Integration of customizable 3D printed mixing printheads for controlled and tuneable material and stiffness gradient fabrication with high cell viability in extrusion-based bioprinting

The fabrication of native tissue-like structures with gradual transitions in material properties, cell types and growth factors remains a major challenge in biofabrication. Particularly the complex hierarchical organization, present in living tissues, has to be mimicked as close as possible for the created models to fulfil the desired function. However, the fabrication of gradual structures incorporating several materials ensuring high cell survival and subsequent unaffected tissue maturation is highly challenging. To get a step closer to the goal of generating tissue models containing controlled gradient structures, here we show a novel approach combining extrusion- based 3D bioprinting with static mixing, using self-designed Digital Light Processing (DLP) printed mixing units to fabricate defined gradual structures. Two passive mixing geometries, sinusoidal and obstacle-based, are designed and fabricated and benchmarked against commercially available static mixers. The mixing performance is assessed pixelwise using dyed alginate solutions that are extruded through the mixers using an adapted 3D printer equipped with cavity pumps. The obstacle structure exhibits the highest mixing rate compared to the sinusoidal design and commercially available static mixers. Beyond mixing efficiency, the biological compatibility of the systems is assessed by evaluating the viability of U87 and NIH3T3 cells after extrusion. Two distinct polymer solutions of differing viscosities, composed of allyl-modified gelatin (gelAGE), polyethylene glycol dithiol (PEG-2-SH), and Matrigel, are extruded through the static mixer, revealing significantly higher cell viability in the self-designed printheads compared to commercial mixers. Subsequently, graded structures mimicking the mechanical profile of native brain tissue are fabricated and mechanically characterized using nanoindentation, confirming the successful generation of continuous stiffness gradients. In summary, this study demonstrates that combining extrusion-based 3D printing with customizable mixing units enables the controlled fabrication of tissue-like gradient structures with enhanced mixing rates and cell viability. The design flexibility and rapid fabrication of DLP-printed printheads enable the adaptation to specific tissue requirements, providing a robust platform for future developments in biofabrication and tissue engineering. This work serves as foundation for the creation of increasingly complex and functional tissue models.