Multiphoton Lithography as a Promising Tool for Biomedical Applications

Multiphoton lithography (MPL) is a powerful and useful structuring tool capable of generating 2D and 3D arbitrary micro- and nanometer features of various materials with high spatial resolution down to nm-scale. This technology has received tremendous interest in tissue engineering and medical device manufacturing, due to its ability to print sophisticated structures, which is difficult to achieve through traditional printing methods. Thorough consideration of two-photon photoinitiators (PIs) and photoreactive biomaterials is key to the fabrication of such complex 3D micro- and nanostructures. In the current review, different types of two-photon PIs are discussed for their use in biomedical applications. Next, an overview of biomaterials (both natural and synthetic polymers) along with their crosslinking mechanisms is provided. Finally, biomedical applications exploiting MPL are presented, including photocleaving and photopatterning strategies, biomedical devices, tissue engineering, organoids, organ-on-chip, and photodynamic therapy. This review offers a helicopter view on the use of MPL technology in the biomedical field and defines the necessary considerations toward selection or design of PIs and photoreactive biomaterials to serve a multitude of biomedical applications.     

Bi‐Layered Tubular Microfiber Scaffolds as Functional Templates for Engineering Human Intestinal Smooth Muscle Tissue

Designing biomimetic scaffolds with in vivo–like microenvironments using biomaterials is an essential component of successful tissue engineering approaches. The intestinal smooth muscle layers exhibit a complex tubular structure consisting of two concentric muscle layers in which the inner circular layer is orthogonally oriented to the outer longitudinal layer. Here, a 3D bi‐layered tubular scaffold is presented based on flexible, mechanically robust, and well aligned silk protein microfibers to mimic the native human intestinal smooth muscle structure. The scaffolds are seeded with primary human intestinal smooth muscle cells to replicate intestinal muscle tissues in vitro. Characterization of the tissue constructs reveals good biocompatibility and support for cell alignment and elongation in the different scaffold layers to enhance cell differentiation and functions. Furthermore, the engineered smooth muscle constructs support oriented neurite outgrowth, a requisite step to achieve functional innervation. These results suggest these microfiber scaffolds as functional templates for in vitro regeneration of human intestinal smooth muscle systems. The scaffolding provides a crucial step toward engineering functional human intestinal tissue in vitro, as well as engineering other types of smooth muscles in terms of their similar phenotypes. Such utility may lead to a better understanding of smooth muscle associated diseases and treatments.     

3D Microperiodic Hydrogel Scaffolds for Robust Neuronal Cultures

Three-dimensional (3D) microperiodic scaffolds of poly(2-hydroxyethyl methacrylate) (pHEMA) have been fabricated by direct-write assembly of a photopolymerizable hydrogel ink. The ink is initially composed of physically entangled pHEMA chains dissolved in a solution of HEMA monomer, comonomer, photoinitiator and water. Upon printing 3D scaffolds of varying architecture, the ink filaments are exposed to UV light, where they are transformed into an interpenetrating hydrogel network of chemically cross-linked and physically entangled pHEMA chains. These 3D microperiodic scaffolds are rendered growth compliant for primary rat hippocampal neurons by absorption of polylysine. Neuronal cells thrive on these scaffolds, forming differentiated, intricately branched networks. Confocal laser scanning microscopy reveals that both cell distribution and extent of neuronal process alignment depend upon scaffold architecture. This work provides an important step forward in the creation of suitable platforms for in vitro study of sensitive cell types.     

Multifunctional Bioinstructive 3D Architectures to Modulate Cellular Behavior

Biological structures control cell behavior via physical, chemical, electrical, and mechanical cues. Approaches that allow us to build devices that mimic these cues in a combinatorial way are lacking due to there being no suitable instructive materials and limited manufacturing procedures. This challenge is addressed by developing a new conductive composite material, allowing for the fabrication of 3D biomimetic structures in a single manufacturing method based on two‐photon polymerization. The approach induces a combinatorial biostimulative input that can be tailored to a specific application. Development of the 3D architecture is performed with a chemically actuating photocurable acrylate previously identified for cardiomyocyte attachment. The material is made conductive by impregnation with multiwalled carbon nanotubes. The bioinstructive effect of 3D nano‐ and microtopography is combined with electrical stimulation, incorporating biochemical, and electromechanical cues to stimulate cells in serum‐free media. The manufactured architecture is combined with cardiomyocytes derived from human pluripotent stem cells. It is demonstrated that by mimicking biological occurring cues, cardiomyocyte behavior can be modulated. This represents a step change in the ability to manufacture 3D multifunctional biomimetic modulatory architectures. This platform technology has implications in areas spanning regenerative medicine, tissue engineering to biosensing, and may lead to more accurate models for predicting toxicity.     

Nanoengineering Hybrid Supramolecular Multilayered Biomaterials Using Polysaccharides and Self‐Assembling Peptide Amphiphiles

Developing complex supramolecular biomaterials through highly dynamic and reversible noncovalent interactions has attracted great attention from the scientific community aiming key biomedical and biotechnological applications, including tissue engineering, regenerative medicine, or drug delivery. In this study, the authors report the fabrication of hybrid supramolecular multilayered biomaterials, comprising high‐molecular‐weight biopolymers and oppositely charged low‐molecular‐weight peptide amphiphiles (PAs), through combination of self‐assembly and electrostatically driven layer‐by‐layer (LbL) assembly approach. Alginate, an anionic polysaccharide, is used to trigger the self‐assembling capability of positively charged PA and formation of 1D nanofiber networks. The LbL technology is further used to fabricate supramolecular multilayered biomaterials by repeating the alternate deposition of both molecules. The fabrication process is monitored by quartz crystal microbalance, revealing that both materials can be successfully combined to conceive stable supramolecular systems. The morphological properties of the systems are studied by advanced microscopy techniques, revealing the nanostructured dimensions and 1D nanofibrous network of the assembly formed by the two molecules. Enhanced C2C12 cell adhesion, proliferation, and differentiation are observed on nanostructures having PA as outermost layer. Such supramolecular biomaterials demonstrate to be innovative matrices for cell culture and hold great potential to be used in the near future as promising biomimetic supramolecular nanoplatforms for practical applications.     

Liquid Metals-Assisted Synthesis of Scalable 2D Nanomaterials: Prospective Sediment Inks for Screen-Printed Energy Storage Applications

The advents in flexible and smart technology like wearable electronics have accelerated the demand for high-performance energy-storage devices. These devices could significantly reduce the size of the next-generation wearable smart electronics. A selection of suitable printing technology and its product typically offer a reasonable manufacturing pathway like high deposition rate, low materials waste, scalable fabrication, and high-performance production. Therefore, the production of novel functional inks with desirable rheological properties that authorize high-resolution printing, are some major challenges of this technology. This work has an emphasis on the recent advancements in supporting and utilizing liquid metals chemistry to synthesis high-quality and scalable 2D nanomaterials by liquid-phase free exfoliation and facile sonication-assisted methods. These are novel concepts in synthesizing 2D nanomaterials particularly for those which either have not intrinsic layered crystal structures or those with strong interaction between their crystal layers which are difficult to synthesized using conventional approaches. It also provides some potentials to make sustainable ink formulation of such 2D nanostructures for the fabrication of high-quality screen-printed patterns for sustainable energy applications. Subsequently, it deals with the possibilities and challenges of printing such 2D nanomaterials (namely, 2D metal oxides) for micro-supercapacitor and micro-battery applications on an industrially viable scale.     

Micro/Nanometer‐Structured Scaffolds for Regeneration of Both Cartilage and Subchondral Bone

Treatment of osteochondral defects remains a great challenge in clinical practice because cartilage and subchondral bone possess significantly different physiological properties. In this study, the controlled surface micro/nanometer structure of bioactive scaffolds in a combination of biomaterial chemistry is harnessed to address this issue. Model bioactive biomaterials, bredigite (BRT) scaffolds, with controlled surface micro/nanostructure are successfully fabricated by combining 3D printing with a hydrothermal process. It is found that the growth of micro/nano–calcium phosphate crystals on the surface of BRT scaffolds notably enhances their compressive strength by healing the microcracks on the strut surface. The micro/nanostructured surface distinctly facilitates the spread and differentiation of chondrocytes by activating integrin αvb1 and α5b1 heterodimers, regulates cell morphology, and promotes osteogenic differentiation of rabbit bone marrow stromal cells (rBMSCs) through the synergetic effect of integrin α5b1 and RhoA, in which the microrod surface demonstrates the highest stimulatory effect on the differentiation of chondrocytes and rBMSCs. The in vivo study shows that the micro/nanostructured surface of the 3D printed scaffolds obviously promotes the regeneration of both cartilage and subchondral bone tissues. This study suggests that the construction of controlled micro/nanostructured surface in porous 3D scaffolds offers a smart strategy to induce bilineage bioactivities for osteochondral regeneration.     

A Bifunctional Biomaterial with Photothermal Effect for Tumor Therapy and Bone Regeneration

Malignant bone tumor is one of the major bone diseases. The treatment of such a bone disease typically requires the removal of bone tumor and regeneration of tumor‐initiated bone defects simultaneously. To address this issue, it is required that implanted biomaterials should combine the bifunctions of both therapy and regeneration. In this work, a bifunctional graphene oxide (GO)‐modified β‐tricalcium phosphate (GO‐TCP) composite scaffold combining a high photothermal effect with significantly improved bone‐forming ability is prepared by 3D‐printing and surface‐modification strategies. The prepared GO‐TCP scaffolds exhibit excellent photothermal effects under the irradiation of 808 nm near infrared laser (NIR) even at an ultralow power density of 0.36 W cm^?2, while no photothermal effects are observed for pure β‐TCP scaffolds. The photothermal temperature of GO‐TCP scaffolds can be effectively modulated in the range of 40–90 °C by controlling the used GO concentrations, surface‐modification times, and power densities of NIR. The distinct photothermal effect of GO‐TCP scaffolds induces more than 90% of cell death for osteosarcoma cells (MG‐63) in vitro, and further effectively inhibits tumor growth in mice. Meanwhile, the prepared GO‐TCP scaffolds possess the improved capability to stimulate the osteogenic differentiation of rabbit bone mesenchymal stem cells (rBMSCs) by upregulating bone‐related gene expression, and significantly promote new bone formation in the bone defects of rabbits as compared to pure β‐TCP scaffolds. These results successfully demonstrate that the prepared GO‐TCP scaffolds have bifunctional properties of photothermal therapy and bone regeneration, which is believed to pave the way to design and fabricate novel implanting biomaterials in combination of therapy and regeneration functions.     

Complex‐Shaped Cellulose Composites Made by Wet Densification of 3D Printed Scaffolds

Cellulose is an attractive material resource for the fabrication of sustainable functional products, but its processing into structures with complex architecture and high cellulose content remains challenging. Such limitation has prevented cellulose‐based synthetic materials from reaching the level of structural control and mechanical properties observed in their biological counterparts, such as wood and plant tissues. To address this issue, a simple approach is reported to manufacture complex‐shaped cellulose‐based composites, in which the shaping capabilities of 3D printing technologies are combined with a wet densification process that increases the concentration of cellulose in the final printed material. Densification is achieved by exchanging the liquid of the wet printed material with a poor solvent mixture that induces attractive interactions between cellulose particles. The effect of the solvent mixture on the final cellulose concentration is rationalized using solubility parameters that quantify the attractive interparticle interactions. Using X‐ray diffraction analysis and mechanical tests, 3D printed composites obtained through this process are shown to exhibit highly aligned microstructures and mechanical properties significantly higher than those obtained by earlier additively manufactured cellulose‐based materials. These features enable the fabrication of cellulose‐rich synthetic structures that more closely resemble the exquisite designs found in biological materials grown by plants in nature.     

3D Printing of Functional Magnetic Materials: From Design to Applications

The research of functional magnetic materials has become a hot topic in the past few years due to their fast, long-range, and precise response in diverse environments. Functional magnetic devices using different magnetic materials and structure designs have been developed and demonstrated good advantages to enable various applications. However, the required magnetic materials and structure designs for diverse functions also increase the fabrication difficulties while developing such devices. 3D printing technology presents a powerful and promising manufacturing approach to rapidly fabricate functional magnetic devices of complex geometries in multiple materials and scales. Here, various 3D printing strategies and the underlying mechanisms of functional magnetic materials for several primary applications are systematically reviewed, including, magnetic anisotropy for property enhancement, magnetic robots, magnetic components in electronics, and magneto-thermal devices. Finally, the current challenges and future perspectives in engineering 3D printed functional magnetic devices are discussed.     

Piezoelectric Nanotopography Induced Neuron‐Like Differentiation of Stem Cells

The biophysical characteristics of the extracellular matrix, such as nanotopography and bioelectricity, have a profound influence on cell proliferation, adhesion, differentiation, etc. Recognition of the function of a certain biophysical cue and fabrication of biomaterial scaffolds with specific properties would have important implications and significant applications in tissue engineering. Herein, nanotopographic and piezoelectric biomaterials are fabricated and the combination effect of and individual contribution to proliferation, adhesion, and neuron‐like differentiation of rat bone marrow‐derived mesenchymal stem cells (rbMSCs) are clarified via nanotopography and piezoelectricity. Piezoelectric polyvinylidene fluoride with nanostripe array structures is fabricated, which can generate a surface piezoelectric potential up to millivolt by cell movement and traction. The results reveal a more favorable effect on neuron‐like differentiation of rbMSCs from the combination of piezoelectricity and nanotopography rather than nanotopography alone, whereas nanotopography can increase cellular adhesion. This research provides a new insight into designing biomaterials for the potential application in neural tissue engineering.     

Diels–Alder Reversible Thermoset 3D Printing: Isotropic Thermoset Polymers via Fused Filament Fabrication

This study presents a new 3D printing process, the Diels–Alder reversible thermoset (DART) process, and a first generation of printable DART resins, which exhibit thermoset properties at use temperatures, ultralow melt viscosity at print temperatures, smooth part surface finish, and as‐printed isotropic mechanical properties. This study utilizes dynamic covalent chemistry based on reversible furan‐maleimide Diels–Alder linkages in the polymers, which can be decrosslinked and melt‐processed during printing between 90 and 150 °C, and recrosslinked at lower temperatures to their entropically favored state. This study compares the first generation of DART materials to commonly 3D printed high‐toughness thermoplastics. Parts printed from typical fused filament fabrication compatible materials exhibit anisotropy of more than 50% and sometimes upward of 98% in toughness when deformed along the build direction, while the first generation of DART materials exhibit less than 4% toughness reduction when deformed along the build direction. At room temperature, the toughest DART materials exhibit baseline toughness of 18.59 ± 0.91 and 18.36 ± 0.57 MJ m^?3 perpendicular and parallel to the build direction, respectively. DART printing will enable chemists, polymer engineers, materials scientists, and industrial designers to translate new robust materials possessing targeted thermomechanical properties, multiaxial toughness, smooth surface finish, and low anisotropy.     

Directly drawn ZnO semiconductors and MWCNT/PSS electrodes via electrohydrodynamic jet printing for use in thin-film transistors: The ideal combination for reliable device performances

Solution-processed deposition of conducting and semiconducting materials enables the fabrication of large-area and low-cost electronic devices without the use of high-vacuum equipment. To obtain the possibility of commercializing solution-processed devices such as thin-film transistors (TFTs), easy and simple patterning process of each component become an important issue. In this study, we prepare directly patterned semiconductors and electrodes with the electrohydrodynamic (EHD) printing technique and utilize them in reliable n-type TFTs. By utilizing EHD printing technique, straight lines of zinc oxide (ZnO) semiconductor are successfully drawn from the highly soluble precursor, zinc acrylate (ZnA), and used as the active layers of TFTs. The resulting devices exhibit good TFT characteristics, and doping with a small amount of indium can enhance their performances. Furthermore, we print three different conducting materials on pre-patterned ZnO substrates for the realization of ZnO TFT arrays consisting of directly-drawn semiconductors and source/drain (S/D) electrodes. Multiwall carbon nanotube/polystyrene sulfonate (MWCNT/PSS) electrodes are found to form stable lines and their solution-processed TFTs display reliable operation with negligible hysteresis.     

Engineering Natural Pollen Grains as Multifunctional 3D Printing Materials

The development of multifunctional 3D printing materials from sustainable natural resources is a high priority in additive manufacturing. Using an eco-friendly method to transform hard pollen grains into stimulus-responsive microgel particles, we engineered a pollen-derived microgel suspension that can serve as a functional reinforcement for composite hydrogel inks and as a supporting matrix for versatile freeform 3D printing systems. The pollen microgel particles enabled the printing of composite inks and improved the mechanical and physiological stabilities of alginate and hyaluronic acid hydrogel scaffolds for 3D cell culture applications. Moreover, the particles endowed the inks with stimulus-responsive controlled release properties. The suitability of the pollen microgel suspension as a supporting matrix for freeform 3D printing of alginate and silicone rubber inks was demonstrated and optimized by tuning the rheological properties of the microgel. Compared with other classes of natural materials, pollen grains have several compelling features, including natural abundance, renewability, affordability, processing ease, monodispersity, and tunable rheological features, which make them attractive candidates to engineer advanced materials for 3D printing applications.     

A Hierarchical 3D Graft Printed with Nanoink for Functional Craniofacial Bone Restoration

An ideal craniofacial bone repair graft shall not only focus on the repair ability but also the regeneration of natural architecture with occlusal loads-related function restoration. However, such functional bone tissue engineering scaffold has rarely been reported. Herein, a hierarchical 3D graft is proposed for rebuilding craniofacial bone with both natural structure and healthy biofunction reconstruction. Inspired by the bone healing process, an organic–inorganic nanoink with ultrasmall calcium phosphate oligomers and bone morphogenetic protein-2 incorporated is developed for spatiotemporal guidance of new bone. Based on such homogeneous nanoink, a biomimetic graft, including a cortical layer containing Haversian system, and a cancellous layer featured with triply periodic minimum surface macrostructures, is fabricated via projection-based 3D printing method, and the layers are loaded with distinct concentrations of bioactive factors for regenerating new bone with gradient density. The graft exhibits excellent osteogenic and angiogenic potential in vitro, and accelerates revascularization and reconstructs neo-bone with original morphology in vivo. Benefiting from such natural architecture, loading force is widely transferred with reduced stress concentration around the inserted dental implant. Taken from native physiochemical and structural cues, this wstudy provides a novel strategy for functional tissue engineering through designing function-oriented biomaterials.     

Materials Design Innovations in Optimizing Cellular Behavior on Melt Electrowritten (MEW) Scaffolds

The field of melt electrowriting (MEW) has seen significant progress, bringing innovative advancements to the fabrication of biomaterial scaffolds, and creating new possibilities for applications in tissue engineering and beyond. Multidisciplinary collaboration across materials science, computational modeling, AI, bioprinting, microfluidics, and dynamic culture systems offers promising new opportunities to gain deeper insights into complex biological systems. As the focus shifts towards personalized medicine and reduced reliance on animal models, the multidisciplinary approach becomes indispensable. This review provides a concise overview of current strategies and innovations in controlling and optimizing cellular responses to MEW scaffolds, highlighting the potential of scaffold material, MEW architecture, and computational modeling tools to accelerate the development of efficient biomimetic systems. Innovations in material science and the incorporation of biologics into MEW scaffolds have shown great potential in adding biomimetic complexity to engineered biological systems. These techniques pave the way for exciting possibilities for tissue modeling and regeneration, personalized drug screening, and cell therapies.     

Light‐Emitting Electrochemical Cells Based on Color‐Tunable Inorganic Colloidal Quantum Dots

Large‐area, ultrathin light‐emitting devices currently inspire architects and interior and automotive designers all over the world. Light‐emitting electrochemical cells (LECs) and quantum dot light‐emitting diodes (QD‐LEDs) belong to the most promising next‐generation device concepts for future flexible and large‐area lighting technologies. Both concepts incorporate solution‐based fabrication techniques, which makes them attractive for low cost applications based on, for example, roll‐to‐roll fabrication or inkjet printing. However, both concepts have unique benefits that justify their appeal. LECs comprise ionic species in the active layer, which leads to the omission of additional organic charge injection and transport layers and reactive cathode materials, thus LECs impress with their simple device architecture. QD‐LEDs impress with purity and opulence of available colors: colloidal quantum dots (QDs) are semiconducting nanocrystals that show high yield light emission, which can be easily tuned over the whole visible spectrum by material composition and size. Emerging technologies that unite the potential of both concepts (LEC and QD‐LED) are covered, either by extending a typical LEC architecture with additional QDs, or by replacing the entire organic LEC emitter with QDs or perovskite nanocrystals, still keeping the easy LEC setup featured by the incorporation of mobile ions.     

Development of Thermoinks for 4D Direct Printing of Temperature-Induced Self-Rolling Hydrogel Actuators

4D printing has emerged as an important technique for fabricating 3D objects from programmable materials capable of time-dependent reshaping. In the present investigation, novel 4D thermoinks composed of laponite (LAP), an interpenetrating network of poly(N-isopropylacrylamide) (PNIPAAm), and alginate (ALG) are developed for direct printing of shape-morphing structures. This approach consists of the design and fabrication of 3D honeycomb-patterned hydrogel discs self-rolling into tubular constructs under the stimulus of temperature. The shape morphing behavior of hydrogels is due to shear-induced anisotropy generated via 3D printing. The compositionally tunable hydrogel discs can be programmed to exhibit different actuation behaviors at different temperatures. Upon immersion in 12 °C water, singly crosslinked sheets roll up into a tubular construct. When transferred to 42 °C water, the tubes first rapidly unfold and then slightly curve up in the opposite direction. Through a dual photocrosslinking of PNIPAAm, it is possible to inverse temperature-dependent shape morphing and induce self-folding at higher and unrolling at lower temperatures. The extensive self-assembling motion is essential to developing thermal actuators with broad applications in, e.g., soft robotics and active implantology, whereas controllable self-rolling of planar hydrogels is of the highest interest to biomedical engineering as it allows for effective fabrication of hollow tubes.     

Elastomeric Fibrous Hybrid Scaffold Supports In Vitro and In Vivo Tissue Formation

Biomimetic materials with biomechanical properties resembling those of native tissues while providing an environment for cell growth and tissue formation, are vital for tissue engineering (TE). Mechanical anisotropy is an important property of native cardiovascular tissues and directly influences tissue function. This study reports fabrication of anisotropic cell‐seeded constructs while retaining control over the construct's architecture and distribution of cells. Newly synthesized poly‐4‐hydroxybutyrate (P4HB) is fabricated with a dry spinning technique to create anelastomeric fibrous scaffold that allows control of fiber diameter, porosity, and rate ofdegradation. To allow cell and tissue ingrowth, hybrid scaffolds with mesenchymalstem cells (MSCs) encapsulated in a photocrosslinkable hydrogel were developed. Culturing the cellularized scaffolds in a cyclic stretch/flexure bioreactor resulted in tissue formation and confirmed the scaffold's performance under mechanical stimulation. In vivo experiments showed that the hybrid scaffold is capable of withstanding physiological pressures when implanted as a patch in the pulmonary artery. Aligned tissue formation occurred on the scaffold luminal surface without macroscopic thrombus formation. This combination of a novel, anisotropic fibrous scaffold and a tunable native‐like hydrogel for cellular encapsulation promoted formation of 3D tissue and provides a biologically functional composite scaffold for soft‐tissue engineering applications.     

3D‐Architected Soft Machines with Topologically Encoded Motion

The limited range of mechanical responses achievable by materials compatible with additive manufacturing hinders the 3D printing of continuum soft robots with programmed motion. This paper describes the rapid design and fabrication of low‐density, 3D‐architected soft machines (ASMs) by combining Voronoi tessellation and additive manufacturing. On tendon‐based actuation, ASMs deform according to the topologically encoded buckling of their structure to produce a wide range of motions (contraction, twisting, bending, and cyclic motion). ASMs exhibiting densities as low as 0.094 g cm^?3 (≈8% of bulk polymer) can be rapidly built by the stereolithographic 3D printing of flexible photopolymers or the injection molding of elastomers. The buckling of ASMs can be programmed by inducing gradients in the thickness of their flexible beams or by the localized enlargement of the Voronoi cells to generate complex motions such as multi‐finger gripping or quadrupedal locomotion. The topological architecture of these low‐density soft robots confers them with the stiffness necessary to recover their original shape even after ultrahigh compression (400%) and extension (500%). ASMs expand the range of mechanical properties currently achievable by 3D printed or molded materials to enable the fabrication of soft machines with auxetic mechanical metamaterial properties.