Department of Materials Science and Engineering, University of Delaware, Newark, Delaware 19716, United States.
Department of Biological Sciences, University of Delaware, Newark, Delaware 19716, United States.
ACS Biomater Sci Eng. 2021 Sep 13;7(9):4305-4317. doi: 10.1021/acsbiomaterials.0c01741. Epub 2021 Feb 26.
There is a critical need for the establishment of an engineered model of the vocal fold epithelium that can be used to gain understanding of its role in vocal fold health, disease, and facilitate the development of new treatment options. Toward this goal, we isolated primary vocal fold epithelial cells (VFECs) from healthy porcine larynxes and used them within passage 3. Culture-expanded VFECs expressed the suprabasal epithelial marker cytokeratin 13 and intercellular junctional proteins occludin, E-cadherin, and zonula occludens-1. To establish the engineered model, we cultured VFECs on a hyaluronic acid-derived synthetic basement membrane displaying fibronectin-derived integrin-binding peptide (RGDSP) and/or laminin 111-derived syndecan-binding peptide AG73 (RKRLQVQLSIRT). Our results show that matrix stiffness and composition cooperatively regulate the adhesion, proliferation, and stratification of VFECs. Cells cultured on hydrogels with physiological stiffness (elastic shear modulus, ' = 1828 Pa) adopted a cobblestone morphology with close cell-cell contacts, whereas those on softer matrices (' = 41 Pa) were spindle shaped with extensive intracellular stress fibers. The development of stratified epithelium with proliferating basal cells and additional (1-2) suprabasal layers requires the presence of both RGDSP and AG73 peptide signals. Supplementation of cytokines produced by vimentin positive primary porcine vocal fold fibroblasts in the VFEC culture led to the establishment of 4-5 distinct cell layers. The engineered vocal fold epithelium resembled native tissue morphologically; expressed cytokeratin 13, mucin 1, and tight/adherens junction markers; and secreted basement membrane proteins collagen IV and laminin 5. Collectively, our results demonstrate that stiffness matching, cell-matrix engagement, and paracrine signaling cooperatively contribute to the stratification of VFECs. The engineered epithelium can be used as a versatile tool for investigations of genetic and molecular mechanisms in vocal fold health and disease.
目前迫切需要建立一种工程化的声带上皮模型，以便深入了解其在声带健康、疾病中的作用，并促进新治疗方法的开发。为此，我们从健康猪的喉中分离出原代声带上皮细胞（VFEC），并在第 3 代时使用它们。培养的 VFEC 表达了细胞角蛋白 13 等基底层上皮标志物和紧密连接蛋白 Occludin、E-cadherin 和 Zonula occludens-1。为了建立这个工程化模型，我们将 VFEC 培养在含有纤维连接蛋白衍生的整合素结合肽（RGDSP）和/或层粘连蛋白 111 衍生的 syndecan 结合肽 AG73（RKRLQVQLSIRT）的透明质酸衍生的合成基底膜上。我们的结果表明，基质硬度和组成协同调节 VFEC 的黏附、增殖和分层。在具有生理弹性（剪切弹性模量 '= 1828 Pa）的水凝胶上培养的细胞呈现鹅卵石样形态，细胞间接触紧密，而在较软的基质（'= 41 Pa）上培养的细胞呈梭形，细胞内有大量应力纤维。只有存在 RGDSP 和 AG73 肽信号，才能形成具有增殖的基底细胞和额外的（1-2）层基底层的分层上皮。在 VFEC 培养物中补充波形蛋白阳性的原代猪声带成纤维细胞产生的细胞因子，可建立 4-5 个不同的细胞层。工程化的声带上皮在形态上类似于天然组织；表达细胞角蛋白 13、粘蛋白 1 和紧密/黏附连接标志物；并分泌基底膜蛋白 IV 型胶原和层粘连蛋白 5。总之，我们的结果表明，硬度匹配、细胞-基质相互作用和旁分泌信号协同促进 VFEC 的分层。该工程化的上皮组织可作为一种通用工具，用于研究声带健康和疾病中的遗传和分子机制。
