Table_1_Senolytic Peptide FOXO4-DRI Selectively Removes Senescent Cells From in vitro Expanded Human Chondrocytes.DOCX

Autologous chondrocyte implantation (ACI) is a procedure used to treat articular cartilage injuries and prevent the onset of post-traumatic osteoarthritis. In vitro expansion of chondrocytes, a necessary step in ACI, results in the generation of senescent cells that adversely affect the quality and quantity of newly formed cartilage. Recently, a senolytic peptide, fork head box O transcription factor 4-D-Retro-Inverso (FOXO4-DRI), was reported to selectively kill the senescent fibroblasts. In this study, we hypothesized that FOXO4-DRI treatment could remove the senescent cells in the expanded chondrocytes, thus enhancing their potential in generating high-quality cartilage. To simulate the in vitro expansion for ACI, chondrocytes isolated from healthy donors were expanded to population doubling level (PDL) 9, representing chondrocytes ready for implantation. Cells at PDL3 were also used to serve as the minimally expanded control. Results showed that the treatment of FOXO4-DRI removed more than half of the cells in PDL9 but did not significantly affect the cell number of PDL3 chondrocytes. Compared to the untreated control, the senescence level in FOXO4-DRI treated PDL9 chondrocytes was significantly reduced. Based on the result from standard pellet culture, FOXO4-DRI pre-treatment did not enhance the chondrogenic potential of PDL9 chondrocytes. However, the cartilage tissue generated from FOXO4-DRI pretreated PDL9 cells displayed lower expression of senescence-relevant secretory factors than that from the untreated control group. Taken together, FOXO4-DRI is able to remove the senescent cells in PDL9 chondrocytes, but its utility in promoting cartilage formation from the in vitro expanded chondrocytes needs further investigation.

自体软骨细胞移植（Autologous chondrocyte implantation, ACI）是一种用于治疗关节软骨损伤、预防创伤后骨关节炎发生的临床手术方案。体外扩增软骨细胞是ACI流程中的关键步骤，但该过程会诱导衰老细胞产生，进而对新生软骨的质量与数量造成不利影响。近期有研究报道，一类衰老裂解肽——叉头框O转录因子4-D-逆转反向肽（FOXO4-DRI）可选择性清除衰老成纤维细胞。本研究提出如下假说：采用FOXO4-DRI处理可清除扩增后的软骨细胞中的衰老细胞，进而提升其合成高质量软骨的潜能。为模拟ACI的体外扩增过程，研究人员将健康供体分离得到的软骨细胞扩增至群体倍增水平（PDL）9，以代表准备用于移植的软骨细胞；同时选取处于PDL3的细胞作为低扩增程度的对照组。实验结果表明，FOXO4-DRI处理可清除PDL9组中超过半数的细胞，但对PDL3组软骨细胞的数量无显著影响。与未处理的对照组相比，经FOXO4-DRI处理的PDL9软骨细胞的衰老水平显著降低。通过标准微团培养实验结果分析可知，FOXO4-DRI预处理并未提升PDL9软骨细胞的成软骨潜能。然而，经FOXO4-DRI预处理的PDL9细胞所生成的软骨组织，其衰老相关分泌因子的表达水平显著低于未处理的对照组。综上，FOXO4-DRI能够清除PDL9软骨细胞中的衰老细胞，但其在促进体外扩增软骨细胞生成功能性软骨方面的应用价值仍需进一步深入研究。