Abstract
Background: Obtaining sufficient chondrocytes by monolayer expansion in vitro is used for articular cartilage tissue engineering. However, chondrocytes lose their chondrogenic phenotype after monolayer expansion via mitochondrial dysfunction-induced senescence. Adipose-derived stem cell mitochondrial transfer (ADSC-MT) improves senescent cell function. We hypothesise that ADSC-MT improves the chondrogenic phenotype of senescent chondrocytes. Methods: After monolayer expansion in vitro, chondrocytes were subjected to ADSC-MT. Cell senescence was evaluated via analysis of p16 and p21 expression and senescence-associated β-galactosidase (SA-β-gal) staining. The chondrogenic phenotype was evaluated by measuring collagen type II (Col-II) and collagen type I (Col-I) levels. Oxidative stress was assessed by determining the mitochondrial superoxide and 8-hydroxydeoxyguanosine (8-OHdG) levels. Mitochondrial dysfunction was assessed by determining the mitochondrial membrane potential (MMP) and PGC-1α levels. Finally, SOD-2, SIRT-1, SIRT-3, TFAM, MFN-1, MFN-2, OPA-1, PINK-1 and Parkin levels were used to assess mitochondrial quality control (MQC). Results: ADSC-MT-recipient chondrocytes exhibited alleviated senescence with decreased p16 and p21 expression and SA-β-gal staining. The increased Col-II and decreased Col-I expression indicated that the chondrogenic phenotype of the chondrocytes was restored. Decreased mitochondrial superoxide and 8-OHdG levels indicated alleviated oxidative stress. The increased MMP indicated alleviation of mitochondrial dysfunction. For MQC, SOD‑2, PGC‑1α, TFAM, SIRT-1, and SIRT-3 were upregulated, indicating that antioxidant defences and mitochondrial biogenesis in MQC were increased in ADSC‑MT–recipient chondrocytes. PINK‑1 and Parkin were downregulated, suggesting that damaged mitochondria were reduced through mitophagy. In contrast, MFN‑1, MFN‑2, and OPA‑1 were not changed, indicating that mitochondrial dynamics were not affected. Conclusions: ADSC-MT improves the chondrogenic phenotype of senescent chondrocytes by ameliorating mitochondrial dysfunction.