3D cell entrapment in crosslinked thiolated gelatin-poly(ethylene glycol) diacrylate hydrogels

Abstract

The combined use of natural ECM components and synthetic materials offers an attractive alternative to fabricate hydrogel-based tissue engineering scaffolds to study cell-matrix interactions in three-dimensions (3D). A facile method was developed to modify gelatin with cysteine via a bifunctional PEG linker, thus introducing free thiol groups to gelatin chains. A covalently crosslinked gelatin hydrogel was fabricated using thiolated gelatin and poly(ethylene glycol) diacrylate (PEGdA) via thiol-ene reaction. Unmodified gelatin was physically incorporated in a PEGdA-only matrix for comparison. We sought to understand the effect of crosslinking modality on hydrogel physicochemical properties and the impact on 3D cell entrapment. Compared to physically incorporated gelatin hydrogels, covalently crosslinked gelatin hydrogels displayed higher maximum weight swelling ratio (Q(max)), higher water content, significantly lower cumulative gelatin dissolution up to 7 days, and lower gel stiffness. Furthermore, fibroblasts encapsulated within covalently crosslinked gelatin hydrogels showed extensive cytoplasmic spreading and the formation of cellular networks over 28 days. In contrast, fibroblasts encapsulated in the physically incorporated gelatin hydrogels remained spheroidal. Hence, crosslinking ECM protein with synthetic matrix creates a stable scaffold with tunable mechanical properties and with long-term cell anchorage points, thus supporting cell attachment and growth in the 3D environment.

Figure 1

Scaffold structure of covalently crosslinked Gel-PEG-Cys hydrogel via thiol-ene photopolymerization (A) and crosslinked PEGdA hydrogel with physically incorporated gelatin (B).

Figure 2

Synthesis scheme of Gel-PEG-Cys

Figure 3

GPC Chromatogram of Gel-PEG-Cys (dark grey solid line), unmodified gelatin (light grey solid line), and PEG-diol 3.4 KDa (black dash line).

Figure 4

Swelling and degradation profiles of physically incorporated gelatin hydrogels (A and C) versus covalently crosslinked Gel-PEG-Cys hydrogels (B and D) at 37 °C. (n=3)

Figure 5

Gelatin dissolution behavior over time. Physically incorporated gelatin hydrogels (A, PEGdA 575 Da; C, PEGdA 3400 Da) versus covalently crosslinked Gel-PEG-Cys hydrogels (B, PEGdA 575 Da; D, PEGdA 3400 Da) at 37 °C. Data represented as mean ± S.D. (n=3).

Figure 6

Bulk rheology of covalently crosslinked gelatin-based hydrogels G_cys10P₃₄₀₀10, G_cys10P₃₄₀₀15, G_cys10P₃₄₀₀20, G_cys10P₅₇₅10, G_cys10P₅₇₅15, G_cys10P₅₇₅20 and physically incorporated gelatin hydrogels G10P₃₄₀₀10, G10P₃₄₀₀15, G10P₃₄₀₀20, G10P₅₇₅10, G10P₅₇₅15, G10P₅₇₅20 at R.T. and 37 °C. Shear storage modulus G′ at R.T. (●), 37 °C ( ); loss modulus G″ at R.T. (○), 37 °C (◊). *, significantly different compared to corresponding nonswollen formulation with p < 0.05.

Figure 7

Bulk rheology of covalently crosslinked gelatin-based hydrogels (A) and physically incorporated gelatin hydrogels (C) at swollen state. Shear storage modulus G′ of G_cys10P₃₄₀₀10 and G10P₃₄₀₀10 ( ), G_cys10P₃₄₀₀15 and G10P₃₄₀₀15 (●), G_cys10P₃₄₀₀20 and G10P₃₄₀₀20 ( ); loss modulus G″ of G_cys10P₃₄₀₀10 and G10P₃₄₀₀10 (◊), G_cys10P₃₄₀₀15 and G10P₃₄₀₀15 (○), G_cys10P₃₄₀₀20 and G10P₃₄₀₀20 (□). Complex shear moduli of covalently crosslinked Gel-PEG-Cys hydrogels (B) and physically incorporated gelatin hydrogels (D). *, significantly different compared to corresponding nonswollen formulation with p < 0.05.

Figure 8

Bulk rheology of covalently crosslinked gelatin hydrogel G_cys10P₃₄₀₀10 (A) and physically incorporated gelatin hydrogel G10P₃₄₀₀10 (B) with 60 min of swelling in PBS at R.T. Shear storage modulus G′ and loss modulus G″.

Figure 9

(A) Viability and morphology of human dermal fibroblasts encapsulated in hydrogels. Covalently crosslinked gelatin hydrogels increase cell cytoplasmic spreading in 3D environment. Fibroblasts were stained with calcein-AM for live cell (green) and EthD for dead cell (red). (Magnification 10×) (B) Normalized live cell percentages of encapsulated fibroblasts in G0P₃₄₀₀15 (■), G10P₃₄₀₀15 (□), G_cys10P₃₄₀₀15 ( ) at various culture times. (C) Fibroblasts proliferation in 3D conditions at day 1 (■) and day 7 (□) of culture. Fluorescence intensities of cells entrapped in covalently crosslinked G_cys10P₃₄₀₀15 hydrogels and physically incorporated G10P₃₄₀₀15 hydrogels were normalized to that of PEGdA-only hydrogel G0P₃₄₀₀15. *, significantly different with p < 0.05.

Figure 10

2D adherent fibroblasts on hydrogel surfaces fabricated using different crosslinking modalities, including G0P₃₄₀₀15, G10P₃₄₀₀15, and G_cys10P₃₄₀₀15. (Magnification 10×)

Figure 11

Morphology of fibroblasts encapsulated within G_cys10P₃₄₀₀15, G10P₃₄₀₀15, and G0P₃₄₀₀15 after 14 d. A: F-actin (green) and vinculin (red) were stained and imaged with CLSM. B: co-localization of F-actin and Integrin α5β1.