Calcification of Various Bioprosthetic Materials in Rats: Is It Really Different?

Abstract

The causes of heart valve bioprosthetic calcification are still not clear. In this paper, we compared the calcification in the porcine aorta (Ao) and the bovine jugular vein (Ve) walls, as well as the bovine pericardium (Pe). Biomaterials were crosslinked with glutaraldehyde (GA) and diepoxide (DE), after which they were implanted subcutaneously in young rats for 10, 20, and 30 days. Collagen, elastin, and fibrillin were visualized in non-implanted samples. Atomic absorption spectroscopy, histological methods, scanning electron microscopy, and Fourier-transform infrared spectroscopy were used to study the dynamics of calcification. By the 30th day, calcium accumulated most intensively in the collagen fibers of the GA-Pe. In elastin-rich materials, calcium deposits were associated with elastin fibers and localized differences in the walls of Ao and Ve. The DE-Pe did not calcify at all for 30 days. Alkaline phosphatase does not affect calcification since it was not found in the implant tissue. Fibrillin surrounds elastin fibers in the Ao and Ve, but its involvement in calcification is questionable. In the subcutaneous space of young rats, which are used to model the implants' calcification, the content of phosphorus was five times higher than in aging animals. We hypothesize that the centers of calcium phosphate nucleation are the positively charged nitrogen of the pyridinium rings, which is the main one in fresh elastin and appears in collagen as a result of GA preservation. Nucleation can be significantly accelerated at high concentrations of phosphorus in biological fluids. The hypothesis needs further experimental confirmation.

Keywords: bioprosthetic materials; calcification; collagen; cross-linking; elastin.

Figure 1

Different fresh biomaterial tissue structures: aortic wall (A), vein wall (B), and pericardium (C). Picro Mallory staining (A–C): collagen is blue, elastin is yellow, and smooth muscle cells are red. Scale bars are 100 μm. Calcium content dynamics in these biomaterials are preserved with GA and DE (D).

Figure 2

Calcium phosphate deposits in biomaterials 10, 20, and 30 days after implantation. Arrows show direction to surfaces: In—intimal; A—adventitial; S—serous; F—fibrous. No sign of calcification in DE-Pe. Scale bars 550 μm.

Figure 3

EDS analyses of the implants 10 days after implantation. SEM images in back-scattering electrons and EDS spectra of DE-Ve surface (left column, scale bar 80 μm) and cross-section (right column, scale bar 40 μm). Left spectrum 1 belongs to the external particle; spectrum 10 is hypothetically obtained from a carbonate-apatite deposit with a high content of CaCO₃; all the other spectra belong to calcium phosphate with different Ca/P ratios.

Figure 4

Results of EDS analysis at 10, 20, and 30 days after implantation. Ca and P content (normalized %) and Ca/P atomic ratios in electron-dense conglomerates located on the surface and in deep layers of tissue samples. Color labeling of biomaterials is similar to Figure 1.

Figure 5

High-resolution SEM images of the biomaterials were explanted on post-implantation days 10 and 30. Elastin (1) and collagen (2) fibers calcification in 10 and 30 days. Collagen fibers completely retain the 3D structure, being totally replaced by calcium phosphates. The increase in the mass of HAP crystals and the formation of large HAP conglomerates after 30 days. Scale bars 500 nm.

Figure 6

FTIR difference spectra as a result of subtraction of the non-implanted biomaterials spectra from spectra of implants after 10 (blue line), 20 (pink), and 30 (red) days of implantation. Bottom—spectra of calcium carbonate (yellow), octacalcium phosphate (ocp, green), and hydroxyapatite (hap, black).

Figure 7

Histochemical visualization of ALP (deep blue spots) localized in connective tissue capsules but not in implants’ tissues. Light blue color marks implant tissues; deep blue lines show connective tissue capsules. Scale bars 200 μm.

Figure 8

Fibrillin in porcine aortic (left column) and bovine jugular vein (right column) walls. Fibrillin is found in elastin fibers throughout the entire wall thickness (top row) and envelops the fibers outside (bottom row). Scale bars are 50 μm.

Figure 9

Ca (red) and P (blue) mapping of dry subcutaneous fascia excised from adult (A) and young (B) rats; EDS spectra of subsequent maps (C,D); calcium is visualized with red dots, phosphorus with blue ones. Electron-dense particles on the fascial surfaces: adult (E) and young (F) rats; spectra corresponding to particles 1 and 2 of each image (G,H). Scale bars 100 μm.