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

Abstract

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, β‐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.

Rapid and cytocompatible cell-laden silk hydrogel formation via riboflavin-mediated crosslinking

“…Silk fibroin hydrogels can be fabricated by inducing conformational changes within the silk chains, leading to tough materials with low elasticity; however, the photo-crosslinking, as previously described, 20 might lower the formation of crystalline regions within the silk chains resulting in materials with higher elasticity. To test the elastic behaviour, the developed silk fibroin hydrogels were subjected to cyclic oscillation tests by alternating low/high shear strain (Fig.…”

“…This suggests that the di-tyrosine crosslinks and the silk fibroin content do not promote the formation of b-sheet crystalline domains. 20…”

“…Previous reports have shown the visible lightinduced crosslinking of silk fibroin hydrogels using ruthenium as photo-initiator. 19,20 The biocompatibility of ruthenium is unknown and under certain conditions might be highly toxic, 21,22 therefore a photo-initiator of known biocompatibility is needed. Riboflavin (RB), also known as vitamin B2, is a non-toxic photoinitiator recently used for the preparation of silk fibroin hydrogels upon exposure to visible light.…”

Cells, Materials, and Fabrication Processes for Cardiac Tissue Engineering

“…Alginate, a sugar-based natural hydrogel obtained from algae, (Orive et al, 2006) has been employed due to their tailorable mechanical properties and simple polymerisation, mediated by cations such as Ca or Mg, albeit lacking biological binding motifs. Silk and its derivatives have also been processed into hydrogels, (Holland et al, 2019) and modified to incorporate electrically active particles (Barreiro et al, 2019) or photocrosslinkable chemical groups (Cui et al, 2020). Allergic and anti-inflammatory side reactions have been reported with some silk derivatives, so care should be taken when incorporating them into an engineered tissue.…”

Tailoring Gelation Mechanisms for Advanced Hydrogel Applications

“…[234] This process has also been exploited to create templated microchannels to support nutrient transport in 3D printed structures. [235,236] A similar approach used the reversible thermosetting of gelatin to fabricate interconnected channel networks throughout GelMA hydrogels. As a cytocompatible material, the gelatin bioink could be preloaded with endothelial cells to form uniform vascular networks without needing to postseed cells into channels.…”