Hydrogels are excellent mimetics of mammalian extracellular matrices and have found widespread use in tissue engineering. Nanoporosity of monolithic bulk hydrogels, however, limits mass transport of key biomolecules. Microgels used in 3D bioprinting achieve both custom shape and vastly improved permissivity to an array of cell functions, however spherical‐microbead‐based bioinks are challenging to upscale, are inherently isotropic, and require secondary crosslinking. Here, bioinks based on high‐aspect‐ratio hydrogel microstrands are introduced to overcome these limitations. Pre‐crosslinked, bulk hydrogels are deconstructed into microstrands by sizing through a grid with apertures of 40–100 µm. The microstrands are moldable and form a porous, entangled structure, stable in aqueous medium without further crosslinking. Entangled microstrands have rheological properties characteristic of excellent bioinks for extrusion bioprinting. Furthermore, individual microstrands align during extrusion and facilitate the alignment of myotubes. Cells can be placed either inside or outside the hydrogel phase with >90% viability. Chondrocytes co‐printed with the microstrands deposit abundant extracellular matrix, resulting in a modulus increase from 2.7 to 780.2 kPa after 6 weeks of culture. This powerful approach to deconstruct bulk hydrogels into advanced bioinks is both scalable and versatile, representing an important toolbox for 3D bioprinting of architected hydrogels.

31Hydrogels are an excellent biomimetic of the extracellular matrix and have found great 32 use in tissue engineering. Nanoporous monolithic hydrogels have limited mass transport, 33 restricting diffusion of key biomolecules. Structured microbead-hydrogels overcome some 34 of these limitations, but suffer from lack of controlled anisotropy. Here we introduce a 35 novel method for producing architected hydrogels based on entanglement of microstrands. 36 The microstrands are mouldable and form a porous structure which is stable in water. 37 Entangled microstrands are useable as bioinks for 3D bioprinting, where they align during 38 the extrusion process. Cells co-printed with the microstrands show excellent viability and 39 augmented matrix deposition resulting in a modulus increase from 2.7 kPa to 780.2 kPa 40 after 6 weeks of culture. Entangled microstands are a new class of bioinks with 41 unprecedented advantages in terms of scalability, material versatility, mass transport, 42 showing foremost outstanding properties as a bioink for 3D printed tissue grafts. 43 44 45 46 47 48 49 50 129 130 131 Results 132 133 Entangled Microstrand Materials are Mouldable, Stable in Water and Macroporous 134 135Here we report on a robust and versatile method for preparing 'entangled' microstrands. 136 Bulk hyaluronan-methacrylate (HA-MA) hydrogels were mechanically pressed through a 137

Achieving regeneration of articular cartilage is challenging due to the low healing capacity of the tissue. Appropriate selection of cell source, hydrogel, and scaffold materials are critical to obtain good integration and long-term stability of implants in native tissues. Specifically, biomechanical stability and in vivo integration can be improved if the rate of degradation of the scaffold material matches the stiffening of the sample by extracellular matrix secretion of the encapsulated cells. To this end, a novel 3D-printed lactide copolymer is presented as a reinforcement scaffold for an enzymatically crosslinked hyaluronic acid hydrogel. In this system, the biodegradable properties of the reinforced scaffold are matched to the matrix deposition of articular chondrocytes embedded in the hydrogel. The lactide reinforcement provides stability to the soft hydrogel in the early stages, allowing the composite to be directly implanted in vivo with no need for a preculture period. Compared to pure cellular hydrogels, maturation and matrix secretion remain unaffected by the reinforced scaffold. Furthermore, excellent biocompatibility and production of glycosaminoglycans and collagens are observed at all timepoints. Finally, in vivo subcutaneous implantation in nude mice shows cartilage-like tissue maturation, indicating the possibility for the use of these composite materials in one-step surgical procedures.

Microcantilevers are widely employed as mass sensors for biological samples, from single molecules to single cells. However, the accurate mass quantification of living adherent cells is impaired by the microcantilever's mass sensitivity and cell migration, both of which can lead to detect masses mismatching by ≫50%. Here, we design photothermally actuated microcantilevers to optimize the accuracy of cell mass measurements. By reducing the inertial mass of the microcantilever using a focused ion beam, we considerably increase its mass sensitivity, which is validated by finite element analysis and experimentally by gelatin microbeads. The improved microcantilevers allow us to instantly monitor at much improved accuracy the mass of both living HeLa cells and mouse fibroblasts adhering to different substrates. Finally, we show that the improved cantilever design favorably restricts cell migration and thus reduces the large measurement errors associated with this effect.

Hydrogels are one of the most widespread biomaterials used in tissue engineering. However, they possess weak mechanical properties and are often unstable in load‐bearing applications in vivo. A novel class of flexible Ti–6Al–4V titanium alloy lattices manufactured using laser powder bed fusion (L‐PBF) serves as a tunable reinforcement for hydrogels, providing them with additional mechanical stability and flexibility, while ensuring biocompatibility. A study on the design parameters of the structural elements of the lattices is performed to evaluate their influence on the mechanical properties of the structure. Mechanical testing of Ti–6Al–4V lattices shows a compressive modulus ranging from 38.9 to 895.5 kPa in the flexible direction. In the other two directions, the lattices are designed to have minimal flexibility. Lattices embedded in a 1% agarose hydrogel show a strain‐rate‐dependent, viscoelastic behavior given by the hydrogel component with the additional stiffness of the titanium lattice. Stress distribution upon loading is simulated using finite element analysis (FEA) and compared to experimental data using multiple regression statistical analysis. As a proof of concept, an intervertebral spinal disc implant is designed with mechanical properties matching the compressive moduli of the nucleus pulposus and anulus fibrosus reported in the literature.

Soft hydrogels have a porous structure that promotes viability and growth of resident cells. However, due to their low structural stability, these materials are fragile and difficult to culture in vitro. Here we present a novel approach for the 3D culture of such materials, where a shape-defining, semi-permeable hydrogel shell is used to provide mechanical stability. These thin hydrogel shells enclose and stabilize the soft materials while still permitting gas and nutrient exchange. Custom alginate-shaped shells were prepared using a thermosetting, ion-eluting hydrogel mold. In a second step, the hydrogel shells were filled with cell-laden infill materials. As an example of the versatility of this technique, materials previously not available for tissue engineering, such as non-annealed microgels or low crosslinked and mechanically unstable hydrogels, were used for tissue culture. Primary human chondrocytes were cultured using this platform, to evaluate its potential for cartilage tissue engineering. To prove the scalability of this technique, anatomically-shaped ears were cultured for 3 weeks. This novel approach has the potential to radically change the material property requirements in the field of tissue engineering: thanks to the shape definition and stability provided by the hydrogel shells, a wide range of materials previously inaccessible for the manufacture of 3D tissue grafts can be re-evaluated.

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