Shape-defining alginate shells as semi-permeable culture chambers for soft cell-laden hydrogels.

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 culturein 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.

Tailoring the Sensitivity of Microcantilevers To Monitor the Mass of Single Adherent Living Cells.

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.

3D-Printed Reinforcement Scaffolds with Targeted Biodegradation Properties for the Tissue Engineering of Articular Cartilage.

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.

3D Bioprinting of Macroporous Materials Based on Entangled Hydrogel Microstrands.

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.