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3.  RESULTS

3.1. In Vitro Experiments

3.1.1. Histology

Isolated auricular cartilage tissue stained positive for elastic and collagen fibers, indicating the presence of pure elastic cartilage. Tissue culture cells derived from isolated auricular cartilage tissue at zero passage stained positive for elastic and collagen fibers, indicating the presence of chondrocytes (Figure 3).

FIGURE 3 Chondrocytes (Cc) growing from a piece of elastic cartilage (Ec) at zero passage. Tissue culture cells (Cc) derived from isolated auricular cartilage tissue (Ec) at zero passage stained positive for elastic and collagen fibers, indicating the presence of chondrocytes.


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3.1.2.  Immunocytochemistry

Elastic cartilage tissue and cells at second passage stained positive for collagen II, indicating the presence of chondrocytes (Figure 4). Cells treated with cell culture medium supplemented with sodium ascorbate showed much stronger staining of collagen II, indicating improved collagen synthesis.
FIGURE 4 Immunocytochemistry of elastic cartilage (left) and chondrocytes in tissue culture of second passage (right). Positive staining for collagen II indicated the presence of chondrocytes.


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3.1.3.  Seeding Efficiency

The luminal surfaces of the four LVAD’s, consisting of sintered titanium and polyurethane, were seeded twice with autologous auricular chondrocytes during the first seeding procedure and the second seeding procedure. The first seeding efficiency was 92.66 ± 10.08% and the second seeding efficiency was 98.14 ± 0.93%.The calculated total seeding efficiency was 95.11% ± 4.23% (n = 4) (Figure 5).

FIGURE 5 Seeding efficiency. The first seeding efficiency of four LVAD’s was 92.66 ± 10.08% and the second seeding efficiency of four LVAD’s was 98.14 ± 0.93%. Total seeding efficiency of four LVAD’s was 95.11 ± 4.23%.


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3.1.4.  Cumulative Cell Loss During Preconditioning

During the 12 hours of preconditioning under flow conditions in vitro, the average cumulative cell loss was 11.45± 0.21% (n = 4). The average cumulative cell loss was 2.22± 0.32% after 30 minutes, 5.13± 0.15% after 3 hours, 7.47± 0.20% after 5 hours, and 9.78± 0.35% after 7 hours (Figure 6).

FIGURE 6 Cumulative cell loss (n = 4). The cumulative cell loss during preconditioning under flow conditions on the in vitro flow loop was 2.22 ± 0.32% after 30 minutes, 5.13 ± 0.15% after 3 hours, 7.47 ± 0.20% after 5 hours, 9.78 ± 0.35% after 7 hours and 11.45 ± 0.21 after 9 hours.


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3.2.  .In Vivo Experiments

3.2.1. Necropsy and Gross Observations

Gross examination ofthe luminal surfaces of the LVAD revealed an intact cell layer and complete coverage of both artificial surfaces after 7 days of implantation (Figure 7). There was no evidence of infection or thrombus on either luminal surface, within the graft, or within the aortic anastomoses. Lungs, liver, rumen, spleen, kidney, rete mirabile, adrenal glands, and brain showed no gross and no histological evidence of emboli, ischemia, or infarction.

FIGURE 7 Gross appearance of the implanted LVAD’s biomaterial surfaces after 7 days of implantation in vivo. (Left) Textured polyurethane surface; (right) sintered titanium surface. The luminal surfaces of the LVAD are completely covered with an intact cell layer.


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3.2.2.  SEM and TEM

SEM revealed an extensive amount of extracellular matrix components and an intact, well-incorporated cellular lining on the sintered titanium and polyurethane surfaces of the implanted LVAD (Figure 8). TEM revealed a well-established monolayer of chondrocytes (Figure 9). No endothelial cells were seen.

FIGURE 8 Scanning electron microscopy of the implanted LVAD’s biomaterial surfaces after 7 days of implantation in vivo . An extensive amount of extracellular matrix and an intact, well-incorporated cellular coating (arrows) were noted on the textured polyurethane (left) and sintered titanium (right) surfaces of the implanted LVAD.


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FIGURE 9 Transmission electron microscopy of the implanted LVAD’s biomaterial surfaces after 7 days of implantation in vivo . (Left) Textured polyurethane surface; (right) sintered titanium surface. A well-established monolayer of chondrocytes was revealed. No endothelial cells were seen.


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