Characterizing nanoscale topography of the aortic heart valve basement membrane for tissue engineering heart valve scaffold design

Abstract

A fully effective prosthetic heart valve has not yet been developed. A successful tissue-engineered valve prosthetic must contain a scaffold that fully supports valve endothelial cell function. Recently, topographic features of scaffolds have been shown to influence the behavior of a variety of cell types and should be considered in rational scaffold design and fabrication. The basement membrane of the aortic valve endothelium provides important parameters for tissue engineering scaffold design. This study presents a quantitative characterization of the topographic features of the native aortic valve endothelial basement membrane; topographical features were measured, and quantitative data were generated using scanning electron microscopy (SEM), atomic force microscopy (AFM), transmission electron microscopy (TEM), and light microscopy. Optimal conditions for basement membrane isolation were established. Histological, immunohistochemical, and TEM analyses following decellularization confirmed basement membrane integrity. SEM and AFM photomicrographs of isolated basement membrane were captured and quantitatively analyzed. The basement membrane of the aortic valve has a rich, felt-like, 3-D nanoscale topography, consisting of pores, fibers, and elevations. All features measured were in the sub-100 nm range. No statistical difference was found between the fibrosal and ventricular surfaces of the cusp. These data provide a rational starting point for the design of extracellular scaffolds with nanoscale topographic features that mimic those found in the native aortic heart valve basement membrane.

FIG. 1

Schematic of the steps in characterizing the basement membrane topography of the porcine aortic heart valve.

FIG. 2

Histological sections of control (a) and de-endothelialized (b) cusps stained with H&E. There is a noticeable absence of endothelial cells on the surface of the de-endothelialized tissue. Cells on the surface of the control are indicated by arrows. (magnification × 200)

FIG. 3

Sections of control (a) and de-endothelialized (b) cusps stained with Alcian blue with periodic acid Schiff. The basement membrane is intact in both cases, as shown by the continuous dark line (indicated by arrows). Note the beaded appearance of the endothelial cells over the basement membrane in (a). (magnification × 200)

FIG. 4

(a) SEM micrographs showing the three layers of the aortic valve cusp; some cells are remaining following EDTA treatment, basement membrane, and the underlying extracellular matrix. (b) A higher magnification SEM image of the basement membrane, typical of those used for stereo pairs.

FIG. 5

A tapping mode atomic force microscopy image of 1 μm² area of basement membrane.