Squarate Cross-Linked Gelatin Hydrogels as Three-Dimensional Scaffolds for Biomedical Applications

Abstract

Hydrogels are useful platforms as three-dimensional (3D) scaffolds for cell culture, drug-release systems, and regenerative medicine applications. Here, we propose a novel chemical cross-linking approach by the use of 3,4-diethoxy-3-cyclobutene-1,2-dione or diethyl squarate for the preparation of 5 and 10% w/v gelatin-based hydrogels. Hydrogels showed good swelling properties, and the 5% gelatin-based hydrogel proved suitable as a 3D cell culture scaffold for the chondrocyte cell line C28/I2. In addition, diffusion properties of different sized molecules inside the hydrogel were determined.

Figure 1

(a) Homo cross-linking of gelatin by 3,4-diethoxy-3-cyclobutene-1,2-dione (DES) and (b) gelatin cross-linked hydrogel.

Figure 2

(a) Reaction between DES and AP; (b) FTIR spectra in the 1900–800 cm^–1 region; (c) second derivatives of spectra collected in “b”; and (d) relative intensity of the 1804 cm^–1 band.

Figure 3

SEM images of the superficial portion of (a) Gel-DES 5% and (b) Gel-DES 10%.

Figure 4

(a) 5% Gel-DES (full circles) and 10% Gel-DES (full squares) SD_t vs time, full lines are best fit to eq 2; (b) 5% Gel-DES swelling curves normalized to ESD as a function of time, the continuous line is the fit to eq 2. Inset: zoom on the early stages of the swelling kinetic. The continuous line is the best fit according to eq 4.

Figure 5

5% Gel-DES and 10% Gel-DES enzymatic degradation by collagenase type I from C. histolyticum.

Figure 6

Cell colonization of 5% Gel-DES: DAPI emission at 485/30 nm is shown in cyan, and TRIC emission at 600/40 nm is shown in green. Panels a, b: C28/I2 chondrocytes; panels c, d: HEK293 cells. Bar size is 57 μm for panels (a,c), 28 μm for panel (b), and 23 μm for panel (d).

Figure 7

(a) Rhodamine uptake expressed as the ratio between the concentration at time t and the equilibrium concentration as a function of time (black line is the fit curve); (b) Rhodamine release normalized to equilibrium concentration as a function of time.

Figure 8

Rhodamine diffusion in 5% Gel-DES. (a) ACF average curve vs time lag for Rhodamine 6G in water (dotted, black) and in 5% Gel-DES (solid, red); (b) ACF average curve vs time lag for GFP in phosphate buffer (dotted, black) and in 5% Gel-DES (solid, red). The continuous lines represent the best fit with eq 5 in both panels.

Figure 9

GNSs in 5% Gel-DES after 24 h of swelling. The luminescence is detected using an EMCCD camera and promoted by two-photon excitation at 800 nm at 10 mW on the sample. (a–c) Illustrative frames (out of 1000 frames acquired) at three arbitrary times; (d) average intensity over all frames. Field of view, 16 × 16 μm².