Developing_VVG a_AT1 tissue-engineered_JJ model_NN1 of_IO the_AT human_JJ bronchiole_NN1 1_MC1 ._. 
Introduction_NN1 Although_CS substantial_JJ progress_NN1 has_VHZ been_VBN made_VVN in_II the_AT study_NN1 of_IO airway_NN1 remodelling_NN1 ,_, the_AT initiation_NN1 and_CC progression_NN1 of_IO chronic_JJ respiratory_JJ disease_NN1 is_VBZ not_XX well_RR understood_VVN ,_, due_II21 to_II22 its_APPGE dynamic_JJ nature_NN1 in_RR21 vivo_RR22 ._. 
A_AT1 tissue-engineered_JJ model_NN1 of_IO the_AT bronchioles_NN2 is_VBZ a_AT1 potentially_RR powerful_JJ approach_NN1 to_II studying_VVG airway_NN1 remodelling_NN1 ,_, as_CSA cell-cell_JJ and_CC cell-matrix_JJ interactions_NN2 can_VM be_VBI considered_VVN ._. 
Indeed_RR ,_, several_DA2 models_NN2 of_IO the_AT airway_NN1 wall_NN1 have_VH0 been_VBN developed_VVN to_TO investigate_VVI epithelial-stromal_JJ communication_NN1 (_( Tschumperlin_NP1 and_CC Drazen_NP1 ,_, 2001_MC ;_; Agarwal_NP1 et_RA21 al._RA22 ,_, 2003_MC ;_; Choe_NP1 et_RA21 al._RA22 ,_, 2006_MC )_) ,_, but_CCB none_PN have_VH0 incorporated_VVN the_AT native_JJ cylindrical_JJ geometry_NN1 of_IO the_AT in_JJ21 vivo_JJ22 bronchiole_NN1 nor_CC both_DB2 fibroblasts_NN2 and_CC smooth_JJ muscle_NN1 cells_NN2 ._. 
Furthermore_RR ,_, tissue_NN1 engineering_NN1 has_VHZ recently_RR been_VBN proposed_VVN as_II an_AT1 important_JJ avenue_NN1 to_II understanding_NN1 tissue_NN1 physiology_NN1 ,_, as_II31 opposed_II32 to_II33 solely_RR developing_JJ tissues_NN2 for_IF implantation_NN1 or_CC replacement_NN1 (_( Griffith_NP1 and_CC Swartz_NP1 ,_, 2006_MC )_) ._. 
As_II an_AT1 experimental_JJ model_NN1 to_TO investigate_VVI airway_NN1 remodelling_NN1 ,_, we_PPIS2 have_VH0 developed_VVN a_AT1 bioreactor_NN1 system_NN1 to_TO fabricate_VVI cylindrical_JJ airways_NN2 ,_, which_DDQ maintains_VVZ the_AT bronchioles_NN2 under_II mechanical_JJ stimulation_NN1 and_CC humidified_JJ air_NN1 flow_NN1 ,_, and_CC allows_VVZ us_PPIO2 to_TO manipulate_VVI the_AT growth_NN1 environment_NN1 ._. 
The_AT overall_JJ intent_NN1 is_VBZ to_TO develop_VVI a_AT1 tissue_NN1 engineered_JJ bronchiole_NN1 model_NN1 of_IO airway_NN1 remodelling_NN1 that_CST approximates_VVZ the_AT behaviour_NN1 of_IO native_JJ tissue_NN1 ._. 
This_DD1 model_NN1 system_NN1 may_VM advance_VVI understanding_NN1 of_IO the_AT cumulative_JJ effects_NN2 of_IO individual_JJ factors_NN2 associated_VVN with_IW remodelling_NN1 of_IO human_JJ bronchioles_NN2 ._. 
Native_JJ bronchioles_NN2 are_VBR affected_VVN by_II many_DA2 factors_NN2 ._. 
Lung_NN1 fibroblasts_NN2 ,_, smooth_JJ muscle_NN1 cells_NN2 and_CC epithelial_JJ cells_NN2 are_VBR influenced_VVN by_II cell-cell_JJ signalling_NN1 and_CC interactions_NN2 with_IW the_AT extracellular_JJ matrix_NN1 ._. 
Stimulation_NN1 of_IO one_MC1 cell_NN1 type_NN1 has_VHZ been_VBN found_VVN to_TO influence_VVI the_AT behaviour_NN1 of_IO other_JJ cells_NN2 types_NN2 that_CST are_VBR in_II close_JJ proximity_NN1 (_( Zhang_NP1 et_RA21 al._RA22 ,_, 1999_MC )_) ._. 
The_AT extracellular_JJ matrices_NN2 bind_VV0 soluble_JJ regulatory_JJ molecules_NN2 that_CST also_RR mediate_VV0 cell_NN1 behaviour_NN1 ._. 
Mechanical_JJ forces_NN2 exerted_VVN on_II the_AT matrix_NN1 and_CC cells_NN2 during_II respiration_NN1 influence_VV0 pathophysiological_JJ conditions_NN2 (_( Hirst_NP1 et_RA21 al._RA22 ,_, 2000_MC ;_; Swartz_NP1 et_RA21 al._RA22 ,_, 2001_MC ;_; Black_NP1 et_RA21 al._RA22 ,_, 2003_MC )_) ._. 
Cytoskeletal-mediated_JJ contraction_NN1 of_IO the_AT airway_NN1 is_VBZ equilibrated_VVN dynamically_RR ,_, affecting_VVG the_AT adaptability_NN1 of_IO the_AT airway_NN1 smooth_JJ muscle_NN1 in_II31 response_II32 to_II33 mechanical_JJ changes_NN2 (_( An_AT1 and_CC Fredberg_NP1 ,_, 2007_MC )_) ._. 
Shear_VV0 stress_NN1 and_CC pressure_NN1 exerted_VVN by_II air_NN1 flow_NN1 through_II the_AT lumen_NN1 also_RR influence_VV0 cell_NN1 behaviour_NN1 (_( Liu_NP1 et_RA21 al._RA22 ,_, 1999_MC )_) ._. 
As_II part_NN1 of_IO a_AT1 novel_JJ approach_NN1 to_TO model_VVI the_AT human_JJ bronchiole_NN1 ,_, we_PPIS2 have_VH0 developed_VVN an_AT1 in_JJ21 vitro_JJ22 model_NN1 that_CST mimics_VVZ bronchiole_NN1 wall_NN1 physiology_NN1 ._. 
The_AT engineered_JJ bronchioles_NN2 are_VBR composed_VVN of_IO a_AT1 collagen_NN1 scaffold_NN1 containing_VVG embedded_JJ lung_NN1 fibroblasts_NN2 ._. 
The_AT exterior_JJ surface_NN1 is_VBZ surrounded_VVN by_II multiple_JJ layers_NN2 of_IO airway_NN1 smooth_JJ muscle_NN1 (_( ASM_NP1 )_) cells_NN2 ,_, and_CC the_AT inner_JJ surface_NN1 (_( lumen_NN1 )_) is_VBZ lined_VVN with_IW bronchial_JJ epithelial_JJ cells_NN2 with_IW an_AT1 air_NN1 interface_NN1 ._. 
The_AT human_JJ airway_NN1 cells_NN2 are_VBR in_II close_JJ proximity_NN1 to_II one_PPX121 another_PPX122 to_TO promote_VVI cell-cell_JJ communications_NN2 ._. 
The_AT bioreactor_NN1 environment_NN1 can_VM be_VBI manipulated_VVN to_TO focus_VVI on_II various_JJ aspects_NN2 of_IO airway_NN1 remodelling_NN1 ,_, such_II21 as_II22 subepithelial_JJ fibrosis_NN1 (_( Woodruff_NP1 and_CC Fahy_NP1 ,_, 2002_MC )_) ,_, smooth_JJ muscle_NN1 hyperplasia_NN1 and_CC hypertrophy_NN1 (_( Hirst_NP1 ,_, 1996_MC )_) and_CC epithelial_JJ cell_NN1 metaplasia_NN1 (_( Woodruff_NP1 and_CC Fahy_NP1 ,_, 2001_MC ;_; Doherty_NP1 and_CC Broide_NP1 ,_, 2007_MC )_) ,_, all_DB of_IO which_DDQ are_VBR key_JJ components_NN2 of_IO airway_NN1 remodelling._NNU 2.3_MC ._. 
Tissue-engineered_JJ bronchiole_NN1 stimulation_NN1 The_AT bioreactor_NN1 system_NN1 stimulates_VVZ the_AT tissue-engineered_JJ bronchioles_NN2 in_II two_MC ways_NN2 ._. 
First_MD ,_, mechanical_JJ stimulation_NN1 is_VBZ applied_VVN through_II the_AT radial_JJ distension_NN1 of_IO the_AT tissue_NN1 construct_VV0 during_II the_AT contractile_JJ phase_NN1 ._. 
Second_MD ,_, humidified_VVD air_NN1 flow_NN1 through_II the_AT epithelialized_JJ lumen_NN1 of_IO the_AT bronchiole_NN1 also_RR causes_VVZ a_AT1 slight_JJ distension_NN1 in_II the_AT radial_JJ direction_NN1 ._. 
These_DD2 force_NN1 applications_NN2 and_CC the_AT geometry_NN1 of_IO the_AT engineered_JJ bronchiole_NN1 may_VM provide_VVI a_AT1 better_JJR understanding_NN1 of_IO the_AT effect_NN1 of_IO mechanotransduction_NN1 on_II cell_NN1 behaviour_NN1 ._. 
Mechanical_JJ stimulation_NN1 is_VBZ applied_VVN to_II the_AT contracting_JJ tissues_NN2 during_II the_AT first_MD 13_MC days_NNT2 after_II tissue_NN1 fabrication_NN1 (_( contraction_NN1 phase_NN1 ,_, Figure_NN1 3_MC )_) ._. 
The_AT fibroblasts_NN2 embedded_VVN in_II collagen_NN1 matrix_NN1 contract_NN1 around_II the_AT silicone_NN1 rubber_NN1 tubing_NN1 ._. 
During_II the_AT initial_JJ mechanical_JJ stimulation_NN1 phase_NN1 ,_, the_AT thin-walled_JJ silicone_NN1 rubber_NN1 tubing_NN1 is_VBZ pulsed_VVN at_II a_AT1 rate_NN1 of_IO 15_MC pulses/min_FU with_IW a_AT1 radial_JJ distension_NN1 of_IO approximately_RR 2%_NNU and_CC distension_NN1 velocity_NN1 of_IO 0.015_MC mm/s_FU ._. 
The_AT diameter_NN1 of_IO the_AT bronchiole_NN1 increases_VVZ by_RP about_RG 60_MC &mu;_NULL m_NNO ,_, which_DDQ causes_VVZ biaxial_JJ (_( circumferential_JJ and_CC axial_JJ )_) forces_VVZ to_TO act_VVI on_II the_AT cells_NN2 ._. 
Mechanical_JJ stimulation_NN1 is_VBZ secondarily_RR applied_VVN to_II the_AT engineered_JJ bronchioles_NN2 by_II flowing_VVG humidified_JJ air_NN1 through_II the_AT epithelialized_JJ lumen_NN1 ._. 
Although_CS mechanical_JJ stimulation_NN1 was_VBDZ applied_VVN to_TO determine_VVI whether_CSW the_AT engineered_JJ bronchioles_NN2 could_VM be_VBI exposed_VVN to_II radial_JJ distension_NN1 during_II the_AT contraction_NN1 phase_NN1 (_( days_NNT2 1-14_MCMC )_) ,_, these_DD2 bronchioles_NN2 were_VBDR not_XX pulsed_VVN with_IW physiologically_RR normal_JJ air_NN1 flow_NN1 after_II the_AT epithelial_JJ monolayer_NN1 was_VBDZ formed_VVN ._. 
The_AT bronchioles_NN2 were_VBDR pulsed_VVN with_IW near-static_JJ humidified_JJ air_NN1 with_IW pressure_NN1 not_XX greater_JJR than_CSN 4_MC mmHg._NNU 2.4_MC ._. 
Tissue-engineered_JJ bronchiole_NN1 phenotype_NN1 analyses_VVZ The_AT engineered_JJ bronchioles_NN2 were_VBDR sampled_VVN at_II 7_MC ,_, 14_MC ,_, 28_MC and_CC 60_MC days_NNT2 post-fabrication_JJ ._. 
Immunohistochemistry_NN1 was_VBDZ performed_VVN in_BCL21 order_BCL22 to_TO assess_VVI changes_NN2 in_II the_AT tissue_NN1 through_II protein_NN1 expression_NN1 ._. 
The_AT engineered_JJ bronchioles_NN2 were_VBDR fixed_VVN in_II 10%_NNU neutral_JJ buffered_JJ formalin_NN1 ,_, graded_VVD ethanol_NN1 dehydrated_VVD ,_, embedded_VVN in_II paraffin_NN1 and_CC then_RT sectioned_VVD (_( 5_MC &mu;_NULL m_NNO thick_JJ )_) ._. 
Fluorescent_JJ staining_NN1 was_VBDZ accomplished_VVN by_II blocking_VVG with_IW 2%_NNU goat_NN1 serum_NN1 for_IF 30_MC min_NNU at_II room_NN1 temperature_NN1 ,_, primary_JJ antibody_NN1 application_NN1 for_IF 1_MC1 h_ZZ1 ,_, three_MC 1_MC1 min_NNU washes_NN2 in_II PBS_NP1 and_CC then_RT application_NN1 of_IO the_AT secondary_JJ antibody_NN1 for_IF 30_MC min_NNU ._. 
ASM_NP1 cells_NN2 were_VBDR labelled_VVN using_VVG primary_JJ antibodies_NN2 against_II smooth_JJ muscle_NN1 &alpha;_NULL -actin_NN1 (_( 1_MC1 :_: 100_MC ;_; DAKO_NP1 ,_, M0851_FO )_) and_CC smooth_JJ muscle_NN1 myosin_NN1 heavy_JJ chain_NN1 (_( 1_MC1 :_: 100_MC ;_; DAKO_NP1 ,_, M3558_FO )_) with_IW Alexa_NP1 Fluor_NP1 488_MC (_( Invitrogen_NP1 ,_, A21121_FO )_) secondary_NN1 ,_, and_CC vimentin_NN1 (_( 1_MC1 :_: 200_MC ;_; DAKO_NP1 ,_, M7020_FO )_) with_IW Alexa_NP1 Fluor_NP1 594_MC secondary_NN1 (_( Invitrogen_NP1 ,_, A21135_FO )_) ._. 
Fibroblasts_NN2 were_VBDR labelled_VVN for_IF FITC_NP1 conjugated_VVD &beta;_NULL -tubulin_NN1 (_( 1_MC1 :_: 200_MC ;_; Sigma_NN1 ,_, T4026_FO )_) with_IW DAPI_NP1 (_( 1_MC1 :_: 2500_MC ;_; Invitrogen_NP1 ,_, D1306_FO )_) nuclear_JJ labelling_NN1 ._. 
The_AT epithelial_JJ cells_NN2 were_VBDR labelled_VVN for_IF cytokeratin-19_MC (_( 1_MC1 :_: 100_MC ;_; Sigma_NN1 ,_, C6930_FO )_) and_CC collagen_NN1 IV_MC (_( 1_MC1 :_: 500_MC ;_; Sigma_NN1 ,_, C1926_FO )_) with_IW TRITC_NP1 (_( 1_MC1 :_: 100_MC ;_; Sigma_NN1 ,_, T2659_FO )_) secondary_NN1 ;_; and_CC &beta;_NULL -tubulin_JJ and_CC mucin_NN1 (_( 1_MC1 :_: 100_MC ;_; Abcam_NP1 ,_, ab7874_FO )_) with_IW Alexa_NP1 Fluor_NP1 488_MC secondary_JJ and_CC DAPI_JJ nuclear_JJ stain_NN1 ._. 
The_AT fluorescently_RR labelled_VVN airway_NN1 cross-sections_NN2 were_VBDR documented_VVN using_VVG a_AT1 Zeiss_NN1 Axiovert_NN1 200_MC microscope_NN1 with_IW a_AT1 digital_JJ camera_NN1 ._. 
Bright-field_JJ microscopy_NN1 was_VBDZ performed_VVN for_IF macroscopic_JJ observations_NN2 ._. 
Haematoxylin_NN1 and_CC eosin_NN1 (_( H_ZZ1 &E);_NULL was_VBDZ used_VVN to_TO view_VVI collagen_NN1 fibres_NN2 and_CC the_AT structure_NN1 of_IO the_AT engineered_JJ bronchioles_NN2 ._. 
Apoptosis_NN1 and_CC proliferating_JJ cell_NN1 nuclear_JJ antigen_NN1 (_( PCNA_NP1 )_) were_VBDR performed_VVN on_II the_AT fibroblasts_NN2 for_IF cell_NN1 viability_NN1 and_CC proliferation_NN1 ._. 
For_IF apoptosis_NN1 ,_, an_AT1 ApopTAG_NN1 kit_NN1 (_( S7100_FO ,_, Chemicon_NP1 )_) was_VBDZ used_VVN ._. 
Proliferating_JJ cells_NN2 were_VBDR identified_VVN with_IW a_AT1 primary_JJ antibody_NN1 against_II PCNA_NP1 (_( 1_MC1 :_: 300_MC ;_; DAKO_NP1 )_) and_CC secondary_JJ antibody_NN1 conjugated_VVD with_IW horseradish_NN1 peroxidase_NN1 ,_, using_VVG a_AT1 VectaStain_JJ ABC_NN1 Elite_NN1 kit_NN1 and_CC DAB_NP1 (_( PK-6102_MC and_CC SK-4100_MC ,_, Vector_NN1 Laboratories_NN2 )_) ._. 
3_MC ._. 
Results_NN2 3.1_MC ._. 
Bioreactor_NN1 design_NN1 and_CC feature_NN1 optimization_NN1 The_AT biomaterials_NN2 for_IF construction_NN1 of_IO the_AT bioreactor_NN1 system_NN1 were_VBDR chosen_VVN to_TO promote_VVI ease_NN1 of_IO sterilization_NN1 and_CC low_JJ cost_NN1 ,_, minimize_VV0 the_AT quantity_NN1 of_IO biological_JJ agents_NN2 and_CC provide_VV0 long-term_JJ use_NN1 without_IW defects_NN2 ._. 
The_AT current_JJ version_NN1 of_IO the_AT bioreactor_NN1 is_VBZ very_RG userfriendly_RR (_( Figure_NN1 1_MC1 )_) ._. 
The_AT bioreactor_NN1 insert_NN1 can_VM be_VBI completely_RR disassembled_VVN for_IF cleaning_VVG or_CC part_NN1 replacement_NN1 and_CC easily_RR reassembled_VVN ._. 
The_AT flint_NN1 glass_NN1 chamber_NN1 is_VBZ economical_JJ and_CC can_VM also_RR be_VBI replaced_VVN ._. 
The_AT bioreactor_NN1 insert_NN1 is_VBZ assembled_VVN with_IW the_AT PTFE_NN1 moulds_NN2 in_II place_NN1 and_CC then_RT the_AT entire_JJ reactor_NN1 is_VBZ sterilized_VVN ._. 
This_DD1 minimizes_VVZ the_AT risk_NN1 of_IO contamination_NN1 ._. 
The_AT pump_NN1 system_NN1 affords_VVZ the_AT most_RGT unique_JJ attribute_NN1 of_IO the_AT bioreactor_NN1 system_NN1 (_( Figure_NN1 2a_FO )_) ._. 
It_PPH1 supplies_VVZ the_AT engineered_JJ bronchioles_NN2 with_IW pulsed_JJ air_NN1 to_TO mechanically_RR stimulate_VVI the_AT tissue_NN1 during_II the_AT contractile_JJ phase_NN1 and_CC distributes_VVZ humidified_JJ air_NN1 to_II the_AT epithelialized_JJ lumen_NN1 during_II the_AT differentiation_NN1 phase_NN1 (_( Figure_NN1 2b_FO )_) ._. 
The_AT peristaltic_JJ pump_NN1 was_VBDZ selected_VVN for_IF its_APPGE slow_JJ rotation_NN1 (_( 1-100_MCMC rpm_NNU )_) and_CC pump_NN1 head_NN1 selection_NN1 for_IF the_AT number_NN1 of_IO rollers_NN2 ._. 
The_AT PharMed_JJ L/S_ZZ1 18_MC tubing_NN1 has_VHZ a_AT1 7.9_MC mm_NNU inner_JJ diameter_NN1 (_( i.d._NNU )_) that_CST forces_VVZ a_AT1 large_JJ volume_NN1 of_IO air_NN1 into_II the_AT three-way_JJ splitter_NN1 and_CC through_II the_AT L/S_NP1 13_MC tubing_NN1 (_( 0.8_MC mm_NNU i.d_NNU ._. )_) ._. 
Pump_NN1 speed_NN1 was_VBDZ varied_VVN to_II pulse_NN1 the_AT thin-walled_JJ silicone_NN1 rubber_NN1 tubing_NN1 15_MC times/min_FU ._. 
The_AT roller_NN1 positions_NN2 in_II the_AT pump_NN1 head_NN1 and_CC the_AT diameter_NN1 of_IO the_AT tubing_NN1 allowed_VVN for_IF a_AT1 2%_NNU radial_JJ distension_NN1 ,_, which_DDQ increased_VVD the_AT diameter_NN1 of_IO the_AT bronchiole_NN1 by_II 60_MC &mu;_NULL m_NNU (_( from_II 3_MC mm_NNU to_II approximately_RR 3.06_MC mm_NNU )_) ._. 
3.2_MC ._. 
Determination_NN1 of_IO tissue_NN1 fabrication_NN1 parameters_NN2 Fabrication_NN1 of_IO the_AT tissue-engineered_JJ airways_NN2 involved_VVD parameter_NN1 optimization_NN1 of_IO matrix_NN1 concentration_NN1 ,_, fabrication_NN1 mechanics_NN2 ,_, cell_NN1 density_NN1 and_CC seeding_VVG methods_NN2 ,_, medium_JJ composition_NN1 and_CC cell_NN1 phenotype_NN1 ._. 
Proper_JJ proportioning_NN1 of_IO these_DD2 parameters_NN2 was_VBDZ necessary_JJ for_IF the_AT creation_NN1 of_IO a_AT1 stable_JJ engineered_JJ bronchiole_NN1 (_( Figure_NN1 4a_FO )_) ._. 
3.2.1_MC ._. 
Matrix_NN1 composition_NN1 The_AT matrix_NN1 concentration_NN1 was_VBDZ determined_VVN by_II modifying_VVG a_AT1 pre-existing_JJ protocol_NN1 (_( Agarwal_NP1 et_RA21 al._RA22 ,_, 2003_MC )_) ._. 
Utilizing_VVG 5_MC mg/ml_NNU collagen_NN1 for_IF the_AT cylindrical_JJ bronchiole_NN1 maintained_VVD a_AT1 tubular_JJ shape_NN1 after_II the_AT PTFEmould_NN1 was_VBDZ removed_VVN ._. 
Lower_JJR concentrations_NN2 of_IO collagen_NN1 tended_VVD to_TO deform_VVI or_CC tear_NN1 during_II mould_NN1 removal._NNU 3.2.2_MC ._. 
Cell_NN1 seeding_VVG density_NN1 Optimal_JJ seeding_JJ densities_NN2 were_VBDR determined_VVN to_TO achieve_VVI the_AT goal_NN1 of_IO a_AT1 monolayer_NN1 of_IO epithelial_JJ cells_NN2 and_CC a_AT1 multilayer_NN1 of_IO ASM_NN1 cells_NN2 ._. 
ASM_NP1 cell-seeding_JJ density_NN1 was_VBDZ initially_RR estimated_VVN by_II calculating_VVG the_AT exterior_JJ surface_NN1 area_NN1 of_IO the_AT engineered_JJ bronchiole_NN1 and_CC estimating_VVG the_AT number_NN1 of_IO ASM_NN1 cells_NN2 to_TO cover_VVI the_AT surface_NN1 with_IW three_MC layers_NN2 of_IO cells_NN2 ._. 
Seeding_VVG densities_NN2 of_IO 0.5_MC ,_, 1_MC1 ,_, 2_MC ,_, and_CC 3_MC million_NNO cells/bronchiole_FU were_VBDR investigated_VVN (_( n_ZZ1 =_FO 10_MC ,_, using_VVG three_MC lots_PN of_IO ASM_NN1 cells_NN2 )_) ._. 
ASM_NP1 cells_NN2 were_VBDR dynamically_RR seeded_VVN by_II suspending_VVG the_AT cells_NN2 in_II SmGM-2_MC ._. 
After_II 48_MC h_ZZ1 ,_, the_AT entire_JJ medium_NN1 volume_NN1 was_VBDZ removed_VVN and_CC a_AT1 cell_NN1 count_NN1 was_VBDZ done._NNU 3.4_MC ._. 
Tissue-engineered_JJ bronchiole_NN1 analyses_VVZ Preliminary_JJ trials_NN2 were_VBDR conducted_VVN at_II 5_MC ,_, 7_MC ,_, 10_MC ,_, 14_MC ,_, 21_MC and_CC 28_MC days_NNT2 to_TO determine_VVI the_AT fabrication_NN1 timeline_NN1 and_CC to_TO select_VVI sampling_NN1 times_NNT2 (_( n_ZZ1 =_FO 6_MC bronchioles_NN2 )_) ._. 
Subsequently_RR ,_, tissue_NN1 characteristics_NN2 were_VBDR determined_VVN at_II 7_MC ,_, 14_MC ,_, 28_MC and_CC 60_MC days_NNT2 ._. 
Each_DD1 bronchiole_NN1 comprises_VVZ 1_MC1 cmof_NN1 testable_JJ tissue_NN1 (_( Figure_NN1 4a_FO )_) ._. 
Immunohistochemistry_NP1 provided_VVD information_NN1 regarding_II protein_NN1 expression_NN1 and_CC location_NN1 (_( Figure_NN1 3_MC )_) ,_, to_TO evaluate_VVI the_AT phenotype_NN1 of_IO the_AT fibroblasts_NN2 ,_, ASM_NP1 cells_NN2 and_CC epithelial_JJ cells_NN2 ._. 
ASM_NP1 cell_NN1 phenotype_NN1 was_VBDZ evaluated_VVN by_II the_AT presence_NN1 of_IO contractile_JJ proteins_NN2 ._. 
The_AT intensity_NN1 of_IO vimentin_NN1 was_VBDZ more_RRR pronounced_VVN during_II the_AT first_MD 10_MC days_NNT2 (_( Figure_NN1 5a-c_FO )_) ,_, as_II31 opposed_II32 to_II33 day_NNT1 28_MC ._. 
Vimentin_NN1 expression_NN1 indicates_VVZ that_CST the_AT ASM_NN1 cells_NN2 are_VBR functioning_VVG synthetically_RR ._. 
As_II the_AT ASM_NN1 cells_NN2 establish_VV0 themselves_PPX2 on_II the_AT tissue_NN1 construct_VV0 ,_, the_AT cells_NN2 made_VVD the_AT transition_NN1 from_II the_AT synthetic_JJ to_II the_AT contractile_JJ phenotype_NN1 ,_, as_CSA shown_VVN by_II the_AT increased_JJ expression_NN1 of_IO smooth_JJ muscle_NN1 myosin_NN1 heavy_JJ chain_NN1 (_( MHC_NP1 )_) after_II day_NNT1 14_MC (_( Figure_NN1 5d-f_NNU )_) ._. 
Smooth_JJ muscle_NN1 &alpha;_NULL -actin_NN1 intensity_NN1 for_IF the_AT ASM_NN1 layer_NN1 remained_VVD constant_JJ from_II day_NNT1 7_MC to_II day_NNT1 14_MC ,_, but_CCB increased_VVD slightly_RR by_II day_NNT1 28_MC (_( Figure_NN1 5g-i_NNU )_) ._. 
The_AT small_JJ airway_NN1 epithelial_JJ cells_NN2 were_VBDR seeded_VVN in_II the_AT lumen_NN1 of_IO the_AT tissue_NN1 to_TO create_VVI an_AT1 epithelialized_JJ bronchiole_NN1 ._. 
The_AT cells_NN2 were_VBDR seeded_VVN on_II day_NNT1 14_MC ,_, which_DDQ was_VBDZ after_II the_AT construct_NN1 had_VHD finished_VVN contracting_JJ ._. 
When_CS epithelial_JJ cells_NN2 were_VBDR seeded_VVN 3-9_MCMC days_NNT2 post-fabrication_JJ ,_, the_AT epithelial_JJ cells_NN2 were_VBDR compressed_VVN and_CC shed_VVN from_II the_AT luminal_NN1 surface_NN1 ._. 
The_AT epithelial_JJ cells_NN2 stained_VVD positively_RR for_IF the_AT epithelial_JJ specific_JJ marker_NN1 cytokeratin-19_MC (_( Figure_NN1 6a_FO )_) ._. 
The_AT presence_NN1 of_IO K19_FO implies_VVZ that_DD1 differentiation_NN1 is_VBZ not_XX being_VBG inhibited_VVN by_II retinoic_JJ acid_NN1 ._. 
At_II low_JJ quiescent_JJ retinoic_JJ acid_NN1 concentrations_NN2 EGF_VV0 can_VM suppress_VVI mucin_NN1 ,_, while_CS higher_JJR concentrations_NN2 of_IO retinoic_JJ acid_NN1 override_NN1 EGF_NN1 and_CC increase_NN1 mucous_NN1 differentiation_NN1 (_( Denning_NP1 and_CC Verma_NP1 ,_, 2001_MC ;_; Gray_NP1 et_RA21 al._RA22 ,_, 2001_MC )_) ._. 
4_MC ._. 
Discussion_NN1 A_ZZ1 bioreactor_NN1 system_NN1 for_IF culturing_VVG bronchiole_NN1 tissues_NN2 ,_, which_DDQ comprise_VV0 three_MC different_JJ cell_NN1 types_NN2 ,_, has_VHZ been_VBN described_VVN ._. 
Human_JJ lung_NN1 fibroblasts_NN2 ,_, airway_NN1 smooth_JJ muscle_NN1 cells_NN2 and_CC bronchiole_NN1 epithelial_JJ cells_NN2 can_VM be_VBI grown_VVN in_II close_JJ proximity_NN1 to_II one_PPX121 another_PPX122 in_II the_AT same_DA culture_NN1 environment_NN1 and_CC exhibit_VV0 evidence_NN1 of_IO proper_JJ cellular_JJ behaviour_NN1 ._. 
Tissue_NN1 fabrication_NN1 protocols_NN2 have_VH0 been_VBN established_VVN and_CC engineered_JJ bronchiole_NN1 stability_NN1 has_VHZ been_VBN shown_VVN through_II phenotypic_JJ analyses_NN2 ,_, both_DB2 protein_NN1 expression_NN1 and_CC morphology_NN1 ,_, and_CC prolonged_JJ culture_NN1 times_NNT2 of_IO 60_MC days_NNT2 (_( Figure_NN1 3_MC )_) ._. 
The_AT stability_NN1 of_IO the_AT bronchiole_NN1 structures_NN2 and_CC their_APPGE cellular_JJ composition_NN1 allows_VVZ these_DD2 constructs_NN2 to_TO be_VBI used_VVN to_TO study_VVI cell-cell_JJ interactions_NN2 and_CC airway_NN1 remodelling_NN1 events_NN2 while_CS maintaining_VVG in_RR21 vivo_RR22 geometrical_JJ dimensions_NN2 and_CC relationships_NN2 ._. 
Currently_RR ,_, treatments_NN2 for_IF asthma_NN1 focus_NN1 on_II the_AT underlying_JJ airway_NN1 inflammatory_JJ and_CC constrictive_JJ processes_NN2 ._. 
Although_CS bronchodilators_NN2 ,_, anti-inflammatories_NN2 and_CC longacting_VVG &beta;_NULL 2_MC agonists_NN2 can_VM improve_VVI lung_NN1 function_NN1 ,_, these_DD2 medications_NN2 only_RR act_VV0 to_TO relieve_VVI ,_, prevent_VV0 and_CC control_NN1 symptoms_NN2 ,_, respectively_RR (_( Kumar_NP1 ,_, 2001_MC )_) ._. 
Neither_RR the_AT initiation_NN1 and_CC progression_NN1 of_IO airway_NN1 remodelling_NN1 nor_CC its_APPGE contribution_NN1 to_II irreversible_JJ airway_NN1 obstruction_NN1 in_II asthma_NN1 is_VBZ well_RR defined_VVN ._. 
Biopsies_NN2 almost_RR always_RR reveal_VV0 airway_NN1 remodelling_NN1 associated_VVN with_IW asthma_NN1 (_( Woodruff_NP1 and_CC Fahy_NP1 ,_, 2001_MC )_) ;_; however_RR ,_, it_PPH1 is_VBZ not_XX always_RR clinically_RR demonstrated_VVN (_( Beasley_NP1 et_RA21 al._RA22 ,_, 2002_MC )_) ._. 
The_AT severity_NN1 of_IO asthma_NN1 varies_VVZ so_RG greatly_RR that_DD1 with_IW its_APPGE onset_NN1 ,_, the_AT clinical_JJ evidence_NN1 of_IO remodelling_NN1 can_VM occur_VVI after_CS only_RR a_AT1 few_DA2 months_NNT2 or_CC as_RG much_DA1 as_CSA several_DA2 decades_NNT2 later_RRR (_( Beasley_NP1 et_RA21 al._RA22 ,_, 2002_MC )_) ._. 
A_AT1 tissue-engineered_JJ bronchiole_NN1 model_NN1 of_IO airway_NN1 remodelling_NN1 may_VM lead_VVI to_II understanding_VVG that_CST could_VM produce_VVI therapeutic_JJ agents_NN2 to_TO inhibit_VVI or_CC control_NN1 airway_NN1 remodelling_NN1 ._. 
