Introduction 6 17 33 26 26 19 16 , 30 1 , 15 , 23 ex vivo 13 −2 4 31 29 , 31 , 32 15 8 in vivo ex vivo Material and Methods Tissue Harvest Fresh porcine aortic valves were obtained from a local abattoir (Holifield Farms, Covington, GA) following on-site dissection of the hearts within 30 min of slaughter. The valves were then transported to the laboratory in sterile, ice-cold Dulbecco’s Phosphate Buffered Saline (DPBS; Sigma, St. Louis, MO). 1 10 −1 2 25 , 26 in vivo. 25 −1 Figure 1. Preparation of aortic valve leaflets for the experiment. Three sections were cut out in the circumferential direction from the base, belly and tip regions of the aortic valve leaflet. These sections were randomly chosen as stretched, fresh and static leaflet groups. Fresh sections were processed immediately. Static control sections were incubated for 48 h in DMEM. Stretched sections were stretched for 48 h.  Figure 2. 25 Ex vivo  Stretch Bioreactor System and Validation 2 et al 10 2 12 In order to determine if the stretch bioreactor induced a shear stress on the leaflet surface due to the relative motion of the leaflet with respect to the surrounding media, a computational fluid dynamics (CFD) model was designed, validated and used to predict the leaflet wall shear stress under the imposed loading curve in the stretch bioreactor. −1 −1 2 Tissue Processing TM TM TM Valve leaflet samples from additional experiments were isolated for analysis of tissue morphology and cell phenotype. For histological and immunohistochemical analyses, leaflet sections were fixed in 10% neutral buffered formalin (Fisher Scientific, Suwanee, GA) for 24 h, saturated in 70% ethanol, processed in a tissue processor (Shandon Pathcenter enclosed Tissue Processor) in descending grades of ethanol, embedded in paraffin and cut into 5 μm sections. Morphological Characterization Routine Hematoxylin and Eosin (H & E) stain was used to examine gross tissue structure and indicate any changes in the structural compositions of the leaflets. H & E staining was done on an autostainer (Leica, Vashaw Scientific, Norcross, GA). Picrosirius red staining was performed to examine collagen fiber structure and morphology. The protocol was as follows. Deparaffinized slides were incubated in Weigert’s hematoxylin (Sigma) followed by picrosirius red solution for one hour. The slides were then washed in acidified water, three changes of 100% ethanol, and xylene before being coverslipped. These slides were viewed at 100× using a Nikon imaging microscope (Nikon Inc., Melville, NY). A Q-imaging camera, Retiga 1300C (I-Cube, Glen Burnie, MD) and its bundled software Q-Capture Pro was used to acquire images of the tissue sections. H & E images were recorded using normal white light while picrosirius red images were recorded with circularly polarized light. Characterization of Cell Phenotype −1 Image Analysis 2 Immunoblotting Analysis −1 Analysis of Tissue Culture Medium TM Statistical Analysis p t p Results Validation of Stretch Bioreactor p n −2 −2 28 1 p n p n p n p n p n 3 Figure 3. p n  p n −1 Cell Morphology and Phenotype 4 p n 5 Figure 4. (a) Cyclic stretch did not damage tissue structure and morphology. H & E stained images of fresh, static and stretched aortic valve leaflets are depicted here. Cytoplasm was stained pink and cell nuclei were stained blue. The representative images show the three-layered morphology of the leaflets was intact in all three groups (F – fibrosa, S – spongiosa, V – ventricularis). (b) Cyclic stretch maintained native collagen architecture. Picrosirius red images of fresh, static and stretched aortic valve leaflets are shown. Mature collagen fibers were stained orange-red. Layered collagen morphology of leaflets was observed and crimp was preserved in stretched leaflets (F – fibrosa). (c) Cyclic stretch increased α-smooth muscle actin (α-SMA)-positive cells in the ventricularis side of the aortic valve leaflets. Fresh, static and stretched leaflets were examined by α-SMA IHC. Actin was stained red and cell nuclei were counterstained blue. Increased expression of α-SMA was observed in stretched leaflets, and α-SMA was reduced in static leaflets (V – ventricularis).  Figure 5. p n  4 5 p n p n 4 2 5 p n p n 6 p n p n Figure 6. p n  Discussion Effect of Cyclic Stretch on ECM Components ex vivo It was revealed that the collagen content of the aortic valve leaflets stretched to 15% was increased when compared to fresh and static control leaflets, while sGAG content was decreased in stretched leaflets compared to fresh leaflets. Furthermore, elastin content was comparable between the stretched and fresh leaflets. This suggests that exposure to isolated effects of circumferential cyclic stretch at physiological levels has altered the extracellular matrix composition, consequently, altering the valve remodeling. 18 , 26 5 et al 14 26 7 et al 11 13 27 5 Effect on Cell Phenotype 24 2 21 , 22 13 , 29 , 31 5 5 6 13 et al 20 3 ,  9 Conclusion ex vivo