Abstract: BACKGROUND: In the repair of large bone defects, a variety of factors such as seed cell passaging can cause senescence of osteoblasts, leading to a reduction in osteogenic differentiation activity after implantation of tissue-engineered bone. In recent years, a novel mechanism involving primary cilia in cell senescence has been widely studied, but the primary cilia-related mechanism of "passage senescence - reduced osteogenic activity" is not fully understood.
OBJECTIVE: To explore the possible mechanisms by which primary cilia regulate the senescence of MC3T3-E1 cells.
METHODS: The osteoblast precursor cell lines MC3T3-E1 were passaged to 10th generation cells (early passage) and 40th generation cells (late passage). siRNA was used to silence IFT88 to inhibit primary cilia formation. The cells were than grouped into passage 10 group, passage 40 group, passage 10+siRNA IFT88 group, and passage 40+siRNA IFT88 group. RT-PCR and western blot assays were used to detect the expression of the aging marker P16 (CDKN2A), the osteogenic activity markers bone morphogenetic protein 2 and alkaline phosphatase, and the Hedgehog pathway IHH expression. Alizarin red staining and primary cilia immunofluorescence staining were performed. Spearman correlation analysis was conducted to analyze primary cilia positive rate and IHH and bone morphogenetic protein 2 expression. 
RESULTS AND CONCLUSION: (1) The expression of CDKN2A (P16) in the passage 10 group was significantly higher than in the passage 40 group, but the difference disappeared after siRNA IFT88 intervention. (2) Meanwhile, the positive rate of primary cilia cells in the passage 10 group were higher than in the passage 40 group, while siRNA IFT88- significantly inhibited the expression of primary cilia in both passage 10 and passage 40 cells. (3) The transcriptional activity and protein expression of bone morphogenetic protein 2 and alkaline phosphatase in the passage 10 group were higher than those in the passage 40 group. After inhibiting the expression of primary cilia with siRNA, the above differences were reduced or disappeared. (4) The positive rate of primary cilia cells was correlated with IHH and bone morphogenetic protein 2 protein expression. To conclude, primary cilia mediate the replicative senescence of osteogenic MC3T3-E1 cells and regulate osteogenic differentiation ability. 

中国组织工程研究杂志出版内容重点：组织构建；骨细胞；软骨细胞；细胞培养；成纤维细胞；血管内皮细胞；骨质疏松；组织工程

Key words: MC3T3-E1 cells, primary cilia, senescence, osteogenic differentiation, passage, bone morphogenetic protein 2, Hedgehog pathway, tissue- engineered cells