Apatite precipitation after incubation of biphasic calcium-phosphate ceramic in various solutions: influence of seed species and proteins

The dissolution-precipitation process for calcium-phosphate ceramics in contact with biological fluid was studied by incubating blocks of biphasic calcium phosphate composed of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP) in different solutions: ionic simulated body fluid (SBF) without protein or SBF that contained various proteins and macromolecules separately (fibronectin, vitronectin, albumin, and poly-L-glutamic acid). Transmission electron microscopy studies revealed that apatite-precipitated microcrystals appeared around ceramic crystals as a result of secondary nucleation; microcrystals were in continuity with the lattice planes of the HA crystals but in a different direction from that of beta-TCP; the size of the precipitates was smaller when fibronectin, vitronectin, and poly-(L-glutamic acid) were present in SBF as compared to SBF without protein; and fibronectin and vitronectin initiated crystal nucleation in the void spaces between the ceramic crystals.     

Lateralisation of speech dominance by spectral analysis of evoked potentials

The case of a 72-yr-old woman with osteoarthritis of the shoulder, is described. A mixture of crystals of hydroxyapatite and calcium pyrophosphate dihydrate was identified in the joint tissues using analytical electron microscopy and infra-red spectroscopy.     

Role of radiology in the diagnosis of joint chondrocalcinosis. Calcium pseudogout

Chondrocalcinosis is a really frequent clinicopathologic entity, which is caused by the penetration of calcium pyrophosphate dihydrate microcrystals into the structures of the joint, including hyaline cartilages and fibrocartilages--hense its name--as well as the synovial fluid and membrane. Calcium gout, which preferentially appears in the knees, is the most spectacular and characteristic symptom of chondrocalcinosis, expressing a crisis of acute microcrystal synovitis, of which it has all the usual clinical features, and thus simulating uratic gout. The positive diagnosis is based on: a) the radiologic demonstration of articular calcifications in the lining cartilages, forming a continuous or fragmented opaque border on the subchondral bone, from which it is separated by a light space, and/or in the fibrocartilaginous structures (most often the menisci, the symphysis pubis, the disk of the inferior radioulnar joint) where they appear as small, irregular clusters with blurred or cloudy margins. The knee is the most frequent site of calcium impregnation images, both in hyaline cartilages and in fibrocartilages. b) the presence of calcium pyrophosphate microcrystals in the synovial fluid; their nature is usually demonstrated convincingly enough with a conventional light microscope; c) needle biopsy findings of microcrystalline clusters embedded in the synovial membrane, that can be easily identified with routine staining. In practice, demonstrating radiologic signs, when these are characteristic and can be detected in their preferred sites, allows recognizing diffuse chondrocalcinosis in satisfactory safety conditions after a calcium gout crisis, as well as in the presence of the many atypical or misleading symptomatic aspects of this microcystal arthropathy, that will be the subject of a further paper.     

Calcification of cervical ligamentum flavum--analysis of calcium compounds and histopathological findings

We operated 3 patients with cervical myelopathy due to calcification of ligamentum flavum (CLF). Specimens collected surgically were subjected to X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Raman spectroscopy (Raman) analysis and also histopathological examination. The calcium compounds deposited were calcium pyrophosphate dihydrate (CPPD) in Case 1, apatite in Case 2, and a double-layer structure with an outer CPPD layer and an inner carbonate apatite layer in Case 3. Histopathologically, CPPD deposition in Case 1 could be distinguished from apatite deposition in Case 2 and 3 by hematoxylin-eosin stain. Chondrocytes were observed in all 3 cases, suggesting the chondrocytes may have played a role in calcification in these three cases. To date, 62 cases of CLF (including the present cases) have been reported, and analysis of calcium compound has been performed for 29 of them. Of these 29, 15 were analyzed as calcium phosphate compounds, 9 as CPPD and 4 cases as mixed crystals like Case 3. However the analysis method and result about CPPD are no problem, the analysis result of calcium phosphate compounds depends on the using methods. Calcium phosphate or undetectable compounds was identified by XRD as hydroxyapatite (HAp), and by FTIR as calcium phosphate or undetectable compound. Analysis of calcium phosphate compounds should be condacted and identified by XRD, FTIR, and Raman. We propose two possible mechanisms for the formation of the double-layer structure in Case 3: one is formation of apatite first followed by deposition of CPPD outside, and the other is formation of CPPD first followed by conversion of CPPD in the central region to apatite. What the process of formation of the double-layer was in this cases remains unclear.     

Superoxide anion production and phagocytosis of crystals by cultured endothelial cells

OBJECTIVE: To show that cultured human umbilical vein endothelial cells (HUVEC) are capable of phagocytizing inflammation-causing crystals and of generating superoxide anion (SOA) during phagocytosis. METHODS: The superoxide dismutase-inhibitable reduction of nitroblue tetrazolium (NBT) dye was used as a measure of SOA production. Phagocytosis was quantified by light microscopy and confirmed by transmission electron microscopy. Cytochrome C was also studied but was found to undergo spontaneous reduction by monosodium urate (MSU) without cells. RESULTS: Crystals of MSU, calcium oxalate, hydroxyapatite, and calcium pyrophosphate dihydrate (CPPD) were phagocytized and, except for the CPPD crystals, induced NBT reduction. Cholesterol and cholesterol monohydrate were neither phagocytized nor did they induce NBT reduction. CONCLUSIONS: Endothelial cells may be a significant source of oxygen radicals in crystal-associated and other arthritides.     

Synthesis and characterization of branched yttrium hydroxide fluoride microcrystals with hierarchical tubular structure

Hexagonal yttrium hydroxide fluoride microcrystals were prepared by a two-step hydrothermal route using yttrium nitrate, sodium hydroxide and sodium fluoride as raw materials to react in propanetriol solvent. The samples were characterized by powder X-ray diffraction (XRD), energy dispersive spectrum (EDS), scanning electron microscopy (SEM), transmission electron microscopy (TEM), Fourier trans-form infrared spectroscopy (FT-IR), thermogravimetre and differential-thermogravimetric analysis (TG-DTA), which revealed that Y(OH)2.14F0.86 microcrystals were multi-branched and that the branches of Y(OH)2.14F0.86 microcrystals were composed of hierarchical tubes. This novel multi-branched and intriguing hierarchical tubular structure of yttrium hydroxide fluoride maybe has a potential application in photoelectric crystals. The formation of branched Y(OH)2.14F0.86 microcrystals with hierarchical tubular structure were due to the substitution reaction and Oswald ripening.     

Pediatric onychomycosis treated with oral antifungal drugs

A compact, high-resolution, laser-plasma, x-ray contact microscopy method using a table-top Nd:glass laser system has been developed. This x-ray microscopy system was applied for the observation of macrophage ultrastructures. These images were produced using proximity imaging in which a 5-ns pulse of soft x-rays with wavelengths near and inside the water windows (23A-44A) produced by the laser-plasma were absorbed by the specimen and then registered on a photo resist. The x-ray images imprinted on the photo resist were then developed and analyzed with an atomic force microscope (AFM). Mouse thioglycollate-elicited peritoneal macrophages in suspension were examined by this new x-ray microscope. The x-ray images of the macrophages were compared with those observed by conventional transmission electron microscopy (TEM). The x-ray images showed no obvious organelles, including the nucleus and endoplasmic reticulum, as can be seen with TEM, but high- and low-contrast structures caused by mass distribution of carbon were observed. Thus, using the x-ray microscopy we visualized the first x-ray images of macrophage ultrastructures. The successful x-ray imaging of macrophage ultrastructure indicates that proximity x-ray microscopy may be of value in studying physiology linked to the dynamics of a cell.     

The nucleation and growth of calcium phosphate crystals at protein and phosphatidylserine liposome surfaces

The kinetics of calcium phosphate crystal growth at the surfaces of proteins and phospholipids has been investigated using free drift and constant composition methods in supersaturated calcium phosphate solutions (relative supersaturations: with respect to hydroxyapatite, HAP, sigma HAP = 15.0, and with respect to octacalcium phosphate, OCP, sigma OCP = 1.9). Fibrinogen and collagen molecules adsorbed at hydrophobic surfaces as well as uncross-linked collagen fibrils induce ion binding and subsequent nucleation of calcium phosphate. The formation of OCP on phosphatidylserine vesicles introduced to highly supersaturated calcium phosphate solutions probably involves the interaction of the calcium ions with the ionized carboxylic groups of the phospholipid.     

Pelvic pseudotumoral calcium pyrophosphate dihydrate deposition: an ultrastructural study

We analyzed large periarticular calcifications of the hips and pubis in a woman with diffuse primary chondrocalcinosis, symptomatic in the knees. Serial articular and periarticular biopsies were obtained during surgery for osteoporotic hip fracture. Histologic examination showed diffuse nonbirefringent clumps in red alizarin stained cartilage, capsula and tendon. Scanning electron microscopy with microanalysis indicated a calcium/phosphorus atomic ratio of about 1. Transmission electron microscopy, electron diffraction and Raman spectroscopy assessed calcium pyrophosphate dihydrate (CPPD) in cartilage, synovia, capsula, tendon and muscle. The cause of these hard to observe, bulky CPPD periarticular deposits, which resembled basic calcium phosphate on radiography, is still unclear.     

Nucleation of hydroxyapatite by bone sialoprotein

Bone sialoprotein (BSP) and osteopontin, the major phosphorylated proteins of mammalian bone, have been proposed to function in the initiation of mineralization. To test this hypothesis, the effects of BSP and osteopontin on hydroxyapatite crystal formation were determined by using a steady-state agarose gel system. At low calcium phosphate concentrations, no accumulation of calcium and phosphate occurred in control gels or gels containing osteopontin. Gels containing BSP at 1-5 micrograms/ml, however, exhibited a visible precipitation band and significantly elevated Ca + PO4 contents. By powder x-ray diffraction, the precipitate formed in the presence of BSP was shown to be hydroxyapatite. These findings suggest that bone sialoprotein may be involved in the nucleation of hydroxyapatite at the mineralization front of bone.     

Phosphates precipitating from artificial urine and fine structure of phosphate renal calculi

Phosphates precipitating from artificial urine in the pH range 6-8 were identified using X-ray diffraction, chemical analysis and scanning electron microscopy. The influence of magnesium and citrate on phases precipitating from urine was established. From urine containing a normal quantity of magnesium (around 70 ppm), brushite accompanied by hydroxyapatite (HAP) precipitated at pH < or = 7.0 and struvite with HAP at pH > 7.0. HAP was formed exclusively from magnesium deficient urine at pH 7.0. Newberyite, octacalcium phosphate and whitlockite were not identified. The chemical and phase composition and inner fine structure of 14 phosphate calculi were studied. Three types of stones were distinguished based on their magnesium content: (i) stones rich in magnesium composed of struvite, hydroxyapatite and abundant organic matter, (ii) stones with low magnesium content constituted by calcium deficient hydroxyapatite, up to 5% of struvite, considerable amount of organic matter and occasionally brushite, and (iii) calculi without magnesium consisting of brushite, hydroxyapatite and little organic matter. Conditions prevaling during stone-formation assessed for each type of stone were confirmed by corresponding urinary biochemical data and corroborate the in vitro studies of phosphates precipitation.     

Setting reaction and hardening of an apatitic calcium phosphate cement

The combination of self-setting and biocompatibility makes calcium phosphate cements potentially useful materials for a variety of dental applications. The objective of this study was to investigate the setting and hardening mechanisms of a cement-type reaction leading to the formation of calcium-deficient hydroxyapatite at low temperature. Reactants used were alpha-tricalcium phosphate containing 17 wt% beta-tricalcium phosphate, and 2 wt% of precipitated hydroxyapatite as solid phase and an aqueous solution 2.5 wt% of disodium hydrogen phosphate as liquid phase. The transformation of the mixture was stopped at selected times by a freeze-drying techniques, so that the cement properties at various stages could be studied by means of x-ray diffraction, infrared spectroscopy, and scanning electron microscopy. Also, the compressive strength of the cement was measured as a function of time. The results showed that: (1) the cement setting was the result of the alpha-tricalcium phosphate hydrolysis, giving as a product calcium-deficient hydroxyapatite, while beta-tricalcium phosphate did not participate in the reaction; (2) the extent of conversion of alpha-TCP was nearly 80% after 24 hr; (3) both the extent of conversion and the compressive strength increased initially linearly with time, subsequently reaching a saturation level, with a strong correlation observed between them, indicating that the microstructural changes taking place as the setting reaction proceeded were responsible for the mechanical behavior of the cement; and (4) the microstructure of the set cement consisted of clusters of big plates with radial or parallel orientations in a matrix of small plate-like crystals.     

An in vitro diffusion model for the study of calcification of bovine pericardium tissue

Bovine pericardium (BP) is extensively used for the production of heart valve bioprostheses. BP has excellent mechanical properties but a limited lifespan because of intrinsic subsurface calcification in vivo. In this study, the in vitro mineralization of BP was investigated by a novel diffusion cell model. In two sets of experiments, glutaraldehyde-treated BP membranes were placed between two compartments, both of which contained calcium phosphate solutions made by equilibration of octacalcium phosphate (Exp I) or dicalcium phosphate dihydrate (Exp II) in phosphoric acid. The movement of calcium (Ca), phosphate (P), and protons through the BP membrane was followed throughout the diffusion process. Histology, scanning electron microscopy, wet chemical analysis, and energy dispersive X-ray analyses provided good evidence of subsurface mineralization of BP that resembled in vivo mineral deposition. Energy dispersive x-ray microanalyses found a Ca/P heterogeneity of the early subsurface mineral that formed in the membrane. The use of a diffusion cell to model BP calcification under well-characterized conditions has led to in vitro mineralization that more closely matches that observed in vivo. The results suggest that this in vitro diffusion model can be used to study the mechanism of pathological mineralization. This model has the potential to provide rapid, inexpensive, basic information about the mineralization process.     

Formation of hydroxyapatite in new calcium phosphate cements

Tetracalcium phosphate (TTCP) has been shown previously to be an essential component of self-setting calcium phosphate cements that form hydroxyapatite (HA) as the only end-product. We report herein on a new self-setting calcium phosphate cement that does not contain TTCP. These cements consist of dicalcium phosphate anhydrous (DCPA), dicalcium phosphate dihydrate (DCPD), alpha-tricalcium phosphate, or amorphous calcium phosphate and, as an additional source of calcium, calcium hydroxide or calcium carbonate. These cements require the use of a phosphate (0.2 moll(-1) or higher) solution or a high pH solution as the cement liquid. The cements harden in relatively short time (5-30 min) and form HA as the dominant end-product in 24 h. The diametral tensile strengths of the 24-h samples are in the range of 0.2 to 7.5 MPa. Results from X-ray diffraction studies suggest that the cement setting is caused by rapid HA formation induced by the high phosphate concentration of the cement liquid. Because DCPA and DCPD are highly soluble at pH values above 12.7, which is the pK3 of phosphoric acid, high phosphate concentration in the slurry solution was also attainable by using a highly alkaline solution as the cement liquid. The physicochemical properties of these cements are comparable to those of TTCP-containing cements, and the new cements may be expected to have in vivo characteristics similar to those of TTCP-containing cements as well.     

Scanning electron microscopy and microcrystals in articular diseases

The value of scanning electron microscopy (SEM) in the study of crystals in articular diseases is underlined in several cases of examination of joint fluid, or crystal deposits in articular or periarticular tissues obtained by percutaneous or surgical treatment with chemical and crystallographic correlations. Apatite crystals. Two deposits of hydroxyapatite of the rotator cuff were studied by SEM, crystallographic techniques and chemical analysis. SEM study showed spherical aggregates of various size. Urate crystals. Three tophi were observed by SEM, with crystallographic techniques and chemical analysis. Their needle-shape and their great size (20 m) were characteristic. Calcium pyrophosphate crystals. In a case of typical clinical and radiological features, examination of joint fluid, with chemical correlation showed shorter and thicker crystals than those or urate. The precise identification of crystals is based on sophisticated crystallographic techniques such as X-ray diffraction, although SEM allows an accurate and quite simple morphologic study, most often sufficient.     

Cervical spine pseudogout with myelopathy and Charcot joints

Myelopathy due to calcium pyrophosphate dihydrate (CPPD) crystals in the cervical spine is rare. Neuropathic-like joints are also uncommon manifestations of CPPD crystal disease. We describe a 79-year-old woman with multiple neuropathic joints containing CPPD crystals. She also had a large mass containing CPPD crystals impinging on the cervical spinal cord, causing a myelopathy. She appears to be the first patient with this combination of unusual clinical features with CPPD disease.     

Morphology and microstructure of electrochemically deposited calcium phosphates in a modified simulated body fluid

Calcium phosphates were deposited on a pure titanium plate for various loading times under 1.3 and 12.9 mA/cm2 in a modified simulated body fluid at 52-92 degrees C. The plate-like crystals formed under 1.3 mA/cm2 were identified to be octacalcium phosphate (OCP) and/or carbonate-containing apatite. OCP converted to carbonate-containing apatite with the elapsing time of the loading current. The needle-like precipitates formed under 12.9 mA/cm2 were identified to be carbonate-containing apatite crystals elongated parallel to the c-axis direction. The degrees of crystallinity of the deposits formed under 1.3 mA/cm2 showed the highest value around 72 degrees C, whereas those under 12.9 mA/cm2 increased with the electrolyte temperature. The carbonate content of the deposits decreased with the electrolyte temperature and the flow current. The degree of crystallinity of the electrochemically deposited calcium phosphates decreased with the carbonate content.     

Physicochemical properties of calcific deposits isolated from porcine bioprosthetic heart valves removed from patients following 2-13 years function

The purpose of this study was to characterize the physicochemical properties of calcific deposits that cause the failure of tissue-derived heart valve bioprostheses. This was done in an effort to understand the mechanism of pathologic biomineralization in the cardiovascular system and potentially prevent deterioration of bioprostheses. Calcific deposits taken from 10 failed bioprosthetic valves that had been implanted in patients for 2-13 years were characterized by chemical analysis, x-ray diffraction, FTIR spectroscopy, scanning electron microscopy, polarized light microscopy, and solubility measurements. The combined results identified the biomineral as an apatitic calcium phosphate salt with substantial incorporation of sodium, magnesium and carbonate. The average Ca/PO4 ratio for this "young" pathologic biomineral was approximately 1.3, considerably lower than approximately 1.7 found in mature atherosclerotic plaque biomineral and mature skeletal biomineral, both of which approximate hydroxyapatite in composition. Deproteinated calcific deposits from bioprostheses had thermodynamic solubilities comparable to those of both atherosclerotic plaque, typical pathologic biomineral and hydrolyzed octacalcium phosphate (OCP, Ca4H(PO4)3 x 2.5 H2O), a proposed precursor phase to biomineral apatite. This later finding, together with chemical composition and structural details of the bioprosthetic deposits themselves, supports a mechanism of cardiovascular calcification in which OCP plays a crucial role in the formation of the final apatitic phase. This suggests an approach toward prevention of bioprosthetic tissue calcification through control of the formation of the kinetically favored OCP precursor and/or its transformation into bioapatite.     

Acute sacroiliitis as a manifestation of calcium pyrophosphate dihydrate crystal deposition disease. A report of two cases

Whereas radiographic lesions of the sacroiliac joints are common in patients with calcium pyrophosphate deposition crystal disease, they are rarely accompanied with clinical symptoms. We report two cases of acute sacroiliitis probably due to calcium pyrophosphate dihydrate deposition disease. The patients were a 53-year-old man and an 82-year-old woman with chondrocalcinosis in other joints and presence on computed tomography studies of the sacroiliac joints of sclerosis and irregularities of the joint margins with a thin linear calcific deposit within the joint. Both patients recovered fully under therapy with colchicine, analgesics and rest. These two cases suggest that acute sacroiliitis can be caused by calcium pyrophosphate dihydrate crystal deposition disease.