<i>N</i>-Acetylcysteine and pro-adrenomedullin dual-crosslinked gelatin–chitosan hydrogels with enhanced mechanical and mineralization performance

Bone regeneration requires coordination between bone formation, vascularization, and inflammatory regulation. However, current biomaterials often fail to provide mechanical stability and sustained bioactivity while supporting cell viability. This study presents the development and characterization of hydrogels composed of methacrylated gelatin (GelMA) and chitosan methacrylate (ChMA), crosslinked by photopolymerization (GC hydrogels). These were evaluated for their mineralization potential <i>in vitro</i> and <i>ex vivo</i> when loaded with <i>N</i>-acetylcysteine (NAC), a bioactive antioxidant (GCN); a pro-angiogenic peptide derived from adrenomedullin (PAMP, GCP); or both compounds (GCNP). FT-IR spectroscopy confirmed successful polymer methacrylation and the interaction of NAC with the polymer network. Scanning electron microscopy revealed that NAC increased the pore size from 24.49 ± 14.19 μm (GC) to 200.49 ± 80.42 μm (GCN). NAC also enhanced mechanical performance, with GCN exhibiting the highest compressive strength (151.79 ± 44.81 kPa) and GCNP the highest stiffness (Young's modulus: 55.26 ± 5.79 kPa). NAC-containing hydrogels degraded faster than GC, enabling biphasic release over 14 days. <i>In vitro</i> and <i>ex vivo</i> assays using pre-osteoblastic cells and a calvarial defect model demonstrated that GCNP hydrogels significantly enhanced cell viability and mineralization, increasing calcium deposition by 2.5-fold compared to GC (<i>p</i> < 0.01). These findings suggest that NAC not only reinforces the mechanical strength of hydrogel scaffolds designed for temporary support in non-load-bearing bone defects, but also acts as a bioactive agent upon release. Its combination with the pro-adrenomedullin peptide (PAMP) results in synergistic effects on mineralization. GCNP hydrogels are therefore promising candidates for drug delivery and bone tissue regeneration.