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Developing a tissue-engineered bronchiole model

Figure 5. The ASM layer labels for (a–c) vimentin during the first 7–14 days, showing a synthetic phenotype that recedes by day 28. An increase in (d–f) MHC and (g–i) α-actin expression, indicating a change from synthetic to contractile phenotype. Bars = 10 µm unless indicated otherwise

60 days. Each bronchiole comprises 1 cm of testable tissue (Figure 4a).

Immunohistochemistry provided information regarding protein expression and location (Figure 3), to evaluate the phenotype of the fibroblasts, ASM cells and epithelial cells. ASM cell phenotype was evaluated by the presence of contractile proteins. The intensity of vimentin was more pronounced during the first 10 days (Figure 5a–c), as opposed to day 28. Vimentin expression indicates that the ASM cells are functioning synthetically. As the ASM cells establish themselves on the tissue construct, the cells made the transition from the synthetic to the contractile phenotype, as shown by the increased expression of smooth muscle myosin heavy chain (MHC) after day 14 (Figure 5d–f). Smooth muscle α-actin intensity for the ASM layer remained constant from day 7 to day 14, but increased slightly by day 28 (Figure 5g–i).

The small airway epithelial cells were seeded in the lumen of the tissue to create an epithelialized bronchiole. The cells were seeded on day 14, which was after the construct had finished contracting. When epithelial cells were seeded 3–9 days post-fabrication, the epithelial cells were compressed and shed from the luminal surface. The epithelial cells stained positively for the epithelial- specific marker cytokeratin-19 (Figure 6a). The presence

Figure 6. The epithelium is positive for (a) K-19 and establishes a basement membrane of (b) collagen IV (arrow denotes epithelial cell side of membrane). When air-lifted for 7–14 days, the epithelial cells produce (c) cilia (green) and (d) mucus (green) by day 28

of K19 implies that differentiation is not being inhibited by retinoic acid. At low quiescent retinoic acid concentrations EGF can suppress mucin, while higher concentrations

Copyright 2010 John Wiley & Sons, Ltd.

J Tissue Eng Regen Med (2010). DOI: 10.1002/term

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