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of retinoic acid override EGF and increase mucous differentiation (Denning and Verma, 2001; Gray et al., 2001). Collagen IV labelling showed that the epithelial cells synthesized a basement membrane of collagen IV (Figure 6b) over the collagen I tissue construct. Once the SAGM was removed from the lumen of the bronchiole around day 21, humidified air flowed at a nearly static rate through the lumen of the engineered bronchiole. The air interface promoted the production of cilia (Figure 6c) and mucin (Figure 6d) by day 28 post-fabrication, when luminal pressures up to 4 mmHg were applied. The cells remained viable as long as the air flow did not dry the cells.

4. Discussion

A bioreactor system for culturing bronchiole tissues, which comprise three different cell types, has been described. Human lung fibroblasts, airway smooth muscle cells and bronchiole epithelial cells can be grown in close proximity to one another in the same culture environment and exhibit evidence of proper cellular behaviour. Tissue fabrication protocols have been established and engineered bronchiole stability has been shown through phenotypic analyses, both protein expression and morphology, and prolonged culture times of 60 days (Figure 3). The stability of the bronchiole structures and their cellular composition allows these constructs to be used to study cell–cell interactions and airway remodelling events while maintaining in vivo geometrical dimensions and relationships.

Currently, treatments for asthma focus on the under- lying airway inflammatory and constrictive processes. Although bronchodilators, anti-inflammatories and long- a c t i n g β 2 a g o n i s t s c a n i m p r o v e l u n g f u n c t i o n , t h e s medications only act to relieve, prevent and control symp- toms, respectively (Kumar, 2001). Neither the initiation and progression of airway remodelling nor its contribu- tion to irreversible airway obstruction in asthma is well defined. Biopsies almost always reveal airway remodelling associated with asthma (Woodruff and Fahy, 2001); how- ever, it is not always clinically demonstrated (Beasley et al., 2002). The severity of asthma varies so greatly that with its onset, the clinical evidence of remodelling can occur after only a few months or as much as several decades later (Beasley et al., 2002). A tissue-engineered bronchiole model of airway remodelling may lead to understanding that could produce therapeutic agents to inhibit or control airway remodelling. e

Immunohistological and morphological evidence, bol- stered by extensive culture protocol optimization, sup- ports a two-phase fabrication protocol of 28 days. Bron- chiole construction and ASM cell seeding during the first 48 h of tissue fabrication, followed by a 7 day contraction period, appeared optimal for producing the bronchiole construct. Epithelial cell seeding too early in the fab- rication process caused compression of the monolayer

Copyright 2010 John Wiley & Sons, Ltd.

C. Miller et al.

with ongoing tissue contraction, thus leading to epithe- lial sloughing. Epithelial cells seeded at day 14 proved effective to maintain cellular viability and allow for sub- sequent differentiation. Once the epithelial cells formed a monolayer and established a basement membrane, the cells were exposed to humidified air flow; after which they produced cilia and mucus.

Since cells can be easily manipulated in vitro to produce behaviour that is not like cellular behaviour under in vivo conditions, phenotypic modulation was deemed important to create a viable bronchiole that can be used as a model of airway remodelling. Determination of phenotypic behaviour is also informative to analyse cell–cell interactions, since the fibroblasts, airway smooth muscle cells and epithelial cells are within 200 µm of one another (Figures 3, 4c). ASM cell expression of vimentin, myosin heavy chain and α-actin changed during the first 28 days of tissue differentiation. By day 28, cell viability and markers of differentiation were stable through day 60.

Preliminary studies are currently under way to determine the parameters to induce airway remodelling. Various studies have shown that remodelling can be induced using growth factors (Stewart et al., 1994; Vignola et al., 1997; Shute, 2001; Chu et al., 2004), cytokines (De et al., 1992; Bonner and Brody, 1995; Cohn et al., 1998; Grunig et al., 1998; Wills-Karp et al., 1998; Renauld, 2001; Elias et al., 2003) and chemokines (Noveral and Grunstein, 1992; Holgate et al., 2003). Manipulation of the growth environment provides a format to utilize these triggers. Supplementation of the growth environment of the bioreactor may improve our understanding of remodelling events associated with ASM, fibroblasts and epithelial cells. This model can be tailored to study individual components of remodelling such as subepithelial fibrosis, smooth muscle hyperplasia and hypertrophy, and epithelial cell metaplasia. By doing a piece-wise investigation of airway remodelling, we may arrive at a better understanding of the individual contributors that initiate and cause the progression of remodelling associated with chronic respiratory disease.


A grant from the National Heart, Lung, and Blood Institute (to Ruth L. Kirschstein) National Research Service Award for Individual Postdoctoral Fellows (No. F32 HL-69646) is gratefully acknowledged.


Agarwal A, Mih J, George S. 2003; Expression of matrix proteins in an in vitro model of airway remodeling in asthma. Allergy Asthma Proc 24(1): 35. An S, Fredberg J. 2007; Biophysical basis for airway hyper- responsiveness. Can J Physiol Pharmacol 85(7): 700–714. Beasley R, Page C, Lichtenstein L. 2002; Airway remodeling in asthma. Clin Exp All Rev 2: 109. Black JL, Burges JK, Johnson P. 2003; Airway smooth muscle – its relationship to the extracellular matrix. Respir Physiol Neurobiol 137: 339.

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

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