Visible-Light Activated Tissue-Derived ECM Bioinks for Printing Biofunctional Tissue Equivalents in Centimeter-Scale

Abstract

In the field of tissue engineering, decellularized extracellular matrix (dECM) has emerged as a highly biomimetic material, comprising of a complex of tissue-specific proteins and growth factors. Recent studies have presented that tissue or organ-derived dECM can be utilized as a bioink for 3D cell printing to reproduce complex tissue structures as well as biophysical and biochemical cues for tissue-specific function and maturation. Yet, dECM bioinks have been challenged with respect to their printability, shape fidelity and physical properties, resulting in limited scalability. Here, we report dECM-based bioinks that can be mechanically reinforced by the secondary crosslinking with visible-light irradiation (400–450 nm). The mechanism of crosslinking is mainly related to tyrosine radicals, generated by the visible light, coupled with nearby radicals inherent to the dECM to rapidly produce dityrosine, of which synthesis was monitored by autofluorescence measurement. This rapid reaction did not affect bioink viscosity profile but enhanced the compressive and complex moduli of the new bioinks by 4.3 and 10.8 times, respectively, compared with those of original dECM bioinks. Photopolymerized dECM bioinks enabled successful fabrication of cylindrical constructs to 86% of the pre-designed height, 5.5 mm, whilst non-photocrosslinked constructs could only be fabricated to approximately 23% of original height. In addition, geometrically complex and large constructs (e.g., the human ear and a hollow pyramid) could also be fabricated without any additional supports. Biocompatibility and tissue-specific functionality of the new bioinks were then confirmed based on the cornea and the heart. Encapsulated keratocytes in cornea-derived dECM (Co-dECM) bioinks with crosslinker showed similar level of functions to the cells in the original Co-dECM bioink, upregulated mRNA levels of cornea-specific genes (KERA and ALDH), and downregulated the level of the myofibroblast marker, ACTA2. The residual crosslinkers were washed by immersion of the printed constructs into the basal medium for 30 min, indicating the preservation of transparency and light transmittance of the 3D-printed cornea. In addition, iPSC-derived cardiomyocytes encapsulated in the heart-derived dECM (hdECM) bioinks with crosslinker maintained their viability higher than 80% at day 2, with the similar level of cardiac-specific marker gene expression such as cTnT, MYH6, CACNA1A, and ATP2A2 to that of the control group. Taken together, the visible-light activated dECM bioinks enable bioprinting of tissue constructs with complex and scalable geometry and biofunctional capacity emulating native tissues, which may serve as a platform for a wider biofabrication window in the field of tissue engineering.