Aim of this study was to investigate heart valve calcification process by different biomineralogical techniques to provide morphological and chemical features of the ectopic deposit extracted from patients with severe mitral and aortic valve stenosis, to better evaluate this pathological process. Polarized light microscopy and scanning electron microscopy analyses brought to light the presence of nodular and massive mineralization forms characterized by different levels of calcification, as well as the presence of submicrometric calcified globular cluster, micrometric cavities containing disorganized tissue structures, and submillimeter pockets formed by organic fibers very similar to amyloid formations. Electron microprobe analyses showed variable concentrations of Ca and P within each deposit and the highest content of Ca and P within calcified tricuspid aortic valves, while powder X-ray diffraction analyses indicated in the nanometer range the dimension of the pathological bioapatite crystals. These findings indicated the presence of highly heterogeneous deposits within heart valve tissues and suggested a progressive maturation process with continuous changes in the composition of the valvular tissue, similar to the multistep formation process of bone tissue. Moreover the micrometric cavities represent structural stages of the valve tissue that immediately precedes the formation of heavily mineralized deposits such as bone-like nodules.
Several physiological and pathological mineralized deposits are present in the human body. The major mineralized products (enamel, dentin, and bone) are formed by well-controlled processes of mineralization [
The calcified deposits are composed of mineral and organic components and represent inorganic phases produced by the activity of living organisms, better known as biominerals [
Calcified aortic valves (tricuspid type,
Patients characteristics, experimental analyses performed (SEM-EDS, scanning electron microscopy with energy dispersive spectrometry; PXRD, powder X-ray diffraction; EMP, electron microprobe analysis; PLM, polarized light microscopy), and average content of calcium (Ca) and phosphorus (P) in different types of calcified valve. Ca and P contents are expressed as oxide-weight percent values and as atomic Ca : P ratio. Values are mean ± SD.
Characteristic | Overall |
Tricuspid aortic valve |
Bicuspid aortic valve |
Mitral valve |
---|---|---|---|---|
Age, y | 72.4 ± 10 | 74.5 ± 7.8 | 55 ± 15 | 69 ± 9 |
Males | 25 (69.4%) | 20 (69%) | 3 (100%) | 2 (50%) |
SEM-EDS | 20 (55.5%) | 13 (45%) | 3 (100%) | 4 (100%) |
PXRD | 27 (75%) | 23 (79%) | 2 (66%) | 2 (50%) |
EMP | 5 (14%) | 2 (7%) | 2 (66%) | 1 (25%) |
PLM | 5 (14%) | 2 (7%) | 2 (66%) | 1 (25%) |
CaO | 40 ± 5.7 | 44.3 ± 4.3 | 35.3 ± 8.2 | 40.7 ± 3.4 |
P2O5 | 28.1 ± 4.2 | 30.85 ± 3.3 | 25 ± 6 | 28.85 ± 2.3 |
Ca : P | 1.8 ± 0.08 | 1.82 ± 0.05 | 1.8 ± 0.105 | 1.78 ± 0.09 |
As the dehydration treatment of the samples gave to the calcified valves the consistency of a soft rock (Figure
Macroscopic images of a large mineralized deposit on a valve leaflet after the mineralogical preparation (dehydration in absolute alcohol and exposure to UV radiation) of the sample. The white deposit represents the biomineral formation grown within the organic tissue (in yellow) of the human heart valve.
PTS were studied with an Olympus BX60 microscope with transmitted- and reflected-polarized light (PLM). Cross polarized light was used to test birefringence of samples. PTS were then coated with a thin layer of graphite, sputter-coated with an Edwards E306A High Vacuum System, and analyzed with a Cameca SX 50 EMP equipped with four wavelength dispersive spectrometers (WDS) and one energy dispersive spectrometer (EDS), at a beam current of 15.0 nA and an accelerating voltage of 15 kV. The samples were investigated using a defocused beam, as described by Ballirano et al. [
A FEI Quanta 400 MK2 scanning electron microscope (SEM) was used for morphological investigations. Samples were fixed on a graphite stub with double-faced tape and investigated in high- and low-vacuum modes. In order to monitor that no artifacts associated with sputter coating were formed, morphological investigations were conducted on coated and uncoated samples. Samples were coated with a thin layer of graphite, as for the PTS, only for high-vacuum mode analysis. To investigate the internal structure of pathological mineralized deposits, some samples were fractured to allow analysis of freshly broken surfaces. X-ray microanalysis was carried out with an EDS Genesis XM2, manufactured by EDAX, and several measurements were taken for each sample. Four PTS were investigated by SEM as well, and X-ray maps of individual chemical elements were acquired at a beam energy of 15 kV using the K
Powder X-ray diffraction (PXRD) patterns were collected on a parallel X-ray beam Bruker AXS D8 FOCUS automated diffractometer, equipped with the parabolically shaped Göbel Mirror, using the Cu K
The crystallite size as expression of mean diameter of the coherent-scattering domains (
Computations were performed with SPSS 19 (IBM, Armonk, NY, USA). Continuous variables are reported as mean ± standard deviation and categorical variables as
Under PLM, the ectopic deposit within heart valve tissue appeared as nodules of variable dimensions from 500
PLM analysis of valve nodules. Appearance of nodules of variable dimensions embedded in the valve tissue of a mineralized bicuspid aortic valve (Tv20ab) under transmitted PLM (a, c). (b, d) Under XPL the presence of extinguished zones is indicative of amorphous component. Zones having the typical first-order gray interference color are suggestive for apatite within the nodule. Contact between organic tissue and the epoxy resin was characterized by the presence of anomalous colors due to reaction between the two (b).
Backscattered electron (BSE) images on PTS, obtained by either EMP or SEM-EDS analyses, confirmed the presence of millimeter nodular formations completely embedded in the heart valve tissue that instead appeared to be not mineralized. Within the millimeter nodules we observed areas with different brightness showing a grayscale range indicative of differences in mean atomic number (Figure
BSE image and EDS analyses of PTS (sample Tv20ab). (a) Millimeter nodule embedded in the heart valve tissue. The nodule appears to be formed by a massive mineral formation with a low brightness in which smaller nodules with higher brightness are visible. The EDS spectra of these zones with different brightness are given in panels (b) and (c); these show that Ca, P, and O are the main chemical elements that made up the heart valve mineralized tissues, confirming their calcium phosphate nature. Mg, Na, and S were also detected; the massive mineral formation characterized by a lower brightness (spot a) than micrometric nodules (spot b) had a greater content of carbon (C) and sulphur (S) and represents a partially mineralized tissue while the micrometric nodules represent fully mineralized areas. The darkest zones represent the organic matrix not mineralized (lowest mean atomic number), while the black zones represent voids in the embedding material.
BSE-SEM analyses of PTS (sample Tv20ab). (a) High magnification image of a micrometric nodule formed by two different stages of mineralization. The lower zone of the nodule having lower brightness had greater content of C and S (spot a) (b) than the upper zone of the nodule (spot b) (c). This indicates a partially mineralized organic matrix for the lower zone of the nodule and a fully mineralized tissue for the upper zone.
Elemental mapping confirmed the presence of high concentrations of Ca and P within the brightest zones of Figure
Elemental maps (sample Tv20ab). (a) BSE-SEM image of the area used to generate elemental maps. (b) Distribution of Ca. (c) Distribution of P. (d) Distribution of S. The arrows in panels (b) and (c) indicate completely mineralized areas characterized by very high concentrations of Ca and P. The arrows in panel (d) indicate high concentrations of S associated only with the extracellular organic matrix surrounding the pathological deposit.
Investigations on PTS brought to light also the presence of circular cavities (Figure
BSE-SEM analysis of circular cavities (sample Tv16 m). (a) Low magnification image of a PTS. The brightest areas represent fully mineralized zones. On the left, a massive and homogeneous deposit is visible, while in the center micrometric circular cavities are visible. (b, c) Magnified view of the small cavities indicated in panel (a); fragments of disorganized and mineralized collagen are visible within the cavities.
(a) SEM image (sample Tv12a) of a pocket filled only with proteins,
At high magnifications SEM analyses revealed the presence of coalescent spherical particles (Figure
SEM analysis of pathological biomineralization morphology. (a) Agglomeration of variably sized (micrometer range) calcified nanoparticles, indicated by the arrows (12000x, mitral valve sample). (b) Agglomeration of spherical calcified nanoparticles in a framework of organic filaments (bicuspid aortic valve sample), formed by extracellular matrix proteins. (c) Magnified view (25000x) of individual spherical nanoparticle along a mineralized filament, indicated by the arrow (mitral valve sample). (d) Agglomeration of spherical calcified nanoparticles in the nanometer range at 40000x; the smallest measured nanoparticle was 262.7 nm in diameter (bicuspid aortic valve sample).
High-resolution images also revealed a strict relationship between fine collagen fibers and spherical particles (Figure
EMP data indicated that the chemical variability of each sample was great; Ca and P concentrations, as well as the atomic Ca : P ratio, changed significantly from point to point. In addition, there was substantial chemical variability also in the different types of heart valve studied. Calcified tricuspid aortic valves had the highest and most uniformly distributed Ca and P concentrations and a high Ca : P ratio; bicuspid aortic valves had the lowest Ca and P concentrations, with less uniform distribution of Ca and P within the calcified deposits; the mitral valve analyzed presented Ca and P concentrations similar to those in tricuspid aortic valves. The chemical variability of Ca and P is given in Figure
Chemical variability of Ca and P for three different calcified valves. The concentrations of Ca and P indicated in the histograms represent the concentrations determined in oxide-weight percent (CaO, P2O5) for fifteen punctual analyses (indicated as spot) taken within the same calcified deposit. Histograms showed that bicuspid aortic valves (b) were characterized by a lower content of Ca and P and by greater variations in Ca : P concentrations. In contrast, mitral (c) and tricuspid aortic valves (a) were characterized by a higher content of Ca and P and less variation in Ca and P concentration.
Comparison between two different types of calcified aortic valve. Histograms showed that the tricuspid aortic valve had a major content of Ca and P (expressed as mean weight %) compared to bicuspid aortic valve. The mean value was calculated on thirty spots taken within each valve. The vertical bars represent the standard deviation.
Despite the variable concentrations of Ca and P in each sample, the atomic Ca : P ratio for each calcified valve was relatively uniform (Table
X-ray diffraction patterns of the untreated powders showed a diffuse background due to organic component (Figure
Experimental XRD patterns relative to three different types of mineralized human cardiac valves. (a) X-ray diffraction pattern of the untreated powders. (b) X-ray powder diffraction pattern of the biomineralization after the enzymatic attack. The main characteristic peaks match the hydroxylapatite pattern numbers 9–432 from the ICDS. The broadening of the peaks indicates a small crystal size within the nanometer range. a = aortic valve, ab = bicuspid aortic valve, and m = mitral valve.
Several difficulties arise when studying the mineral component of calcified tissue and its organic matrix through purely mineralogical techniques. The main difficulties are linked to (1) the application of complementary experimental techniques to obtain reliable and interpretable results, (2) the very small amount of material, not allowing application of all experimental techniques, (3) the sample preparation procedures, and (4) the specific features of the pathological deposit under investigation; indeed calcium phosphate biominerals possess peculiar characteristics that make them very difficult to be analyzed. For example, these are soft and beam-sensitive materials and can be subjected to phase instability and artifacts formation. Therefore an accurate biomineralogical study such as this work is not easy.
To date application of EMP for studying standard geological materials such as inorganic crystals formed in abiotic systems is a routine analytical procedure; thus for calcium phosphate phases of geological environment recommended protocols and various operating conditions are indicated in the literature. Conversely, analytical protocols and sample preparation procedures for EMP analysis of specific biominerals are not still well defined. Therefore we made an accurate samples selection to evaluate the more suitable samples for each type of experimental analysis and then an evaluation of the optimal analytical conditions to obtain correct information. We used the same criterion for SEM analyses; moreover as the preparation of organic material for SEM investigations can introduce a variety of damage and artifacts, we made several morphological investigations, as described in the text, to monitor the formation of artifacts associated with the organic component of the samples.
The first detailed report of aortic valve calcification was published by Moenckeburg in 1904, who proposed two different mechanisms to explain this phenomenon: (1) degeneration of the valve leaflet layers, originating near the sinuses of Valsalva and propagating toward the tips of the cusps, and (2) a sclerotic process of the aortic wall involving the cusps. Since then, a growing scientific body of evidence has been collected on the cellular and molecular mechanisms underlying calcification [
One mechanism involved in the pathogenesis of valve calcification seems to be based on the activation of normally quiescent VICs in response to different environmental stimuli and on their differentiation into osteoblastic VICs [
BSE images revealed the presence of different levels of calcification. We observed a heavily mineralized tissue, characterized by nodular formations, inside a mineral-deficient tissue in which the organic component was still dominant. The areas around the nodule formations represented this type of calcification. We consider these different levels of calcification as the expression of two different stages of the calcification process. In this sense, the mineral-deficient tissue appears to be in an initial stage of calcification and is indicative of an ongoing maturation process. We hypothesize that pathological mineral formation on valve tissue is a continuous process, similar to the multistep formation process of bone tissue [
We also identified the presence of small cavities in the calcifications. High magnification images revealed the presence of disorganized tissue and in some cases of calcified and fragmented fibers, within these cavities. We consider these cavities of particular interest because they clearly show in three dimensions the degradation and abnormal remodeling of the ECM involved in the calcification process. We suggest that these localized zones, which are characterized by a disorganized matrix, represent a stage immediately preceding the formation of a heavily mineralized formation, such as bone-like nodule formations [
Calcification appears to be morphologically organized in globular clusters of spherical particles. To date, the nature and mechanisms of nucleation and growth of spherical calcium phosphate particles in pathologic states are still unclear. It is still uncertain if these spherical particles are self-replicating life forms or if they derive from a physicochemical phenomenon without any relation to living organisms.
However, complementary microanalytical techniques applied to harder minerals, such as focused ion beam, transmission electron microscopy, and laser ablation [
To the best of our knowledge, this is the first study to highlight the quantitative difference in Ca and P tissue concentrations between tricuspid and bicuspid aortic valves. The bicuspid aortic valve (BAV) is the most common congenital valve anomaly, with a prevalence of 2% in the general population and a male preponderance ratio of 2 : 1 [
Understanding the features of pathological crystals is important if we are to obtain new insight into the mechanisms involved in valve calcification. This is fundamental for the development of therapies targeting the biomineralization process in order to improve the durability of surgically and transcatheter-implanted bioprosthetic valves. Invasive and costly surgical intervention and transcatheter implantation are today the only effective treatments of calcific valve stenosis.
We are aware of the fact that a small sample size is the major limitation of this study. As described above this limitation is strictly linked to the methods of sample preparation for specific mineralogical techniques that require a very big amount of calcification, first of all to obtain accurate experimental results and to make reproducible analyses in order to verify their reliability when specific analytical protocols are missing. However, the large number of different and accurate analyses performed and the magnitude of the results are of relevance. Moreover, to our knowledge, this is the first report using pure mineralogical techniques applied to the study of soft bioapatite in humans. Studies comparing data from ultrastructural analyses, inflammatory markers, and biomineralogical data are ongoing and will be the object of future publications.
Bicuspid aortic valve
Backscattered electron microscopy
Extracellular matrix
Energy dispersive spectrometer/spectrometry
Electron microprobe
Polarized light microscope/microscopy
Polished thin sections
Powder X-ray diffraction
Tricuspid aortic valve
Secondary electron
Scanning electron microscope/microscopy
Valve interstitial cells
Wavelength dispersive spectrometer/spectrometry
Cross polarized light.
The authors declare no conflict of interests.
The authors wish to thank the cardiac surgery staff of Treviso Regional Hospital (Italy) for assistance during the surgical interventions and preserving valves for this study. They are grateful to technical staff of CNR-IGAG, La Sapienza section, for assistance with EMP and SEM. This work was supported by the University of Rome La Sapienza (Project C26A14258W), the Research Project of National Interest (PRIN 2010-2011) “Minerals-Biosphere Interaction: Environmental and Health Consequences,” and “Gli Amici del Cuore” Association, Vicenza, Italy.