(355a) Impacts of Beta-Sheet Crystallinity on the Mechanical Properties of Chemically and Physically Crosslinked Silk Fibroin Hydrogels

Statement of Purpose: Silk fibroin (SF), obtained from Bombyx mori silkworm cocoons, is a natural biopolymer used in the formation of biomaterials due to its biocompatibility, controllable degradation rate, and tunable physical properties.¹ SF can be made into many biomaterial formats including films, nanoparticles, sponges, and hydrogels.¹ In SF hydrogels, molecular weight, concentration, and crystallinity are all tunable parameters that affect the mechanical and physical properties of the resulting structures.² SF is known to form physical crosslinks via secondary structure formation within the protein, resulting in ordered beta-sheet crystalline regions.² Organization of beta-sheet structures via hydrogen bonding yields crystalline regions amongst amorphous polymer chains, decreasing optical transparency and increasing mechanical strength over time.^{2, 3} Crystalline beta-sheet structures can be intentionally induced via application of shear forces or by increasing the temperature, but will form even without intentionality as the biopolymers interact within the hydrogel, aiming to minimize free energy.^{4, 5} To interfere with the free energy landscape, we investigated the impact of solution parameters (concentration and molecular weight) on resulting hydrogel mechanical properties through shear rheology.⁶ We also aimed to interrupt the dynamics of beta-sheet formation using chemical crosslinking strategies. Understanding the shear parameters and time scales of spontaneous stiffening within solution and hydrogel form will allow for optimization of silk fibroin hydrogels toward disease modeling where changing mechanics can mimic disease progression.^{7, 8}

Methods: Silk fibroin solution was isolated from the cocoons of Bombyx mori silkworms.¹ Briefly, cocoons were cut into small pieces and boiled (degummed) for 15, 30, 60, or 90 minutes in sodium carbonate solution and the resulting silk fibroin mat was left to dry. The mat was then solubilized in 9.3 M lithium bromide. After undergoing dialysis, the silk was diluted with ultrapure water to 3% or 5% (wt/v) silk fibroin solution. Gel electrophoresis (SDS-PAGE) and HPLC were used to assess molecular weight and amino acid content, respectively. Two methods of physically crosslinked hydrogels were used. Spontaneous physically crosslinked hydrogels were formed by leaving the silk solution to spontaneously form hydrogels over time though beta-sheet formation. Ultrasonicated physically crosslinked hydrogels were formed by sonicating silk fibroin solution for 15 seconds at 15% amplitude. Silk fibroin was chemically modified to enable photocrosslinking, through the azo-based amino tyrosine premodification followed by the addition of a norbornene.^{9, 10} Resulting physical crosslinking (beta-sheet content) was assessed over time through Fourier transform infrared (FTIR) spectroscopy and optical transparency. Mechanical properties of silk fibroin precursor solutions and final hydrogels were assessed via rheology and dynamic mechanical assessment though shear measurements over time to obtain viscosity and modulus values.

Results: Increasing the degumming time of the polymer extraction decreases the molecular weight due to denaturation of the polymer during boiling.¹¹ During the degumming process, thermal degradation results in changes to the distribution of molecular weights as well as primary amino acid structures. We hypothesized that the shorter degumming times and higher polymer concentration would lead to more beta-sheet structures and polymer interactions, which would result in larger mechanical property values. Traditional non-crystalline polymer trends predict that the larger molecular weight conditions (15-minute degumming) will have the highest viscosity, and viscosity will decrease as degumming time increases. However, this trend did not hold for all SF solutions due in part to differences in inter- and intra-molecular interactions from differences in polymer entanglement of the semi-crystalline polymer. We did find that all conditions show shear thinning behavior over low (0.1 to 100 sec^-1) shear rates. This phenomenon also had an impact on beta-sheet structure formation found via deconvolution of FTIR spectra. We hypothesized that the higher molecular weight hydrogels would result in more beta-sheet structures. However, we found that the physical crosslinking method had a larger impact on mechanical properties and that gelation by sonication generates lower initial beta-sheet content. However, over time, the beta-sheet content increases due to spontaneous formation still occurring. Results suggest that the time scale of hydrogel formation in sonicated hydrogels has too short of a gelation time for the beta-sheet structures to organize in their lowest energy state, whereas the spontaneous hydrogel has longer gelation times allowing the polymer chains to rearrange and form more organized secondary structures. Results confirm that SF molecular weight has the greatest impact on viscosity and the greatest impact on physical crosslinking dynamic moduli.

Similarly, preliminary work using chemically modified silk fibroin was used to form hydrogels via photopolymerization, yet physical crosslinks still occurred over time. Most importantly, the dynamics of these changes (timescale) were substantially different from the timescales measured with ultrasonicated hydrogels. Modulating temporal control through chemical crosslinking methods allows for alterations in gelation time and the resulting beta-sheet content over different time scales through more the addition of more flexible chemical linkers. Understanding the spontaneous beta-sheet formation in SF hydrogels enables temporal control over mechanical properties for future hydrogel applications, such as 3D printing or soft tissue engineering, including in vitro tumor modeling.

References:

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