Carbonate Apatite Crystals Research Articles

Phosphate and buffer capacity effects on biomimetic carbonate apatite

With the increasing use of carbonated apatite (CAp) in biomedical devices, it has become necessary to understand how this thermodynamically unstable mineral interacts with varying surrounding body fluids. Despite existing knowledge about the evolution of other calcium phosphates in bio-like fluids, it is unknown how CAp reacts in these solutions. Therefore, our goal was to determine how solution phosphate concentration and the buffer capacity (BC) affects CAp dissolution/recrystallization. To do so, CAp powder was first synthesized through an aqueous precipitation reaction and then exposed to one of the following solutions for 3 days: (1) 0 mM PO4, (2) 8 mM PO4, (3) 16 mM PO4, (4) low BC, or (5) high BC. Afterwards, the powders were analyzed for mass loss, composition via Raman, crystal structure via XRD, and size via TEM. The solutions were evaluated for Ca, P, Na, and K through ICPOES. Our results suggest that increased phosphate in the solution, regardless of BC, created larger, more crystalline CAp crystals, indicating crystal maturation. In addition, the crystals had fewer carbonate substitutions and more phosphate ion uptake from the solution after pH equilibrium. This data offers insight on how CAp in biomaterials may mature and change composition and material properties in the body after implantation.

An effective route to the synthesis of carbonated apatite crystals with controllable morphologies and their growth mechanism

CHAp powders with controllable morphologies and sizes were synthesized using HMT as a hydroxide anion-generating agent in a phosphate-surplus solution.

Large scale production of yolk-shell β-tricalcium phosphate powders, and their bioactivities as novel bone substitutes.

This paper proposes the production of yolk-shell structured β-tricalcium phosphate (β-TCP) powders using a spray-drying method, suitable for commercial scale production. Spray-dried precursor powders, consisting of calcium-phosphate salts and each of the various carbon source materials, are combusted in an oxygen atmosphere to obtain a yolk-shell structure. Only dextrin among the carbon source materials investigated shows promise in the production of β-TCP yolk-shell powders. By evaluating their apatite-forming capacity in simulated body fluid, the outstanding bioactivity of β-TCP yolk-shell powders is confirmed: numerous acicular and newly formed hydroxyl carbonate apatite crystals cover the entire β-TCP surface after a single day of soaking. These crystals are observed on both the outer and inner surfaces of the shells, and on the outer surface of the core, which is encouraging for its potential use as a bone grafting material.

SP0013 Biomineralization: the Mechanism of Crystal Formation in Bone and Other Mineralized Tissues. Considerations on the Relevance to Gout

Crystals of minerals and organic materials are deposited physiologically by the large majority of organisms. The deposition of carbonated apatite crystals in bone and teeth is probably the best known...

Comparative in vitro studies on disodium EDTA effect with and without Proteus mirabilis on the crystallization of carbonate apatite and struvite

Effect of disodium EDTA (salt of ethylenediamine tetraacetic acid) on the crystallization of struvite and carbonate apatite was studied. To evaluate such an effect we performed an experiment of struvite and carbonate apatite growth from artificial urine. The crystallization process was induced by Proteus mirabilis to mimic the real urinary tract infection, which usually leads to urinary stone formation. The results demonstrate that disodium EDTA exhibits the effect against P. mirabilis retarding the activity of urease – an enzyme produced by these microorganisms. The spectrophotometric results demonstrate that, with and without P. mirabilis, the addition of disodium EDTA increases the induction time and decreases the growth efficiency compared to the baseline (without disodium EDTA). These results are discussed from the standpoint of speciation of complexes formed in the solution of artificial urine in the presence of disodium EDTA. The size of struvite crystals was found to decrease in the presence of disodium EDTA. However, struvite crystals are larger in the presence of bacteria while the crystal morphology and habit remain unchanged.

Advanced yolk–shell hydroxyapatite for bone graft materials: kilogram-scale production and structure-in vitro bioactivity relationship

This paper introduces a facile method for synthesizing a new structured hydroxyapatite [HAp, Ca10(PO4)6(OH)2] material, named “yolk–shell”, by a simple spray drying process. The spray-dried precursor powders, consisting of Ca and P salts and dextrin, are transformed into powders of yolk–shell HAp by a simple combustion process under an oxygen atmosphere. For comparison, filled-structure HAp powders are prepared by spray pyrolysis in the absence of dextrin. A few small apatite crystals are found to be formed on the surfaces of the filled-structure HAp grains after 7 days of soaking in simulated body fluid (SBF). On the other hand, small acicular apatite crystals are observed on the yolk–shell grain surfaces after only 9 hours of soaking in SBF. The entire specimen surface is covered by numerous acicular and newly-formed hydroxyl carbonate apatite crystals after 1 day of soaking. These crystals are observed both at the outer and inner surfaces of the shell and the outer surface of the core. Inductively coupled plasma analysis shows that the dissolution of calcium and hydroxyl ions from yolk–shell HAp is notably increased compared with a filled-structure HAp. These results indicate that yolk–shell-structured HAp powders possess an enhanced in vitro bioactivity, which is encouraging for its potential use as a bone grafting material.

Ectopic calcification: importance of common nanoparticle scaffolds containing oxidized acidic lipids

The term nanobacteria, sometimes referred to as nanobacteria-like particles (NLPs), is presently recognized as a misnomer for inert calcified nanoparticles. However, misinterpretation of its propagation as a living organism still continues. Ultrastructural and elemental analyses, combining immuno-electron microscopy with an original NLP isolate (P-17) derived from urinary stones, and an IgM monoclonal antibody (CL-15) raised against P-17 have now revealed that, oxidized lipids with acidified functional groups were key elements in NLP propagation. Lamellar structures composed of acidic/oxidized lipids provided structural scaffolds for carbonate apatite crystals. During in vitro culture, lipid peroxidation induced by γ-irradiation of FBS was a major cause of accelerated NLP propagation. In pathological tissue samples from hyperlipidemic atherosclerosis-prone mice, CL-15 co-localized with fatty plaques, macrophage infiltrates and osteocalcin staining of aortic valve lesions. These observations indicate that naturally occurring NLP composed of mineralo-oxidized lipids complexes are generated as by-products rather than etiological agents of chronic inflammation. From the Clinical EditorThe term “nanobacteria-like particles (NLPs)” is presently recognized as a misnomer for inert calcified nanoparticles as opposed to living organisms. This study convincingly demonstrates that naturally occurring NLPs composed of mineralo-oxidized lipid complexes are generated as by-products rather than etiological agents of chronic inflammation.

Composition and morphological characteristics of sialoliths

Sialolithiasis refers to pathological minerilazation in the salivary glands and ducts. Aiming to a better understanding of the formation phenomena, structural and morphological analysis of a relatively large ensemble of sialoliths extracted from 22 patients via sielendoscopy was performed. Characterization methods, including Fourier Transform Infrared (FTIR) Spectroscopy, X‐Ray Diffraction, Raman spectroscopy and Scanning Electron Microscopy (SEM), showed that the large majority of 90% of sialoliths were composed of carbonate apatite as the inorganic phase, while SEM imaging revealed the presence of bacteria in 13 cases. Furthermore, carbonate apatite crystals in the form of parallel or randomly oriented sheets were observed and in some cases rhombohedral calcite‐type nanocrystals were detected. The present findings may contribute to a better understanding of the formation mechanisms in vivo and and contribute to the treatment of sialolithiasis.

Influence of disodium EDTA on the nucleation and growth of struvite and carbonate apatite

The effect of disodium EDTA, as an additive, on the crystallization of struvite and carbonate apatite was studied. The growth of struvite crystals and carbonate apatite occurred in the solution of artificial urine at 37°C and at the condition emulating real urinary tract infection. The results demonstrate that the addition of disodium EDTA increases the induction time and decreases the growth efficiency compared to the baseline (without disodium EDTA). The struvite crystal mean and median diameters were found to decrease in the presence of disodium EDTA but the crystal morphology and habit remain almost unchanged. Disodium EDTA has demonstrated its potential to be further investigated in the presence of bacteria and in vivo conditions.

In vitro bioactivity and degradability of β‐tricalcium phosphate porous scaffold fabricated via selective laser sintering

Porous scaffolds consisting of β-tricalcium phosphate (β-TCP) were successfully fabricated via selective laser sintering. The scaffolds had a controlled microstructure and totally interconnected porous structure. The microstructure and mechanical properties were studied. The bioactivity and degradability of scaffolds were evaluated through the simulated body fluid (SBF) cultivation experiment. The formation of a biologically active carbonate apatite layer on the surface after immersion in SBF was demonstrated using scanning electron microscope, energy dispersive X-ray, and Fourier transform infrared spectroscopy. Fast nucleation and growth of the carbonate apatite crystals were observed to occur all through the specimen surfaces. The phenomenon was explained in terms of the distribution and dispersion of inorganic phases in the scaffolds and the ionic activity products of the apatite in the SBF. The calculation results of weight loss and Ca/P molar ratio also suggest the good bioactivity and degradability of the scaffolds. These indicate that the β-TCP porous ceramic scaffold is a potential candidate scaffold for bone tissue engineering.

Gradient Organic Inorganic Nanocomposites for Tissue Repair at the Cartilage/Bone Interface

Damages to articular cartilage that are caused by trauma, age-related diseases (arthritis, arthrosis) and/or physical stress pose major medical problems. A possible solution is to introduce a biodegradable sponge-like scaffold containing cartilage-forming cells. In the current work we developed a model for a partially calcified functional biomedical membrane with a gradient of calcium phosphate crystal density to form the interface between bone and a sponge-like cell containing scaffold for cartilage regeneration. The membrane consists of a biocompatible, biodegradable, partially calcified hydrogel, in our case gelatin was used. One part is an organic-inorganic nanocomposite consisting of nanocrystalline calcium phosphate particles, formed in situ within the hydrogel, while the other part is the hydrogel without inorganic crystals. The experimental method used was one-dimensional single diffusion. Gelatin gels containing calcium or phosphate ions, respectively, were exposed from the upper side to a solution of the other constituent ion (i.e. a sodium phosphate solution was allowed to diffuse into a calcium containing gel and vice versa). Scanning electron microscopy (E-SEM), EDX, XRD and ATR-FTIR spectroscopy confirmed the existence within the gel of a density gradient of carbonate apatite crystals, with a dense top layer extending several microns into the gel. Ca/P atomic ratios were in the range characteristic of calcium deficient apatites. The effect of different experimental parameters on the calcification process within the gelatin membranes is discussed.

Bioactivity and Osteoconductivity of Biphasic Calcium Phosphates

Bioactivity and osteoconductivity of calcium phosphates as a function of ?-tricalcium phosphate contents, that is, biodegradability, were investigated. Three calcium phosphates, pure hydroxyapatite, biphasic calcium phosphates with different mixing ratios between hydroxyapatite, and ?-tricalcium phosphate (60HAp:40?- TCP and 40HAp:60?-TCP in wt.%), were synthesized through the precipitation method using calcium hydroxide and phosphoric acid as starting reactants. After drying, they were sintered at 1100 °C for 3 hours, and then bioactivity and osteoconductivity tests were carried out in simulated body fluid (SBF) and calvarial defect of New Zealand white rabbits, respectively. SBF exposure resulted in the deposition of a layer of carbonate apatite crystals on the surfaces of bicalcium phosphates but not on the hydroxyapatite. New bone forming capacity of the biphasic calcium phosphate with 60HAp:40?-TCP granules was the best, whereas that of the pure HAp was the worst. To this end, it can be concluded that the calcium phosphate with moderate biodegradability is good for producing good osteoconductivity.

Apatite Coating of Electrospun PLGA Fibers Using a PVA Vehicle System Carrying Calcium Ions

A novel method to coat electrospun poly(D,L-lactic-co-glycolic acid) (PLGA) fiber surfaces evenly and efficiently with low-crystalline carbonate apatite crystals using a poly(vinyl alcohol) (PVA) vehicle system carrying calcium ions was presented. A non-woven PLGA fabric was prepared by electrospinning: a 10 wt% PLGA solution was prepared using 1,1,3,3-hexafluoro-2-propanol as a solvent and electrospun under a electrical field of 1 kV/cm using a syringe pump with a flowing rate of 3 ml/h. The non-woven PLGA fabric, 12 mm in diameter and 1 mm in thickness, was cut and then coated with a PVA solution containing calcium chloride dihydrate (specimen PPC). As controls, pure non-woven PLGA fabric (specimen P) and fabric coated with a calcium chloride dihydrate solution without PVA (specimen PC) were also prepared. Three specimens were exposed to simulated body fluid for 1 week and this exposure led to form uniform and complete apatite coating layer on the fiber surfaces of specimen PPC. However, no apatite had formed to the fiber surfaces of specimen P and only inhomogeneous coating occurred on the fiber surfaces of specimen PC. These results were explained in terms of the calcium chelating and adhesive properties of PVA vehicle system. The practical implication of the results is that this method provides a simple but efficient technique for coating the fiber surface of an initially non-bioactive material with low-crystalline carbonate apatite.

Influences of electrolytes and glucose on formulation of carbonate apatite nanocrystals for efficient gene delivery to mammalian cells

Genetic manipulation of human cells through delivery of a functional gene or a gene-silencing element is an attractive approach to treat critical diseases very precisely and effectively. Extensive research on the genetic basis of human diseases with complete sequencing of human genome has revealed many vital genes as possible targets in gene therapy programs. On the other hand, to facilitate cell- or tissue-directed delivery of genes and gene-silencing nucleic acid sequences, both genetic and chemical engineering approaches have led to the generation of various viral and nonviral carriers. However, considering the issues of both safety and efficacy, none of the existing vectors is an ideal candidate for clinical use. We recently established pH-sensitive inorganic nanocrystals of carbonate apatite with capability of efficient intracellular delivery and release of associated DNA molecules for subsequent protein expression. Here we show a new synthetic approach for carbonate apatite crystals with stronger affinity toward DNA, leading to significant increment in both transgene delivery and expression. Moreover, CaCl2 and NaCl, existing as the major electrolytes in the bicarbonate-buffered solution, dose-dependently govern particle size and eventually internalization and expression of particle-associated DNA.

In vitro and in vivo evaluation of the effects of demineralized bone matrix or calcium sulfate addition to polycaprolactone–bioglass composites

The objective of this study was to improve the efficacy of polycaprolactone/bioglass (PCL/BG) bone substitute using demineralized bone matrix (DBM) or calcium sulfate (CS) as a third component. Composite discs involving either DBM or CS were prepared by compression moulding. Bioactivity of discs was evaluated by energy dispersive X-ray spectroscopy (ESCA) and scanning electron microscopy (SEM) following simulated body fluid incubation. The closest Calcium/Phosphate ratio to that of hydroxyl carbonate apatite crystals was observed for PCL/ BG/DBM group (1.53) after 15 day incubation. Addition of fillers increased microhardness and compressive modulus of discs. However, after 4 and 6-week PBS incubations, PCL/BG/DBM discs showed significant decrease in modulus (from 266.23 to 54.04 and 33.45 MPa, respectively) in parallel with its highest water uptakes (36.3 and 34.7%). Discs preserved their integrity with only considerable weight loss (7.5-14.5%) in PCL/BG/DBM group. In vitro cytotoxicity tests showed that all discs were biocompatible.

Superplastic Deformation in Carbonate Apatite Ceramics under Constant Compressive Loading for Near-net-shape Production of Bioresorbable Bone Substitutes

To produce carbonate apatite (CAP) ceramics with the desired complex shapes using superplastic deformation, deformation behavior of CAP ceramics under constant loading as well as physical properties after deformation were evaluated. Sintered CAP ceramics were plastically deformed in an electric furnace attached to a universal hydraulic testing machine under a constant load. CAP ceramics subjected to an initial compressive pressure of 10 MPa showed an appreciable amount of plastic deformation at temperatures ranging from 720 to 800 degrees C. Plastic deformation increased with increasing temperature from about 10% to 70% after two hours of loading. X-ray diffraction analysis and SEM observation further revealed that some CAP crystals were elongated and aligned with the c-axis normal to the loading direction during superplastic deformation. It was thus concluded that a marked plastic deformation of about 70% at 800 degrees C would be sufficient for near-net-shape production of bioresorbable CAP bone substitutes with complex shapes.

The associations between mineral crystallinity and the mechanical properties of human cortical bone

It is well known that the amount of mineralization renders bone its stiffness. However, besides the mere amount of the mineral phase, size and shape of carbonated apatite crystals are postulated to affect the mechanical properties of bone tissue as predicted by composite mechanics models. Despite this predictive evidence, there is little experimental insight on the relation between the characteristics of mineral crystals and hard tissue mechanics. In this study, Raman spectroscopy was used to provide information on the crystallinity of bone's mineral phase, a parameter which is an overall indicator of mineral crystal size and stoichiometric perfection. Raman scans and mechanical tests (monotonic and fatigue; n = 64 each) were performed on the anterior, medial, lateral and posterior quadrant sections of 16 human cadaveric femurs (52 y.o.–85 y.o.). The reported coefficient of determination values ( R 2) were adjusted for the effects of age to bring out the unbiased contribution of crystallinity. Crystallinity was able to explain 6.7% to 48.3% of the variation in monotonic mechanical properties. Results indicated that the tissue-level strength and stiffness increased with increasing crystallinity while the ductility reduced. Crystallinity explained 11.3% to 63.5% of the variation in fatigue properties. Moduli of specimens with greater crystallinity degraded at a slower rate and, also, they had longer fatigue lives. However, not every anatomical quadrant displayed these relationships. In conclusion, these results acknowledge crystal properties as an important bone quality factor and raise the possibility that aberrations in these properties may contribute to senile osteoporotic fractures.

Carbonate apatite crystals in primary normophosphatemic tumoral calcinosis

AbstractA 66‐year‐old woman developed firm, painless, slowly growing nodular masses over her elbows, fingers, toes, and left hip over four years. Aspiration of the elbow mass revealed a white chalky material that was shown to be carbonate apatite on infrared spectroscopy and energy dispersive X‐ray spectroscopy. We discuss the classification of tumoral calcinosis and the nature of the calcium deposits. Tumoral calcinosis should be differentiated from tophaceous gout and calcium pyrophosphate dihydrate crystal deposition disease. Polarizing light microscopy and crystal analysis by X‐ray and infrared spectroscopy, electron or X‐ray diffraction will confirm the diagnosis. Secondary causes of tumoral calcinosis should also be excluded.

Supramolecular Assembly of Amelogenin Nanospheres into Birefringent Microribbons

Although both tooth enamel and bone are composed of organized assemblies of carbonated apatite crystals, enamel is unusual in that it does not contain collagen nor does it remodel. Self-assembly of amelogenin protein into nanospheres has been recognized as a key factor in controlling the oriented and elongated growth of carbonated apatite crystals during dental enamel biomineralization. We report the in vitro formation of birefringent microribbon structures that were generated through the supramolecular assembly of amelogenin nanospheres. These microribbons have diffraction patterns that indicate a periodic structure of crystalline units along the long axis. The growth of apatite crystals orientated along the c axis and parallel to the long axes of the microribbons was observed in vitro. The linear arrays (chains) of nanospheres observed as intermediate states before the microribbon formation give an important indication as to the function of amelogenin in controlling the oriented growth of apatite crystals during enamel mineralization.

Interactions of amelogenins with octacalcium phosphate crystal faces are dose dependent.

Amelogenins, the major protein components of the enamel extracellular matrix, are postulated to be involved in controlling the elongated and oriented growth of enamel carbonated apatite crystals. In order to clarify the functional role of amelogenin during the early stage of enamel biomineralization, octacalcium phosphate (OCP) crystals, known to be potent precursors of hydroxyapatite, were grown in 1-10% (w/w) native bovine and two recombinant murine amelogenins. Amelogenins were solution-like at 1% and formed gel at 10%, while 5% amelogenins became gel after reaction and it was inhomogeneous and porous. Morphological changes of OCP crystals were evaluated as the function of amelogenin concentration by analyzing the mean values of length, width, thickness, their reduction ratios (L/Lc, W/Wc, T/Tc) as well as L/W and W/T ratios. Length, width, and thickness decreased in a does-dependent manner. Length decreased almost linearly in 1%-10%, whereas width decreased drastically in 1%-5% while the decrease from 5% to 10% was small. As a result, elongated morphology of OCP crystal was most emphasized in 5% bovine amelogenins and rM166 and 2%-5% rM179. The size reduction was in the order of W/Wc < L/Lc < T/Tc. We therefore concluded that amelogenin interaction with crystal faces was in the order (010) > (001) > (100). At all concentrations, W/ Wc was significantly the smallest. This indicated that the primary role of amelogenin was to decrease the width of OCP by blocking the hydrophobic (010) faces. We suggest that the drastic decrease of crystal width is the result of interaction of the densely packed nanospheres in 5%-10% amelogenin.