Cyclic tensile strain affects the response of human periodontal ligament stromal cells to tumor necrosis factor-α

Abstract

Objectives: Orthodontic treatment in adult patients predisposed to mild or severe periodontal disease is challenging for orthodontists. Orthodontic malpractice or hyper-occlusal forces may aggravate periodontitis-induced destruction of periodontal tissues, but the specific mechanism remains unknown. In the present study, the combined effect of mechanical stress and tumor necrosis factor (TNF)-α on the inflammatory response in human periodontal ligament stromal cells (hPDLSCs) was investigated.

Materials and methods: hPDLSCs from 5 healthy donors were treated with TNF-α and/or subjected to cyclic tensile strain (CTS) of 6% or 12% elongation with 0.1 Hz for 6- and 24 h. The gene expression of interleukin (IL)-6, IL-8 and cell adhesion molecules VCAM and ICAM was analyzed by qPCR. The protein levels of IL-6 and IL-8 in conditioned media was measured by ELISA. The surface expression of VCAM-1 and ICAM-1 was quantified by immunostaining followed by flow cytometry analysis.

Results: TNF-α-induced IL-6 gene and protein expression was inhibited by CTS, whereas TNF-α-induced IL-8 expression was decreased at mRNA expression level but enhanced at the protein level in a magnitude-dependent manner. CTS downregulated the gene expression of VCAM-1 and ICAM-1 under TNF-α stimulation, but the downregulation of the surface expression analyzed by flow cytometry was observed chiefly for VCAM-1.

Conclusions: Our findings show that mechanical force differentially regulates TNF-α-induced expression of inflammatory mediators and adhesion molecules at the early stage of force application. The effect of cyclic tensile strain is complex and could be either anti-inflammatory or pro-inflammatory depending on the type of pro-inflammatory mediators and force magnitude.

Clinical relevance: Orthodontic forces regulate the inflammatory mediators of periodontitis. The underlying mechanism may have significant implications for future strategies of combined periodontal and orthodontic treatment.

Keywords: Human periodontal ligament stromal cells; Inflammatory cytokine; Mechanical loading; Orthodontic force; Periodontitis.

Fig. 1

Schematic of mechanical force loading. (a) BioFlex® Cell Seeders were applied before seeding to confine the cell suspension in the central area of a well in the culture plates. (b) The culture plates were transferred onto the loading post after seeding hPDLSCs at a density of 1 × 10⁵ cells/well in the central region of the flexible membrane using BioFlex® Cell Seeders. (c) Cells were stretched by a cyclic tensile strain (equibiaxial, 6 or 12% elongation, sinusoidal curve, 0.1 Hz, 6 or 24 h) when the vacuum was applied

Fig. 2

Effects of TNF-α on gene and protein expression of IL-6 and IL-8 on unstretched hPDLSCs. Primary hPDLSCs were treated with 10 ng/ml TNF-α without mechanical stretching. After 6- or 24 h of incubation, gene expression levels of IL-6 (a), IL-8 (b) were measured using RT-qPCR. The Y-axes show the n-fold changes in mRNA expression compared to untreated control after 6 h (n-fold expression = 1). The corresponding protein levels of IL-6 (c) and IL-8 (d) in conditioned media were quantified by ELISA. The groups in the absence or presence of TNF-α are indicated as -TNF-α or + TNF-α, respectively. The data are presented as the mean ± standard deviation. *p < 0.05 compared to corresponding control. ^#p < 0.01 compared between groups as indicated

Fig. 3

Effects of CTS on IL-6 expression in the absence / presence of TNF-α in hPDLSCs. Primary hPDLSCs were subjected to either 6% or 12% cyclic stretching, in the absence or presence of 10 ng/ml TNF-α for 6 or 24 h. IL-6 gene expression levels were measured with RT-qPCR after 6 (a) or 24 (b) hours. TNF-α-induced IL-6 protein levels after 24 h (c) were quantified by ELISA. Y-axes show the effect of CTS on IL-6 expression as % of the values observed in unstretched cells with or without TNF-α. For RT-qPCR (a, b), n-fold gene expression was calculated first using the 2^−ΔΔCt method and then the data were normalized to those observed in unstretched cells (100%, dashed line). For ELISA (c), the values were calculated as % of the values measured in unstretched cells (100%, dashed line). The data are presented as the mean ± standard deviation. * and ** — significantly different compared to unstretched control with p < 0.05 and p < 0.01, respectively

Fig. 4

Effects of CTS on IL-8 expression in the absence / presence of TNF-α in hPDLSCs. Primary hPDLSCs were subjected to either 6% or 12% cyclic stretching, in the absence or presence of 10 ng/ml TNF-α. IL-8 gene expression levels were measured with RT-qPCR after 6 (a) or 24 (b) hours. TNF-α-induced IL-8 protein levels after 6 (c) and 24 (d) hours were quantified by ELISA. Y-axes show the effect of CTS on IL-8 expression as % of the values observed in unstretched cells with or without TNF-α. For RT-qPCR (a, b), n-fold gene expression was calculated first using the 2^−ΔΔCt method and then the data were normalized to those observed in unstretched cells (dashed line). For ELISA (c, d), the values were calculated as % of the values measured in unstretched cells (100%, dashed line). The data are presented as the mean ± standard deviation. * and ** — significantly different compared to unstretched control with p < 0.05 and p < 0.01, respectively

Fig. 5

Effects of TNF-α on gene and protein expression of VCAM-1 and ICAM-1 in unstretched hPDLSCs. Primary hPDLSCs were treated with 10 ng/ml TNF-α without mechanical stretching. The groups in the absence or presence of TNF-α are indicated as -TNF-α or + TNF-α, respectively. After 6- or 24 h of incubation, gene expression levels of VCAM-1 (a) and ICAM-1 (b) were measured using RT-qPCR. The Y-axes show the n-fold changes in mRNA expression compared to untreated control after 6 h (n-fold expression = 1). The corresponding protein levels of VCAM-1 (c) and ICAM-1 (d) were quantified by flow cytometry. The Y-axes show the percentage of positive cells and the corresponding mean fluorescence intensity (MFI) of the positive cell population, respectively. No positively stained VCAM-1 and ICAM-1 cells were observed in the absence of TNF-α. The data are presented as the mean ± standard deviation. *p < 0.05 compared to corresponding control. ^#p < 0.01 compared between groups as indicated

Fig. 6

Effects of CTS on VCAM-1 expression in the absence/presence of TNF-α in hPDLSCs. Primary hPDLSCs were subjected to either 6% or 12% cyclic stretching, in the absence or presence of 10 ng/ml TNF-α. VCAM-1 gene expression levels were measured with RT-qPCR using the 2^−ΔΔCt method after 6 (a) or 24 (b) hours. TNF-α-induced VCAM-1 protein levels after 6 (c) and 24 (d) hours were quantified by flow cytometry. Y-axes show the effect of CTS on VCAM-1 expression as % of the values observed in unstretched cells with or without TNF-α. For RT-qPCR (a, b), n-fold gene expression was calculated first using the 2^−ΔΔCt method and then the data were normalized to those observed in unstretched cells (dashed line). For protein expression (c, d), Y-axes show the percentage of positive cells and the mean fluorescence intensity (MFI) of the positive cell population, in % of the values measured in unstretched cells. The data are presented as the mean ± standard deviation. * and ** — significantly different compared to unstretched control with p < 0.05 and p < 0.01, respectively

Fig. 7

Effects of CTS on ICAM-1 expression in the absence / presence of TNF-α in hPDLSCs. Primary hPDLSCs were subjected to either 6% or 12% cyclic stretching, in the absence or presence of 10 ng/ml TNF-α. Unstretched hPDLSCs served as control. ICAM-1 gene expression levels were measured with RT-qPCR after 6 (a) or 24 (b) hours. TNF-α-induced ICAM-1 protein levels after 6 (c) and 24 (d) hours were quantified by flow cytometry. Y-axes show the effect of CTS on ICAM-1 expression as % of the values observed in unstretched cells with or without TNF-α. For RT-qPCR (a, b), n-fold gene expression was calculated first using the 2^−ΔΔCt method and then the data were normalized to those observed in unstretched cells (dashed line). For protein expression (c, d) the Y-axes show the percentage of positive cells and the mean fluorescence intensity (MFI) of the positive cell population, in % of the values measured in unstretched cells. The data are presented as the mean ± standard deviation. * and ** — significantly different compared to unstretched control with p < 0.05 and p < 0.01, respectively