On-chip fabrication and in-flow 3D-printing of microgel constructs: from chip to scaffold materials in one integral process.

Bioprinting has evolved into a thriving technology for the fabrication of cell-laden scaffolds. Bioinks are the most critical component for bioprinting. Recently, microgels have been introduced as a very promising bioink, enabling cell protection and the control of the cellular microenvironment. However, the fabrication of the bioinks involves the microfluidic production of the microgels, with a subsequent multistep process to obtain the bioink, which so far has limited its application potential. Here we introduce a direct coupling of microfluidics and 3D-printing for the continuous microfluidic production of microgels with direct in-flow printing into stable scaffolds. The 3D-channel design of the microfluidic chip provides access to different hydrodynamic microdroplet formation regimes to cover a broad range of droplet and microgel diameters. After exiting a microtubing the produced microgels are hydrodynamically jammed into thin microgel filaments for direct 3D-printing into two- and three-dimensional scaffolds. The methodology enables the continuous on-chip encapsulation of cells into monodisperse microdroplets with subsequent in-flow cross-linking to produce cell-laden microgels. The method is demonstrated for different cross-linking methods and cell lines. This advancement will enable a direct coupling of microfluidics and 3D-bioprinting for scaffold fabrication.

A Print-and-Fuse Strategy for Sacrificial Filaments Enables Biomimetically Structured Perfusable Microvascular Networks with Functional Endothelium Inside 3D Hydrogels.

A facile and flexible approach for the integration of biomimetically branched microvasculature within bulk hydrogels is presented. For this, sacrificial scaffolds of thermoresponsive poly(2-cyclopropyl-2-oxazoline) (PcycloPrOx) are created using melt electrowriting (MEW) in an optimized and predictable way and subsequently placed into a customized bioreactor system, which is then filled with a hydrogel precursor solution. The aqueous environment above the lower critical solution temperature (LCST) of PcycloPrOx at 25 °C swells the polymer without dissolving it, resulting in fusion of filaments that are deposited onto each other (print-and-fuse approach). Accordingly, an adequate printing pathway design results in generating physiological-like branchings and channel volumes that approximate Murray's law in the geometrical ratio between parent and daughter vessels. After gel formation, a temperature decrease below the LCST produces interconnected microchannels with distinct inlet and outlet regions. Initial placement of the sacrificial scaffolds in the bioreactors in a pre-defined manner directly yields perfusable structures via leakage-free fluid connections in a reproducible one-step procedure. Using this approach, rapid formation of a tight and biologically functional endothelial layer, as assessed not only through fluorescent dye diffusion, but also by tumor necrosis factor alpha (TNF-α) stimulation, is obtained within three days.

Freeform printing of thermoresponsive poly(2-cyclopropyl-oxazoline) as cytocompatible and on-demand dissolving template of hollow channel networks in cell-laden hydrogels.

Conventional additive-manufacturing technologies rely on the vertical stacking of layers, whereas each layer provides the structural integrity for the upcoming one. This inherently gives rise to limitations in freedom of design especially when structures containing large voids or truly 3D pathways for printed filaments are aspired. An especially interesting technique, which overcomes these layer limitations, is freeform printing, where thermoplastic materials are printed in 3D through controlling the temperature profile such that the polymer melt solidifies right when it exits the nozzle. In this study, we introduce freeform printing for thermoresponsive polymers at the example of poly(2-cyclopropyl-oxazoline) (PcycloPrOx). This material is especially interesting for biofabrication, as poly(oxazoline)s are known to provide excellent cytocompatibility. Furthermore, (PcycloPrOx) scaffolds provide adequate stability, so that the printed structures can be embedded in cell-laden hydrogels and sufficient time remains for the gel to form around the scaffold before dissolution via temperature reduction. This ensures accuracy and prevents channel collapse for the creation of cell-laden hydrogels with an embedded three-dimensionally interconnected channel network without the need of any additional processing step such as coating.

Influence of charged groups on the cross-linking efficiency and release of guest molecules from thiol-ene cross-linked poly(2-oxazoline) hydrogels.

We describe the preparation of hydrogels using highly functionalized poly(oxazoline) based polymeric precursors and cross-linking via UV mediated radical thiol-ene chemistry. Random copolymers were synthesized based on the combination of the more hydrophilic 2-methyl-2-oxazoline or the less hydrophilic monomer 2-ethyl-2-oxazoline with 2-(3-butenyl)-2-oxazoline. These copolymers were functionalized via a post-polymerization technique with thiol or cysteine functionality at the side chain. Hence, hydrogels were obtained, for which the thermo-responsive behavior, network density and correlated properties such as swelling and mechanics, as well as the possibility of electrostatic interaction, can be tuned. Cell culture tests demonstrated good cytocompatibility of the synthesized copolymers and hydrogels. A study with two low molecular weight substances, methylene blue and fluorescein sodium, was performed to investigate how the thermo-responsive behavior or the positive charge incorporated by cysteine could influence the interaction with the compounds. It was found that the interaction with the hydrogel network was strongly influenced by the chemical properties of the dye. A hydrophilic and positively charged hydrogel network was shown to be a promising candidate for the uptake and prolonged release of negatively charged low molecular weight substances.