Temporal regulation of ascorbic acid synergizes with matrix microenvironment directs chondrogenic commitment and limits hypertrophic differentiation.

The avascular and alymphatic nature of articular cartilage severely limits its intrinsic repair capacity. Even when spontaneous healing occurs, it inevitably culminates in fibrocartilage formation, which lacks the biomechanical functionality of native hyaline cartilage. Mesenchymal stem cells (MSCs) have emerged as a promising therapeutic candidate owing to their regenerative potential. However, direct intra-articular transplantation exposes MSCs to a hostile microenvironment characterized by excessive reactive oxygen species and pro-inflammatory cytokines, leading to extensive apoptosis and substantially compromised therapeutic efficacy. Cell-derived decellularized extracellular matrix (dE) has been shown to enhance chondrogenic potential, yet it fails to suppress hypertrophic differentiation during chondrogenic induction, a major hurdle in cartilage engineering. To overcome this bottleneck, we integrated ascorbic acid (AA) with dE preconditioning. Notably, concurrent dE and AA combination synergistically enhanced antioxidant capacity during expansion, while significantly attenuating hypertrophic markers and matrix catabolism (MMP13, IL-1β) during chondrogenic induction. Mechanistically, this strategy sustained TGF-β receptor I expression alongside endogenous TGF-β1 ligand downregulation, optimizing canonical signaling without pathological overactivation. Strikingly, antecedent AA priming reversed these benefits, compromising chondrogenic potential and exacerbating hypertrophy during chondrogenic induction, underscoring a stringent temporal dependency. Collectively, this biomaterial-guided preconditioning strategy generates chondroprogenitors with reduced hypertrophic markers and improved hyaline phenotype in vitro, warranting further preclinical in vivo investigation.