Distinguish fatty acids impact survival, differentiation and cellular function of periodontal ligament fibroblasts

Abstract

Alveolar bone (AB) remodeling is necessary for the adaption to mechanical stimuli occurring during mastication and orthodontic tooth movement (OTM). Thereby, bone degradation and assembly are strongly regulated processes that can be altered in obese patients. Further, increased fatty acids (FA) serum levels affect bone remodeling cells and we, therefore, investigated whether they also influence the function of periodontal ligament fibroblast (PdLF). PdLF are a major cell type regulating the differentiation and function of osteoblasts and osteoclasts localized in the AB. We stimulated human PdLF (HPdLF) in vitro with palmitic (PA) or oleic acid (OA) and analyzed their metabolic activity, growth, survival and expression of osteogenic markers and calcium deposits. Our results emphasize that PA increased cell death of HPdLF, whereas OA induced their osteoblastic differentiation. Moreover, quantitative expression analysis of OPG and RANKL revealed altered levels in mechanically stimulated PA-treated HPdLF. Furthermore, osteoclasts stimulated with culture medium of mechanical stressed FA-treated HPdLF revealed significant changes in cell differentiation upon FA-treatment. For the first time, our results highlight a potential role of specific FA in the function of HPdLF-modulated AB remodeling and help to elucidate the complex interplay of bone metabolism, mechanical stimulation and obesity-induced alterations.

Figure 1

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.

Figure 2

Oleic acid promotes the osteogenic differentiation of HPdLF. (a) Quantitative expression analysis of the osteogenic marker genes RUNX2, osteocalcin (OCN), osteopontin (OSP), cementum protein 1 (CEMP1) and alkaline phosphatase (ALP) in fatty acid-cultured HPdLF compared to BSA controls. (b) Representative microphotographs of alizarin red staining of calcium deposits in HPdLF cultured in fatty acids and displayed in (c). *P < 0.05; **P < 0.01; ***P < 0.001; One-Way ANOVA and post-hoc test (Tukey). Scale bars: 50 μm in (b). BSA, bovine serum albumin; OA, oleic acid; PA, palmitic acid; RNE, relative normalized expression.

Figure 3

Fatty acid cultivation of mechanically stressed HPdLF affect genes relevant for extracellular matrix degradation as well as influence osteoclast differentiation. (a) Quantitative expression analysis of extracellular matrix (ECM)-remodeling genes MMP-3 and MMP-9 as well as VEGFA coding for an vascularization factor in HPdLF treated with palmitic acid (PA) or oleic acid (OA) and stimulated with compressive force over six hours compared to unstimulated BSA controls. (b) Quantitative expression analysis of osteoclastogenesis-relevant genes OPG and RANKL in HPdLF treated with fatty acids and stimulated with compressive force over six hours compared to unstimulated BSA controls. (c) Quantitative expression analysis of osteoclast-related genes Trap and Rank in mouse osteoclasts stimulated with the media supernatant of mechanically compressed HPdLF cultured in fatty acids compared to BSA (stim BSA, stim PA, stim OA) and only BSA-containing media as control (ctrl BSA). The results are normalized to ctrl BSA. (d) Representative microphotographs of TRAP-staining of osteoclasts stimulated with the medium supernatant of fatty acid treated and mechanically compressed HPdLF quantified as TRAP-positive area of the total area in (e). *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 (c) Ctrl, control; BSA, bovine serum albumin; HPdLF, human periodontal ligament fibroblast; OA, oleic acid; PA, palmitic acid; RNE, relative normalized expression; stim, stimulation.