Characterization of silk-hyaluronic acid composite hydrogels towards vitreous humor substitutes.

Multiple ophthalmic pathologies, such as retinal detachment and diabetic retinopathy, require the removal and replacement of the vitreous humor. Clinical tamponades such as silicone oil and fluorinated gases are utilized but limited due to complications and toxicity. Therefore, there is a need for biocompatible, stable, vitreous humor substitutes. In this study, enzymatically crosslinked silk-hyaluronic acid (HA) hydrogels formed using horseradish peroxidase and H2O2 were characterized for use as vitreous humor substitutes. The composite network structure was characterized with dynamic light scattering. In addition, the rheological, optical, and swelling properties of hydrogels with varying silk to HA ratios and crosslinking densities controlled via H2O2 were determined over time. Hydrogels had refractive indexes of 1.336 and were clear with 75-91% light transmission. Hydrogel shear storage modulus ranged between ~6 and 240 Pa where increased H2O2 increased the modulus. After 1 month of aging, there were no changes in modulus for hydrogels with lower silk ratios, while those with higher silk ratios exhibited a significant increase in modulus. Decreasing H2O2 concentration in the reactions led to increased hydrogel volume during swelling, with higher silk ratios returning to their original size after 15 days. Dynamic light scattering results show three diffusive modes, revealing the possible structures of the hydrogel composite and are consistent with the mechanical properties and swelling results. The normalized intraocular pressure of ex vivo porcine eyes after injecting hydrogels were comparable with those treated with silicone oil showing the potential clinical utility of the hydrogels as vitreous substitutes. The versatility of the silk-HA hydrogel system, the tunable swelling properties, and the stability of hydrogels with lower silk ratios show the benefit of utilizing silk-HA hydrogels as vitreous substitutes.

Ex vivo pregnant-like tissue model to assess injectable hydrogel for preterm birth prevention.

Cervical insufficiency (CI) is an important cause of preterm birth, which leads to severe newborn complications. Standard treatment for CI is cerclage, which has variable success rates, resulting in a clinical need for alternative treatments. Our objective was to develop an ex vivo model of softened cervical tissue to study an injectable silk-based hydrogel as a novel alternative treatment for CI. Cervical tissue from nonpregnant women was enzymatically treated and characterized to determine tissue hydration, collagen organization, and mechanical properties via unconfined compression. Enzymatic treatment led to an 86 ± 7.9% decrease in modulus, which correlated to a decrease in collagen organization as observed by differences in collagen birefringence. The softened tissue was injected with a crosslinked silk-hyaluronic acid composite hydrogel. After injection, the mechanical properties and volume increase of the hydrogel-treated tissue were measured resulting in a 54 ± 16% volume increase with minimal effect on tissue mechanical properties. In addition, cervical fibroblasts on silk-hyaluronic acid hydrogels remained viable and exhibited increased proliferation and metabolic activity over 5 days. Overall, this study developed an ex vivo pregnant-like human tissue model to assess cervical augmentation and showed the potential of silk-based hydrogels as an alternative treatment for cervical insufficiency.

Silk Hydrogels Crosslinked by the Fenton Reaction.

Here, the Fenton reaction is used to prepare silk hydrogels through oxidation of tyrosine residues in silk fibroin, leading to dityrosine crosslinking. At pH 5.7, gelation occurs rapidly within 30 s, and the resultant opaque gels show soft properties with a storage modulus of ≈100 Pa. The addition of ascorbic acid to the Fenton reaction increases the dityrosine bonds in the hydrogels but has little effect on the rheological or mechanical properties. The results indicate that Fe(III) ions significantly interacted with silk fibroin during the Fenton reaction, most likely binding to sites such as tyrosine, glutamate, and aspartate residues, triggering the formation of ß-sheet structures that may impede dityrosine bond formation due to steric hindrance. The use of an iron chelator or the operation of the Fenton reaction at pH 9.2 enables control over the interaction of Fe(III) ions with silk fibroin, achieving a hydrogel with improved optical properties and enhanced dityrosine bond formation. Hydrogels prepared by the Fenton reaction are cytocompatible as L929 mouse fibroblasts remain viable and are proliferative when seeded on the hydrogels. The results offer a useful approach to generate chemically crosslinked silk fibroin hydrogels without the use of enzyme-catalyzed reactions for biomedical applications.

Silk Molecular Weight Influences the Kinetics of Enzymatically Cross-linked Silk Hydrogel Formation.

We transform reconstituted silk solutions into robust hydrogels through covalent dityrosine cross-linking resulting from an enzymatic reaction. The bulk rheological properties and the covalent dityrosine bond formation of these gels are measured during polymerization. We compare the time-resolved bond formation to the mechanical properties, where we find that the gelation process is consistent with a model of percolation. The molecular weight of the protein determines whether a secondary mode of growth postpercolation exists, indicating that molecular weight changes affect the mechanisms by which these gels polymerize.

Enzymatically crosslinked silk-hyaluronic acid hydrogels.

In this study, silk fibroin and hyaluronic acid (HA) were enzymatically crosslinked to form biocompatible composite hydrogels with tunable mechanical properties similar to that of native tissues. The formation of di-tyrosine crosslinks between silk fibroin proteins via horseradish peroxidase has resulted in a highly elastic hydrogel but exhibits time-dependent stiffening related to silk self-assembly and crystallization. Utilizing the same method of crosslinking, tyramine-substituted HA forms hydrophilic and bioactive hydrogels that tend to have limited mechanics and degrade rapidly. To address the limitations of these singular component scaffolds, HA was covalently crosslinked with silk, forming a composite hydrogel that exhibited both mechanical integrity and hydrophilicity. The composite hydrogels were assessed using unconfined compression and infrared spectroscopy to reveal of the physical properties over time in relation to polymer concentration. In addition, the hydrogels were characterized by enzymatic degradation and for cytotoxicity. Results showed that increasing HA concentration, decreased gelation time, increased degradation rate, and reduced changes that were observed over time in mechanics, water retention, and crystallization. These hydrogel composites provide a biologically relevant system with controllable temporal stiffening and elasticity, thus offering enhanced tunable scaffolds for short or long term applications in tissue engineering.