Biofabrication of anisotropic constructs using extrusive chaotic printing

Abstract

Aligned tissue constitutes a considerable percentage of the body mass, and it is this anisotropic characteristic which confers certain mechanical and functional properties to the tissue. For creating tissue-like structures that resembles the body, one relevant challenge lies on using biomaterials and shaping them to create aligned structures. These constructs serve as scaffolding materials that promote cell proliferation and differentiation that can eventually become a working tissue. In this study, continuous chaotic printing was used to fabricate highly oriented printed scaffolds and bioprinted cell-laden constructs. First, we assessed the effectiveness of a chaotic extrusion printhead, containing a Kenics Static Mixer (KSM), as a tool to align fibrillar inks. In short, soft fibrillar materials (i.e., alginate-cellulose and collagen) were chaotically printed into 1 mm thick filaments and scaffold anisotropy was assessed in terms of printed microstructures orientation. Filaments showed orientation up to 68% in a -15° to 15° region where the main axis (i.e., aligned fiber) correspond to 0°. Moreover, we assessed the capability of chaotic bioprinting to produce aligned and pre-vascularized skeletal-muscle-like tissues. Briefly, fibers were bioprinted using three inks: a hydrogel loaded with myoblasts (C2C12 cells), a non-crosslinkable hydrogel to create inner vessels inside the fiber, and a high viscosity hydrogel loaded with mesoporous bioactive glass (MBG) to provide mechanical robustness to the fiber. A comparison was made between homogeneous (pre mixed) fibers and pre-vascularized fibers with a layered inner structure. The constructs were cultured up to 21 days and demonstrated high viability (>85%) and a significant relation in the orientation trend of the F-actin filaments with the stratification. Overall, we demonstrated that chaotic printing is a practical tool for fabricating anisotropic constructs with both, fibrillar inks and cell-laden constructs.