Comparative analysis of scaffold-free tissue-engineered cartilage derived from different seed cells: in vitro and in vivo studies

Objective The development of cartilage tissue engineering offers a promising strategy for effective cartilage regeneration. This study aims to synthesize scaffold-free tissue-engineered cartilage (TEC) using articular chondrocytes (ACs), costal chondrocytes (CCs), bone marrow mesenchymal stem cells (BMSCs), and synovial-derived mesenchymal stromal cells (SDSCs) as seed cells, comparing their cartilage matrix production in vitro and repair capabilities in vivo to guide seed cell selection. Methods ACs, CCs, BMSCs, and SDSCs were seeded at high density (4.0 × 105 cells/cm2) in 24-well plates with 0.2 mM Ascorbate 2-phosphate and cultured to form scaffold-free TEC. In vitro, we performed real-time polymerase chain reaction (RT-qPCR), biological analyses, and histochemical staining to assess structural characteristics and cartilage-related gene expression. In vivo, a rat model of knee cartilage defect was used to evaluate tissue repair capabilities, followed by histological assessments and statistical analysis. Results In vitro, ACs and CCs showed significantly higher GAG/DNA ratios, with CCs exhibiting the highest chondrogenic gene expression. Histological analysis revealed AC and CC TECs were positive for Safranin-O, Alcian blue, and Col2 staining. In vivo, CC-derived TEC maintained a uniform distribution of Col2, while BMSCs and SDSCs primarily showed Col1 distribution, demonstrating better integration with cartilage and in situ differentiation by 12 weeks. Conclusion ACs and CCs excelled in cartilage matrix synthesis; however, AC TEC exhibited phenotypic instability. CC TEC sustained a hyaline chondrocyte phenotype but had later connectivity issues, while BMSCs and SDSCs integrated better but primarily produced fibrous tissue.