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. 2020 Sep 24;10:15706. doi: 10.1038/s41598-020-72736-7

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.

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The cell survival of human periodontal ligament fibroblasts is impaired through palmitic acid cultivation. (a) Analysis of the metabolic activity of human periodontal ligament fibroblasts (HPdLF) cultured either with DMEM, DMEM containing BSA, palmitic acid (PA) or oleic acid (OA). Both fatty acids were coupled to BSA, which is therefore used as control condition. The metabolic activity is displayed in relation to the DMEM control. (b) Representative microphotographs of HPdLF at day 0 and day 8 of culturing with fatty acids with actin cytoskeleton (green) stained with phalloidin-coupled Alexa488 and DAPI-stained cell nuclei (blue). Day 0 is 6 h after cell seeding. Cell number per square millimeter is quantified in (c). (d) Representative images showing scratch sizes on HPdLF monolayers at day 2 and day 6 after scratch test quantified in (e) as relative size to the initial scratch size at day 0 for each fatty acids culture condition. Day 0 is the day the scratch was carried out. (f) Representative microphotographs of HPdLF cultured with fatty acids and stained for Ki67 (red). Cell nuclei are visualized by DAPI-staining (blue). The number of Ki67-positive cells in relation to the total cell number is displayed in (g). (h) Microphotographs of HPdLF cultured with fatty acids and stained for defects in the plasma membrane integrity by EthD-1 (red) referring as dead cells in relation to the total cell number of living cells (green) visualized by their esterase activity. The amount of dead cells was quantified in (i) in relation to the total number of living cells. (j) Representative microphotographs of HPdLF cultured with fatty acids showing senescent cells (CellEvent, green) and an cell nuclei (DAPI, blue). Arrow heads indicate senescent cells, which are analyzed as percentage of DAPI-positive cells in (k). For all conditions, cells from biological triplicates were analyzed. *P < 0.05; **P < 0.01; ***P < 0.001; One-Way ANOVA and post-hoc test (Tukey). Photographs were analyzed with Fiji software (https://imagej.net/Fiji). Scale bars: 50 μm in (b, d), 20 µm in (h), 10 μm in (f, j). BSA, bovine serum albumin; HPdLF, human periodontal ligament fibroblast; OA, oleic acid; PA, palmitic acid.