Experimental model.

<p><b>(A)</b> Bird view of a PDMS well containing five mini-wells. <b>(B)</b> Fluorescent micrographs of a representative well cultured for 60 hours in the absence of strain, stained with DAPI to indicated nuclei (blue, left) and peanut agglutinin lectin (PNA) to indicate condensations (green, right). Scale bar is 500μm. <b>(C)</b> Fluorescent micrographs of representative cultures after 60 hours of culture in the absence of strain. Cells are stained with DAPI (blue), peanut agglutinin lectin (PNA, green), and an anti-avian fibronectin (FN) antibody to indicate deposited FN (red). Lower magnification (top row): scale bar is 100μm. Higher magnification (bottom row): scale bar is 50μm. <b>(D)</b> Schematic representation of the time line of the experiments. Cells are freshly isolated and directly plated onto the PDMS wells. Continuous strain is subsequently initiated at 0hr, 20hr, or 40hr, for a total of 25% strain over 20hours. Cultures are terminated at 60 hours.</p

Mesenchymal condensation.

<p><b>(A)</b> Fluorescent micrographs of samples cultured for 60 hours. Cultures are stained with DAPI (blue, top row) and peanut agglutinin lectin (PNA, green, bottom row). Scale bar is 200μm. <b>(B)</b> The number of condensations per squared mm is quantified at 60 hours, using images of PNA staining. Data represent means ± standard deviations, n≥7. *, p < 0.05. <b>(C)</b> The number of condensations per squared mm for the three strained conditions is corrected for the applied strain, thus multiplied by a factor of 1.25. The graph displays the number of condensations per squared mm of original surface area. Data represent means ± standard deviations, n≥7. *, p < 0.05.</p

Proliferation.

<p><b>(A)</b> Total DNA content is measured after 60 hrs. Data are normalized to the non-strained condition and represent means ± standard deviations, n≥8. <b>(B)</b> Percentages of proliferating cells, indicated by the incorporation of EdU, were measured at 48 hour of culture. Data are normalized to the non-strained condition and represent means ± standard deviations, n≥5. <b>(C)</b> Fluorescent micrographs of 48-hour cultures stained for nuclei (DAPI, blue) and EdU (red), after incubation with EdU for 4 hours. Scale bar is 100μm.</p

Quantitative cell biology.

<p>Cell viability (above; live cells/total cells +SD) and cell density (below; mean total cells/mm² +SD) as percentage of day 0 for each loading condition in the nucleus and outer annulus region after 7, 14 and 21 days of culture in the LDCS. <i>P</i> values comparing experimental groups with day 0 group in a linear mixed model with Bonferroni <i>post-hoc</i> testing: *<i>p</i><0.05; ^#<i>p</i><0.01; ^&<i>p</i><0.001.</p

Histology of IVD specimens.

<p>Midsagittal paraffin sections of IVD specimens (anterior side of IVDs facing right) at day 0 and after 21 days of culture at unloaded, LDL and SPL loading conditions. The left column shows safranin-O stained sections for the proteoglycan distribution (red). In the right column Masson's trichrome staining for distribution of the collagen (blue) is shown.</p

Primer sequences used for PCR.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033147#pone-0033147-t003" target="_blank">Table 3</a>. Primers used for the gene expression analyses showing the oligonucleotide sequences, annealing temperature and product size.</p

IVD stiffness during culture.

<p>Mean IVD stiffness (N/mm) ±SD as calculated from disc loading and displacement measurements during the culture period in the LDCS. The gray dotted line represents IVD stiffness from the discs cultured under LDL condition. The black lines represent the SPL loaded IVD stiffness, respectively during the rest phase (triangles) and active phase (squares) of the diurnal SPL regime.</p

Gene expression.

<p>Relative gene expression (log means ± SEM, normalized to YWHAZ) in the baseline (day 0; grey), unloaded (blue), LDL (green) and SPL (red) group. Shown in the left column is the data for the nucleus region and in the right column for the outer annulus region. Graph rows from top to bottom show respectively the anabolic, remodeling and inflammation-related genes. Brackets indicate significant statistical differences between groups when comparing in a linear fixed model with Bonferroni post-hoc testing. <i>P</i> values are indicated by: ^*<i>p</i><0.05; ^#<i>p</i><0.01; ^&<i>p</i><0.001.</p

Histological sections.

<p>Representative images (2,5× magnification) of midsagittal sections of IVD specimens after 21 days of culture under SPL (upper row), high dynamic (middle) and high static (lower) loading conditions. The left column shows the transitional zone between the nucleus and posterior annulus region (posterior annulus facing left) in Safranin-O stained sections. In the right column the anterior annulus (inner annulus left, outer annulus right) of the IVDs is shown in sections stained with Masson's trichrome.</p

Cell Viability and Cell Density.

<p><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0033147#pone-0033147-t002" target="_blank">Table 2</a> Mean cell viability (percentage live cells ±SD) and mean cell density (cells/mm2 ±SD) per experimental group, region and test duration. <i>P</i> values comparing experimental groups with day 0 group in a linear mixed model with Bonferroni post-hoc testing: *<i>p</i><0.05; ^#<i>p</i><0.01; ^&<i>p</i><0.001.</p