Photoinhibiting via simultaneous photoabsorption and free-radical reaction for high-fidelity light-based bioprinting

Article https://doi.org/10.1038/s41467-023-38838-2 Photoinhibiting via simultaneous photoabsorption and free-radical reaction for high-fidelity light-based bioprinting Received: 8 July 2022 Check for updates 1234567890():,; 1234567890():,; Accepted: 17 May 2023 Ning He 1,2, Xiaonan Wang3, Liyang Shi 3, Jing Li 1, Lan Mo4, Feng Chen Yuting Huang5, Hairong Liu5, Xiaolong Zhu 1, Wei Zhu 1, Yiqi Mao1 & Xiaoxiao Han 1,2 1 , Light-based 3D bioprinting is now employed widely to fabricate geometrically complex constructs for various biomedical applications. However, the inherent light scattering defect creates significant challenges in patterning dilute hydrogels to form high-fidelity structures with fine-scale features. Herein, we introduce a photoinhibiting approach that can effectively suppress the light scattering effect via a mechanism of simultaneous photoabsorption and freeradical reaction. This biocompatible approach significantly improves the printing resolution (~1.2 - ~2.1 pixels depending on swelling) and shape fidelity (geometric error less than 5%), while minimising the costly trial-and-error procedures. The capability in patterning 3D complex constructs using different hydrogels is demonstrated by manufacturing various scaffolds featuring intricate multi-sized channels and thin-walled networks. Importantly, cellularised gyroid scaffolds (HepG2) are fabricated successfully, exhibiting high cell proliferation and functionality. The strategy established in this study promotes the printability and operability of light-based 3D bioprinting systems, allowing numerous new applications for tissue engineering. Three-dimensional (3D) bioprinting technology, which allows cells and biomaterials to be patterned precisely, is a promising tool for fabricating highly sophisticated constructs for biomedical applications, such as tissue engineering, drug testing and surgical implants1–6. Among various 3D bioprinting modalities, digital light processingbased (DLP) printing has recently gained immense popularity due to its fine resolution (a few micrometres), rapid printing speed (seconds to minutes printing time) and predominant spatiotemporal controllability1. This technology converts liquid photocurable prepolymer into structured objects in a layer-wise fashion7,8 or via volumetric projection9 and has advanced bioprinting technology to create more elaborate structures, multi-vascular networks10, cell-laden scaffolds with multiscale channels11 and organ-on-a-chip systems12 for instance. In addition, DLP printers with high optical resolution (typically 1–50 μm) offer the possibility for fabricating 3D hydrogel constructs embedded with fine-scale features necessary to adequately recapitulate the complexities within the native tissue microenvironment and thus achieve appropriate biological behaviours in vitro13–16. However, recent studies1,10,17–19 have highlighted that the inherent physical light scattering defect can significantly deteriorate the printing resolution (defined as the printable minimal feature size relative to the printer’s optical resolution in pixel) of DLP and thus the capability in patterning architecturally-complex devices, hindering its application for more advanced tissue engineering. Bioink for DLP printing typically consists of a photo-cross-linkable hydrogel (e.g., gelatin methacryloyl (GelMA)20, poly(ethylene glycol) 1 National Engineering Research Centre for High Efficiency Grinding, Hunan University, 410082 Changsha, China. 2State Key Laboratory of Advanced Design and Manufacture for Vehicle Body, Hunan University, 410082 Changsha, China. 3College of Biology, Hunan University, 410082 Changsha, China. 4College of Food Science and Technology, Hunan Agricultural University, 410128 Changsha, China. 5College of Material Science and Engineering, Hunan University, e-mail: ; 410082 Changsha, China. Nature Communications | (2023)14:3063 1 Article diacrylate (PEGDA)21 and silk fibroin modified by glycidyl methacrylate (Sil-MA)7), a photoinitiator (e.g., lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP)) and cells. GelMA, a natural protein biopolymer, is commonly used in 3D bioprinting due to its exceptional biocompatibility and cell-adhesive capability. However, it possesses poor mechanical performance and low viscosity, and a mixture of other materials (e.g., alginate/methylcellulose22 and PEGDA23) is usually implemented to enhance the printability of the bioink and the mechanical performance of printed objects. During the printing process, light scattering occurs at the interface where the refractive index is mismatched when light transmits within the bioink or the solidified polymer layers. This effect deviates propagating light to unilluminated regions (voids and channels, for instance), leading to out-of-target solidification and thus substantially deteriorating the printing resolution and geometric accuracy (referred to as fidelity) of the fabricated objects1,10,17,24. The hydrogel polymer can induce the scattering effect per se due to the formation of crosslinked networks that can increase the spatial inhomogeneity of hydrogels19. When cells, in the form of suspended particles, are encapsulated in the bioink, the scattering effect will be further enhanced because of the discrepancy in refractive index between the cytoplasm and external hydrogel environment25,26. It has been observed that particle-induced scattering can broaden the curing width by delivering more light radiation sideways, significantly deteriorating lateral resolution27. Another critical factor affecting pattern fidelity is the curing depth, which is vital in determining axial printing resolution. If the curing depth exceeds the optical depth of focus, the out-of-focus plane will polymerise, resulting in a deteriorated printing resolution and blockage of hollow structures10,17,19. Consequently, eliminating the undesired polymerisation caused by scattering and excessive light penetration is crucial for high-fidelity light-based 3D bioprinting. A commonly used approach to resolve the issues above is doping the bioink with photoabsorbers, such as food dyes (e.g., tartrazine28 and Ponceau 4R29, anionic azo dye30 and 2-hydroxy-4-methoxy benzophenone-5-sulfonic acid (HMBS)31 that have enabled the reproduction of convoluted constructs embedded with multi-channels and vascular network23,29,32. However, the performance of the standard photoabsorbers is limited, deteriorating the capacity of the DLP printers they should possess1. Such an effect is more pronounced for platforms with a higher optical resolution33, particularly when cells are encapsulated25, creating significant challenges for patterning 3D cellularised constructs with fine-scale features (such as intricate microsized channels and thin-walled networks) that are of great importance to nutrient transportation and oxygen permeation. Notably, the existing photoabsorbing-based approach requires intensive trial-anderror efforts to optimise (...truncated)