Effects of serial expansion of septal chondrocytes on tissue-engineered neocartilage composition

OBJECTIVES: Cartilage grafts for reconstructive surgery may someday be created from harvested autologous chondrocytes that are expanded and seeded onto biodegradable scaffolds in vitro. This study sought to quantify the biochemical composition of neocartilage engineered from human septal chondrocytes and to examine the effects of cell multiplication in monolayer culture on the ultimate composition of the neocartilage. METHODS: Human septal chondrocytes from 10 donors were either seeded immediately after harvest (passage 0 (P-0)) onto polyglycolic acid (PGA) scaffolds or underwent multiplication in monolayer culture before scaffold seeding at passage 1 (P-1) and passage 2 (P-2). Cell/scaffold constructs were grown in vitro for 7, 14, and 28 days. Neocartilage constructs underwent histologic analysis for matrix sulfated glycosaminoglycan (S-GAG) and type II collagen as well as quantitative assessment of cellularity (Hoescht 33258 assay), S-GAG content (dimethylmethylene blue assay), and collagen content (hydroxyproline assay). RESULTS: Histologic sections of constructs seeded with P-0 cells stained strongly for S-GAG and type II collagen, whereas decreased staining for both matrix components was observed in constructs derived from P-1 and P-2 cells. Cellularity, S-GAG content, and total collagen content of constructs increased significantly from day 7 to day 28. S-GAG accumulation in P-0 constructs was higher than in either P-1 (P < 0.05) or P-2 (P < 0.01) constructs, whereas cellularity and total collagen content showed no difference between passages. CONCLUSION: Neocartilage created from chondrocytes that have undergone serial passages in monolayer culture exhibited decreased matrix S-GAG and type II collagen, indicative of cellular dedifferentiation. SIGNIFICANCE: The alterations of matrix composition produced by dedifferentiated chondrocytes may limit the mechanical stability of neocartilage constructs.