Nanoindentation of histological specimens: Mapping the elastic properties of soft tissues

Abstract

Although alterations in the gross mechanical properties of dynamic and compliant tissues have a major impact on human health and morbidity, there are no well-established techniques to characterize the micromechanical properties of tissues such as blood vessels and lungs. We have used nanoindentation to spatially map the micromechanical properties of 5-mum-thick sections of ferret aorta and vena cava and to relate these mechanical properties to the histological distribution of fluorescent elastic fibers. To decouple the effect of the glass substrate on our analysis of the nanoindentation data, we have used the extended Oliver and Pharr method. The elastic modulus of the aorta decreased progressively from 35 MPa in the adventitial (outermost) layer to 8 MPa at the intimal (innermost) layer. In contrast, the vena cava was relatively stiff, with an elastic modulus >30 MPa in both the extracellular matrix-rich adventitial and intimal regions of the vessel. The central, highly cellularized, medial layer of the vena cava, however, had an invariant elastic modulus of ~20 MPa. In extracellular matrix-rich regions of the tissue, the elastic modulus, as determined by nanoindentation, was inversely correlated with elastic fiber density. Thus, we show it is possible to distinguish and spatially resolve differences in the micromechanical properties of large arteries and veins, which are related to the tissue microstructure.

FIG. 1

(a) Typical indent separations are represented by the grid. The distance between the points is 15 μm. (b) Typical load-displacement curve with the portion of the unloading curve used for the effective indenter fit indicated on the plot.

FIG. 2

Elastic modulus, determined by nanoindentation, and histological tissue organization visualized by fluorescence microscopy of H&E stained (a) ferret aorta and (b) vena cava. The surface of the adventitia (outermost) region of the vessels is located at position zero. Error bars represent standard deviation.

FIG. 3

Fluorescent images showing (a) H&E stained ferret aorta section. (b) H&E stained ferret vena cava section. (c) Unstained ferret aorta section showing elastic fiber distribution due to elastin autofluorescence. (d) Unstained ferret vena cava section showing elastic fiber distribution due to elastin autofluorescence. Note the darker region in the central portion of the vena cava images, which indicates the presence of little elastin. Scale marker represents 100 μm in each image. The adventitial layer is leftmost in each image with the intimal layer rightmost.

FIG. 4

Typical line profile showing elastic fiber fluorescence in (a) H&E stained ferret aorta section and (b) H&E stained ferret vena cava. Maximal pixel intensity was observed at the intimal surface (linear regression analysis is shown with the red line) for the aorta sample whereas for the vena cava pixel intensity was greatest at the adventitial and intimal surfaces. Mean pixel intensity binned at 15-μm intervals for (c) aorta and (d) vena cava tissue sections. Error bars indicate standard error of the mean.

FIG. 5

Correlation between pixel intensity and elastic modulus. Note that the elastin-rich adventitial and intimal layers of the vena cava fit the regression line, whereas the cellularized medial layer has a lower elastic modulus and elastic fiber density (fluorescence signal).

FIG. 6

Elastic moduli of aorta and ECM components estimated at macroscopic, microscopic, and nanoscopic (molecular) length scales. With the exception of elastin, the component ECM molecules are stiffer than the microscopic tissue structures tested in this study. These microscopic structures are, in turn, stiffer than macroscopic regions of the whole tissue. The references for the values in this figure are taken from: 1. Gozna et al., 2. Reddy., 3. Laurent et al., 4 and 5. values from this study, 6. Gosline (elastin), 7. Sherratt et al. (fibrillin microfibrils), 8. Gosline et al. (fibrillar collagen), and 9. Yang et al. (single collagen fibrils).