Reactive oxygen/nitrogen species scavenging and inflammatory regulation by renal-targeted bio-inspired rhodium nanozymes for acute kidney injury theranostics.

Acute kidney injury (AKI) results from the rapid deterioration of renal function, which is mainly treated by transplantation and dialysis, and has a high mortality rate. Inflammation induced by excess reactive oxygen/nitrogen species (RONS) plays a crucial role in AKI. Although small molecule antioxidants have been utilized to alleviate AKI, low bioavailability and side-effect of these drugs tremendously limit their clinical use. Hence, we successfully construct ultra-small (2-4 nm) rhodium nanoparticles modified with l-serine (denoted as Rh-Ser). Our results show that Rh-Ser with multiple enzyme-mimicking activities, allows remove various RONS to protect damaged kidney cells. Additionally, the ultrasmall size of Rh-Ser is conducive to enrichment in the renal tubules, and the modification of l-serine enables Rh-Ser to bind to kidney injury molecule-1, which is highly expressed on the surface of damaged renal cells, thereby targeting the damaged kidney and increasing the retention time. Moreover, Rh-Ser allows the production of oxygen at the inflammatory site, thus further improving hypoxia and inhibiting pro-inflammatory macrophages to relieve inflammation, and increasing the survival rate of AKI mice from 0 to 80%, which exhibits a better therapeutic effect than that of small molecule drug. Photoacoustic and fluorescence imaging can effectively monitor and evaluate the enrichment and therapeutic effect of Rh-Ser. Our study provides a promising strategy for the targeted treatment of AKI via RONS scavenging and inflammatory regulation.

Rapid Volumetric Bioprinting of Decellularized Extracellular Matrix Bioinks.

Decellularized extracellular matrix (dECM)-based hydrogels are widely applied to additive biomanufacturing strategies for relevant applications. The extracellular matrix components and growth factors of dECM play crucial roles in cell adhesion, growth, and differentiation. However, the generally poor mechanical properties and printability have remained as major limitations for dECM-based materials. In this study, heart-derived dECM (h-dECM) and meniscus-derived dECM (Ms-dECM) bioinks in their pristine, unmodified state supplemented with the photoinitiator system of tris(2,2-bipyridyl) dichlororuthenium(II) hexahydrate and sodium persulfate, demonstrate cytocompatibility with volumetric bioprinting processes. This recently developed bioprinting modality illuminates a dynamically evolving light pattern into a rotating volume of the bioink, and thus decouples the requirement of mechanical strengths of bioprinted hydrogel constructs with printability, allowing for the fabrication of sophisticated shapes and architectures with low-concentration dECM materials that set within tens of seconds. As exemplary applications, cardiac tissues are volumetrically bioprinted using the cardiomyocyte-laden h-dECM bioink showing favorable cell proliferation, expansion, spreading, biomarker expressions, and synchronized contractions; whereas the volumetrically bioprinted Ms-dECM meniscus structures embedded with human mesenchymal stem cells present appropriate chondrogenic differentiation outcomes. This study supplies expanded bioink libraries for volumetric bioprinting and broadens utilities of dECM toward tissue engineering and regenerative medicine.

Advances in volumetric bioprinting.

The three-dimensional (3D) bioprinting technologies are suitable for biomedical applications owing to their ability to manufacture complex and high-precision tissue constructs. However, the slow printing speed of current layer-by-layer (bio)printing modality is the major limitation in biofabrication field. To overcome this issue, volumetric bioprinting (VBP) is developed. VBP changes the layer-wise operation of conventional devices, permitting the creation of geometrically complex, centimeter-scale constructs in tens of seconds. VBP is the next step onward from sequential biofabrication methods, opening new avenues for fast additive manufacturing in the fields of tissue engineering, regenerative medicine, personalized drug testing, and soft robotics, etc. Therefore, this review introduces the printing principles and hardware designs of VBP-based techniques; then focuses on the recent advances in VBP-based (bio)inks and their biomedical applications. Lastly, the current limitations of VBP are discussed together with future direction of research.

A 3D Bioprinted Gut Anaerobic Model for Studying Bacteria-Host Interactions.

The role of the human intestinal tract in host-microbe interactions has been highlighted in recent years. Several 3-dimensional (3D) models have been developed to reproduce the physiological characteristics of the human gut and to investigate the function of the gut microbiota. One challenge for 3D models is to recapitulate the low oxygen concentrations in the intestinal lumen. Moreover, most earlier 3D culture systems used a membrane to physically separate bacteria from the intestinal epithelium, which has sometimes made the studies of bacteria adhering to or invading cells less feasible. We report the establishment of a 3D gut epithelium model and cultured it at high cell viability under an anaerobic condition. We further cocultured intestinal bacteria including both commensal and pathogen directly with epithelial cells in the established 3D model under the anaerobic condition. We subsequently compared the gene expression differences of aerobic and anaerobic conditions for cell and bacterial growth via dual RNA sequencing. Our study provides a physiologically relevant 3D gut epithelium model that mimics the anaerobic condition in the intestinal lumen and supplies a powerful system for future in-depth gut-microbe interactional investigations.

Liquid-embedded (bio)printing of alginate-free, standalone, ultrafine, and ultrathin-walled cannular structures.

While there has been considerable success in the three-dimensional bioprinting of relatively large standalone filamentous tissues, the fabrication of solid fibers with ultrafine diameters or those cannular featuring ultrathin walls remains a particular challenge. Here, an enabling strategy for (bio)printing of solid and hollow fibers whose size ranges could be facilely adjusted across a broad spectrum, is reported, using an aqueous two-phase embedded (bio)printing approach combined with specially designed cross-linking and extrusion methods. The generation of standalone, alginate-free aqueous architectures using this aqueous two-phase strategy allowed freeform patterning of aqueous bioinks, such as those composed of gelatin methacryloyl, within the immiscible aqueous support bath of poly(ethylene oxide). Our (bio)printing strategy revealed the fabrication of standalone solid or cannular structures with diameters as small as approximately 3 or 40 µm, respectively, and wall thicknesses of hollow conduits down to as thin as <5 µm. With cellular functions also demonstrated, we anticipate the methodology to serve as a platform that may satisfy the needs for the different types of potential biomedical and other applications in the future, especially those pertaining to cannular tissues of ultrasmall diameters and ultrathin walls used toward regenerative medicine and tissue model engineering.

Volumetric additive manufacturing of pristine silk-based (bio)inks.

Volumetric additive manufacturing (VAM) enables fast photopolymerization of three-dimensional constructs by illuminating dynamically evolving light patterns in the entire build volume. However, the lack of bioinks suitable for VAM is a critical limitation. This study reports rapid volumetric (bio)printing of pristine, unmodified silk-based (silk sericin (SS) and silk fibroin (SF)) (bio)inks to form sophisticated shapes and architectures. Of interest, combined with post-fabrication processing, the (bio)printed SS constructs reveal properties including reversible as well as repeated shrinkage and expansion, or shape-memory; whereas the (bio)printed SF constructs exhibit tunable mechanical performances ranging from a few hundred Pa to hundreds of MPa. Both types of silk-based (bio)inks are cytocompatible. This work supplies expanded bioink libraries for VAM and provides a path forward for rapid volumetric manufacturing of silk constructs, towards broadened biomedical applications.

An Adhesive Bioink toward Biofabrication under Wet Conditions.

Three-dimensional (3D) bioprinting is driving significant innovations in biomedicine over recent years. Under certain scenarios such as in intraoperative bioprinting, the bioinks used should exhibit not only cyto/biocompatibility but also adhesiveness in wet conditions. Herein, an adhesive bioink composed of gelatin methacryloyl, gelatin, methacrylated hyaluronic acid, and skin secretion of Andrias davidianus is designed. The bioink exhibits favorable cohesion to allow faithful extrusion bioprinting in wet conditions, while simultaneously showing good adhesion to a variety of surfaces of different chemical properties, possibly achieved through the diverse bonds presented in the bioink formulation. As such, this bioink is able to fabricate sophisticated planar and volumetric constructs using extrusion bioprinting, where the dexterity is further enhanced using ergonomic handheld bioprinters to realize in situ bioprinting. In vitro experiments reveal that cells maintain high viability; further in vivo studies demonstrate good integration and immediate injury sealing. The characteristics of the bioink indicate its potential widespread utility in extrusion bioprinting and will likely broaden the applications of bioprinting toward situations such as in situ dressing and minimally invasive tissue regeneration.

Nanofiber Aerogels with Precision Macrochannels and LL-37-Mimic Peptides Synergistically Promote Diabetic Wound Healing.

Fast healing of diabetic wounds remains a major clinical challenge. Herein, this work reports a strategy to combine nanofiber aerogels containing precision macrochannels and the LL-37-mimic peptide W379 for rapid diabetic wound healing. Nanofiber aerogels consisting of poly(glycolide-co-lactide) (PGLA 90:10)/gelatin and poly-p-dioxanone (PDO)/gelatin short electrospun fiber segments were prepared by partially anisotropic freeze-drying, crosslinking, and sacrificial templating with three-dimensional (3D)-printed meshes, exhibiting nanofibrous architecture and precision micro-/macrochannels. Like human cathelicidin LL-37, W379 peptide at a concentration of 3 µg/mL enhanced the migration and proliferation of keratinocytes and dermal fibroblasts in a cell scratch assay and a proliferation assay. In vivo studies show that nanofiber aerogels with precision macrochannels can greatly promote cell penetration compared to aerogels without macrochannels. Relative to control and aerogels with and without macrochannels, adding W379 peptides to aerogels with precision macrochannels shows the best efficacy in healing diabetic wounds in mice in terms of cell infiltration, neovascularization, and re-epithelialization. The fast re-epithelization could be due to upregulation of phospho-extracellular signal-regulated kinase (p38 MAPK) after treatment with W379. Together, the approach developed in this work could be promising for the treatment of diabetic wounds and other chronic wounds.

Microfluidic bioprinting of tough hydrogel-based vascular conduits for functional blood vessels.

Three-dimensional (3D) bioprinting of vascular tissues that are mechanically and functionally comparable to their native counterparts is an unmet challenge. Here, we developed a tough double-network hydrogel (bio)ink for microfluidic (bio)printing of mono- and dual-layered hollow conduits to recreate vein- and artery-like tissues, respectively. The tough hydrogel consisted of energy-dissipative ionically cross-linked alginate and elastic enzyme-cross-linked gelatin. The 3D bioprinted venous and arterial conduits exhibited key functionalities of respective vessels including relevant mechanical properties, perfusability, barrier performance, expressions of specific markers, and susceptibility to severe acute respiratory syndrome coronavirus 2 pseudo-viral infection. Notably, the arterial conduits revealed physiological vasoconstriction and vasodilatation responses. We further explored the feasibility of these conduits for vascular anastomosis. Together, our study presents biofabrication of mechanically and functionally relevant vascular conduits, showcasing their potentials as vascular models for disease studies in vitro and as grafts for vascular surgeries in vivo, possibly serving broad biomedical applications in the future.

Freeform Cell-Laden Cryobioprinting for Shelf-Ready Tissue Fabrication and Storage.

One significant drawback of existing bioprinted tissues is their lack of shelf-availability caused by complications in both fabrication and storage. Here, we report a cryobioprinting strategy for simultaneously fabricating and storing cell-laden volumetric tissue constructs through seamlessly combining extrusion bioprinting and cryopreservation. The cryobioprinting performance was investigated by designing, fabricating, and storing cell-laden constructs made of our optimized cryoprotective gelatin-based bioinks using a freezing plate with precisely controllable temperature. The in situ freezing process further promoted the printability of cell-laden hydrogel bioinks to achieve freeform structures otherwise inconvenient with direct extrusion bioprinting. The effects of bioink composition on printability and cell viability were evaluated. The functionality of the method was finally investigated using cell differentiation and chick ex ovo assays. The results confirmed the feasibility and efficacy of cryobioprinting as a single-step method for concurrent tissue biofabrication and storage.

Molecularly cleavable bioinks facilitate high-performance digital light processing-based bioprinting of functional volumetric soft tissues.

Digital light processing bioprinting favors biofabrication of tissues with improved structural complexity. However, soft-tissue fabrication with this method remains a challenge to balance the physical performances of the bioinks for high-fidelity bioprinting and suitable microenvironments for the encapsulated cells to thrive. Here, we propose a molecular cleavage approach, where hyaluronic acid methacrylate (HAMA) is mixed with gelatin methacryloyl to achieve high-performance bioprinting, followed by selectively enzymatic digestion of HAMA, resulting in tissue-matching mechanical properties without losing the structural complexity and fidelity. Our method allows cellular morphological and functional improvements across multiple bioprinted tissue types featuring a wide range of mechanical stiffness, from the muscles to the brain, the softest organ of the human body. This platform endows us to biofabricate mechanically precisely tunable constructs to meet the biological function requirements of target tissues, potentially paving the way for broad applications in tissue and tissue model engineering.

A multifunctional micropore-forming bioink with enhanced anti-bacterial and anti-inflammatory properties.

Three-dimensional (3D) bioprinting has emerged as an enabling tool for various biomedical applications, such as tissue regeneration and tissue model engineering. To this end, the development of bioinks with multiple functions plays a crucial role in the applications of 3D bioprinting technologies. In this study, we propose a new bioink based on two immiscible aqueous phases of gelatin methacryloyl (GelMA) and dextran, further endowed with anti-bacterial and anti-inflammatory properties. This micropore-forming GelMA-dextran (PGelDex) bioink exhibited excellent printability with vat-polymerization, extrusion, and handheld bioprinting methods. The porous structure was confirmed after bioprinting, which promoted the spreading of the encapsulated cells, exhibiting the exceptional cytocompatibility of this bioink formulation. To extend the applications of such a micropore-forming bioink, interleukin-4 (IL-4)-loaded silver-coated gold nanorods (AgGNRs) and human mesenchymal stem cells (MSCs) were simultaneously incorporated, to display synergistic anti-infection behavior and immunomodulatory function. The results revealed the anti-bacterial properties of the AgGNR-loaded PGelDex bioink for both Gram-negative and Gram-positive bacteria. The data also indicated that the presence of IL-4 and MSCs facilitated macrophage M2-phenotype differentiation, suggesting the potential anti-inflammatory feature of the bioink. Overall, this unique anti-bacterial and immunomodulatory micropore-forming bioink offers an effective strategy for the inhibition of bacterial-induced infections as well as the ability of immune-regulation, which is a promising candidate for broadened tissue bioprinting applications.

Digital Light Processing Based Bioprinting with Composable Gradients.

Recapitulation of complex tissues signifies a remarkable challenge and, to date, only a few approaches have emerged that can efficiently reconstruct necessary gradients in 3D constructs. This is true even though mimicry of these gradients is of great importance to establish the functionality of engineered tissues and devices. Here, a composable-gradient Digital Light Processing (DLP)-based (bio)printing system is developed, utilizing the unprecedented integration of a microfluidic mixer for the generation of either continual or discrete gradients of desired (bio)inks in real time. Notably, the precisely controlled gradients are composable on-the-fly by facilely by adjusting the (bio)ink flow ratios. In addition, this setup is designed in such a way that (bio)ink waste is minimized when exchanging the gradient (bio)inks, further enhancing this time- and (bio)ink-saving strategy. Various planar and 3D structures exhibiting continual gradients of materials, of cell densities, of growth factor concentrations, of hydrogel stiffness, and of porosities in horizontal and/or vertical direction, are exemplified. The composable fabrication of multifunctional gradients strongly supports the potential of the unique bioprinting system in numerous biomedical applications.

Vertical Extrusion Cryo(bio)printing for Anisotropic Tissue Manufacturing.

Due to the poor mechanical properties of many hydrogel bioinks, conventional 3D extrusion bioprinting is usually conducted based on the X-Y plane, where the deposited layers are stacked in the Z-direction with or without the support of prior layers. Herein, a technique is reported, taking advantage of a cryoprotective bioink to enable direct extrusion bioprinting in the vertical direction in the presence of cells, using a freezing plate with precise temperature control. Of interest, vertical 3D cryo-bioprinting concurrently allows the user to create freestanding filamentous constructs containing interconnected, anisotropic microchannels featuring gradient sizes aligned in the vertical direction, also associated with enhanced mechanical performances. Skeletal myoblasts within the 3D-cryo-bioprinted hydrogel constructs show enhanced cell viability, spreading, and alignment, compared to the same cells in the standard hydrogel constructs. This method is further extended to a multimaterial format, finding potential applications in interface tissue engineering, such as creation of the muscle-tendon unit and the muscle-microvascular unit. The unique vertical 3D cryo-bioprinting technique presented here suggests improvements in robustness and versatility to engineer certain tissue types especially those anisotropic in nature, and may extend broad utilities in tissue engineering, regenerative medicine, drug discovery, and personalized therapeutics.

Uniaxial and Coaxial Vertical Embedded Extrusion Bioprinting.

The 3D bioprinting technologies have attracted increasing attention due to their flexibility in producing architecturally relevant tissue constructs. Here, a vertical embedded extrusion bioprinting strategy using uniaxial or coaxial nozzles is presented, which allows formation of vertical structures of homogeneous or heterogeneous properties. By adjusting the bioprinting parameters, the characteristics of the bioprinted vertical patterns can be precisely controlled. Using this strategy, two proof-of-concept applications in tissue biofabrication are demonstrated. Specifically, intestinal villi and hair follicles, two liner-shaped tissues in the human body, are successfully generated with the vertical embedded bioprinting method, reconstructing some of their key structures as well as restoring partial functions in vitro. Caco-2 cells in the bioprinted intestinal villus constructs proliferated and aggregated properly, also showing functional biomarker expressions such as ZO-1 and villin. Moreover, preliminary hair follicle structures featuring keratinized human keratinocytes and spheroid-shaped human dermal papilla cells are formed after vertical bioprinting and culturing. In summary, this vertical embedded extrusion bioprinting technique harnessing a uniaxial or coaxial format will likely bring further improvements in the reconstruction of certain human tissues and organs, especially those with a linear structure, potentially leading to wide utilities in tissue engineering, tissue model engineering, and drug discovery.

Engineering (Bio)Materials through Shrinkage and Expansion.

Although various (bio)fabrication technologies have achieved revolutionary progress in the past decades, engineered constructs still fall short of expectations owing to their inability to attain precisely designable functions. Shrinkable and expandable (bio)materials feature unique characteristics leading to size-/shape-shifting and thus have exhibited a strong potential to equip current engineering technologies with promoted capacities toward applications in biomedicine. In this progress report, the advances of size-/shape-shifting (bio)materials enabled by various stimuli, are evaluated; furthermore, representative biomedical applications associated with size-/shape-shifting (bio)materials are also exemplified. Toward the future, the combination of size-/shape-shifting (bio)materials and 3D/4D fabrication technologies presents a wide range of possibilities for further development of intricate functional architectures.

Faithful Fabrication of Biocompatible Multicompartmental Memomicrospheres for Digitally Color-Tunable Barcoding.

Barcodes have attracted widespread attention, especially for the multiplexed bioassays and anti-counterfeiting used toward medical and biomedical applications. An enabling gas-shearing approach is presented for generating 10-faced microspherical barcodes with precise control over the properties of each compartment. As such, the color of each compartment could be programmatically adjusted in the 10-faced memomicrospheres by using pregel solutions containing different combinations of fluorescent nanoparticles. During the process, three primary colors (red, green, and blue) are adopted to obtain up to seven merged fluorescent colors for constituting a large amount of coding as well as a magnetic compartment, capable of effective and robust high-throughput information-storage. More importantly, by using the biocompatible sodium alginate to construct the multicolor microspherical barcodes, the proposed technology is likely to advance the fields of food and pharmaceutics anti-counterfeiting. These remarkable properties point to the potential value of gas-shearing in engineering microspherical barcodes for biomedical applications in the future.

Bioprinted Injectable Hierarchically Porous Gelatin Methacryloyl Hydrogel Constructs with Shape-Memory Properties.

Direct injection of cell-laden hydrogels shows high potentials in tissue regeneration for translational therapy. The traditional cell-laden hydrogels are often used as bulk space fillers to tissue defects after injection, likely limiting their structural controllability. On the other hand, patterned cell-laden hydrogel constructs often necessitate invasive surgical procedures. To overcome these problems, herein, we report a unique strategy for encapsulating living human cells in a pore-forming gelatin methacryloyl (GelMA)-based bioink to ultimately produce injectable hierarchically macro-micro-nanoporous cell-laden GelMA hydrogel constructs through three-dimensional (3D) extrusion bioprinting. The hydrogel constructs can be fabricated into various shapes and sizes that are defect-specific. Due to the hierarchically macro-micro-nanoporous structures, the cell-laden hydrogel constructs can readily recover to their original shapes, and sustain high cell viability, proliferation, spreading, and differentiation after compression and injection. Besides, in vivo studies further reveal that the hydrogel constructs can integrate well with the surrounding host tissues. These findings suggest that our unique 3D-bioprinted pore-forming GelMA hydrogel constructs are promising candidates for applications in minimally invasive tissue regeneration and cell therapy.

Gas-Shearing Fabrication of Multicompartmental Microspheres: A One-Step and Oil-Free Approach.

Multicompartmental microparticles (MCMs) have attracted considerable attention in biomedical engineering and materials sciences, as they can carry multiple materials in the separated phases of a single particle. However, the robust fabrication of monodisperse, highly compartmental MCMs at the micro- and nanoscales remains challenging. Here, a simple one-step and oil-free process, based on the gas-flow-assisted formation of microdroplets ("gas-shearing"), is established for the scalable production of monodisperse MCMs. By changing the configuration of the needle system and gas flow in the spray ejector device, the oil-free gas-shearing process easily allows the design of microparticles consisting of two, four, six, and even eight compartments with a precise control over the properties of each compartment. As oils and surfactants are not used, the gas-shearing method is highly cytocompatible. The versatile applications of such MCMs are demonstrated by producing a magnetic microrobot and a biocompatible carrier for the coculturing of cells. This research suggests that the oil-free gas-shearing strategy is a reliable, scalable, and biofriendly process for producing MCMs that may become attractive materials for biomedical applications.