Effects of ethanol on human periodontal ligament fibroblasts subjected to static compressive force

Accepted Manuscript Effects of ethanol on human periodontal ligament fibroblasts subjected to static compressive force Agnes Schröder, Dr. rer. nat, Erika Calvano Küchler, DDS, Professor Dr. (PhD), Marjorie Omori, DDS, Gerrit Spanier, MD, DDS, Dr., Peter Proff, MD, DDS, Professor Dr. Dr. (PhD), Christian Kirschneck, DDS, Priv.-Doz. Dr. (PhD) PII:

S0741-8329(18)30124-1

DOI:

10.1016/j.alcohol.2018.10.004

Reference:

ALC 6864

To appear in:

Alcohol

Received Date: 27 April 2018 Revised Date:

8 October 2018

Accepted Date: 9 October 2018

Please cite this article as: Schröder A., Küchler E.C., Omori M., Spanier G., Proff P. & Kirschneck C., Effects of ethanol on human periodontal ligament fibroblasts subjected to static compressive force, Alcohol (2018), doi: https://doi.org/10.1016/j.alcohol.2018.10.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Title:

Effects of ethanol on human periodontal ligament fibroblasts subjected to static compressive force

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Authors: Agnes Schröder, Erika Calvano Küchler, Marjorie Omori, Gerrit Spanier, Peter Proff, Christian Kirschneck

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Agnes Schröder, Dr. rer. nat. (corresponding author) Department of Orthodontics, University Hospital Regensburg Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany Phone: +49 941 944 4991 Facsimile: +49 941 944 6169 E-mail: [email protected]

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Erika Calvano Küchler, DDS, Professor Dr. (PhD) Professor of the Post-graduation Program in Pediatric Dentistry School of Dentistry of Ribeirão Preto, University of São Paulo, Brasil Avenida do Café, s/n - Campus da USP Ribeirão Preto/SP - CEP: 14040-904 E-mail: [email protected]

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Marjorie Omori, DDS School of Dentistry of Ribeirão Preto, University of São Paulo, Brasil Avenida do Café, s/n - Campus da USP Ribeirão Preto/SP - CEP: 14040-904 E-mail: [email protected]

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Gerrit Spanier, MD, DDS, Dr. Department of Cranio-Maxillo-Facial Surgery, University Hospital Regensburg Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany E-mail: [email protected] Peter Proff, MD, DDS, Professor Dr. Dr. (PhD) Department of Orthodontics, University Hospital Regensburg Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany E-mail: [email protected] Christian Kirschneck, DDS, Priv.-Doz. Dr. (PhD) Department of Orthodontics, University Hospital Regensburg Franz-Josef-Strauss-Allee 11, 93053 Regensburg, Germany E-mail: [email protected]

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Abstract (251/300 words) Consumption of toxic substances like alcohol is widespread in the general population and thus also in patients receiving orthodontic treatment. Since human periodontal ligament (hPDL) fibroblasts play a key role in orthodontic tooth movement (OTM) by expressing cytokines and chemokines, we wanted to clarify, whether ethanol modulates the physiological activity and

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expression pattern of hPDL fibroblasts during static compressive force application. We preincubated hPDL fibroblasts for 24h with different ethanol concentrations, corresponding to casual (0.41‰) and excessive (1.79‰) alcohol consumption. At each ethanol concentration we incubated the cells for another 48h with and without an additional physiological

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compressive force of 2 g/cm2 occurring during orthodontic tooth movement in compression areas of the periodontal ligament. Thereafter we analyzed expression and secretion of genes

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and proteins involved in OTM regulation by RT-qPCR and ELISA. We also performed coculture experiments to observe hPDL-fibroblast-mediated osteoclastogenesis. We observed no effects of ethanol on cytotoxicity or cell viability of hPDL fibroblasts in the applied concentrations. Ethanol showed an enhancing effect on angiogenesis and activity of alkaline phosphatase. Simultaneously, ethanol reduced the induction of IL-6 and increased prostaglandin E2 synthesis as well as hPDL-fibroblast-mediated osteoclastogenesis without

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affecting the RANK-L/OPG-system. hPDL fibroblasts thus seem to be a cell type quite resistant to ethanol, as no cytotoxic effects or influence on cell viability were detected. High ethanol concentrations, however, seem to promote bone formation and angiogenesis. Ethanol at 1.79‰ also enhanced hPDL-induced osteoclastogenesis, indicating increased bone

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resorption and thus tooth movement velocity to be expected during orthodontic therapy.

Keywords: ethanol, alcohol, periodontal ligament fibroblast, PDL, orthodontics, compressive force

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Introduction The orthodontic correction of malpositioned teeth is of considerable medical importance, as malocclusions of teeth are associated with an increased prevalence for caries and periodontitis [22, 51], which are designated by the WHO as "the most important global oral health burden" and have a high prevalence of up to 90% [1], leading to long-term malfunction, impaired

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phonetics, dental pain and loss of teeth [60]. The dental specialty of orthodontics has an important prophylactic function for the development and progression of these oral diseases. For orthodontic tooth movement (OTM), which serves to correct tooth misalignment for functional and aesthetic reasons, a mechanical force is applied to teeth in the direction of

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movement by means of removable or fixed orthodontic intraoral or extraoral appliances [24]. This force creates tensile and compression zones within the periodontal ligament (PDL), a

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fibrous and cellular connective tissue linking the tooth to the surrounding alveolar bone. PDL fibroblasts constitute the main cell population within the PDL, which regulate tissue homeostasis, synthesize collagenous structural proteins and perform regulatory functions in innate immune defense [31]. PDL fibroblasts respond to these orthodontic compressive or tensile forces with a release of prostaglandins by increased activity of the induced cyclooxygenase 2 (COX2). These act in an auto- and paracrine manner on PDL fibroblasts

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and osteoblasts leading to an increased formation of soluble and membrane-linked RANK-L (receptor activator of nuclear kappa b ligand) [31] and the secretion of proinflammatory cytokines like interleukin-1 (IL-1), IL-6 and tumor necrosis factor α (TNF α) as well as

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chemokines (IL-8). These potentiate RANK-L formation, which is essential for bone resorption [45]. Furthermore, they release matrix metalloproteinases [54], which promote degradation of the extracellular matrix [41] and thus induce the transformation of the PDL for

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tooth movement [18].

Toxic substances like nicotine or alcohol are regularly consumed by many patients during OTM [33, 35]. These pharmacologically active substances might therefore unintentionally inhibit tooth movement, cause undesirable root resorptions or induce loss of periodontal attachment and bone [5, 8, 33, 35] by modulating the molecular and cellular processes enabling OTM. Investigating these pharmacological side effects on orthodontic therapy is of increasing clinical interest [5, 8]. The consumption of ethanol as a major component of alcoholic beverages is still quite common, particularly in young people, despite a certain decline in recent years [39]. 54.4% of adolescents up to the age of 17 regularly consume alcohol with excessive consumption by 15.8% and binge drinking by 11.5% of adolescents

ACCEPTED MANUSCRIPT [38]. In particular, alcoholic soft drinks (alco-pops) have been quite popular for many years, often leading to chronic alcohol consumption, which is a serious health risk [2, 23, 37, 46, 58]. To date, however, the effect of ethanol on PDL fibroblasts in the context of mechanical stress or orthodontic tooth movement has not been clarified. Periodontal bone loss and an increase

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of inflammatory processes were already observed during chronic alcohol consumption in an animal model [17, 30, 44, 49, 50, 53]. These studies suggest that ethanol has a direct stimulating effect on osteoclasts and osteoclastogenesis [14, 29], whereas osteoblast markers were impaired in the rat [16]. Bannach et al. [4] report significant periodontal bone loss in a

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rat model after the administration of ethanol during the growth phase. Reduced bone density and inhibition of root cement formation have also been observed [16], which could promote

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tooth root resorption by the lack of reparative action of cementoblasts to regenerate defects. Alcohol consumption also has been reported to increase the risk of periodontitis at a young age [27]. These observations, however, are in contrast to also reported inhibitory effects of alcohol (ethanol and propanolol) on osteoclasts and osteoclast activity [3, 43]. Due to the contradictory and ambiguous data situation, the effect of ethanol on periodontal tissue and during orthodontic tooth movement (OTM) is still unclear and requires further

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research. In this study, we investigated the effect of ethanol on PDL fibroblasts during compressive force application in vitro, as PDL fibroblasts play a crucial role in OTM by the secretion of cytokines, chemokines and growth factors [18, 31]. Since available studies

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suggest that the effect of alcohol is dose-dependent, different ethanol concentrations were

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used in this study corresponding to casual and excessive alcohol consumption.

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Material and methods Origin and cultivation of hPDL fibroblasts We used a pool of primary human periodontal ligament (hPDL) fibroblast cell lines from six patients (3 male, 3 female, age: 17-27 years), cultivated from periodontal connective tissue of

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human wisdom teeth free of carious lesions, which were extracted due to medical reasons in our facility. Ethical approval was obtained by the ethics committee of the University of Regensburg, Germany (approval number 12-170-0150). Tissue samples were scraped off the middle third of the wisdom tooth roots and grown in 6-well plates under standard cell culture

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conditions (37°C, 5% CO2, 100% H2O) in full medium (DMEM high glucose, D5796, SigmaAldrich, Munich, Germany), supplemented with 10% FCS (P30-3306, PAN-Biotech, Aidenbach, Germany), 1% L-glutamine (SH30034.01, GE Healthcare Europe, Munich,

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Germany), 100 µM ascorbic acid (A8960, Sigma-Aldrich) and 1% antibiotics/antimycotics (A5955, Sigma-Aldrich). Cells were characterized by expression of PDL-specific marker genes and their spindle-shaped morphology [32] (Supplementary Figure and Table). Until use they were stored in liquid nitrogen (90% FCS, 10% DMSO, freezing 1°C/minute).

Experimental design

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We seeded pooled hPDL fibroblasts (3-5th passage) onto 6-well cell culture plates at a density of 70,000 cells in 2 mL DMEM per well. hPDL fibroblasts were incubated with either 0‰ (control), 0.41‰ or 1.79‰ ethanol (603-002-00-5, Honeywell, Bucharest, Romania) for 72h

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with or without (3/3 wells per plate) compressive orthodontic force application of 2g/cm2 for 48h after a 24h preincubation phase by means of a glass disc according to a published and well established protocol for the simulation of static compressive force application [31, 32]

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(Fig. 1).

Relative gene expression via quantitative real-time polymerase chain reaction (RT-qPCR) We performed RNA isolation, quality assessment, cDNA synthesis and RT-qPCR as described previously according to MIQE guidelines [2]. Normalization of target genes for assessment of relative gene expression was based on two reference genes (RPL22/ PPIB), which were validated before for hPDL fibroblasts and the in vitro model used [2]. We calculated relative gene expression as 2-∆Cq [3] with ∆Cq = Cq (target gene) – Cq (mean RPL22/PPIB). RT-qPCR primer design and validation were performed according to MIQE

ACCEPTED MANUSCRIPT quality criteria as described before [2] (Table 1). RT-qPCR analysis was performed three times for three biological replicates per group (N=3, n=9).

Flow-cytometry-based detection of apoptosis and necrosis To investigate apoptosis and necrosis induced by different ethanol concentrations, we

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performed FITC (Fluorescin isothiocyanate) annexin V (marker for apoptosis) and propodium iodide (PI, marker for necrosis) stainings according to the manufacturer’s instructions (FITC Annexin V Apoptosis Detection Kit I, 556547, BD Pharmingen, Heidelberg, Germany), followed by FACS (fluorescence-activated cell sorting, BD FACSCalibur, Heidelberg,

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Germany). To this aim, we washed the membrane-adherent hPDL fibroblasts after the indicated time periods twice PBS (phosphate-buffered saline) of 4°C and immediately resuspended the fibroblasts at a concentration of 106 cells/pro ml (Z2 cell counter, Beckman

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Coulter, Krefeld, Germany) in 10x binding buffer (0.1 M HEPES/NaOH pH7.4, 1.4 M NaCl, 25 mM CaCl2). We transferred 100 µl of this solution to a 5 ml FACS tube and added 5 µl FITC annexin V and 5 µl PI. After gently vortexing the fibroblasts, we incubated them for 15 min at room temperature in the dark. We added 400 µl binding buffer to each tube and analyzed them by flow cytometry (FSC: 5V, SSC: 349 V, FITC: 350V, PerCP-Cy5-5: 450V,

FSC Area Scaling 1.08).

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Threshold FSC: 5000; Laser Delay Blue 0.00 Red 24.10; Area scaling Blue 1.79 Red 1.80; FACS analysis was performed three times for two biological

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replicates per group (N=3, n=6).

Assessment of cell number

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To obtain the cell number, hPDL fibroblasts were thoroughly scraped from the respective well of the 6-well plate in 1 ml PBS and then 100µl cell suspension were counted in 10 ml isotonic solution with a Beckman Coulter Counter (Z2 cell counter) according to manufacturer’s instructions.

Measurement of cytotoxicity via LDH assay For cytotoxicity assessment, we performed lactate dehydrogenase (LDH) assays (04744926001, Roche, Mannheim, Germany) using respective cell supernatants of all six groups according to the manufacturer’s instructions. We mixed 100 µl of the supernatant with 100 µl of freshly prepared LDH solution (22 µl catalyst mixed with 1 mL dye) and incubated

ACCEPTED MANUSCRIPT for 30 min at room temperature in the dark. Then we added 50 µl of stop solution and measured absorbance at 490 nm (LDH activity) with an ELISA reader (Multiscan GO Microplate Spectrophotometer, Thermo Fisher Scientific), subtracting background absorbance at 690 nm. We calculated LDH activity for 100.000 cells. LDH assays were evaluated two

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times for three biological replicates per group (N=2, n=6).

Measurement of cell viability (mitochondrial enzymatic activity) via MTT assay

To assess cell viability (mitochondrial enzymatic activity) of hPDL fibroblasts under the different experimental conditions evaluated, we performed MTT (3-(4,5-Dimethylthiazol-2-

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yl)-2,5-diphenyltetrazoliumbromid) assays. For the final five hours of incubation, 400µl MTT solution in PBS (5 mg/ml, 4022.1, Carl Roth GmbH & Co. KG) were added per well. We then

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removed the medium and added 1 ml DMSO per well. hPDL fibroblasts were incubated for another 5 min at 37°C and absorbance was measured at 550nm with an ELISA reader (Multiscan GO Microplate Spectrophotometer, Thermo Fisher Scientific), corresponding to cell viability. We calculated cell viability for 100.000 cells. MTT assays were performed three times for three biological replicates per group (N=3, n=9).

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Perforation assay (crystal violet staining)

To quantify dead cells, we performed a perforation assay with crystal violet. To this aim, we washed hPDL fibroblasts with PBS and added 400 µl crystal violet solution per well (2.5 g

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crystal violet, 20 ml formaldehyde, 150 ml EtOH, 0.85 g NaCl, H2Odd added to 500 ml). We incubated the cells for 15 min at 37°C. Afterwards we washed the stained fibroblasts three times with tap water and dried the cells for 30 min at 37°C. Then we added 350µl 33% acetic

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acid and measured crystal violet perforation into the cells relative to 100.000 cells at 595 nm with an ELISA reader (Multiscan GO Microplate Spectrophotometer, Thermo Fisher Scientific). Crystal violet assays were evaluated two times for three biological replicates per group (N=2, n=6).

TUNEL assay hPDL fibroblasts were fixed with freshly prepared fixation solution (4% paraformaldehyde in PBS, pH 7.4) for 1 h at room temperature after an appropriate incubation time. Slides were rinsed three times with PBS and incubated with freshly prepared permeabilisation solution (0.1% Triton in 0.1% sodium citrate) for 2 min on ice. Slides were washed twice with PBS.

ACCEPTED MANUSCRIPT We incubated the slides with 50µl TUNEL reaction mixture - 5 µl TUNEL-Enzyme (11767305001, Sigma-Aldrich, Munich, Germany), mixed with 45 µl TUNEL-Label (11767291910, Sigma-Aldrich) - for 1 h at 37°C in the dark. Slides were rinsed three times

with PBS, mounted with vectashield (H-1200, Vector) and analyzed with a fluorescence microscope at a magnification of x100 (AxioScope.A1, AxioCamMR3/AxioVision 4.8.1, Carl

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Zeiss, Oberkochen, Germany). TUNEL assays were evaluated for five (N=5) biological replicates per group assessing the mean of five random fields of view.

LIVE/DEAD staining

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We used LIVE/DEAD viability/cytoxicity Kit (L3224, Invitrogen) according to the manufacturer’s instructions. Briefly, we seeded hPDL fibroblasts onto a sterile glass

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coverslip. After an appropriate incubation time, we washed the cells to remove the medium. We added 150µl LIVE/DEAD assay reagents (4µM EthD-1 solution, 2µM calcein in PBS) to the glass coverslip and incubated hPDL fibroblasts for 30 min at room temperature. We analyzed the stained cells with a fluorescence microscope at a magnification of x100 (AxioScope.A1, AxioCamMR3/AxioVision 4.8.1, Carl Zeiss, Oberkochen, Germany). We obtained DEAD (red) and LIVE (green) cells and calculated the ratio of dead cells compared

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to live cells. LIVE/DEAD staining was evaluated for five (N=5) biological replicates per group assessing the mean of five random fields of view.

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Enzyme linked immunosorbent assay (ELISA) For quantification of alkaline phosphatase (ALPL), collagen 1 alpha (COL1), interleukin 6

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(IL6), prostaglandin E2 (PGE2), osteoprotegerin (OPG) and vascular endothelial growth factor A (VEGF-A) protein secretion in the hPDL cell supernatant, we used commercially available ELISA kits according to manufacturers’ instructions (ALPL: OKEH00757; Aviva system biology; COL1: ab210966, Abcam; IL6: CSB-E4638h, Cusabio; PGE2: 514010; Cayman chemical; OPG: EHTNFRSF11B, Thermo Fisher Scientific; VEGF-A: RAB0507; Sigma Aldrich). Protein expression per well was related to the respective number of hPDL fibroblasts, counted with a Beckman Coulter Counter (Z2 cell counter).

Immunofluorescence staining of RANK-L

ACCEPTED MANUSCRIPT To investigate RANK-L expression, we fixed, lysed and blocked the cell layer with 4% paraformaldehyde in PBS for 10 min, 0.05% Triton X-100 (Sigma-Aldrich) and 4% goat serum in PBS for 20 min at room temperature. A primary RANK-L antibody coupled with AF488 (1:100, sc-377079, Santa Cruz Biotech, Heidelberg, Germany) was incubated overnight at 4°C with cell nuclei counterstained by 4,6-diamino-2-phenylindole (DAPI; T-

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9284, Sigma-Aldrich; 1:1000). Reliability was confirmed by non-antibody controls. Immunofluorescence of RANK-L and DAPI were detected separately with a fluorescence microscope at a magnification of x200 (AxioScope.A1, AxioCamMR3/AxioVision 4.8.1, Carl Zeiss, Oberkochen, Germany). RANK-L-positive area per hPDL fibroblast in pixel was

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determined in four random fields of view per biological sample by dividing total RANK-Lpositive area, as defined by color thresholding (Image J: Hue 0–255, Saturation 0–255, Brightness 25–255; Default method, Color space HSB) by the number of cell nuclei (DAPI)

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and the arithmetic mean per biological sample was used for further analysis [34]. RANK-L immunofluorescence was evaluated for six (N=6) biological replicates per group assessing the mean of four random fields of view.

Determination of RANK-L protein expression via western blot

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We isolated total protein from hPDL fibroblasts using 100µl CellLytic M (C2978; SigmaAldrich) per well, supplemented with proteinase inhibitors (Carl Roth GmbH & Co. KG). To reduce proteinase activity, proteins were kept on ice for the whole time. Determination of protein concentration was performed with RotiQuant (K015.3; Carl Roth GmbH & Co. KG)

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according to the manufacturer’s instructions. For immunoblotting, we separated equal amounts of total protein on a 12% SDS-polyacrylamide gel under reducing conditions and

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electro-blotted the proteins onto a polyvinylidene diflouride (PVDF) membrane. We blocked the membranes with 5% nonfat milk in tris-buffered saline and 0.1% Tween 20, pH 7.5 (TBST) at 4°C over night and incubated the membranes with anti-RANK-L (ABIN500805, antibodies-online, Aachen, Germany) diluted 1:2,000 in 0.5% milk in TBS-T, or 1:500 antiHSP90 (Santa Cruz Biotech, Heidelberg, Germany) for 1h. After washing three times in TBST, we incubated the blots for 1 h with horseradish peroxidase-conjugated anti-rabbit IgG (Pierce, Rockford, USA), diluted 1:5000 in 0.5% milk in TBS-T at room temperature. We visualized antibody binding using an enhanced chemiluminescence system (Pierce, Rockford, USA). Densitometric quantification of specific bands was performed with ImageJ (ver. 1.47, Wayne Rasband, National Institutes of Health, USA). RANK-L western blot was evaluated for seven (N=7) biological replicates per group.

ACCEPTED MANUSCRIPT TRAP-histochemistry (hPDL-mediated osteoclastogenesis) At the end of incubation, we washed stimulated hPDL fibroblasts with PBS and a macrophage osteoclast-precursor cell line (immortal RAW264.7 cells, CLS Cell Lines Service, Eppelheim, Germany) was added at a concentration of 70,000 cells per well, thus avoiding a possible

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force induction of RAW cell differentiation [34]. The resulting co-culture was then incubated for another 72 h under cell culture conditions [34, 35], enabling RANK-L-mediated osteoclastogenesis induced by hPDL fibroblasts [15]. Histochemical TRAP-staining (Tartrateresistant acid phosphatase, red) was used to detect differentiated osteoclast-like cells [15]. A

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blinded observer counted TRAP-positive cells at a magnification of ×100 with an Olympus IX50 microscope (Olympus Deutschland) in ten random fields of view per well (biological

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replicate) and the arithmetic mean was used for further analysis [34]. Coculture experiments were evaluated two times for three biological replicates (N=2, n=6) per group assessing the mean of ten random fields of view.

Statistical analysis

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Prior to statistical analysis, all absolute data values were divided by the respective arithmetic mean of the pressure-untreated 0‰ ethanol controls to obtain normalized data values relative to these controls, set to 1. Using the software application SPSS® Statistics 24 (IBM®,

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Armonk, NY, USA), all data were tested for normal distribution (Shapiro-Wilk test) and homogeneity of variance (Levene’s test). Descriptive statistics are given as mean ± standard deviation. The experimental groups were independently compared by one-way ANOVAs,

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which were validated by applying Welch’s test, since homogeneity of variance not always present. We used Games–Howell post hoc test for heterogeneous variances for pairwise comparisons. All differences were considered statistically significant at p ≤ 0.05.

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Results Cytotoxicity of ethanol and cell viability of hPDL fibroblasts after compressive force application First, we focused on cytotoxicity of compressive force application and ethanol treatment. We

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found a significantly lower cell number after compressive force application without any additional ethanol effect (Fig. 2 A). Based on LDH assays, we detected cytotoxic cell-lytic effects of compressive force, but not of ethanol on hPDL fibroblasts at the examined concentrations (Fig. 2 B). Furthermore, we observed reduced cell viability (MTT assays, Fig.

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2 C) and enhanced perforation (crystal violet staining, Fig. 2 D) during compressive force application at each ethanol concentration, indicating increased cell death. Both tested ethanol concentrations, however, had no effect on cell viability without or with additional pressure

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application. In contrast, we observed an enhanced perforation with the higher ethanol concentration with compressive force application (Fig. 2 D). Surprisingly, we found no apoptotic or necrotic effects of different ethanol concentrations with or without additional orthodontic pressure application with Annexin V / PI FACS analysis. With that method we also observed no significant differences in apoptosis (annexin V; Q4; p = 0.203) or necrosis (propodium iodide; Q2; p = 0.549) either with the low (0.41‰) or the high (1.79‰) ethanol

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concentration (Table 2). An additional application of a physiological orthodontic compressive force also did not cause significant apoptosis or necrosis in hPDL fibroblasts (Table 2). We additionally performed TUNEL assays to further investigate the effect of ethanol and pressure

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application on apoptosis. We found more apoptotic cells after compressive force application without any additional effect of ethanol (Fig. 2 E). In line with this result, LIVE/DEAD staining revealed more dead cells after mechanical strain application (Fig. 2 F). Both tested

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ethanol concentrations, however, showed no effect (Fig. 2 F)

Effects of ethanol and compressive force application on bone formation, angiogenesis and collagen synthesis

Alkaline phosphatase (ALPL) is involved in bone formation and its gene expression in hPDL fibroblasts was significantly enhanced after stimulation with compressive force at each ethanol concentration (Fig. 3 A). Ethanol at 1.79‰ also significantly induced ALPL gene expression without additional pressure application (Fig. 3 A). ELISA data confirmed the increased secretion of ALPL protein during compressive force application at each ethanol concentration (Fig. 3 A), whereas there was no ethanol effect of ALPL secretion detectable.

ACCEPTED MANUSCRIPT Vascular endothelial growth factor A (VEGF-A) is involved in tissue neoformation, vasodilatation and growth of blood vessels. Compression of hPDL fibroblasts led to a significant increase of VEGF-A mRNA expression and protein secretion independently of the added ethanol concentration (Fig. 3 B). Both tested ethanol concentrations enhanced VEGF-A mRNA expression and protein secretion significantly without additional pressure application

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compared to the 0‰ ethanol control (Fig. 3 B). There was no inductive effect of the tested ethanol concentrations during pressure application on VEGF-A expression. We next investigated the effects of ethanol on collagen synthesis. The collagen-1-α-2 (COL1A2) gene encodes for the pro-alpha 2 chain of collagen type I. There was a significant increase of

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COL1A2 gene expression after stimulation with compressive force at all tested ethanol concentrations (Fig. 3 C). Ethanol at 0.41‰ increased COL1A2 gene expression without additional pressure application compared to 0‰ ethanol (Fig. 3 C). Ethanol at 1.79‰

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however had a significant inductive effect on COL1A2 gene expression both without and with additional orthodontic compressive force application compared to 0‰ ethanol (Fig. 3 C). In contrast to mRNA expression data COL1A protein secretion was reduced during compressive force application. Both tested ethanol concentrations showed no additional effect (Fig. 3 C).

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Effects of ethanol and orthodontic compressive force application on the expression and secretion of proinflammatory factors

Next we focused on the expression of the proinflammatory genes interleukin 6 (IL-6) and

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cyclooxygenase 2 (COX2) in hPDL fibroblasts. Without ethanol stimulation IL-6 mRNA expression and protein secretion was significantly increased after application of compressive

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force (Fig. 4 A). This pressure effect was partly prevented by the low and high ethanol concentrations (Fig. 4 A). COX2 gene expression was elevated by compressive force application, with no effects of ethanol detectable (Fig. 4 B). Prostaglandin E2 (PGE2) secretion was triggered significantly by pressure application at each ethanol concentration (Fig. 4 B). The high ethanol concentration of 1.79‰ significantly further induced PGE2 protein secretion during additional compressive force application compared to 0‰ ethanol (Fig. 4 B).

Effects of ethanol and compressive force on the RANK-L/OPG system and hPDL-mediated osteoclastogenesis.

ACCEPTED MANUSCRIPT Receptor activator of NFκB ligand (RANK-L) immunofluorescence increased after compression in the control group and ethanol-treated groups in hPDL fibroblasts (Fig. 5 A, C), whereas ethanol at both tested concentrations did not affect RANK-L secretion. These results were confirmed by RANK-L western blotting (Fig. 5 D), which showed significant induction of RANK-L protein expression after compressive force application, but no effect of

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the ethanol concentrations applied. Compression reduced osteoprotegerin (OPG) secretion (Fig. 5 E), whereas additional ethanol application had no effect (Fig. 5 E). We next investigated hPDL-fibroblast-mediated osteoclastogenesis and observed a significant pressure effect in all investigated ethanol concentration groups (Fig. 5 B, F), as more osteoclast-like

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without pressure application significantly (Fig. 5 B, F).

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cells could be counted. The high ethanol concentration of 1.79‰ increased osteoclastogenesis

Discussion

In this study we investigated the effects of ethanol on hPDL fibroblasts during compressive force application. We observed no effects of ethanol at a concentration of 0.41‰ or 1.79‰ on apoptosis, cytotoxicity or cell viability. Ethanol, however, increased angiogenesis and activity

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of alkaline phosphatase. In addition, ethanol reduced expression of IL-6 and increased secretion of prostaglandin E2 as well as hPDL-fibroblast-mediated osteoclastogenesis. The ethanol concentrations tested in this study were chosen to correspond to typical blood

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serum concentrations found in man during casual or excessive alcohol consumption, corresponding to the concentrations found in the periodontal ligament fluid surrounding the hPDL fibroblasts, since this fluid is a blood plasma exudate. Based on current epidemiological

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data, the average ethanol consumption per capita per day is 20.5 g [21] for casual and of 88.5 g ethanol for excessive alcohol consumption [6]. This correlates to ethanol blood plasma ஺

concentrations of 0.41‰ and 1.79‰ according to the Widmark formula [57] ‫ = ݓ‬௠•௥, based on an average weight of a male (m = 70.8 kg [56]) and the distribution factor r = 0.7. Our study revealed no effects of ethanol on apoptosis or necrosis in hPDL fibroblasts. There are many studies available dealing with an apoptotic effect of ethanol in many different cell lineages. Most of them deal with fetal alcohol spectrum disorders and therefore investigate the apoptotic effects of ethanol on neural crest cells [10, 12, 12, 13, 54, 65]. In contrast to our study, these studies report that ethanol induced apoptosis in neural crest cells. This difference is probably due to differing cell type and the higher ethanol concentration of about 2.3‰ or

ACCEPTED MANUSCRIPT even 9.2‰ used [12, 13, 54, 65]. Other groups investigated the effect of ethanol on apoptosis in T-cells and also detected significantly increased apoptosis with 1.15‰ ethanol [49]. These data would indicate that hPDL fibroblasts are more resistant to the apoptotic effect of ethanol than T-cells. In contrast, experiments with human hepatic adenocarcinoma cell lines also reported no ethanol-induced apoptosis even at a high ethanol concentration of 4.6‰ [28].

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Apart from absence of increased apoptosis or necrosis, we also did not observe cytotoxic effects of ethanol on hPDL fibroblasts in the concentrations tested. This is in line with Bhopale et al. [7], who reported ethanol-induced apoptosis, which was not accompanied by increased cytotoxicity, even though quite high ethanol concentrations were used.

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Cell viability by contrast was significantly reduced during compressive force application. Ethanol, however, had no effect on cell viability in hPDL fibroblasts in the tested

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concentrations. Neural crest cells also seem to be more sensitive to ethanol, as 2.3‰ also reduced viability of these cells significantly [13]. In summary, hPDL fibroblasts seem to be quite resistant to ethanol-induced apoptosis, cytotoxicity or reduced cell viability. This would indicate that ethanol-induced effects on the expression pattern of hPDL fibroblasts observed during orthodontic compressive forces are independent of cell viability or vitality, which is not impaired at the concentrations corresponding to casual or extensive ethanol consumption.

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Perforation assays using crystal violet staining revealed increased cell death due to compressive force application, which was potentiated by the higher ethanol concentration. In this study, we wanted to investigate the role of ethanol in the context of OTM. One known

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biological response to orthodontic forces is the modification of the surrounding bone architecture. Alkaline phosphatase (ALPL) is linked with bone formation and has a higher

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activity in the periodontal ligament as in other connective tissues [61]. ALPL expression can be induced by osteoprotegerin (OPG) and promote pre-osteoblast maturation [25, 62]. Stimulation of hPDL fibroblasts with 1.79‰ ethanol increased ALPL gene expression significantly, indicating a bone-forming effect of high ethanol concentrations in hPDL fibroblasts.

These cells also show some similarities to osteoblasts, which are not only

involved in bone-formation, but also osteoclastogenesis by their ability to express RANK-L [31, 45, 59]. An increased ALPL expression by hPDL fibroblasts during compressive force application in the presence of ethanol could thus lead to increased osteoblastogenesis and enhanced RANK-L synthesis, which in turn could promote osteoclastogenesis in compression areas of the periodontal ligament during OTM.

ACCEPTED MANUSCRIPT VEGF-A is a growth factor responsible for angiogenesis and vasodilatation of blood vessels [19]. OTM is known to induce formation of new blood vessels and to increase vasodilatation of pre-existing vessels in the periodontal ligament [42]. We thus observed an increased VEGF-A expression and secretion after compressive force application. Both tested ethanol concentrations increased VEGF-A expression significantly, indicating an improved

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circulation in the periodontal ligament. This observation is supported by the finding that moderate ethanol concentrations increase VEGF-A expression in artery vascular smooth muscle cells [26]. The effect of ethanol on hPDL fibroblasts could thus help to resume the circulation at compression areas, which is disturbed during tooth movement.

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hPDL fibroblasts also have an important role in the reorganization of the periodontal ligament itself to allow its adaptation to orthodontic forces by alteration of collagen synthesis [41, 54].

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In this study, we observed an effect of both tested ethanol concentrations on COL1A2 gene expression, but this was not accompanied by enhanced COL1A protein secretion. A previous study performed in rats did not find any changes in type I collagen after ethanol treatment and OTM [3], but these authors particularly intended to investigate the effect of binge pattern alcohol consumption and therefore tested quite high ethanol concentrations [3]. One reason for this result could be that OTM is a multicellular process and other cells beyond hPDL

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fibroblasts play a role in extracellular matrix reorganization.

hPDL fibroblasts synthesize prostaglandins as a primary response to orthodontic forces [31] by enhanced expression of inducible cyclooxygenase 2 (COX2). Ethanol had no effect on

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COX2 expression, but there was an increased secretion of PGE2. This enhanced PGE2 expression could influence hPDL-fibroblast-mediated osteoclastogenesis, as clinical and animal studies have reported an important role of COX2-synthesised prostaglandins for

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stimulating bone resorption [31]. Especially PGE2 seems to mediate inflammatory responses and to induce bone resorption by activating osteoclastic cells [41]. RANK-L and its decoy receptor OPG are essential in the regulation of bone remodeling, OTM and also undesired OTM-associated tooth root resorption [48]. RANK-L expression promotes the differentiation of osteoclast progenitor cells to mature, bone-resorbing osteoclasts in compression areas and OPG as its soluble decoy receptor is able to negate the effects of RANK-L by binding soluble and membrane-associated RANK-L [45, 48]. Neither RANK-L nor OPG expression were distinctly impaired by the low and high ethanol concentrations of 0.41‰ and 1.79‰ tested. Osteoclastogenesis, however, was increased at the high ethanol concentration of 1.79‰ without additional pressure application, indicating

ACCEPTED MANUSCRIPT increased bone resorption under the influence of ethanol. These results also suggest that the increased differentiation of osteoclast progenitor cells to active osteoclasts observed is directly promoted by ethanol via a RANK-L-independent mechanism and not via hPDLmediated RANK-L/OPG expression. These data are in line with Iitsuka et al., who reported an stimulating effect of ethanol on osteoclastogenesis in rats [29]. These authors, however, also

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observed an increased RANK-L expression in the proximal tibia of ethanol-fed rats, thus the direct effect of ethanol on osteoclastogenesis requires further investigation. In contrast, Araujo et al. reported a significant reduction of osteoclasts by ethanol during OTM in rats [3]. These differences might be explained by the different study design of the animal experiment.

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Araujo et al. investigated the effects of binge-pattern alcohol consumption on OTM [3]. Thus they administrated ethanol only on 4 days per week, followed by 3 days without ethanol application. Iitsuka et al. on the other hand used a Lieber-DeCarli ethanol-containing liquid

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diet with 35% of the calories derived from ethanol to constantly feed ethanol to the animals. Ethanol is also reported to induce osteopenia and osteoporosis [36, 40, 55], due to inhibition of osteoblastogenesis [20] and by alteration of remodeling-related genes [9, 11]. OTM is a multicellular process, which depends not only on the activity of hPDL fibroblasts in the compression zone, but also on osteoclasts, macrophages, lymphocytes and osteoblast

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activity [41]. One limitation of our in vitro-study is the fact that our research of the effects of ethanol was focused on hPDL fibroblasts and osteoclast-precursor cells only. It should be considered in future studies to investigate the effects of ethanol on the other mentioned cell

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types relevant during OTM as well.

In conclusion, ethanol had no cytotoxic effects and no influence on apoptosis/necrosis on hPDL fibroblasts, indicating that this cell type is quite ethanol-resistant. High ethanol

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concentrations, however, seem to promote bone formation, angiogenesis and collagen synthesis, which may also influence OTM. Ethanol showed anti-inflammatory effects, but also increased induction of prostaglandins, which are responsible for pain, inflammation and trigger

osteoclastogenesis.

High

ethanol

concentrations

increased

hPDL-mediated

osteoclastogenesis, indicating enhanced bone resorption. This may accelerate orthodontic tooth movement, but could also increase the risk for undesirable root resorption or periodontal bone loss.

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Acknowledgements The authors wish to thank Mrs Eva Zaglauer for their support in performing the experiments as well as Prof. Dr. Martin Rosentritt, Department of Prosthodontics, University of Regensburg Dental School, for generously providing the AxioScope.A1 microscope and corresponding equipment. The authors also thank the German Orthodontic Society (DGKFO)

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for their financial support and funding (Kirschneck 12–01-2015).

Appendix Supplementary Figure

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Supplementary Table

Compliance with ethical standards Conflict of interest

The authors report no financial or other conflict of interest relevant to this article, which is the intellectual property of the authors. Furthermore, no part of this article has been published

Funding

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before or is considered for publication elsewhere.

This work was supported by the German Orthodontic Society (DGKFO) [grant number

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Kirschneck 01/12/2015].

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Data availability statement

All data are publically available either as supplementary information to this article or upon request.

Ethical approval

All procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. Approval for the collection and usage of hPDL fibroblasts was obtained from the ethics committee of the University of Regensburg, Germany [approval number 12-170-0150]. This article does not contain any studies with animals.

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ACCEPTED MANUSCRIPT Fig. 1. In vitro simulation of compressive force application to hPDL fibroblasts. DMEM = Dulbecco’ modified eagle medium, hPDL = human periodontal ligament.

Fig. 2. Evaluation of cytotoxicity and viability of hPDL fibroblasts under the influence of ethanol and orthodontic compressive force. (A) Assessment of cell number; N=2; n=6. (B)

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Evaluation of ethanol cytotoxicity via LDH assay; N=2, n=6. (C) Measurement of cell viability via MTT assay; N=3, n=9. (D) Perforation assay (crystal violet staining); N=2, n=6. (E) TUNEL assay; N=5. (F) LIVE/DEAD staining; N=5. Results shown as normalized x-fold induction relative to 0‰ ethanol without pressure application. Pressure effect: *** p ≤ 0.001;

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[AU] arbitrary units; error bars: standard deviation SD; PerCP-Cy5-5-H = Peridininchlorophyll-protein complex channel (propidiumiodide, necrosis); FITC-H = fluorescein

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thiocyanate channel (annexin V, apoptosis); LDH = lactate dehydrogenase; MTT = 3-(4,5Dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromid.

Fig. 3. Induction of ALPL (A), VEGF-A (B) and COL1 (C) gene expression and protein secretion shown as normalized x-fold induction relative to 0‰ ethanol without pressure

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application. Pressure effect: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; Ethanol effect: # p ≤ 0.05, ## p ≤ 0.01. RT-qPCR: N = 3, n = 9; ELISAs: N=2, n=6; [AU] arbitrary units, error bars: standard deviation SD; RT-qPCR = real-time reverse transcription quantitative polymerase

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chain reaction, ELISA = enzyme-linked immune-sorbent assay.

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Fig. 4. Induction of IL-6 (A) and COX2 (B) gene expression and protein secretion shown as normalized x-fold induction relative to 0‰ ethanol without pressure application. Pressure effect: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; Ethanol effect: # p ≤ 0.05. RT-qPCR: N = 3, n = 9; ELISAs: N=2, n=6; [AU] arbitrary units, error bars: standard deviation SD; RT-qPCR = real-time reverse transcription quantitative polymerase chain reaction, ELISA = enzymelinked immune-sorbent assay.

Fig. 5. RANK-L/OPG system and hPDL-fibroblast-induced osteoclastogenesis. (A) Representative pictures of RANK-L immunofluorescence (IF) staining and (B) TRAP staining after co-culture with osteoclast progenitor cells. Osteoclast-like cells are stained in

ACCEPTED MANUSCRIPT red. Induction of (C) RANK-L immunofluorescence (N=6) and (D) protein expression (N=7) relative to 0‰ ethanol without pressure application. (E) OPG protein secretion relative to 0‰ ethanol without pressure (N=2, n=6). (F) Induction of osteoclastogenesis relative to 0‰ ethanol without pressure (N=2, n=6). Pressure effect: * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001; Ethanol effect: # p ≤ 0.05. [AU] arbitrary units; Error bars: standard deviation SD. ELISA =

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enzyme-linked immune-sorbent assay. TRAP: Tartrate-resistant acid phosphatase

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Table 1. RT-qPCR gene, primer, target and amplicon specifications for references genes (PPIB, RPL22) and target genes. Gene name (Homo sapiens)

Accession Number (NCBI GenBank)

5´-forward primer-3´ (length / Tm / %GC / max. ∆G Hairpin &Self-Dimer / Self-Comp. / Self-3’-Comp.)

5´-reverse primer-3´ (length / Tm / %GC / max. ∆G Hairpin &Self-Dimer / Self-Comp. / Self-3’-Comp.)

Primer Location (max. ∆G Cross-Dimer)

PPIB

peptidylprolyl isomerase A

NM_000942.4

TTCCATCGTGTAATCAAGGACTTC (24bp / 61.3°C / 41.7% / -1.3 / 4 / 2)

GCTCACCGTAGATGCTCTTTC (21bp / 61.2°C / 52.4% / -0.7 / 4 / 0)

exon 3/4 (-2.1)

RPL22

ribosomal protein L22

NM_000983.3

TGATTGCACCCACCCTGTAG (20bp / 62.2°C / 55.0% / -3.4 / 4 / 2)

GGTTCCCAGCTTTTCCGTTC (20bp / 61.8°C / 55.0% / -3.0 / 4 / 0)

exon 2/3 (-1.5)

ALPL

alkaline phosphatase, liver/bone/kidney

NM_000478.4

ACAAGCACTCCCACTTCATCTG (22bp / 60.3°C / 50.0% / -0.5 / 3 / 2)

GGTCCGTCACGTTGTTCCTG (20bp / 61.4°C / 60.0% / -3.3 / 5 / 1)

exon 7-8/9 (-2.1)

COL1A2

collagen, type I, alpha 2

NM_000089.3

AGAAACACGTCTGGCTAGGAG (21bp / 59.8°C / 52.4% / -3.3 / 4 / 2)

GCATGAAGGCAAGTTGGGTAG (21bp / 59.8°C / 52.4% / -2.3 / 5 / 0)

exon 50/51 (-0.7)

IL6

interleukin 6

NM_000600.3

TGGCAGAAAACAACCTGAACC (21bp / 57.9°C / 47.6% / -1.1 / 3 / 0)

CCTCAAACTCCAAAAGACCAGTG (23bp / 60.6°C / 47.8% / -0.8 / 3 / 3)

exon 2/3 (-1.5)

COX2

prostaglandinendoperoxide synthase 2 (prostaglandin G/H synthase and cyclooxygenase)

NM_000963.3

GAGCAGGCAGATGAAATACCAGTC (24bp / 62.7°C / 50.0% / 0.0 / 2 / 2)

TGTCACCATAGAGTGCTTCCAAC (23bp / 60.6°C / 47.8% / -1.3 / 4 / 0)

exon 8/9 (-3.2)

VEGFA

vascular endothelial growth factor A

NM_001171623.1

TGCAGACCAAAGAAAGATAGAGC (23bp / 58.9°C / 43.5% / -3.4 / 4 / 2)

ACGCTCCAGGACTTATACCG (20bp / 59.4°C / 55.0% / -1.3 / 5 / 2)

exon 5-6/7 (-3.3)

Amplicon location (bp of Start/Stop)

Intronspanning (length)

446/533

Yes (3194bp)

115/212

Yes (4597bp)

1045/1176

Yes (3290bp)

4139/4243

Yes (710bp)

370/486

Yes (704bp)

131bp, 42.0%, 82.9°C, no SSAT

1457/1587

Yes (486bp)

Yes (BLAST/ UCSC)

Yes

107bp, 43.9%, 83.7°C, no SSAT

1426/1532

No

Yes (BLAST/ UCSC)

Yes

Amplicon (length, %GC, Tm, SSAT) 88bp, 53.4%, 86.1°C, no SSAT 98bp, 44.9%, 83.8°C, no SSAT 132bp, 56.1%, 89.5°C, no SSAT 105bp, 44.8%, 83.3°C no SSAT 117bp, 43.6%, 83.7°C, no SSAT

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Gene symbol

In silico qPCR specifity Yes (BLAST/ UCSC) Yes (BLAST/ UCSC) Yes (BLAST/ UCSC) Yes (BLAST/ UCSC) Yes (BLAST/ UCSC)

Variants targeted (Transcript /Splice) Yes

Yes

Yes

Yes

Yes

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Tm = melting temperature of primer/specific qPCR product (amplicon); %GC = guanine/cytosine content; bp = base pairs; Comp. = Complementarity; SSAT = secondary structure at annealing temperature

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Table 2. Flow-cytometry-based detection of apoptosis (annexin V) and necrosis (propidium iodide). All data are represented as mean ± standard deviation (N=3, n=6). 0‰ ethanol

Positive cells relative to total cells [%]

0.41‰ ethanol

1.79‰ ethanol

without with without with without with pressure pressure pressure pressure pressure pressure 4.6 ± 1.7

5.9 ± 1.0

7.2 ± 0.8

6.9 ± 0.6

5.4 ± 0.4

5.8 ± 1.9

Apoptosis (Fluorescin isothiocyanate)

2.5 ± 1.4

3.5 ± 1.8

2.9 ± 1.2

4.0 ± 0.8

2.9 ± 1.4

4.5 ± 1.6

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EP

TE D

M AN U

SC

RI PT

Necrosis (Propodium iodide)

ACCEPTED MANUSCRIPT

static compressive force

37°C / 5% CO2 / 100% φH2O

37°C / 5% CO2 / 100% φH2O

RI PT

untreated physiological control

SC

glass disc

M AN U

Ø33mm / 17.1g

2 g/cm2

TE D

hPDL fibroblast layer (initially 70,000/well; 70% confluence)

AC C

EP

2ml mod. DMEM high glucose

ACCEPTED MANUSCRIPT

***

***

***

1.0 0.5

0.0 Pressure: Ethanol:

C

-

+ 0‰

-

+

0.41‰

-

+

1.79‰

LDH ***

3

*** 2 1

0 Pressure: Ethanol:

D

MTT

*

RI PT

1.5

Induction [AU]

Induction [AU]

B

Cell number

A

-

+

-

0‰

-

+

0.41‰

+

1.79‰

Crystal violett

+ 0‰

-

+

0.41‰

TUNEL assay 3

*

**

1

-

-

+

AC C

+ 0‰

Induction [AU]

+

1.79‰

*

2

0 Pressure: Ethanol:

-

TE D

E

-

0.41‰

1.0

***

0.5

0.0 Pressure: Ethanol:

F

-

-

+

0‰

-

+

0.41‰

+

1.79‰

LIVE/DEAD staining

4

*

* **

3 2 1

+

0 Pressure:

1.79‰

Ethanol:

-

***

*

M AN U

0.5

0.0 Pressure: Ethanol:

Induction [AU]

***

Induction [AU]

*** 1.0

1.5

SC

***

1.5

EP

Induction [AU]

#

-

+ 0‰

-

+

0.41‰

-

+

1.79‰

ACCEPTED MANUSCRIPT

ALPL (RT-qPCR)

ALPL (ELISA)

20

***

**

*

Induction [AU]

10

0 Pressure: + Ethanol: 0‰

B

-

+

0.41‰

-

+

1.79‰

1

-

+

0.41‰

-

#

3 2

*

* *

-

+

0.41‰

AC C

0 Pressure: + Ethanol: 0‰

EP

1

TE D

#

-

+

0.41‰

-

+

1.79‰

##

##

4 2

***

0 Pressure: Ethanol:

-

+

0‰

*

*

-

+

0.41‰

-

+

1.79‰

COL1 (ELISA)

#

4

+

1.79‰

COL1 (RT-qPCR)

6

M AN U

***

2

C

Induction [AU]

**

0 Pressure: + Ethanol: 0‰

+

0‰

VEGF-A (ELISA)

#

**

-

Ethanol:

-

+

1.79‰

Induction [AU]

Induction [AU]

2

VEGF-A (RT-qPCR) 4

**

** *

0 Pressure:

##

Induction [AU]

3

SC

Induction [AU]

#

30

RI PT

A

**

1.2

*

**

0.8 0.4

0.0 Pressure: Ethanol:

-

+ 0‰

-

+

0.41‰

-

+

1.79‰

ACCEPTED MANUSCRIPT

IL-6 (RT-qPCR)

IL-6 (ELISA) #

#

**

4

**

**

2

3

1 0

+

0.41‰

0‰

-

+

Pressure:

1.79‰

Ethanol:

**

*** ***

4 2 0 0‰

-

+

0.41‰

+

0.41‰

-

-

+

1.79‰

#

+

Pressure:

1.79‰

Ethanol:

-

**

*

*

+

0‰

EP

+

5 4 3 2 1 0

AC C

Ethanol:

-

-

+ 0‰

PGE2 (ELISA)

COX2 (RT-qPCR) 6

-

M AN U

-

Induction [AU]

Induction [AU]

+

TE D

-

Ethanol:

Pressure:

*

*

2

0 Pressure:

B

#

**

RI PT

#

6

4

SC

8

Induction [AU]

Induction [AU]

A

-

+

0.41‰

-

+

1.79‰

ACCEPTED MANUSCRIPT

*

8 4

**

1

M AN U

+

1.79‰

(western blot) *

SC

2

-

+

0.41‰

RI PT

RANK-L 3

-

0 Pressure: + Ethanol: 0‰

-

+

*

-

0.41‰

+

1.79‰

RANK-L

200x

200x

without pressure

with pressure

HSP90

E

OPG

100x

F 100x

100x

(ELISA)

*

*

**

0.8 0.4

0.0 Pressure: Ethanol:

Induction [AU]

EP

100x

100x

Induction [AU]

1.2

AC C

0.41‰ ethanol

200x

*

*

12

0 Pressure: + Ethanol: 0‰

Induction [AU]

200x

100x

1.79‰ ethanol

RANK-L (IF)

D

0‰ ethanol

B

200x

TE D

1.79‰ ethanol

0.41‰ ethanol

200x

Induction [AU]

C

with pressure

without pressure

0‰ ethanol

A

6 4

-

-

+ 0‰

+

0.41‰

-

+

1.79‰

TRAP (osteoclastogenesis) #

***

*

**

2

0 Pressure: + Ethanol: 0‰

-

+

0.41‰

-

+

1.79‰

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

• hPDL fibroblasts play a key mediating role in orthodontic tooth movement. • 0.41‰ and 1.79‰ of ethanol did not affect hPDL fibroblast viability or vitality. • Ethanol may increase alveolar angiogenesis, bone formation and collagen synthesis. • Ethanol increased hPDL-fibroblast-mediated osteoclastogenesis. • Ethanol may increase tooth movement and bone loss during orthodontic treatment.