ONLINE APPENDIX
TISSUE HARVEST, PREPARATION, AND ANALYSIS. At the completion of a study
period, each animal was euthanized using Fatal-Plus solution (phenobarbital 390 mg/ml,
propylene glycol 0.01 mg/ml, ethyl alcohol 0.29 mg/ml, benzyl alcohol 0.2 mg/ml) 1 ml/10 lbs.
Tricuspid valve tissue was collected en bloc to include the papillary muscles as well as both
atrial and ventricular tissue around the native annulus. Explanted valve tissue was inspected for
any gross thrombus, leaflet defects, or calcification. Each valve was prepared in Dulbecco's
phosphate buffered saline (Hyclone, GE Healthcare, Little Chalfont, Buckinghamshire, United
Kingdom) and supplemented with protease inhibitor (1:100, Sigma-Aldrich Co., LLC, St. Louis,
Missouri) until it was divided into 3 pieces, each with a papillary attachment: 1 fresh piece was
used for biomechanical testing, 1 fixed piece for histopathologic evaluation, and the third piece
was preserved for electron microscopy, as previously described (1, 2). The same leaflet was used
for the same experiments each time to maintain consistency. Leaflets were sectioned at various
portions of the valve including annulus, papillary attachments, and commissure; however, all the
comparisons were made between the same areas of the valve leaflet.
Histologic evaluation included hematoxylin and eosin (H&E) to show cellular
infiltration, alizarin red to show mineralization, and Movat’s pentachrome to show ECM
organization. Immunohistochemistry was performed using antibodies directed against CD31
(1:50; Abcam plc, Cambridge, United Kingdom) to identify endothelial cells, CD 68 (1:100,
Dako, Carpinteria, California) to identify macrophages, fetal liver kinase 1/vascular endothelial
growth factor receptor 2 (FLK/VEGFR2; 1:500; 55B11) to identify cell signaling, and alpha
smooth muscle actin (SMA, 1:500, Sigma) to identify resident valve interstitial cells (VIC).
Ultrastructure analysis was performed using a Hitachi 7600 (Hitachi High Technologies
America, Inc., Schaumburg, Illinois) electron microscope. In order to visualize collagens and
elastic fibers, sections were counter stained with aqueous solutions of 5% tannic acid followed
by 1% uranyl acetate, and then counterstained with lead citrate, as described previously (3).
Mechanical testing of valve tissue was performed using micropipette aspiration as previously
described (2). This was done at 3 distinct points on the leaflet (close to annulus, equidistance
from annulus and papillary attachment and close to papillary attachment). All these points were
homogenous across the biomechanical experiments. The valve surface was accessed from the
ventricular side using a micromanipulator and glass micropipette drawn to an inner diameter of
100 μm; upon contact, aspiration pressure was applied in a stepwise manner (4). Videos of tissue
aspiration were recorded and the aspiration length and radius were measured. Linear regression
was used to curve fit the data. Young’s modulus (E) as a measure of tissue stiffness was
determined using a half-space model equation (5,6). One-way analysis of variance tests were
used to determine effects on Young’s modulus by valve type (SIS-ECM, NV), valve leaflet
region (proximal, distal), and age (3 months post-implant, 8 months post-implant).
References:
1. Hinton RB Jr, Lincoln J, Deutsch GH, et al. Extracellular matric remodeling and
organization in developing and diseased aortic valves. Circ Res 2006;98:1431-8.
2. Krishnamurthy VK, Guilak F, Narmoneva DA, Hinton RB. Regional structure-function
relationships in mouse aortic valve tissue. J Biomech 2011;44:77-83.
3. Hinton RB, Adelman-Brown J, Witt S, et al. Elastin haploinsufficiency results in
progressive aortic valve malformation and latent valve disease in a mouse model. Circ
Res 2010;107:549-57.
4. Jones WR, Ting-Beall HP, Lee GM, Kelley SS, Hochmuth RM, Guilak F. Alterations in
the Young’s modulus and volumetric properties of chondrocytes isolated from normal
and osteoarthritic human cartilage. J Biomech 1999;32:119-27.
5. Guilak F, Alexopoulos LG, Haider MA, Ting-Beall HP, Setton LA. Zonal uniformity in
mechanical properties of the chondrocyte pericellular matrix: micropipette aspiration of
canine chondrons isolated by cartilage homogenization. Ann Biomed Eng 2005;33:131218.
6. Theret DP, Levesque MJ, Sato M, Nerem RM, Wheeler LT. The application of a
homogenous half-space model in the analysis of endothelial cell micropipette
measurements. J Biomec Eng 1988;110:190-9.