Department of Conservative Dentistry and Periodontology, University Clinic of Dentistry, Medical University of Vienna, Vienna, Austria.
Austrian Cluster for Tissue Regeneration, Vienna, Austria.
J Tissue Eng Regen Med. 2020 Sep;14(9):1307-1317. doi: 10.1002/term.3102. Epub 2020 Jul 24.
Collagen membranes and bone substitute are popular biomaterials in guided tissue regeneration for treatment of traumatized or diseased periodontal tissue. Development of these biomaterials starts in monolayer cell culture, failing to reflect in vivo tissue organization. Spheroid cultures potentially mimic in vivo tissues in structure and functionality. This study aims to compare gingiva cell (GC) monolayers and spheroids to ex vivo gingiva. Human GC monolayers, spheroids and gingiva ex vivo tissues were cultured on plastic surfaces, collagen membranes or bone substitute. Hematoxylin-eosin (HE) staining, immunohistochemistry for KI67 and caspase 3 (CASP3), resazurin-based toxicity assays, quantitative polymerase chain reaction for collagen I (COL1A1), vascular endothelial growth factor (VEGF), angiogenin (ANG), interleukin (IL)6 and IL8 and ELISA for COL1A1, VEGF, ANG, IL6 and IL8 were performed in all cultures. Morphology was different in all culture set-ups. Staining of KI67 was positive in monolayers and staining of CASP3 was positive in spheroids. All culture set-ups were viable. COL1A1 production was modulated in monolayers and ex vivo tissues at mRNA levels, VEGF in monolayers and ex vivo tissues at mRNA levels and in spheroids at protein levels, ANG in spheroids at mRNA levels and in monolayers and spheroids at protein levels, IL6 in monolayers and spheroids at mRNA levels and in spheroids and ex vivo tissues at protein levels and IL8 in monolayers and ex vivo tissues at mRNA levels. Modulations were surface-dependent. In conclusion, each culture model is structurally and functionally different. Neither GC monolayers nor spheroids mimicked gingiva ex vivo tissue in all measured aspects.
胶原膜和骨替代物是引导组织再生中用于治疗创伤或患病牙周组织的常用生物材料。这些生物材料的开发始于单层细胞培养，无法反映体内组织的组织。球体培养有可能在结构和功能上模拟体内组织。本研究旨在比较牙龈细胞 (GC) 单层和球体与离体牙龈。人 GC 单层、球体和离体牙龈组织在塑料表面、胶原膜或骨替代物上进行培养。对所有培养物进行苏木精-伊红 (HE) 染色、Ki67 和半胱氨酸天冬氨酸蛋白酶 3 (CASP3) 的免疫组织化学染色、基于 Resazurin 的毒性测定、I 型胶原 (COL1A1)、血管内皮生长因子 (VEGF)、血管生成素 (ANG)、白细胞介素 (IL)6 和 IL8 的定量聚合酶链反应以及 COL1A1、VEGF、ANG、IL6 和 IL8 的 ELISA 检测。所有培养物的形态均不同。Ki67 染色在单层中呈阳性，CASP3 染色在球体中呈阳性。所有培养物均具有活力。COL1A1 的产生在单层和离体组织中在 mRNA 水平上受到调节，VEGF 在单层和离体组织中在 mRNA 水平上和在球体中在蛋白水平上受到调节，ANG 在球体中在 mRNA 水平上和在单层和球体中在蛋白水平上受到调节，IL6 在单层和球体中在 mRNA 水平上和在球体和离体组织中在蛋白水平上受到调节，IL8 在单层和离体组织中在 mRNA 水平上受到调节。调节取决于表面。总之，每种培养模型在结构和功能上都不同。GC 单层和球体都没有在所有测量的方面模拟离体牙龈组织。
