A significant expansion of autologous chondrocytes in vitro is required for cell-based cartilage repair. However, the in vitro expansion of chondrocytes under standard culture conditions inevitably leads to the dedifferentiation of chondrocytes and contributes to suboptimal clinical outcomes. To address this challenge, we focused our efforts on developing an improved in vitro expansion protocol, which shortens the expansion time with decreased dedifferentiation. It is known that the tissue microenvironment plays a critical role in regulating the cellular functions of resident cells and provides guidance in tissue-specific regeneration. We hypothesized that chondrocyte extracellular matrix (ECM) mimics a native microenvironment and that it may support chondrocyte expansion in vitro. To test this hypothesis, we prepared decellularized ECMs from allogeneic human articular chondrocytes (HAC) (AC-ECM) and bone marrow stromal cells (BM-ECM) and studied their effects on the in vitro expansion of primary HAC. The differential composition and physical properties of these two ECMs were revealed by mass spectrometry and atomic force microscopy. Compared with standard tissue culture polystyrene (TCP) or BM-ECM, HAC cultured on AC-ECM proliferated faster and maintained the highest ratio of COL2A1/COL1A1. Furthermore, a pellet culture study demonstrated that cells expanded on AC-ECM produced a more cartilage-like ECM than cells expanded on BM-ECM or TCP. This is the first report on modulating chondrocyte expansion and dedifferentiation using cell type-specific ECM and on identifying AC-ECM as a preferred substrate for in vitro expansion of HAC cell-based therapies. STATEMENT OF SIGNIFICANCE: To reduce the dedifferentiation of chondrocytes during in vitro expansion, cell type-specific extracellular matrix (ECM), which mimics a native microenvironment, was prepared from human articular chondrocytes (AC-ECM) or bone marrow stromal cells (BM-ECM). As demonstrated by mass spectrometry and atomic force microscopy, AC-ECM and BM-ECM have differential ECM compositions and physical characteristics. Human articular chondrocytes (HAC) expanded faster and maintained a better chondrocyte phenotype on AC-ECM than on BM-ECM or a standard culture surface. AC-ECM has potential to be developed for expanding HAC for cell-based therapies.

中文翻译：

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源自软骨细胞的细胞外基质可促进人原代软骨细胞在体外快速扩增，并具有减少的去分化能力。

基于细胞的软骨修复需要体外自体软骨细胞的显着扩增。但是，在标准培养条件下软骨细胞的体外扩增不可避免地导致软骨细胞去分化，并导致临床效果欠佳。为了应对这一挑战，我们将精力集中在开发改进的体外扩增方案上，该方案可缩短扩增时间并减少去分化的时间。已知组织微环境在调节驻留细胞的细胞功能中起关键作用，并在组织特异性再生中提供指导。我们假设软骨细胞细胞外基质（ECM）模仿一个自然的微环境，它可能支持体外软骨细胞扩增。为了检验这个假设，我们从同种异体人类关节软骨细胞（HAC）（AC-ECM）和骨髓基质细胞（BM-ECM）中制备了脱细胞的ECM，并研究了它们对原代HAC体外扩增的影响。质谱和原子力显微镜揭示了这两种ECM的不同组成和物理性质。与标准组织培养聚苯乙烯（TCP）或BM-ECM相比，在AC-ECM上培养的HAC增殖更快，并保持最高的COL2A1 / COL1A1比例。此外，一项沉淀培养研究表明，在AC-ECM上扩增的细胞比在BM-ECM或TCP上扩增的细胞产生更多的软骨样ECM。这是有关使用细胞类型特异性ECM调节软骨细胞扩增和去分化以及鉴定AC-ECM作为基于HAC细胞的体外疗法的优选底物的第一份报道。意义声明：为了减少体外扩增过程中软骨细胞的去分化，从人关节软骨细胞（AC-ECM）或骨髓基质细胞（BM- ECM）。正如质谱和原子力显微镜所证明的，AC-ECM和BM-ECM具有不同的ECM组成和物理特性。与BM-ECM或标准培养表面相比，AC-ECM上的人关节软骨细胞（HAC）扩张更快，并保持更好的软骨表型。