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.

Translation of Collagen Ultrastructure to Biomaterial Fabrication for Material-Independent but Highly Efficient Topographic Immunomodulation.

Supplement-free induction of cellular differentiation and polarization solely through the topography of materials is an auspicious strategy but has so far significantly lagged behind the efficiency and intensity of media-supplementation-based protocols. Consistent with the idea that 3D structural motifs in the extracellular matrix possess immunomodulatory capacity as part of the natural healing process, it is found in this study that human-monocyte-derived macrophages show a strong M2a-like prohealing polarization when cultured on type I rat-tail collagen fibers but not on collagen I films. Therefore, it is hypothesized that highly aligned nanofibrils also of synthetic polymers, if packed into larger bundles in 3D topographical biomimetic similarity to native collagen I, would induce a localized macrophage polarization. For the automated fabrication of such bundles in a 3D printing manner, the strategy of "melt electrofibrillation" is pioneered by the integration of flow-directed polymer phase separation into melt electrowriting and subsequent selective dissolution of the matrix polymer postprocessing. This process yields nanofiber bundles with a remarkable structural similarity to native collagen I fibers, particularly for medical-grade poly(ε-caprolactone). These biomimetic fibrillar structures indeed induce a pronounced elongation of human-monocyte-derived macrophages and unprecedentedly trigger their M2-like polarization similar in efficacy as interleukin-4 treatment.

Easy-to-Prepare Coating of Standard Cell Culture Dishes for Cell-Sheet Engineering Using Aqueous Solutions of Poly(2-n-propyl-oxazoline).

Cell-sheet technology is a well-known method by which cells are grown on thermoswitchable substrates that become nonadhesive upon cooling, such that a complete layer of adherent cells, along with the produced extracellular matrix, detaches as a sheet. Polymers that exhibit a lower critical solution temperature (LCST) below physiological temperature in water, commonly poly(N-isopropylacrylamide) (PNIPAM), are covalently grafted or, for block copolymers, physisorbed onto substrates in a monomolecular thin film to achieve this. Consequently, such substrates, and the polymers required for film formation, can only be prepared in a chemical lab with profound macromolecular expertise. In this study, we present an easy and robust method to coat standard cell culture dishes with aqueous solutions of commercially available poly(2-n-propyl-2-oxazoline) (PnPrOx), a polymer that exhibits LCST behavior. Different standard cell culture dishes were repeatedly coated with 0.1 wt % aqueous solutions of PnPrOx and dried in an oven to create a fully covered and thermoresponsive surface. Using this PnPrOx surface a variety of cell types including endothelial cells, mesenchymal stem cells, and fibroblasts, were seeded and cultured until confluency. By decreasing the temperature to 16 °C, viable cell sheets were detached within cell-type dependent time frames and could be harvested for biological analysis. We show that the cytoskeleton rearranges, leading to a more contracted morphology of the cells in the detached cell sheet. The cellular junctions between single cells within the sheet could be detected using immunostainings, indicating that strong and intact intracellular contacts are preserved in the harvested sheets.