Department of Health Sciences, University of Jaén, Jaén E-23071, Spain.
Osteoarthritis Cartilage. 2013 Jan;21(1):246-58. doi: 10.1016/j.joca.2012.10.007. Epub 2012 Oct 18.
Infrapatellar fat pad of patients with osteoarthritis (OA) contains multipotent and highly clonogenic adipose-derived stem cells that can be isolated by low invasive methods. Moreover, nuclear and cytoplasmic cellular extracts have been showed to be effective in induction of cell differentiation and reprogramming. The aim of this study was to induce chondrogenic differentiation of autologous mesenchymal stem cells (MSCs) obtained from infrapatellar fat pad (IFPSCs) of patients with OA using cellular extracts-based transdifferentiation method.
IFPSCs and chondrocytes were isolated and characterized by flow cytometry. IFPSCs were permeabilized with Streptolysin O and then exposed to a cell extract obtained from chondrocytes. Then, IFPSCs were cultured for 2 weeks and chondrogenesis was evaluated by morphologic and ultrastructural observations, immunologic detection, gene expression analysis and growth on 3-D poly (dl-lactic-co-glycolic acid) (PLGA) scaffolds.
After isolation, both chondrocytes and IFPSCs displayed similar expression of MSCs surface makers. Collagen II was highly expressed in chondrocytes and showed a basal expression in IFPSCs. Cells exposed to chondrocyte extracts acquired a characteristic morphological and ultrastructural chondrocyte phenotype that was confirmed by the increased proteoglycan formation and enhanced collagen II immunostaining. Moreover, chondrocyte extracts induced an increase in mRNA expression of chondrogenic genes such as Sox9, L-Sox5, Sox6 and Col2a1. Interestingly, chondrocytes, IFPSCs and transdifferentiated IFPSCs were able to grow, expand and produce extracellular matrix (ECM) on 3D PLGA scaffolds.
We demonstrate for the first time that extracts obtained from chondrocytes of osteoarthritic knees promote chondrogenic differentiation of autologous IFPSCs. Moreover, combination of transdifferentiated IFPSCs with biodegradable PLGA 3D scaffolds can serve as an efficient system for the maintenance and maturation of cartilage tissue. These findings suggest its usefulness to repair articular surface in OA.
骨关节炎（OA）患者的髌下脂肪垫含有多能且高克隆性的脂肪来源干细胞，可通过微创方法分离。此外，已证明核和细胞质细胞提取物在诱导细胞分化和重编程方面有效。本研究旨在使用基于细胞提取物的转分化方法诱导 OA 患者髌下脂肪垫（IFPSCs）来源的自体间充质干细胞（MSCs）的软骨分化。
通过流式细胞术分离和表征 IFPSCs 和软骨细胞。用链球菌溶血素 O 透化 IFPSCs，然后使其暴露于从软骨细胞获得的细胞提取物中。然后，将 IFPSCs 培养 2 周，并通过形态和超微结构观察、免疫检测、基因表达分析以及在 3D 聚（DL-丙交酯-共-乙交酯）（PLGA）支架上的生长来评估软骨生成。
分离后，软骨细胞和 IFPSCs 均表现出相似的间充质干细胞表面标志物表达。胶原蛋白 II 在软骨细胞中高度表达，并在 IFPSCs 中呈现基础表达。暴露于软骨细胞提取物的细胞获得了特征性的形态和超微结构软骨细胞表型，这通过增加糖胺聚糖形成和增强胶原蛋白 II 免疫染色得到证实。此外，软骨细胞提取物诱导软骨形成基因如 Sox9、L-Sox5、Sox6 和 Col2a1 的 mRNA 表达增加。有趣的是，软骨细胞、IFPSCs 和转分化的 IFPSCs 能够在 3D PLGA 支架上生长、扩增和产生细胞外基质（ECM）。
我们首次证明，从 OA 膝关节软骨细胞获得的提取物促进自体 IFPSCs 的软骨分化。此外，转分化的 IFPSCs 与可生物降解的 3D PLGA 支架的组合可作为维持和成熟软骨组织的有效系统。这些发现表明其在修复 OA 关节表面方面的有用性。
