Cellular localization of type II and type I collagen after culture in static or dynamic conditions.

<p>Representative high magnification observations of HACs cultured for 21 days in static or dynamic conditions using either program 1 (n = 4) or program 2 (n = 6) in the presence of BIT (scale bars 10 μm). N: nucleus. The dotted lines indicate the cell border.</p

Experimental design and dynamic compression profile.

<p>Chondrocytes cultured in agarose for 6 days underwent dynamic compression using the FX-4000C Flexercell Compression Plus System (Flexcell International). Chondrocyte-agarose constructs underwent cyclical compression ranging from 20 kPa to 40 kPa at a frequency of 0.5 Hz for 5, 15 or 30 min. Signalling proteins were analysed by Western blot at each of the three time points. DNA microarray analysis was performed to compare 30 min-compression constructs to uncompressed constructs.</p

The down-regulation of type I collagen production in dynamic conditions is independent from the presence of BIT.

<p>(a) Representative WB analysis of type II and type I collagen after 21 days in static or dynamic conditions (program 1) in the absence (CTR) or in the presence of BIT. The positions of mature collagen chains (mature), unprocessed (pro) or processing intermediates of the procollagen containing the aminopropetide (pN) or the carboxypropetide (pC) are indicated. The upper band represents dimers (β) of collagen molecules. Actin bands are shown as a control for equivalent loading of the extracts. Please note that because type II collagen was relatively more abundantly produced in the presence of BIT, bands are smeared. (b) Quantification of type I collagen through the densitometric analysis of WB data. Total type I collagen (i.e. the unprocessed chains, the processing intermediates of the procollagen and the mature collagen chains) was normalized to actin. The values given in the dot plot represent single data points with mean (n = 3, a: <i>p</i> < 0.05).</p

The Oscillating Perfusion Bioreactor (OPB).

<p>(a) The tissue culture unit is a closed loop of gas-permeable silicone tubing connected to a chamber (asterisk). A single collagen sponges is fixed within a scaffold holder inserted in the chamber to avoid fluid flow around the construct. (b) Eighteen independent closed-loop chambers are mounted on a motorized frame oscillating the chambers around their central axis in a pendulum-like motion. (c) Time sequences of the two perfusion programs used with the OPB.</p

Type I collagen production is down-regulated by dynamic culture.

<p>(a) Representative WB analysis of type II and type I collagen after 21 days in static or dynamic conditions (program 2) in the presence of BIT. The positions of mature collagen chains (mature), unprocessed (pro) or processing intermediates of the procollagen containing the aminopropetide (pN) or the carboxypropetide (pC) are indicated. The upper band represents dimers (β) of collagen molecules. Actin bands are shown as a control for equivalent loading of the extracts. Please note that because type I collagen was more abundantly produced in static conditions, bands are smeared. (b) Quantification of type I collagen and of type II/type I collagen ratio through the densitometric analysis of WB data. Total type I and total type II collagen (i.e. the unprocessed chains, the processing intermediates of the procollagen, and the mature collagen chains) were normalized to actin. The values given in the dot plots represent single data points with mean (n = 6, a: <i>p</i> < 0.05).</p

Interstitial perfusion enhances ECM production in the scaffold core.

<p>HACs were cultured for 21 days within collagen sponges in static or dynamic conditions using either program 1 (n = 4) or program 2 (n = 6) in the presence of BIT. Representative pictures show parallel sections of the scaffold core stained for type II and type I collagen by IHC (scale bars 10 μm).</p

The combination of interstitial perfusion and BIT improves ECM neo-synthesis and distribution.

<p>(a) Representative low-magnification pictures showing cell and ECM distribution in collagen sponges cultured for 21 days in static or dynamic conditions (program 1, n = 4) in the absence (CTR) or in the presence of BIT (HE staining, scale bars 100 μm). Inserts in the left low corner show the Safranin O staining in the scaffold core (scale bars 10 μm). (b) High-magnification pictures showing cell and matrix distribution in the scaffold core (HE staining, scale bars 100 μm).</p

HACs undergo dedifferentiation during the amplification phase.

<p>(a) Representative phase-contrast micrographs showing changes in cell morphology during HAC expansion in monolayer (scale bars 50 μm). (b) Transcriptional levels of type I and type II collagen measured by Real-time PCR. Data were normalized on <i>GAPDH</i> using the 2^-ΔCt method, expressed relatively to data obtained for P0 HACs36 hours after isolation (P0-36h, reference value = 1) and reported as single data points with mean in the dot plots (n = 10, a: <i>p</i> < 0.05, significant effects versus P0-36h HACs; b: <i>p</i> < 0.05, significant effect versus P0 HACs).</p

Identification of major candidate mechanosensitive genes.

<p>Gene expression levels of compressed samples were compared to uncompressed controls. (A) DNA microarray analysis was performed on four independent pairs of uncompressed/compressed experiments. Expression level differences were sorted to identify highly responsive genes (fold change >2), resulting in a list of 20 transcripts. Bars represent the fold change in gene expression upon compression, i.e. up-regulation (red) or down-regulation (green) (p<0.01). Exact modulation factors and associated p-values are detailed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0036964#pone-0036964-t001" target="_blank">Table 1</a>. (B) Real-time PCR analysis on three independent experiments confirmed DNA microarray results for eight selected genes. Bars represent the compression-induced gene expression modulation (mean +/− SD), either up-regulation (red) or down-regulation (green) (* p<0.05, ** p<0.01).</p

Chondrocytes embedded in agarose gel maintain a well-differentiated phenotype.

<p>Expression of extracellular matrix proteins, integrins and the Sox9 transcription factor were analysed on Western blots of chondrocytes cultured in 3D for 3 days (d3) or 6 days (d6). The presence or absence patterns of proteins at day 6 are representative of 3 independent experiments. A: Type II (Col II) and type IX (Col IX) collagens accumulate and become cross-linked by day 6. Procollagen II forms (pro) and mature collagen II chains [α1(II)] are present. Beta (β) indicates cross-linked α1(II) dimers. Collagen IX chains [α1(IX)] are present. Asterisks (*) indicate cross-linked collagens. B: Type I collagen (Col I) and α11 integrin (Itgα11) are not or only faintly detected, whereas the chondrocyte-specific α10 integrin (Itgα10) is present at day 6. Passaged chondrocytes cultured in monolayer were used as positive controls (Ctrl) for Col I and α11 integrin immunorevelations. Procollagen I (pro) and mature collagen I chains α1(I) and α2(I) are indicated. C: Sox9 chondrogenic transcription factor increases with the duration of culture.</p