Pfeifer João Pedro Hübbe, Stievani Fernanda de Castro, Fernandes Célio J da Costa, Rosa Gustavo Dos Santos, Apolonio Emanuel Vitor Pereira, Rossi Mariana Correa, Zambuzzi Willian Fernando, Alves Ana Liz Garcia
Regenerative Medicine Lab, Veterinary Surgery and Animal Reproduction Department, School of Veterinary Medicine and Animal Science, São Paulo State University - UNESP, Botucatu, Brazil.
Biophysics and Pharmacology Department, Institute of Biosciences, São Paulo State University - UNESP, Botucatu, Brazil.
Front Bioeng Biotechnol. 2024 Aug 7;12:1416694. doi: 10.3389/fbioe.2024.1416694. eCollection 2024.
In osteoarthritis (OA), articular homeostasis is regulated by microRNA-140 that inhibits ADAMTS-5, an enzyme that cleaves aggrecan and stimulates the synthesis of other inflammatory mediators. This study aims to evaluate the expression of microRNA-140 in extracellular vesicles (EVs) derived from equine synovial-membrane-derived mesenchymal stem cells (eqSMMSCs) cultured in monolayer (2D) and three-dimensional (3D) culture models under an inflammatory environment.
Four experimental groups of eqSMMSC cultures were defined for isolation of the EVs. The 2D and 3D control groups were cultured in a conventional cell culture medium, while the 2D-OA and 3D-OA treatment groups were exposed to an OA-like medium containing IL-1β and TNFα. The culture media samples were collected at 24 h, 72 h, and 120 h time points for EV isolation and characterization using nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). Reverse transcription quantitative polymerase chain reaction was employed to assess the expressions of microRNA-140 in both the cells and EVs. All statistical analyses were conducted at the 5% significance level.
Encapsulation of the eqSMMSCs protected the cells from the inflammatory media compared to the monolayer cultures. EVs were found in higher concentrations in the 3D-OA cultures. Additionally, higher expressions of microRNA-140 were observed in the cells of the 3D-OA group at 24 and 72 h, whereas microRNA-140 expressions in the EVs were higher in the 3D group at 72 h and in the 2D-OA group at 120 h ( < 0.001). However, the 3D-OA culture showed higher expression of the mRNA in the EVs at 120 h.
The responses of the eqSMMSCs to inflammatory stimuli involve intracellular expression of microRNA-140 and its subsequent transportation via the EVs, with quicker responses observed in the 3D than 2D cultures. This study sheds light on the behaviors of stem cells in restoring homeostasis in osteoarthritic joints.
在骨关节炎（OA）中，微小RNA - 140调节关节内环境稳定，它可抑制ADAMTS - 5，ADAMTS - 5是一种能切割聚集蛋白聚糖并刺激其他炎症介质合成的酶。本研究旨在评估在炎症环境下，单层（2D）和三维（3D）培养模型中培养的马滑膜间充质干细胞（eqSMMSCs）衍生的细胞外囊泡（EVs）中微小RNA - 140的表达情况。
定义四组eqSMMSC培养实验组用于分离EVs。2D和3D对照组在常规细胞培养基中培养，而2D - OA和3D - OA处理组暴露于含有IL - 1β和TNFα的类OA培养基中。在24小时、72小时和120小时时间点收集培养基样本，用于通过纳米颗粒跟踪分析（NTA）和透射电子显微镜（TEM）分离和鉴定EVs。采用逆转录定量聚合酶链反应评估细胞和EVs中微小RNA - 140的表达。所有统计分析均在5%显著性水平下进行。
与单层培养相比，eqSMMSCs的包封保护细胞免受炎症培养基的影响。在3D - OA培养物中发现EVs浓度更高。此外，在24小时和72小时时，3D - OA组细胞中观察到更高的微小RNA - 140表达，而在72小时时3D组的EVs中微小RNA - 140表达更高，在120小时时2D - OA组的EVs中微小RNA - 140表达更高（<0.001）。然而，在120小时时，3D - OA培养物的EVs中mRNA表达更高。
eqSMMSCs对炎症刺激的反应涉及微小RNA - 140的细胞内表达及其随后通过EVs的转运，3D培养比2D培养观察到更快的反应。本研究揭示了干细胞在恢复骨关节炎关节内环境稳定中的行为。
