The College of Stomatology, Guangxi Medical University, Nanning, 530021, China.
Biomater Sci. 2018 May 29;6(6):1556-1568. doi: 10.1039/c8bm00317c.
Sox9 is a transcription factor that regulates chondrogenesis, but its role in the chondrogenic differentiation of mesenchymal stem cells (MSCs) triggered by materials is poorly understood. In this study, we investigated the effect of Sox9 interference on collagen-induced chondrogenesis and further collagen-based therapies for cartilage defects. In this paper, MSCs were infected with a vector carrying the Sox9 promoter and related markers were detected. A lentivirus-mediated vector targeting the silencing of the Sox9 gene was used in bone marrow-derived MSCs prior to being encapsulated in a collagen hydrogel. The collagen hydrogel as a sole inducer was also compared with transforming growth factor-β1 (TGF-β1). Before being implanted into the articular cartilage defect in rats, the cell-hydrogel pellets were cultured in vitro for 14 days. The effect of Sox9 transfection on cell proliferation was evaluated by measuring the total DNA content. Safranin-O staining and a biochemistry assay were performed to assess the synthesis and secretion of glycosaminoglycan (GAG) of MSCs. The real-time fluorescent quantitative polymerase chain reaction (RT-PCR) was performed to detect the gene expression levels of Col1a1, Col2a1, Acan and Sox9. The protein expression of collagen type II and collagen type I was analyzed by immunohistochemical analysis. Collagen alone significantly increased the luciferase activity of the Sox9 promoter, which was in parallel with the upregulation of cartilage specific markers. In vitro, the chondrogenic differentiation ability of MSCs was greatly inhibited after Sox9 interference, both in the collagen and TGF-β1-induced groups. In vivo, a further study showed that cartilage regeneration was arrested by using transfected MSCs with an injectable collagen gel or induced by TGF-β1. The results indicated that collagen may mediate Sox9 expression by providing a biomimetic microenvironment favoring cell condensation prior to chondrogenesis. The role of Sox9 regulation by materials is similar to that by growth factors, suggesting that well-designed scaffolds may replace growth factors in chondrogenesis. Thus, interventions targeting Sox9 may help improve articular cartilage repair.
Sox9 是一种转录因子，可调节软骨生成，但人们对其在材料引发的间充质干细胞（MSCs）软骨分化中的作用知之甚少。在这项研究中，我们研究了 Sox9 干扰对胶原诱导软骨生成的影响，以及进一步用胶原基疗法治疗软骨缺损。本文中，我们通过感染携带 Sox9 启动子和相关标记物的载体来检测 MSCs。在骨髓来源的 MSCs 中，我们使用慢病毒介导的 Sox9 基因沉默载体，然后将其包裹在胶原水凝胶中。我们还将胶原水凝胶与转化生长因子-β1（TGF-β1）进行了比较。在将细胞-水凝胶球植入大鼠关节软骨缺损之前，我们在体外培养它们 14 天。通过测量总 DNA 含量来评估 Sox9 转染对细胞增殖的影响。我们通过番红 O 染色和生化分析来评估 MSCs 糖胺聚糖（GAG）的合成和分泌。我们通过实时荧光定量聚合酶链反应（RT-PCR）检测 Col1a1、Col2a1、Acan 和 Sox9 的基因表达水平。我们通过免疫组化分析来分析Ⅱ型和Ⅰ型胶原的蛋白表达。单独的胶原显著增加了 Sox9 启动子的荧光素酶活性，这与软骨特异性标记物的上调呈平行关系。在体外，Sox9 干扰后 MSCs 的软骨分化能力大大受到抑制，无论是在胶原和 TGF-β1 诱导的组中。在体内，进一步的研究表明，使用可注射胶原凝胶或 TGF-β1 诱导转染的 MSCs 会导致软骨再生停滞。结果表明，胶原可能通过提供有利于细胞凝聚的仿生微环境来介导 Sox9 的表达，然后启动软骨生成。材料对 Sox9 的调节作用与生长因子相似，这表明设计良好的支架可能替代生长因子在软骨生成中的作用。因此，针对 Sox9 的干预措施可能有助于改善关节软骨修复。
