Developing a tissue-engineered model of the human bronchiole
1. Introduction
Although substantial progress has been made in the study of airway remodelling, the initiation and progression of chronic respiratory disease is not well understood, due to its dynamic nature in vivo. A tissue-engineered model of the bronchioles is a potentially powerful approach to studying airway remodelling, as cell-cell and cell-matrix interactions can be considered. Indeed, several models of the airway wall have been developed to investigate epithelial-stromal communication (Tschumperlin and Drazen, 2001; Agarwal et al., 2003; Choe et al., 2006), but none have incorporated the native cylindrical geometry of the in vivo bronchiole nor both fibroblasts and smooth muscle cells. Furthermore, tissue engineering has recently been proposed as an important avenue to understanding tissue physiology, as opposed to solely developing tissues for implantation or replacement (Griffith and Swartz, 2006).
As an experimental model to investigate airway remodelling, we have developed a bioreactor system to fabricate cylindrical airways, which maintains the bronchioles under mechanical stimulation and humidified air flow, and allows us to manipulate the growth environment. The overall intent is to develop a tissue engineered bronchiole model of airway remodelling that approximates the behaviour of native tissue. This model system may advance understanding of the cumulative effects of individual factors associated with remodelling of human bronchioles. Native bronchioles are affected by many factors. Lung fibroblasts, smooth muscle cells and epithelial cells are influenced by cell-cell signalling and interactions with the extracellular matrix. Stimulation of one cell type has been found to influence the behaviour of other cells types that are in close proximity (Zhang et al., 1999). The extracellular matrices bind soluble regulatory molecules that also mediate cell behaviour. Mechanical forces exerted on the matrix and cells during respiration influence pathophysiological conditions (Hirst et al., 2000; Swartz et al., 2001; Black et al., 2003). Cytoskeletal-mediated contraction of the airway is equilibrated dynamically, affecting the adaptability of the airway smooth muscle in response to mechanical changes (An and Fredberg, 2007). Shear stress and pressure exerted by air flow through the lumen also influence cell behaviour (Liu et al., 1999).
As part of a novel approach to model the human bronchiole, we have developed an in vitro model that mimics bronchiole wall physiology. The engineered bronchioles are composed of a collagen scaffold containing embedded lung fibroblasts. The exterior surface is surrounded by multiple layers of airway smooth muscle (ASM) cells, and the inner surface (lumen) is lined with bronchial epithelial cells with an air interface. The human airway cells are in close proximity to one another to promote cell-cell communications. The bioreactor environment can be manipulated to focus on various aspects of airway remodelling, such as subepithelial fibrosis (Woodruff and Fahy, 2002), smooth muscle hyperplasia and hypertrophy (Hirst, 1996) and epithelial cell metaplasia (Woodruff and Fahy, 2001; Doherty and Broide, 2007), all of which are key components of airway remodelling.
2.3. Tissue-engineered bronchiole stimulation
The bioreactor system stimulates the tissue-engineered bronchioles in two ways. First, mechanical stimulation is applied through the radial distension of the tissue construct during the contractile phase. Second, humidified air flow through the epithelialized lumen of the bronchiole also causes a slight distension in the radial direction. These force applications and the geometry of the engineered 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 &mu;m, 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 near-static humidified air with pressure not greater than 4 mmHg.
2.4. Tissue-engineered bronchiole phenotype analyses
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 &mu;m 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 &alpha;-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 	&beta;-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 	&beta;-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.
Bright-field microscopy was performed for macroscopic observations. Haematoxylin and eosin (H&E) was used to view collagen fibres and the structure of the engineered bronchioles. Apoptosis and proliferating cell nuclear antigen (PCNA) were performed on the fibroblasts for cell viability and proliferation. For apoptosis, an ApopTAG kit (S7100, Chemicon) was used. Proliferating cells were identified with a primary antibody against PCNA (1 : 300; DAKO) and secondary antibody conjugated with horseradish peroxidase, using a VectaStain ABC Elite kit and DAB (PK-6102 and SK-4100, Vector Laboratories).
3. Results
3.1. Bioreactor design and feature optimization
The biomaterials for construction of the bioreactor system were chosen to promote ease of sterilization and low cost, minimize the quantity of biological agents and provide long-term use without defects.
The current version of the bioreactor is very userfriendly (Figure 1). The bioreactor insert can be completely disassembled for cleaning or part replacement and easily reassembled. The flint glass chamber is economical and can also be replaced. The bioreactor insert is assembled with the PTFE moulds in place and then the entire reactor is sterilized. This minimizes the risk of contamination.
The pump system affords the most unique attribute of the bioreactor system (Figure 2a). It supplies the engineered bronchioles with pulsed air to mechanically stimulate the tissue during the contractile phase and distributes humidified air to the epithelialized lumen during the differentiation phase (Figure 2b). The peristaltic pump was selected for its slow rotation (1-100 rpm) and pump head selection for the number of rollers. The PharMed L/S 18 tubing has a 7.9 mm inner diameter (i.d.) that forces a large volume of air into the three-way splitter and through the L/S 13 tubing (0.8 mm i.d.). Pump speed was varied to pulse the thin-walled silicone rubber tubing 15 times/min. The roller positions in the pump head and the diameter of the tubing allowed for a 2% radial distension, which increased the diameter of the bronchiole by 60 &mu;m (from 3 mm to approximately 3.06 mm).
3.2. Determination of tissue fabrication parameters
Fabrication of the tissue-engineered airways involved parameter optimization of matrix concentration, fabrication mechanics, cell density and seeding methods, medium composition and cell phenotype. Proper proportioning of these parameters was necessary for the creation of a stable engineered bronchiole (Figure 4a).
3.2.1. Matrix composition
The matrix concentration was determined by modifying a pre-existing protocol (Agarwal et al., 2003). Utilizing 5 mg/ml collagen for the cylindrical bronchiole maintained a tubular shape after the PTFEmould was removed. Lower concentrations of collagen tended to deform or tear during mould removal.
3.2.2. Cell seeding density
Optimal seeding densities were determined to achieve the goal of a monolayer of epithelial cells and a multilayer of ASM cells. ASM cell-seeding density was initially estimated by calculating the exterior surface area of the engineered bronchiole and estimating the number of ASM cells to cover the surface with three layers of cells. Seeding densities of 0.5, 1, 2, and 3 million cells/bronchiole were investigated (n = 10, using three lots of ASM cells). ASM cells were dynamically seeded by suspending the cells in SmGM-2. After 48 h, the entire medium volume was removed and a cell count was done.
3.4. Tissue-engineered bronchiole analyses
Preliminary trials were conducted at 5, 7, 10, 14, 21 and 28 days to determine the fabrication timeline and to select sampling times (n = 6 bronchioles). Subsequently, tissue characteristics were determined at 7, 14, 28 and 60 days. Each bronchiole comprises 1 cmof 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 &alpha;-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 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 of retinoic acid override EGF and increase mucous differentiation (Denning and Verma, 2001; Gray et al., 2001).
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 underlying airway inflammatory and constrictive processes. Although bronchodilators, anti-inflammatories and longacting 	&beta;2 agonists can improve lung function, these medications only act to relieve, prevent and control symptoms, respectively (Kumar, 2001). Neither the initiation and progression of airway remodelling nor its contribution to irreversible airway obstruction in asthma is well defined. Biopsies almost always reveal airway remodelling associated with asthma (Woodruff and Fahy, 2001); however, 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.