Paradiso, Alessia (Warsaw University of Technology )


Recently, tissue engineering still lacks thorough vasculature, which represents a major drawback in developing physiologically relevant tissue constructs. Among others, the design and the biofabrication of blood vessels at the microscale remain challenging, due to their role in nutrient and oxygen exchange, but also waste removal. Parallelly, fiber-based biofabrication techniques such as 3D-(bio)printing are considered time-consuming approaches in the field of microvascular tissue engineering. In this work, vessel-like 3D core-shell bundles have been rapidly fabricated using a novel wet-spinning system, allowing for the collection of cell-laden hydrogel-based fibers onto a rotating drum to reproduce the native architecture of the microvascular network.
Therefore, two different hydrogel formulations were optimized. First, a fibrinogen- and an alginate-based solutions were studied in terms of material characterization. Rheological measurements on the pre-polymer solutions and swelling test on the fibrous scaffolds were carried out to investigate the behavior of these biomaterials. Then, SEM analysis was assessed to evaluate the microstructure of the core-shell yarns. Afterwards, the fibrinogen-based formulation loaded with a co-culture of human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs), and the alginate biomaterial ink were simultaneously extruded from a microfluidic co-axial nozzle immersed in a CaCl2 coagulation bath to produce tissue-specific core and supporting tubular shell structures, respectively. Both motor speed and flow rates were adjusted and tuned to create fibers with a diameter dimension of around 300 μm. Upon instantaneous gelation of alginate, wet-spun fibers were collected to form densely packed fascicles. To further stabilize the bundles, a secondary crosslinking was performed by immersing the yarns in a thrombin solution to induce the enzymatic polymerization of the core. Thus, the engineered constructs were incubated at cell culture conditions for up to 21 days. The metabolic activity of encapsulated cells was evaluated by means of proliferation assay. Subsequently, cell-laden scaffolds were investigated in terms of morphological characterization. Finally, immunocytochemistry will be performed to prove the formation of vessel-like structures.
The material characterization of the proposed formulations exhibited a Newtonian-like behavior, proving the suitability of non-shear thinning hydrogel-based bioinks for wet-spinning. Tissue-specific wet-spun core-shell fibers supported cell adhesion, migration, and alignment over the culture time, generating packed 3D cell-laden constructs that may recapitulate the microvascular network. The proliferation assay confirmed consistent metabolic activity during the cell-culture period. In addition, scaffolds would likely reveal their endothelialization role, highlighting the potential of the two proposed bioinks in the frame of microvascular tissue engineering.
In conclusion, wet-spun fibers were produced from a novel co-axial needle and collected by using functionalized hydrogels, thus validating the system for the (bio)fabrication of Newtonian-like hydrogel-based constructs. Herein, encapsulated MSC-HUVEC migrated within the cell-laden hydrogel core towards the wall of the alginate shell, thus aligning along the direction of the microfibers axis to form a cellular layer. This study aims to highlight a new model to promote microvascular networks. The proposed wet-spinning platform can be considered as a potential alternative to 3D-(bio)printed engineered microvascularized constructs.


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