Silk Hydrogels of Tunable Structure and Viscoelastic Properties Using Different Chronological Orders of Genipin and Physical Cross-Linking

Abstract

Catering the hydrogel manufacturing process toward defined viscoelastic properties for intended biomedical use is important to hydrogel scaffolding function and cell differentiation. Silk fibroin hydrogels may undergo "physical" cross-linking through β-sheet crystallization during high pressure carbon dioxide treatment, or covalent "chemical" cross-linking by genipin. We demonstrate here that time-dependent mechanical properties are tunable in silk fibroin hydrogels by altering the chronological order of genipin cross-linking with β-sheet formation. Genipin cross-linking before β-sheet formation affects gelation mechanics through increased molecular weight, affecting gel morphology, and decreasing stiffness response. Alternately, genipin cross-linking after gelation anchored amorphous regions of the protein chain, and increasing stiffness. These differences are highlighted and validated through large amplitude oscillatory strain near physiologic levels, after incorporation of material characterization at molecular and micron length scales.

Keywords: genipin; high pressure carbon dioxide; large amplitude oscillatory strain; porous hydrogel; silk fibroin.

Figure 1

Preparation protocols and physical appearances of the gel samples. All samples were prepared from 3% w/v concentration of silk fibroin. The protocols show the order and degree of genipin cross-linking (GCX) with shaded blocks. Processing temperature during each 24 h block is listed, along with the cumulative time of processing. Herein, the control gels were from pure fibroin solution with 24 h PCT-induced crystallization, whereas all the other samples contained 1 mM concentration of genipin. Representative pictures of gels illustrating sample coloration and shape are shown on the right.

Figure 2

Gel permeation chromatography results, showing an increase in both M_N and M_W for gels with genipin cross-linking before gelation.

Figure 3

Genipin fluorescence emission results. Lyophilized and rehydrated samples were examined for genipin cross-linking (GCX) efficiencies, illustrated with the average intensities from confocal images. Results show the fluorescent signals from all the cross-linked gels are statistically (p < 0.10) different from control. Also, Pre48CX and CCX show statistically significant (p < 0.10) differences from all other cross-linked gels (labeled with “*”).

Figure 4

Primary amine sites quantified by ninhydrin assay. Results show the fraction of primary amine quantity in GCX gels (y-axis), when compared to control gels. All gels show decreased fraction in primary amine groups. Higher reduction in primary amine groups suggests increased genipin reactivity at bonding sites along single fibroin chains, rather than reaction between two fibroin molecules.

Figure 5

Infrared spectra (FTIR) showing similar peak patterns among gel samples treated with PCT. (A) All gels presented high similarity in all major peaks of FTIR spectra, which showed the tight, intermolecular β-sheet crystals (1622 cm⁻¹) and weaker β-sheet structures (1700 cm⁻¹) of fibroin. (B) Lyophilized fibroin solutions without or with genipin cross-linking, undergoing no PCT gelation, are compared to the Control with PCT gelation. Both fibroin solutions present random coil (1645 cm⁻¹) and tyrosine side chain peak shift (1516 cm⁻¹), common in amorphous fibroin. Additionally, there is a peak at 1532 cm⁻¹ in fibroin with genipin cross-linking, possibly resulting from in-plane deformation of the genipin bicyclic, fused ring structure.

Figure 6

SEM micrographs showing the gel microstructure and morphology. (A) Control gel showed uniform pores with limited connectivity. (B) Pre48CX gels presented greater average pore size and more fibrous structure with high connectivity. (C) Pre24CX gels presented even larger pores with connectivity lower than Pre48CX. (D) CCX gels slightly increased pore size and connectivity when compared to the Control. (E) Post24CX gels presented little change in morphology from the Control. Scale bar equals 10 μm.

Figure 7

Comparison of Control (top) and Pre48CX (bottom) microstructures. The images on the right panel illustrate analysis of pore changes in the pore structure (red) and connecting perforation (blue). Scale bar equals 3 μm.

Figure 8

SEM (left) and confocal microscopy (right) images, showing the gels in dry and wet environments, respectively. These correlated images demonstrate that the gel microstructure retained after hydration. Confocal imaging was performed by illuminating pores with Rhodamine 123. (A) Control; (B) Pre48CX; (C) Pre24CX; (D) CCX; (E) Post24CX. Scale bar equals 100 μm.

Figure 9

Storage (E₁′) and loss (E₁″) moduli as functions of frequency. Results in control gels indicate decreased overall stiffness at lower frequencies. Additionally, E₁″ appears lower than E₁′ for all frequency values, and appears decreasing at similar or faster rate than E₁′. (A) Comparison of Control (n = 3), Pre48CX (n = 3), and Pre24CX (n = 3) shows decreased stiffness for both E₁′ and E₁″ at all frequencies. (B) Post24CX (n = 2) shows increased stiffness compared to Control, whereas CCX (n = 4) shows lower transition frequency. Key: ●○, Control E₁′ E₁″; ■□, Pre48CX E₁′ E₁″; ◆ ◇, Pre24CX E₁′ E₁″; ▲△, CCX E₁′ E₁″; ++, Post24CX E₁′ E₁″.

Figure 10

Energy dissipation per cycle was calculated from hysteresis curves, and plotted against frequency. (A) Comparisons of Control (n = 3), Pre48CX (n = 3), Pre24CX (n = 3) show decreased energy dissipation in GCX cross-linked gels. (B) Comparisons of Control, CCX (n = 4), and Post24CX (n = 2) show increased energy dissipation from Post24CX gels, and peak shift to lower transition frequency. Key: ●, Control; ■, Pre48CX; ◆, Pre24CX; ▲, CCX; +, Post24CX.