posted on 2023-01-04, 16:35authored byCarmen Radeke, Raphaël Pons, Marko Mihajlovic, Jonas R. Knudsen, Sarkhan Butdayev, Paul J. Kempen, Charis-Patricia Segeritz, Thomas L. Andresen, Christian K. Pehmøller, Thomas E. Jensen, Johan U. Lind
For
three-dimensional (3D) bioprinting to fulfill its promise and
enable the automated fabrication of complex tissue-mimicking constructs,
there is a need for developing bioinks that are not only printable
and biocompatible but also have integrated cell-instructive properties.
Toward this goal, we here present a scalable technique for generating
nanofiber 3D printing inks with unique tissue-guiding capabilities.
Our core methodology relies on tailoring the size and dispersibility
of cellulose fibrils through a solvent-controlled partial carboxymethylation.
This way, we generate partially negatively charged cellulose nanofibers
with diameters of ∼250 nm and lengths spanning tens to hundreds
of microns. In this range, the fibers structurally match the size
and dimensions of natural collagen fibers making them sufficiently
large to orient cells. Yet, they are simultaneously sufficiently thin
to be optically transparent. By adjusting fiber concentration, 3D
printing inks with excellent shear-thinning properties can be established.
In addition, as the fibers are readily dispersible, composite inks
with both carbohydrates and extracellular matrix (ECM)-derived proteins
can easily be generated. We apply such composite inks for 3D printing
cell-laden and cross-linkable structures, as well as tissue-guiding
gel substrates. Interestingly, we find that the spatial organization
of engineered tissues can be defined by the shear-induced alignment
of fibers during the printing procedure. Specifically, we show how
myotubes derived from human and murine skeletal myoblasts can be programmed
into linear and complex nonlinear architectures on soft printed substrates
with intermediate fiber contents. Our nanofibrillated cellulose inks
can thus serve as a simple and scalable tool for engineering anisotropic
human muscle tissues that mimic native structure and function.