Chronic cellular senescence contributes to fibrocartilage repair in a rat model of bone marrow stimulation

Bone marrow stimulation techniques are frequently used to treat focal lesions in articular cartilage, yet poor long-term outcomes necessitate further study into the mechanisms driving inferior fibrocartilage repair. Cellular senescence has been implicated in the progression of post-traumatic osteoarthritis, but its role in pre-osteoarthritic cartilage repair is unclear. Our objective was to determine whether senescent cells contribute to fibrocartilage repair using a rodent model of marrow stimulation.Design: A 1 mm osteochondral defect was generated in the trochlear groove of young adult and middle-aged Lewis rats, modeling the age range when humans receive marrow stimulation. Some rats were treated by intra-articular (IA) injection of senolytic drugs navitoclax or dasatinib plus quercetin. We demonstrated expression of multiple senesence markers within repair tissue throughout the first months of repair, though not within adjacent, intact articular cartilage. The IA delivery of senolytic drugs reduced p16Ink4a-immunopositive cell numbers, although the selected regimens did not enhance chondrogenesis. The number of these cells were similar between young adult and middle-aged rats; in contrast, proteoglycan deposition decreased markedly with increased age. Bulk RNA sequencing (RNA-seq) of middle-aged repair tissue demonstrated sustained upregulation of factors associated with the senescence-associated secretory phenotype. Single cell RNA-seq revealed heterogeneity in mesenchymal cells within the early repair tissue, confirmed differences in senescence marker expression between newly-formed chondrocytes and those within adjacent intact cartilage, and established differences in oxidative stress response between these populations.