Chaotic bioprinting: A powerful and simple biofabrication strategy to produce pre-vascularized hydrogel-based constructs

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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Trujillo-de Santiago, Grissel (Tecnológico de Monterrey)


"The biofabrication of living constructs containing hollow channels is critical for manufacturing thick tissues. Current technologies are limited in their effectiveness in the fabrication of channels with diameters smaller than dozens of micrometers.
Here we present advances in continuous chaotic bioprinting, an additive manufacturing technique that enables the creation of complex biological structures at an unprecedented level of resolution and throughput. Chaotic printing uses deterministic chaotic advection for producing structures with internal layers. Here, we showcase its use to print multichannel filaments using permanent and sacrificial inks.
In continuous chaotic printing, two or more free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a filament with a well-defined internal multilayer microarchitecture at high-throughput (>1.0 m min-1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal layers, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (~102 cm2 cm-3) at high resolution (~10 µm). Comparison of experimental and computational results demonstrates that continuous chaotic 3D printing is a robust process with predictable output.
In this study, we demonstrate that the co-extrusion of cell-laden hydrogels and sacrificial materials through printheads containing a set of KSM elements enables the one-step fabrication of hydrogel filaments containing dozens of hollow microchannels. The KSM elements induce the generation of intercalated layers of the print fluids as they are continuously extruded through the printhead. We report the fabrication of hydrogel filaments 1 mm in diameter containing up to 31 inner hollow channels with diameters as small as a single cell. Scanning electron microscopy micrographs showed that channels with widths from 100 µm to 22 µm could be produced using a printhead with 3 or 6 KSM elements. We bioprinted pre-vascularized skeletal muscle-like filaments by loading C2C12 cells in GelMA-alginate hydrogels and using hydroxyethyl cellulose (HEC) as a sacrificial material. We observed higher viability and metabolic activity in filaments with hollow multi-channels than in solid constructs. The presence of hollow channels promoted the expression of Ki67 (a proliferation biomarker), mitigated the expression of hypoxia-inducible factor 1 alpha (HIF1-α), and markedly enhanced cell alignment. Moreover, after seven days of culture, 82% of muscle myofibrils aligned (in ±10°) to the main direction of the microchannels contained in the hydrogel filament. The emergence of sarcomeric α-actin was verified through immunofluorescence and gene expression. Overall, this work presents what we envision will be an effective and practical tool for the fabrication of multi-layer and vascularized engineered tissues. The simplicity and high resolution of continuous chaotic printing strongly support its potential use for bioprinting complex and functional tissues."

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