Abstract
Biofabrication via light-based 3D printing offers superior resolution and ability to generate free-form architectures, compared to conventional extrusion technologies. While extensive efforts in the design of new hydrogel bioinks lead to major advances in extrusion methods, the accessibility of lithographic bioprinting is still hampered by a limited choice of cell-friendly resins. Herein, we report the development of a novel set of photoresponsive bioresins derived from ichthyic-origin gelatin, designed to print high-resolution hydrogel constructs with embedded convoluted networks of vessel-mimetic channels. Unlike mammalian gelatins, these materials display thermal stability as pre-hydrogel solutions at room temperature, ideal for bioprinting on any easily-accessible lithographic printer. Norbornene- and methacryloyl-modification of the gelatin backbone, combined with a ruthenium-based visible light photoinitiator and new coccine as a cytocompatible photoabsorber, allowed to print structures resolving single-pixel features (∼50 μm) with high shape fidelity, even when using low stiffness gels, ideal for cell encapsulation (1–2 kPa). Moreover, aqueous two-phase emulsion bioresins allowed to modulate the permeability of the printed hydrogel bulk. Bioprinted mesenchymal stromal cells displayed high functionality over a month of culture, and underwent multi-lineage differentiation while colonizing the bioresin bulk with tissue-specific neo-deposited extracellular matrix. Importantly, printed hydrogels embedding complex channels with perfusable lumen (diameter <200 μm) were obtained, replicating anatomical 3D networks with out-of-plane branches (i.e. brain vessels) that cannot otherwise be reproduced by extrusion bioprinting. This versatile bioresin platform opens new avenues for the widespread adoption of lithographic biofabrication, and for bioprinting complex channel-laden constructs with envisioned applications in regenerative medicine and hydrogel-based organ-on-a-chip devices.
Highlights
The automation of the in vitro production of living tissues and the generation of clinically relevant, centimeter-scale constructs remains a major hope towards the availability of implantable grafts for regenerative medicine, as well as for manufacturing advanced in vitro models for drug discovery [1].The intricate structure of biological tissue is a result of a precise sequence of cell-driven processes occurring during tissue development, which, in a laboratory settings, to date can only be partially recapitulated within miniaturized-scale structures originated by stem cells, such as organoids [2]
All samples demonstrated a relatively low sol fraction (
This result indicates that the crosslinking efficiency was similar for LTS-GelMA and LTS-GelNB hydrogels, despite them undergoing fundamentally different crosslinking mechanisms, namely chain-growth or step-growth reactions, respectively
Summary
The automation of the in vitro production of living tissues and the generation of clinically relevant, centimeter-scale constructs remains a major hope towards the availability of implantable grafts for regenerative medicine, as well as for manufacturing advanced in vitro models for drug discovery [1]. Recent developments have demonstrated ultra-fast volumetric bioprinting of complex centimeter-scale 3D cell-laden constructs in less than 30 s, by tomographic light patterning [18] Despite such promising advances, only few hydrogel-based bioresins are currently available, the vast majority of which are based on synthetic polymers, prevalently acrylate-derivatives of polyethylene glycol, which, in their native form, lack cell instructive cues [15,19,20,21,22]. A particular attention was directed towards designing a system to enable prints with high shape fidelity microchannels, even when using hydrogels displaying very low stiffness (~1 kPa) Such system would overcome the current limitations experience in the field of DLP (bio)printing, which is mostly reliant on stiff high polymer content materials [28], and can open new opportunities for material scientists, engineers and biologists to build the generation of 3D constructs with ideal properties for cell encapsulation, tissue engineering and regenerative medicine. Together with this superior printing resolution and freedom of design, further modification of the bioresin composition was investigated in order to produce hydrogel-built structures with tunable bulk diffusion properties
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