The exceptional properties of natural structures with density gradients (e.g. bone, sponges, bamboo) have stimulated the interest in reproducing such complex architectures harnessing biopolymer functionality. However, the possibility to generate a hierarchical structure comprising multiple density gradient has not yet demonstrated, mainly due to the lack of technological advancements in engineering of new emulsion materials and rapid fabrication platforms.
In the current work, we reported the 3D printing of porosity-controlled dextran methacrylate (DexMA) oil-in-water (o-w) emulsions using a microfluidic circuit and a fluid-gel support bath. The fabrication of density gradient scaffolds within a supporting gel overcomes the problems associated with low-viscosity bioink extrusion in 3D printing, supporting density gradient structures that would be otherwise impossible to print in-air. The density gradient was engineered using a flow-focusing printhead. The characterisation of the emulsions demonstrated how the regulation of the continuous and dispersed phases by using microfluidic pumps allowed the controlled and automated tuning of the material final porosity. Therefore, we proved that a higher droplet diameter is obtained by increasing the flow rate of the oil phase with a direct and significant proportionality between the diameter and the volume fraction of the dispersed phase (p<0.0001). The rheological characterisation of the emulsions revealed a decrease in viscosity as the applied shear rate increased. The continuous phase of DexMA and Pluronic F-68 exhibited a Newtonian fluid-like trend, while the emulsions presented an increasingly pseudoplastic behaviour with expanding dispersed phase volume fraction.
To show the effectiveness of the developed methodology, we realised complex geometries consisting of porous biopolymer fibres, as well as porous scaffolds with axial (two, four and alternate) and radial density obtain differential regions within a single construct. The inclusion of photo-radical initiators in the outer phase of the inks enabled the crosslinking of the structure, following printing, directly into the supporting fluid-gel medium.
The 3D printed porous scaffolds exhibited high-end mechanical properties and elastic response to applied strains. Furthermore, morphological characterisation allowed the observation of the hierarchical internal porous architecture of the scaffolds using X-ray computed micro-tomography (μCT), scanning electron (SEM) and laser scanning confocal microscopy (LSCM), confirming the ability of the novel bioprinting platform to deposit high-resolution density gradient constructs in 3D.
Moreover, we demonstrated the possibility to print highly complex density gradient structures (e.g. free-standing stairs, inverted pyramids, hollow structures) with extremely low viscosity using an agarose fluid-gel. Furthermore, we investigated the printing of a combination of materials (DexMA and GelMA; DexMA and nHA) by a multi-inlet flow-focusing printhead, resulting in density gradient structures with hierarchical mechanical properties and swelling ability.
Altogether, this work outlines the potential of combining microfluidics and rapid prototyping techniques with the use of a suspending medium, providing a viable alternative for optimally 3D printing of biphasic systems with low viscosities and controlled densities.