TY - JOUR
AU - Kletsas,, Dimitris
AB - Summary Background Cyclic tensile stretching (CTS) induces osteoblastic differentiation of periodontal ligament fibroblasts (PDLF). On the other hand, increased concentrations of tumour necrosis factor-α (TNF-α) are found in inflammatory conditions, leading to periodontal disease and tooth loss. Accordingly, our aim was to investigate the short- and long-term effect of TNF-α on the response of human PDLF to CTS and its implication on osteoblastic differentiation. Methods PDLF were either pre-incubated for 4 hours or were repeatedly exposed to TNF-α for up to 50 days and then subjected to CTS. Gene expression was determined by quantitative real-time polymerase chain reaction. Activation of mitogen-activated protein kinase (MAPK) was monitored by western analysis and cell proliferation by bromodeoxyuridine incorporation. Intracellular reactive oxygen species were determined by the 2´, 7´-dichlorofluorescein-diacetate assay and osteoblastic differentiation by Alizarin Red-S staining after an osteo-inductive period of 21 days. Results CTS of PDLF induced an immediate upregulation of the c-fos transcription factor and, further downstream the overexpression of alkaline phosphatase and osteopontin, two major osteoblast marker genes. A 4-hour pre-incubation with TNF-α repressed these effects. Similarly, long-term propagation of PDLF along with TNF-α diminished their osteoblastic differentiation capacity and suppressed cells’ CTS-elicited responses. The observed phenomena were not linked with TNF-α-induced premature senescence or oxidative stress. While CTS induced the activation of MAPKs, involved in mechanotransduction, TNF-α treatment provoked a small delay in the phosphorylation of extracellular signal-regulated kinase and c-Jun N-terminal kinase. Conclusion Increased concentrations of TNF-α, such as those recorded in many inflammatory diseases, suppress PDLF’s immediate responses to mechanical forces compromising their osteoblastic differentiation potential, possibly leading to tissue’s impaired homeostasis. Introduction Chronic inflammatory conditions, such as rheumatoid arthritis, lead to a progressive destruction of articular cartilage and bone (1–3). In this context, rheumatoid arthritis patients exhibit a higher incidence of periodontal disease and tooth loss (4) as it has previously been reported (5–6). Moreover, animal studies have provided evidence for the association of inflammatory conditions with periodontal ligament’s breakdown (7). Many pro-inflammatory cytokines are in abundance in the tissues of patients afflicted by chronic inflammatory diseases, such as chronic periodontitis (8) or rheumatoid arthritis. However, in the case of rheumatoid arthritis, it is generally considered that tumour necrosis factor-α (TNF-α) is the dominant regulator of all cytokines (9). As such, TNF-α plays a major role in the pathogenesis of arthritis-associated bone destruction (2–3) as it is implicated in the disruption of the balance in bone formation and resorption processes. For instance, TNF-α has been reported to suppress differentiation-associated increase in key marker genes of osteogenesis, such as alkaline phosphatase (ALP) and osteopontin (OPN) (10). Furthermore, numerous studies have demonstrated the role of TNF-α in reactive oxygen species (ROS) generation (11–13), as well as the implication of oxidative stress in the inhibition of osteoblastic differentiation of competent cells (14–15). Finally, in multiple cell types, a long-term exposure to TNF-α provokes premature senescence (11); the latter being a negative factor for osteoblastic differentiation (16–18). Periodontal ligament (PDL) is a highly specialized, dense connective tissue that resides between the tooth root and the alveolar bone. Amongst other properties, it mediates the transmission of mechanical stimuli applied to the tissue during mastication, occlusion, as well as orthodontic tooth movement (19–20). PDL fibroblasts (PDLF) represent the most abundant cellular type of the tissue, which respond to mechanical signals undergoing an osteoblastic differentiation (21), thus sustaining the integrity of the tissue through repair and remodelling processes. More specifically, transmission of mechanical stimuli applied to PDL is converted into a cellular response through the activation of signalling pathways, such as the family of mitogen-activated protein kinase (MAPK) cascades [i.e. the extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK)], mediated by a complex network of mechanical sensing molecules (collectively termed mechanotransduction) (22–23). Further downstream, MAPK signalling cascades activate several transcription factors, such as members of the fos family, while are involved in the regulation of the expression of osteoblast-specific genes, among them being ALP and OPN (24–25). In particular, it has been shown that mechanical stimulation-induced c-fos up-regulation is mediated by ERK and JNK activation, while is inhibited by p38 MAPK (23). Having in mind that mechanical forces, such as those applied to periodontal ligament during orthodontic therapy, ignite the osteoblastic differentiation of competent cells, that is, PDLFs, thus preserving tissue’s integrity and given TNF-α’s implication in PDLF’s deterioration of osteoblastic differentiation potential, as well as tissue’s destruction, the aim of the present work was to investigate the short- and long-term effects of TNF-α on the response of human PDL fibroblasts to cyclic mechanical loading in terms of the expression of c-fos transcription factor and osteoblast marker genes, as well as to elucidate the mechanisms mediating this effect. With the extension of lifespan, increasing occurrence of age-related inflammatory diseases, such as rheumatoid arthritis, has been recorded. Accordingly, this affects the outcome of orthodontic treatment as rheumatoid arthritis impairs connective tissue’s homeostasis, thus rendering the current study of great clinical implication and interest. Materials and methods Cells and culture conditions Human teeth of consenting healthy donors were extracted in the course of orthodontic treatment under local research Bioethics Committee’s approval (No 240/2013-1640). PDL tissue explants were used to develop primary cultures of fibroblasts as previously described (26). PDL fibroblasts isolated from PDL tissue explants have been characterized by their spindle-like (fibroblastic) morphology by the expression of fibroblastic surface markers, such as vimentin, and their ability of osteoblastic differentiation upon their long-term incubation with an osteogenic medium. PDLF were routinely cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with penicillin (100 U/ml), streptomycin (100μg/ml; both from Biochrom AG, Berlin, Germany) and 10 per cent (v/v) foetal bovine serum (FBS, Gibco BRL, Invitrogen, Paisley, UK). Cells were subcultured when confluent using a trypsin/citrate (0.25/0.30 per cent w/v) solution and cultures were preserved at 37°C under conditions of 5 per cent CO2 and 85 per cent humidity. Early passage cells were used at Passages 3–6. Application of cyclic tensile stress and TNF-α treatment protocol It is generally accepted in the literature that the mechanical loading applied by orthodontic appliances generates two different strains in the periodontal ligament, that is, compressive and tensile strain. Furthermore, according to pressure-tension theory, force-subjected PDL progenitor cells differentiate into compression-associated osteoclasts and tension-associated osteoblasts, leading to bone resorption and apposition, respectively (27). In contrast to other studies that used compressive force-generating in vitro loading models (28–29), in the current study, PDL fibroblasts were subjected to uniaxial cyclic tensile loading by using a specially designed device equipped with a rotary motor and a camshaft (30). As previously described (23), the cells were plated onto silicone dishes pre-coated with fibronectin (Sigma, St. Louis, Missouri, USA; 2 ng/ml in 0.5-M NaCl–50-mM Tris-HCl, pH 7.5) in DMEM supplemented with 10 per cent (v/v) FBS and maintained for 48 hours in order to adapt to culture conditions. Twenty-four hours prior to mechanical loading application, the medium was replaced by a fresh one. Subsequently, cells were subjected to uniaxial cyclic strain falling within the range of physiological tissue deformation during orthodontic tooth movement, that is, extension 8 per cent and frequency 1 Hz (31–32), for the indicated time periods, that is, 15 and 30 minutes or 1, 3, 6 and 18 hours. The maximal PDL strains for horizontal displacements of a human maxillary central incisor under physiological loading conditions lie in the vicinity of 8–25 per cent, depending upon the apico-crestal position (32). In order to simulate in vitro the inflammatory microenvironment, cells were exposed to TNF-α (10 ng/ml) (Peprotech, Rocky Hill, New Jersey, USA) 4 hours prior to mechanical loading application. When indicated, 1 hour prior to TNF-α addition, cells were treated with 2-mM of N-acetyl-L-cysteine (NAC, Sigma) in order to evaluate the possible impact of ROS. For the investigation of the long-term effect of TNF-α on human PDLF, confluent cultures of early passage cells were repeatedly exposed to 10 ng/ml of TNF-α three times a week for up to 20 treatments. Cells were subcultured every five treatments and, only when cultures reached confluence again, that is, after 2 to 3 days, cells were exposed to the next five additional treatments with TNF-α. The above stimulation period, that lasted for about 50 days, was followed by an additional recovery period of 4 days in 10 per cent FBS (v/v) DMEM in the absence of TNF-α so as to avoid the immediate and determine solely the permanent effect of the cytokine on the cells. Hereafter, these PDL fibroblasts will be referred to as ‘pretreated cells and then in the absence of TNF-α (p/a TNF-α cells)’. A schematic presentation of short- and long-term TNF-α treatment and mechanical stimulation protocol, as well as of subsequently performed assays is provided in Figure 1. Figure 1. Open in new tabDownload slide Schematic presentation of short- and long-term tumour necrosis factor-α (TNF-α) treatment and mechanical stimulation protocol. Figure 1. Open in new tabDownload slide Schematic presentation of short- and long-term tumour necrosis factor-α (TNF-α) treatment and mechanical stimulation protocol. Estimation of intracellular levels of ROS Impact of TNF-α on PDLF’s intracellular ROS levels were evaluated using the 2´, 7´-dichlorofluorescein-diacetate (DCFH-DA; Sigma) assay as previously described (33). In brief, cells were plated in 96-well plates in DMEM containing 10 per cent (v/v) FBS. When cultures became confluent, TNF-α was added in culture medium at a concentration of 10 ng/ml along with 10 μM of DCFH-DA. Fluorescence intensity (excitation wavelength: 485 nm, emission wavelength: 520 nm) was monitored using an Infinite 200 Tecan micro-titre-plate photometer (Tecan Trading AG, Switzerland). In order to assess the effect of repetitive exposures to TNF-α on intracellular ROS levels, pre-treated PDLF were plated and maintained for 7 days in 96-well plates with DMEM supplemented with 10 per cent (v/v) FBS in the absence of TNFα before the addition of DCFH-DA in each well. Western immunoblot analysis Protein expression was assessed by Western immunoblot analysis as previously described (16). Analysis was performed with primary antibodies against p38, phospho-p38 (Thr180/Tyr182), phospho-JNK (Thr183/Tyr185) and JNK (Cell Signaling Technology, Hertfordshire, UK). The antibodies against ERK and phospho-ERK1/2 (Thr202/Tyr204) were from BD Pharmingen (Bedford, MA); the antibody against glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The goat anti-mouse and goat anti-rabbit secondary horseradish peroxidase-conjugated antibodies were purchased from Sigma. The immunoreactive bands were visualized on KODAK-X-OMAT AR film by chemiluminescence (ECL kit) according to the manufacturer’s instructions (GenScript, Piscataway, New Jersey, USA). In all cases, GAPDH expression was used as a loading control. Real-time reverse transcriptase polymerase chain reaction Gene expression was evaluated by quantitative real-time polymerase chain reaction (qRT-PCR) as previously described (11). Accordingly, total RNA was isolated using Trizol (Invitrogen, Paisley, UK). RNA samples’ quality (A260/280 ratio ranging from 1.86 to 1.96), as well as concentration, was determined spectrophotometrically in a Nanodrop ND-1000 spectrophotometer (Nanodrop Technologies, Wilmington, DE). First-strand complementary DNA (cDNA) was synthesized from 500 ng of the total RNA using PrimeScript™ RT Reagent Kit according to the manufacturer’s instructions (Takara Bio Inc, Tokyo, Japan). Real-time PCR experiments were performed in 20 μl using cDNA in a dilution ratio of 1:100 and the qPCRBIO SyGreen Mix Lo-ROX (PCR Biosystems Ltd, London, UK) in a MX3000 P cycler (Stratagene, La Jolla, California, USA) under the following conditions: denaturation program (95°C for 2 minutes), amplification and quantification program (95°C for 5 seconds, 60°C for 20 seconds and 72°C for 10 seconds) repeated 45 times, melting curve program (60–95°C with a heating rate of 0.1°C/second and a continuous fluorescence measurement) and finally a cooling step to 55°C. Cycle threshold (Ct) values of each target gene were normalized to that of the house-keeping gene GAPDH. Relative gene expression was estimated using the 2-ΔΔCt method (34). Primers’ specificity check was performed using the NCBI PrimerBlast and confirmed by melting curve analysis. Efficiencies of used primers’ ranged from 92 to 98 per cent. The sequences of the qPCR primers used were c-fos: forward 5’-AGA ATC CGA AGG GAA AGG AA-3′, reverse 5’-CTT CTC CTT CAG GTT GG-3′, ALP: forward 5’-GGC CAT TGG CAC CTG CCT TA-3′, reverse 5’-ACC CAT CCC ATC TCC CAG GAA-3′,OPN forward 5′-GCC GTG GGA AGG ACA GTT ATG-3′, reverse 5′-TGC TCA TTG CTC TCA TCA TTG G-3′, p16INK4a: forward 5´-TGA GCA CTC ACG CCC TAA GC-3´, reverse 5´-TAG CAG TGT GAC TCA AGA GAA GCC-3´, p21Cip1/Waf1: forward 5´-CTG GAG ACT CTC AGG GTC GAA-3´, reverse 5´-CCA GGA CTG CAG GGT TCC T-3´ and GAPDH: forward 5′-GAG TCC ACT GGG GTC TTC-3′, reverse 5′-GCA TTG CTG ATG ATC TTG GG-3′. Of note, similar results were obtained when peptidylprolyl isomerase A (cyclophilin B; PPIB) and ribosomal protein L22 (PRL22) were used as house-keeping genes (29). Estimation of cell proliferation by 5-bromo-2´-deoxyuridine incorporation In order to estimate the proliferative ability of PDL fibroblasts, novel DNA synthesis was measured with dual labelling with 5-bromo-2´-deoxyuridine (BrdU) and 4´, 6-diamino-2-phenylindole (DAPI) dihydrochloride (Sigma) as previously described (16). In brief, TNFα-pre-treated (p/a TNF-α) cells were plated sparsely on glass coverslips and allowed to attach for 48 hours prior to 50-μΜ BrdU labelling in DMEM containing 10 per cent (v/v) FBS. After an additional 48-hour incubation, cells were fixed with freshly prepared 4 per cent (w/v) formaldehyde in phosphate buffered saline (PBS), blocked for 30 minutes with 0.5 per cent (v/v) cold water fish gelatine in PBS and finally incubated overnight at 4°C with anti-BrdU fluorescein isothiocyanate-conjugated antibody (Roche Diagnostics GmbH, Mannheim, Germany). Subsequently, cells were counterstained with 2.5 μg/ml DAPI in PBS for 20 minutes. DAPI- and BrdU-positive nuclei were observed under a Zeiss Axioplan 2 fluorescent microscope (Carl Zeiss, Germany). Mineralization assay The effect of TNF-α on PDL fibroblasts’ ability towards osteoblastic differentiation was investigated by using the Alizarin Red-S staining assay. Briefly, after 20 consecutive exposures to TNFα, PDL fibroblasts were plated in a 12-well dish as previously described (16). When cultures reached ~90 per cent confluence, medium was aspirated and replaced with the commercial osteogenic medium StemPro® Osteogenesis Differentiation kit (Gibco BRL, Invitrogen, Paisley, UK) for 21 days with medium changes every 3 days either along with TNF-α (10 ng/ml) or not. Parallel to the long-term TNF-α-treated PDL fibroblasts, their naive (TNF-α untreated) counterparts were either incubated with the osteo-inductive medium for the whole 21 days’ period or were cultured for the same period in DMEM containing 10 per cent (v/v) FBS. At the end of the osteo-inductive period, cells were fixed with 4 per cent (w/v) formaldehyde for 30 minutes and stained for 20 minutes with 40 mM Alizarin Red-S pH 4.2. Quantification of calcium mineral content was estimated after a destaining procedure by extraction of Alizarin Red-S stain with 10 per cent (w/v) cetylpyridinium chloride (CPC; Sigma) in 10 mM sodium phosphate, pH 7.0 (35), for 15 minutes at room temperature followed by absorbance measurement at 562 nm on an Infinite 200 Tecan micro-titre plate photometer. Values of absorbance were normalized to the number of cells in each culture and osteogenic differentiation potential of each culture was expressed as a percentage ratio of the cells cultured in DMEM containing 10 per cent (v/v) FBS. Statistical analysis Normality assumptions were checked through Shapiro–Wilk tests and visual inspection through q–q plots. When non-normal distribution of the residuals was detected, transformation of variables or non-parametric statistics (Mann–Whitney U-tests) were used as appropriate. Descriptive statistics were used to present outcome variables across cyclic stress and TNF-α presence conditions. Multivariable linear regression with observed coefficients and 95 per cent confidence intervals (CIs) was used to assess the effect of TNF-α and cyclic stress on the examined outcome variables (i.e. expression of c-fos, OPN, and ALP). Standard errors were calculated using the bootstrap method with 1000 replications. A likelihood ratio test for interaction (LR test) between TNF-α and cyclic stress was performed. The level of statistical significance was pre-specified at P < 0.05. Statistical analyses were performed with STATA version 15.1 software (Stata Corporation, College Station, Texas, USA). Results Short-term treatment with TNF-α alters uniaxial cyclic tensile stretching (CTS) induced upregulation of MAPK and diminishes the expression of genes involved in osteoblastic differentiation. It was previously shown that cyclic stretching provokes in PDLF an immediate activation of all three MAPKs and subsequently the upregulation of immediate-early and effector genes involved in osteoblastic differentiation (23). Here, we first studied the immediate effect of TNF-α on these events. As can be seen in Figure 2A, cyclic stretching induces the upregulation of ERK, p38 and JNK with a peak at 0.5 hours of stimulation. On the other hand, in cells treated with TNF-α, while p38 phosphorylation is immediately observed, there is a delay of ERK and JNK activation (with a peak at 1 hour after stimulation) and the time frame of activation is shortened (especially for ERK). Following that we have found that cyclic tensile stress resulted in an immediate and intense upregulation of the transcription factor c-fos, a major cellular response to mechanical loading (β = 1.59; 95 per cent CI: 1.35, 1.82; P < 0.001; Table 1). On the other hand, when the cells were exposed to TNF-α, the tensile stress-induced c-fos upregulation was significantly decreased (β = −0.52; 95 per cent CI: −0.77, −0.28; P < 0.001; Table 1; Figure 2B). Table 1. Multivariable linear regression with observed coefficients and 95% confidence intervals (95% CIs) for the short-term effect of tumour necrosis factor-α (TNF-α) and cyclic stress (CTS) on the regulation of c-fos (as a logarithm of the original value), osteopontin (OPN) and alkaline phosphatase (ALP) Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α− Reference TNF-α+ −0.52 −0.77, −0.28 <0.001 CTS− Reference CTS+ 1.59 1.35, 1.82 <0.001 LR test (TNF-α × CTS) 0.88 OPN TNF-α−, CTS− Reference TNF-α +, CTS− 0.18 0.00, 0.35 0.05 CTS−, TNF-α− Reference CTS+, TNF-α− 1.11 0.93, 1.28 <0.001 TNF-α−, CTS+ Reference TNF-α+, CTS+ −0.27 −0.44, −0.10 0.002 LR test (TNF-α × CTS) <0.001 ALP TNF-α−, CTS− Reference TNF-α+, CTS− −0.01 −0.41, 0.21 0.53 CTS−, TNF-α− Reference CTS+, TNF-α− 0.45 0.17, 0.72 0.001 TNF-α−, CTS+ Reference TNF-α+, CTS+ −0.52 −0.78, −0.26 <0.001 LR test (TNF- α × CTS) 0.03 Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α− Reference TNF-α+ −0.52 −0.77, −0.28 <0.001 CTS− Reference CTS+ 1.59 1.35, 1.82 <0.001 LR test (TNF-α × CTS) 0.88 OPN TNF-α−, CTS− Reference TNF-α +, CTS− 0.18 0.00, 0.35 0.05 CTS−, TNF-α− Reference CTS+, TNF-α− 1.11 0.93, 1.28 <0.001 TNF-α−, CTS+ Reference TNF-α+, CTS+ −0.27 −0.44, −0.10 0.002 LR test (TNF-α × CTS) <0.001 ALP TNF-α−, CTS− Reference TNF-α+, CTS− −0.01 −0.41, 0.21 0.53 CTS−, TNF-α− Reference CTS+, TNF-α− 0.45 0.17, 0.72 0.001 TNF-α−, CTS+ Reference TNF-α+, CTS+ −0.52 −0.78, −0.26 <0.001 LR test (TNF- α × CTS) 0.03 Standard errors were calculated using the bootstrap method with 1000 replications. + denotes presence; − denotes absence. LR, likelihood ratio. Open in new tab Table 1. Multivariable linear regression with observed coefficients and 95% confidence intervals (95% CIs) for the short-term effect of tumour necrosis factor-α (TNF-α) and cyclic stress (CTS) on the regulation of c-fos (as a logarithm of the original value), osteopontin (OPN) and alkaline phosphatase (ALP) Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α− Reference TNF-α+ −0.52 −0.77, −0.28 <0.001 CTS− Reference CTS+ 1.59 1.35, 1.82 <0.001 LR test (TNF-α × CTS) 0.88 OPN TNF-α−, CTS− Reference TNF-α +, CTS− 0.18 0.00, 0.35 0.05 CTS−, TNF-α− Reference CTS+, TNF-α− 1.11 0.93, 1.28 <0.001 TNF-α−, CTS+ Reference TNF-α+, CTS+ −0.27 −0.44, −0.10 0.002 LR test (TNF-α × CTS) <0.001 ALP TNF-α−, CTS− Reference TNF-α+, CTS− −0.01 −0.41, 0.21 0.53 CTS−, TNF-α− Reference CTS+, TNF-α− 0.45 0.17, 0.72 0.001 TNF-α−, CTS+ Reference TNF-α+, CTS+ −0.52 −0.78, −0.26 <0.001 LR test (TNF- α × CTS) 0.03 Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α− Reference TNF-α+ −0.52 −0.77, −0.28 <0.001 CTS− Reference CTS+ 1.59 1.35, 1.82 <0.001 LR test (TNF-α × CTS) 0.88 OPN TNF-α−, CTS− Reference TNF-α +, CTS− 0.18 0.00, 0.35 0.05 CTS−, TNF-α− Reference CTS+, TNF-α− 1.11 0.93, 1.28 <0.001 TNF-α−, CTS+ Reference TNF-α+, CTS+ −0.27 −0.44, −0.10 0.002 LR test (TNF-α × CTS) <0.001 ALP TNF-α−, CTS− Reference TNF-α+, CTS− −0.01 −0.41, 0.21 0.53 CTS−, TNF-α− Reference CTS+, TNF-α− 0.45 0.17, 0.72 0.001 TNF-α−, CTS+ Reference TNF-α+, CTS+ −0.52 −0.78, −0.26 <0.001 LR test (TNF- α × CTS) 0.03 Standard errors were calculated using the bootstrap method with 1000 replications. + denotes presence; − denotes absence. LR, likelihood ratio. Open in new tab Figure 2. Open in new tabDownload slide Effect of short-term exposure of human periodontal ligament fibroblasts (PDLF) to tumour necrosis factor-α (TNF-α). (A) Early passage human PDLF were subjected to cyclic tensile stretching (CTS) in the absence or presence of TNF-α (CTS + TNF-α) and total and phosphorylated forms of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were assessed, after 0.25, 0.5, 1, 3 and 6 hours of CTS application, by western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. A representative experiment, among two similar ones with similar results, is presented here. (B) Box plot for c-fos gene expression after application of CTS for 30 minutes. (C) Box plot for osteopontin (OPN) gene expression after application of CTS for 18 hours. (D) Box plot for alkaline phosphatase (ALP) gene expression after application of CTS for 18 hours. Quantitative real-time polymerase chain reaction (qRT-PCR) experiments were performed on samples from three different donors and for at least three times in triplicates. Figure 2. Open in new tabDownload slide Effect of short-term exposure of human periodontal ligament fibroblasts (PDLF) to tumour necrosis factor-α (TNF-α). (A) Early passage human PDLF were subjected to cyclic tensile stretching (CTS) in the absence or presence of TNF-α (CTS + TNF-α) and total and phosphorylated forms of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were assessed, after 0.25, 0.5, 1, 3 and 6 hours of CTS application, by western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. A representative experiment, among two similar ones with similar results, is presented here. (B) Box plot for c-fos gene expression after application of CTS for 30 minutes. (C) Box plot for osteopontin (OPN) gene expression after application of CTS for 18 hours. (D) Box plot for alkaline phosphatase (ALP) gene expression after application of CTS for 18 hours. Quantitative real-time polymerase chain reaction (qRT-PCR) experiments were performed on samples from three different donors and for at least three times in triplicates. Further downstream, cyclic stress exerted an intense upregulation of the osteoblastic marker OPN (β = 1.11; 95 per cent CI: 0.93, 1.28; P < 0.001; Table 1). In the presence of TNF-α but without cyclic stress, the basal levels of OPN were retained at the same levels with untreated and mechanically unstimulated cells (P = 0.14; Figure 2C). In addition, TNF-α downregulated also the expression of OPN after mechanical stimulation (β= −0.27; 95 per cent CI: −0.44, -0.10; P = 0.002; Table 1; Figure 2C). Finally, cyclic stress upregulated also the expression of ALP, another classical marker of osteoblastic differentiation (β = 0.45, 95 per cent CI: 0.17, 0.72; P = 0.001; Table 1). Furthermore, similarly to OPN expression, while the basal levels of ALP in the presence of TNF-α were retained at the same level, TNF-α decreased the expression of ALP in mechanically stimulated cells (β = −0.52; 95 per cent CI: −0.78, −0.26; P < 0.001: LR test, P = 0.03; Table 1; Figure 2D). Of note, similar results were obtained when peptidylprolyl isomerase A (cyclophilin B; PPIB) and ribosomal protein L22 (PRL22) were used as house-keeping genes, as according to Kirschneck et al., they were identified as reliable and suitable reference genes for normalization in relative qRT-PCR gene expression studies on human periodontal ligament fibroblasts (data not shown) (29). Table 1 presents the stratum specific estimates for the effect of different levels of TNF-α and tensile stress on the regulation of OPN and ALP. Prolonged exposure to TNF-α compromises human PDLF’s osteoblastic differentiation ability Next, we investigated the long-term effect of the presence of increased concentration of TNF-α on human PDLF. Accordingly, PDLF were cultured in the presence of TNF-α as described in Materials and methods. At the end of this period, TNF-α was eliminated by medium change and the cells were cultured in the absence of TNF-α for at least 4 days (p/a TNF-α cells), so as to evaluate only the permanent (and not the short-term) effect of this inflammatory cytokine. First, we assessed the basal levels of the expression of the genes under study and found that, in comparison to cells cultured for the same period in the absence of TNF-α, while the levels of c-fos were practically unchanged, ALP and OPN levels were dramatically reduced (Figure 3A). In agreement with the attenuation of these osteoblastic effectors, we observed that, while untreated cells underwent to an osteoblastic differentiation in the presence of an osteo-inductive medium, the differentiation process is significantly inhibited in TNF-α-treated cells as shown by Alizarin Red-S staining (Figure 3B). Figure 3. Open in new tabDownload slide Effect of long-term exposure to tumour necrosis factor-α (TNF-α) on the osteoblastic potential of human periodontal ligament fibroblasts (PDLF). (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of alkaline phosphatase (ALP), osteopontin (OPN) and c-fos messenger RNA (mRNA) basal levels of PDLF after repetitive exposures to TNF-α followed by a recovery period of at least 4 days in the absence of TNF-α (p/a TNF-α). Mean cycle threshold (Ct) values of all three genes were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative gene expression was calculated as the ratio of the expression level of p/a TNF-α cells to that of untreated control. Box plots of the three genes’ expression are presented. qRT-PCR experiments were performed on samples from three different donors and for at least three times in triplicates. (B) Quantification of calcium mineral content of Alizarin Red-S extracts of long-term treated with TNF-α cells (TNF-α pretreated) after incubation with an osteogenic medium along with TNF-α (+) or not (−). Osteoblastic differentiation is expressed as a % ratio of the cells cultured in 10% (v/v) fetal bovine serum (FBS) Dulbecco’s modified Eagle’s medium (DMEM). TNF-α–untreated osteogenic-induced cells (CTRL). Figure 3. Open in new tabDownload slide Effect of long-term exposure to tumour necrosis factor-α (TNF-α) on the osteoblastic potential of human periodontal ligament fibroblasts (PDLF). (A) Quantitative real-time polymerase chain reaction (qRT-PCR) analysis of alkaline phosphatase (ALP), osteopontin (OPN) and c-fos messenger RNA (mRNA) basal levels of PDLF after repetitive exposures to TNF-α followed by a recovery period of at least 4 days in the absence of TNF-α (p/a TNF-α). Mean cycle threshold (Ct) values of all three genes were normalized to those of glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Relative gene expression was calculated as the ratio of the expression level of p/a TNF-α cells to that of untreated control. Box plots of the three genes’ expression are presented. qRT-PCR experiments were performed on samples from three different donors and for at least three times in triplicates. (B) Quantification of calcium mineral content of Alizarin Red-S extracts of long-term treated with TNF-α cells (TNF-α pretreated) after incubation with an osteogenic medium along with TNF-α (+) or not (−). Osteoblastic differentiation is expressed as a % ratio of the cells cultured in 10% (v/v) fetal bovine serum (FBS) Dulbecco’s modified Eagle’s medium (DMEM). TNF-α–untreated osteogenic-induced cells (CTRL). The effect of long-term propagation of human PDLF in the presence of TNF-α on the cellular responses to cyclic mechanical stimulation Next, we studied the long-term effect of TNF-α on the mechanical loading-elicited responses of PDLF. First, we studied the regulation of MAPK activation of p/a TNF-α-cells in response to mechanical stimulation. As can be seen in Figure 4A, the activation of p38 is acute while ERK and JNK phosphorylation is short and delayed as compared with the response of naïve cells, similarly to what was found after the short-term treatment with TNF-α as shown in Figure 2A. Figure 4. Open in new tabDownload slide Impact of prolonged propagation with tumour necrosis factor-α (TNF-α) on human periodontal ligament fibroblasts’ (PDLF) responses to mechanical stimulation. (A) PDLF repeatedly exposed to TNF-α were subjected to cyclic tensile strain in the absence of TNF-α and total and phosphorylated forms of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were assessed, after 0.25, 0.5, 1, 3 and 6 hours of cyclic tensile strain application, by western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. A representative experiment, among three similar ones with similar results, is presented here. (B–D) TNF-α-pre-treated human PDLF were subjected to cyclic tensile stretching (p/a TNF-α + CTS) in the absence of TNF-α for 30 minutes (B) or 18 hours (C, D) and messenger RNA (mRNA) levels of c-fos, alkaline phosphatase (ALP) and osteopontin (OPN), respectively, were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Box plots for c-fos, OPN and ALP gene expression are presented. qRT-PCR experiments were performed on samples from three different donors and for at least three times in triplicates. Figure 4. Open in new tabDownload slide Impact of prolonged propagation with tumour necrosis factor-α (TNF-α) on human periodontal ligament fibroblasts’ (PDLF) responses to mechanical stimulation. (A) PDLF repeatedly exposed to TNF-α were subjected to cyclic tensile strain in the absence of TNF-α and total and phosphorylated forms of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were assessed, after 0.25, 0.5, 1, 3 and 6 hours of cyclic tensile strain application, by western blot analysis. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as the loading control. A representative experiment, among three similar ones with similar results, is presented here. (B–D) TNF-α-pre-treated human PDLF were subjected to cyclic tensile stretching (p/a TNF-α + CTS) in the absence of TNF-α for 30 minutes (B) or 18 hours (C, D) and messenger RNA (mRNA) levels of c-fos, alkaline phosphatase (ALP) and osteopontin (OPN), respectively, were assessed by quantitative real-time polymerase chain reaction (qRT-PCR). Box plots for c-fos, OPN and ALP gene expression are presented. qRT-PCR experiments were performed on samples from three different donors and for at least three times in triplicates. Subsequently, we investigated the activation of downstream genes. We found, in the absence of TNF-α (naïve cells), an intense c-fos upregulation after the application of mechanical stretching (β = 2.05; 95 per cent CI: 1.46, 2.64; P < 0.001; Table 2). However, this stimulation was significantly reduced in p/a TNF-α fibroblasts by a mean value of −0.83 (95 per cent CI: −1.59, −0.08; P = 0.03; Figure 4B). Table 2 presents the stratum specific estimates for c-fos regulation under pre-treatment or not with TNF-α and after mechanical stretching. Table 2. Multivariable linear regression with observed coefficients and 95% confidence intervals (95% CIs) for the long-term effect of tumour necrosis factor-α (TNF-α; + pre-treatment, − no treatment) and cyclic stress (CTS) on the regulation of c-fos (as a logarithm of the original value) Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α pre-treatment−, CTS− Reference TNF-α pre-treatment+, CTS− −0.01 −0.26, 0.24 0.95 CTS−, TNF-α pre-treatment− Reference CTS+, TNF-α pre-treatment− 2.05 1.46, 2.64 <0.001 CTS+, TNF-α pre-treatment− Reference CTS+, TNF-α pre-treatment+ −0.83 −1.59, −0.08 0.03 Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α pre-treatment−, CTS− Reference TNF-α pre-treatment+, CTS− −0.01 −0.26, 0.24 0.95 CTS−, TNF-α pre-treatment− Reference CTS+, TNF-α pre-treatment− 2.05 1.46, 2.64 <0.001 CTS+, TNF-α pre-treatment− Reference CTS+, TNF-α pre-treatment+ −0.83 −1.59, −0.08 0.03 Standard errors were calculated using the bootstrap method with 1000 replications. Likelihood ratio test for interaction (TNF-α pre-treatment × CTS), P = 0.04; + denotes presence; − denotes absence. Open in new tab Table 2. Multivariable linear regression with observed coefficients and 95% confidence intervals (95% CIs) for the long-term effect of tumour necrosis factor-α (TNF-α; + pre-treatment, − no treatment) and cyclic stress (CTS) on the regulation of c-fos (as a logarithm of the original value) Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α pre-treatment−, CTS− Reference TNF-α pre-treatment+, CTS− −0.01 −0.26, 0.24 0.95 CTS−, TNF-α pre-treatment− Reference CTS+, TNF-α pre-treatment− 2.05 1.46, 2.64 <0.001 CTS+, TNF-α pre-treatment− Reference CTS+, TNF-α pre-treatment+ −0.83 −1.59, −0.08 0.03 Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α pre-treatment−, CTS− Reference TNF-α pre-treatment+, CTS− −0.01 −0.26, 0.24 0.95 CTS−, TNF-α pre-treatment− Reference CTS+, TNF-α pre-treatment− 2.05 1.46, 2.64 <0.001 CTS+, TNF-α pre-treatment− Reference CTS+, TNF-α pre-treatment+ −0.83 −1.59, −0.08 0.03 Standard errors were calculated using the bootstrap method with 1000 replications. Likelihood ratio test for interaction (TNF-α pre-treatment × CTS), P = 0.04; + denotes presence; − denotes absence. Open in new tab We also found that mechanical stretching leads to significant upregulation of both OPN and ALP genes (Mann–Whitney U-test, P = 0.002, for both genes; Figure 4C and 4D). However, in p/a TNF-α fibroblasts, this stretching-induced upregulation was significantly alleviated (Mann–Whitney U-test, P = 0.002, for both genes; Figure 4C and 4D). These data are analogous to the response of naïve cells in the short-term presence of TNF-α (Figure 2A) and suggest similar but permanent molecular alterations after a long-term treatment with this cytokine. Finally, as in the case of short-term TNF-α-treated and mechanically stimulated cells, similar results were obtained when peptidylprolyl isomerase A (cyclophilin B) (PPIB) and ribosomal protein L22 (PRL22) were used as house-keeping genes (data not shown). Prolonged exposure to TNF-α does not lead human PDLF to premature senescence or to an increased oxidative burden. In an effort to investigate permanent changes that may explain the observed phenomena, we first checked for a possible induction of premature senescence by the prolonged exposure to TNF-α as described in other cell types. However, as shown in Figure 5A, there were no signs of increased senescence as the percentage of cells incorporating BrdU (a marker of the cell’s proliferative capacity) is unaltered between p/a TNF-α and untreated cells. The same stands for the expression of two robust markers of senescence, the cell cycle inhibitors p16Ink4a and p21WAF1 (Figure 5B). Figure 5. Open in new tabDownload slide Influence of repetitive exposures to tumour necrosis factor-α (TNF-α) on the induction of premature senescence of human periodontal ligament fibroblasts (PDLF). (A) 5-bromo-2´-deoxyuridine (BrdU) incorporation of human PDLF repeatedly exposed to TNF-α and left to recover for at least 4 days in the absence of TNF-α (p/a TNF-α), performed as described in Materials and methods, is expressed as a % ratio of positive cells in the culture. (B) Box plots of messenger RNA (mRNA) levels of p16INK4a and p21WAF1 senescence marker genes of human PDLF repeatedly exposed to TNF-α (p/a TNF-α) as estimated by quantitative real-time polymerase chain reaction (qRT-PCR) as in Figure 3. Experiments were performed in triplicates and at least twice. Figure 5. Open in new tabDownload slide Influence of repetitive exposures to tumour necrosis factor-α (TNF-α) on the induction of premature senescence of human periodontal ligament fibroblasts (PDLF). (A) 5-bromo-2´-deoxyuridine (BrdU) incorporation of human PDLF repeatedly exposed to TNF-α and left to recover for at least 4 days in the absence of TNF-α (p/a TNF-α), performed as described in Materials and methods, is expressed as a % ratio of positive cells in the culture. (B) Box plots of messenger RNA (mRNA) levels of p16INK4a and p21WAF1 senescence marker genes of human PDLF repeatedly exposed to TNF-α (p/a TNF-α) as estimated by quantitative real-time polymerase chain reaction (qRT-PCR) as in Figure 3. Experiments were performed in triplicates and at least twice. Finally, we checked for changes in the oxidative burden in treated cells. We found that the short-term treatment with TNF-α, at the pathological concentrations used, was unable to provoke an immediate increase in the intracellular levels of ROS (data not shown). In addition, the prolonged exposure to TNF-α does not lead to increased ROS levels in p/a TNF-α cells (Mann–Whitney U-test, P = 0.40; Figure 6A). In agreement, a classical antioxidant agent N-acetyl-cysteine (NAC) was unable to change the expression of c-fos (β = −0.09; 95 per cent CI: −0.44, 0.27; P = 0.63; Figure 6B; Table 3) and ALP genes (β = 0.04; 95 per cent CI: −0.05, 0.14; P = 0.38; Figure 6C; Table 3) in control and mechanically stimulated PDLF. Figure 6. Open in new tabDownload slide Role of oxidative stress on tumour necrosis factor-α (TNF-α) treated human periodontal ligament fibroblasts’ (PDLF) impaired responses to mechanical stimulation. (A) Estimation of intracellular reactive oxygen species (ROS) levels of human PDLF repeatedly exposed to TNF-α and left to recover for at least 4 days in the absence of TNF-α (p/a TNF-α cells) as assessed by 2´, 7´-dichlorofluorescein-diacetate (DCFH-DA) assay. Intracellular ROS levels are expressed as a % ratio of the untreated control. (B) Box plots for messenger RNA (mRNA) levels of c-fos of mechanically stimulated for 30 minutes and repeatedly exposed to TNF-α PDLF in the presence or absence of 2-mM N-acetyl-cysteine (NAC) as assessed by quantitative real-time polymerase chain reaction (qRT-PCR). (C) Box plots for mRNA levels of alkaline phosphatase (ALP) of mechanically stimulated for 18 hours and repeatedly exposed to TNF-α PDLF in the presence or absence of 2-mM NAC. A representative experiment, among two similar ones performed in duplicates, is presented here. CTRL, unstimulated untreated control cells; CTS, stimulated cells; TNF-α, unstimulated TNF-α-treated cells; CTS + TNF-α, stimulated TNFα-treated cells; NAC, unstimulated NAC-treated cells; CTS + NAC, stimulated NAC-treated cells; NAC + TNF-α, unstimulated NAC- and TNF-α-treated cells; CTS + NAC + TNF-α, stimulated NAC- and TNF-α-treated cells. Figure 6. Open in new tabDownload slide Role of oxidative stress on tumour necrosis factor-α (TNF-α) treated human periodontal ligament fibroblasts’ (PDLF) impaired responses to mechanical stimulation. (A) Estimation of intracellular reactive oxygen species (ROS) levels of human PDLF repeatedly exposed to TNF-α and left to recover for at least 4 days in the absence of TNF-α (p/a TNF-α cells) as assessed by 2´, 7´-dichlorofluorescein-diacetate (DCFH-DA) assay. Intracellular ROS levels are expressed as a % ratio of the untreated control. (B) Box plots for messenger RNA (mRNA) levels of c-fos of mechanically stimulated for 30 minutes and repeatedly exposed to TNF-α PDLF in the presence or absence of 2-mM N-acetyl-cysteine (NAC) as assessed by quantitative real-time polymerase chain reaction (qRT-PCR). (C) Box plots for mRNA levels of alkaline phosphatase (ALP) of mechanically stimulated for 18 hours and repeatedly exposed to TNF-α PDLF in the presence or absence of 2-mM NAC. A representative experiment, among two similar ones performed in duplicates, is presented here. CTRL, unstimulated untreated control cells; CTS, stimulated cells; TNF-α, unstimulated TNF-α-treated cells; CTS + TNF-α, stimulated TNFα-treated cells; NAC, unstimulated NAC-treated cells; CTS + NAC, stimulated NAC-treated cells; NAC + TNF-α, unstimulated NAC- and TNF-α-treated cells; CTS + NAC + TNF-α, stimulated NAC- and TNF-α-treated cells. Table 3. Multivariable linear regression with observed coefficients and 95% confidence Intervals (95% CIs) for the effect of TNF-α (+ presence, − absence), cyclic stress (CTS) and N-acetyl-cysteine (NAC) on the regulation of c-fos (as a logarithm of the original value) and alkaline phosphatase [sqrt(ALP), as a square root of the original value] Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α− Reference TNF-α+ −0.84 −1.20, −0.49 <0.001 CTS− Reference CTS+ 2.45 2.08, 2.82 <0.001 NAC− Reference NAC+ −0.09 −0.44, 0.27 0.63 LR test (TNF-α × stress) 0.07 Sqrt(ALP) TNF-α− Reference TNF-α+ −0.50 −0.59, −0.40 <0.001 CTS− Reference CTS+ 0.20 0.10, 0.30 <0.001 NAC− Reference NAC+ 0.04 −0.05, 0.14 0.38 LR test (TNF-α × stress) 0.96 Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α− Reference TNF-α+ −0.84 −1.20, −0.49 <0.001 CTS− Reference CTS+ 2.45 2.08, 2.82 <0.001 NAC− Reference NAC+ −0.09 −0.44, 0.27 0.63 LR test (TNF-α × stress) 0.07 Sqrt(ALP) TNF-α− Reference TNF-α+ −0.50 −0.59, −0.40 <0.001 CTS− Reference CTS+ 0.20 0.10, 0.30 <0.001 NAC− Reference NAC+ 0.04 −0.05, 0.14 0.38 LR test (TNF-α × stress) 0.96 Standard errors were calculated using the bootstrap method with 1000 replications. + denotes presence; − denotes absence. LR, likelihood ratio. Open in new tab Table 3. Multivariable linear regression with observed coefficients and 95% confidence Intervals (95% CIs) for the effect of TNF-α (+ presence, − absence), cyclic stress (CTS) and N-acetyl-cysteine (NAC) on the regulation of c-fos (as a logarithm of the original value) and alkaline phosphatase [sqrt(ALP), as a square root of the original value] Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α− Reference TNF-α+ −0.84 −1.20, −0.49 <0.001 CTS− Reference CTS+ 2.45 2.08, 2.82 <0.001 NAC− Reference NAC+ −0.09 −0.44, 0.27 0.63 LR test (TNF-α × stress) 0.07 Sqrt(ALP) TNF-α− Reference TNF-α+ −0.50 −0.59, −0.40 <0.001 CTS− Reference CTS+ 0.20 0.10, 0.30 <0.001 NAC− Reference NAC+ 0.04 −0.05, 0.14 0.38 LR test (TNF-α × stress) 0.96 Log(c-fos) . Observed coefficient β . 95% CIs . P-value . TNF-α− Reference TNF-α+ −0.84 −1.20, −0.49 <0.001 CTS− Reference CTS+ 2.45 2.08, 2.82 <0.001 NAC− Reference NAC+ −0.09 −0.44, 0.27 0.63 LR test (TNF-α × stress) 0.07 Sqrt(ALP) TNF-α− Reference TNF-α+ −0.50 −0.59, −0.40 <0.001 CTS− Reference CTS+ 0.20 0.10, 0.30 <0.001 NAC− Reference NAC+ 0.04 −0.05, 0.14 0.38 LR test (TNF-α × stress) 0.96 Standard errors were calculated using the bootstrap method with 1000 replications. + denotes presence; − denotes absence. LR, likelihood ratio. Open in new tab Discussion Rheumatoid arthritis, among many other chronic inflammatory diseases, exerts damaging effects in bone tissue as it is characterized by progressive tissue deterioration leading to a generalized osteoporosis (36). Moreover, these patients are most likely to be afflicted by periodontal disease and suffer from tooth loss (4). Abundant data demonstrate that the severe outcomes of the disease are mainly driven by the overproduction of TNF-α, as this cytokine is implicated in both local joint damage and systemic bone loss (2, 37). In this context, multiple studies indicated the negative effect of TNF-α on the osteoblastic differentiation capacity of competent cells (10, 38–41), such as PDL fibroblasts, known to play a pronounced role in the maintenance of PDL tissue homeostasis. An integral part of PDL homeostasis is the continuous exposure to mechanical stimulation and several studies indicate that these forces activate molecular events leading to osteoblastic differentiation (23). Accordingly, here we investigated the influence of short- and long-term treatment of these cells with TNF-α on their response to cyclic mechanical stretching and the induction of osteoblastic differentiation. This is important for orthodontic patients afflicted by systematic diseases affecting the connective tissue homeostasis, such as rheumatoid arthritis. In this context and in contrast to the study of Kirschneck et al. (29) that used a compression-generating loading model, we used a specially designed device that enabled the application of uniform and uniaxial cyclic tensile strain on PDL fibroblasts (30). The applied uniaxial tensile strain falls within the range of physiological tissue deformation during orthodontic tooth movement (31–32, 42). In accordance with previous studies demonstrating mechanical loading-induced activation of c-fos under normal conditions (22–23, 25), we found a notable upregulation of c-fos expression evoked by cyclic tensile strain application. However, TNF-α significantly repressed c-fos activation. In addition, mechanical strain triggers the upregulation of ALP and OPN expression, two major osteoblast marker genes (42–43), which is also diminished in the presence of the inflammatory cytokine. On the other hand, in still unpublished studies, we have found that the expression of Runx2, another transcription factor related to osteoblastic differentiation, is not affected under the current experimental conditions, that is, under application of cyclic tensile strain (8 per cent elongation, 1Hz), at least up to 18 hours after stimulation. This is in accordance to the results of Sun et al., which indicated that under normal conditions, Runx2 gene expression of mechanically stimulated PDL cells is practically unaffected (44). Taken together, the above results indicate that short-term exposure of PDL fibroblasts to TNF-α compromises their immediate response to mechanical strains and jeopardizes their osteoblastic differentiation potential. We also investigated the impact of prolonged propagation, that is, up to 20 consecutive treatments that lasted for about 50 days, of human PDLF within this inflammatory microenvironment. Accordingly, after this prolonged treatment, the cells were studied after a 4-day recovery period in order to avoid the immediate effect of TNF-α. We found that the basal levels of ALP and OPN osteoblast marker genes were significantly downregulated after repetitive exposures of cells to TNF-α and, in accordance, mineral nodule formation capacity of TNF-α-treated PDLF was also notably decreased in agreement with previous reports indicating an inflammation-related impairment of the osteogenic potential after a 4-weeks’ incubation with an osteo-inductive medium, as well as a decrease in ALP activity of human PDL cells exposed to a combination of the inflammatory cytokines TNF-α and IL-6 (45). Recently, Sun et al., in agreement with our data, have reported that the osteogenesis of PDL cells is suppressed under inflammatory microenvironments upon the short-term application of uniaxial tensile strength by using a more complex inflammatory environment (i.e. TNF-α + IL-6) (44). Moreover, here, we demonstrated, for the first time, that long-term treatment upon repetitive exposure of cells to TNF-α led to a notable repression of cyclic tensile strain-induced activation of c-fos, as well as ALP and OPN osteoblast-specific genes, even after a few days’ recovery period of cells from the inflammatory mediator. These data suggest that a long-term treatment with TNF-α provokes permanent changes inhibiting tensile strain-elicited osteoblastic differentiation. In order to identify molecular alterations that may explain the observed phenomena, we studied the role of premature senescence and of the intracellular oxidative burden. TNF-α-induced premature senescence has been previously reported in many cell types and it is further considered to be mediated by ROS generation (11, 46–47). Nevertheless, in the current study, no such effect was observed. In particular, the proliferative potential of PDLF long-term treated with TNF-α (up to 20 consecutive treatments) was uncompromised compared to their untreated counterparts. In addition, the expression of major cellular senescence gene markers, that is, p16INK4a and p21WAF1, exhibited no increase. This may indicate that PDLF may be resistant to the TNF-α-mediated premature senescence and possibly a longer period of treatment is needed for this effect. However, even this exposure to TNF-α leads to a distinct impairment of osteoblastic differentiation. The activation of MAPK is considered an immediate response to mechanical stimulation in PDLF and is involved in the regulation of signalling networks leading to osteoblastic differentiation (23). Interestingly, we observed in both short- and long-term treatment of these cells a change in MAPK regulation, that is, the activation of both ERK and JNK is delayed and of shorter duration. Knowing that ERK and JNK are responsible for the stretching-induced c-fos expression in these cells (23), as well as the role of the kinetics of such pathways’ activation in bone remodelling (48), these changes may represent the molecular basis of the observed phenomena. However, further investigation of this hypothesis is pending. Finally, a previously published study indicated that patients with periodontitis and ischaemic heart disease presented lower serum and salivary vitamin C and anti-oxidant levels compared to healthy subjects (49), suggesting the implication of oxidative stress in chronic inflammatory diseases. However, neither short-term nor long-term exposure of PDLF to TNF-α provoked any changes in intracellular ROS levels. In agreement with the above, incubation of TNF-α-treated PDLF with the antioxidant NAC could not restore CTS-induced upregulated levels of c-fos transcription factor and of ALP, suggesting that TNF-α-evoked deteriorative effects on the immediate responses of PDLF during mechanical stretching are likely not mediated by an oxidative stress. Conclusion Taken together, the current study’s data indicate that increased concentrations of TNF-α, such as those confronted by PDLF in many inflammatory conditions, such as rheumatoid arthritis, constitute a potent suppressor of cells’ immediate responses to mechanical forces and compromise their capacity for an osteoblastic differentiation leading to the impairment of tissue repair and remodelling. Nonetheless, additional experimental approaches are needed, so as to access the effect of enforced tooth movement at the tissue level in patients suffering from inflammatory diseases. Author contributions AP contributed to conception, design, data acquisition, analysis and interpretation of the data, drafting of the article and critical revision of the manuscript; AC contributed to conception, design, data acquisition, analysis and interpretation of the data, drafting of the article and critical revision of the manuscript; DK contributed to analysis, interpretation of the data and critical revision of the manuscript; TE contributed to conception, design and interpretation of the data and critically revised the manuscript; DK contributed to conception, design, interpretation of the data, drafting of the article and critically revised the manuscript. All authors have approved the final article. Funding This work has been partly supported by the project ‘Target Identification and Development of Novel Approaches for Health and Environmental Applications’ (MIS 5002514), which is implemented under the Action for the Strategic Development on the Research and Technological Sectors, funded by the Operational Programme ‘Competitiveness, Entrepreneurship and Innovation’ (NSRF 2014–2020) and co-financed by Greece and the European Union (European Regional Development Fund). Conflict of interest The authors declare no potential conflicts of interest with respect to the authorship and/or the publication of this article. References 1. Corrado , A. , Maruotti , N. and Cantatore , F.P. ( 2017 ) Osteoblast role in rheumatic diseases . International Journal of Molecular Sciences , 18 , 1272. OpenURL Placeholder Text WorldCat 2. Keffer , J. , Probert , L., Cazlaris , H., Georgopoulos , S., Kaslaris , E., Kioussis , D. and Kollias , G. 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TI - Short- and long-term treatment with TNF-α inhibits the induction of osteoblastic differentiation in cyclic tensile-stretched periodontal ligament fibroblasts
JF - The European Journal of Orthodontics
DO - 10.1093/ejo/cjaa042
DA - 2020-09-11
UR - https://www.deepdyve.com/lp/oxford-university-press/short-and-long-term-treatment-with-tnf-inhibits-the-induction-of-OWcoLeTnAn
SP - 396
EP - 406
VL - 42
IS - 4
DP - DeepDyve
ER -