Salivary gland cell differentiation and organization on micropatterned PLGA nanofiber craters.

There is a need for an artificial salivary gland as a long-term remedy for patients suffering from salivary hypofunction, a leading cause of chronic xerostomia (dry mouth). Current salivary gland tissue engineering approaches are limited in that they either lack sufficient physical cues and surface area needed to facilitate epithelial cell differentiation, or they fail to provide a mechanism for assembling an interconnected branched network of cells. We have developed highly-ordered arrays of curved hemispherical “craters” in polydimethylsiloxane (PDMS) using wafer-level integrated circuit (IC) fabrication processes, and lined them with electrospun poly-lactic-co-glycolic acid (PLGA) nanofibers, designed to mimic the three-dimensional (3-D) in vivo architecture of the basement membrane surrounding spherical acini of salivary gland epithelial cells. These micropatterned scaffolds provide a method for engineering increased surface area and were additionally investigated for their ability to promote cell polarization. Two immortalized salivary gland cell lines (SIMS, ductal and Par-C10, acinar) were cultured on fibrous crater arrays of various radii and compared with those grown on flat PLGA nanofiber substrates, and in 3-D Matrigel. It was found that by increasing crater curvature, the average height of the cell monolayer of SIMS cells and to a lesser extent, Par-C10 cells, increased to a maximum similar to that seen in cells grown in 3-D Matrigel. Increasing curvature resulted in higher expression levels of tight junction protein occludin in both cell lines, but did not induce a change in expression of adherens junction protein Ecadherin. Additionally, increasing curvature promoted polarity of both cell lines, as a greater apical localization of occludin was seen in cells on substrates of higher curvature. Lastly, substrate curvature increased expression of the water channel protein aquaporin-5 (Aqp-5) in Par-C10 cells, suggesting that curved nanofiber substrates are more suitable for promoting differentiation of salivary gland cells.

对于患有唾液功能减退（慢性口干的主要病因）的患者，需要一种人造唾液腺作为长期治疗方法。目前的唾液腺组织工程方法存在局限性，要么缺乏促进上皮细胞分化所需的足够物理信号和表面积，要么无法提供一种构建相互连接的分支细胞网络的机制。我们利用晶圆级集成电路制造工艺在聚二甲基硅氧烷（PDMS）中开发了高度有序的弯曲半球形“凹坑”阵列，并用静电纺丝的聚乳酸 - 羟基乙酸共聚物（PLGA）纳米纤维排列在其内部，旨在模拟唾液腺上皮细胞球形腺泡周围基底膜的三维体内结构。这些微图案化支架为构建增加的表面积提供了一种方法，并且还对其促进细胞极化的能力进行了研究。将两种永生化唾液腺细胞系（SIMS，导管细胞；Par - C10，腺泡细胞）培养在不同半径的纤维凹坑阵列上，并与在平坦的PLGA纳米纤维基底以及三维基质胶中培养的细胞进行比较。研究发现，通过增加凹坑曲率，SIMS细胞的单层细胞平均高度增加，Par - C10细胞在较小程度上也增加，最大高度与在三维基质胶中生长的细胞相似。增加曲率导致两种细胞系中紧密连接蛋白occludin的表达水平升高，但未引起黏附连接蛋白E - 钙黏蛋白表达的变化。此外，增加曲率促进了两种细胞系的极性，因为在曲率较高的基底上的细胞中观察到occludin的顶端定位更明显。最后，基底曲率增加了Par - C10细胞中水通道蛋白水通道蛋白 - 5（Aqp - 5）的表达，这表明弯曲的纳米纤维基底更适合促进唾液腺细胞的分化。