Enzyme-mediated crosslinking of silk fibroin/ N-acetylcysteine-modified chitosan/hydroxyapatite hydrogel composites for subchondral bone regeneration.

Composite hydrogels based on N-acetylcysteine-modified chitosan (CHS-NAC), silk fibroin (SF), and hydroxyapatite (HAp) were developed via horseradish peroxidase (HRP)-mediated enzymatic crosslinking for subchondral bone regeneration. The novelty of the system lies in integrating NAC-modified chitosan, characterised by reactive -SH groups and antioxidant activity, with SF and HAp within a single enzymatically crosslinked matrix, while directly comparing synthesised (HAp-syn) and commercial (HAp-com) mineral phases. CHS-NAC contained 225.35 μmol g-1 -SH groups and exhibited antioxidant activity of 13.91 ± 0.71 μg Trolox equivalents per g-1of materials. Although SF reduced antioxidant activity by ∼25%, incorporating either HAp restored it to levels comparable to CHS-NAC. The hydrogels exhibited interconnected porosity, composition-dependent swelling, stability in simulated physiological media, and predominantly elastic behaviour (G' > G″). HAp markedly increased rheological stiffness, with CHS-NAC_SF_5HAp-syn showing the highest G' and the greatest critical strain among HAp-containing composites. HAp-syn also enabled more controlled modulation of surface roughness than HAp-com. Biological evaluation confirmed the absence of sustained cytotoxic effects and progressive MC3T3-E1 proliferation over 7 days. Osteogenic performance was assessed by ALP activity at 7, 14, and 21 days, Alizarin Red S mineralisation, and quantification of OCN and RUNX2. CHS-NAC_SF_5HAp-syn showed the highest mineralisation, while selected HAp-com formulations produced the highest OCN levels. NBT assays further revealed composition- and cell phenotype-dependent modulation of superoxide anion generation in THP-1 monocytes and macrophages. Overall, composites incorporating HAp-syn showed the most balanced combination of controlled surface properties, rheological reinforcement, no sustained cytotoxic effects, and enhanced mineralisation, supporting their potential for subchondral bone regeneration.