Riccardo Rizzo

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Rizzo

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Riccardo

ORCID

0000-0001-8297-6776

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Publications 1 - 10 of 12

Light-based vat-polymerization bioprinting
Levato, Riccardo; Dudaryeva, Oksana; Garciamendez-Mijares, Carlos Ezio; et al. (2023)
Nature Reviews Methods Primers
Light-based vat-polymerization bioprinting enables computer-aided patterning of 3D cell-laden structures in a point-by-point, layer-by-layer or volumetric manner, using vat (vats) filled with photoactivatable bioresin (bioresins). This collection of technologies — divided by their modes of operation into stereolithography, digital light processing and volumetric additive manufacturing — has been extensively developed over the past few decades, leading to broad applications in biomedicine. In this Primer, we illustrate the methodology of light-based vat-polymerization 3D bioprinting from the perspectives of hardware, software and bioresin selections. We follow with discussions on methodological variations of these technologies, including their latest advancements, as well as elaborating on key assessments utilized towards ensuring qualities of the bioprinting procedures and products. We conclude by providing insights into future directions of light-based vat-polymerization methods.
FLight Biofabrication Supports Maturation of Articular Cartilage with Anisotropic Properties
Puiggalí-Jou, Anna; Rizzo, Riccardo; Bonato, Angela; et al. (2024)
Advanced Healthcare Materials
Tissue engineering approaches that recapitulate cartilage biomechanical properties are emerging as promising methods to restore the function of injured or degenerated tissue. However, despite significant progress in this research area, the generation of engineered cartilage constructs akin to native counterparts still represents an unmet challenge. In particular, the inability to accurately reproduce cartilage zonal architecture with different collagen fibril orientations is a significant limitation. The arrangement of the extracellular matrix (ECM) plays a fundamental role in determining the mechanical and biological functions of the tissue. In this study, it is shown that a novel light-based approach, Filamented Light (FLight) biofabrication, can be used to generate highly porous, 3D cell-instructive anisotropic constructs that lead to directional collagen deposition. Using a photoclick-based photoresin optimized for cartilage tissue engineering, a significantly improved maturation of the cartilaginous tissues with zonal architecture and remarkable native-like mechanical properties is demonstrated.
Macroporous Aligned Hydrogel Microstrands for 3D Cell Guidance
Rizzo, Riccardo; Bonato, Angela; Chansoria, Parth; et al. (2022)
ACS Biomaterials Science & Engineering
Tissue engineering strongly relies on the use of hydrogels as highly hydrated 3D matrices to support the maturation of laden cells. However, because of the lack of microarchitecture and sufficient porosity, common hydrogel systems do not provide physical cell-instructive guidance cues and efficient transport of nutrients and oxygen to the inner part of the construct. A controlled, organized cellular alignment and resulting alignment of secreted ECM are hallmarks of muscle, tendons, and nerves and play an important role in determining their functional properties. Although several strategies to induce cellular alignment have been investigated in 2D systems, the generation of cell-instructive 3D hydrogels remains a challenge. Here, we report on the development of a simple and scalable method to efficiently generate highly macroporous constructs featuring aligned guidance cues. A precross-linked bulk hydrogel is pressed through a grid with variable opening sizes, thus deconstructing it into an array of aligned, high aspect ratio microgels (microstrands) with tunable diameter that are eventually stabilized by a second photoclick cross-linking step. This method has been investigated and optimized both in silico and in vitro, thereby leading to conditions with excellent viability and organized cellular alignment. Finally, as proof of concept, the method has been shown to direct aligned muscle tissue maturation. These findings demonstrate the 3D physical guidance potential of our system, which can be used for a variety of anisotropic tissues and applications.
Development and Application of Photosensitive Bioresins for 3D Biofabrication Strategies: From Volumetric Printing to Two-Photon Stereolithography
Rizzo, Riccardo (2023)
From Free-Radical to Radical-Free: A Paradigm Shift in Light-Mediated Biofabrication
Rizzo, Riccardo; Petelinsek, Nika; Bonato, Angela; et al. (2023)
Advanced Science
In recent years, the development of novel photocrosslinking strategies and photoactivatable materials has stimulated widespread use of light-mediated biofabrication techniques. However, despite great progress toward more efficient and biocompatible photochemical strategies, current photoresins still rely on photoinitiators (PIs) producing radical-initiating species to trigger the so-called free-radical crosslinking/polymerization. In the context of bioprinting, where cells are encapsulated in the bioink, the presence of radicals raises concerns of potential cytotoxicity. In this work, a universal, radical-free (RF) photocrosslinking strategy to be used for light-based technologies is presented. Leveraging RF uncaging mechanisms and Michael addition, cell-laden constructs are photocrosslinked by means of one- and two-photon excitation with high biocompatibility. A hydrophilic coumarin-based group is used to cage a universal RF photocrosslinker based on 4-arm-PEG-thiol (PEG4SH). Upon light exposure, thiols are uncaged and react with an alkene counterpart to form a hydrogel. RF photocrosslinker is shown to be highly stable, enabling potential for off-the-shelf products. While PI-based systems cause a strong upregulation of reactive oxygen species (ROS)-associated genes, ROS are not detected in RF photoresins. Finally, optimized RF photoresin is successfully exploited for high resolution two-photon stereolithography (2P-SL) using remarkably low polymer concentration (<1.5%), paving the way for a shift toward radical-free light-based bioprinting.
Optimized Photoclick (Bio)Resins for Fast Volumetric Bioprinting
Rizzo, Riccardo; Ruetsche, Dominic; Liu, Hao; et al. (2021)
Advanced Materials
Volumetric printing (VP) is a light-mediated technique enabling printing of complex, low-defect 3D objects within seconds, overcoming major drawbacks of layer-by-layer additive manufacturing. An optimized photoresin is presented for VP in the presence of cells (volumetric bioprinting) based on fast thiol-ene step-growth photoclick crosslinking. Gelatin-norbornene (Gel-NB) photoresin shows superior performance, both in physicochemical and biocompatibility aspects, compared to (meth-)acryloyl resins. The extremely efficient thiol-norbornene reaction produces the fastest VP reported to date (approximate to 10 s), with significantly lower polymer content, degree of substitution (DS), and radical species, making it more suitable for cell encapsulation. This approach enables the generation of cellular free-form constructs with excellent cell viability (approximate to 100%) and tissue maturation potential, demonstrated by development of contractile myotubes. Varying the DS, polymer content, thiol-ene ratio, and thiolated crosslinker allows fine-tuning of mechanical properties over a broad stiffness range (approximate to 40 Pa to approximate to 15 kPa). These properties are achieved through fast and scalable methods for producing Gel-NB with inexpensive, off-the-shelf reagents that can help establish it as the gold standard for light-mediated biofabrication techniques. With potential applications from high-throughput bioprinting of tissue models to soft robotics and regenerative medicine, this work paves the way for exploitation of VPs unprecedented capabilities.
Multiscale Hybrid Fabrication: Volumetric Printing Meets Two-Photon Ablation
Rizzo, Riccardo; Rütsche, Dominic; Liu, Hao; et al. (2023)
Advanced Materials Technologies
Multiscale printing of 3D perfusable geometries holds great potential for a range of applications, from microfluidic systems to organ-on-a-chip. However, the generation of freeform designs spanning from centimeter to micrometer features represents an unmet challenge for a single fabrication method and thus may require the convergence of two or more modalities. Leveraging the great advances in light-based printing, herein a hybrid strategy is introduced to tackle this challenge. By combining volumetric printing (VP) and high-resolution two-photon ablation (2PA), the possibility to create multiscale models with features ranging from mesoscale (VP) to microscale (2PA) is demonstrated. To successfully combine these two methods, micrometer-size defects generated during the VP process due to optical modulation instability and self-focusing phenomena are first eliminated. By optical tuning the refractive index of the photoresin, defect-free VP that can then be combined with 2PA is demonstrated. To facilitate the 2PA process and meet VP requirements, a purely protein-based photoclick photoresin combining gelatin-norbornene and gelatin-thiol is introduced. Finally, the possibility to generate complex organotypic 3D vasculature-like constructs with features ranging from ≈400 µm of VP to ≈2 µm of 2PA is demonstrated. This hybrid strategy opens new possibilities for multiscale printing, with particular potential for microfluidics and organ/tissue-on-a-chip technologies.
Filamented Light (FLight) Biofabrication of Highly Aligned Tissue-Engineered Constructs
Liu, Hao; Chansoria, Parth; Delrot, Paul; et al. (2022)
Advanced Materials
Cell-laden hydrogels used in tissue engineering generally lack sufficient 3D topographical guidance for cells to mature into aligned tissues. A new strategy called filamented light (FLight) biofabrication rapidly creates hydrogels composed of unidirectional microfilament networks, with diameters on the length scale of single cells. Due to optical modulation instability, a light beam is divided optically into FLight beams. Local polymerization of a photoactive resin is triggered, leading to local increase in refractive index, which itself creates self-focusing waveguides and further polymerization of photoresin into long hydrogel microfilaments. Diameter and spacing of the microfilaments can be tuned from 2 to 30 mu m by changing the coherence length of the light beam. Microfilaments show outstanding cell instructive properties with fibroblasts, tenocytes, endothelial cells, and myoblasts, influencing cell alignment, nuclear deformation, and extracellular matrix deposition. FLight is compatible with multiple types of photoresins and allows for biofabrication of centimeter-scale hydrogel constructs with excellent cell viability within seconds (<10 s per construct). Multidirectional microfilaments are achievable within a single hydrogel construct by changing the direction of FLight projection, and complex multimaterial/multicellular tissue-engineered constructs are possible by sequentially exchanging the cell-laden photoresin. FLight offers a transformational approach to developing anisotropic tissues using photo-crosslinkable biomaterials.
Guiding Lights: Tissue Bioprinting Using Photoactivated Materials
Lee, Mihyun; Rizzo, Riccardo; Surman, František; et al. (2020)
Chemical Reviews
Photo-activated materials have found widespread use in biological and medical applications and are playing an increasingly important role in the nascent field of three-dimensional (3D) bioprinting. Light can be used as a trigger to drive the formation or the degradation of chemical bonds, leading to unprecedented spatiotemporal control over a material’s chemical, physical, and biological properties. With resolution and construct size ranging from nanometres to centimeters, light-mediated biofabrication allows multicellular and multimaterial approaches. It promises to be a powerful tool to mimic the complex multiscale organization of living tissues including skin, bone, cartilage, muscle, vessels, heart, and liver, among others, with increasing organotypic functionality. With this review, we comprehensively discuss photochemical reactions, photo-activated materials, and their use in state-of-the-art deposition-based (extrusion and droplet) and vat polymerization-based (one- and two-photon) bioprinting. By offering an up-to-date view on these techniques, we identify emerging trends, focusing on both the chemistry and instrument aspects, thereby allowing the readers to select the best-suited approach. Starting with photochemical reactions and photo-activated materials, we then discuss principles, applications, and limitations of each technique. With a critical eye to the most recent achievements, the reader is guided through this exciting, emerging field with special emphasis on cell-laden hydrogel constructs.
Factor XIII Cross-Linked Adhesive Chitosan Hydrogels
Berg, Ingrid; Rizzo, Riccardo; Lee, Mihyun; et al. (2021)
ACS Biomaterials Science & Engineering
Biomedical adhesives have been found to be an attractive alternative to suturing in several circumstances. However, to date most of the clinically approved formulations are based on synthetic and highly reactive toxic chemicals. In this work, we aimed to combine for the first time the bioactive properties of the cationic polysaccharide chitosan and its intrinsic electrostatic binding to negatively charged tissues with the biocompatible and clinically compliant enzymatic cross-linking scheme of fibrin glue. This synergistic activity led to the generation of a transglutaminase Factor XIII cross-linkable chitosan formulation with fast gelation kinetics, tunable mechanical properties, antibacterial activity, and strong adhesion to cartilage. © 2021 American Chemical Society

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Riccardo Rizzo

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