Mobile micro-and nanorobots have been proposed for future biomedical applications, such as diagnostics and targeted delivery. For their translation to clinical practice, biocompatibility and biodegradability of micro-and nanorobots are required aspects. The fabrication of smallscale robots with non-cytotoxic biodegradable soft components will allow for enhanced device assimilation, optimal tissue interaction and minimized immune reactions. We report the 3D microfabrication of biodegradable soft helical microswimmers via two-photon polymerization of the nontoxic photocrosslinkable hydrogel gelatin methacryloyl (GelMA). GelMA microswimmers are fabricated with user-defined geometry and rendered magnetically responsive by decorating their surface with magnetic nanoparticles. In contrast to previous rigid helical microrobots, our soft helical microswimmers can corkscrew above the step-out frequency with relatively high values of forward velocity, suggesting an unprecedented selfadaptive behavior. Cytotoxicity assays show the toxicity of GelMA is at least three orders of

The natural extracellular matrix (ECM) represents a complex and dynamic environment. It provides numerous spatio‐temporal signals mediating many cellular functions including morphogenesis, adhesion, proliferation and differentiation. The cell–ECM interaction is bidirectional. Cells dynamically receive and process information from the ECM and remodel it at the same time. Theses complex interactions are still not fully understood. For better understanding, it is indispensable to deconstruct the ECM up to the point of investigating isolated characteristics and cell responses to physical, chemical and topographical cues. Two‐photon polymerization (2PP) allows the exact reconstruction of cell specific sites in 3D at micro‐ and nanometer precision. Processing biocompatible synthetic and naturally‐derived hydrogels, the microenvironment of cells can be designed to specifically investigate their behavior in respect to key chemical, mechanical and topographical attributes. Moreover, 3D manipulation can be performed in the presence of cells, guiding biological tissue formation in all stages of its development. Here, advances in 2PP microfabrication of synthetic and naturally based hydrogels are reviewed. Key components of photopolymerizable hydrogel precursors, their structure–property relationships and their polymerization mechanisms are presented. Furthermore, it is shown how biocompatible 2PP fabricated constructs can act as biologically relevant matrices to study cell functions and tissue development.

Neurodegenerative diseases generally result in irreversible neuronal damage and neuronal death. Cell therapy shows promise as a potential treatment for these diseases. However, the therapeutic targeted delivery of these cells and the in situ provision of a suitable microenvironment for their differentiation into functional neuronal networks remain challenging. A highly integrated multifunctional soft helical microswimmer featuring targeted neuronal cell delivery, on-demand localized wireless neuronal electrostimulation, and post-delivery enzymatic degradation is introduced. The helical soft body of the microswimmer is fabricated by two-photon lithography of the photocurable gelatin-methacryloyl (GelMA)-based hydrogel. The helical body is then impregnated with composite multiferroic nanoparticles displaying magnetoelectric features (MENPs). While the soft GelMA hydrogel chassis supports the cell growth, and is degraded by enzymes secreted by cells, the MENPs allow for the magnetic transportation of the bioactive chassis, and act as magnetically mediated electrostimulators of neuron-like cells. The unique combination of the materials makes these microswimmers highly integrated devices that fulfill several requirements for their future translation to clinical applications, such as cargo delivery, cell stimulation, and biodegradability. The authors envision that these devices will inspire new avenues for targeted cell therapies for traumatic injuries and diseases in the central nervous system.

The two-photon polymerization (2PP) of photosensitive gelatin in the presence of living cells is reported. The 2PP technique is based on the localized cross-linking of photopolymers induced by femtosecond laser pulses. The availability of water-soluble photoinitiators (PI) suitable for 2PP is crucial for applying this method to cell-containing materials. Novel PIs developed by our group allow 2PP of formulations with up to 80% cell culture medium. The cytocompatibility of these PIs was evaluated by an MTT assay. The results of cell encapsulation by 2PP show the occurrence of cell damage within the laser-exposed regions. However, some cells located in the immediate vicinity and even within the 2PP-produced structures remain viable and can further proliferate. The control experiments demonstrate that the laser radiation itself does not damage the cells at the parameters used for 2PP. On the basis of these findings and the reports by other groups, we conclude that such localized cell damage is of a chemical origin and can be attributed to reactive species generated during 2PP. The viable cells trapped within the 2PP structures but not exposed to laser radiation continued to proliferate. The live/dead staining after 3 weeks revealed viable cells occupying most of the space available within the 3D hydrogel constructs. While some of the questions raised by this study remain open, the presented results indicate the general practicability of 2PP for 3D processing of cell-containing materials. The potential applications of this highly versatile approach span from precise engineering of 3D tissue models to the fabrication of cellular microarrays.

Hydrogels are polymeric materials with water contents similar to that of soft tissues. Due to their biomimetic properties, they have been extensively used in various biomedical applications including cell encapsulation for tissue engineering. The utilization of photopolymers provides a possibility for the temporal and spatial controlling of hydrogel cross-links. We produced three-dimensional (3-D) hydrogel scaffolds by means of the two-photon polymerization (2PP) technique. Using a highly efficient water-soluble initiator, photopolymers with up to 80 wt.% water were processed with high precision and reproducibility at a writing speed of 10  mm/s. The biocompatibility of the applied materials was verified using Caenorhabditis elegans as living test organisms. Furthermore, these living organisms were successfully embedded within a 200×200×35 μm³ hydrogel scaffold. As most biologic tissues exhibit a window of transparency at the wavelength of the applied femtosecond laser, it is suggested that 2PP is promising for an in situ approach. Our results demonstrate the feasibility of and potential for bio-fabricating 3-D tissue constructs in the micrometre-range via near-infrared lasers in direct contact with a living organism.

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Monomers for radical photopolymerization based on vinyl esters (VEs) have recently been identified as suitable alternatives to (meth)acrylates on account of their low irritancy and cytotoxicity. The drawback of most VEs with abstractable hydrogens is their relatively low reactivity compared with (meth)acrylates. Within this article, we proved by photo-differential scanning calorimetry measurements and real-time Fourier transform infrared spectroscopy that the thiol-ene concept is able to improve the photoreactivity of these VEs to a large extent to a level between those of acrylates and methacrylates.Other VEs have now a reactivity of at least the level of similar acrylates. Mechanical properties as determined by Dynamic Mechanical Analysis and Charpy impact tests showed significant toughening of these materials. Furthermore, we were able to confirm low toxicity of all components by osteoblast cell culture experiments.

Advanced hydrogel systems that allow precise control of cells and their 3D microenvironments are needed in tissue engineering, disease modeling, and drug screening. Multiphoton lithography (MPL) allows true 3D microfabrication of complex objects, but its biological application requires a cell-compatible hydrogel resist that is sufficiently photosensitive, cell-degradable, and permissive to support 3D cell growth. Here, an extremely photosensitive cell-responsive hydrogel composed of peptide-crosslinked polyvinyl alcohol (PVA) is designed to expand the biological applications of MPL. PVA hydrogels are formed rapidly by ultraviolet light within 1 min in the presence of cells, providing fully synthetic matrices that are instructive for cell-matrix remodeling, multicellular morphogenesis, and protease-mediated cell invasion. By focusing a multiphoton laser into a cell-laden PVA hydrogel, cell-instructive extracellular cues are site-specifically attached to the PVA matrix. Cell invasion is thus precisely guided in 3D with micrometer-scale spatial resolution. This robust hydrogel enables, for the first time, ultrafast MPL of cell-responsive synthetic matrices at writing speeds up to 50 mm s . This approach should enable facile photochemical construction and manipulation of 3D cellular microenvironments with unprecedented flexibility and precision.