Design and Characterization of a Composite Bioink derived from Decellularized Liver Matrix for Liver Tissue Regeneration

Abstract

Liver transplantation remains the only curative option for end-stage liver failure, but its limitations necessitate alternative regenerative strategies. Three-dimensional (3D) bioprinting offers a promising approach, contingent on the development of bioinks that are mechanically stable, biocompatible, and capable of supporting hepatic function and cell growth. This study is aimed to design and characterize composite bioink incorporating gelatin(G), alginate(A), tannic acid (TA), eggshell membrane (ESM) powder, and decellularized liver extracellular matrix (DTM) powder for liver tissue engineering applications and perform physicochemical characterisation of the same, along with in vitro cell studies using HepG2 cell line. For this purpose, ESM and DTM powder were prepared successfully and characterized by scanning electron microscopy and histological staining. SEM images confirmed fibrous morphology of ESM powder with fibre diameters ranging from 1.2μm – 2.5μm and in case of DTM powder, the images revealed a well-preserved fibrous morphology with interconnected fibres, thus confirming structural integrity post decellularization. Histological staining of decellularised tissue with Haematoxylin and Eosin confirmed decellularization was successful and staining with Picrosirius red confirmed preservation of collagen content in the same. Four different compositions of bioink using the aforementioned materials were prepared. Rheological analysis demonstrated shear-thinning behaviour across all bioink formulations. Among the compositions being currently optimized, GA-TA-ESM2 exhibited viscosity (7.5 ± 0.25 Pa·s) closer to the desirable range for liver bioinks. MTT cytotoxicity assays using L929 and HepG2 cells indicated high biocompatibility (>80 % viability for L929 cells) and (>90% viability for HepG2 cells) across all formulations. For checking printability, filament extrusion confirmed good filament forming tendency. Overall, the current findings highlight the potential of composite bioinks, particularly those incorporating ESM and DTM, in mimicking liver specific microenvironments. Further optimization of the bioink composition is expected to yield a formulation with ideal mechanical properties and printability. The bioink properties are being studied further to achieve enhanced cytocompatibility and long-term functionality for liver tissue regeneration.