Bastioli, M. | bioRxiv Science

bioRxiv2025

Aqueous two-phase bioinks for discrete packing and compartmentalisation of 3D bioprinted cells

The unparalleled ability of aqueous two-phase systems (ATPS) to reproduce microscale cellular and biomaterial compartmentalisation to selectively modulate cell behaviour and functionality is ideal for tissue engineering and regenerative medicine (TERM) purposes. Herein, we introduce new ATPS biomaterial inks for 3D bioprinting of water-in-water (W/W) emulsions, enabling precise cellular crowding for tissue regeneration in vitro and ex vivo. Gelatin methacryloyl (GelMA) was hierarchically dispersed in an alginic acid phase depending on sodium chloride (NaCl) concentration (0-36 g/L). Emulsion droplet size (12.8{+/-}2.6 {micro}m to 52.4{+/-}11.4 {micro}m) influenced degradation and spatial cell localisation (A549, C2C12, MG63). A microfluidic-assisted 3D bioprinting approach allowed fine-tuning of fibre structure adjusting ATPS deposition by modulating flow rates and printing speed. Rheological properties supported the findings of the two-phase partitioning, aiding the selection of the ATPS ink formulation for functional cell-laden construct fabrication. Encapsulation of C2C12 cells revealed enhanced cytoskeletal remodelling at higher salt concentrations. Increased GelMA phase promoted human bone marrow stromal cells (HBMSCs) crowding, mineral deposition and skeletal differentiation. In ovo studies demonstrated degradation control and vascular infiltration via salt modulation. Altogether, ATPS bioinks offer a versatile platform for the assembling of complex, hierarchical tissues with microscale precision, expanding biofabrication strategies for TERM applications.

bioengineering↗

bioRxiv2025

Engineering a microfluidic-assisted 3D bioprinting approach for the hierarchical control deposition and compartmentalisation of graded bioinks

The advent of 3D bioprinting has revolutionised tissue engineering and regenerative medicine (TERM). Today, tissues of single cell type can be printed with extreme resolution and printing fidelity. However, the ultimate functionality of the desired tissue is limited, due to the absence of a multicellular population and diversity in micro-environment distribution. Currently, 3D bioprinting technologies are facing challenges in delivering multiple cells and biomaterials in a controlled fashion. The use of interchangeable syringe-based systems has often favoured the delamination between interfaces, greatly limiting the fabrication of interconnected tissue constructs. Microfluidic-assisted 3D bioprinting platforms have been found capable of rescuing the fabrication of tissue interfaces, but often fails to guarantee printing fidelity, cell density control and compatimentalisation. Herein, we present the convergence of microfluidic and 3D bioprinting platforms into a new deposition system capable of harnessing a microfluidic printhead for the continuous rapid fabrication of interconnected functional tissues. The use of flow-focusing and passive mixer printhead modules allowed for the rapid and dynamic modulation of fibre diameter and material composition, respectively. Cells were compartmentalised into discrete three-dimensional layers with defined density patterns, confirming the punctual control of the presented microfluidic platform in arranging cells and materials in 3D. In ovo and in vivo studies demonstrated the functionality of 3D bioprinted constructs with patterned vascular endothelial growth factor (VEGF) and transforming growth factor-{beta}1 (TGF-{beta}1), respectively. This, in turn, facilitated the simulation of diverse cellular environments and proliferation pathways within a single construct, which is currently unachievable with conventional 3D bioprinting techniques, offering new opportunities for the fabrication of functionally graded materials and physiologically-relevant skeletal tissue substitutes.

bioengineering↗