Synthesis and characterization of hybrid hyaluronic acid-gelatin hydrogels

Abstract

Biomimetic hybrid hydrogels have generated broad interest in tissue engineering and regenerative medicine. Hyaluronic acid (HA) and gelatin (hydrolyzed collagen) are naturally derived polymers and biodegradable under physiological conditions. Moreover, collagen and HA are major components of the extracellular matrix (ECM) in most of the tissues (e.g., cardiovascular, cartilage, neural). When used as a hybrid material, HA-gelatin hydrogels may enable mimicking the ECM of native tissues. Although HA-gelatin hybrid hydrogels are promising biomimetic substrates, their material properties have not been thoroughly characterized in the literature. Herein, we generated hybrid hydrogels with tunable physical and biological properties by using different concentrations of HA and gelatin. The physical properties of the fabricated hydrogels including swelling ratio, degradation, and mechanical properties were investigated. In addition, in vitro cellular responses in both two and three-dimensional culture conditions were assessed. It was found that the addition of gelatin methacrylate (GelMA) into HA methacrylate (HAMA) promoted cell spreading in the hybrid hydogels. Moreover, the hybrid hydrogels showed significantly improved mechanical properties compared to their single component analogs. The HAMA-GelMA hydrogels exhibited remarkable tunability behavior and may be useful for cardiovascular tissue engineering applications.

Figure 1

Mass swelling ratio of HAMA-GelMA hybrid hydrogels at different concentrations. The swelling behavior of HAMA-GelMA hybrid hydrogels was tunable (NA: Not applicable, error bars: ±SD, ***p<0.001).

Figure 2

Degradation of HAMA-GelMA hybrid hydrogels at different concentrations by 2.5 U/mL collagenase. The increase in the concentration of GelMA degrades the gels slower demonstrating the tunable degradation behavior of HAMA-GelMA hybrid hydrogels.

Figure 3

Mechanical characterization of HAMA-GelMA hybrid hydrogels at different concentrations. The compressive moduli for HAMA-GelMA hybrid hydrogels are found to be mechanically tunable (NA: Not applicable, error bars: ±SD, ***p<0.001).

Figure 4

Cytoskeleton and nuclei staining (F-actin/DAPI) for HUVEC-seeded HAMA-GelMA hybrid hydrogels in 2D and quantification of cell spreading on the hybrid hydrogels. Percent (%) area occupied by the cells on day 3 was calculated at different conditions. a-c) The hydrogels were crosslinked at different UV exposure times and seeded with different cell densities to study tunability of cell spreading (data is taken at day 3). Scale bars represent 100 um; d) UV time: 30 sec, cell density: 0.6×10⁵ cells/cm²; e) UV time: 120 sec, cell density: 0.6×10⁵ cells/cm²; f) UV time: 120 sec, cell density: 1.8×10⁵ cells/cm² (NA: Not applicable, error bars: ±SD, * p<0.05, **p<0.01, and ***p<0.001).

Figure 5

Fluorescent imaging for HUVEC-laden HAMA-GelMA hybrid hydrogels in 3D. Cell spreading data is given for days 0, 3, and 7. Scale bars represent 100 um.

Figure 6

Cytoskeleton and nuclei staining (F-actin/DAPI) for HUVEC-laden HAMA-GelMA hybrid hydrogels in 3D. Cell spreading images are taken at day 7. Scale bars represent 100 um.

Figure 7

Proliferation of HUVECs within HAMA-GelMA hybrid hydrogels at different hydrogel conditions. Alamar Blue values are provided as the fluorescence reading at 544/590 nm (Ex/Em) (error bars: ±SD, **p<0.01, and ***p<0.001).