A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro
A Bruch's membrane substitute fabricated from silk fibroin supports the function of retinal pigment epithelial cells in vitro
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DOI：
10.1002/term.2089
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发表时间：
2017-06-01
影响因子：
3.3
通讯作者：
Harkin, Damien G.
中科院分区：
文献类型：
--
作者：
Shadforth, Audra M. A.;Suzuki, Shuko;Harkin, Damien G.
丝素蛋白为眼组织重建提供了一种有前途的生物材料，包括患有年龄相关性黄斑变性（AMD）的患者受损的外血视网膜屏障。本研究的目的是在体外评估视网膜色素上皮（RPE）细胞在厚度与布鲁赫膜（3μm）相似的丝心蛋白膜上生长时的功能。在丝心蛋白膜上建立 RPE 细胞 (ARPE-19) 的汇合培养物，并在旨在促进成熟的条件下维持 4 个月以上。对照培养物在聚酯细胞培养孔插入物(Transwell(R))上生长。在这两种材料上建立的培养物在 12 周内就形成了鹅卵石形态，并有部分色素沉着。 16 周时的免疫细胞化学显示，F-肌动蛋白、ZO-1、埃兹蛋白、细胞角蛋白对 8/18、RPE-65 和 Na+/K+-ATP 酶在培养物之间具有相似的分布模式。电子显微镜显示，在丝心蛋白上生长的培养物显示出更圆的顶端表面和更密集的微绒毛分布。两种培养物都大量摄入涂有玻连蛋白和牛血清白蛋白 (BSA) 的荧光微球，但仅涂有 BSA 的对照则不然。在从两种膜类型上方和下方收集的条件培养基中检测到 VEGF 和 PEDF。两种膜上的 PEDF 水平均显着高于 VEGF，并且观察到心尖区室中 PEDF 含量较高的趋势。这些发现表明，丝心蛋白膜上的 RPE 细胞功能与标准测试材料（聚酯膜）中观察到的功能相同。因此，这些研究支持临床前模型中 RPE 细胞植入丝心蛋白膜上的研究的进展。版权所有 (c) 2015 约翰·威利父子有限公司
Silk fibroin provides a promising biomaterial for ocular tissue reconstruction, including the damaged outer blood-retinal barrier of patients afflicted with age-related macular degeneration (AMD). The aim of the present study was to evaluate the function of retinal pigment epithelial (RPE) cells in vitro, when grown on fibroin membranes manufactured to a thickness similar to that of Bruch's membrane (3 mu m). Confluent cultures of RPE cells (ARPE-19) were established on fibroin membranes and maintained under conditions designed to promote maturation over 4months. Control cultures were grown on polyester cell culture well inserts (Transwell((R))). Cultures established on either material developed a cobblestone morphology, with partial pigmentation, within 12weeks. Immunocytochemistry at 16weeks revealed a similar distribution pattern between cultures for F-actin, ZO-1, ezrin, cytokeratin pair 8/18, RPE-65 and Na+/K+-ATPase. Electron microscopy revealed that cultures grown on fibroin displayed a rounder apical surface with a more dense distribution of microvilli. Both cultures avidly ingested fluorescent microspheres coated with vitronectin and bovine serum albumin (BSA), but not controls coated with BSA alone. VEGF and PEDF were detected in the conditioned media collected from above and below the two membrane types. Levels of PEDF were significantly higher than for VEGF on both membranes and a trend was observed towards larger amounts of PEDF in apical compartments. These findings demonstrated that RPE cell functions on fibroin membranes are equivalent to those observed for standard test materials (polyester membranes). As such, these studies support advancement to studies of RPE cell implantation on fibroin membranes in a preclinical model. Copyright (c) 2015 John Wiley & Sons, Ltd.