Grdgspc

Four-arm PEG-4MAL macromer (molecular weight, 22,000 or 10,000; Laysan Bio) was dissolved in 1× phosphate-buffered saline (PBS) containing 10 mM Hepes (pH 7.4). Cell adhesive peptides (GRGDSPC, GRDGSPC, CGGEGYGEGYIGSR, and CGGKAFDITYVRLKF; >95% purity; GenScript) were dissolved in 1× PBS containing 10 mM Hepes and added to PEG-4MAL solution to produce functionalized PEG-4MAL precursors. Freshly isolated MuSCs were then added to the solution containing functionalized PEG-4MAL precursors. To synthesize cell-encapsulated hydrogels, the solution containing functionalized PEG-4MAL precursors and cells was mixed with protease-degradable cross-linking peptide (GCRDVPMSMRGGDRCG; GenScript) or nondegradable hexa(ethylene glycol) dithiol (Sigma-Aldrich) dissolved in 1× PBS containing 10 mM Hepes and subsequently polymerized at 37°C/5% CO₂ for 5 min before adding MuSC growth media (F10 containing 1% penicillin/streptomycin, 1% GlutaMAX, and 20% horse serum). Recombinant human FGF-2 (25 ng ml⁻¹; PeproTech) was supplemented daily. To prime the MuSCs to differentiate, the growth medium was replaced with differentiation media (DMEM containing 1% penicillin/streptomycin, 1% GlutaMAX, and 2% horse serum) on days 5 and 6 of culture. Cells were cultured in the differentiation media for an additional 4 days.