Vasileios D. Trikalitis

Dynamic growth factor presentation influences how individual endothelial cells assemble into complex vascular networks. Here, programmable bioinks are developed that facilitate dynamic vascular endothelial growth factor (VEGF) presentation to guide vascular morphogenesis within 3D‐bioprinted constructs. Aptamer's high affinity is leveraged for rapid VEGF sequestration in spatially confined regions and utilized aptamer‐complementary sequence (CS) hybridization to tune VEGF release kinetics temporally, days after bioprinting. It is shown that spatial resolution of programmable bioink, combined with CS‐triggered VEGF release, significantly influences the alignment, organization, and morphogenesis of microvascular networks in bioprinted constructs. The presence of aptamer‐tethered VEGF and the generation of instantaneous VEGF gradients upon CS‐triggering restricted hierarchical network formation to the printed aptamer regions at all spatial resolutions. Network properties improved as the spatial resolution decreased, with low‐resolution designs yielding the highest network properties. Specifically, CS‐treated low‐resolution designs exhibited significant vascular network remodeling, with an increase in vessel density(1.35‐fold), branching density(1.54‐fold), and average vessel length(2.19‐fold) compared to non‐treated samples. The results suggest that CS acts as an external trigger capable of inducing time‐controlled changes in network organization and alignment on‐demand within spatially localized regions of a bioprinted construct. It is envisioned that these programmable bioinks will open new opportunities for bioengineering functional, hierarchically self‐organized vascular networks within engineered tissues.

The functional principle behind extrusion‐based printing is the capability of flowing material through a nozzle on demand, which must solidify upon deposition, a behavior exhibited only by some materials. Embedded printing offers a solution to maintain shape fidelity during the deposition of a wider range of materials. However, the use of a moving nozzle in a support bath can lead to bath disturbance and the spreading of the ink. In this study, a novel embedded printing technique that eliminates the need for a nozzle by employing a magnetic sphere as the plotting moiety is introduced. The externally steered sphere creates a path by locally fluidizing the bath, allowing the simultaneously injected ink to flow into the space behind it. The method is benchmarked using water as an ink, achieving free‐form printing without additional stabilization methods. The creation of solid structures is also demonstrated by printing a photocurable ink that is crosslinked and removed from the bath. Moreover, the plotting magnet can be incorporated into the printed part during the crosslinking, thus giving place to a magnetically responsive structure. This advancement paves the way for innovations in fields such as tissue engineering and microrobotics by enabling the fabrication of intricate and functional designs.

Dynamic growth factor presentation influences how individual endothelial cells assemble into complex vascular networks. Here, we developed programmable bioinks that facilitate dynamic VEGF presentation to guide vascular morphogenesis within 3D-bioprinted constructs. We leveraged aptamer’s high affinity for rapid VEGF sequestration in spatially confined regions and utilized aptamer-complementary sequence (CS) hybridization to tune VEGF release kinetics temporally, days after bioprinting. We show that spatial resolution of programmable bioink, combined with CS-triggered VEGF release, significantly influences alignment, organization, and morphogenesis of microvascular networks in bioprinted constructs. The presence of aptamer-tethered VEGF and the generation of instantaneous VEGF gradients upon CS-triggering restricted hierarchical network formation to the printed aptamer regions at all spatial resolutions. Network properties improved as the spatial resolution decreased, with low-resolution designs yielding the highest network properties. Specifically, CS-treated low-resolution designs exhibited significant vascular network remodeling, with increase in vessel density(1.35-fold), branching density(1.54-fold), and average vessel length(2.19-fold) compared to non-treated samples. Our results suggests that CS acts as an external trigger capable of inducing time-controlled changes in network organization and alignment on-demand within spatially localized regions of a bioprinted construct. We envision that these programmable bioinks will open new opportunities for bioengineering functional, hierarchically self-organized vascular networks within engineered tissues.

In order to fabricate functional organoids and microtissues, a high cell density is generally required. As such, the placement of cell suspensions in molds or microwells to allow for cell concentration by sedimentation is the current standard for the production of organoids and microtissues. Even though molds offer some level of control over the shape of the resulting microtissue, this control is limited as microtissues tend to compact towards a sphere after sedimentation of the cells. 3D bioprinting on the other hand offers complete control over the shape of the resulting structure. Even though the printing of dense cell suspensions in the ink has been reported, extruding dense cellular suspensions is challenging and generally results in high shear stresses on the cells and a poor shape fidelity of the print. As such, additional materials such as hydrogels are added in the bioink to limit shear stresses, and to improve shape fidelity and resolution. The maximum cell concentration that can be incorporated in a hydrogel-based ink before the ink’s rheological properties are compromised, is significantly lower than the concentration in a tissue equivalent. Additionally, the hydrogel components often interfere with cellular self-assembly processes. To circumvent these limitations, we report a simple and inexpensive xanthan bath based embedded printing method to 3D print dense functional linear tissues using dilute particle suspensions consisting of cells, spheroids, hydrogel beads, or combinations thereof. Using this method, we demonstrated the self-organization of functional cardiac tissue fibers with a layer of epicardial cells surrounding a body of cardiomyocytes.