Thiol–Gelatin–Norbornene Bioink for Laser‐Based High‐Definition Bioprinting

Abstract

Two‐photon polymerization (2PP) is a lithography‐based 3D printing method allowing the fabrication of 3D structures with sub‐micrometer resolution. This work focuses on the characterization of gelatin–norbornene (Gel–NB) bioinks which enables the embedding of cells via 2PP. The high reactivity of the thiol‐ene system allows 2PP processing of cell‐containing materials at remarkably high scanning speeds (1000 mm s−1) placing this technology in the domain of bioprinting. Atomic force microscopy results demonstrate that the indentation moduli of the produced hydrogel constructs can be adjusted in the 0.2–0.7 kPa range by controlling the 2PP processing parameters. Using this approach gradient 3D constructs are produced and the morphology of the embedded cells is observed in the course of 3 weeks. Furthermore, it is possible to tune the enzymatic degradation of the crosslinked bioink by varying the applied laser power. The 3D printed Gel–NB hydrogel constructs show exceptional biocompatibility, supported cell adhesion, and migration. Furthermore, cells maintain their proliferation capacity demonstrated by Ki‐67 immunostaining. Moreover, the results demonstrate that direct embedding of cells provides uniform distribution and high cell loading independently of the pore size of the scaffold. The investigated photosensitive bioink enables high‐definition bioprinting of well‐defined constructs for long‐term cell culture studies.

Bioprinting Neural Systems to Model Central Nervous System Diseases

“…≈90% (day 6) [36] Optic-based Laser-induced forward transfer (LIFT) ≈200 µm [39] E15 rat primary dorsal root ganglia (DRG) [39] Live-Dye /PI: ≈85% (24 h post printing) [39] • Successful prints performed with hyaluronic acid and Matrigel [40] • Neurite growths reported [39] • Proven in situ differentiation of bioprinted cells [34] • Low cell density: ≈80 cells per drop [39] • Limited manufacturer diversity might affect device accessibility [41] Not assessed [40] hiPSC [40] Trypan Blue: ≈82% (2-3 h post printing) [40] Stereolithography (SLA) ≈190 µm [42] Mouse NSCs (NE-4C) [42] Calcein AM/PI: ≈100-70%, 40-120 mW laser power) [42] • ≈5 µm micrometer-scale resolution a chievable [43] • Custom devices a vailable [44] • Combined with 3D printing using PCL fibers [42] • Used with conductive graphene-loaded bioinks [45] • Potential cell damage due to UV light exposure…”

“…• Laser output affects cell survival [42] • Potential toxicity from photosensitive resins and initiators [27,31] ≈1k µm [45] Mouse NSCs [45] CCK-8: Proliferation over 5 days of culture [45] Digital light processing (DLP) 50-100 µm [46] No current report on the bioprinting of cells of neuronal lineage • Delivery rate: ≈20 mm 3 min −1 [27] • High Resolutions <100 µm achievable [46] • Easily accessible: Commercial projectors [46a] • Biocompatible polyethylene glycol diacrylate (PEGDA) and Gel-MA resins already available [47] • Potential cell damage due to UV light exposure…”

High-Resolution 3D Bioprinting of Photo-Cross-linkable Recombinant Collagen to Serve Tissue Engineering Applications

“…2PP is a HD 3D-printing technique that enables the production of scaffolds with a spatial resolution in the submicrometer range due to the nonlinear nature of the 2PP process, thereby often using near-infrared femtosecond pulsed laser irradiation. 13 In addition, we also investigated whether the recombinant protein could potentially be applied as a bioink, which implies the production of 3D constructs produced in the presence of living cells. 14 To this end, RCPhC1 was functionalized with different photo-cross-linkable functionalities, including methacrylamide, norbornene, and thiol moieties, which can be cross-linked via chain-growth (methacrylamide) versus step-growth (thiol-norbornene) polymerization in the presence of a suitable photoinitiator upon irradiation using a tightly focused femtosecond laser.…”

Tuning Surface Morphology of Fluorescent Hydrogels Using a Vortex Fluidic Device

“…Our results demonstrate a novel technique, facilitating a one-step fabrication of PVA hydrogels with different surface morphologies. However, more studies are required to examine the effectiveness of the proposed method for the fabrication of a series of hydrogels, with their creation relying on the formation of hydrogen bonds including, but not limited to, PVA, gelatin, starch, cellulose, guar gum, and their composites [27][28][29]. Another direction for future studies is to develop an understanding of the mechanism of the formation of hydrogels.…”

“…This technique applies to photo-crosslinkable hydrogels with no control over the depth of crosslinking. Recent laser-based technologies, including stereolithography, digital light projection, and two-photon polymerization, are alternative photopatterning systems advancing the fabrication of micropatterned hydrogels with a higher resolution, where no physical mask is used, contrasting with photomask patterning counterparts [29]. Lithographic patterning techniques relying on the selective elimination of materials to create topographies with preferred shape and size do not need further assembly steps.…”