Background: Formation and functionality of microvascular networks plays a critical role in continuous and homogeneous supply of oxygen and nutrients for surrounding cells. Current biofabrication technologies heavily rely on fabrication of hollow channels and post-fabrication endothelialization by intraluminal colonization, which could be inefficient and time consuming. Herein, we report a novel approach for fabrication of ultrafine microvessel systems with great geometrical flexibility, with the possibility to be fully pre-endothelilized prior to embedment within a tissue-mimicking hydrogel matrix.
Methods: Poly(2-oxazoline)s (POx), a class of thermoresponsive polymers were used as sacrificial materials to create vessel-like networks. The networks with different levels of complexities were fabricated using Melt Electrowriting (MEW) and optimized by Response Surface Methodology (RSM). Printed structures were processed by different types of surface modifications to improve the stability and handling under cell culture conditions. Primary human umbilical vein endothelial cells (HUVECs) were seeded on the scaffolds and the formation of endothelial monolayer was observed using confocal microscopy. Metabolic activity of cells was measured by WST-8 assay.
Results: The sacrificial POx structures with controlled levels of complexity and branching were fabricated and the correlations between MEW process parameters and resultant fiber diameters were modeled by RSM. The sacrificial structures with diameters ranging from below 100 to near to 300 µm were produced. In order to control the plasticizing effect of water during cell culture, a dip-coating method was employed to develop a thin protective layer of PLGA over the POx surfaces. Three different time points (D3, D7, D14) were chosen for detailed determination of endothelial cell layer formation on the scaffolds. Confocal microscopy confirmed the presence of a cell friendly environment for primary HUVECs. Full endothelialization of sacrificial structures was achieved within only 2 weeks of culture.
Conclusions: Results of this study showed that micron-scaled thermoresponsive scaffolds produced by MEW could be used as a novel platform for generation of complex vascular structures, with the advantage of being fully endothelialized prior to embedment within any hydrogel matrices. We speculate that combination of this approach with the current perfusion systems could be used to produce standardized in vitro models.