To prepare the substrates with different stiffnesses, 3 g of gelatin (type A, Sigma-Aldrich, USA) was dissolved in 10 ml of ultrapure water at 50°C under stirring to obtain a 30 weight % (wt %) gelatin aqueous solution. Microbial transglutaminase (0.4 g; mTG, Pangbo, China) was dissolved in 10 ml of ultrapure water at room temperature to obtain a 4 wt % aqueous solution. Gelatin and mTG aqueous solutions were filtered and then mixed with different concentration, respectively (gelatin aqueous solution: 10, 15, 10, 10, 15, and 20%; mTG aqueous solution: 0.5, 0.5, 1.5, 2, 2, and 2%) in petri dishes at 37°C for 3 hours to obtain hydrogels with gradient stiffness of 5, 15, 25, 45, 65, and 125 kPa, respectively. The stiffness was measured by an atomic force microscope (Agilent Technologies, USA). After soaking in phosphate-buffered saline for 24 hours, 3T3 cells were seeded onto gelatin hydrogels at a concentration of 1 × 106 cells/ml.

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