Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Bld 75, Cnr Cooper and College Rds, Queensland 4072, Australia.
Biomaterials. 2011 Aug;32(24):5600-14. doi: 10.1016/j.biomaterials.2011.04.033. Epub 2011 May 17.
Due to its limited healing potential within the inner avascular region, functional repair of the meniscus remains a significant challenge in orthopaedic surgery. Tissue engineering of a meniscus implant using meniscal cells offers the promise of enhancing the reparative process and achieving functional meniscal repair. In this work, using quantitative real-time reverse transcriptase polymerase chain reaction (RT-qPCR) analysis, we show that human fibrochondrocytes rapidly dedifferentiate during monolayer expansion on standard tissue culture flasks, representing a significant limit to clinical use of this cell population for meniscal repair. Previously, we have characterized and described the feasibility of a tailored biomimetic surface (C6S surface) for reversing dedifferentiation of monolayer-expanded rat meniscal cells. The surface is comprised of major meniscal extracellular matrix (ECM) components in the inner region, namely collagen I/II (at a 2:3 ratio) and chondroitin-6-sulfate. We thus have further evaluated the effects of the C6S surface, alongside a number of other tailored surfaces, on cell adhesion, proliferation, matrix synthesis and relevant marker gene expression (collagen I, -II, aggrecan and Sox-9 etc) of passaged human fibrochondrocytes in 2D (coated glass coverslips) and 3D (surface-modified polymeric scaffolds) environments. We show that the C6S surface is permissive for cell adhesion, proliferation and ECM synthesis, as demonstrated using DNA quantification, 1,9-dimethylmethylene blue (DMMB) assay, histology and immunohistochemistry. More importantly, RT-qPCR analyses corroborate the feasibility of the C6S surface for reversing phenotypic changes, especially the downregulation of collagen II, of dedifferentiated human fibrochondrocytes. Furthermore, human fibrochondrocyte redifferentiation was enhanced by hypoxia in the 3D cultures, independent of hypoxia inducible factor (HIF) transcriptional activity and was shown to potentially involve the transcriptional activation of Sox-9.
由于半月板内的血管区域有限，其功能修复仍然是骨科手术中的一个重大挑战。使用半月板细胞对半月板植入物进行组织工程学处理，有望增强修复过程并实现功能性半月板修复。在这项工作中，我们通过定量实时逆转录聚合酶链反应（RT-qPCR）分析表明，人纤维软骨细胞在标准组织培养瓶的单层扩张过程中迅速去分化，这是临床应用该细胞群体进行半月板修复的一个重大限制。此前，我们已经对定制仿生表面（C6S 表面）逆转单层扩增的大鼠半月板细胞去分化的特性和可行性进行了描述和表征。该表面由内层主要的半月板细胞外基质（ECM）成分组成，即胶原 I/II（比例为 2:3）和硫酸软骨素-6-硫酸盐。因此，我们进一步评估了 C6S 表面以及其他一些定制表面对传代人纤维软骨细胞在 2D（涂层玻璃盖玻片）和 3D（表面改性聚合物支架）环境中的细胞黏附、增殖、基质合成和相关标记基因表达（胶原 I、-II、聚集蛋白聚糖和 Sox-9 等）的影响。我们表明，C6S 表面有利于细胞黏附、增殖和 ECM 合成，这可以通过 DNA 定量、1,9-二甲基亚甲蓝（DMMB）测定、组织学和免疫组织化学来证明。更重要的是，RT-qPCR 分析证实了 C6S 表面逆转去分化人纤维软骨细胞表型变化的可行性，尤其是胶原 II 的下调。此外，3D 培养中的缺氧增强了人纤维软骨细胞的再分化，而与缺氧诱导因子（HIF）转录活性无关，并显示出可能涉及 Sox-9 的转录激活。
