The mechanical properties and cytotoxicity of cell-laden double-network hydrogels based on photocrosslinkable gelatin and gellan gum biomacromolecules

Abstract

A major goal in the application of hydrogels for tissue engineering scaffolds, especially for load-bearing tissues such as cartilage, is to develop hydrogels with high mechanical strength. In this study, a double-network (DN) strategy was used to engineer strong hydrogels that can encapsulate cells. We improved upon previously studied double-network (DN) hydrogels by using a processing condition compatible with cell survival. The DN hydrogels were created by a two-step photocrosslinking using gellan gum methacrylate (GGMA) for the rigid and brittle first network, and gelatin methacrylamide (GelMA) for the soft and ductile second network. We controlled the degree of methacrylation of each polymer so that they obtain relevant mechanical properties as each network. The DN was formed by photocrosslinking the GGMA, diffusing GelMA into the first network, and photocrosslinking the GelMA to form the second network. The formation of the DN was examined by diffusion tests of the large GelMA molecules into the GGMA network, the resulting enhancement in the mechanical properties, and the difference in mechanical properties between GGMA/GelMA single networks (SN) and DNs. The resulting DN hydrogels exhibited the compressive failure stress of up to 6.9 MPa, which approaches the strength of cartilage. It was found that there is an optimal range of the crosslink density of the second network for high strength of DN hydrogels. DN hydrogels with a higher mass ratio of GelMA to GGMA exhibited higher strength, which shows promise in developing even stronger DN hydrogels in the future. Three dimensional (3D) encapsulation of NIH-3T3 fibroblasts and the following viability test showed the cell-compatibility of the DN formation process. Given the high strength and the ability to encapsulate cells, the DN hydrogels made from photocrosslinkable macromolecules could be useful for the regeneration of load-bearing tissues.

Fig. 1

Synthesis scheme of (A) gellan gum methacrylate (GGMA) (pictured as above for simplicity, although methacrylic anhydride can react with any hydroxyl group in gellan gum) and (B) gelatin methacrylamide (GelMA). (C) Fabrication of DN hydrogels through a two-step photocrosslinking process.

Fig. 2

Formation of double-network (DN) hydrogels. (A–D) Diffusion of FITC-GelMA molecules into GGMA hydrogels over time. (A) 1hr, (B) 2hrs, (C) 3hrs, (D) 5hrs. Scale bars represent 1 mm. (E) Vertical fluorescence profile of the cross-section of hydrogels over time. (F) Compressive modulus, (G) failure strain, and (H) failure stress of DN hydrogels with varying 2^nd crosslinking time. 0.5% GGMA hydrogels crosslinked for 120 seconds and 20% GelMA(DM: 14.7%) solutions were used for (A)–(H). (*) indicates significant difference (P < 0.05).

Fig. 3

(A) FTIR spectra of GGMA and dried GGMA hydrogels crosslinked for varying time. The shoulder peak appearing around 1640cm⁻¹ corresponds to the unreacted C=C bonds. (B) Stress-strain curves of GGMA/GelMA SN and DN hydrogels with the same mass ratio (GelMA/GGMA = 8.2). Every crosslinking time was 120 seconds, and GelMA (DM: 32.3%) was used. The number in parenthesis refers to the polymer content of the hydrogels.

Fig. 4

(A) Stress-strain curves for SN and DN hydrogels under uniaxial compression. (B) Polymer content, (C) compressive modulus, (D) failure strain, and (E) failure stress of SN and DN hydrogels. (†) indicates the stress under which the majority of GelMA SN gels started to break, and the strain at that stress. Every crosslinking time was 120 seconds, and GelMA (DM: 32.3%) was used for (A)–(E). (*) indicates significant difference (P < 0.05).

Fig. 5

(A) Degree of methacrylation (DM) of GelMA with varying amount of methacrylic anhydride added to the reaction. (Inset in B) Polymer content of DN hydrogels with varying DM of GelMA. Effect of DM of GelMA on (B) compressive modulus, (C) failure strain, and (D) failure stress of DN hydrogels. Every crosslinking time was 120 seconds. 0.5% GGMA hydrogels and 20% GelMA(each DM) solutions were used for (A)–(E). (*) indicates significant difference (P < 0.05).

Fig. 6

Mass ratio (GelMA/GGMA), polymer content, compressive modulus, failure strain, and failure stress of DN hydrogels with varying concentration of either component: (A) varying concentration of GGMA hydrogels + 20% GelMA solution, and (B) 0.5% GGMA hydrogels + varying concentration of GelMA solution. GelMA (DM: 32.3%) was used for (A)–(B). (*) indicates significant difference (P < 0.05).

Fig. 7

Fluorescence images of live/dead stained NIH-3T3 fibroblasts encapsulated in DN hydrogels: (A) day 0 and (B) day 3 of culture after DN hydrogel formation. Scale bars represent 200μm. (C) Viability of 3T3 fibroblasts encapsulated in SN and DN hydrogels. (*) indicates significant difference (P < 0.05). 0.5% GGMA hydrogels and 20% GelMA (DM: 14.7%) solutions were used for (A)–(C).