3D Hydrogel Research Articles

Hydrogel Ink for 3D Printing with High and Widely Tunable Mechanical Properties via the Salting‐Out Effect

AbstractDue to the difficulty of simultaneously ensuring printability and controlling their mechanical properties, 3D printed hydrogels thus far have low modulus, far less than that of many biological tissues and organs. Therefore, their feasibility toward various biomimetic applications is currently limited. Herein, Direct‐Ink‐Writing (DIW) based 3D printable hydrogel ink with broadly adjustable mechanical properties is introduced. The salting‐out effect is utilized to effectively lower the gelation temperature of hydrogel inks with a wide range of modulus (0.193–1.072 MPa), making them all 3D printable. As a proof of concept, multi‐layered synthetic blood vessels that mimic the mechanical properties of biological blood vessels are printed, and their practicability toward surgical training is demonstrated. The versatility and simplicity of this technique make it highly promising for use in various 3D‐printed hydrogel biomaterials applications.

Efficient Generation of Skin Organoids from Pluripotent Cells via Defined Extracellular Matrix Cues and Morphogen Gradients in a Spindle-Shaped Microfluidic Device.

Pluripotent stem cell-derived skin organoids (PSOs) emerge as a developmental skin model that is self-organized into multiple components, such as hair follicles. Despite their impressive complexity, PSOs are currently generated in the absence of 3D extracellular matrix (ECM) signals and have several major limitations, including an inverted anatomy (e.g., epidermis inside/dermis outside). In this work, a method is established to generate PSOs effectively in a chemically-defined 3D ECM environment. After examining various dermal ECM molecules, it is found that PSOs generated in collagen-type I (COLI) supplemented with laminin 511 (LAM511) exhibit increased growth compared to conventional free-floating conditions, but fail to induce complete skin differentiation due in part to necrosis. This problem is addressed by generating the PSOs in a 3D bioprinted spindle-shaped hydrogel device, which constrains organoid growth longitudinally. This culture system significantly reduces organoid necrosis and leads to a twofold increase in keratinocyte differentiation and an eightfold increase in hair follicle formation. Finally, the system is adapted as a microfluidic device to create asymmetrical gradients of differentiation factors and improves the spatial organization of dermal and epidermal cells. This study highlights the pivotal role of ECM and morphogen gradients in promoting and spatially-controlling skin differentiation in the PSO framework.

Development and validation of 3-dimensional simulators for penile prosthesis surgery.

The acquisition of skills in penile prosthesis surgery has many limitations mainly due to the absence of simulators and models for training. Three-dimensional (3D) printed models can be utilized for surgical simulations, as they provide an opportunity to practice before entering the operating room and provide better understanding of the surgical approach. This study aimed to evaluate and validate a 3D model of human male genitalia for penile prosthesis surgery. This study included 3 evaluation and validation stages. The first stage involved verification of the 3D prototype model for anatomic landmarks compared with a cadaveric pelvis. The second stage involved validation of the improved model for anatomic accuracy and teaching purposes with the Rochester evaluation score. The third stage comprised validation of the suitability of the 3D prototype model as a surgical simulator and for skill acquisition. The third stage was performed at 3 centers using a modified version of a pre-existing, validated questionnaire and correlated with the Rochester evaluation score. We sought to determine the suitability of 3D model for training in penile prosthesis surgery in comparison with the available cadaveric model. The evaluation revealed a high Pearson correlation coefficient (0.86) between questions of the Rochester evaluation score and modified validated questionnaire. The 3D model scored 4.33 ± 0.57 (on a Likert scale from 1 to 5) regarding replication of the relevant human anatomy for the penile prosthesis surgery procedure. The 3D model scored 4.33 ± 0.57 (on a Likert scale from 1 to 5) regarding its ability to improve technical skills, teach and practice the procedure, and assess a surgeon's ability. Furthermore, the experts stated that compared with the cadaver, the 3D model presented greater ethical suitability, reduced costs, and easier accessibility. A validated 3D model is a suitable alternative for penile prosthesis surgery training. This is the first validated 3D hydrogel model for penile prosthesis surgery teaching and training that experts consider suitable for skill acquisition. Because specific validated guidelines and questionnaires for the validation and verifications of 3D simulators for penile surgery are not available, a modified questionnaire was used. The current 3D model for penile prosthesis surgery shows promising results regarding anatomic properties and suitability to train surgeons to perform penile implant surgery. The possibility of having an ethical, easy-to-use model with lower costs and limited consequences for the environment is encouraging for further development of the models.

Study of chondrogenesis of umbilical cord mesenchymal stem cells in curdlan- poly(vinyl alcohol) composite hydrogels and its mechanical properties of freezing-thawing treatments

The curdlan gel is a natural material produced by bacteria. It utilizes chemical cross-linking reactions to form a 3D porous composite hydrogel, increasing its porosity and water content, and improving its mechanical properties. It can be used in tissue repair and regenerative medicine. Curdlan-Poly(vinyl alcohol) (PVA) composite hydrogel can rapidly swell within 1 min due to its porous structure. Compression tests confirmed that it still maintains its original mechanical strength, even after five repeated freeze-thaw (FT) processes, making it suitable for long-term cryopreservation. The purpose of this study is to transplant umbilical cord mesenchymal stem cells (UC-MSCs) on Curdlan-PVA composite hydrogel and observe the chondrocytes on the material. The results of using 4′,6-diamidino-2-phenylindole (DAPI), hematoxylin and eosin (H&E), calcein-acetoxymethyl ester (calcein AM), and Collagen type II–Fluorescein isothiocyanate (FITC) staining, confirmed that UC-MSCs can attach and differentiate into chondrocytes on 3D Curdlan-PVA composite hydrogel.

Chitin nanofibrils assisted 3D printing all-chitin hydrogels for wound dressing

The direct ink writing technique used in 3D printing technology is generally applied to designing biomedical hydrogels. Herein, we proposed a strategy for preparing all-chitin-based inks for wound dressing via direct ink writing technique. The β-chitin nanofibers (MACNF) with a high aspect ratio were applied as a nanofiller to modulate the rheological properties of the alkaline dissolved chitin solution. The printing fidelity significantly depends on the MACNF introduction amount to the composite ink. 5–10 wt% MACNF ratio showed superior printing performance. The printed scaffold showed a uniform micron-sized pore structure and a woven network of nanofibers. Due to the good biocompatibility of chitin and the stereoscopic spatial skeleton, this scaffold showed excellent performance as a wound dressing, which can promote cell proliferation, collagen deposition and the angiogenesis of wounds, demonstrating its potential in biomedical applications. This approach successfully balanced the chitinous printability and biofunctions.

Clay Sculpture-Inspired 3D Printed Microcage Module Using Bioadhesion Assembly for Specific-Shaped Tissue Vascularization and Regeneration.

3D bioprinting techniques have enabled the fabrication of irregular large-sized tissue engineering scaffolds. However, complicated customized designs increase the medical burden. Meanwhile, the integrated printing process hinders the cellular uniform distribution and local angiogenesis. A novel approach is introduced to the construction of sizable tissue engineering grafts by employing hydrogel 3D printing for modular bioadhesion assembly, and a poly (ethylene glycol) diacrylate (PEGDA)-gelatin-dopamine (PGD) hydrogel, photosensitive and adhesive, enabling fine microcage module fabrication via DLP 3D printing is developed. The PGD hydrogel printed micocages are flexible, allowing various shapes and cell/tissue fillings for repairing diverse irregular tissue defects. In vivo experiments demonstrate robust vascularization and superior graft survival in nude mice. This assembly strategy based on scalable 3D printed hydrogel microcage module could simplify the construction of tissue with large volume and complex components, offering promise for diverse large tissue defect repairs.

Preclustering Gelatin for Faster-Forming Injectable Hydrogels: A Strategy for Fabricating 3D Hydrogel Scaffolds with Improved Cell Dispersion.

Gelatin-based injectable hydrogels capable of encapsulating cells are pivotal in tissue engineering because they can conform to any geometry and exhibit physical properties similar to those of living tissues. However, the slow gelation process observed in these cell-encapsulating hydrogels often causes an uneven dispersion of cells. This study proposes an approach for achieving fast gelation of unmodified gelatin under physiological conditions through gelatin preclustering. By using tetrafunctional succinimidyl-terminated poly(ethylene glycol) as a clustering agent, the gelation process is successfully expedited fivefold without requiring chemical modifications, effectively addressing the associated challenges of uneven cell distribution.

Localized compaction of collagen hydrogels using acoustic droplet vaporization promotes osteogenic differentiation of mesenchymal stem cells

The lineage specification of mesenchymal stem cells (MSCs) is dependent on matrix stiffness, with osteogenesis promoted on or within stiff microenvironments. Unlike native extracellular matrix, conventional hydrogels have relatively static and uniform mechanical properties, thereby limiting the study of how spatiotemporally controlled mechanical cues impact MSC behavior. Here, we spatiotemporally control MSC differentiation in 3D hydrogels by actively modulating matrix stiffness using acoustic droplet vaporization (ADV). An acoustically responsive scaffold (ARS) was generated by co-encapsulating MSCs and perfluorohexane-based emulsion (13 μm) within type I collagen. ADV was used to generate bubbles within the ARS. Bubble diameter and the width of the compacted matrix region increased over time following ADV. Atomic force microscopy measurements demonstrated that the hydrogel region proximal to the bubble was significantly stiffer than the distal regions. Significantly greater levels of Runx2 and osteocalcin expression were observed in MSCs proximal to bubbles compared to distal on days 7 and 14. Higher alkaline phosphatase activity also validated the above findings. The significant upregulation of these osteogenic biomarkers suggests ADV-induced, mechanical changes in ARSs were sufficient to enhance osteogenic differentiation of MSCs. This approach is a significant step towards controlling the 3D differentiation of MSCs in a spatially localized, non-invasive, and on-demand manner.

A facile method to fabricate versatile keratin cryogels for tissue engineering applications

Human hair keratin (HHK) has been extensively explored as a biomaterial for soft tissue regeneration due to their excellent bioactivity and biocompatibility. The possibility to fabricate HHK into three-dimensional (3D) hydrogels with physical properties resembling soft tissues has been well demonstrated. However, conventional keratin hydrogels often exhibit a dense architecture that could hinder cell filtration. In the present study, HHK-based cryogels were fabricated using a freeze-thaw (FT) method, where oxidized dopamine (ODA) was employed to covalently crosslink thiol/amine rich-keratin molecules at sub-zero temperatures. The obtained HHK-ODA cryogels have micron-sized pores ranging between 100 and 200 μm and mechanical properties that can be tuned by varying the crosslinking density between ODA and HHK. Through optimization of the weight content of ODA and the number of FT cycles, the compressive strengths and stiffnesses of these cryogels achieved 15-fold increments from ∼1.5 kPa to ∼22 kPa and ∼300 Pa to ∼5000 Pa, respectively. The HHK-ODA cryogels competently supported human dermal fibroblast spreading and proliferation. Overall, this study exhibited a facile method to fabricate mechanically superior keratin-based cryogels with cell compatible microarchitecture, circumventing the need for complicated chemical modifications and the use of cytotoxic crosslinkers.

Understanding the Role of Biofilms in Acute Recurrent Tonsillitis through 3D Bioprinting of a Novel Gelatin-PEGDA Hydrogel.

Acute recurrent tonsillitis is a chronic, biofilm-related infection that is a significant burden to patients and healthcare systems. It is often treated with repeated courses of antibiotics, which contributes to antimicrobial resistance. Studying biofilms is key to understanding this disease. In vitro modelling using 3D bioprinted hydrogels is a promising approach to achieve this. A novel gelatin-PEGDA pseudomonas fluorescens-laden bioink was developed and bioprinted in a 3D hydrogel construct fabricated using computer-aided design to mimic the tonsillar biofilm environment. The bioprinted constructs were cultured at 37 °C in lysogeny broth for 12 days. Bacterial growth was assessed by spectrophotometry. Cellular viability analysis was conducted using optical fluorescence microscopy (FDA/PI staining). A biocompatible 3D-printed bacteria-laden hydrogel construct was successfully fabricated. Bacterial growth was observed using optical fluorescence microscopy. A live/dead cellular-staining protocol demonstrated bacterial viability. Results obtained after the 12-day culture period showed higher bacterial growth in the 1% gelatin concentration construct compared to the 0% control. This study demonstrates the first use of a bacteria-laden gelatin-PEGDA hydrogel for biofabrication of a 3D-printed construct designed to model acute recurrent tonsillitis. Initiating a study with clinically relevant ex vivo tonsil bacteria will be an important next step in improving treatment of this impactful but understudied disease.

Self-Organization of Long-Lasting Human Endothelial Capillary-Like Networks Guided by DLP Bioprinting.

Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.

A dual-crosslinking electroactive hydrogel based on gelatin methacrylate and dibenzaldehyde-terminated telechelic polyethylene glycol for 3D bio-printing

Gelatin was widely used as scaffold materials in 3D bio-printing due to its excellent bioactivity and availability and especially that their arginine–glycine–aspartic acid (RGD) sequences could efficiently promote cell adhesion and proliferation. In this study, an electroactive and 3D bio-printable hydrogel was prepared through a two-step chemical cross-linking process. Specifically, residual free amino groups of methacrylated gelatin (GelMA) were cross-linked with the aldehyde groups of dibenzaldehyde-terminated telechelic polyethylene glycol (DF-PEG) via Schiff base bonds, forming a gel at 37 °C. During the subsequent 3D bio-printing process, GelMA underwent UV curing, forming a secondary cross-linked network to the mechanical strength and stability of the printed structure. The uniform dispersion of carbon nanotubes (CNTs) in the GelMA/DF-PEG composite hydrogel significantly increased its conductivity. The optimized GelMA/DF-PEG composite hydrogel, i.e., 30% GelMA and 25% DF-PEG (G30D25-CNTs), exhibited superior bio-printability. When the content of CNTs was above 4%, the conductivity of G30D25-CNTs hydrogel exceeded 10–2 S/m, which satisfied the needs of cells for micro-current stimulation. Furthermore, the pore microstructures, swelling behavior, degradation ability and cell toxicity of G30D25-CNTs electroactive hydrogels were thoroughly evaluated. Thus, the G30D25-CNTs hydrogel with 4% MWCNTs could be considered for further application in electrical stimulation of tissue regeneration such as muscle and cardiac nerve tissue repair.

3D Bioprinted Xanthan Hydrogels with Dual Antioxidant and Chondrogenic Functions for Post-traumatic Cartilage Regeneration.

Intra-articular trauma typically initiates the overgeneration of reactive oxidative species (ROS), leading to post-traumatic osteoarthritis and cartilage degeneration. Xanthan gum (XG), a branched polysaccharide, has shown its potential in many biomedical fields, but some of its inherent properties, including undesirable viscosity and poor mechanical stability, limit its application in 3D printed scaffolds for cartilage regeneration. In this project, we developed 3D bioprinted XG hydrogels by modifying XG with methacrylic (MA) groups for post-traumatic cartilage therapy. Our results demonstrated that the chemical modification optimized the viscoelasticity of the bioink, improved printability, and enhanced the mechanical properties of the resulting scaffolds. The XG hydrogels also exhibit decent ROS scavenging capacities to protect stem cells from oxidative stress. Furthermore, XGMA(H) (5% MA substitution) exhibited superior chondrogenic potential in vitro and promoted cartilage regeneration in vivo. These dual-functional XGMA hydrogels may provide a new opportunity for cartilage tissue engineering.

3D micro/nano hydrogel structures fabricated by two-photon polymerization for biomedical applications.

Hydrogels are three-dimensional natural or synthetic cross-linked networks composed of polymer chains formed by hydrophilic monomers. Due to the ability to simulate many properties of natural extracellular matrix, hydrogels have been widely used in the biomedical field. Hydrogels can be obtained through a variety of polymerization strategies such as heating and redox. However, photochemistry is one of the most interesting methods for researchers in this field. Gelatin-methacryloyl (GelMA) inherits the biological activity of gelatin and has become one of the gold standards in the field of biomaterials. GelMA, as a photopolymerizable hydrogel precursor, can be used to fabricate 3D porous structures for biomedical applications through two-photon polymerization. We report a new formulation of GelMA-based photoresist and used it to manufacture a series of two-photon polymerization structures, with a maximum resolution less than 120nm. The influence of process parameters on 3D structures manufacturing is studied by adjusting the scanning speed, laser power, and layer spacing values in two-photon polymerization processing. In vitro biological tests show that the 3D hydrogel produced by two-photon polymerization in this paper is biocompatible and suitable for MC3T3-E1 cell.

Engineering Anisotropic Cell Models: Development of Collagen Hydrogel Scaffolds with Magneto‐Responsive PEG Microgels for Tissue Engineering Applications

AbstractMimicking tissue‐oriented organization in vitro has been extensively studied in recent years, using both natural and synthetic materials in combination with external magnetic fields to establish anisotropic conditions. Here, a new combination between magneto‐responsive anisometric PEG microgels and collagen hydrogels is explored to establish anisotropic in vitro models. Different sizes of PEG microgels are tested to assess the impact of both width and aspect ratio on the formation and alignment of collagen hydrogels. Results show that the key properties of collagen hydrogels, regarding fibrillogenesis, rheological properties, and fiber diameter are kept consistent upon the combination with PEG microgels. Furthermore, partial collagen fiber alignment is observed when larger (width 10 µm) PEG microgels are employed and magnetically aligned. In vitro studies show cell alignment within the anisotropic collagen hydrogels from the first day in culture. Interestingly, PEG microgels with higher width and length tend to induce less hydrogel contraction even after 7 days in culture. The results demonstrate the ability to establish a 3D unidirectional collagen hydrogel by magnetically aligning anisometric microgels during the gelation process, which can be promising for different tissue engineering applications.

Functionality of macrophages encapsulated in porcine decellularized adipose matrix hydrogels and interaction with Candida albicans

Extracellular matrix hydrogels are considered one of the most suitable biomaterials for tissue regeneration due to their similarity with the extracellular microenvironment of the native tissue. Their properties are dependent on their composition, material concentration, fiber density and the fabrication approaches, among other factors. The encapsulation of immune cells in this kind of hydrogels, both in absence or presence of a pathogen, represents a promising strategy for the development of platforms that mimic healthy and infected tissues, respectively. In this work, we have encapsulated macrophages in 3D hydrogels of porcine decellularized adipose matrices (pDAMs) without and with the Candida albicans fungus, as 3D experimental models to study the macrophage immunocompetence in a closer situation to the physiological conditions and to mimic an infection scenario. Our results indicate that encapsulated macrophages preserve their functionality within these pDAM hydrogels and phagocytose live pathogens. In addition, their behavior is influenced by the hydrogel pore size, inversely related to the hydrogel concentration. Thus, larger pore size promotes the polarization of macrophages towards M2 phenotype along the time and enhances their phagocytosis capability. It is important to point out that encapsulated macrophages in absence of pathogen showed an M2 phenotype, but macrophages coencapsulated with C. albicans can switch towards an M1 inflammatory phenotype to resolve the infection, depending on the fungus quantity. The present study reveals that pDAM hydrogels preserve the macrophage plasticity, demonstrating their relevance as new models for macrophage-pathogen interaction studies that mimic an infection scenario with application in regenerative medicine research.

Hydrogel Based on Chitosan/Gelatin/Poly(Vinyl Alcohol) for In Vitro Human Auricular Chondrocyte Culture.

Three-dimensional (3D) hydrogels provide tissue-like complexities and allow for the spatial orientation of cells, leading to more realistic cellular responses in pathophysiological environments. There is a growing interest in developing multifunctional hydrogels using ternary mixtures for biomedical applications. This study examined the biocompatibility and suitability of human auricular chondrocytes from microtia cultured onto steam-sterilized 3D Chitosan/Gelatin/Poly(Vinyl Alcohol) (CS/Gel/PVA) hydrogels as scaffolds for tissue engineering applications. Hydrogels were prepared in a polymer ratio (1:1:1) through freezing/thawing and freeze-drying and were sterilized by autoclaving. The macrostructure of the resulting hydrogels was investigated by scanning electron microscopy (SEM), showing a heterogeneous macroporous structure with a pore size between 50 and 500 μm. Fourier-transform infrared (FTIR) spectra showed that the three polymers interacted through hydrogen bonding between the amino and hydroxyl moieties. The profile of amino acids present in the gelatin and the hydrogel was determined by ultra-performance liquid chromatography (UPLC), suggesting that the majority of amino acids interacted during the formation of the hydrogel. The cytocompatibility, viability, cell growth and formation of extracellular matrix (ECM) proteins were evaluated to demonstrate the suitability and functionality of the 3D hydrogels for the culture of auricular chondrocytes. The cytocompatibility of the 3D hydrogels was confirmed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, reaching 100% viability after 72 h. Chondrocyte viability showed a high affinity of chondrocytes for the hydrogel after 14 days, using the Live/Dead assay. The chondrocyte attachment onto the 3D hydrogels and the formation of an ECM were observed using SEM. Immunofluorescence confirmed the expression of elastin, aggrecan and type II collagen, three of the main components found in an elastic cartilage extracellular matrix. These results demonstrate the suitability and functionality of a CS/Gel/PVA hydrogel as a 3D support for the auricular chondrocytes culture, suggesting that these hydrogels are a potential biomaterial for cartilage tissue engineering applications, aimed at the regeneration of elastic cartilage.

Epigenetic Priming Enhances Chondrogenic Potential of Expanded Chondrocytes.

Expansion of chondrocytes presents a major obstacle in the cartilage regeneration procedure, such as matrix-induced autologous chondrocyte implantation. Dedifferentiation of chondrocytes during the expansion process leads to the emergence of a fibrotic (chondrofibrotic) phenotype that decreases the chondrogenic potential of the implanted cells. We aim to (1) determine the extent that chromatin architecture of H3K27me3 and H3K9me3 remodels during dedifferentiation and persists after the transfer to a three-dimensional (3D) culture; and (2) to prevent this persistent remodeling to enhance the chondrogenic potential of expanded bovine chondrocytes, used as a model system. Chromatin architecture remodeling of H3K27me3 and H3K9me3 was observed at 0 population doublings, 8 population doublings, and 16 population doublings (PD16) in a two-dimensional (2D) culture and after encapsulation of the expanded chondrocytes in a 3D hydrogel culture. Chondrocytes were treated with inhibitors of epigenetic modifiers (epigenetic priming) for PD16 and then encapsulated in 3D hydrogels. Chromatin architecture of chondrocytes and gene expression were evaluated before and after encapsulation. We observed a change in chromatin architecture of epigenetic modifications H3K27me3 and H3K9me3 during chondrocyte dedifferentiation. Although inhibiting enzymes that modify H3K27me3 and H3K9me3 did not alter the dedifferentiation process in 2D culture, applying these treatments during the 2D expansion did increase the expression of select chondrogenic genes and protein deposition of type II collagen when transferred to a 3D environment. Overall, we found that epigenetic priming of expanded bovine chondrocytes alters the cell fate when chondrocytes are later encapsulated into a 3D environment, providing a potential method to enhance the success of cartilage regeneration procedures.

Fabrication of gelatin-heparin based cartilage models: enhancing spatial complexity through refinement of stiffness properties and oxygen availability

The intricate nature of native cartilage, characterized by zonal variations in oxygen levels and ECM composition, poses a challenge for existing hydrogel-based tissue models. Consequently, these 3D models often present simplified renditions of the native tissue, failing to fully capture its heterogenous nature. The combined effects of hydrogel components, network properties, and structural designs on cellular responses are often overlooked. In this work, we aim to establish more physiological cartilage models through biofabrication of photopolymerizable allylated-gelatin (GelAGE) and Thiolated Heparin (HepSH) constructs with tailorable matrix stiffness and customized architectures. This involves systematically studying how the native glycosaminoglycan Heparin together with hydrogel stiffness, and oxygen availability within 3D structures influence chondrogenic differentiation and regional heterogeneity. A comprehensive library of 3D hydrogel constructs was successfully developed, encompassing GelAGE-HepSH hydrogels with three distinct stiffness levels: 12, 55 and 121 kPa, and three unique geometries: spheres, discs, and square lattices. In soft GelAGE-HepSH hydrogels, the localization of differentiating cells was observed to be irregular, while stiff hydrogels restricted the overall secretion of ECM components. The medium-stiff hydrogels were found to be most applicable, supporting both uniform tissue formation and maintained shape fidelity. Three different 3D architectures were explored, where biofabrication of smaller GelAGE-HepSH spheres without oxygen gradients induced homogenous, hyaline cartilage tissue formation. Conversely, fabrication of larger constructs (discs and lattices) with oxygen gradients could be utilized to design heterogenous cartilage tissue models. Similarly, temporal oxygen gradients were observed to drive interconnected deposition of glycosaminoglycans (GAGs). Control samples of GelAGE without HepSH did not exhibit any notable changes in chondrogenesis as a function of stiffness, architectures, or oxygen concentrations. Overall, the incorporation of HepSH within GelAGE hydrogels was observed to serve as an amplifier for the biological effects from both stiffness and oxygen cues. In conclusion, fabrication of GelAGE-HepSH constructs designed to impose limitations on oxygen availability induce more zone-specific cartilage tissue alignment. This systematic study of matrix components, network stiffness, and oxygen levels in 3D biofabricated structures contributes to the development of more physiologically relevant cartilage models while further enhancing our overall understanding of cartilage tissue engineering.

Photocuring 3D Printing of Hydrogels: Techniques, Materials, and Applications in Tissue Engineering and Flexible Devices.

Photocuring 3D printing of hydrogels, with sophisticated, delicate structures and biocompatibility, attracts significant attention by researchers and possesses promising application in the fields of tissue engineering and flexible devices. After years of development, photocuring 3D printing technologies and hydrogel inks make great progress. Herein, the techniques of photocuring 3D printing of hydrogels, including direct ink writing (DIW), stereolithography (SLA), digital light processing (DLP), continuous liquid interface production (CLIP), volumetric additive manufacturing (VAM), and two photon polymerization (TPP) are reviewed. Further, the raw materials for hydrogel inks (photocurable polymers, monomers, photoinitiators, and additives) and applications in tissue engineering and flexible devices are also reviewed. At last, the current challenges and future perspectives of photocuring 3D printing of hydrogels are discussed.