Cell culture and strain application
Primary PDL fibroblasts (PDLF) were derived from the ligament tissues of periodontally healthy, non-carious human premolar teeth, extracted from juvenile donors (12-14 years) for orthodontic reasons with informed consent, and this study was approved by the institutional ethic committee of the Medical Faculty, University of Heidelberg (Vote number 148/2003; renewal 30.09.2005). Small tissue fragments were established as explant cultures by means of DME medium (PAA, Cölbe, Germany) supplemented with 10% foetal calf serum (FCS; Biochrom, Berlin, Germany), 2 mM L-glutamine (Invitrogen, Karlsruhe, Germany), and antibiotics (kanamycin, 50 mg/ml; Roche, Mannheim, Germany). After nearly reaching confluence, cells were used for strain experiments between passages 8 and 12. Strain application was carried out according to the method described by Hasagawa . Briefly, following trypsination, 3.5 × 103/cm2 PDL cells were seeded on flexible-bottomed dishes (Greiner Bio-One, Frickenhausen, Germany), coated with coating medium, and grown until near-confluence. As an approach to the composition of the extracellular matrix (ECM) environment of PDLF in vivo, the coating medium in addition to 1% BSA (Sigma, Munich, Germany) comprises 20 μg/ml native collagen type-I (IBM, Leipzig, Germany) and 10 μg/ml fibronectin (Biomol, Hamburg, Germany). Both molecules are essentially found in the PDL's ECM . The bottom of each dish was strained by induction of a continuous average strain of 2.5%  for periods of 0.25, 0.5, 1, 3, and 6 hours concerning the western blot experiments, and 0.5, 3 and 6 hours in case of RT-Profiler qPCR experiments, respectively. Irrespective from the modus operandi, unstrained cells served as controls. Concerning the forces acting on the PDLF, the continuous stretch mimics forces which are applied during orthodontic tooth movement using fixed appliances. The chosen time periods, mentioned above, reflect initial stages of therapeutically applied mechanical forces. Depending on the type of tooth and the type of tooth movement, orthodontic forces in a range of 0.15 - 2.5 Newton (N) are usual. Although this force range is of therapeutic significance, it is noteworthy to mention that it is not possible up till now to predict neither the exact force which acts on the periodontium on the single cell level nor the optimal force for an individual tooth . Concerning the forces used in our study, in a first approach, translation of the average strain of 2.5% onto the single cell level reveals a force amount of approximately 30 nN.
For MAP-kinases inhibition experiments, PDL cells were additionally treated with protein kinase-specific inhibitors, SB202190 for phosphorylation inhibition of phosphor-p38 and UO-126 for specific inhibition of phospho-p42/44. Both inhibitors were purchased from Calbiochem (Merck, Darmstadt, Germany). PDL cells were incubated with U0126 (10 μM) or SB202190 (10 μM) for 1 h prior to strain application. After the incubation period, fresh DMEM medium was added to the cell cultures followed by strain application for the above mentioned time periods.
RNA-isolation and quantitative RT-PCR-Profiler
Total RNA was isolated from nearly confluent human PDL cells from lumox dishes (Greiner bio-one, Frickenhausen, Germany) after periods of 0.5, 3, and 6 hours strain, by using the RNasy system (Qiagen, Hilden, Germany), and treated with RNase-free DNase (Qiagen, Hilden, Germany) for 15 min at room temperature (24°C). RNA concentration was measured with Bio-Rad electrophoresis system (Experion™ System, Bio-Rad, Munich, Germany) according to the manufacturer's instructions. Total RNA (1 μg) was reverse-transcribed into first strand cDNA using RT2 First Strand Synthesis Kit (C-03) (SA Bioscience Corporation, Frederick, MD, USA) according to the manufacturer's instructions. cDNA was mixed with instrument-specific and ready-to-use RT2-qPCR master mix (RT2-SYBR® Green/Fluorescein qPCR master mix, SA Bioscience Corporation, Frederick, MD, USA), and 25 μl of the master mix were pipetted into each well containing pre-dispensed gene-specific primer pairs. The pathway-specific qPCR experiments were performed for human extracellular matrix and integrins, human growth factors, and MAP-kinase signalling and cell cycle pathways (SA Biosciences Corporation, Frederick, MD, USA) and amplification was done after an initial cycle of 10 min at 95°C to activate the HotStart DNA polymerase, followed by the manufacture's specific PCR protocol. The annealing conditions for the pre-validated and pre-dispersed primer pairs were set to 55°C according to the manufacture's instructions. Relative gene expression levels were analysed using a modification of the ΔΔCT equation, which allows counting for differences in efficiencies (E = 101/slope) between the PCR reactions . The ΔCT values were calculated using excel plug-in provided with the pathway-specific profilers (SA Biosciences Corporation, Frederick, MD, USA). The data were obtained from two individual experiments and normalised to the CT of the experimental internal set of 4 different housekeeping genes (B2M, HPRT1, RPL13A, GAPDH, ACTB). The relative expression levels were subjected to means t-test and only p-values less than 0.01 were considered statistically significant and plotted in Figure 5.
After strain application, PDL cells were washed with ice cold PBS, fixed for 5 min with ethanol (96%, -20°C), and subsequently air-dried. For antibody treatment flexible membranes were cut in pieces and separated into inner and outer membrane parts. Thereafter, alcohol-fixed membrane pieces were incubated overnight with the primary antibodies directed against integrin subunits β1 and β3 (both mouse monoclonal, (wd) 1:50 in PBT (PBS containing 0.5% BSA, 0.5 Tween-20, and 0.02% NaN3), Santa Cruz Biotechnology, Heidelberg, Germany). Then, membrane pieces were washed in PBS for three times (5 min each), followed by incubation with the secondary fluorochrome-conjugated antibody (Alexa FluorTM 488, Mo Bi Tec Göttingen, Germany, IgG (H+L) goat anti-mouse, wd 1:100) for 1 hour at room temperature. To allow for nucleus counterstain, DAPI-staining (Sigma, Deisenhofen, Germany; wd 1:1000) was performed for 10 min at RT. After air drying, membrane pieces were embedded in mounting medium ProlongGold antifade reagent (Invitrogen, Karlsruhe, Germany), and documented by using a Leica digital camera (DFC300 FX), and the fluorescence microscope with Leica Application Suit 2.4.0 software (DMRE, Leica TCS/NT, Leica, Bensheim, Germany). All images were taken with equal exposure times within the experimental set up for each antibody.
Western blot analysis
After strain periods of 0.25, 0.5, 1, 3, and 6 hours, cells were washed with PBS and lysed on ice with cold RIPA buffer (Sigma-Aldrich, Steinheim, Germany) containing protease inhibitors (Complete Mini, Roche Diagnostics, Mannheim, Germany). After centrifugation (8000 g, 10 min), protein amounts were measured with the experion system (Experion™ Pro260, Pro260 Chips, Bio-Rad Laboratories, Munich, Germany) according to manufacturer's instructions. In all western blot experiments equal protein aliquots of 25 μg of protein were diluted in NuPAGE LDS sample buffer and NuPAGE sample reducing agent (Invitrogen, Karlsruhe, Germany), heated at 70°C for 5 min and separated by NuPAGE 4-12% Bis-Tris gradient gels (Invitrogen, Karlsruhe, Germany). For immunoblotting, the separated proteins were transferred onto a PVDF membrane (Invitrolon™ PVDF Filter Paper Sandwich, Invitrogen, Karlsruhe, Germany) in a Trans-Blot electrophoretic transfer cell (BioRad, Munich, Germany). Subsequently, immunodetection was performed by incubation the PDVF membranes with primary antibodies directed against focal adhesion kinase (FAK), phospho-specific FAKTyr576 (rabbit monoclonal, (wd) 1:1000 and 1:500 Santa Cruz Biotechnology, Heidelberg, Germany), MMP-13 (mouse monoclonal, (wd) 1:1000, RD Systems, Wiesbaden-Nordenstadt, Germany), p44/42 MAP-kinase, phospho-specific p44/42 MAP-kinase (Thr202/Tyr204) (rabbit polyclonal, (wd) 1:1000 and 1:500, Cell Signalling Technology, Danvers, MA, USA), p38 MAP-kinase, phospho-specific p38 MAP-kinase(Thr180/Tyr182) (rabbit monoclonal, (wd) 1:1000 and 1:500, Epitomics, Burlingame, CA, USA), and integrin β3 (mouse monoclonal, (wd) 1:1000, Santa Cruz Biotechnology, Heidelberg, Germany) in PBT for one hour at room temperature. Specific proteins were revealed by the WesternBreeze Chromogenic Immunodetection System (Invitrogen, Karlsruhe, Germany). The developed blots were documented by a digital camera (Casio Digital Camera EX-P700, Casio Europe GmbH, Norderstedt, Germany) and images were analysed using the image analysis software (BioDocAnalyzer 2.1, Biometra, Goettingen, Germany) for quantification of different band gray pixel values. To exclude that the observed changes in band intensities might be within the experimental noise range, analysis was performed by direct import of the digital image of each blot into the software, thereby subtracting the background with a standard algorithm to minimise experimental noise ratio. The gray pixel values of modulated protein expression following strain application were always normalised with the expression of unstrained control PDL cells, and plotted into graphs. To get a maximum of reproducibility, the experimental western blot setup was standardised concerning the protein amount loaded on the gel, equal time period for development of the western blot membranes with substrate and the same camera documentation setup, which includes the camera-membrane distance and the exposure time. This standardised western blot protocol allows detection of band intensity changes in independent experiments below 10%. Data of the western blot experiments represent the mean of three independent experiments (n = 3, mean ± SD), and for proper statistical analysis the means were subjected to the Students T-test (MedCalc® 184.108.40.206, Mariakerke, Belgium). Hereby, all compared mean values with p < 0.01 were denoted as statistical significant (Table 1) and were marked with an asterisk in the plotted graphs.