Amorphous Calcium Phosphate Phase Research Articles

Influence of glass composition on the network structure and mineralization of europium containing mesoporous bioactive glass nanoparticles

A mesoporous bioactive glass nanoparticle (MBN) containing functional elements effectively enhances its healing effect in medical applications. The MBN mainly comprises SiO2 structure and controls the composition by incorporating functional ions into the glass network, which affects the network structure and bioactivity. Europium has already been identified to have luminescence properties for drug delivery systems and positively affects osteogenic properties. Therefore, in this study, we recognize the changes in the glass network according to different compositions with the same amount of europium oxide (10 mol%).The MBN compositions with SiO2–CaO–P2O5–Eu2O3 were synthesized by sol-gel method, and we control the contents of SiO2 and CaO, respectively. The obtained Eu-containing MBNs had spherical morphology with mesoporous structure. Changes in bioactivity and mineralization properties would be investigated to apply the dental products. After soaking in simulated body fluid (SBF), Eu-containing MBNs with substituted SiO2 form an amorphous calcium phosphate phase on their surface. The results indicate that incorporating Eu in the bioactive glass network is an effective strategy for mineralization and developing novel multifunctional MBNs for bone tissue and drug delivery carriers.

Direct observations of nanoscale brushite dissolution by the concentration-dependent adsorption of phosphate or phytate

With the development of agricultural intensification, phosphorus (P) accumulation in croplands and sediments has resulted in the increasingly widespread interaction between inorganic and organic P species, which has been, previously, underestimated or even ignored. We quantified the nanoscale dissolution kinetics of sparingly soluble brushite (CaHPO4·2H2O, DCPD) over a broad range of phosphate and/or phytate concentrations by using in situ atomic force microscopy (AFM). Compared to water, we found that low concentrations of phosphate (1–1000 µM) or phytate (1–100 µM) inhibited brushite dissolution by slowing single step retraction. However, with increasing phosphate or phytate concentrations to 10 mM, there was a reverse effect of dissolution promotion at brushite-water interfaces. In situ observations of the coupled dissolution-reprecipitation showed that phosphate precipitated more readily than phytate on brushite surfaces, with the formation of amorphous calcium phosphate (ACP). For a fundamental understanding, zeta potential and in situ Raman spectroscopy (RS) revealed that the concentration-dependent dissolution is attributed to the reverse of outer-sphere to inner-sphere adsorption with increasing phosphate or phytate concentrations. In addition, the mineralization of phytate with outer-sphere adsorption by phytase was higher than that with inner-spere adsorption, and the presence of phytate delayed ACP phase transformation to hydroxylapatite (HAP). These in situ observations and analyses may fill the knowledge gaps of interaction between inorganic and organic P species in P-rich terrestrial and aquatic environments, thereby implicating their biogeochemical cycling and the associated availability.

Triggered metabolism of adenosine triphosphate as an explanation for the chemical heterogeneity of heterotopic ossification

Heterotopic ossification (HO), the pathological formation of bone in soft tissues, is a debilitating condition, as well as one of the few instances of de novo bone formation in adults. Chemical mapping of HO tissue showed distinct islands of calcium phosphate within phosphate-deficient, calcium-rich regions, suggesting a transition to apatitic bone mineral from a non-phosphatic precursor. The transition of amorphous calcium carbonate (ACC), a generally suggested bone-mineral precursor, in physiological conditions was thus investigated. Here, we show that adenosine triphosphate (ATP), present in high amounts in forming bone, stabilised ACC for weeks in physiological conditions and that enzymatic degradation of ATP triggered rapid crystallisation into apatite, through an amorphous calcium phosphate phase. It is suggested that this localised enzymatic degradation could explain the chemical heterogeneity seen in HO and may also represent a pathway to physiological bone mineralisation.

Substrate dependent structural variations of biomimetic carbonated hydroxyapatite deposited on glass, Ti and sputtered ZnO thin films

The formation of hydroxyapatite films on glass, titanium (Ti), and sputtered ZnO is attempted to understand the crystal growth and its manipulation by substrates of a different kind. The substrates used are commercially available glass and polished Ti (99.9% purity), as-deposited, and annealed nanostructured ZnO thin films developed by pulsed DC magnetron sputtering, which are immersed in the calcifying solution for the nucleation and growth of the hydroxyapatite. The calcifying solution is obtained by mixing equal volumes of aqueous calcium (5 mM) and carbonate-containing phosphate (3 mM) solutions prepared at 37 °C and with a pH of ~7.4. X-ray diffraction, Raman spectroscopy, and Field Emission Scanning & Transmission Electron Microscopy (FESEM & FETEM) analyses reveal that the structure and morphology of the hydroxyapatite films are dependent on the growth duration, substrate type, and even on the post-deposition annealing of the sputtered ZnO substrates. The Ti substrate induces greater crystallinity in hydroxyapatite, whereas sputtered ZnO thin films cause a significantly reduced crystallinity. Moreover, the thicknesses of HAp are exacerbated by increasing deposition time irrespective of the substrate. FETEM analyses reveal the formation of needle-shaped hydroxyapatite nanocrystals grown on Ti and ZnO thin films. Thicker needle-shaped hydroxyapatite nanocrystals are observed when grown on Ti (~6–12 nm) than ZnO thin films (~4–6 nm). Further, the amorphous calcium phosphate phase coexists with nanocrystalline hydroxyapatite when ZnO thin films deposited at 50 and 70 W sputtering powers are used as the substrates. Both charged surfaces and the supersaturation of the calcifying solution impact the nucleation of hydroxyapatite onto substrates.

A new multiphase calcium phosphate graft material improves bone healing-An in vitro and in vivo analysis.

This study aims to evaluate the potential of a novel biomaterial synthesized from amorphous calcium phosphate (ACP), octacalcium phosphate (OCP), and hydroxyapatite (HA) to repair critical-sized defects (CSD) in rabbit calvaria. In vitro analyses of cell viability, cell proliferation, formation of mineral nodules, and cell differentiation using qPCR were performed for comparing experimental calcium phosphate (ECP), deproteinized bovine bone (DBB), and beta-tricalcium phosphate (β-TCP). Bilateral CSDs were created in 45 rabbit calvaria. Six groups were evaluated: ECP, ECP + fibrin sealant (ECP + S), coagulum, autogenous bone, DBB, and β-TCP. Euthanasia was performed at 2, 4, and 8 weeks, followed by micro-computed tomography and histological and immunohistochemical analyses. Results from in vitro analyses revealed similar biocompatibility for all tested materials and a tendency for higher gene expression of some bone markers in the ECP group than in β-TCP and DBB groups at 7 days. In contrast to that in DBB and β-TCP groups, ECP displayed growing bone volume over total volume percentage (BV/TV%) with time in vivo. Histological analysis revealed a greater number of giant cells and reduced size of grafted particles in ECP during all periods of analysis. RUNX-2 expression was statistically lower in ECP than DBB at 2 and 4 weeks. Despite no statistical significance, ECP presented the highest absolute values for ALP-expression at 2, 4, and 8 weeks compared with other groups. Together, our findings indicate that a combination of the ACP, OCP, and HA phases into ECP is beneficial and promising for bone regeneration.

Pure hydroxyapatite synthesis originating from amorphous calcium carbonate

We report a synthesis strategy for pure hydroxyapatite (HAp) using an amorphous calcium carbonate (ACC) colloid as the starting source. Room-temperature phosphorylation and subsequent calcination produce pure HAp via intermediate amorphous calcium phosphate (ACP). The pre-calcined sample undergoes a competitive transformation from ACC to ACP and crystalline calcium carbonate. The water content, ACC concentration, Ca/P molar ratio, and pH during the phosphorylation reaction play crucial roles in the final phase of the crystalline phosphate compound. Pure HAp is formed after ACP is transformed from ACC at a low concentration (1 wt%) of ACC colloid (1.71 < Ca/P < 1.88), whereas Ca/P = 1.51 leads to pure β-tricalcium phosphate. The ACP phases are precursors for calcium phosphate compounds and may determine the final crystalline phase.

Structure and formation of amorphous calcium phosphate and its role as surface layer of nanocrystalline apatite: Implications for bone mineralization

We provide a critical review of the chemical composition and structure of synthetic and biogenic (bone/dentin mineral) nanocrystalline hydroxy-carbonate apatite (HCA). Such particles exhibit a “core–shell” organization, where an ordered HCA core is coated by a surface layer, whose nature is best captured by amorphous calcium phosphate (ACP), which is known to be a precursor phase of synthetic HCA, but whose role of bone/dentin mineralization has remained a most controversial subject. After reviewing the structure of each HCA and ACP component, as well as the most recent findings on their in vitro formation mechanisms, we examine the core–shell HCA organization further, with a focus on the disordered surface (“shell”) domain. In most of recent literature, the surface portion is often referred to as the “hydrated surface layer”, but without identifying its shared chemical and structural features of (synthetic) ACP. Unfortunately, that missing surface-layer/ACP equivalence obscures that the surface layer at the synthetic/biogenic nanocrystallites may simply constitute a remnant of the ACP phase from which the ordered HCA “core” nucleated. Although many topics reviewed herein have been investigated for more than six decades, several remain unsettled and heavily debated. Notably, decades-old articles offer suggestions that have passed unnoticed by the younger generations of researchers; we contrast and discuss both the latest and early contributions of this field, as well as highlighting several unsettled topics that should be revisited to improve our understanding of the ACP and HCA structures and in vitro/in vivo formation mechanisms.

Structural Role and Spatial Distribution of Carbonate Ions in Amorphous Calcium Phosphate

The local structures of a series of amorphous calcium phosphate (ACP) phases with increasing carbonate contents (2–14 wt %) were studied by multinuclear 1H, 13C, 23Na, and 31P magic-angle spinning ...

Effect of aspartic acid on the crystallization kinetics of ACP and dentin remineralization

Type I collagen and non-collagen proteins are the main organic components of dentin. This study aimed to investigate the biomimetic remineralization of demineralized dentin by aspartic acid (Asp), which is abundant in non-collagenous proteins (NCPs). Asp was added to a mineralizing solution containing polyacrylic acid (PAA) to explore the mechanism of Asp regulating the pure amorphous calcium phosphate (ACP) phase transition process. The remineralization process and superstructure of the remineralized layer of demineralized dentin were evaluated and analyzed by transmission electron microscope (TEM) and scanning electron microscope (SEM), and the biological stability of the remineralized layer was investigated by collagenase degradation experiment. It demonstrated that Asp promoted the crystallization kinetics of PAA-stabilized amorphous calcium phosphate to hydroxyapatite (HAP), and shortened the remineralization time of demineralized dentin from 7 days to 2 days. The newly formed remineralized dentin had similar morphology and biological stability to the natural dentin layer. The presence of a large number of Asp residues in NCPs promoted the phase transformation of ACP, and further revealed the mechanism of action of NCPs in dentin biomineralization. This experiment also showed that Asp promoted the biomimetic remineralization of dentin; the morphology and hierarchical structure of remineralized layer was similar to that of natural teeth, and had good biological properties.

Nucleation of Brushite and Hydroxyapatite from Amorphous Calcium Phosphate Phases Revealed by Dynamic In Situ Raman Spectroscopy

Since the 1970s, it has been shown that calcium phosphate crystals nucleate from one or several early amorphous calcium phosphate phases into several in vivo and in vitro systems. However, the prec...

Visualizing different crystalline states during the infrared imaging of calcium phosphates

Methods utilizing relatively simple mathematical operations during physical analyses to enable the visualization of otherwise invisible correlations and effects are of particular appeal to researchers and students in pedagogical settings. At the same time, discerning the amorphous phase from its crystalline counterpart in materials is challenging with the use of vibrational spectroscopy and is nowhere as straightforward as in phase composition analytical methods such as X-ray diffraction. A method is demonstrated for the use of first- and second-order differentiation of Fourier transform infrared spectra of calcium phosphates to distinguish their amorphous states from the crystalline ones based on the exact line positioning rather than on comparatively vaguer band broadening and splitting effects. The study utilizes a kinetic approach, focusing on the comparison of spectral features of amorphous precursors annealed in air at different temperatures or aged for different periods of time in an aqueous solution until transforming to one or a mixture of crystalline phases, including hydroxyapatite and α- and β-tricalcium phosphate. One of the findings challenges the concept of the nucleation lag time preceding the crystallization from amorphous precursors as a “dead” period and derives a finite degree of constructive changes occurring at the atomic scale in its course. The differential method for highlighting spectral differences depending on the sample crystallinity allows for monitoring in situ the process of conversion of the amorphous calcium phosphate phase to its crystalline analogue(s). One such method can be of practical significance for synthetic solid state chemists testing for the chemical stability and/or concentration of the reactive amorphous phase in these materials, but also for biologists measuring the maturity of bone and medical researchers evaluating its phase composition and, thus, the state of metabolic and mechanical stability.

Insights into OCP identification and quantification in the context of apatite biomineralization

Monitoring apatite formation throughin situRAMAN andex situssNMR spectroscopy.

Synthesis, microstructure and mechanical properties of tricalcium phosphate–hydroxyapatite (TCP/HA) composite ceramic

Tricalcium phosphate-hydroxyapatite (TCP/HA) is an important composite ceramic ubiquitously applied in biomedicine research. In this work, a novel design and synthesizing method for preparation of TCP/HA composites is proposed consisting in suspension mixing amorphous calcium phosphate (ACP) and hydroxyapatite (HA) ceramic powders with different weight ratios, and their compaction under a uniaxial pressure of 200 MPa and sintering at 1150 °C for 2 h. The obtained sintered bodies exhibited optimum hardness and fracture toughness of 5 GPa and 1.27 MPa m1/2, and values significantly improved as compared with those obtained with the traditional mechanical mixing method. The mechanical enhancement mechanism was mainly attributed to the good ACP and HA phase dispersion and ACP crystallization. SEM and HRTEM observations showed the suspension mixing method to allow amorphous spherical ACP and crystalline rod-like HA particles with size around 50 nm to uniformly disperse at the nano scale in the composite powder, which contributes to the excellent sinter ability. Furthermore, XRD results indicate that after sintering, nearly 100% of amorphous spherical ACP transformed to the crystalline β-TCP, which results in the nanoparticle shrinkage, porosity reduction and composite densification.

Electrically Polarized Hydroxyapatite: Influence of the Polarization Process on the Microstructure and Properties.

Semipermanently polarized hydroxyapatite, named SP/HAp(w), is obtained by applying a constant dc electric field of 1-10 kV/cm at 300-850 °C to the samples previously sintered in water vapor, while permanently polarized hydroxyapatite, PP/HAp(a), is produced by applying a dc electric field of 3 kV/cm at 1000 °C to the samples sintered in air. SP/HAp(w) has been used for biomedical applications, while PP/HAp(a) has been proved to be a valuable catalyst for N2 and CO2 fixation. In this work, structural differences between SP/HAp(w) and PP/HAp(a) have been ascertained using Raman microscopy, wide-angle X-ray diffraction, scanning electronic microscopy, high-resolution transmission electron microscopy, and grazing incidence X-ray diffraction. Results prove the existence of crystal distortion in the form of amorphous calcium phosphate and β-tricalcium phosphate (β-TCP) phases close to the surface because of the atmosphere used in the sintering process. The existence of an amorphous layer in the surface and the phase transition through β-TCP of SP/HAp(w) are the structural factors responsible for the differences with respect to PP/HAp(a). Moreover, a superstructure has been identified in PP/HAp(a) samples, which could be another structural factor associated with enhanced conductivity, permanent polarization, and catalytic activity of this material.

Formation of stable strontium-rich amorphous calcium phosphate: Possible effects on bone mineral

Bone, tooth enamel, and dentin accumulate Sr2+, a natural trace element in the human body. Sr2+ comes from dietary and environmental sources and is thought to play a key role in osteoporosis treatments. However, the underlying impacts of Sr2+on bone mineralization remain unclear and the use of synthetic apatites (which are structurally different from bone mineral) and non-physiological conditions have led to contradictory results. Here, we report on the formation of a new Sr2+-rich and stable amorphous calcium phosphate phase, Sr(ACP). Relying on a bioinspired pathway, a series of Sr2+ substituted hydroxyapatite (HA) that combines the major bone mineral features is depicted as model to investigate how this phase forms and Sr2+ affects bone. In addition, by means of a comprehensive investigation the biomineralization pathway of Sr2+ bearing HA is described showing that not more than 10 at% of Sr2+, i.e. a physiological limit incorporated in bone, can be incorporated into HA without phase segregation. A combination of 31P and 1H solid state NMR, energy electron loss spectromicroscopy, transmission electron microscopy, electron diffraction, and Raman spectroscopy shows that Sr2+ introduces disorder in the HA culminating with the unexpected Sr(ACP), which co-exists with the HA under physiological conditions. These results suggest that heterogeneous Sr2+ distribution in bone is associated with regions of low structural organization. Going further, such observations give clues from the physicochemical standpoint to understand the defects in bone formation induced by high Sr2+ doses. Statement of SignificanceUnderstanding the role played by Sr2+ has a relevant impact in physiological biomineralization and provides insights for its use as osteoporosis treatments. Previous studies inspired by the bone remodelling pathway led to the formation of biomimetic HA in terms of composition, structures and properties in water. Herein, by investigating different atomic percentage of Sr2+ related to Ca2+ in the synthesis, we demonstrate that 10% of Sr2+ is the critical loads into the biomimetic HA phase; similarly to bone. Unexpectedly, using higher amount leads to the formation of a stable Sr2+-rich amorphous calcium phosphate phase that may high-dose related pathologies. Our results provide further understanding of the different ways Sr2+ impacts bone.

Stabilisation of amorphous calcium phosphate in polyethylene glycol hydrogels

Acellular polymer-calcium phosphate composites are promising bone graft materials. Hydrogels are suitable for providing a temporary matrix, while calcium phosphate minerals serve as ion depots for calcium and phosphate required for de novo bone formation. Crystalline calcium phosphates are stable under biological conditions and are commonly used in such scaffolds. However, the low solubility of these phases reduces the availability of free ions and potentially obstructs the remodelling necessary for the formation of mineralised tissue. Here, we investigate two different strategies to stabilise amorphous calcium phosphates in a synthetic polyethylene glycol-based hydrogel matrix.In vitro experiments mimicking an injectable application showed that amorphous calcium phosphate (ACP) of variable stability was formed in the hydrogel matrices. In additive-free composites, ACP transformed into brushite within minutes. Citrate or zinc additives were found to stabilise the formed ACP phase to different degrees. In the presence of citrate, ACP was stable for at least 2 h before it transformed into hydroxyapatite within 3–20 days. Partial calcium substitution with zinc (Zn/Ca = 10%) produced zinc-doped ACP of high stability that did not show signs of crystallisation for at least 20 days.The presented methods and findings open new possibilities for the design of novel injectable synthetic bone graft materials. The possibility to produce ACP with tailorable stability promises great potential for creating temporary scaffolds with good osteogenic properties. Statement of significanceSynthetic hydrogel-calcium phosphate (CaP) composites are promising biomaterials to replace human- and animal-derived bone scaffolds. Most reported hydrogel-CaP composite materials employ crystalline CaP phases that lack the osteoinductive properties of autograft. Stabilising amorphous calcium phosphates (ACP) could overcome this limitation, readily delivering calcium and phosphate ions and facilitating remodelling into new bone tissue. The design of synthetic hydrogel-ACP scaffolds, however, requires more understanding of the mineralisation processes in such matrices. This study presents a model system to characterise the complex mineral formation and transformation processes within a hydrogel matrix. We demonstrate a facile route to produce self-mineralising injectable synthetic hydrogels and prove two different strategies to stabilise ACP for different periods within the formed composites.

Underlying Role of Brushite in Pathological Mineralization of Hydroxyapatite.

The majority of human kidney stones are composed of multiple calcium oxalate crystals with variable amounts of brushite [dicalcium phosphate dihydrate (DCPD)] and hydroxyapatite (HAP) as a nucleus, in which fluid-mediated dissolution and reprecipitation may result in the phase transformation of DCPD to HAP. However, the underlying mechanisms of the phase transition and its modulation by natural inhibitors, such as osteopontin (OPN) proteins, remain poorly understood. Here, the in vitro formation of new phases on the DCPD (010) surface is observed in situ using atomic force microscopy in a simulated hypercalciuria milieu. We demonstrate the presence of an acidic amorphous calcium phosphate (ACP) phase with a characteristic Raman band of ν1HPO42- and the octacalcium phosphate (OCP)-like phase during the transformation process. High-resolution transmission electron microscopy analyses also confirm the existence of OCP and HAP within an amorphous matrix phase. In support of clinical observations, we further demonstrate the inhibitory effect of OPN peptide segments on the dissolution of DCPD and reprecipitation of acidic ACP. The definition of respective roles of DCPD and OPN thereby provides insights into the control of nucleus formation and subsequent inhibition of pathological mineralization.

Effect of strontium substitution on the material properties and osteogenic potential of 3D powder printed magnesium phosphate scaffolds

3D powder printing is a versatile method for the fabrication of individual bone implants and was used for the processing of in vivo degradable ceramic scaffolds based on ammonium magnesium phosphate hexahydrate (struvite). In this study, synergetic effects could be achieved by the substitution of magnesium phosphate cements with strontium carbonate. This substitution resulted in 8.2 wt%, 16.4 wt%, and 24.6 wt% Sr2+ doped scaffolds, with a 1.9–3.1 times increased radiopacity compared to pure struvite. The maximal compressive strength of (16.1 ± 1.1) MPa found for strontium substituted magnesium phosphate was in the range of cancelleous bone, which makes these 3D printed structures suitable for medical application in low-load-bearing bone areas. In an ion release study over a course of 18 days, the release of strontium, magnesium, calcium, and phosphate ions from scaffolds was analyzed by means of inductively coupled plasma mass spectrometry. Independent of the scaffold composition the Mg2+ concentrations (83–499 mg/l) continuously increased in the cell media. The Sr2+ release varied between 4.3 μg/day and 15.1 μg/day per g scaffold, corresponding to a Sr2+ concentration in media between 1.14 mg/l and 7.24 mg/l. Moreover, decreasing calcium and phosphate concentrations indicated the precipitation of an amorphous calcium phosphate phase. The superior osteogenic properties of strontium substituted magnesium phosphate, e.g. the increase of osteoblast activity and cell number and the simultaneous suppression of osteoclast differentiation could be verified in vitro by means of WST-assay, TRAP-staining, and SEM imaging.

Effects of applied voltage on the microstructure and properties of hydroxyapatite bioceramic coatings formed on Ti7Cu5Sn titanium alloy by micro-arc oxidation

ABSTRACTThe effects of the applied voltage on the morphology, composition and corrosion behaviour of Ti7Cu5Sn coated were investigated. At applied voltages lower than 250 V, the composite coatings consist of anatase-TiO2, rutile-TiO2, DCPD(CaHPO4·2H2O) and a small amount of amorphous calcium phosphate phase. When the applied voltage is increased, the ceramic coatings transform from DCPD (CaHPO4·2H2O) to HA (Ca10(PO4)6(OH)2, 300 V), and new phases of Ca2P2O7, CaTiO3 and TCP(Ca3(PO4)2) form at 350 V. The passive current densities at body potential are one order of magnitude lower than that of the uncoated sample, indicating better corrosion resistance. The MAO film is a tri-layer system: a compact inner layer, a mesosphere porous oxide layer, and an outer layer.

The effect of osteoblasts on the surface oxidation processes of biodegradable Mg and Mg-Ag alloys studied by synchrotron IR microspectroscopy

High-resolution analytical methods, including synchrotron infrared microspectroscopy combined with wavelength-dispersive X-ray emission spectroscopy were applied to study the structure and chemical composition of the oxidized layer of pure and Ag-alloyed Mg exposed to cell culture media without and with osteoblasts. Comparative analysis has been done on pure Mg immersed in two different cell culture media: Dulbecco's Modified Eagle's Medium (DMEM) and Roswell Park Memorial Institute medium (RPMI), whereas Mg-xAg binary alloys (x = 2, 4, 6, 8 wt%) were studied after immersion in DMEM. It is shown that the physicochemical formation of degradation products as well as the activity of the biological component is influenced by the addition of silver. It could be demonstrated that the presence of Ag in the Mg alloy enhances the chemical reaction between Mg and C to form amorphous and/or crystalline MgCO3 on account of CaCO3. As a consequence, the further available P and Ca react easily to form Mg-poor amorphous calcium phosphate phases. Osteoblasts actively adjusted these phases towards hydroxyapatite-like phases.