(81d) Designing Injectable, Shear Thinning Hydrogels Based on Charged Silk Protein Conjugates for 3D Mammalian Cell Culture

Polymeric hydrogels are three-dimensional, highly hydrated structures that may replicate key characteristics of the extracellular matrix, making them ideal for applications in tissue engineering. Silk protein biomaterials are considered as prominent materials in biomedicine due to their biodegradability, biocompatibility, and versatility to be processed into various formats, including films, scaffolds, hydrogels, and microparticles. Silk fibroin, extracted from the fibers of the Bombyx mori silkworm’s cocoon, is a high molecular weight protein composed of hydrophilic amorphous domains, which can be chemically modified through various aqueous and organic phase chemistries, and repeating hydrophobic crystalline domains, which form beta sheet structures via hydrogen bonding. Silk fibroin’s hydrogen bonding serves to physically cross-link the polymer chains, providing unique potential to tune the mechanical and degradation properties. Silk hydrogels have been generated using different strategies, such as covalent cross-linking via dityrosine bond formation or by inducing beta sheet structures through ultrasonication, thermal modulation, organic solvent application, or pH adjustments. This work was motivated by a need for biocompatible methods to encapsulate cells within silk hydrogels that would also provide opportunity to recover the cells from the hydrogels to facilitate their analysis. This work reports on the synthesis of electrostatically crosslinked hydrogels using charged silk conjugates that can be reversibly converted to solution and studies the effect of shearing on the structural properties of silk hydrogels.

This work uses an efficient synthetic route to carboxylate silk fibroin in an ionic liquid, a reaction that generates higher amounts of negatively charged silk conjugate materials. The reaction employs succinic anhydride in the presence of urea to modify the hydroxyl groups of the serine and threonine residues, as well as amine groups of lysine, arginine, and histidine residues, resulting in the enrichment of carboxylic acid functional groups on the fibroin. The addition of urea during the reaction led to a two-fold increase in the degree of protein carboxylation compared to existing methods, a result that is attributed to the disruption of hydrogen bonding to expose reactive sites within the hydrophobic regions of the protein. The increase in charged groups facilitated the formation of hydrogels via electrostatic interactions when mixed with oppositely charged molecules. Linear and branched amine containing molecules, such as a diamine derived from a propylene oxide-capped poly(ethylene glycol) (Jeffamine ED-2003) and an 8-arm poly(ethylene glycol) amine, were mixed with carboxylated silk in a 1:1 molar ratio for charged groups, resulting in the formation of hydrogels via electrostatic crosslinking at physiological conditions. The gelation kinetics were found to be more rapid as the concentration of carboxylated silk increased. The hydrogels were also synthesized in different media, including water, PBS buffer, and cell culture media, to investigate the effect of salt concentration on the formation of the networks. This mode of crosslinking enables the reversible transition from gel phase to sol phase by tuning electrostatic interactions and by shearing. The gel to solution transition of the hydrogels can be induced in different ways, including by changing the stoichiometric ratio of amine to carboxyl groups and by mechanical shearing applied by passing the gel through a pipette tip. The shear thinning behavior of the electrostatically crosslinked hydrogels was quantified using dynamic shear rheology. The hydrogels of all compositions were demonstrated to flow into a solution and then recover their mechanical properties when subjected to oscillatory strain sweeps. No significant induction of beta sheet structures was found in the electrostatically crosslinked silk hydrogels over a period of two weeks. Additionally, the hydrogels were found to be degradable upon exposure to collagenase type IA over a two-week period. Human mesenchymal stem cells (hMSCs) encapsulated in the silk hydrogels exhibited a uniform distribution under a confocal microscope, and the recovery of stem cells was found to be > 90% upon shearing with a 200 ul micropipette tip. The percentage of encapsulated cells recovered from gels was found to be higher for softer hydrogel compositions. Tuning the electrostatic charges on silk fibroin provides a viable route to synthesize electrostatically cross-linked protein hydrogels suitable for the encapsulation and subsequent retrieval of hMSCs for applications in tissue engineering and regenerative medicine.