Filamented Light (FLight) Biofabrication of Highly Aligned Tissue-Engineered Constructs.

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 µm 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.

Controlling Light in Scattering Materials for Volumetric Additive Manufacturing.

3D printing has revolutionized the manufacturing of volumetric components and structures in many areas. Several fully volumetric light-based techniques have been recently developed thanks to the advent of photocurable resins, promising to reach unprecedented short print time (down to a few tens of seconds) while keeping a good resolution (around 100 µm). However, these new approaches only work with homogeneous and relatively transparent resins so that the light patterns used for photo-polymerization are not scrambled along their propagation. Herein, a method that takes into account light scattering in the resin prior to computing projection patterns is proposed. Using a tomographic volumetric printer, it is experimentally demonstrated that implementation of this correction is critical when printing objects whose size exceeds the scattering mean free path. To show the broad applicability of the technique, functional objects of high print fidelity are fabricated in hard organic scattering acrylates and soft cell-laden hydrogels (at 4 million cells mL-1 ). This opens up promising perspectives in printing inside turbid materials with particular interesting applications for bioprinting cell-laden constructs.

Volumetric Bioprinting of Organoids and Optically Tuned Hydrogels to Build Liver-Like Metabolic Biofactories.

Organ- and tissue-level biological functions are intimately linked to microscale cell-cell interactions and to the overarching tissue architecture. Together, biofabrication and organoid technologies offer the unique potential to engineer multi-scale living constructs, with cellular microenvironments formed by stem cell self-assembled structures embedded in customizable bioprinted geometries. This study introduces the volumetric bioprinting of complex organoid-laden constructs, which capture key functions of the human liver. Volumetric bioprinting via optical tomography shapes organoid-laden gelatin hydrogels into complex centimeter-scale 3D structures in under 20 s. Optically tuned bioresins enable refractive index matching of specific intracellular structures, countering the disruptive impact of cell-mediated light scattering on printing resolution. This layerless, nozzle-free technique poses no harmful mechanical stresses on organoids, resulting in superior viability and morphology preservation post-printing. Bioprinted organoids undergo hepatocytic differentiation showing albumin synthesis, liver-specific enzyme activity, and remarkably acquired native-like polarization. Organoids embedded within low stiffness gelatins (<2 kPa) are bioprinted into mathematically defined lattices with varying degrees of pore network tortuosity, and cultured under perfusion. These structures act as metabolic biofactories in which liver-specific ammonia detoxification can be enhanced by the architectural profile of the constructs. This technology opens up new possibilities for regenerative medicine and personalized drug testing.

Needle-free delivery of fluids from compact laser-based jet injector.

Jet injection devices have been studied and developed for transdermal drug delivery to avoid the use of needles. Due to bulky actuation mechanisms, they are limited to body areas that are easy to reach such as skin. Here, we demonstrate a thin and long liquid delivery system (e.g. flexible and 30 cm long with 1.2 mm outer diameter) compatible with minimally invasive surgical procedures. The actuation mechanism is based on optical cavitation in a capillary nozzle where a laser pulse is delivered via a multimode optical fibre. We show good controllability of the jet speed by varying the actuation laser fluence. The generated jets can successfully penetrate into a 1% agarose gel which is representative of the mechanical properties of several soft body tissues. We further observe that when the system is used in a low laser energy regime (<60 µJ), the ejection is in the form of the single droplet which is promising for fluid delivery with high volume precision or drop-on-demand inkjet printing. The jet injection system we propose has the potential to deliver heat-sensitive therapeutics as we show processing of biomolecules without altering their functionality.

Repetitive regime of highly focused liquid microjets for needle-free injection.

Fast liquid jets are investigated for use as a needle-free drug delivery system into an elastic tissue such as skin. Using smaller jet diameters in a repetitive regime can mitigate bruising and pain associated with current injectors. In this study, we aim to unravel the potential of the method to deliver liquids into biological tissues having higher elasticity than healthy skin (i.e >60 kPa). To address this challenge, we have implemented a laser-based jetting system capable of generating supersonic liquid microjets in a repetitive regime. We provide insights on the penetration of microjets into hydrogel samples with elastic modulus ranging from 16 kPa to 0.5 MPa. The unprecedented speeds of injection (>680 m/s) together with a newly introduced repetitive regime opens possibilities for usage in needle-free drug administration into materials with elasticity covering the wide spectrum of biological soft tissues like blood vessels, all skin layers, scarred or dried skin or tumors.

High-resolution tomographic volumetric additive manufacturing.

In tomographic volumetric additive manufacturing, an entire three-dimensional object is simultaneously solidified by irradiating a liquid photopolymer volume from multiple angles with dynamic light patterns. Though tomographic additive manufacturing has the potential to produce complex parts with a higher throughput and a wider range of printable materials than layer-by-layer additive manufacturing, its resolution currently remains limited to 300 µm. Here, we show that a low-étendue illumination system enables the production of high-resolution features. We further demonstrate an integrated feedback system to accurately control the photopolymerization kinetics over the entire build volume and improve the geometric fidelity of the object solidification. Hard and soft centimeter-scale parts are produced in less than 30 seconds with 80 µm positive and 500 µm negative features, thus demonstrating that tomographic additive manufacturing is potentially suitable for the ultrafast fabrication of advanced and functional constructs.

Volumetric Bioprinting of Complex Living-Tissue Constructs within Seconds.

Biofabrication technologies, including stereolithography and extrusion-based printing, are revolutionizing the creation of complex engineered tissues. The current paradigm in bioprinting relies on the additive layer-by-layer deposition and assembly of repetitive building blocks, typically cell-laden hydrogel fibers or voxels, single cells, or cellular aggregates. The scalability of these additive manufacturing technologies is limited by their printing velocity, as lengthy biofabrication processes impair cell functionality. Overcoming such limitations, the volumetric bioprinting of clinically relevant sized, anatomically shaped constructs, in a time frame ranging from seconds to tens of seconds is described. An optical-tomography-inspired printing approach, based on visible light projection, is developed to generate cell-laden tissue constructs with high viability (>85%) from gelatin-based photoresponsive hydrogels. Free-form architectures, difficult to reproduce with conventional printing, are obtained, including anatomically correct trabecular bone models with embedded angiogenic sprouts and meniscal grafts. The latter undergoes maturation in vitro as the bioprinted chondroprogenitor cells synthesize neo-fibrocartilage matrix. Moreover, free-floating structures are generated, as demonstrated by printing functional hydrogel-based ball-and-cage fluidic valves. Volumetric bioprinting permits the creation of geometrically complex, centimeter-scale constructs at an unprecedented printing velocity, opening new avenues for upscaling the production of hydrogel-based constructs and for their application in tissue engineering, regenerative medicine, and soft robotics.

Single-photon three-dimensional microfabrication through a multimode optical fiber.

Two-photon polymerization (TPP) processes have enabled the fabrication of advanced and functional microstructures. However, most TPP platforms are bulky and require the use of expensive femtosecond lasers. Here, we propose an inexpensive and compact alternative to TPP by adapting an endoscopic imaging system for single-photon three-dimensional microfabrication. The wavefront of a visible continuous-wave laser beam is shaped so that it focuses into a photoresist through a 5 cm long ultra-thin multimode optical fiber (∅70 µm, NA 0.64). Using this device, we show that single-photon polymerization can be confined to the phase-controlled focal spot thanks to the non-linearity of the photoresist, likely due to oxygen radical scavenging. Thus, by exploiting this non-linearity with a specific overcuring method we demonstrate single-photon three-dimensional fabrication of solid and hollow microstructures through a multimode fiber with a 1.0-µm lateral and 21.5-µm axial printing resolution. This opens up new possibilities for advanced and functional microfabrication through endoscopic probes with inexpensive laser sources.