Associate Professor of Biomedical Engineering Saint Louis University, United States
Introduction:: Three-dimensional (3D) tissue-engineered scaffolds mimic the physiological environment of cells by providing the essential structural support, biochemical cues, and the mechanical strength needed for cell adhesion, proliferation, migration, and differentiation. Hydrogels like polyethylene glycol diacrylate (PEGDA) are commonly used biomaterials for cell culture due to their affordability, wide range of stiffness, and ability to efficiently transport nutrients and gases. However, PEGDA lacks cell adhesion sites essential for cell proliferation and migration and has limited degradability. Methacrylated gelatin (GelMA), produced from denatured bovine collagen, crosslinks under UV and provides a degradable hydrogel with cell binding sites. Here, we synthesized GelMA with robust degree of methacrylation and coupled GelMA-PEGDA to produce cell scaffolds with tunable mechanical and biochemical properties by varying the ratios of the copolymers. Importantly, we determined polymer ratios that resulted in scaffolds with different mechanical properties but same gelatin (providing cell adhesion and degradation sites) concentrations or different gelatin concentrations but the same mechanical properties. The developed scaffolds are excellent platforms for mechanosensing research in 3D environments.
Materials and Methods::
GelMA was synthesized following the protocol developed by Van Den Bulk et al. Briefly GelMA was synthesized by the metacrylation of bovine gelatin with methacrylic anhydride (MA). A high degree of methacrylation was achieved by adding 10% (w/v) MA to gelatin at a rate of 0.5 mL/min under stirring at 60°C for 2 h and then dialysis for 7 days. Methacrylation was confirmed with 1H nuclear magnetic resonance (Bruker). GelMA-PEGDA hydrogels were crosslinked under UV using Irgacure as the photoinitiator at 0.1% (w/v). Hydrogel mechanical properties were characterized via rheology (TA Instruments), swelling ratio was measured as the ratio of the wet gel mass to dry gel mass, degradation (mass loss) was followed in a 0.2 mg/mL collagenase solution, and gel morphology was imaged under scanning electron microscopy (SEM; Bruker). U87 human glioblastoma cells were seeded at 5x105 cells/mL by adding them directly to the hydrogel precursor solution. Cell viability was measured via live/dead staining (cell tracker green/PI) and cell morphology (spreading area) was measured from confocal microscopy (Leika) images using ImageJ software. Fluorescence correlation spectroscopy (FCS) was performed to study the mesh size, interconnectivity of pores, and transport properties through the hydrogels.
Results, Conclusions, and Discussions:: We tested different ratios of GelMa-PEGDA hydrogel (2%, 5%, and 10% w/v GelMA with either 0%, 3%, 5%, and 10% w/v PEGDA) that provided a stiffness ranging from ~E=3.7 kPa to ~ E=35 kPa as shown in Fig.1A. We specifically focus on hydrogels with the same stiffness but different amounts of adhesion sites: G2P3and G10 for soft gels and G2P10 and G10P10 for the stiff gels. Using the same four conditions, we can study the effect of same concentration of adhesion sites but different stiffness on cell fate. This design of easy and accessible tunable hydrogel systems allows us to interrogate the interplay between biochemical and mechanical properties and how they affect cell spreading, proliferation, and migration. SEM images show that increasing the ratio of GelMA resulted in higher pore size and that the addition of PEGDA led to a slight decrease of hydrogel pore size due to the crosslinking of PEGDA, hence the inhomogeneous pore size in G2P10 and G10P10 (Fig1B). The encapsulation of U87 cells in the GelMA-PEGDA gels showed a uniform cell spreading throughout the scaffold and a high cell viability >90% over 14 days of culture (Fig.1C). Confocal images of U87 encapsulated in G10 show spreading at day 14 (Fig.1D).Current work is focused on evaluating cell morphology as a function of the interplay between gel mechanical and biochemical properties.
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