Increasing mechanical strength of gelatin hydrogels by divalent metal ion removal

Abstract

The usage of gelatin hydrogel is limited due to its instability and poor mechanical properties, especially under physiological conditions. Divalent metal ions present in gelatin such as Ca(2+) and Fe(2+) play important roles in the gelatin molecule interactions. The objective of this study was to determine the impact of divalent ion removal on the stability and mechanical properties of gelatin gels with and without chemical crosslinking. The gelatin solution was purified by Chelex resin to replace divalent metal ions with sodium ions. The gel was then chemically crosslinked by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). Results showed that the removal of divalent metal ions significantly impacted the formation of the gelatin network. The purified gelatin hydrogels had less interactions between gelatin molecules and form larger-pore network which enabled EDC to penetrate and crosslink the gel more efficiently. The crosslinked purified gels showed small swelling ratio, higher crosslinking density and dramatically increased storage and loss moduli. The removal of divalent ions is a simple yet effective method that can significantly improve the stability and strength of gelatin hydrogels. The in vitro cell culture demonstrated that the purified gelatin maintained its ability to support cell attachment and spreading.

Figure 1. The change of Ca²⁺ and Fe²⁺ concentration with respect to time during the purification process.

The ion concentration was expressed as the percentage of initial concentration.

Figure 2. Effect of purification, solvent and EDC crosslinking on swelling ratio of gelatin hydrogels.

(A). Swelling of uncrosslinked hydrogel in DIH₂O at room temperature. (B). Swelling of uncrosslinked hydrogel in PBS at room temperature. (C). Swelling of crosslinked hydrogel in DIH₂O at room temperature. (D). Swelling of crosslinked hydrogel in PBS at room temperature (RT) and 37°C.

Figure 3. FTIR spectra for uncrosslinked and EDC-crosslinked purified and unpurified gelatin hydrogels.

Figure 4. Morphologies of unpurified and purified hydrogels.

Gross appearance of crosslinked unpurified (A) and purified (B) gels after 48 h swelling in PBS at 37 °C. SEM images of transverse cross sections of unpurified (C) and purified (D) gels before EDC crosslinking, and unpurified (E) and purified (F) gels after EDC crosslinking. Scale bar: 150 μm.

Figure 5. The in vitro hydrolysis degradation behavior of purified and unpurified gelatin hydrogels under pH 7.4 and 37°C.

Figure 6. Comparison of storage G′ and loss G″ moduli of freshly prepared gelatin hydrogels before EDC crosslinking.

(A). Purified gelatin gels. (B). Unpurified gelatin gels.

Figure 7. Frequency sweeps of EDC-crosslinked gelatin hydrogels swollen at different time points.

(A). Storage modulus (G′) of purified gelatin gels. (B). Storage modulus (G′) of unpurified gelatin gels. (C). Loss modulus (G″) of purified gelatin gels. (D). Loss modulus (G″) of unpurified gelatin gels.

Figure 8. Effects of exogenously introduced Ca²⁺ ions in the purified gelatin solution on mechanical properties of gelatin hydrogels.

(A). Storage modulus (G′) of uncrosslinked gelatin gels. (B). Loss modulus (G″) of uncrosslinked gelatin gels. (C). Storage modulus (G′) of crosslinked gelatin gels. (D). Loss modulus (G″) of crosslinked gelatin gels. * p < 0.05 compared with samples containing 0.1 mM Ca²⁺.

Figure 9. In vitro hMSC culture on gelatin hydrogels.

F-actin staining of cells on (A). Purified gel at 6 h; (B). Unpurified gel at 6 h; (C). Purified gel at 24 h; (D). Unpurified gel at 24 h. (E). Average cellular area covered by each cell; (F). Cell attachment at 6 and 24 h after seeding. Error bars = means ± SD (n = 3).

Figure 10. Schematic representing the possible interactions between ions and gelatin molecules, formation of hydrogen bonds during swelling and amide bonds during chemical crosslinking in unpurified (A), (B), (C) and purified gels (D), (E), (F).