Engineering a Chemically Defined Hydrogel Bioink for Direct Bioprinting of Microvasculature

Abstract

Vascularizing printed tissues is a critical challenge in bioprinting. While protein-based hydrogel bioinks have been successfully used to bioprint microvasculature, their compositions are ill-defined and subject to batch variation. Few studies have focused on engineering proangiogenic bioinks with defined properties to direct endogenous microvascular network formation after printing. Here, a peptide-functionalized alginate hydrogel bioink with defined mechanical, rheological, and biochemical properties is developed for direct bioprinting of microvascularized tissues. An integrin-binding peptide (RGD) and a vascular endothelial growth factor-mimetic peptide with a protease-sensitive linker are conjugated onto a biodegradable alginate to synergistically promote vascular morphogenesis and capillary-scale endothelial tube formation. Partial ionic crosslinking before printing converts the otherwise unprintable hydrogel into a viscoelastic bioink with excellent printability and cytocompatibility. We use the bioink to fabricate a compartmentalized vascularized tissue construct, wherein we observe pericyte-endothelial cell colocalization and angiogenic sprouting across a tissue interface, accompanied by deposition of fibronectin and collagen in vascular and tissue components, respectively. This study provides a tunable and translational "off-the-shelf" hydrogel bioink with defined composition for vascularized bioprinting.

Figure 1.

Vasculogenic hydrogel matrix design. (A) Chemical schematic of functionalized alginate synthesis via CuAAC chemistry. (B) Schematic illustration of functionalized alginate synthesis with click-able integrin-binding and protease-sensitive VEGF receptor binding peptides. (C) Schematic illustration of cell-matrix interactions and endogenous vascularization within the alginate matrix.

Figure 2.

Characterization of peptide-functionalized alginate hydrogels. (A) LC and MS spectrum of RGDSP peptide and CuAAC catalytic complex before and after Click conjugation to alginate. Blue arrow indicates the corresponding peaks of THPTA (M.W.=434.25 kDa, copper coordinating compound). Red arrow indicates the corresponding peaks of RGDSP peptide (M.W.=896.36 kDa). (B) Storage and loss modulus of peptide-functionalized (2% w/v) alginates measured by oscillatory frequency sweep from 0.1 to 10 Hz at 1% strain; n≥4 for each group; n.s. no significant difference. (C) Representative SEM images of peptide-functionalized alginate hydrogel microstructures and quantification of scaffold percent porosity. Scale bars = 200 μm.

Figure 3.

Vascular morphogenesis in peptide-functionalized alginate hydrogels. (A) Representative day 7 images of RFP-HUVECs cultured with or without ADSCs in 2% (w/v) peptide-functionalized alginates. (B) The quantification of vascular morphogenesis at day 7, n≥4 for each group (* p <0.05, ** p <0.01, n.s. not significant). (C) Representative day 7 images of microvascular networks in RGD+MMPQK hydrogels (2:1 HUVEC:ADSC coculture) with or without VEGF in the culture media. (D) Quantification of vascular morphogenesis with or without VEGF, n≥4 for each group. (E) Microvascularization at day 14 in 2% (w/v) alginates functionalized with RGD and MMPQK. (F) 3D reconstruction and ortho-slices showing a lumenized (*) endothelial tube.

Figure 4.

Tuning the rheological properties of alginate hydrogel precursors via partial ionic crosslinking. (A) Increasing concentrations of CaCl₂ (0–20 mM) were added to 2% (w/v) alginate hydrogel precursors to yield progressively crosslinked solutions. (B) Flow curves for shear stress as a function of shear rate for partially crosslinked alginate solutions, n≥3 for each group. (C) Yield stress measurements of the partially crosslinked alginate solutions as determined using the Herschel-Bulkley model. (D) Flow curves for viscosity as a function of shear rate for partially crosslinked alginate solutions, n≥3 for each group. (E) Zero shear viscosity measurements of the partially crosslinked alginate solutions as determined using the Cross model. (F) Oscillatory rheological behavior of 15 mM solution, n≥3 for each group (**** p <0.0001, significant difference from all groups).

Figure 5.

Printability of the partially crosslinked RGD+MMPQK alginate bioink. (A) Cell sedimentation test in 2% (w/v) alginates that were or were not partially crosslinked with 15 mM CaCl₂. Scale bar = 200 micrometers. (B) Lattice constructs printed with functionalized alginate bioink using a point-to-point fabrication method with or without partial crosslinking. (C) Resolution of printed hydrogel dots, n = 25 for each group (*** p<0.001). (D) Relative print accuracy of construct dimensions compared to the original dimensions of the CAD design. (E) Representative image of a printed construct and quantification of print consistency in four consecutively printed lattices. n.s. not significant. (F) Live/Dead assay immediately post-print and quantification of live cells in printed constructs. Cell viability in three regions of interest (ROIs) in each of three printed constructs was evaluated.

Figure 6.

Fabricating a vascularized Tissue Unit (VTU) with partially crosslinked RGD+MMPQK alginate bioink. (A) Conceptual design of the Vascularized Tissue Unit (VTU). (B) Brightfield image of a bioprinted VTU (V, vascular components; T, tissue components). Scale bars = 100 μm. (C) Representative images of edge region of the VTU at day 7: V, vascular components; T, tissue components. Scale bars = 200 micrometers. (D) Representative images of middle region of the VTU at day 7. Scale bars = 200 micrometers. (E) Slices from a confocal z-stack show RFP-HUVECs colocalized with NG2+ cells in the printed VTU after 7 days. At z=0 um, the nuclei of RFP-HUVECs (red arrows) can be seen. At z=5 um and z=10 um, the nuclei are obscured by NG2+ cells, and the nuclei of NG2+ cells can be seen with clarity at z=15 um (yellow arrow). Scale bars = 50 micrometers.

Figure 7.

ECM deposition in the vascular component of the printed VTU. (A) Fibronectin deposition in the printed VTU after 7 days. (B) Relative fluorescence of fibronectin in vascular and tissue components (** p< 0.01). Scale bars = 200 micrometers.