Advancing Cartilage Regeneration: Hypoxia and Alginate Sulfate Preserve Chondrocyte Phenotype by Promoting ECM Production and Reducing Inflammation

Abstract

Articular cartilage's limited regenerative capacity poses challenges in treating degenerative diseases like osteoarthritis (OA). Autologous chondrocyte transplantation (ACT) offers potential for cartilage repair by reintroducing healthy chondrocytes into damaged tissue. However, chondrocytes often lose their phenotype during expansion on tissue culture plastic (TCP), limiting their regenerative efficacy. This study explores the synergistic effects of basic fibroblast growth factor (FGF-2) and alginate sulfate films under hypoxic conditions to optimize in vitro conditions for preserving the chondrocyte phenotype during expansion. Results showed that alginate sulfate significantly reduces proliferation, prioritizing matrix production over cell division compared to alginate (p < 0.05). It also increases total collagen content (collagen types 1 and 2) under hypoxia (p < 0.05) and reduces catabolic factors such as IL-1β, IL-6, and MMP-13, indicating its role in maintaining cartilage integrity. Conversely, FGF-2 significantly enhances proliferation, nearly doubling cell numbers (p < 0.05), and induces a fibroblastic shift under hypoxia by increasing collagen type I, decreasing collagen type II, and elevating catabolic factors (p < 0.05). Hypoxia reduced proliferation by nearly half (p < 0.05) but promoted a chondrogenic phenotype. It dedifferentiated late-passaged chondrocytes (P3), making them morphologically resemble early-passaged chondrocytes (P1). Hypoxia also enhanced total collagen content and suppressed inflammatory factors when combined with alginate sulfate (p < 0.05), indicating a protective effect on chondrocyte phenotype. These findings suggest that the optimal condition for chondrocyte expansion while maintaining phenotype is alginate sulfate under hypoxic conditions without FGF-2. This approach balances collagen production, enhances cartilage-specific extracellular matrix (ECM), reduces inflammation, and preserves cellular morphology, making it a promising strategy for cartilage tissue engineering.