Facile Biofabrication of Heterogeneous Multilayer Tubular Hydrogels by Fast Diffusion-Induced Gelation

Multilayer (ML) hydrogels are useful to achieve stepwise and heterogeneous control over the organization of biomedical materials and cells. There are numerous challenges in the development of fabrication approaches toward this, including the need for mild processing conditions that maintain the integrity of embedded compounds and the versatility in processing to introduce desired complexity. Here, we report a method to fabricate heterogeneous multilayered hydrogels based on diffusion-induced gelation. This technique uses the quick diffusion of ions and small molecules (i.e., photoinitiators) through gel–sol or gel–gel interfaces to produce hydrogel layers. Specifically, ionically (e.g., alginate-based) and covalently [e.g., gelatin methacryloyl (GelMA-based)] photocross-linked hydrogels are generated in converse directions from the same interface. The ML (e.g., seven layers) ionic hydrogels can be formed within seconds to minutes with thicknesses ranging from tens to hundreds of micrometers. The thicknesses of the covalent hydrogels are determined by the reaction time (or the molecule diffusion time). Multiwalled tubular structures (e.g., mimicking branched multiwalled vessels) are mainly investigated in this study based on a removable gel core, but this method can be generalized to other material patterns. The process is also demonstrated to support the encapsulation of viable cells and is compatible with a range of thermally reversible core materials (e.g., gelatin and Pluronic F127) and covalently cross-linked formulations (e.g., GelMA and methacrylated hyaluronic acid). This biofabrication process enhances our ability to fabricate a range of structures that are useful for biomedical applications

Facile Biofabrication of Heterogeneous Multilayer Tubular Hydrogels by Fast Diffusion-Induced Gelation

Multilayer (ML) hydrogels are useful to achieve stepwise and heterogeneous control over the organization of biomedical materials and cells. There are numerous challenges in the development of fabrication approaches toward this, including the need for mild processing conditions that maintain the integrity of embedded compounds and the versatility in processing to introduce desired complexity. Here, we report a method to fabricate heterogeneous multilayered hydrogels based on diffusion-induced gelation. This technique uses the quick diffusion of ions and small molecules (i.e., photoinitiators) through gel–sol or gel–gel interfaces to produce hydrogel layers. Specifically, ionically (e.g., alginate-based) and covalently [e.g., gelatin methacryloyl (GelMA-based)] photocross-linked hydrogels are generated in converse directions from the same interface. The ML (e.g., seven layers) ionic hydrogels can be formed within seconds to minutes with thicknesses ranging from tens to hundreds of micrometers. The thicknesses of the covalent hydrogels are determined by the reaction time (or the molecule diffusion time). Multiwalled tubular structures (e.g., mimicking branched multiwalled vessels) are mainly investigated in this study based on a removable gel core, but this method can be generalized to other material patterns. The process is also demonstrated to support the encapsulation of viable cells and is compatible with a range of thermally reversible core materials (e.g., gelatin and Pluronic F127) and covalently cross-linked formulations (e.g., GelMA and methacrylated hyaluronic acid). This biofabrication process enhances our ability to fabricate a range of structures that are useful for biomedical applications

Hydrolytically Degradable Hyaluronic Acid Hydrogels with Controlled Temporal Structures

Polysaccharides are being processed into biomaterials for numerous biological applications due to their native source in numerous tissues and biological functions. For instance, hyaluronic acid (HA) is found abundantly in the body, interacts with cells through surface receptors, and can regulate cellular behavior (e.g., proliferation, migration). HA was previously modified with reactive groups to form hydrogels that are degraded by hyaluronidases, either added exogenously or produced by cells. However, these hydrogels may be inhibitory and their applications are limited if the appropriate enzymes are not present. Here, for the first time, we synthesized HA macromers and hydrogels that are both hydrolytically (via ester group hydrolysis) and enzymatically degradable. The hydrogel degradation and growth factor release was tailored through the hydrogel cross-linking density (i.e., macromer concentration) and copolymerization with purely enzymatically degradable macromers. When mesenchymal stem cells (MSCs) were encapsulated in the hydrogels, cellular organization and tissue distribution was influenced by the copolymer concentration. Importantly, the distribution of released extracellular matrix molecules (e.g., chondroitin sulfate) was improved with increasing amounts of the hydrolytically degradable component. Overall, this new macromer allows for enhanced control over the structural evolution of the HA hydrogels toward applications as biomaterials

Rational Design of Network Properties in Guest–Host Assembled and Shear-Thinning Hyaluronic Acid Hydrogels

Shear-thinning hydrogels afford direct injection or catheter delivery to tissues without potential premature gel formation and delivery failure or the use of triggers such as chemical initiators or heat. However, many shear-thinning hydrogels require long reassembly times or exhibit rapid erosion. We developed a shear-thinning hyaluronic acid (HA) hydrogel based on the guest–host interactions of adamantane modified HA (guest macromer, Ad-HA) and β-cyclodextrin modified HA (host macromer, CD-HA). The ability of the guest and host molecules to interact with their counterpart following conjugation to HA was confirmed by ¹H NMR spectroscopy and was similar to that of the native complex. Mixing of Ad-HA and CD-HA resulted in rapid formation of a hydrogel composed of guest–host bonds. The hydrogel physical properties, including mechanics and flow characteristics, were dependent on cross-link density and network structure, which were controlled through macromer concentration, the extent of guest macromer modification, and the molar ratio of guest and host functional groups. The guest–host assembly mechanism permitted both shear-thinning behavior for ease of injection and near-instantaneous reassembly for material retention at the target sight. The hydrogel erosion and release of a model biomolecule were also dependent on design parameters and were sustained for over 60 days. These hydrogels show potential as a minimally invasive injectable hydrogel for biomedical applications

Picrosirius red and polarized light stained images at 4 weeks post-implantation.

<p>Images were collected for NA (A, D), AL (B, E), and CO (C, F) samples at 4 weeks post-implantation as viewed using brightfield (A–C) and polarized light microscopy (D–F). S denotes the scaffold region that was used for quantification and * denotes the edge of the scaffold. Scale bar = 50 µm.</p

Composite scaffold fabrication schematic and representative electrospun scaffold images.

<p>Schematic of dual polymer electrospinning set up (A). Representative fluorescent (Acr-PGS  =  red, PEO  =  blue) and SEM images of NA (B, C), AL (D, E), and CO (F,G) scaffolds following crosslinking and CO scaffolds following PEO removal and lyophilization (H, I). Scale bar = 100 µm (B, D, F, and H) or 20 µm (C, E, G, I).</p

Anisotropy ratio of AL and CO scaffolds.

<p>Anisotropy ratio of AL and CO scaffolds.</p

Quantification of collagen fiber alignment.

<p>Histogram representing the quantification of collagen fiber alignment within the NA (black), AL (white), and CO (grey) scaffold regions at 4 weeks post-implantation.</p

Trichrome stained images of subcutaneous implant samples at 2, 3, and 4 weeks post-implantation.

<p>Images were collected for NA (A, D, G), AL (B, E, H), and CO (C, F, I) samples at 2 (A–C), 3 (D–F), and 4 (G–I) weeks post implantation to evaluate integration. S denotes the scaffold region. Scale bar = 50 µm.</p

Young's moduli for PEO, NA, AL, and CO scaffolds.

<p>Scaffolds were tested in parallel (PA) and perpendicular (PE) fiber directions after photocrosslinking (A, black), after PEO removal and lyophilization (A, white), and after hydration in PBS following PEO removal (B, grey). * denotes p<0.05 for post-wash dry scaffolds (white) versus after crosslinking (black). All scaffolds were significantly different for hydrated scaffolds compared to post-wash dry scaffolds.</p