Advancing Synthetic Hydrogels through Nature-Inspired Materials Chemistry.

Synthetic extracellular matrix (ECM) mimics that can recapitulate the complex biochemical and mechanical nature of native tissues are needed for advanced models of development and disease. Biomedical research has heavily relied on the use of animal-derived biomaterials, which is now impeding their translational potential and convoluting the biological insights gleaned from in vitro tissue models. Natural hydrogels have long served as a convenient and effective cell culture tool, but advances in materials chemistry and fabrication techniques now present promising new avenues for creating xenogenic-free ECM substitutes appropriate for organotypic models and microphysiological systems. However, significant challenges remain in creating synthetic matrices that can approximate the structural sophistication, biochemical complexity, and dynamic functionality of native tissues. This review summarizes key properties of the native ECM, and discusses recent approaches used to systematically decouple and tune these properties in synthetic matrices. The importance of dynamic ECM mechanics, such as viscoelasticity and matrix plasticity, is also discussed, particularly within the context of organoid and engineered tissue matrices. Emerging design strategies to mimic these dynamic mechanical properties are reviewed, such as multi-network hydrogels, supramolecular chemistry, and hydrogels assembled from biological monomers.

Harnessing Macromolecular Chemistry to Design Hydrogel Micro- and Macro-Environments.

Cell encapsulation within three-dimensional hydrogels is a promising approach to mimic tissues. However, true biomimicry of the intricate microenvironment, biophysical and biochemical gradients, and the macroscale hierarchical spatial organizations of native tissues is an unmet challenge within tissue engineering. This review provides an overview of the macromolecular chemistries that have been applied toward the design of cell-friendly hydrogels, as well as their application toward controlling biophysical and biochemical bulk and gradient properties of the microenvironment. Furthermore, biofabrication technologies provide the opportunity to simultaneously replicate macroscale features of native tissues. Biofabrication strategies are reviewed in detail with a particular focus on the compatibility of these strategies with the current macromolecular toolkit described for hydrogel design and the challenges associated with their clinical translation. This review identifies that the convergence of the ever-expanding macromolecular toolkit and technological advancements within the field of biofabrication, along with an improved biological understanding, represents a promising strategy toward the successful tissue regeneration.

3D Volumetric Mechanosensation of MCF7 Breast Cancer Spheroids in a Linear Stiffness Gradient GelAGE.

The tumor microenvironment presents spatiotemporal shifts in biomechanical properties with cancer progression. Hydrogel biomaterials like GelAGE offer the stiffness tuneability to recapitulate dynamic changes in tumor tissues by altering photo-energy exposures. Here, a tuneable hydrogel with spatiotemporal control of stiffness and mesh-network is developed. The volume of MCF7 spheroids encapsulated in a linear stiffness gradient demonstrates an inverse relationship with stiffness (p < 0.0001). As spheroids are exposed to increased crosslinking (stiffer) and greater mechanical confinement, spheroid stiffness increases. Protein expression (TRPV4, ß1 integrin, E-cadherin, and F-actin) decreases with increasing stiffness while showing strong correlations to spheroid volume (r2  > 0.9). To further investigate the role of volume, MCF7 spheroids are grown in a soft matrix for 5 days prior to a second polymerisation which presents a stiffness gradient to equally expanded spheroids. Despite being exposed to variable stiffness, these spheroids show even protein expression, confirming volume as a key regulator. Overall, this work showcases the versatility of GelAGE and demonstrates volume expansion as a key regulator of 3D mechanosensation in MCF7 breast cancer spheroids. This platform has the potential to further investigation into the role of stiffness and dimensionality in 3D spheroid culture for other types of cancers and diseases.

Injection-Free Delivery of MSC-Derived Extracellular Vesicles for Myocardial Infarction Therapeutics.

As emerging therapeutic factors, extracellular vesicles (EVs) offer significant potential for myocardial infarction (MI) treatment. Current delivery approaches for EVs involve either intra-myocardial or intravenous injection, where both have inherent limitations for downstream clinical applications such as secondary tissue injury and low delivery efficiency. Herein, an injection-free approach for delivering EVs onto the heart surface to treat MI is proposed. By spraying a mixture of EVs, gelatin methacryloyl (GelMA) precursors, and photoinitiators followed by visible light irradiation for 30 s, EVs are physically entrapped within the GelMA hydrogel network covering the surface of the heart, resulting in an enhanced retention rate. Moreover, EVs are gradually released from the hydrogel network through a combination of diffusion and/or enzymatic degradation of the hydrogel, and they are effectively taken up by the sprayed tissue area. More importantly, the released EVs further migrate deep into myocardium tissue, which exerts an improved therapeutic effect. In an MI-induced mice model, the group treated with EVs-laden GelMA hydrogels shows significant recovery in cardiac function after 4 weeks. The work demonstrates a new strategy for delivering EVs into cardiac tissues for MI treatment in a localized manner with high retention.

Development and Characterization of Gelatin-Norbornene Bioink to Understand the Interplay between Physical Architecture and Micro-Capillary Formation in Biofabricated Vascularized Constructs.

The principle challenge for engineering viable, cell-laden hydrogel constructs of clinically-relevant size, is rapid vascularization, in order to moderate the finite capacity of passive nutrient diffusion. A multiscale vascular approach, with large open channels and bulk microcapillaries may be an admissible approach to accelerate this process, promoting overall pre-vascularization for long-term viability of constructs. However, the limited availability of bioinks that possess suitable characteristics that support both fabrication of complex architectures and formation of microcapillaries, remains a barrier to advancement in this space. In this study, gelatin-norbornene (Gel-NOR) is investigated as a vascular bioink with tailorable physico-mechanical properties, which promoted the self-assembly of human stromal and endothelial cells into microcapillaries, as well as being compatible with extrusion and lithography-based biofabrication modalities. Gel-NOR constructs containing self-assembled microcapillaries are successfully biofabricated with varying physical architecture (fiber diameter, spacing, and orientation). Both channel sizes and cell types affect the overall structural changes of the printed constructs, where cross-signaling between both human stromal and endothelial cells may be responsible for the reduction in open channel lumen observed over time. Overall, this work highlights an exciting three-way interplay between bioink formulation, construct design, and cell-mediated response that can be exploited towards engineering vascular tissues.

Effect of Photoinitiator on Precursory Stability and Curing Depth of Thiol-Ene Clickable Gelatin.

Recent advances highlight the potential of photopolymerizable allylated gelatin (GelAGE) as a versatile hydrogel with highly tailorable properties. It is, however, unknown how different photoinitiating system affects the stability, gelation kinetics and curing depth of GelAGE. In this study, sol fraction, mass swelling ratio, mechanical properties, rheological properties, and curing depth were evaluated as a function of time with three photo-initiating systems: Irgacure 2959 (Ig2959; 320-500 nm), lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP; 320-500 nm), and ruthenium/sodium persulfate (Ru/SPS; 400-500 nm). Results demonstrated that GelAGE precursory solutions mixed with either Ig2959 or LAP remained stable over time while the Ru/SPS system enabled the onset of controllable redox polymerization without irradiation during pre-incubation. Photo-polymerization using the Ru/SPS system was significantly faster (<5 s) compared to both Ig2959 (70 s) and LAP (50 s). Plus, The Ru/SPS system was capable of polymerizing a thick construct (8.88 ± 0.94 mm), while Ig2959 (1.62 ± 0.49 mm) initiated hydrogels displayed poor penetration depth with LAP (7.38 ± 2.13 mm) in between. These results thus support the use of the visible light based Ru/SPS photo-initiator for constructs requiring rapid gelation and a good curing depth while Ig2959 or LAP can be applied for photo-polymerization of GelAGE materials requiring long-term incubation prior to application if UV is not a concern.

Stepwise Control of Crosslinking in a One-Pot System for Bioprinting of Low-Density Bioinks.

Extrusion-based 3D bioprinting is hampered by the inability to print materials of low-viscosity. In this study, a single initiating system based on ruthenium (Ru) and sodium persulfate (SPS) is utilized for a sequential dual-step crosslinking approach: 1) primary (partial) crosslinking in absence of light to alter the bioink's rheological profile for print fidelity, and 2) subsequent secondary post-printing crosslinking for shape maintenance. Allyl-functionalized gelatin (Gel-AGE) is used as a bioink, allowing thiol-ene click reaction between allyl moieties and thiolated crosslinkers. A systematic investigation of primary crosslinking reveals that a thiol-persulfate redox reaction facilitates thiol-ene crosslinking, mediating an increase in bioink viscosity that is controllable by tailoring the Ru/SPS, crosslinker, and/or Gel-AGE concentrations. Thereafter, subsequent photoinitiated secondary crosslinking then facilitates maximum conversion of thiol-ene bonds between AGE and thiol groups. The dual-step crosslinking method is applicable to a wide biofabrication window (3-10 wt% Gel-AGE) and is demonstrated to allow printing of low-density (3 wt%) Gel-AGE, normally exhibiting low viscosity (4 mPa s), with high shape fidelity and high cell viability (>80%) over 7 days of culture. The presented approach can therefore be used as a one-pot system for printing low-viscous bioinks without the need for multiple initiating systems, viscosity enhancers, or complex chemical modifications.

Rapid Photocrosslinking of Silk Hydrogels with High Cell Density and Enhanced Shape Fidelity.

Silk fibroin hydrogels crosslinked through di-tyrosine bonds are clear, elastomeric constructs with immense potential in regenerative medicine applications. In this study, demonstrated is a new visible light-mediated photoredox system for di-tyrosine bond formation in silk fibroin that overcomes major limitations of current conventional enzymatic-based crosslinking. This photomediated system rapidly crosslinks silk fibroin (<1 min), allowing encapsulation of cells at significantly higher cell densities (15 million cells mL-1 ) while retaining high cell viability (>80%). The photocrosslinked silk hydrogels present more stable mechanical properties which do not undergo spontaneous transition to stiff, ß-sheet-rich networks typically seen for enzymatically crosslinked systems. These hydrogels also support long-term culture of human articular chondrocytes, with excellent cartilage tissue formation. This system also facilitates the first demonstration of biofabrication of silk fibroin constructs in the absence of chemical modification of the protein structure or rheological additives. Cell-laden constructs with complex, ordered, graduated architectures, and high resolution (40 µm) are fabricated using the photocrosslinking system, which cannot be achieved using the enzymatic crosslinking system. Taken together, this work demonstrates the immense potential of a new crosslinking approach for fabrication of elastomeric silk hydrogels with applications in biofabrication and tissue regeneration.