Effect of intraoral mechanical stress application on the expression of a force-responsive prognostic marker associated with system disease progression

Abstract

Objectives

Malocclusion may be corrected nonsurgically by mechanical tooth movement. The plasma protein profiles of human subjects receiving the first phase of orthodontic treatment were examined to test the hypothesis that application of mechanical stresses to teeth induces systemic proteomic alterations.

Methods

Tandem mass tag-based liquid chromatography-mass spectrometry (LC–MS/MS) was used to examine systemic proteomic alterations in subjects undergoing controlled stress application (N = 10) and in volunteers not receiving treatment (N = 7) at 3 time intervals within 24 h. Proteins differentially expressed by the tooth movement group were functionally analyzed with “Gene Ontology” (GO) and “Search Tool to Retrieve Interacting Genes/proteins” (STRING) softwares. Enzyme-Linked Immunosorbent Assay and Western-blot were used to validate the in vivo protein alterations. An in vitro model consisting of human periodontal ligament cells (hPDLCs) under compression was used to validate the force-responsive characteristics of galectin-3 binding protein (LGALS3BP).

Results

Sixteen out of the 294 proteins identified by LC–MS/MS were differentially expressed in the plasma of subjects receiving controlled mechanical stresses for moving teeth. Those proteins were clustered in biological processes related to acute inflammatory response and vesicle-related transportation. Serotransferrin, fibronectin and LGALS3BP were processed for confirmation in vivo ; LGALS3BP was significantly increased in the tooth movement group. In vitro secretion of LGALS3BP in PDLCs was force-responsive.

Conclusions

Regional application of mechanical stresses stimulates systemic proteomic changes. Because serum LGALS3BP is over-expressed in different systemic diseases, including cancer, further work is needed to examine how systemic up-regulation of LGALS3BP affects the progression of those diseases.

Introduction

Induction of tooth movement by mechanical forces may be considered a regional inflammatory process that is characterized by alveolar bone and periodontal ligament remodeling. Both mechanical responses and inflammation are essential for tooth movement clinically . Bone homeostasis and inflammation, as well as related diseases, are regulated by the immune system. Although inflammation induced by mechanical stresses has been studied for several years, little is known about how systemic responses are manifested after the application of orthodontic forces. Preliminary work conducted with a murine model indicates that systemic manifestation of inflammatory monocytes is augmented in the presence of orthodontic forces, and that T-cells are required for controlled tooth movement . These preliminary results infer that systemic responses induced by regional mechanical stresses are responsible for the physiologic processes of bone resorption and deposition that occur during tooth movement. Nevertheless, the exact nature of those systemic responses remains unclear.

Proteomic technologies are important tools for identification of specific proteins involved in biological processes. Liquid chromatography–mass spectrometry (LC–MS) is a combination of two powerful protein separation techniques, namely, liquid chromatography (LC) and mass spectrometry (MS). The use of LC–MS facilitates detection of biomarkers in body fluids . High-throughput LC coupled with tandem mass spectroscopy (MS-MS) is a gel-free, efficient strategy in comparative proteomics. This strategy has been used to identify disease-related protein biomarkers in pathologic processes, such as matrix metalloproteinase-9 in patients with periodontitis and neutrophil gelatinase-associated lipocalin in patients with pancreatic cancer . Other studies utilized MS for analyzing the peptidome profile in the human saliva proteome . Two-dimensional LC–MS has been used to identify changes in protein expression in the gingival crevicular fluid of teeth manifesting external root resorption . The efficacy and sensitivity of proteomic analysis make it an ideal approach for studying the systemic responses in patients undergoing orthodontic treatment.

Recent research on the systemic responses in orthodontic patients has improved the profession’s understanding of the biology of tooth movement. Such information should be helpful for establishing clinical guidelines for orthodontic treatment in individuals with systemic diseases. Patients who suffer from systemic diseases such as diabetes mellitus and asthma have been associated with excessive root resorption after orthodontic treatment for no obvious reason . This finding implies that the prevailing systemic condition of the host may adversely affect orthodontic treatment outcomes. Because limited knowledge is available on systemic responses even in healthy patients undergoing orthodontic treatment, there is a pressing need for investigating systemic responses after regional mechanical stress application.

Accordingly, the objectives of the present study were to identify the proteomic profiles in human subjects who were undergoing the initiation phase of orthodontic treatment and to correlate those profiles with systemic responses manifested by those subjects.

Materials and methods

Participants and ethical statement

Ten human subjects (five males and five females) who required orthodontic treatment as part of their treatment plan were recruited for the study. The criteria for recruitment included: (1) anterior teeth with greater or equal to 8 mm crowding; (2) aged between 20 and 30 years; (3) no medical contraindications and (4) without known oral diseases such as caries, gingivitis, periodontitis, diseases of the oral mucosa or oral cancer. Seven healthy volunteers not receiving orthodontic treatment (four females and three males aged between 20 and 30) were also enrolled in the study (Supplementary Table). Ethical approval for the study was obtained from the Peking University Biomedical Ethics Committee (reference number PKUSSIRB-201311103). All 17 participants consented to participating in the study.

Plasma sample collection

Peripheral blood samples from the ten orthodontic patients were collected during their pre-treatment visit (designated as S0), and at 8 h (designated as S8) and 24 h (designated as S24) after the commencement of initial treatment ( Fig. 1 A). All patients were bonded with MBT brackets (Shi Ye Dental Material, Hang Zhou, China) involving the entire dentition. A 0.014 nickel titanium wire was used as the initial archwire. Bracket attachment and archwire activation were performed by three orthodontists from Peking University School and Hospital of Stomatology.

Fig. 1
Differential proteomic profiles from subjects receiving orthodontic treatment (tooth movement group) and subjects not receiving orthodontic treatment (control group). A. Time intervals of peripheral blood collection in the two groups were prior to orthodontic treatment, 8 h, and 24 h after the application of mechanical stresses to teeth. Peripheral blood samples in the control group were collected at the same time intervals. B. Flow chart of TMT-based proteomics strategy, including serum sample collection and pooling, protein enzymatic digestion, TMT-labeling, LC–MS/MS, annotation and statistical analysis. C. Venn diagram showing the number proteins that were differentially expressed by the tooth movement group (16) or the control group (7), and proteins that were identified in both groups (28).

Peripheral blood samples from the seven healthy subjects who did not receive orthodontic treatment were concomitantly collected to eliminate diurnal protein variations (designated as C0, C8, and C24). The blood samples were stored in anticoagulation tubes containing ethylenediamine tetraacetic acid (BD Biosciences, San Jose, CA, USA) and centrifuged at 3000 g at 4 °C for 10 min to obtain plasma samples. All plasma samples were stored at −80 °C until further analysis.

Peptide fractionation by reversed-phase high performance liquid chromatography

Protein extraction, reduction, alkylation and enzymatic in-solution digestion were performed as previously described ( Fig. 1 B). The peptide mixture was labeled using chemicals from the tandem mass tagged (TMT) reagent (Pierce Biotechnology, ThermoFisher Scientific, Waltham, MA, USA). The proteins were labeled as follows: C0 with 126, C8 with 127, C24 with 128, S0 with 129, S8 with 130, and S24 with 131 isobaric tag. The labeled samples were combined and dried in vacuo .

The mixture was re-dissolved and fractionated using the pH reversed-phase separation technique. An Aquity UPLC system (Waters Corporation, Milford, MA, USA) was connected to a reversed-phase column (BEH C18 column, 2.1 mm × 50 mm, 1.7 μm, 300 Å, Waters Corp.). High pH separation was performed using a linear gradient starting from 0% B (mobile phase solvent B: 20 mM ammonium formate in 90% acetonitrile, pH 10.0; adjusted with ammonium hydroxide) to 45% B in 20 min. The column was re-equilibrated to the initial condition for 2 min. The column flow rate and temperature were maintained at 600 μL/min and 45 °C, respectively. The collected fractions were dried inside a vacuum concentrator.

Low pH nano-HPLC–MS/MS analysis

Each dried fraction was re-suspended in 20 μL solvent C (water with 0.1% formic acid), separated by nano-LC, and analyzed by on-line electrospray tandem mass spectrometry. The experiments were performed with a Nano Aquity UPLC system (Waters Corp.) connected to a Q-Exactive (ThermoFisher Scientific) quadrupole-Orbitrap mass spectrometer that was equipped with an online nano-electrospray ion source. An 8 μL peptide sample was loaded into the trap column (ThermoScientific Acclaim PepMap C18, 100 μm × 2 cm) with a flow rate of 10 μL/min for 3 min and subsequently separated in an analytical column (Acclaim PepMap C18, 75 μm × 50 cm) with a linear gradient from 5% D (mobile solvent D: acetonitrile with 0.1% formic acid) to 30% D in 95 min. The column was re-equilibrated to the initial condition for 15 min. Column flow rate and column temperature were maintained at 300 nL/min and 45 °C, respectively. An electrospray voltage of 1.8 kV was employed against the inlet of the mass spectrometer.
Operation of the Q-Exactive mass spectrometer was conducted in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Full-scan survey MS spectra (350–1200 m / z ) were acquired with mass resolution of 70 K, followed by 15 sequential high-energy collisional dissociation MS/MS scans with mass resolution of 17.5 K. For all scans, one micro-scan was recorded using 30-s dynamic exclusion. The MS/MS fixed first mass was set at 100 m / z .

Database search and quantitative data analysis

Extraction of the tandem mass spectra was conducted with ProteoWizard (ThermoFisher Scientific, version 3.0.5126), using the Proteome Discoverer software (ThermoFisher Scientific, version 1.4.0.288). The MS–MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3; www.matrixscience.com ) , which was set up for searching the UniProt Swiss-Prot database (release 2014_04_10, 20264 reviewed entries) for proteins that simulate the digestive enzyme trypsin. Mascot was set up with a fragment ion mass tolerance of 0.050 Da and a parent-ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine, TMT 6-plex of lysine and N-terminus were specified in Mascot as fixed modifications. Protein quantification was performed using unique peptides only and experimental bias was corrected using the method of normalization for protein median. Percolator algorithm was used to control the peptide level false discovery rate to lower than 1%. The minimum number of proteins that could be observed was set to 200.

Identification of selected proteins with western-blot

Western-blot was used to detect Galectin-3 binding protein in plasma. For each patient, 20 μL of 1/50 phosphate buffered saline-diluted plasma was loaded for 8% acrylamide-sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes . Primary antibodies against human LGALS3BP (sc-374541, Santa Cruz, Dallas, TX, USA) were added at a dilution ratio of 1:1000 after blocking with 5% skimmed milk powder in Tween 20-Tris-buffered saline for 1 h. After overnight incubation with the primary antibody, the blots were washed with Tris-buffered saline, and then treated with a mouse anti-IgG antibody for 1 h. Signals were visualized with an enhanced chemiluminescent substrate (Super Signal West Pico, ThermoFisher Scientific) and auto-radiographic films.

Static compression of human periodontal ligament cells (hPDLCs)

Human periodontal ligament cells (hPDLCs) were isolated from the periodontium of freshly extracted premolars, as described previously . Cell suspensions (1 × 10 4 cells) were seeded in 10-cm diameter culture plates (Costar, Cambridge, MA, USA). The seeded cells were cultured with alpha-modified Eagle’s medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 15% foetal calf serum (Equitech-Bio Inc, Kerrville, TX, USA), 100 μmol/L ascorbic acid 2-phosphate (Wako, Tokyo, Japan), 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Biofluids, Rockville, MD, USA), and incubated at 37 °C in 5% carbon dioxide atmosphere. Mechanical stress was applied to the hPDLCs after four passages . Briefly, a glass cover slip was placed over a 80%-confluent cell layer in 6-well plates. Metal weights were subsequently placed over the cover slip to exert a continuous compressive stress of 1 g/cm 2 on the hPDSCs for 24 h.

Immunofluorescence

Immunofluorescence staining was performed as previously described . The hPDLCs were cultured with or without 1 g/cm 2 of compressive stress for 24 h. The treated hPDLCs were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde. The cells were subsequently stained with anti-LGASAL3BP antibodies (1:100 dilution; Santa Cruz, Dallas, TX, USA) and Alexa Fluor ® 488 Phalloidin for staining F-actin (green fluorescence; 1:200; Millipore Sigma, St. Louis, MO, USA). Stained sections were then incubated with tetramethylrhodamine isothiocyanate-conjugated secondary antibody (red fluorescence; 1:200, Jackson Immuno Research Laboratories, West Grove, PA, USA) to identify LGASAL3BP. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue fluorescence). Images were acquired using a confocal laser scanning microscope (LSM 510, Zeiss, Jena, Germany) and processed using the LSM 5 Release 4.2 software.

Enzyme-linked immunosorbent assay (ELISA)

Supernatants derived from the hPDLCs were collected after compressive stress application. The concentration of LGALS3BP was quantified using human galectin-3BP/MAC-2BP Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA). The concentration of fibronectin was quantified with human Fibronectin Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA). The concentration of serotransferrin was quantified with human serotransferrin ELISA kit (Biosamite, Jijin Chemistry Technology Co. Ltd, Shanghai, China). The aforementioned kits were used in accordance with the manufacturers’ instructions.

Statistical analyses

Kruskal-Wallis analysis of variances (ANOVAs) were used to examine if significant differences in protein profiles were present in the tooth movement group before treatment (0 h; baseline), 8 h and 24 h after initiation of orthodontic treatment. Likewise, Kruskal-Wallis ANOVAs were used to analyze the control group at baseline, 8 h and 24 h. The Benjamini-Hochberg false discovery rate was pre-set at 10% . Post-hoc comparisons were performed using Dunn’s multiple comparison tests. All reported P values were two-tailed. Statistical significance was pre-set at α = 0.05.

Because the normality and equal variance assumptions of the ELISA data were violated, thee data were logarithmically transformed to satisfy those assumptions prior to the use of parametric statistical methods. Two-way repeated measures ANOVA was used to analyze the transformed ELISA results, to examine the effects of controlled mechanical stress application (tooth movement group vs control) and stress application time (0 h vs 8 h vs 24 h) on the expression of serotransferrin, fibronectin or LGALS3BP in the collected plasma. When a significant difference was observed for the respective protein, pairwise comparisons were performed using the Holm- Šidák statistic. One-way repeated measures ANOVA and the Holm- Šidák statistic were used to analyze the Western blot results acquired for LGALS3BP in the plasma of the subjects in the tooth movement group. Statistical analysis of LGALS3BP in the hPDLCs was performed using two-tailed Student’s t -test. For all analyses, statistical significance was pre-set at α = 0.05.

Materials and methods

Participants and ethical statement

Ten human subjects (five males and five females) who required orthodontic treatment as part of their treatment plan were recruited for the study. The criteria for recruitment included: (1) anterior teeth with greater or equal to 8 mm crowding; (2) aged between 20 and 30 years; (3) no medical contraindications and (4) without known oral diseases such as caries, gingivitis, periodontitis, diseases of the oral mucosa or oral cancer. Seven healthy volunteers not receiving orthodontic treatment (four females and three males aged between 20 and 30) were also enrolled in the study (Supplementary Table). Ethical approval for the study was obtained from the Peking University Biomedical Ethics Committee (reference number PKUSSIRB-201311103). All 17 participants consented to participating in the study.

Plasma sample collection

Peripheral blood samples from the ten orthodontic patients were collected during their pre-treatment visit (designated as S0), and at 8 h (designated as S8) and 24 h (designated as S24) after the commencement of initial treatment ( Fig. 1 A). All patients were bonded with MBT brackets (Shi Ye Dental Material, Hang Zhou, China) involving the entire dentition. A 0.014 nickel titanium wire was used as the initial archwire. Bracket attachment and archwire activation were performed by three orthodontists from Peking University School and Hospital of Stomatology.

Fig. 1
Differential proteomic profiles from subjects receiving orthodontic treatment (tooth movement group) and subjects not receiving orthodontic treatment (control group). A. Time intervals of peripheral blood collection in the two groups were prior to orthodontic treatment, 8 h, and 24 h after the application of mechanical stresses to teeth. Peripheral blood samples in the control group were collected at the same time intervals. B. Flow chart of TMT-based proteomics strategy, including serum sample collection and pooling, protein enzymatic digestion, TMT-labeling, LC–MS/MS, annotation and statistical analysis. C. Venn diagram showing the number proteins that were differentially expressed by the tooth movement group (16) or the control group (7), and proteins that were identified in both groups (28).

Peripheral blood samples from the seven healthy subjects who did not receive orthodontic treatment were concomitantly collected to eliminate diurnal protein variations (designated as C0, C8, and C24). The blood samples were stored in anticoagulation tubes containing ethylenediamine tetraacetic acid (BD Biosciences, San Jose, CA, USA) and centrifuged at 3000 g at 4 °C for 10 min to obtain plasma samples. All plasma samples were stored at −80 °C until further analysis.

Peptide fractionation by reversed-phase high performance liquid chromatography

Protein extraction, reduction, alkylation and enzymatic in-solution digestion were performed as previously described ( Fig. 1 B). The peptide mixture was labeled using chemicals from the tandem mass tagged (TMT) reagent (Pierce Biotechnology, ThermoFisher Scientific, Waltham, MA, USA). The proteins were labeled as follows: C0 with 126, C8 with 127, C24 with 128, S0 with 129, S8 with 130, and S24 with 131 isobaric tag. The labeled samples were combined and dried in vacuo .

The mixture was re-dissolved and fractionated using the pH reversed-phase separation technique. An Aquity UPLC system (Waters Corporation, Milford, MA, USA) was connected to a reversed-phase column (BEH C18 column, 2.1 mm × 50 mm, 1.7 μm, 300 Å, Waters Corp.). High pH separation was performed using a linear gradient starting from 0% B (mobile phase solvent B: 20 mM ammonium formate in 90% acetonitrile, pH 10.0; adjusted with ammonium hydroxide) to 45% B in 20 min. The column was re-equilibrated to the initial condition for 2 min. The column flow rate and temperature were maintained at 600 μL/min and 45 °C, respectively. The collected fractions were dried inside a vacuum concentrator.

Low pH nano-HPLC–MS/MS analysis

Each dried fraction was re-suspended in 20 μL solvent C (water with 0.1% formic acid), separated by nano-LC, and analyzed by on-line electrospray tandem mass spectrometry. The experiments were performed with a Nano Aquity UPLC system (Waters Corp.) connected to a Q-Exactive (ThermoFisher Scientific) quadrupole-Orbitrap mass spectrometer that was equipped with an online nano-electrospray ion source. An 8 μL peptide sample was loaded into the trap column (ThermoScientific Acclaim PepMap C18, 100 μm × 2 cm) with a flow rate of 10 μL/min for 3 min and subsequently separated in an analytical column (Acclaim PepMap C18, 75 μm × 50 cm) with a linear gradient from 5% D (mobile solvent D: acetonitrile with 0.1% formic acid) to 30% D in 95 min. The column was re-equilibrated to the initial condition for 15 min. Column flow rate and column temperature were maintained at 300 nL/min and 45 °C, respectively. An electrospray voltage of 1.8 kV was employed against the inlet of the mass spectrometer.
Operation of the Q-Exactive mass spectrometer was conducted in the data-dependent mode to switch automatically between MS and MS/MS acquisition. Full-scan survey MS spectra (350–1200 m / z ) were acquired with mass resolution of 70 K, followed by 15 sequential high-energy collisional dissociation MS/MS scans with mass resolution of 17.5 K. For all scans, one micro-scan was recorded using 30-s dynamic exclusion. The MS/MS fixed first mass was set at 100 m / z .

Database search and quantitative data analysis

Extraction of the tandem mass spectra was conducted with ProteoWizard (ThermoFisher Scientific, version 3.0.5126), using the Proteome Discoverer software (ThermoFisher Scientific, version 1.4.0.288). The MS–MS samples were analyzed using Mascot (Matrix Science, London, UK; version 2.3; www.matrixscience.com ) , which was set up for searching the UniProt Swiss-Prot database (release 2014_04_10, 20264 reviewed entries) for proteins that simulate the digestive enzyme trypsin. Mascot was set up with a fragment ion mass tolerance of 0.050 Da and a parent-ion tolerance of 10.0 ppm. Carbamidomethyl of cysteine, TMT 6-plex of lysine and N-terminus were specified in Mascot as fixed modifications. Protein quantification was performed using unique peptides only and experimental bias was corrected using the method of normalization for protein median. Percolator algorithm was used to control the peptide level false discovery rate to lower than 1%. The minimum number of proteins that could be observed was set to 200.

Identification of selected proteins with western-blot

Western-blot was used to detect Galectin-3 binding protein in plasma. For each patient, 20 μL of 1/50 phosphate buffered saline-diluted plasma was loaded for 8% acrylamide-sodium dodecyl sulfate polyacrylamide gel electrophoresis. The separated proteins were transferred to nitrocellulose membranes . Primary antibodies against human LGALS3BP (sc-374541, Santa Cruz, Dallas, TX, USA) were added at a dilution ratio of 1:1000 after blocking with 5% skimmed milk powder in Tween 20-Tris-buffered saline for 1 h. After overnight incubation with the primary antibody, the blots were washed with Tris-buffered saline, and then treated with a mouse anti-IgG antibody for 1 h. Signals were visualized with an enhanced chemiluminescent substrate (Super Signal West Pico, ThermoFisher Scientific) and auto-radiographic films.

Static compression of human periodontal ligament cells (hPDLCs)

Human periodontal ligament cells (hPDLCs) were isolated from the periodontium of freshly extracted premolars, as described previously . Cell suspensions (1 × 10 4 cells) were seeded in 10-cm diameter culture plates (Costar, Cambridge, MA, USA). The seeded cells were cultured with alpha-modified Eagle’s medium (GIBCO BRL, Grand Island, NY, USA) supplemented with 15% foetal calf serum (Equitech-Bio Inc, Kerrville, TX, USA), 100 μmol/L ascorbic acid 2-phosphate (Wako, Tokyo, Japan), 2 mmol/L glutamine, 100 U/mL penicillin, and 100 μg/mL streptomycin (Biofluids, Rockville, MD, USA), and incubated at 37 °C in 5% carbon dioxide atmosphere. Mechanical stress was applied to the hPDLCs after four passages . Briefly, a glass cover slip was placed over a 80%-confluent cell layer in 6-well plates. Metal weights were subsequently placed over the cover slip to exert a continuous compressive stress of 1 g/cm 2 on the hPDSCs for 24 h.

Immunofluorescence

Immunofluorescence staining was performed as previously described . The hPDLCs were cultured with or without 1 g/cm 2 of compressive stress for 24 h. The treated hPDLCs were washed with phosphate-buffered saline and fixed with 4% paraformaldehyde. The cells were subsequently stained with anti-LGASAL3BP antibodies (1:100 dilution; Santa Cruz, Dallas, TX, USA) and Alexa Fluor ® 488 Phalloidin for staining F-actin (green fluorescence; 1:200; Millipore Sigma, St. Louis, MO, USA). Stained sections were then incubated with tetramethylrhodamine isothiocyanate-conjugated secondary antibody (red fluorescence; 1:200, Jackson Immuno Research Laboratories, West Grove, PA, USA) to identify LGASAL3BP. The nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; blue fluorescence). Images were acquired using a confocal laser scanning microscope (LSM 510, Zeiss, Jena, Germany) and processed using the LSM 5 Release 4.2 software.

Enzyme-linked immunosorbent assay (ELISA)

Supernatants derived from the hPDLCs were collected after compressive stress application. The concentration of LGALS3BP was quantified using human galectin-3BP/MAC-2BP Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA). The concentration of fibronectin was quantified with human Fibronectin Quantikine ELISA kit (R&D Systems, Minneapolis, MN, USA). The concentration of serotransferrin was quantified with human serotransferrin ELISA kit (Biosamite, Jijin Chemistry Technology Co. Ltd, Shanghai, China). The aforementioned kits were used in accordance with the manufacturers’ instructions.

Statistical analyses

Kruskal-Wallis analysis of variances (ANOVAs) were used to examine if significant differences in protein profiles were present in the tooth movement group before treatment (0 h; baseline), 8 h and 24 h after initiation of orthodontic treatment. Likewise, Kruskal-Wallis ANOVAs were used to analyze the control group at baseline, 8 h and 24 h. The Benjamini-Hochberg false discovery rate was pre-set at 10% . Post-hoc comparisons were performed using Dunn’s multiple comparison tests. All reported P values were two-tailed. Statistical significance was pre-set at α = 0.05.

Because the normality and equal variance assumptions of the ELISA data were violated, thee data were logarithmically transformed to satisfy those assumptions prior to the use of parametric statistical methods. Two-way repeated measures ANOVA was used to analyze the transformed ELISA results, to examine the effects of controlled mechanical stress application (tooth movement group vs control) and stress application time (0 h vs 8 h vs 24 h) on the expression of serotransferrin, fibronectin or LGALS3BP in the collected plasma. When a significant difference was observed for the respective protein, pairwise comparisons were performed using the Holm- Šidák statistic. One-way repeated measures ANOVA and the Holm- Šidák statistic were used to analyze the Western blot results acquired for LGALS3BP in the plasma of the subjects in the tooth movement group. Statistical analysis of LGALS3BP in the hPDLCs was performed using two-tailed Student’s t -test. For all analyses, statistical significance was pre-set at α = 0.05.

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Jun 19, 2018 | Posted by in General Dentistry | Comments Off on Effect of intraoral mechanical stress application on the expression of a force-responsive prognostic marker associated with system disease progression
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