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C. Miller et al.

Figure 3. The tissue is fabrication by embedding fibroblasts in collagen I on day 0. The ASM are dynamically seeded and mechanical stimulation is applied on day 2. The tissue contracts over the next 7–10 days and then the epithelial cells are seeded on day 14. As the tissue is modified by the cells and stabilizes, the phenotypic gene expression alters (denoted by shading and protein name) and cell functionality changes

in chronological order, including medium use, seeding timing and tissue behaviour.

near-static humidified air with pressure not greater than 4 mmHg.

2.3. Tissue-engineered bronchiole stimulation

2.4. Tissue-engineered bronchiole phenotype analyses

The bioreactor system stimulates the tissue-engineered bronchioles in two ways. First, mechanical stimulation is applied through the radial distension of the tissue con- struct during the contractile phase. Second, humidified air flow through the epithelialized lumen of the bronchi- ole also causes a slight distension in the radial direction. These force applications and the geometry of the engi- neered bronchiole may provide a better understanding of the effect of mechanotransduction on cell behaviour.

Mechanical stimulation is applied to the contracting tissues during the first 13 days after tissue fabrication (contraction phase, Figure 3). The fibroblasts embedded in collagen matrix contract around the silicone rubber tubing. During the initial mechanical stimulation phase, the thin-walled silicone rubber tubing is pulsed at a rate of 15 pulses/min with a radial distension of approximately 2% and distension velocity of 0.015 mm/s. The diameter of the bronchiole increases by about 60 µm, which causes biaxial (circumferential and axial) forces to act on the cells. Mechanical stimulation is secondarily applied to the engineered bronchioles by flowing humidified air through the epithelialized lumen.

Although mechanical stimulation was applied to determine whether the engineered bronchioles could be exposed to radial distension during the contraction phase (days 1–14), these bronchioles were not pulsed with physiologically normal air flow after the epithelial monolayer was formed. The bronchioles were pulsed with

The engineered bronchioles were sampled at 7, 14, 28 and 60 days post-fabrication. Immunohistochemistry was performed in order to assess changes in the tissue through protein expression. The engineered bronchioles were fixed in 10% neutral buffered formalin, graded ethanol dehydrated, embedded in paraffin and then sectioned (5 µm thick). Fluorescent staining was accomplished by blocking with 2% goat serum for 30 min at room temperature, primary antibody application for 1 h, three 1 min washes in PBS and then application of the secondary antibody for 30 min. ASM cells were labelled using primary antibodies against smooth muscle α-actin (1 : 100; DAKO, M0851) and smooth muscle myosin heavy chain (1 : 100; DAKO, M3558) with Alexa Fluor 488 (Invitrogen, A21121) secondary, and vimentin (1 : 200; DAKO, M7020) with Alexa Fluor 594 secondary (Invitrogen, A21135). Fibroblasts were labelled for FITC- conjugated β-tubulin (1 : 200; Sigma, T4026) with DAPI (1 : 2500; Invitrogen, D1306) nuclear labelling. The epithelial cells were labelled for cytokeratin-19 (1 : 100; Sigma, C6930) and collagen IV (1 : 500; Sigma, C1926) with TRITC (1 : 100; Sigma, T2659) secondary; and β-tubulin and mucin (1 : 100; Abcam, ab7874) with Alexa Fluor 488 secondary and DAPI nuclear stain. The fluorescently labelled airway cross-sections were documented using a Zeiss Axiovert 200 microscope with a digital camera.

Copyright 2010 John Wiley & Sons, Ltd.

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

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