Hydrogel Bioinks Research Articles

Alginate-Lysozyme Nanofibers Hydrogels with Improved Rheological Behavior, Printability and Biological Properties for 3D Bioprinting Applications.

In this study, alginate nanocomposite hydrogel bioinks reinforced with lysozyme nanofibers (LNFs) were developed. Alginate-LNF (A-LNF) suspensions with different LNF contents (1, 5 and 10 wt.%) were prepared and pre-crosslinked with 0.5% (w/v) CaCl2 to formulate A-LNF inks. These inks exhibit proper shear-thinning behavior and good recovery properties (~90%), with the pre-crosslinking step playing a crucial role. A-LNF fully crosslinked hydrogels (with 2% (w/v) CaCl2) that mimic 3D printing scaffolds were prepared, and it was observed that the addition of LNFs improved several properties of the hydrogels, such as the morphology, swelling and degradation profiles, and mechanical properties. All formulations are also noncytotoxic towards HaCaT cells. The printing parameters and 3D scaffold model were then optimized, with A-LNF inks showing improved printability. Selected A-LNF inks (A-LNF0 and A-LNF5) were loaded with HaCaT cells (cell density 2 × 106 cells mL−1), and the cell viability within the bioprinted scaffolds was evaluated for 1, 3 and 7 days, with scaffolds printed with the A-LNF5 bioink showing the highest values for 7 days (87.99 ± 1.28%). Hence, A-LNF bioinks exhibited improved rheological performance, printability and biological properties representing a good strategy to overcome the main limitations of alginate-based bioinks.

Functionalizing multi-component bioink with platelet-rich plasma for customized in-situ bilayer bioprinting for wound healing.

In-situ three-dimensional (3D) bioprinting has been emerging as a promising technology designed to rapidly seal cutaneous defects according to their contour. Improvements in the formulations of multi-component bioink are needed to support cytocompatible encapsulation and biological functions. Platelet-rich plasma (PRP), as a source of patient-specific autologous growth factors, exhibits capabilities in tissue repair and rejuvenation. This study aimed to prepare PRP-integrated alginate-gelatin (AG) composite hydrogel bioinks and evaluate the biological effects in vitro and in vivo. 3D bioprinted constructs embedded with dermal fibroblasts and epidermal stem cells were fabricated using extrusion strategy. The integration of PRP not only improved the cellular behavior of seeded cells, but regulate the tube formation of vascular endothelial cells and macrophage polarization in a paracrine manner, which obtained an optimal effect at an incorporation concentration of 5%. For in-situ bioprinting, PRP integration accelerated the high-quality wound closure, modulated the inflammation and initiated the angiogenesis compared with the AG bioink. In conclusion, we revealed the regenerative potential of PRP, readily available at the bedside, as an initial signaling provider in multi-component bioink development. Combined with in-situ printing technology, it is expected to accelerate the clinical translation of rapid individualized wound repair.

A Guide to Polysaccharide-Based Hydrogel Bioinks for 3D Bioprinting Applications.

Three-dimensional (3D) bioprinting is an innovative technology in the biomedical field, allowing the fabrication of living constructs through an approach of layer-by-layer deposition of cell-laden inks, the so-called bioinks. An ideal bioink should possess proper mechanical, rheological, chemical, and biological characteristics to ensure high cell viability and the production of tissue constructs with dimensional stability and shape fidelity. Among the several types of bioinks, hydrogels are extremely appealing as they have many similarities with the extracellular matrix, providing a highly hydrated environment for cell proliferation and tunability in terms of mechanical and rheological properties. Hydrogels derived from natural polymers, and polysaccharides, in particular, are an excellent platform to mimic the extracellular matrix, given their low cytotoxicity, high hydrophilicity, and diversity of structures. In fact, polysaccharide-based hydrogels are trendy materials for 3D bioprinting since they are abundant and combine adequate physicochemical and biomimetic features for the development of novel bioinks. Thus, this review portrays the most relevant advances in polysaccharide-based hydrogel bioinks for 3D bioprinting, focusing on the last five years, with emphasis on their properties, advantages, and limitations, considering polysaccharide families classified according to their source, namely from seaweed, higher plants, microbial, and animal (particularly crustaceans) origin.

Manufacturing of self-standing multi-layered 3D-bioprinted alginate-hyaluronate constructs by controlling the cross-linking mechanisms for tissue engineering applications

Three-dimensional (3D) bioprinting of self-supporting stable tissue and organ structure is critically important in extrusion-based bioprinting system, especially for tissue engineering and regenerative medicine applications. However, the development of self-standing bioinks with desired crosslinking density, biocompatibility, tunable mechanical strength and other properties like self-healing, in situ gelation, drug or protein incorporation is still a challenge. In this study, we report a hydrogel bioink prepared from alginate (Alg) and hyaluronic acid (HA) crosslinked through multiple crosslinking mechanisms, i.e. acyl-hydrazone, hydrazide interactions and calcium ions. These Alg-HA gels were highly dynamic and shear-thinning with exceptional biocompatibility and tunable mechanical properties. The increased dynamic nature of the gels is mainly chemically attributed to the presence of acyl-hydrazone bonds formed between the amine groups of the acyl-hydrazide of alginate and the monoaldehyde of the HA. Among the different combinations of Alg-HA gel compositions prepared, the A5H5 (Alginate-acyl-hydrazide:HA-monoaldehyde, ratio 50:50) gel showed a gelation time of ∼60 s, viscosity of ∼400 Pa s (at zero shear rate), high stability in various pH solutions and increased degradation time (>50 days) than the other samples. The A5H5 gels showed high printability with increased post-printing stability as observed from the 3D printed structures (e.g. hollow tube (∼100 layers), porous cube (∼50 layers), star, heart-in, meniscus and lattice). The scanning electron microscopy analysis of the 3D constructs and hydrogels showed the interconnected pores (∼181 µm) and crosslinked networks. Further, the gels showed sustained release of 5-amino salicylic acid and bovine serum albumin. Also, the mechanical properties were tuned by secondary crosslinking via different calcium concentrations. In vitro assays confirmed the cytocompatibility of these gels, where the 3D bioprinted lattice and tubular (∼70 layers) constructs demonstrated high cell viability under fluorescence analysis. In in vivo studies, Alg-HA gel showed high biocompatibility (>90%) and increased angiogenesis (threefolds) and reduced macrophage infiltration (twofold decrease), demonstrating the promising potential of these hydrogels in 3D bioprinting applications for tissue engineering and regenerative medicine with tunable properties.

Preparation and Characterization of Graphene-Based 3D Biohybrid Hydrogel Bioink for Peripheral Neuroengineering.

Peripheral neuropathies can occur as a result of axonal damage, and occasionally due to demyelinating diseases. Peripheral nerve damage is a global problem that occurs in 1.5%-5% of emergency patients and may lead to significant job losses. Today, tissue engineering-based approaches, consisting of scaffolds, appropriate cell lines, and biosignals, have become more applicable with the development of three-dimensional (3D) bioprinting technologies. The combination of various hydrogel biomaterials with stem cells, exosomes, or bio-signaling molecules is frequently studied to overcome the existing problems in peripheral nerve regeneration. Accordingly, the production of injectable systems, such as hydrogels, or implantable conduit structures formed by various bioprinting methods has gained importance in peripheral neuro-engineering. Under normal conditions, stem cells are the regenerative cells of the body, and their number and functions do not decrease with time to protect their populations; these are not specialized cells but can differentiate upon appropriate stimulation in response to injury. The stem cell system is under the influence of its microenvironment, called the stem cell niche. In peripheral nerve injuries, especially in neurotmesis, this microenvironment cannot be fully rescued even after surgically binding severed nerve endings together. The composite biomaterials and combined cellular therapies approach increases the functionality and applicability of materials in terms of various properties such as biodegradability, biocompatibility, and processability. Accordingly, this study aims to demonstrate the preparation and use of graphene-based biohybrid hydrogel patterning and to examine the differentiation efficiency of stem cells into nerve cells, which can be an effective solution in nerve regeneration.

Development of a mist-based printhead for droplet-based bioprinting of ionically crosslinking hydrogel bioinks

In this paper, a novel droplet-based printhead is developed for 3D bioprinting of ionically crosslinking hydrogel bioinks. Contrary to previous approaches to droplet-based bioprinting, many of which use a liquid crosslinking agent to construct scaffolds, the developed system delivers the crosslinker in mist form within the printhead. A mechanism is developed to remove excess mist within the printhead, which prevents scaffold instability caused by the accumulation of liquid on the printing stage. The developed printhead is compatible with commercial systems and offers good droplet gelation and co-droplet adhesion. It is shown that the gelation rate can be controlled by adjusting the delivery rate of the mist. As a result, it is shown that the delivery rate of the mist has an impact on the shape fidelity and mechanical properties of the printed constructs. The printability and swelling properties of printed constructs crosslinked using different mist delivery rates are studied. Moreover, the impacts of printing parameters including printhead height, mist outlet pressure and printhead channel dimensions on the mist distribution and therefore the gelation rate within the printhead is investigated. Additionally, using high-speed imaging, the effects of mist concentration and droplet velocity on the dynamics of droplet impact onto the surface is characterized. Finally, it is shown that the printed constructs using the developed printhead show a low level of cytotoxicity and high level of cell viability. This development advances the applicability of droplet-based bioprinting to construct complex and biocompatible scaffolds for tissue and organ regeneration.

Fabrication of SA/Gel/C scaffold with 3D bioprinting to generate micro-nano porosity structure for skin wound healing: a detailed animal in vivo study

Bioprinting has exhibited remarkable promises for the fabrication of functional skin substitutes. However, there are some significant challenges for the treatment of full-thickness skin defects in clinical practice. It is necessary to determine bioinks with suitable mechanical properties and desirable biocompatibilities. Additionally, the key for printing skin is to design the skin structure optimally, enabling the function of the skin. In this study, the full-thickness skin scaffolds were prepared with a gradient pore structure constructing the dense layer, epidermis, and dermis by different ratios of bioinks. We hypothesized that the dense layer protects the wound surface and maintains a moist environment on the wound surface. By developing a suitable hydrogel bioink formulation (sodium alginate/gelatin/collagen), to simulate the physiological structure of the skin via 3D printing, the proportion of hydrogels was optimized corresponding to each layer. These results reveal that the scaffold has interconnected macroscopic channels, and sodium alginate/gelatin/collagen scaffolds accelerated wound healing, reduced skin wound contraction, and re-epithelialization in vivo. It is expected to provide a rapid and economical production method of skin scaffolds for future clinical applications.

Design and Implementation of Anatomically Inspired Mesenteric and Intestinal Vascular Patterns for Personalized 3D Bioprinting

Recent progress in bioprinting has made possible the creation of complex 3D intestinal constructs, including vascularized villi. However, for their integration into functional units useful for experimentation or implantation, the next challenge is to endow them with a larger-scale, anatomically realistic vasculature. In general, the perfusion of bioprinted constructs has remained difficult, and the current solution is to provide them with mostly linear and simply branched channels. To address this limitation, here we demonstrated an image analysis-based workflow leading through computer-assisted design from anatomic images of rodent mesentery and colon to the actual printing of such patterns with paste and hydrogel bioinks. Moreover, we reverse-engineered the 2D intestinal image-derived designs into cylindrical objects, and 3D-printed them in a support hydrogel. These results open the path towards generation of more realistically vascularized tissue constructs for a variety of personalized medicine applications.

In Situ Crosslinkable Collagen-Based Hydrogels for 3D Printing of Dermis-Mimetic Constructs

Recently, several three-dimensional (3D) bioprinting techniques have emerged for the synthesis of 3D tissue analogs. Accordingly, many researchers have focused on the development of novel bioinks that can mimic the natural extracellular matrix with cytocompatibility and biofunctionality. Hyaluronic acid and collagen are the most abundant proteins in the extracellular matrix of the skin and are known to support several cellular behaviors. Herein, we developed tyramine-conjugated hyaluronic acid and collagen (HA-Tyr/Col-Tyr) hydrogel bioinks, which are photocrosslinkable in the presence of riboflavin and ammonium peroxydisulfate, to fabricate dermis-mimetic constructs. The physicochemical properties and 3D printability of the HA-Tyr/Col-Tyr hydrogel were examined. 3D printing of the lattice structure with the HA-Tyr/Col-Tyr hydrogel enabled a sophisticated micron-sized fine structure without any clogging or coagulation. Approximately 80% of normal human dermal fibroblasts (NHDFs) in the printed constructs were alive after 24 h of culture. Moreover, the 3D printed constructs supported 4.57-fold cell proliferation and 4.23-fold f-actin expansion over four days in culture, indicating that HA-Tyr/Col-Tyr hydrogels provide cytocompatible microenvironments. The findings of this study suggest that HA-Tyr/Col-Tyr hydrogels are promising candidates as bioinks for the 3D printing of dermis-mimetic constructs.

Three-Dimensional Printing Self-Healing Dynamic/Photocrosslinking Gelatin-Hyaluronic Acid Double-Network Hydrogel for Tissue Engineering.

Three-dimensional (3D) printing technology has great potential for constructing structurally and functionally complex scaffold materials for tissue engineering. Bio-inks are a critical part of 3D printing for this purpose. In this study, based on dynamic hydrazone-crosslinked hyaluronic acid (HA-HYD) and photocrosslinked gelatin methacrylate (GelMA), a double-network (DN) hydrogel with significantly enhanced mechanical strength, self-healing, and shear-thinning properties was developed as a printable hydrogel bio-ink for extrusion-based 3D printing. Owing to shear thinning, the DN hydrogel bio-inks could be extruded to form uniform filaments, which were printed layer by layer to fabricate the scaffolds. The self-healing performance of the filaments and photocrosslinking of GelMA worked together to obtain an integrated and stable printed structure with high mechanical strength. The in vitro cytocompatibility assay showed that the DN hydrogel printed scaffolds supported the survival and proliferation of bone marrow mesenchymal stem cells. GelMA/HA-HYD DN hydrogel bio-inks with printability, good structural integrity, and biocompatibility are promising materials for 3D printing of tissue engineering scaffolds.

3D Bioprinting of Heterogeneous Constructs Providing Tissue‐Specific Microenvironment Based on Host–Guest Modulated Dynamic Hydrogel Bioink for Osteochondral Regeneration

Abstract3D bioprinting is a promising strategy to develop heterogeneous constructs that mimic osteochondral tissue. However, conventional bioprinted hydrogels suffer from intrinsically weak mechanical strength, limited cell adaptability, and no sustained release of biochemical drugs, restraining their use as bioinks to emulate native osteochondral extracellular matrix. Herein, a novel host–guest modulated dynamic hydrogel is developed for 3D bioprinting heterogeneous cell‐laden constructs for osteochondral regeneration. Apart from gelatin methacryloyl (GelMA), this bioink consists of dopamine‐functionalized GelMA and acrylate β‐cyclodextrin and is crosslinked by host–guest interaction to develop the dynamic network for obtaining promoted cell adaptability, enhanced cell adhesion, reinforced mechanical strength, and tunable modulus. Moreover, based on the sustained drug release provided by the cavity of β‐cyclodextrin, a heterogeneous construct is constructed by employing kartogenin (a chondrogenic factor) into the upper zone with lower Young's modulus and melatonin (an osteogenic factor) into the bottom zone with higher modulus to mimic the osteochondral microenvironment. With the favorable regeneration results in vitro and in vivo, a broad application of this bioink in 3D bioprinting for tissues engineering is expected.

Review on Multicomponent Hydrogel Bioinks Based on Natural Biomaterials for Bioprinting 3D Liver Tissues.

Three-dimensional (3D)-printed in vitro tissue models have been used in various biomedical fields owing to numerous advantages such as enhancements in cell response and functionality. In liver tissue engineering, several studies have been reported using 3D-printed liver tissue models with improved cellular responses and functions in drug screening, liver disease, and liver regenerative medicine. However, the application of conventional single-component bioinks for the printing of 3D in vitro liver constructs remains problematic because of the complex structural and physiological characteristics of the liver. The use of multicomponent bioinks has become an attractive strategy for bioprinting 3D functional in vitro liver tissue models because of the various advantages of multicomponent bioinks, such as improved mechanical properties of the printed tissue construct and cell functionality. Therefore, it is essential to review various 3D bioprinting techniques and multicomponent hydrogel bioinks proposed for liver tissue engineering to suggest future directions for liver tissue engineering. Accordingly, we herein review multicomponent bioinks for 3D-bioprinted liver tissues. We first describe the fabrication methods capable of printing multicomponent bioinks and introduce considerations for bioprinting. We subsequently categorize and evaluate the materials typically utilized for multicomponent bioinks based on their characteristics. In addition, we also review recent studies for the application of multicomponent bioinks to fabricate in vitro liver tissue models. Finally, we discuss the limitations of current studies and emphasize aspects that must be resolved to enhance the future applicability of such bioinks.

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.

Sustained released of bioactive mesenchymal stromal cell-derived extracellular vesicles from 3D-printed gelatin methacrylate hydrogels.

Extracellular vesicles (EVs) represent an emerging class of therapeutics with significant potential and broad applicability. However, a general limitation is their rapid clearance after administration. Thus, methods to enable sustained EV release are of great potential value. Here, we demonstrate that EVs from mesenchymal stem/stromal cells (MSCs) can be incorporated into 3D-printed gelatin methacrylate (GelMA) hydrogel bioink, and that the initial burst release of EVs can be reduced by increasing the concentration of crosslinker during gelation. Further, the data show that MSC EV bioactivity in an endothelial gap closure assay is retained after the 3D printing and photocrosslinking processes. Our group previously showed that MSC EV bioactivity in this assay correlates with pro-angiogenic bioactivity in vivo, thus these results indicate the therapeutic potential of MSC EV-laden GelMA bioinks.

Click Chemistry Hydrogels for Extrusion Bioprinting: Progress, Challenges, and Opportunities.

The emergence of 3D bioprinting has allowed a variety of hydrogel-based "bioinks" to be printed in the presence of cells to create precisely defined cell-loaded 3D scaffolds in a single step for advancing tissue engineering and/or regenerative medicine. While existing bioinks based primarily on ionic cross-linking, photo-cross-linking, or thermogelation have significantly advanced the field, they offer technical limitations in terms of the mechanics, degradation rates, and the cell viabilities achievable with the printed scaffolds, particularly in terms of aiming to match the wide range of mechanics and cellular microenvironments. Click chemistry offers an appealing solution to this challenge given that proper selection of the chemistry can enable precise tuning of both the gelation rate and the degradation rate, both key to successful tissue regeneration; simultaneously, the often bio-orthogonal nature of click chemistry is beneficial to maintain high cell viabilities within the scaffolds. However, to date, relatively few examples of 3D-printed click chemistry hydrogels have been reported, mostly due to the technical challenges of controlling mixing during the printing process to generate high-fidelity prints without clogging the printer. This review aims to showcase existing cross-linking modalities, characterize the advantages and disadvantages of different click chemistries reported, highlight current examples of click chemistry hydrogel bioinks, and discuss the design of mixing strategies to enable effective 3D extrusion bioprinting of click hydrogels.

Stem cell-laden hydrogel bioink for generation of high resolution and fidelity engineered tissues with complex geometries

Recently, 3D bioprinting has been explored as a promising technology for biomedical applications with the potential to create complex structures with precise features. Cell encapsulated hydrogels composed of materials such as gelatin, collagen, hyaluronic acid, alginate and polyethylene glycol have been widely used as bioinks for 3D bioprinting. However, since most hydrogel-based bioinks may not allow rapid stabilization immediately after 3D bioprinting, achieving high resolution and fidelity to the intended architecture is a common challenge in 3D bioprinting of hydrogels. In this study, we have utilized shear-thinning and self-healing ionically crosslinked oxidized and methacrylated alginates (OMAs) as a bioink, which can be rapidly gelled by its self-healing property after bioprinting and further stabilized via secondary crosslinking. It was successfully demonstrated that stem cell-laden calcium-crosslinked OMA hydrogels can be bioprinted into complicated 3D tissue structures with both high resolution and fidelity. Additional photocrosslinking enables long-term culture of 3D bioprinted constructs for formation of functional tissue by differentiation of encapsulated human mesenchymal stem cells.

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.

Advanced Strategies for 3D Bioprinting of Tissue and Organ Analogs Using Alginate Hydrogel Bioinks.

Alginate is a natural polysaccharide that typically originates from various species of algae. Due to its low cost, good biocompatibility, and rapid ionic gelation, the alginate hydrogel has become a good option of bioink source for 3D bioprinting. However, the lack of cell adhesive moieties, erratic biodegradability, and poor printability are the critical limitations of alginate hydrogel bioink. This review discusses the pivotal properties of alginate hydrogel as a bioink for 3D bioprinting technologies. Afterward, a variety of advanced material formulations and biofabrication strategies that have recently been developed to overcome the drawbacks of alginate hydrogel bioink will be focused on. In addition, the applications of these advanced solutions for 3D bioprinting of tissue/organ mimicries such as regenerative implants and in vitro tissue models using alginate-based bioink will be systematically summarized.

Tricomposite gelatin-carboxymethylcellulose-alginate bioink for direct and indirect 3D printing of human knee meniscal scaffold

The development of technologies that could ease the production of customizable patient-specific tissue engineering constructs having required biomechanical properties and restoring function in damaged tissue is the need of the hour. In this study, we report the optimization of composite, bioactive and biocompatible tripolymeric hydrogel bioink, suitable for both direct and indirect printing of customizable scaffolds for cartilage tissue engineering applications. A customized hierarchical meniscal scaffold was designed using solid works software and developed using a negative mould made of polylactic acid (PLA) filament and by a direct 3D printing process. A composite tripolymeric bioink made of gelatin, carboxymethyl cellulose (CMC) and alginate was optimized and characterized for its printability, structural, bio-mechanical and bio-functional properties. The optimized composite hydrogel bioink was extruded into the negative mould with and without live cells, cross-linked and the replica of meniscus structure was retrieved aseptically. The cellular proliferation, apatite formation, and extracellular matrix secretion from negative printed meniscal scaffold were determined using MTT, live/dead and collagen estimation assays. A significant increase in collagen secretion, cellular proliferation and changes in biomechanical properties was observed in the 3D scaffolds with MG63-osteosarcoma cells indicating its suitability for cartilage tissue engineering.

Slide-Ring Structure-Based Double-Network Hydrogel with Enhanced Stretchability and Toughness for 3D-Bio-Printing and Its Potential Application as Artificial Small-Diameter Blood Vessels.

Artificial small-diameter blood vessels (SDBVs) are extremely limited in their thrombosis and still present significant clinical challenges worldwide. In recent years, 3D-bio-printing has offered a powerful technique to fabricate vessel channels in tissue engineering applications. Hydrogels are attractive bio-inks for SDBVs 3D-bio-printing, but they usually present weak mechanical properties. To overcome the weak mechanical properties of hydrogel bio-inks, a printable human umbilical vein endothelial cell (HUVEC)-laden polyrotaxane-alginate (PR-Alg) double-network (DN) hydrogel was fabricated. The PR-Alg DN hydrogel consists of a Ca2+ cross-linked alginate network to form the first network rapidly, and a photo-cross-linked slide-ring network was designed as the second network. By combining special hydrogel structures of slide-ring (SR) and double network (DN), we significantly improved the mechanical properties of hydrogels. The PR-Alg DN hydrogel provides excellent stress (199 ± 20 kPa) and strain (1239 ± 58%), and the fracture energy reaches 668 ± 80 J/m2. Additionally, due to the presence of biocompatible materials and the gentle 3D-bio-printing process, the 3D-bio-printed channels showed outstanding biocompatibility, particularly in HUVECs' survival and proliferation. We anticipate that this work will expand the application of hydrogels with improved mechanical properties in biomedicine, particularly for artificial SDBVs.