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 ).

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.

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.

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.

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 ).

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).

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 .

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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