In-situ ionic crosslinking of 3D bioprinted cell-hydrogel constructs for mechanical reinforcement and improved cell growth

Abstract

Hydrogels are commonly used in 3D bioprinting technology owing to their ability to encapsulate living cells. However, their inherent delicate properties limit their applicability in the fabrication of mechanically reliable tissue engineering constructs. Herein, we propose a novel reinvented layering integration method for the functional enhancement of 3D cell-hydrogel bioprinting. This was implemented by inserting electrospun microfiber sheets with a crosslinker between the 3D bioprinted layers. When surface-modified microfiber sheets were combined with Ca²⁺ ionic crosslinkers, the as-printed cell-hydrogel strand was immediately crosslinked when it contacted the sheet surface. The in-situ crosslinking in the bioprinting process not only improved the overall structural stability, but also reinforced the compressive strength and elastic modulus. The enhanced structural stability guaranteed the shape fidelity of the 3D architecture, which included the internal channel network, resulting in improved perfusion conditions for cell growth. The growth of NIH3T3 fibroblasts in 3D bioconstructs with in-situ crosslinking increased by up to five times compared to that of normally bioprinted constructs. The strengthened structural integrity was distinctly sustainable during the cell culture period owing to the sustained release of Ca²⁺ ions from the embedded microfiber sheets. The synergistic effect of the reinforced mechanical properties with enhanced cell growth is expected to extend the applicability of the proposed hydrogel-based bioprinting technique for soft tissue engineering.

Keywords: 3D bioprinting; Cell growth; Hydrogel; In-situ ionic crosslinking; Mechanical properties.