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PubMed1976

Transmission microscopy of freeze dried, unstained epiphyseal cartilage of the guinea pig.

The question of the initial mineralization in the epiphyseal plate has been investigated to date in specimens prepared by conventional electron microscopical techniques. As conventional techniques can produce artifacts, either a loss of mineral substance or a secondary nucleation, the mineralization process was investigated using freeze dried, vacuum embedded growth cartilage which was neither contrasted not stained and which had a very short contact with water. The prevailing theory that the first mineralization begins within extracellular matrix vesicles and that the mineralization outside these vesicles is a secondary process was confirmed. Mineralized matrix vesicles were found in the fully mineralized long septa down to the opening zone. In several cases a mineralization could be observed in those transverse septa in which organic substance was present between the cells. The typical radial arrangement of the apatitic needles and platelets in the matrix vesicles could be explained by the formation of a mineralization in an ionotropic gel, the orientation of the matrix macromolecules to be produced by a vectorial influx of calcium ions and phosphate groups coming from different directions. Thin strands of mineral substance with low contrast, which follow the direction of the longitudinal septum, were assumed to be the mineralized collagen fibrils. In several needles dot-like formations were seen and the distance between the middle of neighbouring dots was found to lie mainly in the range 30-56 A, while the lateral separation distance between the middle of closely packed parallel chains and needles was found to lie mainly in the range 30-42 A. Parallel periodic structures which could be visualized in apatitic chains and needles 20-40 A in diameter were assumed to be the 8.2 A-(100)-lattice planes of apatite, being an indication that these formations already possess criteria of the apatite lattice.

Animals↗

PubMed1992

An FT-IR microscopic investigation of the effects of tissue preservation on bone.

Fourier transform infrared microscopy is a powerful tool for the characterization of mineral and protein in histologic sections of bone. This study was concerned with determining whether techniques used to preserve these tissues and to prepare them for sectioning had an effect on spectral properties. The nu 1, nu 3 phosphate bands in the 900-1200 cm-1 spectral region were used to evaluate the structure of the apatitic mineral in fresh-frozen, ethanol-fixed, and formalin-fixed 35-day-old rat femurs; fresh-frozen and formalin-fixed 20-day-old fetal rat femurs; ground 35-day-old rat diaphyseal bone samples; and formalin-fixed, methacrylate-embedded ground diaphyseal bone. The crystallinity (crystal size and perfection) of the bone apatite was assessed by a curve-fitting analysis of the nu 1, nu 3 phosphate bands. Results indicate that ethanol or formalin fixation of the 35-day-old intact rat femur, and formalin fixation and embedding of the ground rat bone do not significantly alter the crystallinity of the apatite. However, formalin fixation of the fetal rat bone did alter the structure of the apatite mineral phase. In addition, evaluation of protein secondary structure in the 35-day-old rat femur from the Amide I and Amide II vibrations near 1650 and 1550 cm-1, respectively, revealed that protein conformation was altered by ethanol fixation.

Amides↗

PubMed1992

Calcium biomineralization in the radular teeth of the chiton, Acanthopleura hirtosa.

A method has been devised for isolating the calcium biomineral from the iron biominerals and organic components present in the major lateral teeth of the chiton Acanthopleura hirtosa. Fourier-transform infrared spectroscopy of the calcium biomineral indicated that it was an apatite material containing carbonate and fluoride ions. Carbonate was not found to be present as a separate phase. The apatite was further separated into low and high density fractions, both of which showed crystallinity intermediate between that of bovine tibia cortical bone and human tooth enamel, as indicated by powder X-ray diffraction analysis. The calcified region of the major lateral teeth was also studied in situ using transmission electron microscopy and electron diffraction analysis, revealing a close spatial relationship between the mineral apatite phase and underlying organic matrix. It is suggested that the architectural arrangement of apatite biomineral and fibrous organic constituents imparts specialized mechanical properties to the tooth making it ideally suited for the task of obtaining food from hard surfaces.

Animals↗

PubMed1977

An X-ray crystallographic examination of calcium phosphate formation in Ca(OH)2/H3PO4 mixtures.

Precipitates formed over a 10 day period by adding variable amounts of Ca(OH)2 to H3PO4 were examined chemically and by X-ray diffraction in order to determine and to relate the crystallographic changes observed during calcium phosphate formation in this system to those seen previously in dental plaque. In the lower Ca(OH)2/H3PO4 ratio mixtures the initial precipitates were amorphous or poorly crystalline. The latter deposits showed an apatite-like diffraction pattern which changed to brushite and decreased in Ca/P ratio from approximately 1.2-1.0. Since the amorphous precipitates showed a similar decrease in the Ca/P ratio and a transient appearance of apatite, it was suggested that these initial precipitates, like the poorly crystalline deposits, may contain brushite amorphous to X-rays. Precipitates from the higher Ca(OH)2/H3PO4 ratio mixtures changed from a poorly crystalline to a more crystalline apatite and the Ca/P ratio remained fairly constant. The relation between the pH, Ca and P on the one hand and the brushite and apatite contents and the pattern of crystallization of the precipitates on the other was strikingly similar to the same realtionship observed earlier in dental plaque in situ, suggesting that the system used in this study could be used to study certain aspects of plaque mineralization in vitro.

Apatites↗

PubMed1978

Biological calcium phosphates and their role in the physiology of bone and dental tissues I. Composition and solubility of calcium phosphates.

Variations in the composition of bone and tooth mineral are consistent with the model that the constituents are a mixed microcrystalline apatite (AP)-octocalcium phosphate (OCP) like phase and an amorphous or submicrocrystalline calcium phosphate (ACP) like phase whereby these phases can occur in different proportions. An appropriate model for a description of the variable composition and the solubility behavior of the apatite phase is given by the formula (formula: see text) in which the compositional parameters x, y, z, and u each account for one type of defect mechanism. Other point defects are formed as well by incorporation of minority amounts of ions such as Cl-, K+, and F-; a number of trace elements can substitute for Ca2+ ions under in vivo conditions. It is suggested that the incorporation of ions in or loss from the crystals in contact with aqueous solutions is reversible. Literature data are used to show the direction in which the solubility product of the apatite phase shifts by incorporation of the different physiologically relevant ions. A quantitative evaluation of the available literature data revealed that Na+ and CO3= incorporation is the main cause for shifts in the solubility product of biological apatites.

Apatites↗

PubMed1983

Electron microscopy and microanalysis of a subcutaneous heterotopic calcification.

This paper reports the study of a subcutaneous heterotopic calcification from a patient with Thibierge-Weissenbach's syndrome, a type of creeping scleroderma included in the CRST syndrome. These local deposits, whose origin is still unknown, are commonly considered to be a classic apatite phase. Using SEM, high resolution TEM, electron diffraction, infrared spectrometry, and SEM and TEM microanalysis, it is demonstrated that this material is highly heterogeneous and appears in a nonstoichiometric, carbonated, calcium ion-deficient apatitic solid phase. Our study shows the coexistence of dense globules presenting an ill-organized, more or less amorphous phase (ACP) or microcrystalline (OCP, beta tricalcium phosphate), with scattered apatite crystals, and of interglobular apatite crystals with a good cristallinity.

Aged↗

PubMed1984

Compositional, structural, and phase changes in in vitro laser-irradiated human tooth enamel.

Tooth enamel laser irradiated under certain conditions previously has been shown to have reduced subsurface demineralization rates. Identification of these laser-induced changes has bearing on understanding the dissolution rate reduction mechanism; some of these changes, ones that occur in high temperature regions, were studied in this report. X-ray diffraction and infrared spectroscopy were used to identify changes in enamel of extracted intact human teeth subjected to high energy density (approximately 10,000 J/cm2) 10.6 microns wavelength carbon dioxide laser irradiance. The laser irradiance melted the enamel apatite; this solidified melt was composed of minor phases of alpha-tricalcium phosphate, alpha-Ca3 (PO4)2, and tetracalcium phosphate, Ca4(PO4)2O, and a major phase of modified apatite. The apatite modifications, as compared with the original were (1) reductions in contents of water, protein, carbonate, and chloride (or chloride rearrangement); (2) essentially no change in apatite hydroxide content; (3) possible incorporation of oxide replacing some hydroxide ions; and (4) an uptake of traces of carbon dioxide and cyanate. An infrared band at 434 cm-1 that appears in spectra of hydroxyapatite partially dehydroxylated by thermal treatment was assigned to oxide translation. This band was utilized to search for oxide formation in the laser-irradiated tooth enamel.

Apatites↗

PubMed1999

Metastable equilibrium solubility behavior of bone mineral.

Previous studies have shown that carbonated apatites with a range of carbonate contents and crystallinities exhibit the phenomenon of metastable equilibrium solubility (MES) distributions. The purpose of the present study was to investigate the solubility behavior of bone mineral using the concepts of MES and MES distributions and, together with crystallinity and chemical composition data, examine the similarity of bone mineral to carbonated apatite (CAP). Bone samples were harvested from 1-, 5-, and 8-month-old rats. The organic components of the bone samples were removed by hydrazine deproteination. Carbonated apatite was synthesized by the hydrolysis of dicalcium phosphate dihydrate (DCPD) in a NaHCO3-containing media at 50 degrees C. The MES distributions of bone mineral and CAP were determined by equilibrating predetermined amounts of CAP or bone mineral in a series of 0.1 M acetate buffers containing calculated levels of calcium and phosphate and maintained at essentially constant pHs of 5.0, 5.3, 5.7, and 6.5. From the compositions of the equilibrating buffer solutions, ion activity products based upon the stoichiometries of octacalcium phosphate, hydroxyapatite, and carbonated apatite were calculated in an attempt to determine the function governing the dissolution of CAP and bone mineral. The results of this study demonstrated that the MES distribution phenomenon appeared to hold for bone mineral and that the changes in crystallinity of bone mineral with age correlated well with changes in the MES values. A CAP sample was prepared that was found to be an excellent synthetic prototype closely mimicking the physicochemical behavior of bone mineral from an 8-month-old rat. Another finding of this study was that the ion activity product function based upon the hydroxyapatite stoichiometry well described the MES results obtained with both CAP and bone mineral. The interpretation that a surface complex with hydroxyapatite stoichiometry governs the solubility behavior of bone mineral is, therefore, consistent with the experimental data. Other calcium phosphate stoichiometries for the surface complex showed systematic variations in the MES profiles when the pH of the equilibrating solution was varied.

Age Factors↗

PubMed1989

Solubility, unit cell dimensions and crystallinity of fluoridated human dental enamel.

The aim was to describe the dimensions of the fluorhydroxyapatite unit cell and the solubility of fluorhydroxyapatite as a function of the fluoride concentration in the apatitic lattice. Various amounts of powdered human dental enamel were suspended in a pH 4.5 acetate buffer containing fluoride. The suspensions were equilibrated for 365 days at 20 degrees C. After equilibration the fluoride concentration in the apatite varied from 0.003 to 0.89 (mole fraction). X-ray diffraction showed that the length of the alpha-axis decreased with increasing fluoride concentration, whilst a broadening of the (300) reflection indicated the presence of mixtures of various fluorhydroxyapatites. The solubility of the apatite after the long equilibration decreased slightly with increasing fluoride concentration in the solid. In a few samples the fluoride concentration in the aqueous phase was still discernible, indicating that even after 365 days, equilibrium had not been attained. In these samples the amount of dissolved apatite was extraordinarily low.

Apatites↗

PubMed2004

Interactions of cisplatin with calcium phosphate nanoparticles: in vitro controlled adsorption and release.

A new system for the local delivery of chemotherapy to malignant solid tumors has been developed based on calcium phosphate (CaP) nanoparticles. The adsorption of the anti-neoplastic drug cis-diamminedichloroplatinum (cisplatin) was characterized on three types of apatitic CaP (poorly and well crystallized hydroxyapatite, and carbonated apatite). Adsorption isotherms obtained in chloride-free phosphate solutions at pH = 7.4 (24 and 37 degrees C) indicate that cisplatin adsorption increases with temperature and increases with decreasing crystallinity. Release studies in phosphate buffer saline (containing the chloride ion essential for release) showed that while the cumulative amount of released drug was the same for all apatites at 20 days (approximately 70% of the total bound), the least crystalline material released the drug more slowly. The drug release rate increased slightly with temperature. Cytotoxicity testing was conducted in a K8 clonal murine osteosarcoma cell line to verify that drug activity was retained after adsorption onto the apatite crystals. K8 cells were plated onto dried films of the apatite/cisplatin conjugates and after 24 h, viability was measured with tritiated uridine. The apatite/cisplatin formulations exhibited cytotoxic effects with a dose dependent diminishment of cell viability.

Adsorption↗

PubMed1999

Structural and functional macrophages alterations by ceramics of different composition.

Biomaterials may initiate several and complex biological reactions in host tissues, and the cell-biomaterial interactions can determine the release of mediators including monocytes and lymphocytes chemotactic factors. The present work was aimed to investigate in vitro the macrophage biological reactions of a natural apatite obtained by heat treatment at 400 degrees C of bovine bone, compared to other ceramics usually used for dental and orthopedic applications, using synthetic apatite and three types of alumina as controls. Particles chemotactic activity and powders oxidative burst evidenced no monocyte macrophages sensitivity reaction for natural and synthetic hydroxyapatite powders at great granulometry (> 50 microm); data were confirmed by ultrastructural observations; electron microscopy analysis showed macrophages with the features of healthy cells in the presence of both natural and synthetic apatites while macrophages grown in the presence of alumina seemed to be negatively affected. In conclusion, among all ceramics tested, natural apatite displayed a good compatibility with living cells, being better tolerated than synthetic hydroxyapatite which in turn is better tolerated than alumina.

Aluminum Oxide↗

PubMed2002

Infrared spectroscopy of the mineralogy of coprolites from Brean Down: evidence of past human activities and animal husbandry.

The mineralogy of 11 concretions from the Bronze Age settlement horizons at Brean Down near Weston-super-Mare, Somerset, UK, has been examined by infrared spectroscopy. The concretions are found to contain calcite and apatite and, in some cases, quartz. Four further concretions from the later Iron Age Meare Village, soil samples from Brean Down and mineralised samples of known faecal origin from a cesspit within the Tudor Merchant's house in Tenby have been similarly examined. It is found that all samples contain calcite, but only the concretions and the Tenby cesspit samples contain apatite. None of the soil samples contain apatite, although these are relatively high in quartz. This suggests that the concretions are coprolites and that the apatite has a biological origin in small bone fragments. The infrared study is backed up by scanning electron microscopy which confirms the presence of phosphorus in the coprolite samples and shows a morphology suggestive of the presence of bone fragments; it is likely, therefore, that the coprolites result from a carnivore--most probably from dogs. The findings show the usefulness of infrared spectroscopy for the rapid identification of mineralised coprolitic material from archaeological sites.

Animals↗

PubMed2004

Bioactive metals: preparation and properties.

Some ceramics, such as Bioglass, sintered hydroxyapatite, and glass-ceramic A-W, spontaneously form a bone-like apatite layer on their surface in the living body, and bond to bone through the apatite layer. These materials are called bioactive ceramics, and are clinically important for use as bone-repairing materials. However, they cannot be used at high-load sites, such as is found in femoral and tibial bones, because their fracture toughness values are not as high as that of human cortical bone. Titanium metal and its alloys have high fracture toughness, and form a sodium titanate layer on its surface when soaked in a 5 M-NaOH solution at 60 degrees C for 24 h, followed by a heat treatment at 600 degrees C for 1 h. On moving toward the metal interior, the sodium titanate layer gradually changes into the pure metal within a distance of 1 microm from the surface. The mechanical strength of the titanium metal or a titanium alloy is not adversely affected by these chemical and thermal treatments. The titanium metal and its alloys resulting from the above treatment can release Na+ ions from its surface into a surrounding body fluid via an ion exchange reaction with H3O+ ions, resulting in many Ti-OH groups forming on its surface. These Ti-OH groups initially combine with Ca2+ ions to form amorphous calcium titanate in the body environment, and later the calcium titanate combines with phosphate ions to form amorphous calcium phosphate. The amorphous calcium phosphate eventually transforms into bone-like apatite, and by this process the titanium metals are soon tightly bonded to the surrounding living bone through the bone-like apatite layer. The treated metals have already been subjected to clinical trials for applications in artificial total hip joints. Metallic tantalum has also been found to bond to living bone after it has been subjected to the NaOH and heat treatment to form a sodium tantalate layer on its surface.

Apatites↗

PubMed2004

Bioactivity of ceramic-polymer composites with varied composition and surface topography.

HAPEX trade mark (40 vol % hydroxyapatite in a high-density polyethylene matrix) and AWPEX (40 vol % glass-ceramic apatite-wollastonite in a high-density polyethylene matrix) are composites designed to provide bioactivity and to match the mechanical properties of human cortical bone. HAPEX trade mark has had clinical success in middle ear and orbital implants, and there is great potential for further orthopaedic applications of these materials. However, more detailed in vitro investigations must be performed to better understand the biological interactions of the composites. In this study, the bioactivity of each material was assessed. Specifically, the effects of controlled surface topography and ceramic filler composition on apatite layer formation in acellular simulated body fluid (SBF) with ion concentration similar to those of human blood plasma were examined. Samples were prepared as 1 x 10 x 10 mm(3) tiles with polished, roughened or parallel-grooved surface finishes, and were incubated in 20 ml of SBF at 36.5 degrees C for one, three, seven or 14 days. The formation of an apatite layer on the composite surface after immersion was demonstrated by thin-film X-ray diffraction, environmental scanning electron microscopy and energy dispersive X-ray analysis. Variations in sample weight and solution pH over the period of incubation were also recorded. Significant differences were found between the two materials tested, with greater bioactivity in AWPEX than HAPEX trade mark. Results also showed surface topography to be important, with rougher samples correlated to earlier apatite formation. Osteoblast-like cells proliferated favourably on both composite materials, with many filopodia connections, preferential attachment to ceramic particles and contact guidance effects evident.

Apatites↗

PubMed2003

Synovial fluid features and their relations to osteoarthritis severity: new findings from sequential studies.

OBJECTIVE: Many factors are involved in the osteoarthritic process. It is not yet known which are initiators, promoters or simply results. Thus, we have evaluated some of those potentially important factors in osteoarthritis (OA) as observed sequentially for the first time in synovial fluids. DESIGN: Synovial fluids (SF) obtained between 1992-2002 were all routinely evaluated for gross appearance, leukocyte counts and microscopic examination of wet drop preparations. We used regular and polarized light and alizarin red s stains. We separated out all OA patients, then we looked for patients who had more than two synovial fluid analyses to get sequential information. Time between first and final aspiration ranged from 2 to 7 (3.6+/-1.6) years and number of analyses per patients from 3 to 6 (3.3+/-0.7). We related synovial fluid crystals, fibrils and white blood cell count (WBC) to age, sex, disease duration and radiographic assessment according to the Kellgren-Lawrence radiographic rating system. RESULTS: Of 4523 synovial fluid examinations, we found 855 in patients with knee OA; 330 patients with adequate clinical details for comparison were included in our study. Twenty-six patients (one woman and 25 men) had sequentially examined SF. We found that 52% of those OA patients with effusions studied had crystals identified in their synovial fluid. Twenty-one percent of all the patients had CPPD crystals, 47% had hydroxyapatite, also called basic calcium phosphate (BCP) crystals and 16% had both types of crystals. Microscopically identifiable fibrils were found in 60% of SF. In sequentially examined patients, CPPD crystals and apatite (BCP) were found in 19% and 23%, respectively, at the first aspiration and, in 34% and 58% at the final aspiration. Fibrils were seen in 54% at first examination and 85% later. Apatite and fibrils showed more significant correlation with time (r=0.51,r =0.92) than did CPPD (r=0.32). SF WBC correlated only with CPPD crystals and did not increase with OA duration or severity. CPPD, apatite and fibrils all correlated with higher radiographic grades of OA. CONCLUSIONS: As noted before CPPD and apatite crystals were more common in patients with more severe OA. New findings are that our sequential cases showed that there were some patients with no crystals at onset but that crystals appeared with progression of the disease. Fibril presence in SF also correlated with progression of the disease.

Aged↗

PubMed1998

Nanobacteria: an alternative mechanism for pathogenic intra- and extracellular calcification and stone formation.

Calcium phosphate is deposited in many diseases, but formation mechanisms remain speculative. Nanobacteria are the smallest cell-walled bacteria, only recently discovered in human and cow blood and commercial cell culture serum. In this study, we identified with energy-dispersive x-ray microanalysis and chemical analysis that all growth phases of nanobacteria produce biogenic apatite on their cell envelope. Fourier transform IR spectroscopy revealed the mineral as carbonate apatite. The biomineralization in cell culture media resulted in biofilms and mineral aggregates closely resembling those found in tissue calcification and kidney stones. In nanobacteria-infected fibroblasts, electron microscopy revealed intra- and extracellular acicular crystal deposits, stainable with von Kossa staining and resembling calcospherules found in pathological calcification. Previous models for stone formation have led to an hypothesis that elevated pH due to urease and/or alkaline phosphatase activity is a lithogenic factor. Our results indicate that carbonate apatite can be formed without these factors at pH 7.4, at physiological phosphate and calcium concentrations. Nanobacteria can produce apatite in media mimicking tissue fluids and glomerular filtrate and provide a unique model for in vitro studies on calcification.

Animals↗

PubMed1989

Chemical and structural challenges in remineralization of dental enamel lesions.

The aim of the present paper was to identify some chemical and structural factors which may prevent a full remineralization of caries lesions and to study whether it is possible to overcome such obstacles. Samples of powdered enamel apatite were equilibrated with solutions metastably supersaturated with respect to enamel hydroxyapatite and fluorapatite. After 10 min and 60 min of equilibration at 20 degrees C the suspensions were centrifuged and the calcium and phosphate concentrations and the pH were determined in the supernatant. In parallel studies, 50 75-microns-thick sections of 27 fluorotic teeth of a severity of 5-7 according to Thylstrup and Fejerskov's classification were examined by microradiography and in polarized light using distilled water, and Thoulett's media or seen dry in air. Five obstacles inhibiting remineralization were identified: 1) Although remineralizing solutions or saliva are supersaturated with respect to enamel apatite the total amount of calcium and phosphate dissolved in it is small, so that after precipitation of the dissolved mineral only 1/20,000-1/30,000 of the volume of the mineralizing solution is occupied by mineral. 2) The concentration gradients from the mineralizing solution into the enamel is small, which indicates a slow diffusion into and out of the lesion. 3) The uptake of calcium and phosphate by the enamel apatite crystals is so rapid that the aqueous phase within the pores can be presumed to be only marginally supersaturated in the deeper parts of the lesion. 4) The surface layer of the enamel lesions was found to be a serious obstacle to remineralization so that a subsurface area remains hypomineralized after exposure to salivary remineralization even for a lifetime. 5) Nucleation of new apatite crystals to substitute lost crystals is an unsolved problem.

Apatites↗

PubMed2002

Nuclear magnetic resonance spin-spin relaxation of the crystals of bone, dental enamel, and synthetic hydroxyapatites.

Studies of the apatitic crystals of bone and enamel by a variety of spectroscopic techniques have established clearly that their chemical composition, short-range order, and physical chemical reactivity are distinctly different from those of pure hydroxyapatite. Moreover, these characteristics change with aging and maturation of the bone and enamel crystals. Phosphorus-31 solid state nuclear magnetic resonance (NMR) spin-spin relaxation studies were carried out on bovine bone and dental enamel crystals of different ages and the data were compared with those obtained from pure and carbonated hydroxyapatites. By measuring the 31P Hahn spin echo amplitude as a function of echo time, Van Vleck second moments (expansion coefficients describing the homonuclear dipolar line shape) were obtained and analyzed in terms of the number density of phosphorus nuclei. 31P magnetization prepared by a 90 degree pulse or by proton-phosphorus cross-polarization (CP) yielded different second moments and experienced different degrees of proton spin-spin coupling, suggesting that these two preparation methods sample different regions, possibly the interior and the surface, respectively, of bone mineral crystals. Distinct differences were found between the biological apatites and the synthetic hydroxyapatites and as a function of the age and maturity of the biological apatites. The data provide evidence that a significant fraction of the protonated phosphates (HPO4(-2)) are located on the surfaces of the biological crystals, and the concentration of unprotonated phosphates (PO4(-3)) within the apatitic lattice is elevated with respect to the surface. The total concentration of the surface HPO4(-2) groups is higher in the younger, less mature biological crystals.

Aging↗