Development and characterization of a 3D oral mucosa model as a tool for host-pathogen interactions
Development and characterization of a 3D oral mucosa model as a tool for host-pathogen interactions
复制标题
DOI：
10.1016/j.mimet.2018.07.004
复制
发表时间：
2018-09-01
影响因子：
2.2
通讯作者：
Vergani, Carlos Eduardo
中科院分区：
文献类型：
--
作者：
Dias, Kassia de Carvalho;de Sousa, Denise Lins;Vergani, Carlos Eduardo
本研究的目的是（i）设计、开发和验证实用且生理相关的体外重建口腔粘膜组织模型，以及（ii）评估其在与白色念珠菌和金黄色葡萄球菌的体外宿主-病原体相互作用中的适用性。通过将特定数量的角质形成细胞 (NOK-si) 整合到细胞化的胶原凝胶支架上创建共培养器官型结构，该支架含有在 KGM 培养基中孵育并培养 14 天的人牙龈成纤维细胞。通过组织学（苏木精和伊红 (HE)、高碘酸希夫 (P.A.S.) 和天狼星红）和免疫细胞化学（细胞角蛋白 13、细胞角蛋白 14、Ki-67 和胶原 IV）与正常人牙龈进行比较来评估适当口腔粘膜/上皮结构的检测。通过透射电子显微镜分析重建组织的形态。为了进一步定量组织损伤，测量了组织上清液中的乳酸脱氢酶（LDH）。在牙龈支架上生长的 NOK-si 在体外环境中提供了器官型模型，并表现出通常与正常口腔粘膜相关的结构特征。免疫细胞化学显示在重建的口腔粘膜模型中检测到上皮细胞角蛋白 13 和 14、Col IV 和 Ki-67。 8小时和16小时后检测到感染。这项研究提出了重建口腔粘膜的体外细胞化器官型模型，该模型能够在培养的中期时间内对其结构和分化进行密切控制和表征。
The aim of this study was to (i) design, develop and validate a practical and physiologically relevant reconstituted in vitro oral mucosa tissue model and (ii) to assess its applicability in in vitro host-pathogen interactions with C. albicans and S. aureus. Co-culture organotypic constructions were created by incorporating specific numbers of keratinocytes (NOK-si) onto cellularised, collagen gel scaffolds containing human gingival fibroblasts incubated in KGM media and cultured for 14 days. The detection of the appropriate oral mucosa/epithelial structure was evaluated by histology (hematoxylin and eosin (HE), periodic acid Schiff (P.A.S.) and Picrosirius red), and immunocytochemistry (cytokeratin 13, cytokeratin 14, Ki-67 and collagen IV) compared to a normal human gingiva. The morphology of the reconstituted tissue was analyzed by Transmission Electron Microscopy. To further quantitate tissue damage, lactate dehydrogenase (LDH) was measured in the tissue supernatant. NOK-si grown upon a gingival scaffold provided an organotypic model in an in vitro setting and exhibited structural characteristics typically associated with normal oral mucosa. Immunocytochemistry revealed the detection of epithelial cytokeratins 13 and 14, Col IV and Ki-67 in the reconstituted oral mucosa model. Infection was detected after 8 h and 16 h. This study presents an in vitro cellularised, organotypic model of reconstituted oral mucosa, which enables close control and characterization of its structure and differentiation over a mid-length period of time in culture.