N‐Acetylcysteine‐Loaded Scaffolds and Their Role in Extracellular ROS and Redox‐Related Gene Regulation in Chondrocytes

ABSTRACT Oxidative stress is a key driver of articular cartilage degeneration, disrupting chondrocyte redox homeostasis and the extracellular matrix (ECM). N‐acetylcysteine (NAC) can modulate both extracellular and intracellular redox balance, making it a promising therapeutic agent for cartilage damage. However, delivery of NAC to the damaged cartilage site is limited. Therefore, this study incorporates NAC into polycaprolactone (PCL) fibers, aiming to regulate ROS levels and the genes related to glutathione production and transsulfuration pathways. The scaffolds containing NAC at concentrations of 0%, 0.5%, and 1% (w/v) are fabricated via electrospinning and characterized using FTIR, DPPH assay, and mechanical testing. For in vitro assessments, primary bovine chondrocytes are isolated and cultured on scaffolds for 21 days. The results demonstrate that NAC maintains its stability after exposure to environmental factors. Furthermore, NAC addition significantly increases the antioxidant activity of scaffolds while affecting the fiber diameter and mechanical properties. Seeded chondrocytes maintain viability for 21 days, showcasing no toxic effects of NAC. Critically, gene expression analysis reveals that 1% NAC scaffolds significantly upregulate GCLC, GSS, CBS, and CTH genes, indicating potential activation of the glutathione and transsulfuration pathways. These findings highlight a promising strategy for using redox‐modulating antioxidants in future regenerative medicine applications.