Lunenfeld-Tanenbaum Research Institute, Toronto, Canada.
Institute of Biomaterials and Biomedical Engineering, University of Toronto, Toronto, Canada.
Tissue Eng Part A. 2021 Sep;27(17-18):1140-1150. doi: 10.1089/ten.TEA.2020.0120. Epub 2021 Feb 10.
Transforming growth factor beta (TGFβ) signaling is required for chondrogenesis. In animal models of osteoarthritis (OA), TGFβ receptor alterations are detected in chondrocytes in severe OA cartilage. It is not known whether such changes are dependent on the grade of human OA and if they affect chondrogenesis. Thus, the purpose of this study was to determine if human OA chondrocytes obtained from low-grade or high-grade disease could form cartilage tissue and to assess the role of the co-receptors, endoglin (ENG) and TGFβ receptor 3 (TGFBRIII), in the regulation of this tissue generation . We hypothesized that the grade of OA disease would not affect the ability of cells to form cartilage tissue and that the TGFβ co-receptor, ENG, would be critical to regulating tissue formation. Chondrocytes isolated from low-grade OA or high-grade OA human articular cartilage (AC) were analyzed directly (P0) or passaged in monolayer to P2. Expression of the primary TGFβ receptor ALK5, and the co-receptors ENG and TGFβRIII, was assessed by image flow cytometry. To assess the ability to form cartilaginous tissue, cells were placed in three-dimensional culture at high density and cultured in chondrogenic media containing TGFβ3. ENG knockdown was used to determine its role in regulating tissue formation. Overall, grade-specific differences in expression of ALK5, ENG, and TGFβRIII in primary or passaged chondrocytes were not detected; however, ENG expression increased significantly after passaging. Despite the presence of ALK5, P0 cells did not form cartilaginous tissue. In contrast, P2 cells derived from low-grade and high-grade OA AC formed hyaline-like cartilaginous tissues of similar quality. Knockdown of ENG in P2 cells inhibited cartilaginous tissue formation compared to controls indicating that the level of ENG protein expression is critical for chondrogenesis by passaged articular chondrocytes. This study demonstrates that it is not the grade of OA, but the levels of ENG in the presence of ALK5 that influences the ability of human passaged articular chondrocytes to form cartilaginous tissue in 3D culture. This has implications for cartilage repair therapies. Impact statement These findings are important clinically, given the limited availability of osteoarthritis (OA) cartilage tissue. Being able to use cells from all grades of OA will increase our ability to obtain sufficient cells for cartilage repair. In addition, it is possible that endoglin (ENG) levels, in the presence of ALK5 expression, may be suitable to use as biomarkers to identify cells able to produce cartilage.
转化生长因子-β（TGFβ）信号对于软骨形成是必需的。在骨关节炎（OA）的动物模型中，在严重 OA 软骨的软骨细胞中检测到 TGFβ 受体改变。尚不清楚这些变化是否依赖于人类 OA 的程度，以及它们是否影响软骨形成。因此，本研究的目的是确定低等级或高等级疾病的人 OA 软骨细胞是否能够形成软骨组织，并评估共受体 ENG（内皮糖蛋白）和 TGFβ 受体 3（TGFBRIII）在调节这种组织生成中的作用。我们假设 OA 疾病的程度不会影响细胞形成软骨组织的能力，并且 TGFβ 共受体 ENG 将是调节组织形成的关键。从低等级 OA 或高等级 OA 人关节软骨（AC）中分离的软骨细胞直接（P0）或在单层中传代至 P2 进行分析。通过图像流动细胞术评估主要 TGFβ 受体 ALK5 以及共受体 ENG 和 TGFβRIII 的表达。为了评估形成软骨组织的能力，将细胞在三维高密度培养物中放置并用含有 TGFβ3 的软骨形成培养基培养。使用 ENG 敲低来确定其在调节组织形成中的作用。总体而言，在原代或传代软骨细胞中未检测到 ALK5、ENG 和 TGFβRIII 在特定于等级的表达上的差异；然而，ENG 表达在传代后显著增加。尽管存在 ALK5，但 P0 细胞未形成软骨组织。相比之下，源自低等级和高等级 OA AC 的 P2 细胞形成了具有相似质量的透明样软骨组织。与对照相比，P2 细胞中 ENG 的敲低抑制了软骨组织的形成，表明共受体 ENG 蛋白表达水平对于传代关节软骨细胞的软骨形成至关重要。这项研究表明，影响人传代关节软骨细胞在 3D 培养中形成软骨组织的能力的不是 OA 的等级，而是存在 ALK5 时的 ENG 水平。这对软骨修复疗法具有重要意义。影响声明鉴于骨关节炎（OA）软骨组织的有限可用性，这些发现具有重要的临床意义。能够使用所有等级的 OA 细胞将增加我们获得足够的软骨修复细胞的能力。此外，存在 ALK5 表达时，内皮糖蛋白（ENG）水平可能适合用作鉴定能够产生软骨的细胞的生物标志物。
