Harnessing microfluidic bioprinting to fabricate gradient-like porous 3D constructs via emulsion ink deposition

The exceptional properties of natural structures with density gradients (e.g. bone, sponges,bamboo) have stimulated the interest in reproducing such complex architectures harnessingbiopolymer functionality. However, the possibility to generate a hierarchical structurecomprising multiple density gradient has not yet demonstrated, mainly due to the lack oftechnological advancements in engineering of new emulsion materials and rapid fabricationplatforms.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 supportbath. The fabrication of density gradient scaffolds within a supporting gel overcomes theproblems associated with low-viscosity bioink extrusion in 3D printing, supporting densitygradient structures that would be otherwise impossible to print in-air. The density gradientwas engineered using a flow-focusing printhead. The characterisation of the emulsionsdemonstrated how the regulation of the continuous and dispersed phases by using microfluidicpumps 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 phasewith a direct and significant proportionality between the diameter and the volume fraction ofthe dispersed phase (p<0.0001). The rheological characterisation of the emulsions revealeda decrease in viscosity as the applied shear rate increased. The continuous phase of DexMAand Pluronic F-68 exhibited a Newtonian fluid-like trend, while the emulsions presented anincreasingly pseudoplastic behaviour with expanding dispersed phase volume fraction.To show the effectiveness of the developed methodology, we realised complex geometriesconsisting of porous biopolymer fibres, as well as porous scaffolds with axial (two, four andalternate) and radial density obtain differential regions within a single construct. The inclusion ofphoto-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 responseto applied strains. Furthermore, morphological characterisation allowed the observation ofthe 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 densitygradient 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 viscosityusing an agarose fluid-gel. Furthermore, we investigated the printing of a combination ofmaterials (DexMA and GelMA; DexMA and nHA) by a multi-inlet flow-focusing printhead,resulting in density gradient structures with hierarchical mechanical properties and swellingability.Altogether, this work outlines the potential of combining microfluidics and rapid prototypingtechniques with the use of a suspending medium, providing a viable alternative for optimally 3Dprinting of biphasic systems with low viscosities and controlled densities.