A hydrated crystalline calcium carbonate phase: Calcium carbonate hemihydrate.

As one of the most abundant materials in the world, calcium carbonate, CaCO3, is the main constituent of the skeletons and shells of various marine organisms. It is used in the cement industry and plays a crucial role in the global carbon cycle and formation of sedimentary rocks. For more than a century, only three polymorphs of pure CaCO3-calcite, aragonite, and vaterite-were known to exist at ambient conditions, as well as two hydrated crystal phases, monohydrocalcite (CaCO3·1H2O) and ikaite (CaCO3·6H2O). While investigating the role of magnesium ions in crystallization pathways of amorphous calcium carbonate, we unexpectedly discovered an unknown crystalline phase, hemihydrate CaCO3·½H2O, with monoclinic structure. This discovery may have important implications in biomineralization, geology, and industrial processes based on hydration of CaCO3.

Mobility of hydrous species in amorphous calcium/magnesium carbonates.

Amorphous calcium carbonate (ACC) is commonly found in many biological materials. As ACC readily crystallizes into calcite, stabilizers, such as anions, cations or macromolecules, often occur to avoid or delay unwanted crystallization. In biogenic ACC, magnesium is commonly present as one of the stabilizing agents. It is generally thought that the presence of mobile water in ACC is responsible for its limited stability and that the strong interaction of Mg2+ with water stabilizes the amorphous structure by retarding dehydration of ACC. To test this hypothesis, we studied the mobility of hydrous species in the model materials ACC, amorphous magnesium carbonate (AMC) and amorphous calcium/magnesium carbonate (ACMC), using quasi elastic neutron scattering (QENS) which is highly sensitive to the dynamics of H atoms. We discovered that hydrous species in the considered amorphous materials consist of water and hydroxide ions, as magnesium ions are incorporated in a ratio of 1 to about 0.6 with OH-. Surprisingly, we found that there is no evidence of translational diffusion of water and hydroxides when calcium is present in the samples, showing that hydrous species are highly static. However, we did observe diffusion of water in AMC with similar dynamics to that found for water in clays. Our results suggest that Mg2+-water interactions alone are not the only reason for the high stability of AMC and ACMC. The stabilizing effect of Mg ions, in addition to Mg-water binding, is likely to be caused by binding to hydroxide in amorphous calcium carbonates. In fact, the incorporation of hydroxides into the amorphous phase results in a mineral composition that is incompatible with any of the known Ca/Mg-carbonate crystal phases, requiring large scale phase separation to reach the composition of even the basic magnesium carbonate minerals artinite and hydromagnesite.

Additives influence the phase behavior of calcium carbonate solution by a cooperative ion-association process.

Amorphous calcium carbonate (ACC) has been widely found in biomineralization, both as a transient precursor and a stable phase, but how organisms accurately control its formation and crystallization pathway remains unclear. Here, we aim to illuminate the role of biologically relevant additives on the phase behaviour of calcium carbonate solution by investigating their effects on the formation of ACC. Results show that divalent cations like magnesium (Mg2+) ions and negatively charged small organic molecules like aspartic acid (Asp) have little/no effect on ACC formation. However, the particle size of ACC is significantly reduced by poly(aspartic acid) (pAsp) with long chain-length, but no effect on the position of the phase boundary for ACC formation was observed. Phosphate (PO4 3-) ions are even more effective in reducing ACC particle size, and shift the phase boundary for ACC formation to lower concentrations. These phenomena can be explained by a cooperative ion-association process where the formation of ACC is only influenced by additives that are able to attract either Ca2+ ions or CO3 2- ions and, more importantly, introduce an additional long range interaction between the CaCO complexes and promote the phase separation process. The findings corroborate with our proposed model of ACC formation via spinodal decomposition and provide a more realistic representation of how biology can direct mineralization processes.

A classical view on nonclassical nucleation.

Understanding and controlling nucleation is important for many crystallization applications. Calcium carbonate (CaCO3) is often used as a model system to investigate nucleation mechanisms. Despite its great importance in geology, biology, and many industrial applications, CaCO3 nucleation is still a topic of intense discussion, with new pathways for its growth from ions in solution proposed in recent years. These new pathways include the so-called nonclassical nucleation mechanism via the assembly of thermodynamically stable prenucleation clusters, as well as the formation of a dense liquid precursor phase via liquid-liquid phase separation. Here, we present results from a combined experimental and computational investigation on the precipitation of CaCO3 in dilute aqueous solutions. We propose that a dense liquid phase (containing 4-7 H2O per CaCO3 unit) forms in supersaturated solutions through the association of ions and ion pairs without significant participation of larger ion clusters. This liquid acts as the precursor for the formation of solid CaCO3 in the form of vaterite, which grows via a net transfer of ions from solution according to z Ca2+ + z CO32- → z CaCO3 The results show that all steps in this process can be explained according to classical concepts of crystal nucleation and growth, and that long-standing physical concepts of nucleation can describe multistep, multiphase growth mechanisms.

Control of Polymorph Selection in Amorphous Calcium Carbonate Crystallization by Poly(Aspartic Acid): Two Different Mechanisms.

Poly(aspartic acid) (pAsp) is known to stabilize amorphous calcium carbonate (ACC) and affect its crystallization pathways. However, little is known about the mechanisms behind these phenomena. Here it is shown that ACC is stabilized by pAsp molecules in the solution rather than by the amount of pAsp incorporated into the ACC bulk, and that the effect of pAsp on the polymorph selection is entirely different at low and high concentration of pAsp. At low concentrations, pAsp is more effective in inhibiting the nucleation and growth of vaterite than calcite. At high concentrations, when calcite formation is prevented, the crystallization of vaterite proceeds via a pseudomorphic transformation of ACC nanospheres, where vaterite nucleates on the surface of ACC nanospheres and grows by a local transformation of the bulk ACC phase. These results shed some light on the function of pAsp during an ACC-mediated biomineralization process and provide an explanation for the presence of metastable vaterite at conditions where calcite is thermodynamically favored.

Octacalcium phosphate - a metastable mineral phase controls the evolution of scaffold forming proteins.

The molecular structure of collagen type 1 can be understood as the result of evolutionary selection in the process of formation of calcium phosphate based biocomposites acting as load bearing components in living organisms. The evolutionary selection fulfills the principle of 'survival of the fittest' in a particular biological environment. Disk-like post-nucleation complexes of Ca2(HPO4)3 2- organized in ribbon-like assemblies in the metastable octacalcium phosphate (OCP) phase, and Ca3 triangles in the stable HAP phase had formed the crystallographic motifs in this selection process. The rotational as well as the translational symmetry of the major tropocollagen (TC) helix agree nearly perfectly with the corresponding symmetries of the OCP structure. The sequence of (Gly-X-Y) motifs of the three α chains constituting the TC molecule enables an optimized structural fit for the nucleation of Ca3 triangles, the directed growth of nanostructured OCP, and the subsequent formation of hydroxyapatite (HAP) in collagen macrofibrils by a topotaxial transition. The known connection between genetic defects of collagen type 1 and Osteogenesis imperfecta should motivate the search for similar dependences of other bone diseases on a disturbed molecular structure of collagen on the genetic scale.

Layered growth of crayfish gastrolith: about the stability of amorphous calcium carbonate and role of additives.

Previous studies on pre-molt gastroliths have shown a typical onion-like morphology of layers of amorphous mineral (mostly calcium carbonate) and chitin, resulting from the continuous deposition and densification of amorphous mineral spheres on a chitin-matrix during time. To investigate the consequences of this layered growth on the local structure and composition of the gastrolith, we performed spatially-resolved Raman, X-ray and SEM-EDS analysis on complete pre-molt gastrolith cross-sections. Results show that especially the abundance of inorganic phosphate, phosphoenolpyruvate (PEP)/citrate and proteins is not uniform throughout the organ but changes from layer to layer. Based on these results we can conclude that ACC stabilization in the gastrolith takes place by more than one compound and not by only one of these additives.

The integration of ion potentiometric measurements with chemical, structural, and morphological analysis to follow mineralization reactions in solution.

The solution crystallization of biominerals like calcium carbonate or calcium phosphate is a process that requires a high level of control over reaction kinetics. Ion potentiometric measurements are a way to follow and control reaction kinetics by measuring changes in pH and ion concentration, also allowing quantification of chemical compositions and solubility characteristics. By combining these measurements with various analysis techniques, one can acquire a complete spectrum of chemical, structural, and morphological data, even on metastable precursors. Therefore, in this chapter, the use of potentiometric measurements in standard solution crystallization experiments is described and the integration of these measurements with microscopy, spectroscopic and scattering analyses in such a way that the control over the reaction kinetics is maintained.

Ion-association complexes unite classical and non-classical theories for the biomimetic nucleation of calcium phosphate.

Despite its importance in many industrial, geological and biological processes, the mechanism of crystallization from supersaturated solutions remains a matter of debate. Recent discoveries show that in many solution systems nanometre-sized structural units are already present before nucleation. Still little is known about the structure and role of these so-called pre-nucleation clusters. Here we present a combination of in situ investigations, which show that for the crystallization of calcium phosphate these nanometre-sized units are in fact calcium triphosphate complexes. Under conditions in which apatite forms from an amorphous calcium phosphate precursor, these complexes aggregate and take up an extra calcium ion to form amorphous calcium phosphate, which is a fractal of Ca(2)(HPO(4))(3)(2-) clusters. The calcium triphosphate complex also forms the basis of the crystal structure of octacalcium phosphate and apatite. Finally, we demonstrate how the existence of these complexes lowers the energy barrier to nucleation and unites classical and non-classical nucleation theories.

Injectable calcium phosphate cement with PLGA, gelatin and PTMC microspheres in a rabbit femoral defect.

In this study, we investigated the in vivo degradation properties and tissue response towards injectable calcium phosphate cement (CPC) with no further addition, or calcium phosphate composite cement containing approximately 50 vol.% of microspheres. Three types of spheres were assessed, i.e. poly(lactic-co-glycolic acid) (PLGA), gelatin (GEL) and poly(trimethylene carbonate) (PTMC). The cements were injected into 4.6 mm diameter and 6mm deep cylindrical defects in the femoral condyle of New Zealand white rabbits, hardened in situ and, after wound closure, left to heal for 4, 8 and 12 weeks (n=6 for each composition and time period). After retrieval, specimens were analyzed using histological and histomorphometrical methods. Results showed that non-modified CPCs showed excellent bone contact but only very limited erosion at the surface. The CPC/PLGA implant degraded almost completely, while tissue response significantly improved at each time period. CPC/PTMC showed slower degradation characteristics compared to CPC/PLGA. Finally, at all time periods, there was an evident inflammatory response to the CPC/GEL composite cement. In conclusion, the degradation properties of the CPC/PLGA microspheres composite and its bone response when implanted into the femoral condyles of rabbits were significantly better than those of CPC/gelatin and CPC/PTMC microspheres composites.

Porcine gelatin microsphere/calcium phosphate cement composites: an in vitro degradation study.

Scaffolds for bone tissue engineering preferably should be mechanically stable, osteoconductive, biodegradable and porous. To comply with these characteristics, calcium phosphate cements (CPCs) with porcine (type A) gelatin microspheres were formulated. In this experiment, in vitro degradation of 10 wt % gelatin type A microsphere CPCs (GELA CPCs) was followed for 12 weeks in proteolytic medium. Results showed a gradual decrease in mass, compression strength and E-modulus. Morphology investigation showed that degradation of the spheres started at the surface of the composite and gradually proceeded to the inner part. Overall, porcine gelatin microspheres can be used to generate in situ macroporosity into an injectable CPC.

Effect of polymer molecular weight on the bone biological activity of biodegradable polymer/calcium phosphate cement composites.

Previous studies demonstrated that the addition of biodegradable polymer microparticles to calcium phosphate (CaP) cement improves the cement's degradative behavior without affecting its handling characteristics, especially its injectability and moldability. We investigated the influence of molecular weight of polymeric microparticles included in CaP cement on implant degradation and bone formation in critical-sized defects. Forty rats received cranial defects filled with formulations of CaP cement and poly(DL-lactic-co-glycolic acid) (PLGA) microparticles. Microparticles consisted of 100% high- (HMW) or low-molecular-weight (LMW) PLGA or mixtures of these (25%, 50%, or 75%). Implantation time was 12 weeks. Porosity measurements showed that the 100% HMW group was significantly less porous than the other groups. Histology and histomorphometry revealed significantly greater implant degradation in the 100% LMW group. Defect bridging was mainly seen in the 75% and 100% LMW groups, with the highest amount of bone in the 100% LMW formulation. These results suggest that LMW PLGA microparticles are associated with better bone formation than HMW PLGA, which is most likely explained by the greater degradation of LMW PLGA microparticles. In conclusion, CaP cement composites with high percentages of LMW PLGA microparticles show good bone transductive behavior, with complete defect bridging. The 100% LMW group turned out to be the best formulation.

Introduction of enzymatically degradable poly(trimethylene carbonate) microspheres into an injectable calcium phosphate cement.

Poly(trimethylene carbonate) (PTMC) is an enzymatically degradable polyester with rubber-like properties. Introduction of this polymer into an injectable calcium phosphate bone cement can therefore be used to introduce macroporosity into the cement for tissue engineering purposes as well as to improve mechanical properties. Aim of this study was to investigate calcium phosphate cements with incorporated PTMC microspheres (PTMC CPCs) on their physical/mechanical properties and in vitro degradation characteristics. Therefore, composites were tested on setting time and mechanical strength as well as subjected to phosphate buffered saline (PBS) and enzyme containing medium. PTMC CPCs (12.5 and 25 wt%) with molecular weights of 52.7 kg mol(-1) and 176.2 kg mol(-1) were prepared, which showed initial setting times similar to that of original CPC. Though compression strength decreased upon incorporation of PTMC microspheres, elastic properties were improved as strain-at-yield increased with increasing content of microspheres. Sustained degradation of the microspheres inside PTMC CPC occurred when incubated in the enzymatic environment, but not in PBS, which resulted in an interconnected macroporosity for the 25 wt% composites.