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.