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Technical Forum

Biophysical Analysis of Dystrophic and Osteogenic Models of Valvular Calcification

[+] Author and Article Information
Joseph Chen

Department of Biomedical Engineering,
Vanderbilt University,
2213 Garland Avenue,
Nashville, TN 37232-0493
e-mail: joseph.chen@vanderbilt.edu

Jon R. Peacock

Department of Biomedical Engineering,
Vanderbilt University,
2213 Garland Avenue,
Nashville, TN 37232-0493
e-mail: jon.r.peacock@vanderbilt.edu

Janelle Branch

Department of Civil
and Environmental Engineering,
Vanderbilt University,
400 24th Avenue South,
Nashville, TN 37212
e-mail: janelle.l.branch@vanderbilt.edu

W. David Merryman

Department of Biomedical Engineering,
Vanderbilt University,
2213 Garland Avenue,
Nashville, TN 37232-0493
e-mail: david.merryman@vanderbilt.edu

Manuscript received August 25, 2014; final manuscript received November 13, 2014; published online January 26, 2015. Editor: Victor H. Barocas.

J Biomech Eng 137(2), 020903 (Feb 01, 2015) (6 pages) Paper No: BIO-14-1418; doi: 10.1115/1.4029115 History: Received August 25, 2014; Revised November 13, 2014; Online January 26, 2015

Calcific aortic valve disease (CAVD) is a significant cardiovascular disorder characterized by the formation of calcific nodules (CN) on the valve. In vitro assays studying the formation of these nodules were developed and have led to many significant mechanistic findings; however, the biophysical properties of CNs have not been clearly defined. A thorough analysis of dystrophic and osteogenic nodules utilizing scanning electron microscopy (SEM), energy dispersive spectrometry (EDS), and atomic force microscopy (AFM) was conducted to describe calcific nodule properties and provide a link between calcific nodule morphogenesis in vitro and in vivo. Unique nodule properties were observed for dystrophic and osteogenic nodules, highlighting the distinct mechanisms occurring in valvular calcification.

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Figures

Grahic Jump Location
Fig. 1

Dystrophic and osteogenic CNs exhibit differences in morphology and cell viability. (a) and (d) Dystrophic CNs and Osteogenic CNs stain positive for Alizarin Red. (b) and (e) Bright field images reveal morphological differences between dystrophic and osteogenic nodules. (c) Annexin/PI stain show strong uptake of PI stain in the nodule center with an Annexin V positive ring around dystrophic nodules while Calcein AM/PI stain show living cells within the osteogenic nodules and no uptake of PI stain. Scalebar = 100 μm.

Grahic Jump Location
Fig. 2

EDS reveals significant Ca and P content in CNs. (a) and (d) Backscattered SEM image of CNs with annotations showing regions of the nodules scanned with EDS. A black circle represents an EDS scan on cells; a blue circle represents an EDS scan on the nodule body; and a yellow X marks a point scanned to represent an area of calcification. (b) Representative EDS spectrum of a dystrophic nodule scanned at the yellow X in A. Ca and P scans show a weight percent of 2.8 and 1.5, respectively. (c) Relative percent weight concentration shows significantly higher amounts of Ca and P in regions of calcification. (e) Representative EDS spectrum of an osteogenic nodule scanned at the yellow X in D shows a weight percent of 10.1 and 5.3 for Ca and P, respectively. (f) Relative percent weight concentration shows dramatically higher levels of Ca and P in calcification regions. * indicates p ≤ 0.05

Grahic Jump Location
Fig. 3

Biomechanical analysis reveals nodule heterogeneity. (a) Representative modulus scan distributions for cells and nodules. Inset graph shows average median modulus and reveals no significant difference between sample groups; however, osteogenic nodules had bimodal distribution, which likely skews downward their overall stiffness. (b), (e), and (h) Regions scanned on AVIC, dystrophic, and osteogenic nodules via AFM are represented by the black dotted box. AVIC and osteogenic nodule body scans display consistent homogeneous modulus scans (c) and (i) while dystrophic nodules exhibit heterogeneous modulus scans (f). Osteogenic nodules contain dramatically stiffer regions resembling spherical calcifications on the surface of the nodule body revealing the heterogeneous make up of osteogenic nodules. (d), (g), (j), and (l) 3D topographical maps for AVIC, dystrophic, and osteogenic nodules were overlaid with the modulus map.

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