Biological niches within human calcified aortic valves. Towards understanding of the pathological biomineralization process

Despite recent advances, mineralization site, its microarchitecture, and composition in calcific heart valve remain poorly understood. A multiscale investigation, using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectrometry (EDS), from micrometre up to nanometre, was conducted on human severely calcified aortic and mitral valves, to provide new insights into calcificationp rocess. Our aim was to evaluate the spatial relationship existing between bioapatite crystals, their local growing microenvironment, and the presence of a hierarchical architecture. Here we detected the presence of bioapatite crystals in two different mineralization sites that suggest the action of two different growth processes:a pathological crystallization process that occurs in biological niches and is ascribed to a purely physicochemical process and a matrix- mediated mineralized process in which the extracellular matrix acts as the template for a site-directed nanocrystals nucleation. Different shapes of bioapatite crystallization were observed at micrometer scale in each microenvironment but at the nanoscale level crystals appear to be made up by the same subunit

A multi-scale investigation of biological niches within human calcified aortic valves helps to understand the pathological biomineralization process

Calcific aortic valve stenosis (CAVS) is the most common form of heart valve disease in the industrialized countries, being an important public health problem [1]. Ectopic calcifications within aortic valve leaflets are strictly associated with CAVS, interfering with cusps opening, they lead to ventricular outflow obstruction [2]. Up to date no proven medical therapy stops CAVS course progression, so valve replacement is the only possible treatment of severe CAVS. Unfortunately, the degenerative valve calcification process, affects also bioprosthetic implants [3]. Being the molecular mechanisms leading to valve calcification still not understood, our aim was to carry on a multi-scale investigation using Scanning Electron Microscopy, Transmission Electron Microscopy and Energy Dispersive X-ray Spectrometry, to provide new insights into calcification process. Severely calcified aortic (tricuspid type, n = 29; bicuspid type, n = 3) and mitral valves (n = 4) were obtained from patients of both sexes (males=25) and different ages (mean age 72±10, range 41-90 years old) undergoing valve replacement due to severe aortic and mitral valve stenosis. We detected bioapatite crystals in two different mineralization sites: niches and extracellular matrix. This suggests the action of two different growth processes: the first occurs in biological niches and it is ascribed to a purely physico-chemical process; the second has the extracellular matrix acting ass the template for a site-directed nanocrystals nucleation. Different shapes of bioapatite crystallization were observed at micrometer scale in each microenvironment but at the nanoscale level crystals appear made up by the same subunits. We suggest that bioapatite nanocrystals in heart valve may activate a strong inflammatory process leading to irreversible pathological condition that, once activated,may aggravate the inflammatory response against bioapatite nanocrystals leading to a severe calcification process

Crystal-chemistry of "bioapatite" deposits from valve tissues of the human heart: from macrostructures to nanocrystals

Biomineralization is well-known as the essential process for human skeletal development. It is a complex and multistage process requiring an interaction of many physicochemical factors. It results in highly complex mineral-organic products in which organic and inorganic components are structured on many dimensional scales to form hierarchical architectures. From a mineralogical point of view the concept of biomineral refers not only to mineral produced with the intervention of living organisms but to phases characterized by specific features and properties that distinguish them from their bulk, macroscopic counterparts formed geologically or synthetically. Calcium phosphate phases can be considered the most important class of biominerals. These take place in human body mainly as physiologic products such as bones and teeth but also as pathological products, when their formation occurs outside the normal mineralization sites. In different scientific fields these are generically known with the inappropriate term of calcifications to distinguish them from the products having a precise function in the human body; from a mineralogical point of view this well-defined distinction is not taken to account and all biominerals meet only the criteria for being true minerals. The present thesis addresses the issue of “pathological” calcium phosphate biominerals within valve tissues of the human heart. Nowadays the formation of such biominerals is a worldwide important topic associated with major morbidity, mortality and health economic costs. These represent the leading cause of failure of natural and bioprosthetic heart valves and the major indication for surgical valve replacement. The mechanisms involved in their deposition are still poorly understood and no medical intervention is able to delay or halt “calcification” progression, thus there is a pressing need to deeply understanding the biomineralization processes linked to these “pathological” calcium phosphate phases. Aim of the present thesis is to provide a comprehensive mineralogical characterization of such calcium phosphate biominerals in an effort to obtain new insights into the factors controlling this biomineralization process and hence to supply a better picture on which to base new hypothesis on the nucleation and growth processes linked to these phases. Composition, morphology, crystallite size and structure are all correlated with their growth conditions; an understanding of their morphological and crystal-chemical features allows to gain valuable information on their crystallization pathways. The main debated issues linked to calcium phosphate biominerals will be discussed, starting from a clarification of the term “bioapatite” used in this study to indicate a well-distinct calcium phosphate phase but often used improperly in different scientific fields. Relevant topics concerning specific features of the nanocrystalline bioapatite will be developed. These include: 1) location of the carbonate group CO32- in the bioapatite lattice; 2) carbonate content; 3) hydroxylation degree; 3) bioapatite stoichiometry 4) surface properties; 5) presence of precursor phases; 6) macro- and microstructures; 7) nanocrystals structure. Complementary mineralogical techniques were employed to obtain a comprehensive characterization of this biomineral phase, and a multi-scale investigation, from millimeters to nanometers, has been conducted to define all structural organization levels, typical of biomineral phases. The greatest difficulties linked to the characterization of natural nanocrystalline bioapatites will be also discussed. The complete mineralogical characterization has allowed to determine the lowest units constituting the “pathological” deposits within the valve tissues of the human heart. These are represented by needle- and rod-like nanocrystals showing characteristic aggregation properties in a wide range of crystallite size associated to local growth conditions and to different mineralization sites. The nucleation and growth mechanisms of the investigated phase seem to be mainly regulated by thermodynamic and physicochemical factors while the role of the organic matrix appears to be mainly limited to a spatial template; both homogeneous and heterogeneous nucleation processes appear to be involved in the formation of “pathological” nanocrystals, and in the latter case a surface-induce mineralization process linked to the functionalization of the organic interfaces by negatively charged functional groups can be hypothesized. The presence of the CO32- group both in the bioapatite lattice and as labile ions localized at the nanocrystals surface, as well as the presence of the HPO42- group, suggests a possible involvement of these functional groups in inducing bioapatite nucleation onto organic substrate and a superficial ionic mobility. This can assume an important role for the chemical interactions of the inorganic phase with the organic matrix and the biological fluids representing a relevant feature for ion exchange processes. Finally, at larger length scales, the three-dimensional arrangement of bioapatite nanocrystals in spherulitic shapes located onto and beside the collagen fibrils, or in the form of uniform mineral coating, seems to be linked to the local density and distribution of the organic network, but also to aggregation processes ruled by surface energy minimization

Calcification of the human heart valves: a mineralogical approach

Normal physiologic processes result in development of mineralized tissue. Bones and tooth enamel are the main example of biominerals. Pathologic processes lead to calcification of the atherosclerotic plaques, kidney and salivary stones and other pathologic deposits. Most of these seem to be constituted from a mixture of calcium phosphate phases but their formation mechanisms are not completely known. In cardiac pathology, calcification of heart valves can be advanced by a congenital malformation or an infectious process or related to the senile degeneration. Pathological mineral deposits occurring in human cardiac valves were studied using Polarizing Microscopy, Scanning Electron Microscopy (SEM-EDS), Electron Microprobe (EMPA), X-Ray Powders Diffraction (XRPD), Infrared Spectroscopy (FTIR). Samples were obtained as surgical waste from thirty patients undergoing valvular replacement in case of severe aortic and mitral stenoses. The experimental results showed that the mineral phase grown in human cardiac valves is a calcium phosphate with poor crystallinity. It develops as nodules in the organic matrix. The FT-IR spectra may be used to infer the presence of carbonate group. The carbonate bands in the infrared spectra have a saw-tooth profile similar to sample PC18, a synthetic type A-B CAp but in samples of aortic valves a-parameter is smaller and the c-parameter is greater than those of PC18 [i.e. TV12 a=9.4165(8) Å, c= 6.8951(7) Å; PC18 a=9.4803(3) Å, c=6.8853(3) Å] probably due to substitutional carbonate groups in phosphate positions which cause a shrinkage in the a-parameter. Pathological phase investigated can be considered a bioapatite as the inorganic component of bone and tooth enamel, even if it possesses unusual morphologies for a calcium phosphate and a Ca/P ratio unlike that of normal mineralized tissue

Biominerals: nano-scale characterization of calcium phosphate crystals forming the “calcification” of the human heart valves

Calcium phosphates, mainly ‘bioapatite’, make up the inorganic part of bone and teeth but also occur as pathological deposits. Among pathological biomineralizations, better known in the medical field with the generic term calcification, the deposition of calcium phosphate nanocrystals in the valve tissues of the human heart is a worldwide important topic associated with major morbidity, mortality and health economic costs. Nowadays the mechanisms leading to these pathological products are an open question despite all effort devoted to their comprehension. The nanometer scale structure of such pathological crystals was investigated using a dual beam Zeiss Auriga 405 HR-FESEM with resolution of 1 nm, and low accelerating voltage (< 15 kV) to obtain information about biomineral/organic structure interface. TEM analyses were performed on powdered samples by a Jeol JEM 2010 operating at 200KV with LaB6 source, nominal point resolution of 1.9 Å, and spherical aberration of 0.5 mm. Ultrastructural investigations brought to light the lowest units constituting the pathological deposits of the human valve tissues. These are represented by needle- and rod-like nanocrystals having the typical structure of hexagonal hydroxylapatite, but different chemical composition as [CO3]2- is present both for [PO4]3- and (OH)-. Characteristic aggregation properties of ‘bioapatite’ result in the formation of micrometer sized spherical particles. These latter appear to be also associated to mixed aggregates formed by nanocrystals and organic matrix, and to individual organic structures but without any relation to nanobacteria. All nanocrystals show the typical features of hydroxylapatite crystals precipitated in aqueous solutions, and a wide range of crystallite size, from a few to several hundreds of nanometers, that appears to be associated to local growth conditions and to different mineralization sites. The presence of localized compartments within the organic tissue, similar to “vugs” in rocks, in which a locally and progressive increase of ions concentration can take place, seems to be the pivotal requirement for the bioapatite precipitation. This confirms the important role of purely physicochemical processes in the biomineralization process of the valve tissues of the human heart and allows to ascribe to the organic matrix only the function of spatial template. However the presence of nanocrystals directly formed onto the organic substrate and oriented respect to this latter, also suggests a surface-induced mineralization process and a possible involvement of matrix components inducing ‘bioapatite’ nucleation

Morphological and Chemical Study of Pathological Deposits in Human Aortic and Mitral Valve Stenosis: A Biomineralogical Contribution

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

Pathological biomineralization from human aortic and mitral valve stenosis.

Samples were collected as surgical waste from patients undergoing valvular replacement because of severe aortic (n=6) and mitral (n=2) stenosis. Pathological mineral formations have been investigated with XRPD and SEM-EDS, both in high and in low vacuum conditions. Samples were not coated because of metallic coating artifacts.The a cell parameters were found to be smaller than the a parameter of human dental enamel apatite, while the c parameters were greater. High resolution images show a complex relationship between inorganic component and organic matrix as well as particular morphologies of the pathogenic biomineralization. Bioapatite appears as lamellar crystals, globular aggregated and massive; at high magnification it appears to be constituted of spherical particles of variable size. Bioapatite morphology observed in this study appears to be different from biogenic calcium phosphate crystals and from inorganically produced counterparts. The small spheres could be considered as nanobacterial-like structures (?). This attractive hypothesis has not been confirmed yet

Infrared and Raman studies of bioapatite deposits in human heart valves

The carbonate group is an important constituent of bioapatite, a calcium phosphate close to hydroxyapatite, main constituent of bone and dental enamel. [CO3] can occupy two different sites in the structure (type A and B), and seems to control the growth, evolution, morphology, and physical properties of synthetic nano-carbonated hydroxyapatite. Infrared and Raman spectroscopy were used to evaluate the carbonate substitution in pathological bioapatite from patients undergoing valvular replacement because of severe aortic and mitral stenoses. FTIR spectra were collected in the 4000-400 cm-1 spectral range using a PerkinElmer System 2000, while Raman spectra in the range 4000-200 cm-1 using a Horiba Jobin-Yvon LabRam Confocal Microscope at a resolution of about 3 cm-1. The PO4 asymmetric stretching IR mode appears as an intense band at 1023 cm-1, a shoulder at 1059 cm-1, and a third band at 1104 cm-1. The CO3 asymmetric stretch vibration mode is represented by four bands at 1418, 1450, 1471, 1503 cm-1 while the CO3 out-of-plane bending mode by the band at 872 cm-1. This characteristic IR signature seems to be typical of Na-bearing type A-B carbonate apatite. The band at 1503 cm-1 could indicate the accommodation of the carbonate group in a second channel position (Type A2) usually present in carbonate apatite synthesized at high-pressure. On the contrary the Raman band at 1071 cm-1 due to CO3 symmetric stretching mode is specific of [CO3] substituting [PO4] (type B) and the band observed at 961 cm-1 due to symmetric stretching mode of PO4 is in agreement with the shift assigned to PO4 symmetric stretch mode for bone and synthetic type B carbonate apatite with different carbonate contents. The Raman peaks’ height and area are strongly correlated with weight percent carbonate. The ratio of peak area at 1071 cm-1 and peak area at 961 cm-1 was used to determine the percentage of carbonate in the analyzed samples. Values obtained (4.5-7.0 %) are in good agreement with those of biological apatite from bone

Pathological Biominerals: Raman and Infrared Studies of Bioapatite Deposits in Human Heart Valves

We studied pathological bioapatite from patients undergoing valvular replacement due to severe aortic and mitral stenosis. Three different types of mineralized human cardiac valves were analyzed. We used infrared and Raman spectroscopy to infer the presence of the carbonate group and evaluate the carbonate substitution in bioapatite structure. The Raman spectra showed that the pathological bioapatite is a B-type "carbonate-apatite" (CO32- for PO43-) similar to the major mineralized products derived from normal biomineralization processes occurring in the human body. Fourier transform infrared spectra (FT-IR) confirmed the B-type carbonate substitution (CO32- for PO43-) and showed evidence for the partial replacement of [OH] by [CO3] (A-type substitution). The carbonate content of the samples inferred by the spectroscopic measurements is in good agreement with the range of values estimated for biological apatite. On the contrary, the crystal size of the pathological apatite estimated using the percentage area of the component at 1059 cm(-1) of the infrared spectrum is in the nanometer range and it is significantly smaller than the crystal size of normal mineralized tissues

Biological Niches within Human Calcified Aortic Valves: Towards Understanding of the Pathological Biomineralization Process

Despite recent advances, mineralization site, its microarchitecture, and composition in calcific heart valve remain poorly understood. A multiscale investigation, using scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy dispersive X-ray spectrometry (EDS), from micrometre up to nanometre, was conducted on human severely calcified aortic and mitral valves, to provide new insights into calcification process. Our aim was to evaluate the spatial relationship existing between bioapatite crystals, their local growing microenvironment, and the presence of a hierarchical architecture. Here we detected the presence of bioapatite crystals in two different mineralization sites that suggest the action of two different growth processes: a pathological crystallization process that occurs in biological niches and is ascribed to a purely physicochemical process and a matrix-mediated mineralized process in which the extracellular matrix acts as the template for a site-directed nanocrystals nucleation. Different shapes of bioapatite crystallization were observed at micrometer scale in each microenvironment but at the nanoscale level crystals appear to be made up by the same subunits