Tailored Bio-Hybrid Matrices for 3D Printed Tissue Models

Multi-material micro-extrusion three dimensional (3D) printing, also known as Direct-ink-writing (DIW), has opened new unique opportunities for the design and fabrication of complex bio-devices for in vitro and in vivo applications. Regardless of the application, the formulation of inks that are simultaneously printable and functional is perhaps the most critical challenge. Cellulose is appealing in this regard, as it is a versatile and affordable biomaterial that can be tailored extensively in terms of size, shape, and chemistry towards a specific intended application. In this thesis, I have studied the formulation of novel cellulose-based inks and print procedures towards two diverse applications: i) in vitro models of living mammalian tissue, and ii) oral drug-delivery patches.
Development of enhanced in vitro tissue models is of great interest to improve pre-clinical testing of novel drugs and development of patient-specific disease models. Within this thesis, the major focus was the development of new bio-inks for in vitro tissue models of mammalian tissues, based on logical modifications of microfibrillated cellulose (MFC). MFC has excellent printing properties while its fibrillar structure resembles the fibrillar network of the extracellular matrix (ECM). However, it is not translucent, which complicates microscopy, has no specific cell-adhesive motifs, and it is not miscible with protein biomaterials.
To overcome these shortcomings, we have here developed a procedure for partial carboxymethylation of MFC, to yield transparent, carboxymethylated nanofibrillated cellulose (cNFC) hydrogels, that can be combined with ECM-derived proteins. The procedure relies on careful adjustment of reaction degree by adjusting the polarity of the reaction solvents. The cNFC-derived inks show interesting properties for 3D printing functional tissue models. In addition to serving as rheological modifiers, they display unique cell-guiding abilities. Specifically, we demonstrate their ability to organize human and murine myotubes into anisotropic linear and complex patterns.
We also investigated cNFC as support matrices for embedded printing of cell-laden inks. We speculate, that the fibrillar structure of cNFC hydrogels might enhance cellular migration of cells within cNFC hydrogels compared to commonly used supports based on granular gel microparticles. However, studies are yet inconclusive. Still, we did observe that the spatial resolution of traces printed into cNFC supports was inferior to conventional granular hydrogel supports, likely due to viscous properties dominating.
A secondary project was the development of complex cellulose-based drug-delivery patches for the oral mucosa via DIW. Here, we tailored the design of the patches to increase their flexibility while decreasing the stiffness. Using multi-material printing further allowed us to precisely determine the drug-load per patch. Further, we added effervescent agents for improving delivery of saquinavir in the mouth.
Our studies showcase the versatility of cellulose-derived biomaterials for 3D printed biodevices, and demonstrates how rational modifications in material chemistry and formulation can serve to achieve complex functionalities. It is my hope that my work thus contributes to the ongoing transformation of DIW 3D printing from mainly being research topic to becoming a tool for solving real-world problems and questions.