Double chemical functionalization of gelatin by methacrylation and acetylation of free amino groups enables control over both the viscous behavior of its solutions and the mechanical properties of the resulting hydrogels after photochemical crosslinking. The degree of methacrylation is controlled by the molar excess of methacrylic anhydride applied. Tenfold molar excess leads to highly methacrylated gelatin (GM₁₀), resulting in solutions with low viscosities within the inkjet-printable range (10 wt%: 3.3 ± 0.5 mPa s, 37 °C) and crosslinked hydrogels with high storage moduli G′ (10 wt%: 15.2 ± 6.4 kPa). Twofold excess of methacrylic anhydride leads to less methacrylated gelatin (GM₂) proper for preparation of soft hydrogels (10 wt%: G′ = 9.8 ± 4.6 mPa s) but its solutions are highly viscous (10 wt%: 14.2 ± 1.1 mPa s, 37 °C) and thus prone to clogging printing nozzles. Here we show that additional introduction of acetyl functionalities into GM₂ results in a significant decrease in solution viscosity (10 wt%: 2.9 ± 0.2 mPa s, 37 °C) and prevention of physical gel formation. In such a manner twofold functionalized gelatin can be inkjet-printed while the degree of chemical crosslinking remains low and the resulting gels are soft. Thus, by adjustable twofold modification of gelatin, i.e. inserting photochemically reactive and inert groups, a versatile bioink for inkjet bioprinting is created, which allows for addressing ECM based hydrogel matrices with a broad range of physical properties. Moreover, bioinks are proven to be cytocompatible and proper for inkjet printing of viable mammalian cells.

Free-form fabrication techniques, often referred to as '3D printing', are currently tested with regard to the processing of biological and biocompatible materials in general and for fabrication of vessel-like structures in particular. Such computer-controlled methods assemble 3D objects by layer-wise deposition or layer-wise cross-linking of materials. They use, for example, nozzle-based deposition of hydrogels and cells, drop-on-demand inkjet-printing of cell suspensions with subsequent cross-linking, layer-by-layer cross-linking of synthetic or biological polymers by selective irradiation with light and even laser-induced deposition of single cells. The need of vessel-like structures has become increasingly crucial for the supply of encapsulated cells for 3D tissue engineering, or even with regard to future application such as vascular grafts. The anticipated potential of providing tubes with tailored branching geometries made of biocompatible or biological materials pushes future visions of patient-specific vascularized tissue substitutions, tissue-engineered blood vessels and bio-based vascular grafts. We review here the early attempts of bringing together innovative free-form manufacturing processes with bio-based and biodegradable materials. The presented studies provide many important proofs of concepts such as the possibility to integrate viable cells into computer-controlled processes and the feasibility of supplying cells in a hydrogel matrix by generation of a network of perfused channels. Several impressive results in the generation of complex shapes and high-aspect-ratio tubular structures demonstrate the potential of additive assembly methods. Yet, it also becomes obvious that there remain major challenges to simultaneously match all material requirements in terms of biological functions (cell function supporting properties), physicochemical functions (mechanical properties of the printed material) and process-related (viscosity, cross-linkability) functions, towards the demanding goal of biofabricating artificial blood vessels.

Gelatin methacryloyl (GM) hydrogels have been investigated for almost 20 years, especially for biomedical applications. Recently, strengthening effects of a sequential cross-linking procedure, whereby GM hydrogel precursor solutions are cooled before chemical cross-linking, were reported. It was hypothesized that physical and enhanced chemical cross-linking of the GM hydrogels contribute to the observed strengthening effects. However, a detailed investigation is missing so far. In this contribution, we aimed to reveal the impact of physical and chemical cross-linking on strengthening of sequentially cross-linked GM and gelatin methacryloyl acetyl (GMA) hydrogels. We investigated physical and chemical cross-linking of three different GM(A) derivatives (GM10, GM2A8 and GM2), which provided systematically varied ratios of side-group modifications. GM10 contained the highest methacryloylation degree (DM), reducing its ability to cross-link physically. GM2 had the lowest DM and showed physical cross-linking. The total modification degree, determining the physical cross-linking ability, of GM2A8 was comparable to that of GM10, but the chemical cross-linking ability was comparable to GM2. At first, we measured the double bond conversion (DBC) kinetics during chemical GM(A) cross-linking quantitatively in real-time via near infrared spectroscopy-photorheology and showed that the DBC decreased due to sequential cross-linking. Furthermore, results of circular dichroism spectroscopy and differential scanning calorimetry indicated gelation and conformation changes, which increased storage moduli of all GM(A) hydrogels due to sequential cross-linking. The data suggested that the total cross-link density determines hydrogel stiffness, regardless of the physical or chemical nature of the cross-links.