Morphologic and gene expression analysis of periodontal ligament fibroblasts subjected to pressure

Introduction

Force application (FA) during orthodontic tooth movement is mediated through periodontal ligament (PDL) fibroblasts. FA on deciduous teeth has an inherent risk of root resorption, which is less in permanent teeth. Currently, the root resorption mechanism is poorly understood. We hypothesized that FA alters the morphology and gene expression of PDL fibroblasts. This study was designed to achieve homogenous PDL fibroblast cultures, establish an in-vitro FA model, analyze fibroblast morphology after FA, and compare the gene expressions of PDL fibroblasts of deciduous and permanent teeth after FA.

Methods

Fibroblasts were sorted from primary cultures of deciduous and permanent tooth PDLs. Cell viability was evaluated in the Opticell (Thermo Scientific, Waltham, Mass) FA model. Cellular morphology was analyzed using immunofluorescence staining for actin and focal adhesion complexes. Gene expressions of untreated or pressure-treated PDL fibroblasts of deciduous and permanent teeth were compared by gene array and confirmed by real-time polymerase chain reaction.

Results

Cell sorting resulted in cultures containing 98% of PDL fibroblasts. The Opticell model showed 94% cell survival after FA. FA increased fibroblasts’ adhesion. Gene arrays and real-time polymerase chain reactions indicated greater up-regulation of DKK2 mRNA in untreated PDL fibroblasts of deciduous teeth and greater up-regulation of ADAMTS1 mRNA in pressurized PDL fibroblasts of deciduous and permanent teeth.

Conclusions

Cell sorting is an efficient method to establish homogenous PDL fibroblast cultures. Using the Opticell FA model allows the maintenance of excellent cell viability. FA increased the surface adherence of fibroblasts. Up-regulation of ADAMTS1 after FA may indicate its involvement in the remodeling of the periodontium during orthodontic tooth movement. Understanding root resorption mechanisms under FA will help to prevent it during orthodontic treatment.

Highlights

  • Homogenous primary cultures after immunomagnetic cell sorting are proposed.

  • A new and innovative model for in-vitro hydrostatic pressure application is proposed.

  • DKK2 gene is upregulated in PDL fibroblasts from deciduous teeth with no successors.

  • ADAMTS1 gene is upregulated after pressure application on PDL fibroblasts.

Orthodontic treatment uses applications of mechanical force (orthodontic force) to the tooth, resulting in remodeling of the periodontal ligament (PDL) and the alveolar bone, enabling the transposition of the tooth. Alveolar bone is absorbed, and the PDL is degraded at the side of compression, whereas the bone is reconstructed, and the PDL is regenerated at the tension side. It is assumed that the mechanical force is transduced to biologic signals responsible for changes in gene transcripts, which differentially affect the regulation of the extracellular matrix components of the PDL The interactions between fibroblasts and the extracellular matrix are mediated by integrins—specific cell receptors —that bind specific ligands in the extracellular matrix, sense mechanical changes in the extracellular matrix or cell surface, and consequently trigger intracellular signals leading to altered gene expression. Cell interactions with the extracellular matrix or other cells are mediated by focal adhesion complex proteins, located in actin projections of the cytoskeleton (lamellipodia) and include the vinculin, a key regulator protein in these interactions.

From the “cell-type” perspective, the PDL is a highly heterogeneous tissue, mainly comprising fibroblasts, osteoblasts, and mesenchymal stem cells, as well as other less-abundant cell types such as osteoclasts, cementoblasts, odontoclasts, macrophages, neutrophils, monocytes, plasma cells, eosinophils, mast cells, and epithelial cell rests of Malassez. To date, several models have been proposed for pressure application on PDL culture cells, aiming to simulate orthodontic forces of either tension type (the majority) or pressure type ( Table I ). The major disadvantages of most models are inappropriate culture conditions and decreased cell viability during prolonged experiments, the inability to fix the cells in the model vessel for further experiments, and the costly equipment.

Table I
Summary of published pressure- and tension-type force application models
Force Type of force application References
Pressure Centrifuge Gebken, 1999; Palmon, 2001; Baumert, 2004; Redlich, 2004; Maeda, 2007
Hydrostatic pressure Ngan, 1992; Yousefian, 1995; Nakago-Matsuo, 1996, 2000
Micro gravity in satellite Carmeliet, 1998
Silastic wells Stanford, 2000
Static compressive force Kanzaki, 2002; Mitsui, 2005; Goga, 2006; Mishijima, 2006; Yamaguchi, 2006; De Araujo, 2007; Lee, 2007; Mayahara, 2007; Nakao, 2007; Wongkhantee, 2007
Tension Coated cover glass Peake, 2000
Dynamic negative hydrostatic pressure Yousefian, 1995
Flexcell type I/II culture dishes, Flexercell strain unit Yamaguchi, 1994,1996,1997, 2002; Carvalho, 1995,1996; Harter,1995; Shimizu, 1995,1997; Ozawa, 1997; Brady, 1998; Matsuda, 1998; Kimoto, 1999; Ohzeki, 1999; Bolcato-Bellemin, 2000; Kikuiri, 2000; Miura, 2000; Long, 2001, 2002; Doi, 2003; Myokai, 2003; Tsuji, 2004; Kanzaki, 2006; Kim, 2007; Yamashiro, 2007
Fluid flow pump Joldersma, 2000; Maeda, 2007
Paramagnetic microparticles Bierbaun, 1998; Rychly, 1998; Schmidt, 1998; Pommerenke, 2002
Petriperm culture dishes Andersen, 1991; Saito, 1991; Basdra, 1995, 1996, 1997; Norton, 1995; Kletsas, 1998, 2002; Peverali, 2001; Ziros, 2002; Ritter, 2007
Self-contraction in collagen gels Von den Hoff, 2003
Stretch membrane Carano, 1996; Neidlinger-Wilke, 2001; Ohara, 2006; Yang, 2006
Viscometer Sakai, 1998

The biologic mechanism underlying the process in which a deciduous tooth exfoliates after the eruption of a permanent tooth is not fully elucidated. Clinical data show that application of orthodontic force on a deciduous tooth causes a greater risk for root resorption, compared with a permanent tooth. Interestingly, the roots of a deciduous tooth without a permanent successor are often seen gradually resorbed for decades, despite the absence of the permanent tooth. There are no predictors for the survival of deciduous teeth lacking successors; in some cases, they are maintained well into adulthood, but they can also exfoliate faster.

Since the PDL cells are the mediators of the force applied to the tooth and since deciduous and permanent teeth respond differently to pressure application, we hypothesized that the morphology and gene expression of PDL fibroblasts of deciduous and permanent teeth, before and after force application, should change.

Our aims were (1) to achieve homogenous PDL fibroblast cultures, (2) to establish an in-vitro orthodontic force application model with enhanced cell viability, (3) to analyze fibroblast morphology changes with pressure application, and (4) to compare gene expression responses of PDL fibroblasts from deciduous teeth with no successor and permanent teeth before and after pressure application.

Material and methods

PDL cells were isolated from healthy PDL tissue of permanent premolars from 3 healthy male subjects and from deciduous teeth with no successor from 3 healthy male subjects (12-30 years old), having tooth extractions as part of orthodontic treatment as described by Somerman et al with minor modifications.

Deciduous teeth with no successor were chosen to ensure that no permanent tooth bud cells were present in the culture.

All patients signed an informed consent before providing the samples in accordance with the institutional Helsinki committee guidance of Hadassah-Hebrew University of Jerusalem, Israel, which approved this study.

Experiments were carried out on the PDL cells at passages 3 through 6.

To create homogenous PDL fibroblast cultures from the primary cultures, the fibroblasts were immunomagnetically sorted from the cultures by incubation of the PDL cells with the “Anti-Fibroblast” microbead antibodies (Miltenyi Biotec, Teterow, Germany) as part of the magnetic affinity cell sorting process, according to the manufacturer’s protocol. These microbeads adhere to a 112 kDa membrane molecule (termed D7-FIB) known to be expressed on the surface of the fibroblasts. PDL fibroblasts from permanent and deciduous tooth cultures were used for further experiments.

The magnetic affinity cell sorting-based separation process was verified by fluorescence-activated cell sorter analysis using anti-D7-FIB fluorescence labeled antibody (PE) and isotype-control fluorescence labeled antibody as a negative control (Acris, Herford, Germany).

To apply hydrostatic pressure to cultured cells, we propose a new in-vitro pressure application model based on the Opticell chamber (Thermo Scientific, Waltham, Mass). The Opticell is a closed 10-mL culture chamber, built from a 2-mm thick plastic frame with 2 polystyrene flexible membranes (50 cm 2 ) (transparent and permeable to gas only), 1 on each side, to which the cells can adhere. Since the membranes are flexible, we hypothesized that by placing the Opticell between two 5-mm thick perforated (to allow gas exchange) rigid acrylic plates (Perspex; Darwen, Lancashire, United Kingdom) with weight loads on top, hydrostatic pressure would be generated in the chamber without bending the surface to which the cells adhere ( Fig 1 ) according to the equation of pressure: P = M/A, where P is hydrostatic pressure (g/cm 2 ), M is mass of weights (g), and A is area of weight contact area (cm 2 ).

Fig 1
A new model for in-vitro pressure application. Hydrostatic pressure calibration of the assembled Opticell-based in-vitro pressure application: 1 , Opticell culture flask; 2 , 2 perforated 5-mm plastic plates; 3 , 2500-g lead weight.

To test our model, 1 million PDL fibroblasts of permanent teeth were cultured in a collagen type-I precoated Opticell and incubated for 24 hours to allow cell adhesion. Then a series of weights were placed (500, 1000, 1500, 2500, 5000, and 10,000 g), and the forces were measured with a hydrostatic pressure meter used to measure cerebral spinal fluid pressure in patients ( Fig 1 ).

Cell membrane integrity under pressures of 100 and 200 g/cm 2 for 24 and 48 hours was measured using the trypan blue cell viability assay as previously described. The live and dead cells were automatically counted with the Cellometer (Nexcelom Bioscience, Lawrence, Mass).

Pressure was applied to cultured cells by using our new Opticell-based model. PDL fibroblast cells from permanent and deciduous teeth were separately cultured in a collagen-I precoated Opticell (1 million cells each) and incubated for 24 hours to allow cell adhesion. The medium was then replaced by a serum-free medium for an additional 24 hours (to achieve cell-cycle synchronization) and changed back to regular culture medium; then 100 g/cm 2 pressure was applied to the cultures for 24 and 48 hours in the incubator.

Immunofluorescence staining was performed using the Actin Cytoskeleton and Focal Adhesion Staining Kit (FAK 100; Chemicon International, Temecula, Calif) per the manufacturer’s protocol after 100 g/cm 2 pressure for 24 and 48 hours. Microscopic imaging was performed by using a confocal laser scanning system (LSM 410; Zeiss, Oberkochen, Germany) attached to an Axiovert 135M inverted microscope (Zeiss). Image analysis was performed with Camedia Master Pro 4.1 software (Olympus, Tokyo, Japan).

Twenty sample fields were chosen from every Opticell flask at 40-times magnification. Using the “transparent layer” in the Photoshop software (version 7-0; Adobe, San Jose, Calif), cell areas, number of processes, length of processes, and number of focal adhesion clusters were measured in relation to the main axis of the fibroblasts.

PDL fibroblast cells of permanent and deciduous teeth were suspended in TRIZOL reagent (Molecular Research Center, Cincinnati, Ohio) according to the manufacturer’s protocol, and total RNA was extracted as previously described. After total RNA extraction, the samples were purified with RNase-Free DNase Set (Qiagen, Hilden, Germany).

The quality and concentration of total RNA were analyzed using the Nano-drop (Thermo Scientific, Wilmington, Del) and Agilent Bioanalyzer (Agilent Technologies, Santa Clara, Calif) (data not shown) before further experiments.

The gene expression profiles of the cultures (PDL fibroblasts of deciduous and permanent teeth, before and after 100 g/cm 2 of pressure for 24 hours) were compared by a semiquantitative measurement (2 colors) using whole human genome chip slide (Qiagen) as previously described. The hybridization was measured with the Axon scanner and analyzed with GenePix Pro (version 4.0; Molecular Devices, Sunnyvale, Calif). Bioinformatics analysis was performed with the help of the bioinformatics unit of our campus, aided by Matlab software (version 2017; MathWorks, Natick, Mass). Relevant pathways were defined with ingenuity pathway analysis (Qiagen).

For real-time polymerase chain reaction (PCR) analysis, mRNA was reverse transcribed to cDNA using a cDNA synthetizing kit (Qiagen); real-time PCR was then performed as previously described for ADAMTS1 and DKK2 . These genes were identified in the gene arrays.

Statistical analysis

The 2-group Student t test was used for individual matched group comparisons. Data are expressed as means and standard deviations. Values of P <0.05 were considered statistically significant.

Results

In the fluorescence-activated cell sorter analysis of cultures treated with magnetic affinity cell sorting, on average, the percentage of fibroblasts in the treated cultures reached 98%, whereas that in untreated cultures was only 90% ( P <0.05) ( Fig 2 , A ), indicating high levels of homogeneity.

Fig 2
Yield of D7-FIB positive cells in cultures before and after magnetic affinity cell sorting separation. A, Magnetic affinity cell sorting ( MACS ) treated PDL fibroblast purity reached 98% ( P <0.05) vs 90% in untreated cell culture. B, Fluorescence-activated cell sorter analysis of primary culture (p3-p8) shows the percentage of PDL fibroblasts in untreated PDL primary culture as 83%. C, Fluorescence-activated cell sorter analysis of magnetic affinity cell sorting treated culture shows the percentage of PDL fibroblasts as 98.8%. ∗ p<0.05.

Figure 2 , B and C , shows a representative fluorescence-activated cell sorter analysis of fibroblast primary culture (from 6 cultures) before and after magnetic affinity cell sorting separation treatment. The percentages of fibroblasts were 83% and 98.8%, respectively.

Hydrostatic pressure inside the Opticell chamber was measured in a series of increasing weight loads ( Fig 3 , A ). We applied weights of 500, 1000, 1500, 2500, 5000, and 10,000 g and measured 10 ± 0.05, 20 ± 0.47, 31 ± 0.54, 52 ± 0.99, 103 ± 2.15, and 206 ± 4.14 g/cm 2 force pressures, respectively.

Fig 3
Opticell-based model for in-vitro pressure application. A, Hydrostatic pressure measured inside the Opticell chamber in correlation with weight loads. B, Trypan blue cell viability exclusion test: cell viability was assessed in correlation with weight load for 24 hours of pressure. C, Trypan blue cell viability exclusion test: cell viability was assessed in correlation with weight load for 48 hours of pressure.

In the trypan blue cell viability exclusion test, as shown in Figure 3 , B and C , under 100 and 200 g/cm 2 of pressure for 24 hours, 96.54% ± 3.38% and 92.07% ± 2.16% of cells were viable, respectively; under 100 and 200 g/cm 2 of pressure for 48 hours, 96.41% ± 0.53% and 90.43% ± 3.31% of cells were viable, respectively.

Immunofluorescence staining for fluorescence-activated cells on PDL fibroblasts under pressure for 24 and 48 hours demonstrated lamellipodia, actin, and dominant clustering of fluorescence-activated cells at the end of the actin processes and in untreated and treated cultures ( Fig 4 , A ).

Fig 4
Histology and microscopic morphometric analysis of untreated and pressure-treated PDL fibroblast cultures. A, Immunofluorescence confocal microscopy of actin fibers in the cytoskeleton and focal adhesion complexes (FACs) in PDL fibroblasts. F-actin was stained with TRITC-conjugated phalloidin and appears in red , FACs were stained with anti-vinculin monoclonal antibodies, and the cells’ nuclei were stained with DAPI. B, Cells treated with 100 g/cm 2 of pressure for 24 hours show greater cell areas compared with untreated cells ( P <0.05). C, Distribution of the cells’ area according to the treatment shows a trend in which the area is increasing with any type of force. D, Treated cells generate more processes compared with untreated cells ( P <0.05). E, Treated cells generate longer processes compared with untreated cells ( P <0.05). F, Treated cells generate more vinculin, both generally and within FACs. G, Treated cells generate longer vinculin clusters compared with untreated cells. ∗ p<0.05, ∗∗ p<0.005.

After 24 hours of 100 g/cm 2 pressure application, the cells’ mean area significantly increased from 5189 to 6503 μm 2 ( Fig 4 , B ), and the proportion of the larger cells in the culture (5000-10,000 μm 2 and >10,000 μm2) increased from 31.5% to 42.4% ( Fig 4 , C ). After 48 hours, the cells’ mean area was also greater than that of the control cells (6050 vs 5189 μm 2 ). The cells’ area distribution showed the same trend. This effect might be explained by an immediate response of the cells to pressure application that gradually decreased with time.

The number of the cells’ lamellipodia as well as the mean lamellipodia length significantly increased ( Fig 4 , D and E ). The number of cells’ lamellipodia increased from 1.4 lamellipodia in the control group to 2.35 lamellipodia after the 24-hour pressure application. Moreover, the cells’ number of lamellipodia in the 48-hour group (2.12 vs 1.4 lamellipodia) was still significantly higher than that of the control group. The mean cells’ lamellipodia lengths significantly increased from 172 to 191 μm in the 24-hour group and to 207 μm in the 48-hour group.

Vinculin density significantly increased after 48 hours of pressure application, and the mean vinculin cluster length significantly increased from 4.75 μm in the control group to 5.70 μm after 24 hours and to 5.55 μm after 48 hours of pressure application ( Fig 4 , F and G ). Correlation analysis showed that the proportion of larger vinculin clusters in the cells (5-10 μm 2 and >10 μm 2 ) increased from 32.1% to 45.4% in the 24-hour group and to 47.2% in the 48-hour group as pressure was maintained (data not shown).

Gene arrays are depicted as “volcano” displays ( Fig 5 , A and B ). The raw data from the gene arrays were further analyzed with GenePix Pro (version 4.0). Exclusion criteria included genes that were up- or down-regulated by less than 2 folds or nonsignificantly up- or down-regulated ( P >0.05).

Fig 5
Gene arrays and real-time PCR validations. A, “Volcano” display of up- and down-regulated genes shows that ADAMTS1 mRNA is up-regulated in pressurized permanent PDL fibroblasts (2.14 folds; P <0.05) and pressurized deciduous PDL fibroblasts (4.5 folds; P <0.05) compared with unpressurized cultures. B, “Volcano” display of up- and down-regulated genes shows that DKK2 mRNA is up-regulated in untreated deciduous PDL fibroblasts compared with untreated permanent PDL fibroblasts (23.21 folds; P <0.00005). C, Real-time PCR validation analysis shows that ADAMTS1 mRNA is up-regulated in pressurized PDL fibroblast cells (2.36 folds; P <0.005) compared with untreated PDL fibroblasts. D, Real-time PCR validation analysis shows that DKK2 mRNA is up-regulated in untreated deciduous PDL fibroblasts compared with untreated permanent PDL fibroblasts (64.35 folds). ∗ p<0.05, ∗∗∗ p<0.0005.

Based on these criteria, the lists of genes that were significantly up- or down-regulated by 2 folds or more are shown in Tables II through V .

Table II
Up- and down-regulated genes in untreated deciduous PDLfibroblasts compared with untreated permenent PDL fibroblasts
Block A Block B Block C
Line Gene name Fold P value Gene name Fold P value Gene name Fold P value
1 RPS4Y 116.02 0.016 SLC14A1 3.53 0.019 ITGA6 2.31 0.000
2 DKK2 23.21 0.000 CPM 3.30 0.010 PTGS2 2.23 0.039
3 MME 12.08 0.002 TNFRSF11B 3.18 0.030 C6orf35 2.21 0.043
4 WNT16 6.40 0.038 ASPN 3.07 0.047 DUSP6 2.18 0.023
5 PSG7 6.32 0.004 PENK 2.98 0.026 PTN 2.17 0.022
6 PSG1 6.25 0.047 IL12A 2.95 0.020 MN1 2.16 0.009
7 GTPBP1 5.05 0.016 KDELR1 2.94 0.007 RECK 2.15 0.005
8 LPXN 4.40 0.001 EDG2 2.93 0.019 DPYSL3 2.12 0.033
9 ASPN 4.36 0.009 AMOT 2.87 0.022 IGF2 2.11 0.033
10 ENPP2 4.20 0.048 ITGA8 2.78 0.001 PIK3R1 2.10 0.019
11 ANGPT1 4.18 0.011 SDC1 2.72 0.032 GBP3 2.06 0.039
12 PSG8 4.17 0.027 FST 2.56 0.030 GSK3B 2.03 0.009
13 GAS1 4.14 0.037 MEF2C 2.55 0.007 NTF3 2.03 0.013
14 KYNU 4.09 0.019 VCAM1 2.41 0.016 SHANK2 2.01 0.026
15 FBN2 3.86 0.002 PDE5A 2.35 0.048 QPCT 2.01 0.042
16 ENPP2 3.71 0.010 WNT5B 2.32 0.007 PTGER2 2.00 0.013
17 CSEN 3.64 0.041 BACH1 2.31 0.048
18 DMD 14.67 0.000 EDNRA 4.16 0.001 SLMAP 2.69 0.010
19 EFEMP1 8.44 0.007 EBI2 3.96 0.035 TFPI 2.53 0.007
20 BEX1 8.18 0.012 EBF 3.82 0.032 GARP 2.45 0.019
21 KCNMB1 7.10 0.049 ANK3 3.64 0.006 ECM2 2.43 0.001
22 THBD 7.01 0.010 SDC2 3.44 0.033 IRF7 2.36 0.013
23 ADA 6.60 0.012 LAMB3 3.38 0.038 RAMP1 2.30 0.035
24 ALPL 6.52 0.005 DCAMKL1 3.34 0.031 STAT1 2.29 0.013
25 PCOLCE2 6.44 0.008 TBX2 3.23 0.013 A2M 2.16 0.026
26 PPP1R14A 6.42 0.016 CNN1 3.20 0.007 CSRP2 2.14 0.050
27 CRLF1 6.41 0.014 PGBD3 3.19 0.028 LGALS3BP 2.13 0.047
28 GATA6 5.66 0.021 HES1 3.05 0.034 TPM1 2.12 0.025
29 RGS4 4.73 0.020 HLA-B 3.03 0.029 PPP1R14A 2.07 0.005
30 TRPC4 4.61 0.002 FHL1 2.97 0.015 KDELC1 2.06 0.024
31 WFDC1 4.59 0.005 PRG1 2.79 0.018 TCTE1 2.06 0.016
32 MYH11 4.47 0.001 NR4A3 2.77 0.016 HSPA2 2.02 0.036
33 PRELP 4.18 0.001 KRT18 2.71 0.024
Lines 1-17, up-regulated genes in untreated deciduous PDL fibroblasts compared with untreated permanent PDL fibroblasts.
Lines 18-33, up-regulated genes in untreated permanent PDL fibroblasts compared with untreated deciduous PDL fibroblasts.

Table III
Up- and down-regulated genes in deciduous PDLfibroblasts after 24 hours of pressure compared with untreated deciduous PDLfibroblasts
Block A Block B Block C
Line Gene name Fold P value Gene name Fold P value Gene name Fold P value
34 ADAMTS1 4.50 0.03 KRTAP4-7 3.69 0.05 STK12 2.88 0.03
35 NR4A2 6.93 0.043 RASSF2 3.11 0.028 QSCN6 2.47 0.041
36 COL11A1 6.83 0.029 REV3L 3.01 0.026 RARA 2.33 0.044
37 PDE5A 5.29 0.029 APOL6 3.00 0.045 TMSB4X 2.14 0.041
38 ATRX 3.70 0.048 BTN3A1 2.74 0.045 ZNF42 2.12 0.049
39 C20orf103 3.65 0.011 HMGCS1 2.64 0.029 BPGM 2.05 0.026
40 SLC12A8 3.53 0.032 TNFRSF14 2.64 0.035
41 LUM 3.15 0.045 LDLR 2.62 0.004
Line 34, up-regulated genes in deciduous PDL fibroblasts after 24 hours of pressure compared with untreated deciduous PDL fibroblasts.
Lines 35-41, up-regulated genes in untreated deciduous PDLfibroblasts compared with deciduous PDL fibroblasts after 24 hours of pressure.

The comparison of untreated deciduous PDL fibroblasts with untreated permanent PDL fiborblasts ( Table II ) showed 50 known genes (out of 90) up-regulated in the deciduous group (lines 1-17) and 47 known genes (out of 99) up-regulated in the permanent group (lines 18-33).

Table IV
Up- and down-regulated genes in P-PDLF after 24 hours of pressure compared with untreated P-PDLF
Block A Block B Block C
Line Gene name Fold P value Gene name Fold P value Gene name Fold P value
42 ESM1 17.42 0.017 SOX9 2.93 0.032 KRT19 2.39 0.035
43 AREG 11.31 0.017 MT1H 2.87 0.044 SPAG5 2.37 0.017
44 PRSS2 8.77 0.017 KNSL7 2.80 0.011 RTTN 2.34 0.014
45 PODXL 7.82 0.034 SEMA3A 2.79 0.034 GPR56 2.33 0.022
46 ANGPTL4 5.06 0.000 THBD 2.79 0.015 KIF11 2.24 0.004
47 SERPINB2 4.88 0.012 SLC38A5 2.79 0.035 RFC2 2.23 0.015
48 STC1 4.84 0.020 RRM2 2.76 0.028 BDKRB1 2.23 0.038
49 KRTAP4-7 4.81 0.005 ORC6L 2.75 0.037 NET1 2.22 0.015
50 PCOLCE2 4.70 0.049 TCF19 2.73 0.025 KIF23 2.21 0.042
51 ARHI 4.65 0.042 MT2A 2.71 0.017 CDC45L 2.17 0.011
52 PTGS1 4.35 0.009 RFC3 2.70 0.001 MSH2 2.17 0.026
53 STK31 4.23 0.007 HS3ST3A1 2.70 0.022 EZH2 2.15 0.007
54 RGS20 3.84 0.010 TNFRSF10D 2.68 0.041 ADAMTS1 2.14 0.019
55 CHRNA10 3.83 0.014 FEN1 2.68 0.016 PRIM1 2.14 0.009
56 ZNF367 3.79 0.033 H4FN 2.64 0.018 TM4SF1 2.11 0.007
57 MMD 3.54 0.012 FIGNL1 2.63 0.012 RBBP8 2.10 0.005
58 SLC22A4 3.30 0.004 CENPA 2.60 0.011 CDC25C 2.06 0.019
59 PLAUR 3.12 0.004 STEAP 2.60 0.048 MT1E 2.05 0.036
60 MT1A 3.09 0.020 CENPE 2.58 0.032 CENPJ 2.05 0.007
61 C20orf127 3.00 0.009 SEMA3C 2.58 0.008 POU2F2 2.04 0.022
62 C13orf3 2.99 0.020 HMGA2 2.53 0.010 GPR30 2.03 0.007
63 RETNLB 2.95 0.049 STK12 2.46 0.019 MICA 2.01 0.050
64 IGFBP3 2.94 0.001 KRT18 2.44 0.006
65 ID4 10.41 0.036 SERPINH2 3.18 0.039 ZNF216 2.28 0.043
66 CALD1 4.17 0.021 LMO7 2.86 0.035 RAMP1 2.25 0.036
67 CCL2 4.13 0.003 FBN2 2.80 0.019 FBLN1 2.22 0.047
68 IL6 3.95 0.022 MASP1 2.34 0.013 CYR61 2.19 0.034
69 CALD1 3.32 0.030 G1P2 2.30 0.014 CNN3 2.04 0.011
70 BHLHB2 3.28 0.026 C9orf9 2.30 0.020
71 SYTL2 3.23 0.004 DAB2 2.30 0.016
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Dec 8, 2018 | Posted by in Orthodontics | Comments Off on Morphologic and gene expression analysis of periodontal ligament fibroblasts subjected to pressure
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