Cell for Cells, Santiago, Chile.
Laboratory of Nano-Regenerative Medicine, Faculty of Medicine, Universidad de los Andes, Santiago, Chile.
Theranostics. 2024 Oct 7;14(17):6471-6486. doi: 10.7150/thno.96723. eCollection 2024.
Osteoarthrosis (OA) is a leading cause of disability and early mortality, with no disease modifying treatment. Mitochondrial (MT) dysfunction and changes in energy metabolism, leading to oxidative stress and apoptosis, are main drivers of disease. In reaction to stress, mesenchymal stromal/stem cells (MSCs) donate their MT to damaged tissues. To evaluate the capacity of clinically validated MSCs to spontaneously transfer their MT to human OA chondrocytes (OA-Ch), primary cultured Ch isolated from the articular cartilage of OA patients were co-cultured with MT-labeled MSCs. MT transfer (MitoT) was evidenced by flow cytometry and confocal microscopy of MitoTracker-stained and YFP-tagged MT protein. MT persistence and metabolic analysis on target cells were assessed by direct transfer of MSC-derived MT to OA-Chs (Mitoception), through SNP-qPCR analysis, ATP measurements and Seahorse technology. The effects of MitoT on MT dynamics, oxidative stress and cell viability were gauged by western blot of fusion/fission proteins, confocal image analysis, ROS levels, Annexin V/7AAD and TUNEL assays. Intra-articular injection of MSC-derived MT was tested in a collagenase-induced murine model of OA. Dose-dependent cell-to-cell MitoT from MSCs to cultured OA-Chs was detected starting at 4 hours of co-culture, with increasing MT-fluorescence levels at higher MSC:Ch ratios. PCR analysis confirmed the presence of exogenous MSC-MT within MitoT OA-Chs up to 9 days post Mitoception. MitoT from MSCs to OA-Ch restores energetic status, with a higher ATP production and metabolic OXPHOS/Glycolisis ratio. Significant changes in the expression of MT network regulators, increased MFN2 and decreased p-DRP1, reveal that MitoT promotes MT fusion restoring the MT dynamics in the OA-Ch. Additionally, MitoT increases SOD2 transcripts, protein, and activity levels, and reduces ROS levels, confering resistance to oxidative stress and enhancing resistance to apoptosis. Intra-articular injection of MSC-derived MT improves histologic scores and bone density of the affected joints in the OA mouse model, demonstrating a protective effect of MT transplantation on cartilage degradation. The Mitochondria transfer of MSC-derived MT induced reversal of the metabolic dysfunction by restoring the energetic status and mitochondrial dynamics in the OA chondrocyte, while conferring resistance to oxidative stress and apoptosis. Intra-articular injection of MT improved the disease in collagenase-induced OA mouse model. The restoration of the cellular homeostasis and the preclinical benefit of the intra-articular MT treatment offer a new approach for the treatment of OA.
骨关节炎（OA）是导致残疾和早逝的主要原因，目前尚无疾病修饰治疗方法。线粒体（MT）功能障碍和能量代谢改变，导致氧化应激和细胞凋亡，是疾病的主要驱动因素。为了应对应激，间充质基质/干细胞（MSCs）将其 MT 捐赠给受损组织。为了评估经过临床验证的 MSC 自发将其 MT 转移到人类 OA 软骨细胞（OA-Ch）的能力，从 OA 患者关节软骨中分离的原代培养 Ch 与 MT 标记的 MSC 共培养。通过流式细胞术和共聚焦显微镜对 MitoTracker 染色和 YFP 标记的 MT 蛋白进行分析，证明了 MT 转移（MitoT）。通过 SNP-qPCR 分析、ATP 测量和 Seahorse 技术，直接将 MSC 衍生的 MT 转移到 OA-Ch（Mitoception），评估靶细胞的 MT 持久性和代谢分析。通过融合/裂变蛋白的 Western blot、共聚焦图像分析、ROS 水平、Annexin V/7AAD 和 TUNEL 测定，评估 MitoT 对 MT 动力学、氧化应激和细胞活力的影响。在胶原酶诱导的 OA 小鼠模型中测试了 MSC 衍生的 MT 的关节内注射。从 MSC 到培养的 OA-Ch 的细胞间剂量依赖性 MitoT 从共培养 4 小时开始检测到，随着 MSC:Ch 比例的增加，MT 荧光水平增加。PCR 分析证实，在 Mitoception 后 9 天内，MitoT OA-Ch 中存在外源性 MSC-MT。从 MSC 到 OA-Ch 的 MitoT 恢复能量状态，具有更高的 ATP 产生和代谢 OXPHOS/Glycolisis 比值。MT 网络调节剂的表达发生显著变化，MFN2 增加，p-DRP1 减少，表明 MitoT 促进 MT 融合，恢复 OA-Ch 中的 MT 动力学。此外，MitoT 增加 SOD2 转录本、蛋白质和活性水平，并降低 ROS 水平，赋予氧化应激抗性并增强对细胞凋亡的抗性。MSC 衍生的 MT 的关节内注射改善了 OA 小鼠模型中受影响关节的组织学评分和骨密度，表明 MT 移植对软骨降解具有保护作用。MSC 衍生的 MT 的线粒体转移通过恢复 OA 软骨细胞的能量状态和线粒体动力学来逆转代谢功能障碍，同时赋予氧化应激和细胞凋亡抗性。胶原酶诱导的 OA 小鼠模型中的关节内 MT 注射改善了疾病。细胞内平衡的恢复和关节内 MT 治疗的临床前益处为 OA 的治疗提供了一种新方法。
