery. These inks hold promise for in vivo testing and eventually clinical translation.