MeHA macromolecules were synthesized from sodium hyaluronate powder (molecular weight, ~74 kDa; Lifecore, Chaska, Minnesota, USA), as previously reported (8). Briefly, 100 ml of 1% (w/v) sodium hyaluronate solution was reacted for 24 hours with 4 or 1.5 ml of methacrylic anhydride at pH 9.5, adjusted with 2 M NaOH solution. After complete dialysis and lyophilization, 100% methacrylation or 30% methacrylation was confirmed using proton nuclear magnetic resonance (1H NMR). The RGD peptide (GCGYGRGDSPG) and Foxy5 peptide (MDGCEL) (GenScript, Nanjing, Jiangsu, China) with a cysteine amino acid at the C-terminal end were conjugated to the MeHA backbone with a Michael addition reaction between the methacrylate groups and the thiol groups of each peptide in basic phosphate buffer (pH 8.0) containing 10 μM tris(2-carboxyethyl)phosphine at 37°C. The molar ratio of methacrylate to each peptide thiol was 100:3. RGD-functionalized, Foxy5-conjugated porous MeHA hydrogels were fabricated from 50 μl of the peptide-conjugated MeHA solution (3% w/v, 100% methacrylation) after 2 hours, with 1.51 μmol of DTT as the cross-linker to consume all residual methacrylic groups, in round polyvinyl chloride molds fully packed with a poly(methyl methacrylate) (PMMA) microsphere porogen (Ø 200 μm). The constructs generated were immersed in acetone and shaken at 90 rpm to dissolve the PMMA porogen, sterilized with 75% ethanol for 1 day, and rinsed three times with sterile phosphate-buffered saline (PBS). In the directly Foxy5 peptide–supplemented MeHA (free Foxy5) group and the directly scrambled peptide–supplemented MeHA (free Scram) group, we only used the same Foxy5 + RGD–functionalized MeHA solution or Scram + RGD–functionalized MeHA solution (3% w/v, 100% methacrylation) to fabricate the porous hydrogels, respectively.

We used MeHA with 30% methacrylation supplemented with 0.05% (w/v) photoinitiator I2959 (Sigma-Aldrich, MO, USA) to make Foxy5 + RGD–functionalized MeHA solution, Scram + RGD–functionalized MeHA solution, or RGD-functionalized MeHA solution (3% w/v, 30% methacrylation) and to fabricate 2D hydrogels with different stiffness in polyvinyl chloride molds under 367, 467, 667, or 1067 seconds of UV exposure. All 2D hydrogels were sterilized before cell culture. The surface roughness and modulus were determined by atomic force microscopy (Bruker, MA, USA).

2D-biofunctionalized substrates were fabricated by polymerizing peptide-conjugated MeHA precursor solutions (3% w/v, 30% methacrylation) under UV light (wavelength, 365 nm; intensity, 7 mW/cm2) on methacrylated glass coverslips. The porous MeHA hydrogel constructs (height, ~1.5 mm; Ø 1 mm) were polymerized in molds filled with Ø 200-μm PMMA microbeads to form an interconnected porous structure for in vitro experiments and for in vivo calvarial defect regeneration (Fig. 1, B and C) (9). Homogenous, interconnected spherical structures within the hydrogels were characterized through bright-field images captured using a fluorescence microscope (Nikon, Japan) (Fig. 1D). The Young moduli of the hydrogels were determined using a mechanical tester (Mach-1, Biomomentum Inc., Suite, Canada), and the strain-controlled frequency sweep mechanical tests were performed using a rheometer (Malvern Inc., Malvern, Britain).

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