Expression of stem cell and differentiation markers.

<p>Immunostaining analyses were used to observe the expression of markers for stem cells (c-kit⁺, <b>A</b>), cardiomyocytes (troponin-I⁺ cells, <b>B</b>), smooth muscle cells (SMA⁺ cells, <b>C</b>), or endothelial cells (CD31⁺ cells, <b>D</b>) after 3 days of culture. Representative images of immunostaining are shown in the upper panels. Nuclei were labeled with DAPI. Scale bars represent 100 µm. Quantitative data were derived from six separate experiments using different cells.</p

Morphological changes and cell survival.

<p><b>A</b>) The cells of both groups showed similar density and morphology at baseline. However, when compared to static culture, stretching stimulation decreased the cell density and caused the cells to be arranged parallel to the stretch direction after 24 hrs and 3 days of culture (bar = 100 µm). <b>B</b>) The number of surviving cells was significantly less at 24 hrs and 3 days when the cells were cultured under stretching stimulation compared to under static condition.</p

Experimental designs and time schedule of assessments.

<p>The cells were seeded at a density of 1×10⁵/chamber. After 24 hrs for manipulation, the chambers were continuously stretched at a frequency of 60 cycles/min with 120% elongation (stretch group). Non-stretched chambers were used as controls (static group). Assessments were performed at the indicated time points.</p

Expression of adhesion molecules and extracellular matrix (ECM).

<p>Representative images show the variable expression in stretch and static groups of adhesion molecules and ECM after 3 days of culture with (Stretch) or without (Static) stretching stimulation, as imaged by immunostaining for focal adhesion kinase (<b>A</b>), Integrin-β₁ (<b>B</b>), collagen I (<b>C</b>), and collagen III (<b>D</b>). Nuclei were labeled with DAPI (blue). Semi-quantitative data on the relative intensity of staining was also shown (bar graph on the left). Scale bars represent 100 µm.</p

Morphological observation of cells under scanning electron microscopy.

<p>Cells in the stretch group were more likely to display a spindle shape, in contrast to the flat shape typically observed in the static group.</p

Cell proliferation and apoptosis.

<p>The upper panels show representative images of immunostaining for proliferating cells (Ki67-positive, <b>A</b>) and apoptotic (TUNEL, <b>B</b>) cells. Nuclei were labeled with DAPI. Quantitative analysis showed a significant decrease in the number of proliferative cells (<b>A</b>, lower panel) and a significant increase in the number of apoptotic cells (<b>B</b>, lower panel) in the stretch group compared with the static group. Data represent 4 separate experiments using different cells.</p

ELISA analysis of cytokines and growth factors released from CDCs.

<p>The concentrations of the angiogenic factors, VEGF (<b>A</b>) and bFGF (<b>B</b>), in conditioned media were significantly higher in the stretch group than the static group. The concentrations of the inflammatory cytokines, IL-6 (<b>C</b>) and IL-1β (<b>D</b>), were also significantly higher in the stretch group than the static group. In contrast, the concentrations of IGF-1 (<b>E</b>), HGF (<b>F</b>), SDF-1α (<b>G</b>), and TGF-β₁ (<b>H</b>) were not significantly different between the groups. Data represent 6 separate experiments using different cells.</p