Fluorescently Labeled Gradient Hydrogels Reveal Matrix-Dependent Cell Responses to Substrate Stiffness

Area of Science:

• Biomaterials Science
• Cell Biology
• Tissue Engineering

Background:

• Microfabricated stiffness gradient hydrogels are valuable for mechanobiology and tissue engineering.
• Designing these materials is challenging due to complex processing and difficulties in correlating substrate properties with biological responses.
• Current characterization methods like atomic force microscopy are often cumbersome.

Purpose of the Study:

• To develop a straightforward fabrication strategy for patterning stiffness gradients in hydrogels.
• To enable quantitative assessment and contactless stiffness mapping of gradient hydrogels.
• To investigate the combined effects of substrate stiffness and extracellular matrix composition on cell behavior.

Main Methods:

• A thermophoresis-based fabrication strategy was employed to create stiffness gradients.
• Fluorescein isothiocyanate-labeled hydrogels allowed for polymer concentration-dependent fluorescence readout.
• Standard microscopy imaging was used for quantitative assessment and contactless stiffness mapping.

Main Results:

• The thermophoresis method successfully patterned stiffness gradients in gelatin methacryloyl and Gellan gum hydrogels.
• Quantitative assessment and contactless stiffness mapping of the gradient formation process were achieved.
• Substrate stiffness and extracellular matrix composition were shown to influence 3T3-L1 fibroblast cell morphology and migration, with hydrogel type also playing a role.

Conclusions:

• This work presents a simple, reliable method for fabricating and characterizing stiffness gradient hydrogels.
• The thermophoretic fabrication platform is advanced, offering new possibilities for biomaterial systems.
• The findings contribute to a better understanding and control of cell-material interactions in engineered environments.