School of Clinical Dentistry, Claremont Crescent, University of Sheffield, UK.
Department of Materials Science and Engineering, University of Sheffield, UK.
J Tissue Eng Regen Med. 2018 Mar;12(3):e1636-e1646. doi: 10.1002/term.2590. Epub 2017 Dec 3.
The generation of tissue-engineered epithelial models is often hampered by the limited proliferative capacity of primary epithelial cells. This study aimed to isolate normal tonsillar keratinocytes (NTK) from human tonsils, increase the lifespan of these cells using the Rho kinase inhibitor Y-27632 and to develop tissue-engineered equivalents of healthy and infected tonsil epithelium. The proliferation rate of isolated NTK and expression of c-MYC and p16INK4A were measured in the absence or presence of the inhibitor. Y-27632-treated NTK were used to generate tissue-engineered tonsil epithelium equivalents using de-epidermised dermis that were then incubated with Streptococcus pyogenes to model bacterial tonsillitis, and the expression of pro-inflammatory cytokines was measured by cytokine array and ELISA. NTK cultured in the absence of Y-27632 rapidly senesced whereas cells cultured in the presence of this inhibitor proliferated for over 30 population doublings without changing their phenotype. Y-27632-treated NTK produced a multi-layered differentiated epithelium that histologically resembled normal tonsillar surface epithelium and responded to S. pyogenes infection by increased expression of pro-inflammatory cytokines including CXCL5 and IL-6. NTK can be isolated and successfully cultured in vitro with Y-27632 leading to a markedly prolonged lifespan without any deleterious consequences to cell morphology. This functional tissue-engineered equivalent of tonsil epithelium will provide a valuable tool for studying tonsil biology and host-pathogen interactions in a more physiologically relevant manner.
组织工程化上皮模型的生成常常受到原代上皮细胞增殖能力有限的阻碍。本研究旨在从人扁桃体中分离正常扁桃体角质形成细胞（NTK），使用 Rho 激酶抑制剂 Y-27632 延长这些细胞的寿命，并开发健康和感染的扁桃体上皮组织工程等效物。在不存在或存在抑制剂的情况下，测量分离的 NTK 的增殖率以及 c-MYC 和 p16INK4A 的表达。使用去表皮真皮生成 Y-27632 处理的 NTK 来生成组织工程化的扁桃体上皮等效物，然后将其与酿脓链球菌孵育以模拟细菌性扁桃体炎，并通过细胞因子阵列和 ELISA 测量促炎细胞因子的表达。在不存在 Y-27632 的情况下培养的 NTK 迅速衰老，而在存在该抑制剂的情况下培养的细胞增殖超过 30 个倍增而不改变其表型。Y-27632 处理的 NTK 产生了具有组织学上类似于正常扁桃体表面上皮的多层分化上皮，并通过增加促炎细胞因子（包括 CXCL5 和 IL-6）的表达对酿脓链球菌感染作出反应。可以从扁桃体中分离并成功地在体外使用 Y-27632 培养 NTK，从而显著延长寿命，而不会对细胞形态造成任何有害影响。这种功能性扁桃体上皮的组织工程等效物将为更生理相关的方式研究扁桃体生物学和宿主-病原体相互作用提供有价值的工具。
