Bioprinting can be defined as the art of combining materials and cells to fabricate designed, hierarchical 3D hybrid constructs. Suitable materials, so called bioinks, have to comply with challenging rheological processing demands and rapidly form a stable hydrogel postprinting in a cytocompatible manner. Gelatin is often adopted for this purpose, usually modified with (meth-)acryloyl functionalities for postfabrication curing by free radical photopolymerization, resulting in a hydrogel that is cross-linked via nondegradable polymer chains of uncontrolled length. The application of allylated gelatin (GelAGE) as a thiol-ene clickable bioink for distinct biofabrication applications is reported. Curing of this system occurs via dimerization and yields a network with flexible properties that offer a wider biofabrication window than (meth-)acryloyl chemistry, and without additional nondegradable components. An in-depth analysis of GelAGE synthesis is conducted, and standard UV-initiation is further compared with a recently described visible-light-initiator system for GelAGE hydrogel formation. It is demonstrated that GelAGE may serve as a platform bioink for several biofabrication technologies by fabricating constructs with high shape fidelity via lithography-based (digital light processing) 3D printing and extrusion-based 3D bioprinting, the latter supporting long-term viability postprinting of encapsulated chondrocytes.

Oxygen inhibition is a phenomenon that directly impacts the print fidelity of 3D biofabricated and photopolymerized hydrogel constructs. It typically results in the undesirable physical collapse of fabricated constructs due to impaired cross-linking, and is an issue that generally remains unreported in the literature. In this study, we describe a systematic approach to minimizing oxygen inhibition in photopolymerized gelatin-methacryloyl (Gel-MA)-based hydrogel constructs, by comparing a new visible-light initiating system, Vis + ruthenium (Ru)/sodium persulfate (SPS) to more conventionally adopted ultraviolet (UV) + Irgacure 2959 system. For both systems, increasing photoinitiator concentration and light irradiation intensity successfully reduced oxygen inhibition. However, the UV + I2959 system was detrimental to cells at both high I2959 concentrations and UV light irradiation intensities. The Vis + Ru/SPS system yielded better cell cyto-compatibility, where encapsulated cells remained >85% viable even at high Ru/SPS concentrations and visible-light irradiation intensities for up to 21 days, further highlighting the potential of this system to biofabricate cell-laden constructs with high shape fidelity, cell viability, and metabolic activity.

Bottom-up biofabrication approaches combining micro-tissue fabrication techniques with extrusion-based 3D printing of thermoplastic polymer scaffolds are emerging strategies in tissue engineering. These biofabrication strategies support native self-assembly mechanisms observed in developmental stages of tissue or organoid growth as well as promoting cell-cell interactions and cell differentiation capacity. Few technologies have been developed to automate the precise assembly of micro-tissues or tissue modules into structural scaffolds. We describe an automated 3D bioassembly platform capable of fabricating simple hybrid constructs via a two-step bottom-up bioassembly strategy, as well as complex hybrid hierarchical constructs via a multistep bottom-up bioassembly strategy. The bioassembly system consisted of a fluidic-based singularisation and injection module incorporated into a commercial 3D bioprinter. The singularisation module delivers individual micro-tissues to an injection module, for insertion into precise locations within a 3D plotted scaffold. To demonstrate applicability for cartilage tissue engineering, human chondrocytes were isolated and micro-tissues of 1 mm diameter were generated utilising a high throughput 96-well plate format. Micro-tissues were singularised with an efficiency of 96.0 ± 5.1%. There was no significant difference in size, shape or viability of micro-tissues before and after automated singularisation and injection. A layer-by-layer approach or aforementioned bottom-up bioassembly strategy was employed to fabricate a bilayered construct by alternatively 3D plotting a thermoplastic (PEGT/PBT) polymer scaffold and inserting pre-differentiated chondrogenic micro-tissues or cell-laden gelatin-based (GelMA) hydrogel micro-spheres, both formed via high-throughput fabrication techniques. No significant difference in viability between the construct assembled utilising the automated bioassembly system and manually assembled construct was observed. Bioassembly of pre-differentiated micro-tissues as well as chondrocyte-laden hydrogel micro-spheres demonstrated the flexibility of the platform while supporting tissue fusion, long-term cell viability, and deposition of cartilage-specific extracellular matrix proteins. This technology provides an automated and scalable pathway for bioassembly of both simple and complex 3D tissue constructs of clinically relevant shape and size, with demonstrated capability to facilitate direct spatial organisation and hierarchical 3D assembly of micro-tissue modules, ranging from biomaterial free cell pellets to cell-laden hydrogel formulations.

Multicomponent gelatin-methacryloyl (GelMA) hydrogels are regularly adopted for cartilage tissue engineering (TE) applications, where optimizing chemical modifications for preserving biofunctionality is often overlooked. This study investigates the biological effect of two different modification methods, methacrylation and thiolation, to copolymerize GelMA and heparin. The native bioactivity of methacrylated heparin (HepMA) and thiolated heparin (HepSH) is evaluated via thromboplastin time and heparan sulfate-deficient myeloid cell-line proliferation assay, demonstrating that thiolation is superior for preserving anticoagulation and growth factor signaling capacity. Furthermore, incorporating either HepMA or HepSH in chondrocyte-laden GelMA hydrogels, cultured for 5 weeks under chondrogenic conditions, promotes cell viability and chondrocyte phenotype. However, only GelMA-HepSH hydrogels yield significantly greater differentiation and matrix deposition in vitro compared to GelMA. This study demonstrates that thiol-ene chemistry offers a favorable strategy for incorporating bioactives into gelatin hydrogels as compared to methacrylation while furthermore highlighting GelMA-HepSH hydrogels as candidates for cartilage TE applications.

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