Krishna Lekshmi, Nilawar Sagar, Ponnalagu Murugeswari, Subramani Murali, Jayadev Chaitra, Shetty Rohit, Chatterjee Kaushik, Das Debashish
Stem Cell Research Laboratory, GROW Laboratory, Narayana Nethralaya Foundation, Narayana Nethralaya, Bangalore 560 099, Karnataka, India.
School of Biosciences and Technology, Vellore Institute of Technology, Vellore 632 014, Tamil Nadu, India.
ACS Appl Bio Mater. 2020 Feb 17;3(2):823-837. doi: 10.1021/acsabm.9b00897. Epub 2020 Feb 4.
Biomaterials have significant functions as tissue scaffolds to support cells for regeneration. Nanofibrous scaffolds which mimic the architecture of the extracellular matrix are well suited to support epithelial cells for ocular tissue engineering. This study aimed at investigating the role of scaffold architecture, if any, on the response of ocular epithelial cells. Thus, we have cultured two different types of ocular epithelial cells on nanofibrous scaffolds of two different diameters to evaluate their generic and cell-specific properties. Human adult retinal pigment epithelial (ARPE-19) and human corneal epithelial (HCE-T) cells were cultured on poly(ε-caprolactone) (PCL) nanofibers of different diameters, nominally 500 and 1300 nm. Moduli of the fiber mats were marginally different at 7.4 and 11.1 kPa for 500 and 1300 nm diameter, respectively. The molecular changes in the cells in response to the different fibers were analyzed by qRT-PCR, Western blot, immunofluorescence, ELISA, flow cytometry, MTT assay, and SEM to assess properties such as proliferation, apoptosis, membrane potential, epithelial-mesenchymal transition, stem cell population, VEGF-A secretion, differentiation, and metabolic status of the cells. HCE-T cells revealed characteristic morphology along with higher expression of proliferation, differentiation, and lower apoptotic markers when cultured on PCL nanofibers of 500 nm. However, on nanofibers of 1300 nm, the cells showed higher expression of the corneal stem/progenitor as well as pluripotent stem cell markers. ARPE-19 cells exhibited characteristic hexagonal morphology with elevated expression levels of proliferative markers, phagocytic activity, and lower apoptosis levels. However, on 500 nm nanofibers, they expressed higher levels of pluripotent markers and secretion of VEGF-A. These findings demonstrate that the response can differ markedly from scaffold architecture even if derived from the same tissue and originating from the same germ layer. Furthermore, it paves the way for a target specific outcome and, thereby, for personalized translational medicine.
生物材料作为组织支架具有重要功能，可支持细胞再生。模拟细胞外基质结构的纳米纤维支架非常适合用于眼组织工程中支持上皮细胞。本研究旨在调查支架结构对眼上皮细胞反应的作用（若有）。因此，我们在两种不同直径的纳米纤维支架上培养了两种不同类型的眼上皮细胞，以评估它们的一般特性和细胞特异性特性。将人成年视网膜色素上皮（ARPE - 19）细胞和人角膜上皮（HCE - T）细胞培养在名义直径分别为500和1300 nm的聚（ε-己内酯）（PCL）纳米纤维上。直径为500和1300 nm的纤维垫的模量分别为7.4和11.1 kPa，略有不同。通过qRT - PCR、蛋白质印迹、免疫荧光、酶联免疫吸附测定、流式细胞术、MTT分析和扫描电子显微镜分析细胞对不同纤维的分子变化，以评估细胞的增殖、凋亡、膜电位、上皮 - 间充质转化、干细胞群体、VEGF - A分泌、分化和代谢状态等特性。当在500 nm的PCL纳米纤维上培养时，HCE - T细胞呈现出特征性形态，同时增殖、分化相关标志物表达较高，凋亡标志物表达较低。然而，在1300 nm的纳米纤维上，细胞显示角膜干/祖细胞以及多能干细胞标志物的表达较高。ARPE - 19细胞呈现出特征性的六边形形态，增殖标志物表达水平升高，具有吞噬活性，凋亡水平较低。然而，在500 nm的纳米纤维上，它们表达更高水平的多能标志物和VEGF - A分泌。这些发现表明，即使来自相同组织且起源于相同胚层，细胞对支架结构的反应也可能有显著差异。此外，这为实现靶标特异性结果以及个性化转化医学铺平了道路。
