Growth Of Apatite Crystals Research Articles

Functional Significance of Pyrophosphate in Discrete Forms in Mineralizing Milieus.

The current work aimed to gain a better insight into the molecular states and functional significance of pyrophosphate ions (PPi) in media assimilated to human saliva. PPi exhibited complete inhibition of apatite crystal growth in a concentration range of 10-6M or higher. Experimental data also showed that PPi at corresponding concentrations could evoke precipitation of calcium pyrophosphate dihydrate (Ca2P2O7·2H2O) in the presence of 1mM Ca2, giving rise to the loss of inhibitory activity. Fluoride at concentrations around 1ppm counteracted the inhibition of apatite precipitation by PPi. It is conceivable that increasing the driving force for apatite growth after fluoride addition allowed a growing step to squeeze between the adsorbed PPi molecules and then bury them in the lattice. On the basis of currently available information, we propose a delineated model to explain the behavior of PPi at the crystalsolution boundary in mineralizing milieus.

Improvements of Apatite-Forming Abilities on Pure and Sodium-Containing Diopside Substrates Using Porous Diopside Thin Films as Nucleating Agent

AbstractPure diopside, CaMgSi2O6 and the partly modified new type of crystalline diopside, NaxCa1-xMgSi2O6, were prepared mainly by coprecipitation process These ceramics were synthesized from the inorganic nitrates and the metal alkoxide in ethanol solutions. The sodium contents in the crystal had remarkable effect on the apparent densities and improved the mechanical properties, especially when the hot-isostatic pressing was applied in the final process. The precise measurements of the X-ray diffraction patterns strongly suggested that the Ca positions in the unit cell were partly substituted by the Na atoms. SBF soaking tests showed that the porous sol-gel method diopside induced the higher apatite crystal growth rates than the hot-pressed surface of the pure or the Na-modified diopside.The surface of the coprecipitation process diopside specimens was coated with the porous ceramics for the purpose of the formation of the bioactive diopside films.

Interaction of amelogenin with hydroxyapatite crystals: an adherence effect through amelogenin molecular self-association.

At the secretory stage of tooth enamel formation the majority of the organic matrix is composed of amelogenin proteins that are believed to provide the scaffolding for the initial carbonated hydroxyapatite crystals to grow. The primary objective of this study was to investigate the interaction between amelogenins and growing apatite crystals. Two in vitro strategies were used: first, we examined the influence of amelogenins as compared to two other macromolecules, on the kinetics of seeded growth of apatite crystals; second, using transmission electron micrographs of the crystal powders, based on a particle size distribution study, we evaluated the effect of the macromolecules on the aggregation of growing apatite crystals. Two recombinant amelogenins (rM179, rM166), the synthetic leucine-rich amelogenin polypeptide (LRAP), poly(L-proline), and phosvitin were used. It was shown that the rM179 amelogenin had some inhibitory effect on the kinetics of calcium hydroxyapatite seeded growth. The inhibitory effect, however, was not as destructive as that of other macromolecules tested. The degree of inhibition of the macromolecules was in the order of phosvitin > LRAP > poly(L-proline) > rM179 > rM166. Analysis of particle size distribution of apatite crystal aggregates indicated that the full-length amelogenin protein (rM179) caused aggregation of the growing apatite crystals more effectively than other macromolecules. We propose that during the formation of hydroxyapatite crystal clusters, the growing apatite crystals adhere to each other through the molecular self-association of interacting amelogenin molecules. The biological implications of this adherence effect with respect to enamel biomineralization are discussed.

Effect of Apatite Crystals on the Activity of Amelogenin Degrading Enzymes In Vitro

The objective of the present study was to determine the effect of apatite crystals on the activity of amelogenin degrading enzymes in vitro. Current experimental data, together with previous reports support the view that among the different proteinases present in the enamel extracellular matrix, serine proteinase(s) are responsible for the massive degradation of amelogenins during the maturation stage. For our in-vitro experiments we used the recombinant amelogenin M179 as substrate and a “65%-satd. (NH4)2SO4” fraction of enamel proteins as well as chymotrypsin as sources for serine-proteinase activity. We report preliminary experiments of amelogenin proteolysis in the presence of apatite crystals resulting in a different proteolysis pattern when compared to amelogenin proteolysis without apatite crystals. Quantitative analysis of the HPLC peaks corresponding to the proteolysis products indicates that the presence of apatite crystals in the proteolysis solution inhibits the ability of the serine-proteinases to degrade amelogenin. The present observations support the hypothesis that amelogenin degradation correlates with apatite crystal growth during enamel maturation.

PH and Carbonate Levels in Developing Enamel

Our earlier studies showed that the surface of developing and calcifying enamel changes its pH alternatively along the tooth axis when stained with pH indicating dyes. Based on the pH conditions, the enamel at this stage was distinguished as neutral zone (N1 and N2) and acid zone (A1 and A2). The aim of the present study was to correlate changes of pH with proteolytic activity and crystal size of the calcifying bovine enamel. Specimens of developing bovine enamel were separated into four maturing stages using pH staining methods. Crystal chemistry of the developing enamel was investigated using thermo-gravimetry (TGA), ICP emission spectrometry, X-ray diffractometry (XRD) and infrared spectroscopy (IR). Previous biochemical analysis of proteolytic enzyme activity from enamel indicated that the optimal pH of the major protease was approximately pH 6.0, coinciding with the pH of the Al zone. IR, TGA and XRD analyses showed that most of the organic components of the enamel decomposed at 580°C. Higher levels of carbonate were observed in the secretory stages than in mature enamel. The Ca/P molar ratio of the enamel apatite was lower than the stoichiometric value of 1.67. These results suggest that growth and maturation of enamel apatite crystals is related to a decrease in the carbonate level and appear to be related to the alternative calcification and decomposition of enamel proteins.

アパタイト結晶の成長初期過程の動的光散乱法による研究,その1 : 溶液成長III

アパタイト結晶の成長初期過程の動的光散乱法による研究,その1 : 溶液成長III

Uptake and metabolism of albumin by rodent incisor enamel in vivo and postmortem: implications for control of mineralization by albumin.

The distribution of albumin throughout enamel development in the rat mandibular incisor was investigated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) and Western blotting employing an anti-rat albumin antibody. Intact albumin was detectable at all stages of enamel development but was most evident during late secretion/transition. Its concentration was subsequently reduced during the maturation stage. Albumin degradation products appeared during the transition/early maturation stage indicating that albumin breakdown preceded its removal. As albumin inhibits apatite crystal growth, its degradation and removal may be a necessary prerequisite for normal enamel crystal growth, perhaps reflecting a general mechanism for removal of residual endogenous matrix or adventitious crystal growth inhibitors. Additional studies revealed that the maturation stage was particularly susceptible to albumin influx postmortem. Albumin could therefore form part of the natural crystal growth control process, which, if not removed, could hamper maturation and lead to white spot hypoplasias.

Studies of the Levamisole inhibitory effect on rat stromal‐cell commitment to mineralization

The ability of Levamisole to decrease mineralization in skeletal tissue is usually related to its effect on alkaline phosphatase (ALP). However, Levamisole is also suspected to diminish mineralization by an additional mechanism which is unrelated to the ALP control of apatite crystal growth. To delineate the time in differentiation during which Levamisole inhibits mineralization, a tissue culture model system of bone marrow stromal cells was used. Secondary cultures of stromal cells were propagated in osteoprogenitor cell (OPC) induction medium for three weeks, followed by measurement of calcium precipitation. In situ ALP assays at pH 7.6 were also performed. When cells were cultured with 0.2 mM Levamisole for three weeks, Day 20 values of calcium precipitates were lower than in controls, but Day 20 ALP values were paradoxically higher. The correlation between calcium and ALP within each group was low. The correlation slightly improved, in uninhibited cultures, when Day 21 calcium values were matched with earlier Day 12 ALP values. This suggested the existence of a Levamisole-sensitive mechanism for mineralization inhibition effective prior to the culture's mineralization stage. To focus on this early effect on mineralization Levamisole was added to stromal cultures on different days and removed on Day 12. Levamisole decreased Day 21 mineralization when added on Days 0, 3, 5, and 7, but not when added on Day 9. The Levamisole-induced inhibition of mineralization was accompanied by an increase in Day 12 ALP specific activity, compared to controls, when added from Day 5 and thereafter. The results indicate that part of the ability of stromal cells to mineralize is determined during the first week of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Human amelogenesis: high resolution electron microscopy of nanometer-sized particles.

Growth of inorganic crystals of enamel is described as a two-stage process with growth of ribbon-like crystals in length and width, followed by their development in thickness. In early stages of crystal growth during human amelogenesis nanometer-sized particles with a mean diameter of 1.1 nm were described between ribbon-like crystals. These small particles had a crystalline structure but their lattice parameters did not seem to be directly related to those of calcium phosphates. The nanometer-sized particles appear to correspond to initial stages of apatite crystal growth. Their localization close to ribbon-like crystals and their progressive increase in size and number may indicate that they represent a precursor phase for these crystals. Nucleation areas at both extremities, of elongated ribbon-like crystals could be involved in the two-directional growth of ribbons and/or in nanometer-sized particle nucleation.

Organoapatites: Materials for artificial bone. III. Biological testing

This article reports on the in vivo testing of new artificial bone materials we have termed "organoapatites." These materials consist of mineral networks in which organic polymers are intimately dispersed by nucleation and growth of apatite crystals from a mother liquor containing the organic substances. Organoapatites were tested as implants in adult canine cortical bone for periods in the range from 12-35 weeks and fluorochromes were used in the model to investigate the kinetics of bone growth or repair. The analysis of histological samples was carried out using histomorphometric methods as well as fluorescence microscopy. Results showed excellent apposition of poly(amino acid) organoapatites with mineralized bone and fibrous encapsulation when a synthetic polyelectrolyte was the only organic component. This observation suggests that the molecularly dispersed organic dopant amounting to only 2-3% by weight of the microstructure can play a critical role in the tissue response to the implant. Relative to apatite controls, organoapatites were also found to have greater resistance to fragmentation in vivo and those containing amino acid units revealed interfacial bioerosion accompanied by regeneration of mineralized tissue. Design of organoapatite compositions and microstructures may therefore be useful in achieving the specific rate of biological response which is clinically desired.

Organoapatites: materials for artificial bone. I. Synthesis and microstructure.

We have synthesized a new family of materials we termed organoapatites which may be useful in the formulation of artificial bone. These materials are synthesized by nucleation and growth of apatite crystals in media containing poly(amino acids) or synthetic organic polyelectrolytes using strict atmospheric, temperature, and pH control. The macromolecules used to synthesize the organoapatites include poly(L-lysine), poly(L-glutamic acid), and poly(sodium acrylate). The products were characterized by x-ray diffraction, scanning electron microscopy, surface area measurements, elemental analysis, and spectroscopic techniques. Organoapatites were found to contain large surface area morphologies with small crystallites which mature slowly based on analysis of Ca/P ratios. The organic macromolecules are thought to induce nucleation of crystals but also to quench their growth, thus becoming intimately dispersed in a mineral network. The organomineral particles harvested from the reaction medium contain polymer-netted microcrystals, and for this reason the synthetic approach can be used to modulate crystal maturation and biological response. It is likely that the preparative approach mimics some aspects of natural bone matrix synthesis and could be specially useful in the preparation of mineral implants containing intimate dispersions of small amounts of biomolecules such as growth factors, special drugs, or bioadhesives.

Cristallographic behaviour of fluoridated hydroxyapatites containing Mg 2+ and CO 2−3 ions

Fluoridated hydroxyapatites containing small amounts of magnesium and carbonate ions were synthesized at 80 and 60° C to examine their inhibiting properties regarding apatite crystal growth, in contrast to the promoting action of fluoride. The shortening of a-axis and c-axis dimensions of the apatite crystals, as revealed by X-ray diffraction analysis, suggested that both magnesium and carbonate ions were substituted into the apatite crystals. The a-axis dimensions also decreased with the degree of fluoridation. The infrared spectra due to CO 2− 3 ions at 875 cm −1 were shifted with increasing fluoride content. The overall crystallinity was inhibited in comparison with that of Mg and CO 3-free fluoridated hydroxyapatites, but recovered considerably with increased fluoride content. The apparent solubility of the apatites at pH 4.0 and 37°C was higher than that of Mg and CO 3-free fluoridated hydroxyapatites at lower fluoride contents, but gradually approached the latter at higher fluoride content. After 1 month's incubation, dicalcium phosphate dihydrate was formed from fluoride-free Mg-CO 3 apatite synthesized at 60°C.

Ultrastructural studies on crystal growth of enameloid minerals in elasmobranch and teleost fish

The composition and morphology of crystals formed in fish enameloid were investigated at various developmental stages. Species studied were shark, skate, red seabream, puffer, and carp. For comparative purposes, mammalian enamel samples were obtained from developing porcine teeth and erupted human teeth. Chemical and physical analyses (FTIR, X-ray diffraction, and electron microprobe) indicated that the mineral phase of enameloid in elasmobranch and teleost fish was most adequately characterized as fluoridated carbonatoapatites but that the degree of fluoridation and carbonation of the apatite latice varied among species and, within species, with the developmental stage. High resolution electron microscopy demonstrated differences in the nature and morphology of the initially precipitating crystallites of the enameloids of elasmobranch as compared with those of teleost fish. The elasmobranch enameloid contained high levels of fluoride (2.5% wt or more) at the beginning of precipitation and its mineralization was characterized by the initial formation and subsequent growth of prismatic apatite crystals having hexagonal (frequently equilateral) cross-sectional areas. In contrast, the initially precipitating crystallites in the teleost fish appeared as thin ribbons, like those commonly reported in mammalian enamel. The crystal morphology of the teleost fish enameloid may be related to the low fluoride contents maintained in the early stages of enameloid formation. However, the growth process of enameloid crystallites varied within the teleostei depending on the fluoride accretion during the mineralization stages. In the seabream enameloid (the highfluoride group), the growth on the side planes of the apatitic prisms was accelerated with increasing fluoride concentration in the tissue; the resulting crystallites had equilateral hexagonal cross sections. The morphology and growth of enameloid crystallites in puffer and carp (the low-fluoride group) were similar to those reported in mammalian enamel. However, appreciable differences existed between the enameloid of this fish group and the mammalian enamel with respect to carbonation, fluoridation, and central defects of their crystallites. The overall results support the contention that fluoride significantly affects the morphology and structure of enameloid crystals, most probably by increasing the driving force for carbonatoapatite formation and by accelerating hydrolysis of possible acidic precursors.

Effect of Solution Composition on Morphological and Structural Features of Carbonated Calcium Apatites

The composition of enamel mineral corresponds to that of a calcium carbonato-apatite. For insight to be gained into the precipitation of carbonato-apatites having specific properties (crystal size, morphology, and carbonate incorporation into the crystal lattice), apatites were prepared at 80 degrees C in aqueous systems having various CO3 concentrations and pH values of around 7.5 or 10.5 (+/- 0.5). The various preparations had a wide range (0.005 to 0.19) of CO3/Ca molar ratios that bracket the ratios found in porcine enamel mineral at various developmental stages. Fourier transform infrared spectroscopy (FTIR) and x-ray diffraction analyses showed that the calcium apatites precipitating at neutral pH incorporated the carbonate into both the hydroxyl and phosphate ion sites in their lattices (A,B-types), whereas the preparations made at the alkaline pH (high OH-(-)CO3(2-)-competition) or in the presence of fluoride (F-(-)CO3(2-) competition) yielded only the B-type carbonato-apatite. It was also ascertained that the size and morphology of the carbonato-apatites, assessed by specific surface area determination and high-resolution electron microscopy, were highly dependent on the driving force for precipitation and the presence of regulators (CO3(2-) and F-) in solution. In neutral media, early precipitates were thin-ribbon in appearance, but grew into crystals having flattened-hexagonal cross-sections. In the presence of fluoride or in alkaline media, acicular apatite crystals, precipitated initially, grew into large rod-like carbonato-apatites having a symmetric-hexagonal cross-section. In both neutral and alkaline solutions, carbonate inhibited the growth of apatite crystals along their c axis, leading to the formation of bulkier crystals. The formation of carbonato-apatites at the neutral pH and their properties are consistent with observations made on enamel minerals formed in the early developmental stages.

High-Resolution Electron Microscopy Study of Enamel Crystal Growth in Human Foetus

The mineral component of human dental enamel is formed by nonstoichiometric carbonated hydroxyapatite crystals. The presence of relatively large amounts of impurities in mature enamel crystals as well as their external shape are related to their growing mechanism. Enamel crystal growth was investigated previously directly with low resolution electron microscopy or by comparison with synthetic apatite crystal growth. The growing process of enamel crystals is still unknown and the aim of this study was to investigate foetal enamel growth during secretory stage by high resolution transmission electron microscopy.Developing enamel from 5 month-old human foetuses was fixed for 3 hours in a 2% glutaraldehyde and 2% paraformaldehyde solution in 0.1 M sodium cacodylate buffer. After 2 hour postfixation in 1% OsO4in the same buffer and embedding in Epon, non decalcified ultrathin sections were obtained with a Sorvall-Porter MT-2C microtome equipped with a diamond knife and floated on demineralized water saturated against hydroxyapatite (200mg/l). The sections were observed in a Philips EM 430 transmission electron microscope operating at 300 kV equipped with a double tilt specimen holder. This microscope possessed a Sherzer resolution of 0.19 nm and the absence of drift and astigmatism was checked for each micrograph by an optical diffraction.

Growth and Fusion of Apatite Crystals in the Remineralized Enamel

The present study was caried out to observe the process of growth and fusion of apatite crystals in human enamel remineralized in vitro in a solution containing Ca, 1 mM; P, 0.6 mM; and F, 0.05 mM. The growth of newly formed crystals and the regrowth of pre-existing enamel crystals occurred extensively in remineralized enamel. With advancing growth, the crystals came into contact and fused with each other, forming large crystals with hexagonal outlines. Various kinds of crystalline defects, including edge dislocation, low-angle grain boundary, and lattice shifting, were frequently detected between the fusing crystals. X-ray diffraction and electron-probe studies led to the assumption that the regular-hexagonal crystals were fluoroapatite

Ultrastructure, Morphology and Crystal Growth of Biogenic and Synthetic Apatites

The morphology, structure and crystal growth of apatite crystals isolated from calcified turkey tendon and synthetic carbonated apatites have been examined using high resolution transmission electron microscopy. The biogenic apatite consisted of small (35 x 20 x 5 nm) platelike crystals. Despite their irregular shape and ill-defined edges, individual particles were single domain crystals. Lattice images recorded from isolated turkey tendon crystals indicated that the crystallographic c-axis (0001) of apatite lies in the plane of the plate and parallel to the length of the crystallites. Lattice images suggested that the top face corresponds to the (1100) face of carbonated apatite. Lattice fringes observed in platelike crystallites viewed from the side corresponded to the projection of the apatite structure viewed along the [1120] direction. Thus, it can be argued that crystal growth is constrained along the [1100] direction, extends laterally along the [1120] direction, and is maximal along the [0001] direction. This latter direction is aligned with the collagen fiber axis. A mean length to width ratio (1.7) was determined by systemically measuring the maximum distances parallel and perpendicular to the c-axis identified from lattice images of the crystals. Similar information was obtained from lattice images of crystals located in collagen fibres. This confirmed that the morphological and structural features of isolated turkey tendon apatite crystals correlate directly with the in vivo crystallochemical characteristics of apatite. Crystals of synthetic carbonated apatite prepared at 37 degrees C were also platelike and, although generally much larger, had length to width ratios comparable with the turkey tendon apatite. The synthetic carbonated apatites were noticeably more sensitive to radiolytic damage than the turkey tendon crystals. The crystallographic c-axis of the inorganic particles was aligned parallel with the long, physical axis of the plate and the top face was identified as (1100). Similar data were also obtained from noncarbonated synthetic apatite samples. The results of the present study offer critical information about the crystal growth of individual carbonated apatite crystals in calcified turkey tendon and its relationship to the morphology of the crystallites. As similar growth characteristics are expressed in synthetic analogues, the data bring into question the putative regulatory role of the collagen-based matrix upon the nucleation and growth of biogenic apatite.(ABSTRACT TRUNCATED AT 400 WORDS)

Possible roles of partial sequences at N- and C-termini of amelogenin in protein-enamel mineral interaction.

The purpose of this study was to assess the functional significance of homologous sequences of mammalian amelogenins at their N- and C-termini. A porcine 5-kDa fragment corresponding to the N-terminal 45 residues of amelogenins was purified from the secretory enamel. The decapeptide TDKTKREEVD corresponding to the C-terminal 10 residues of amelogenins was synthesized according to conventional solid-phase procedures. The inhibitory activity of both moieties on apatite crystal growth was determined in a supersaturated solution having an ionic composition similar to that of the fluid phase separated from porcine secretory enamel. The 5-kDa amelogenin fragment was sparingly soluble in neutral solutions and (in condensed forms because of aggregation) showed no significant inhibition of crystal growth, whereas the fragment molecules pre-adsorbed onto the seed crystals yielded modest inhibition of hydroxyapatite precipitation. However, their inhibitory activity was significantly lower than that of parent porcine amelogenin (25-kDa molecular mass). The high solubility of synthesized decapeptide allowed us to determine the adsorption isotherm onto hydroxyapatite at 37 degrees C, at an ionic strength similar to that of the enamel fluid. The obtained adsorption isotherm was described by a Langmuir model; the adsorption affinity and the maximum adsorption sites were 6.2 mL/mumol and 0.53 mumol/m2, respectively. As expected from the low adsorption affinity, the peptide showed a much weaker inhibition of apatite crystal growth than the parent amelogenin.(ABSTRACT TRUNCATED AT 250 WORDS)

Crystal deposition in hypophosphatasia: a reappraisal.

Six subjects (three female, three male; age range 38-85 years) with adult onset hypophosphatasia are described. Three presented atypically with calcific periarthritis (due to apatite) in the absence of osteopenia; two had classical presentation with osteopenic fracture; and one was the asymptomatic father of one of the patients with calcific periarthritis. All three subjects over age 70 had isolated polyarticular chondrocalcinosis due to calcium pyrophosphate dihydrate crystal deposition; four of the six had spinal hyperostosis, extensive in two (Forestier's disease). The apparent paradoxical association of hypophosphatasia with calcific periarthritis and spinal hyperostosis is discussed in relation to the known effects of inorganic pyrophosphate on apatite crystal nucleation and growth.

High resolution electron microscopy of nonstoichiometric apatite crystals.

The application of high resolution electron microscopy, computer image processing, and image simulation techniques to the investigation of synthetic nonstoichiometric apatites has provided new details of apatite crystal growth mechanisms. Under certain precipitation conditions, calcium-deficient apatites with distinct octacalcium phosphate (OCP)-apatite intergrowths have been observed. Apatite crystals with unit-cell thick overgrowths of OCP on their surfaces confirmed the stepwise hydrolysis crystal growth mechanism initially proposed by Brown (Nature 196:1048-1050). However, many crystals also contained a central two-dimensional OCP inclusion one to two unit cells thick, embedded in an apatite matrix. Similar planar defects have been observed in dental enamel, dentin, and bone crystals. We have developed a modified version of Brown's stepwise OCP hydrolysis apatite crystal growth mechanism to explain the formation of crystals with OCP central planar defects. The mechanism involves the nucleation of an OCP seed that grows until it reaches a critical size, rh, before OCP hydrolysis occurs. Apatite subsequently grows epitaxially on the OCP seed, thereby embedding it in the center of an apatite crystal. Apatite growth is facilitated by partial screw dislocations emanating from the planar defect.