Manufacturing of self-standing multi-layered 3D-bioprinted alginate-hyaluronate constructs by controlling the cross-linking mechanisms for tissue engineering applications

Three-dimensional (3D) bioprinting of self-supporting stable tissue and organ structure is critically important in extrusion-based bioprinting system, especially for tissue engineering and regenerative medicine applications. However, the development of self-standing bioinks with desired crosslinking density, biocompatibility, tunable mechanical strength and other properties like self-healing,in situgelation, drug or protein incorporation is still a challenge. In this study, we report a hydrogel bioink prepared from alginate (Alg) and hyaluronic acid (HA) crosslinked through multiple crosslinking mechanisms, i.e. acyl-hydrazone, hydrazide interactions and calcium ions. These Alg-HA gels were highly dynamic and shear-thinning with exceptional biocompatibility and tunable mechanical properties. The increased dynamic nature of the gels is mainly chemically attributed to the presence of acyl-hydrazone bonds formed between the amine groups of the acyl-hydrazide of alginate and the monoaldehyde of the HA. Among the different combinations of Alg-HA gel compositions prepared, the A5H5 (Alginate-acyl-hydrazide:HA-monoaldehyde, ratio 50:50) gel showed a gelation time of ∼60 s, viscosity of ∼400 Pa s (at zero shear rate), high stability in various pH solutions and increased degradation time (>50 days) than the other samples. The A5H5 gels showed high printability with increased post-printing stability as observed from the 3D printed structures (e.g. hollow tube (∼100 layers), porous cube (∼50 layers), star, heart-in, meniscus and lattice). The scanning electron microscopy analysis of the 3D constructs and hydrogels showed the interconnected pores (∼181µm) and crosslinked networks. Further, the gels showed sustained release of 5-amino salicylic acid and bovine serum albumin. Also, the mechanical properties were tuned by secondary crosslinking via different calcium concentrations.In vitroassays confirmed the cytocompatibility of these gels, where the 3D bioprinted lattice and tubular (∼70 layers) constructs demonstrated high cell viability under fluorescence analysis. Inin vivostudies, Alg-HA gel showed high biocompatibility (>90%) and increased angiogenesis (threefolds) and reduced macrophage infiltration (twofold decrease), demonstrating the promising potential of these hydrogels in 3D bioprinting applications for tissue engineering and regenerative medicine with tunable properties.