Hydroxyapatite in the Pathogenesis of Cystic Fibrosis

Submandibular saliva collected from cystic fibrosis patients and control subjects was separated by centrifugation into an insoluble deposit and a clear supernatant. The resulting calcium and phosphorus analyses performed on both fractions warranted a closer investigation as a consistent Ca/P molar ratio of 1·5 was found in the deposit of the cystic fibrosis patients, while no consistent ratio >1·0 was found in the deposit of the control subjects. The expected result, that calcium and phosphorus in the deposit of cystic fibrosis patients is present as the solid phase of hydroxyapatite, was confirmed by a detailed comparison of x-ray powder diffraction patterns of an ashed sample of this deposit and a similarly treated synthetic sample of hydroxyapatite.     

A STUDY OF METASTATIC RENAL CALCIFICATION AT THE CELLULAR LEVEL

Experimental metastatic calcification in the proximal convoluted tubules of rat kidney, produced by large doses of vitamin D, has been studied with a variety of techniques. These techniques include the examination of thin sections of Araldite-embedded material under the electron microscope, selected area electron diffraction, and several histochemical methods. Two types of mineral are found in relation to the proximal convoluted tubule. The first form consists of aggregates of elongated crystals within cytoplasmic vacuoles of the proximal tubular cells. The dimensions of these crystals are consistent with those of hydroxyapatite. The other type of mineral deposit is found in and adjacent to the extracellular phase of the basal infoldings of these tubules. The latter deposits are made up of smaller crystals arranged in layers. These crystals could not be definitely identified by means of selected area electron diffraction. The observations are discussed in relation to calcium transport by the proximal convoluted tubule and also in terms of mechanisms of pathological calcification.     

Effects of cholestanol feeding on corneal dystrophy in mice

A cholestanol-enriched diet administered for 8 months to BALB/c mice produced in 20% two kinds of corneal opacities resembling calcific band keratopathy and Schnyder's crystalline dystrophy in humans. The concentrations of cholestanol in serum, liver and cornea of the corneal opacity bearing mice were 30-40-times higher than those of normal mice. On the other hand, brain cholestanol level increased only 7-times in the opacity group as compared with that of control group. There was no significant difference in the cholesterol concentrations of serum and several tissues among opacity, non-opacity and the control group. The crystal particles were observed between epithelial basement membrane and superficial stroma by the electron microscopy. Energy dispersive analysis of the particles revealed that the deposits were composed principally of calcium and phosphorus with other crystalline materials, which was presumed to be cholestanol. These results suggest that the cholestanol may deposit in the cornea from elevated serum levels. Deposition of cholestanol in cornea and related area may be a cause of corneal dystrophy in CTX.     

Absorption of phosphorus from the spermatophore through the cuticle of the bursa copulatrix of the butterfly, Calpodes ethlius

In lepidoptera the spermatophore is deposited in a bursa copulatrix, which is lined by a cuticle through which solubilized components of the spermatophore must be absorbed by the underlying epithelium. This study follows the route taken by the phosphorus which, with calcium, forms a significant portion of the spermatophore of Calpodes ethlius in the form of homogeneous, amorphous concretions. Lead nitrate was used in the primary fixative to precipitate the phosphorus in order to follow the pathway taken by it through the cuticle and bursal epithelium. The epicuticle-free pits, which cover the bursal cuticle, are identified as the points of entry, and the epicuticular filaments as major pathways for transport of phosphorus and presumably soluble molecules. Lead phosphate deposits also occurred in the epithelium extracellularly between the apical microvilli, lateral membranes and basal folds, in calcium granules and in large compound structures which appear to be made up, at least in part, of calcium granules. The confinement of the phosphorus to extracellular compartments of the epithelium suggests that its function is to be found elsewhere.     

Electron microbeam analysis of calcium distribution in the ciliated protozoan, Spirostomum ambiguum.

Electron microprobe analyses of calcium distribution in the ciliated protozoan, Spirostomum ambiguum, indicated several calcium rich sites. One site was an endoplasmic distribution of calcium coincident with phosphorus which corroborates previous findings of hydroxyapatite deposits within Spirostomum. These apatite deposits were distributed throughout the endoplasm, but not within the nuclei or the contractile vacuole. Calcium was also detected within the cortical region. Cortical calcium was in greater concentration in the anterior portion of the organism and decreased towards the posterior end (region containing the contractile vacuole). Phosphorus and potassium were also detected as gradients from the anterior end, whereas magnesium was detected in the same density throughout the cortical region. Line scans of cortical regions suggested (1) distributions of calcium within mitochondria and/or vesicles, and (2) calcium associated with bundles of microfilaments.     

The formation of intravesicular calcium phosphate deposits in microsomes of smooth muscle

Summary The calcium uptake in the microsomial fraction isolated from the smooth muscle of the antrum of the pig stomach is stimulated by phosphate. The microsomial vesicles which are loaded with calcium phosphate can be purified by differential centrifugation. A purification of 36 times in terms of calcium content was reached. Electron microscopy of the freshly prepared material revealed calcium phosphate deposits in the form of needles of crystalline calcium phosphate. This structure differs from that of the deposits which appear in the fragmented sarcoplasmic reticulum of skeletal muscle. Their morphology is that of non-crystalline calcium phosphate. However, on standing these deposits convert slowly into crystalline calcium phosphate. This difference reflects different kinetics of crystallization of the precipitates in the two preparations. After negative staining of the calcium phosphate loaded microsomes of skeletal and of smooth muscle, only few deposits are preserved because a release of calcium occurs as a consequence of the action of the stain and also of the dilution and warming up of the suspension. Smooth muscle microsomes partially purified by loading with calcium phosphate were studied by freeze etching and rotary replication. Membrane fragments displaying subunit intramembrane particles similar to those observed in sarcoplasmic reticulum of skeletal muscle could be identified. However, in the smooth muscle microsomes the intramembrane particles were much less densely packed. Part of these particles could correspond to calcium transport sites.     

Occurrence of brain calcifications,iron deposits,and tonic-clonic convulsions following experimental infections with Candida albicans

Summary During the prolonged presence ofCandida albicans in the spleen of Wistar rats, two cases of calcium deposits in the brain were observed. In one case there was one big calcium deposit at some distance from the cortex. In this case, thickening in a limited area of the leptomeninges was found. The animal had generalized tonicclonic convulsions which lead to death. In the second case, calcium deposits formed two separate agglomerations in the posterior part of the thalamus. Serial sections made at six micron intervals showed the presence of calcium deposits to a depth of forty-eight microns. At twenty-four microns other calcium deposits were observed in the thalamus symmetrically located to those described above.Small iron deposits were found in the anterior part of the thalamus and other iron deposits were found in the temporal lobe of the brain. They were arranged in a straight line along the capillary vessels and most of them were seen in the macrophages in the vicinity of the vessels.Since calcium deposits in the brain occurred in the same experimental conditions as reported cases of calcinosis, it is probable that the same mechanism is involved in all these pathological calcium deposits.This work was supported by a Damon Runyon Memorial Fund Grant Nr. 720.     

Biomimetic CoUagen/Hydroxyapatite Composite Scaffolds： Fabrication and Characterizations

Biomimetic collagen/hydroxyapatite scaffolds have been prepared by microwave assisted co-titration of phosphorous acid-containing collagen solution and calcium hydroxide-containing solution. The resultant scaffolds have been characterised with respect to their mechanical properties, composition and microstructures. It was observed that the in situ precipitation process could combine collagen fibril formation and hydroxyapatite （HAp） formation in one process step. Collagen fibrils guided hydroxyapatite precipitation to form bone-mimic collagen/hydroxyapatite composite containing both intrafibrillar and interfibrillar hydroxyapatites. The mineral phase was determined as low crystalline calcium-deficient hydroxyapatite with calcium to phosphorus ratio （Ca/P） of 1.4. The obtained 1% （collagen/HAp = 75/25） scaffold has a porosity of 72% and a mean pore size of 69.4 ~tm. The incorporation of hydroxyapatite into collagen matrix improved the mechanical modulus of the scaffold significantly. This could be attributed to hydroxyapatite crystallites in collagen fibrils which restricted the deformation of the collagen fibril network, and the load transfer of the collagen to the higher modulus mineral component of the composite.     

Subcellular localization of calcium deposits in the noble crayfish Astacus astacus spermatophore: Implications for post‐mating spermatophore hardening and spermatozoon maturation

The freshly ejaculated spermatophore of crayfish undergoes a hardening process during post‐mating storage on the body surface of female. The ultrastructural distribution of calcium deposits were studied and compared in freshly ejaculated and post‐mating noble crayfish spermatophores, using the oxalate‐pyroantimonate technique, to determine possible roles of calcium in post‐mating spermatophore hardening and spermatozoon maturation. Small particles of sparsely distributed calcium deposits were visible in the wall of freshly ejaculated spermatophore. Also, large amount of calcium deposits were visible in the membranes of the freshly ejaculated spermatozoon. Five minutes post‐ejaculation, granules in the spermatophore wall appeared as porous formations with numerous electron lucent spaces. Calcium deposits were visible within the spaces and scattered in the spermatophore wall matrix, where smaller calcium deposits combined to form globular calcium deposits. Large numbers of the globular calcium deposits were visible in the wall of the post‐mating spermatophore. Smaller calcium deposits were detected in the central area of post‐mating spermatophore, which contains the sperm mass, and in the extracellular matrix and capsule. While the density of calcium deposits decreased in the post‐mating spermatozoon membranes, numerous small calcium deposits appeared in the subacrosomal zone and nucleus. Substantial changes in calcium deposit distribution in the crayfish spermatophore during post‐mating storage on the body of female may be involved in the processes of the spermatophore hardening and spermatozoon maturation. J. Morphol. 277:445–452, 2016. © 2016 Wiley Periodicals, Inc.     

Connective tissue mineralization in Abcc6-/- mice, a model for pseudoxanthoma elasticum

Pseudoxanthoma elasticum (PXE) is a heritable multisystem disorder characterized by ectopic mineralization. However, the structure of the mineral deposits, their interactions with the connective tissue matrix, and the details of the progressive maturation of the mineral crystals are currently unknown. In this study, we examined the mineralization processes in Abcc6(-/-) mice, a model system for PXE, by energy dispersive X-ray and Fourier transform infrared imaging spectroscopy (FT-IRIS). The results indicated that the principal components of the mineral deposits were calcium and phosphate which co-localized within the histologically demonstrable lesions determined by topographic mapping. The Ca/P ratio increased in samples with progressive mineralization reaching the value comparable to that in endochondral bone. A progressive increase in mineralization was also reflected by increased mineral-to-matrix ratio determined by FT-IRIS. Determination of the mineral phases by FT-IRIS suggested progressive maturation of the mineral deposits from amorphous calcium phosphate to hydroxyapatite. These results provide critical information of the mechanisms of mineralization in PXE, with potential pharmacologic implications.     

Calcium-containing structures in vertebrate glial cells. Ultrastructural and microprobe analysis

Electron probe microanalysis has revealed that vesicular or cisternal structures containing electron-dense material in frog ependymal glial cells contain deposits of calcium and phosphorus. The so-called "osmiophilic particles" in human astrocytes also contain calcium. It is suggested that these organelles are storage sites of calcium.     

Quantitative changes of calcium, phosphorus, and magnesium in common iliac arteries with aging

To elucidate the mechanism of element accumulations in the arteries with aging, the authors investigated the mass ratios among calcium, phosphorus, and magnesium in the common iliac arteries by inductively coupled plasma-atomic emission spectrometry. The subjects consisted of 16 men and 8 women, ranging in age from 65 to 93 yr. It was found that there were extremely significant correlations between calcium and phosphorus contents, between calcium and magnesium contents, and between phosphorus and magnesium contents in the common iliac arteries. In regard to the mass ratio, although the mass ratio of calcium to phosphorus was almost constant, the mass ratios of magnesium to calcium and phosphorus were different at early and advanced stages of the accumulation of calcium and phosphorus. It was found that both the mass ratios of magnesium to calcium and phosphorus were higher at an early stage of the accumulation of calcium and phosphorus in the arteries than at an advanced stage of the accumulation.     

Contribution of Raman spectroscopy to identification of biominerals present in teeth of Acanthopleura rehderi,Acanthopleura curtisiana,and Onithochiton quercinus

Raman spectroscopic investigations of the major lateral teeth of the chitons Acanthopleura rehderi and Acanthopleura curtisiana indicate that, in addition to the magnetite of the cutting surface and a carbonated hydroxyapatite in the central tooth core, these species deposit limonite in place of the lepidocrocite reported for other members of the genus Acanthopleura. A comparison of the spectra from these species with those of Onithochiton quercinus, which also deposits limonite, indicates that the current assignment of these species to Acanthopleura may not be appropriate. Biomineralization of the major lateral teeth may be a useful parameter to include in the taxonomic classification of chiton species.     

The verification of cytochemical tests for ATP-ase activity in plant cells using X-ray microanalysis

Summary Electron microprobe analysis (EMMA 4) was carried out on two types of electron opaque deposit found in thin sections of barley root tips as a result of cytochemical tests for ATP-ase activity.The granular type of deposit, which mainly occurs in radial and tangential cell walls of epidermal and sub-epidermal cells, was shown to contain lead, whereas lead was absent from the opaque globular deposits, which are much more generally distributed and always associated with membrane structures. Thus the latter deposits, which contain osmium and have often been interpreted as ATP-ase reaction products, should be regarded as artifacts of the fixation and rinsing procedures. It is suggested that calcium may play a role in the formation of the osmiophilic deposits.     

Selective calcification of rat brain lesions caused by systemic administration of kainic acid

Dystrophic calcification of previously damaged areas of nervous tissue occurs in a wide range of human diseases. The relationship between astroglial and microglial reactions and deposits of calcium salts was studied for up to five months in rats with a brain lesion produced by systemic administration of kainate. The morphology and atomic composition of the calcium salt deposits was also studied. Two types of lesions, sclerotic and liquefactive, were observed. In sclerotic lesions hyperplasia and hypertrophy of astrocytes partially substituted for the lost neurons, reaching a maximum in about twenty-five days after treatment. In liquefactive lesions, the astrocytic reaction occurred only around the liquefactive area. Microglial reaction was similar in both types of lesion and reached its highest expression in about twenty-five days. Calcium deposits were observed in the sclerotic but not in the liquefactive lesions. Clearly distinguishable granules of calcium salts were observed in sclerotic lesions under scanning electron microscopy after only five days post-injection. The size of calcified granules increased with time reaching 40 micro m or more in diameter at five months. The atomic composition of these deposits, studied by X-ray microanalysis, showed a time-dependent increase in calcium concentration. While there was no clear relationship between astroglial and microglial reactions and calcium salt deposits, the systemic injection of kainate produced progressively larger and more concentrated calcium deposits in sclerotic, but not in liquefactive lesions.     

Magnesium and strontium interaction with carbonate-containing hydroxyapatite in aqueous medium

Crystallographic and spectroscopic studies were performed on the solid products obtained from magnesium and strontium interaction with carbonate-containing hydroxyapatite under aqueous conditions employing a cyclic pH variation technique. Low concentrations of magnesium and strontium make easier the transformation of carbonate-containing hydroxyapatite into β-tricalcium phosphate by heat treatment. At high concentration magnesium stabilizes the β-tricalcium phosphate crystal structure while the apatite crystal structure is maintained when the Ca/Sr molar ratio decreases. It is suggested that these ions are important in determining the structure of the calcium phosphate deposits in biological tissues during the physiological and pathological local pH variations.     

Effect of K+ and Na+ on calcium-dependent electron-dense particles in the monoaminergic synaptic vesicles of rat pineal nerves fixed in Ca2+-containing solutions

The effect of K+ and Na+ on the Ca2+ binding site in the dense core of monoaminergic vesicles of pineal nerves was investigated in the rat. Rat pineal glands, bisected immediately after decapitation, were incubated at room temperature in solutions containing high K+ or high Na+ in the presence or absence of Ca2+. Fixation was performed in glutaraldehyde-osmium tetroxide in collidine buffer, with and without CaCl2. It was confirmed that, after fixation in Ca2+-containing solutions, an electron-dense particle, located in the vesicle core, which can be considered a calcium deposit, appears within the synaptic vesicles. It was observed that this Ca2+ deposit may be modified by incubation in a high K+ or high Na+ milieu before fixation in Ca2+ containing solutions. When the incubation was carried out with high K+ and high Ca2+ simultaneously, Ca2+ deposits were considerably increased. With K+ alone, no Ca2+ deposits were apparent, as when electrical stimulation is applied before fixation. This effect was not observed when the incubation was done in high Na+. Consecutive incubations in high K+ and high Na+, respectively, restored the capability of the vesicle cores to bind Ca2+. Prolonged incubation in high Na+ before fixation increased Ca2+ deposits within the vesicles. These findings are in line with data on the effect of these ions upon the storage and release of biogenic amines and suggest that these ions modify the capability of synaptic vesicles to bind Ca2+.     

The reaction between some dyes and synthetic hydroxyapatite I. The uptake of dyes in relation to their structures

Synopsis The uptake of dyes from dilute solutions by synthetic hydroxyapatite and other sparingly soluble calcium compounds has been determined. About 30 dyes, mostly azo-, dis-azo and anthraquinonoid types were used in 95% ethanol or 0.1 M tris buffer. Many had closely related configurations. Chemical groupings possibly responsible for the adsorption of particular dyes by hydroxyapatite have been deduced from an analysis of the results. The uptake of most dyes from alcoholic solutions was, linearly related to the surface area of hydroxyapatite. Calcium carbonate and secondary calcium phosphate took up less stain than hydroxyapatite of similar surface area. With the simpler anthraquinonoid dyes, the uptake was higher from aqueous than alcoholic solutions, but specificity for hydroxyapatite was much less. The increased uptake of dye by powdered bone or dentine when rendered anorganic was proportional to the increased surface area. It was found that several dyes in common use as stains for bone and calcified tissue were only poorly adsorbed by synthetic hydroxyapatite under the particular conditions of these experiments.The experimental data presented could be used as a basis for the development of histochemical reactions for calcified tissue or inclusions. By suitable choice of dyes, solvent and rinsing solution it ought to be possible to differentiate various forms of calcified material.     

Quantitation of intraplatelet Ca++ deposits as a potential marker of senility

OBJECTIVE: To perform a morphometric evaluation of calcium deposits in human platelets as a quantitative procedure to seek a potential marker of senility in a peripheral cellular model. STUDY DESIGN: In human blood samples from middle-aged, healthy volunteers, the intraplatelet calcium content was cytochemically evidenced by the oxalatepyroantimonate (OPA) reaction. The number and area of OPA aggregates per square micrometer of total sampled area, the area of the deposits per square micrometer of platelet surface and the percentage of positive platelets were the ultrastructural features calculated by computer-assisted image analysis. RESULTS: OPA precipitates were easily identified in all the samples evaluated. The area of OPA deposits per square micrometer of platelet surface was rather constant not only among the measurements performed on the same sample but also comparing the different subjects analyzed. Other OPA deposit features showed higher variabilities; thus, to obtain a representative sample from each patient, several measurements had to be carried out. CONCLUSION: Quantitation of calcium deposits may be of help in evidencing increased Ca++ sequestering activity by platelets, supposedly due to altered calcium homeostasis. The OPA cytochemical procedure visualizes millimolar quantities of Ca++ ions; thus, only high calcium concentration sites (granules) can be detected by morphometric methods.