Application of retinoic acid improves form and function of tissue engineered corneal construct.
Application of retinoic acid improves form and function of tissue engineered corneal construct.
复制标题
视黄酸的应用改善了组织工程角膜结构的形式和功能。
DOI：
10.1080/15476278.2015.1093267
复制
发表时间：
2015
期刊：
影响因子：
2.3
通讯作者：
Connon CJ
中科院分区：
文献类型：
--
作者：
Abidin FZ;Gouveia RM;Connon CJ
维甲酸最近被证明可以控制体外培养的单层角膜基质细胞的表型和细胞外基质组成。本研究旨在研究维甲酸在3D环境中对人角膜基质细胞的影响。将人角膜基质细胞包裹在胶原凝胶中，然后在负荷下压缩，并在添加10µM维甲酸或DMSO载体的无血清培养液中培养30天。用RT-PCR和免疫印迹法检测30天时几种重要的角膜细胞标志物的表达。在水合和收缩分析前后测量胶原结构的重量和大小。维甲酸可促进角膜基质细胞的增殖，直到第30天，而对照培养条件下的细胞在第21天后数量减少。补充维甲酸后，角质细胞特有的蛋白多糖(角蛋白聚糖、鲁米肯和核心蛋白)、角膜晶体蛋白以及I型和V型胶原的基因和蛋白表达均显著增加。维甲酸还显著降低基质金属蛋白酶1、3和9的表达，而不增加α-平滑肌肌动蛋白和纤维连接蛋白的表达。此外，这些效应还与维甲酸显著抑制角质细胞收缩的能力有关，同时允许3D结构内角膜基质细胞外基质的建立。因此，补充维甲酸是一种很有前途的策略，可以改善3D培养的角膜基质细胞的表型，并将其用作角膜生物学和再生医学应用的角膜基质模型。
Retinoic acid has recently been shown to control the phenotype and extracellular matrix composition of corneal stromal cells cultured in vitro as monolayers. This study set out to investigate the effects of retinoic acid on human corneal keratocytes within a 3D environment. Human corneal keratocytes were encapsulated in collagen gels, which were subsequently compressed under load, and cultured in serum-free media supplemented with 10 µM retinoic acid or DMSO vehicle for 30 days. Cell proliferation was quantified on selected days, while the expression of several important keratocytes markers was evaluated at day 30 using RT-PCR and immunoblotting. The weight and size of the collagen constructs were measured before and after hydration and contraction analyses. Retinoic acid enhanced keratocyte proliferation until day 30, whereas cells in control culture conditions showed reduced numbers after day 21. Both gene and protein expressions of keratocyte-characteristic proteoglycans (keratocan, lumican and decorin), corneal crystallins and collagen type I and V were significantly increased following retinoic acid supplementation. Retinoic acid also significantly reduced the expression of matrix metalloproteases 1, 3 and 9 while not increasing α-smooth muscle actin and fibronectin expression. Furthermore, these effects were also correlated with the ability of retinoic acid to significantly inhibit the contractility of keratocytes while allowing the build-up of corneal stromal extracellular matrix within the 3D constructs. Thus, retinoic acid supplementation represents a promising strategy to improve the phenotype of 3D-cultured keratocytes, and their usefulness as a model of corneal stroma for corneal biology and regenerative medicine applications.