Fetuin-A and Albumin Alter Cytotoxic Effects of Calcium Phosphate Nanoparticles on Human Vascular Smooth Muscle Cells

Calcification is a detrimental process in vascular ageing and in diseases such as atherosclerosis and arthritis. In particular, small calcium phosphate (CaP) crystal deposits are associated with inflammation and atherosclerotic plaque de-stabilisation. We previously reported that CaP particles caused human vascular smooth muscle cell (VSMC) death and that serum reduced the toxic effects of the particles. Here, we found that the serum proteins fetuin-A and albumin (≥1 µM) reduced intracellular Ca²⁺ elevations and cell death in VSMCs in response to CaP particles. In addition, CaP particles functionalised with fetuin-A, but not albumin, were less toxic than naked CaP particles. Electron microscopic studies revealed that CaP particles were internalised in different ways; via macropinocytosis, membrane invagination or plasma membrane damage, which occurred within 10 minutes of exposure to particles. However, cell death did not occur until approximately 30 minutes, suggesting that plasma membrane repair and survival mechanisms were activated. In the presence of fetuin-A, CaP particle-induced damage was inhibited and CaP/plasma membrane interactions and particle uptake were delayed. Fetuin-A also reduced dissolution of CaP particles under acidic conditions, which may contribute to its cytoprotective effects after CaP particle exposure to VSMCs. These studies are particularly relevant to the calcification observed in blood vessels in patients with kidney disease, where circulating levels of fetuin-A and albumin are low, and in pathological situations where CaP crystal formation outweighs calcification-inhibitory mechanisms.     

Early calcification patterns of the iliac arteries and their relation to the arterial structure

Summary Gross calcifications of the common iliac and internal iliac arteries represent a common finding in newborn children and infants. In both arteries, the calcific deposits regularly appear in certain areas of the arterial luminal surface only, whereas the other parts of the arterial wall remain free of gross lesions even in cases with a pronounced calcification. In the common iliac artery, the lateral wall of the vessel and the adjacent sectors of the anterior and posterior wall represent the predilection site of calcific deposits. In the internal iliac artery, the gross calcifications have been regularly demonstrated in the dorso-medial wall. The predominant localisation of the calcification in these parts of the vessels and its absence in the others depend on the definite structural features of the arterial tube and different affinity for calcium of the individual structural elements. In both iliac arteries, only the primary internal elastic membrane undergoes early calcification. However, unlike the most muscular arteries, this membrane is not developed in the whole arterial circumference of the common iliac and internal iliac arteries, but is absent in large areas of their arterial luminal layer. In these areas, the subendothelial or subintimal elastic layers are formed by the networks of longitudinally arranged elastic fibers or membraneous elastic structures which arise from the elastic networks with the further growth. These elastic elements always stay free of calcific deposits. The structural features found in both iliac arteries may be important for the development of the later pathological changes.     

Ultrastructure of cardiocyte degeneration and myocardial calcification in the dystrophic hamster

The myocardium of the Bio 14.6 cardiomyopathic hamster was examined with the electron microscope to identify cellular and organelle changes during the acute lesioning stage, a period typified by concomitant cardiocyte destruction and calcium elevation. Most cardiocytes retained their normal histologic and ultrastructural features, but scattered foci of altered and necrotic cells were observed in association with degenerative calcifying lesions. Prenecrotic alterations of myocytes included cellular edema; varying degrees of distension of sarcoplasmic reticulum and T-tubules; contraction bands and other myofibrillar abnormalities; mitochondrial clustering and hyperplasia; a wide spectrum of mitochondrial changes such as altered sizes, shapes, and cristal patterns, and increases in the number and size of osmiophilic matrix inclusions. Morphologic features consistent with substantial calcium excess were not observed in most altered but prenecrotic cells. Instead, calcium deposition within extruded mitochondria and upon degenerating organelle debris was observed only after cardiocyte disruption. Some calcifying cell remnants were phagocytized by macrophages, whereas large calcified plaques and other deposits remained in the interstitium. Mitochondrial calcification in vascular smooth muscle cells and fibroblasts was evident in highly calcified lesions. These observations suggest that most of the morphologically identifiable calcium deposition present in this cardiomyopathy results from secondary calcification subsequent to sarcolemmal disruption.     

A case study of ligation induced calcification in middle cerebral artery in rat

A 90 min ligation of the middle cerebral artery (MCA) followed by 72-hour reperfusion appeared to cause calcium deposition in vascular myocytes of the tunica media and the perivascular tissue of the Sprague Dawley rat. The presence of small ovoid to large irregularly shaped intracellular opaque deposits were demonstrated by light and electron microscopy. Using X-ray elemental analysis the chemical nature of the deposits was found to be calcium phosphate. The functional significance of this first demonstration of acute calcification following transient ligation of the rodent MCA invites further studies.     

Age-related arterial calcification in rats

In man, i) arteries calcify with age and ii) age-linked arterial calcification is amplified by vascular pathology such as hypertension or arteriosclerosis. Age-linked arterial calcification has a bad prognosis but drugs to prevent it are lacking. This is partially due to the lack of appropriate animal models. This paper looks at the extent to which arteries calcify with age in the rat and whether hypertension or arteriosclerosis amplifies such calcification. Total calcium levels were determined by acid digestion and flame spectrophotometry and intracellular calcium levels ([Ca2+]i) by the intracellular calcium-sensitive dye, fura-2. Arteries contained up to 5 times more calcium than other soft tissues. Arteries progressively calcified with age whereas other soft tissues did not. Accumulation of calcium with age was essentially extracellular. Hypertension had no effect on age-related arterial calcification. Calcification of the same order as in man was produced in a rat model of arteriosclerosis (vitamin D plus nicotine treatment). In conclusion, as in man, age-linked, organ-specific arterial calcification does occur in rats but its intensity is far less. Arterial calcification of a similar degree to that observed in man can be obtained in rats by hypervitaminosis D plus nicotine.     

Temperature effects on calcification rate and skeletal deposition in the temperate coral, Plesiastrea versipora (Lamarck)

The geographic range of the coral, Plesiastrea versipora (Lamarck, 1816), extends into temperate waters outside the southern limit for hermatypic corals. In the present study, calcification in Plesiastrea collected from Port Phillip Bay, Victoria was examined over the coral's normal annual temperature range (10-21 °C), which is well below the normal optimum for coral calcification in tropical corals (25-28 °C). Calcification rate in Plesiastrea was considerably lower than in reef corals, but showed a similar pattern in temperature responses, with a trend towards higher rates at ∼18 °C. The light/dark calcification ratio was markedly lower than that in tropical corals. Autoradiography showed that calcification occurred primarily by deposition of calcium carbonate at the upper surfaces of the septo-costae. Scanning electron microscopy (SEM) showed that skeletal deposition in Plesiastrea had a temperature-dependent diel pattern. In the light, calcium carbonate was deposited as small spheroidal crystals and, at higher temperatures, small needle-shaped crystals. In the dark, calcium carbonate deposition appeared to be in the form of an amorphous sheet-like cementation. Compared with other scleractinian corals, calcification rate in Plesiastrea was relatively slow and showed different patterns of skeletal deposition.     

补镁对大鼠血管钙化的影响

目的:在大鼠血管钙化模型上,观察外源性补充硫酸镁对大鼠血管钙化的影响,以探讨硫酸镁在血管钙化中作用及机制。方法:用维生素D3加尼古丁诱导大鼠血管钙化,von Kossa染色、钙含量测定及碱性磷酸酶活性测定判断血管钙化程度;用半定量RT-PCR方法测定血管钙化标志分子骨桥蛋白mRNA水平;用生物化学方法测定血管一氧化氮(NO)、超氧化物歧化酶(SOD)和丙二醛(MDA)含量。结果:钙化组大鼠血压升高,收缩压较对照组高27%(P<0.05);血管von Kossa染色见血管中膜弹性纤维间可见大量棕黑色颗粒沉积,主动脉钙含量及碱性磷酸酶活性分别较对照高3.9倍和3.4倍(P<0.01),钙化血管组织骨桥蛋白mRNA表达上调40%(P<0.01),血管钙化后可加重血管组织过氧化损伤;而诱导钙化后外源性补充硫酸镁可减轻血管钙化程度,与钙化组比较,低、高剂量硫酸镁组均明显缓解上述指标的变化。结论:诱导血管钙化后外源性补充硫酸镁可以减轻大鼠血管钙化和血管损伤程度。     

A model for studying the initiation of normal calcification in vivo

1. Rat costal cartilage was found to begin to calcify normally when the rats weigh 35-45g. 2. The cartilage is suggested as a model for the study in vivo of mechanisms concerned with normal calcification. 3. The model was tested by studying the incorporation of fluoride into the mineral deposited in the tissue. 4. The percentage of inorganic material in cartilage rose from approx. 3% of the dry weight in the uncalcified tissue to 62% in the tissue from rats weighing 300g. 5. Mineral deposited had a calcium/phosphorus molar ratio of 1.65. 6. After the oral administration of sodium fluoride to rats, fluoride was incorporated into cartilage mineral. 7. The concentration of fluoride in cartilage ash increased rapidly with calcification and the mineral became more highly fluoridated than the corresponding rib bone. 8. Fluoridated mineral showed a marked decrease in citrate concentration.     

Sustained expression of osteopontin is closely associated with calcium deposits in the rat hippocampus after transient forebrain ischemia

The present study was designed to evaluate the extent and topography of osteopontin (OPN) protein expression in the rat hippocampus 4 to 12 weeks following transient forebrain ischemia, and to compare OPN expression patterns with those of calcium deposits and astroglial and microglial reactions. Two patterns of OPN staining were recognized by light microscopy: 1) a diffuse pattern of tiny granular deposits throughout the CA1 region at 4 weeks after ischemia and 2) non-diffuse ovoid to round deposits, which formed conglomerates in the CA1 pyramidal cell layer over the chronic interval of 8 to 12 weeks. Immunogold-silver electron microscopy and electron probe microanalysis demonstrated that OPN deposits were indeed diverse types of calcium deposits, which were clearly delineated by profuse silver grains indicative of OPN expression. Intracellular OPN deposits were frequently observed within reactive astrocytes and neurons 4 weeks after ischemia but rarely at later times. By contrast, extracellular OPN deposits progressively increased in size and appeared to be gradually phagocytized by microglia or brain macrophages and some astrocytes over 8 to 12 weeks. These data indicate an interaction between OPN and calcium in the hippocampus in the chronic period after ischemia, suggesting that OPN binding to calcium deposits may be involved in scavenging mechanisms.     

Role of calcium-phosphate deposition in vascular smooth muscle cell calcification

In this work we are studying whether calcium phosphate deposition (CPD) during vascular calcification is a passive or a cell-mediated mechanism. Passive CPD was studied in fixed vascular smooth muscle cells (VSMC), which calcify faster than live cells in the presence of 1.8 mM Ca2(+) and 2 mM P(i). CPD seems to be a cell-independent process that depends on the concentration of calcium, phosphate, and hydroxyl ions, but not on Ca × P(i) concentration products, given that deposition is obtained with 2 × 2 and 4 × 1 Ca × P(i) mM2 but not with 2 × 1 or 1 × 4 Ca × P(i) mM2. Incubation with 4 mM P(i) without CPD (i.e., plus 1 mM Ca) does not induce osteogene expression. Increased expression of bone markers such as Bmp2 and Cbfa1 is only observed concomitantly with CPD. Hydroxyapatite is the only crystalline phase in both lysed and live cells. Lysed cell deposits are highly crystalline, whereas live cell deposits still contain large amounts of amorphous calcium. High-resolution transmission electron microscopy revealed a nanostructure of rounded crystallites of 5-10 nm oriented at random in lysed cells, which is compatible with spontaneous precipitation. The nanostructure in live cells consisted of long fiber crystals, 10-nm thick, embedded in an amorphous matrix. This structure indicates an active role of cells in the process of hydroxyapatite crystallization. In conclusion, our data suggest that CPD is a passive phenomenon, which triggers the osteogenic changes that are involved in the formation of a well organized, calcified crystalline structure.     

Effect of hydrochlorothiazide on urine saturation with brushite,in vitro collagen calcification by urine,and urinary inhibitors of collagen calcification

To clarify further the beneficial effect of thiazide diuretics on recurrent calcium nephrolithiasis, the effect of short-term hydrochlorothiazide therapy on urine saturation with brushite (CaHPO₄·2H₂O), in vitro collagen calcification by urine, and urinary inhibitors of calcification was studied.In 22 patients with idiopathic calcium oxalate/phosphate stones the urine calcium excretion decreased, the urine magnesium excretion increased and the urine magnesium/calcium ratio increased significantly (P < 0.001) during hydrochlorothiazide therapy. Supersaturation of the urine with brushite, which was present in 19 of the 22 patients, was reduced significantly (P < 0.001) in all during thiazide therapy, and to the undersaturated range in 16. The ability of urine to calcify collagen in vitro also decreased significantly (P < 0.001) during thiazide therapy, a change that correlated significantly (r = 0.4513, P < 0.05) with the decrease in brushite saturation. The concentration of urinary inhibitors of calcification, as determined with an in vitro collagen calcification system, was decreased significantly (P < 0.01) by thiazide therapy.It was concluded that, in addition to decreasing urine calcium excretion and increasing urine magnesium excretion, thiazide diuretics decrease the urinary brushite saturation and thus may prevent spontaneous nucleation or crystal growth, or both, of calcium phosphate. The ability of thiazides to decrease collagen calcification in vitro suggests that they may also prevent crystal growth on a nidus of organic matrix. Thiazides do not appear to act by increasing the excretion of urinary inhibitors of calcification.     

The relationship between calcification and photosynthesis in the coccolithophorid Pleurochrysis carterae

Coccolithophorids are one of the dominant groups of marine phytoplankton. They are found in large numbers throughout the surface euphotic zone of the ocean, and are able to form large-scale blooms that persist for long periods of time. Coccolithophorid cells are covered by species-specific calcium carbonate crystals of various structures. In the process of calcification in coccolithophorids, Ca²⁺ is absorbed into cells from the culture medium, and a coccolith unit is formed inside the cell. Then, the coccolith unit extrudes to the cell surface where it is constructed into crystal layers. The formation of these crystals is regulated by cellular metabolism under different environmental conditions. The carbon biogeochemical cycle in the coccolithophorids involves both photosynthetic and calcification processes, which not only play an important role in population dynamics, but also in the global carbon cycle and climate change. However, one important question remains, namely, whether the relationship between photosynthesis and calcification is species-dependent. Previous studies have yielded controversial results, even in the same species. In this paper, we selected Pleurochrysis carterae, a coccolithophore species that frequently blooms in coastal areas, to study the relationship between calcification and photosynthesis. First, we studied population growth in a batch culture over several days. For batch cultures, P. carterae was inoculated into a 10 L bioreactor at an initial cell density of approximately 5 × 10⁴ cells mL^-1. The culture conditions were optimal for cell growth. Dissolved oxygen (DO) was detected during all the culture period, and the rate of photosynthetic oxygen evolution was calculated according the DO changes during the 12-h illumination period. Algal samples (10 mL) were collected during the population growth phases. The calcium carbonate content on the cell surface was determined each day by chemical titration. Next, we studied the relationship between photosynthesis and calcification at the cellular level by observing patterns of recalcification during a 12-h period. In this study, non-calcified cells were obtained by decalcifying calcified cells collected during the exponential growth period in MES-NaOH buffer solution (pH 5.5). The non-calcified cells were inoculated into culture media containing different concentrations of Ca²⁺ (0, 5, 20, 40, 50, or 100 mg L^-1). The rate of recalcification was determined by microscopic analyses in which the number of recalcified cells per 100 cells was counted at 0, 3, 6, 9, and 12 h of culture. Ca²⁺ absorbed into the cell was detected by measuring the fluorescence intensity of Fluo-3/AM labeled Ca²⁺. The rate of photosynthetic oxygen evolution in the non-calcified cell cultures was detected by measuring the changes in dissolved oxygen during the 12-h illumination period. The results showed that during the population growth period, the rate of photosynthetic oxygen evolution was inversely related to the calcium carbonate content per cell. When the amount of calcium carbonate on the cell surface increased, the relative photosynthetic ability (the rate of photosynthetic oxygen evolution) decreased, and vice versa. Both recalcification rates and photosynthetic oxygen evolution were affected by the extracellular calcium concentration. Non-calcified cells showed different recalcification abilities at different extracellular Ca²⁺ concentrations. The recalcification rate of non-calcified cells was positively correlated with the extracellular calcium concentration when [Ca²⁺] in the medium ranged from 0 to 100 mg L^-1. However, photosynthetic oxygen evolution was suppressed at higher cell calcification rates, especially when extracellular [Ca²⁺] was 50–100 mg L^-1. Our analyses of the population growth process and the cell recalcification process confirmed that photosynthesis is inversely related to calcification in P. carterae.     

Reciprocal changes in calcification of the gastrolith and cuticle during the molt cycle of the red claw crayfish Cherax quadricarinatus

Mobilization of calcium during the molt cycle from the cuticle to transient calcium deposits is widely spread in crustaceans. The dynamics of calcium transport to transient calcium deposits called gastroliths and to the cuticle over the course of the molt cycle were studied in the crayfish Cherax quadricarinatus. In this species, calcium was deposited in the gastroliths during premolt and transported back to the cuticle during postmolt, shown by digital X-ray radiograph analysis. The predominant mineral in the crayfish is amorphous calcium carbonate embedded in an organic matrix composed mainly of chitin. Scanning electron micrographs of the cuticle during premolt showed that the endocuticle and parts of the exocuticle were the source of most of the labile calcium, while the epicuticle did not undergo degradation and remained mineralized throughout the molt cycle. The gastroliths are made of concentric layers of amorphous calcium carbonate intercalated between chitinous lamella. Measurements of pH and calcium levels during gastrolith deposition showed that calcium concentrations in the gastroliths, stomach, and muscle were about the same (10 to 11 mmol l(-1)). On the other hand, pH varied greatly, from 8.7+/-0.15 in the gastrolith cavity through 7.6+/-0.2 in muscle to 6.9+/-0.5 in the stomach.     

Synthesis of soluble phosphate polymers by RAFT and their in vitro mineralization

Soluble linear (non-cross-linked) poly(monoacryloxyethyl phosphate) (PMAEP) and poly(2-(methacryloyloxy)ethyl phosphate) (PMOEP) were successfully synthesized through reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization and by keeping the molecular weight below 20 K. Above this molecular weight, insoluble (cross-linked) polymers were observed, postulated to be due to residual diene (cross-linkable) monomers formed during purification of the monomers, MOEP and MAEP. Block copolymers consisting of PMAEP or PMOEP and poly(2-(acetoacetoxy)ethyl methacrylate) (PAAEMA) were successfully prepared and were immobilized on aminated slides. Simulated body fluid studies revealed that calcium phosphate (CaP) minerals formed on both the soluble polymers and the cross-linked gels were very similar. Both the PMAEP polymers and the PMOEP gel showed a CaP layer most probably brushite or monetite based on the Ca/P ratios. A secondary CaP mineral growth with a typical hydroxyapatite (HAP) globular morphology was found on the PMOEP gel. The soluble PMOEP film formed carbonated HAP according to Fourier transform infrared (FTIR) spectroscopy. Block copolymers attached to aminated slides showed only patchy mineralization, possibly due to the ionic interaction of negatively charged phosphate groups and protonated amines.     

Early pattern of calcification in the dorsal carapace of the blue crab, Callinectes sapidus

The pattern of calcium carbonate deposition was observed in the dorsal carapace of premolt (D2-D3) and early postmolt (0-48 h) blue crabs, Callinectes sapidus, using scanning (SEM) and transmission (TEM) electron microscopy. Samples of dorsal carapace for SEM were quick-frozen in liquid nitrogen, subsequently lyophilized, and viewed using secondary and backscattered electrons as well as X-ray maps of calcium. Pieces of lyophilized cuticle were also embedded in epoxy resin and subsequently sectioned and viewed with TEM and SEM. Fresh pieces of dorsal carapace for TEM were also fixed in 2.5% glutaraldehyde in phosphate buffer followed by postfixation in 1% OsO4 in cacodylate buffer. Calcium concentrations were determined using atomic absorption spectrophotometry and quantitative X-ray microanalysis. Calcium accumulation began in the cuticle at 3 h postmolt at the epicuticle/exocuticle boundary and at the distal and proximal margins of the interprismatic septa (IPS). The bidirectional calcification of the IPS continued until the two fronts met at 5-8 h postmolt. The roughly hexagonal walls of the IPS formed a honeycomb-like structure that resulted in a rigid cuticle. The walls of the canal containing sensory neurons also calcified at 3 h, thereby imparting rigidity to the structure and additional strength to the cuticle. Examination of thin sections of lyophilized cuticle and fixed cuticle revealed that the first mineral deposited is more soluble than calcite and is probably amorphous calcium carbonate. The amorphous calcium carbonate is transformed to calcite along a front that follows the original deposition and is probably controlled by a specialized matrix within the IPS. Since amorphous calcium carbonate is isotropic, it would also make the mineral in the exocuticle stronger by an equal distribution of mechanical stress.     

Osteopontin is associated with bioprosthetic heart valve calcification in humans

Calcification of non-osseous tissues such as heart valves or vessels is a major concern in clinical practice. The exact mechanism is still unknown. Numerous studies have shown that mineral deposits of crystalline hydroxyapatite within these tissues were associated with increased non-collagenous protein content. More recently osteopontin was found to be associated with calcification in living tissues such as vessels and native human aortic valves. The aim of this study was to determine whether or not non-collagenous proteins can also be found in non-living tissues such as glutaraldehyde-pretreatedporcine valves after implantation in humans. Thirty-eight glutaraldéhyde pretreated porcine bioprostheses were studied: 16 not implanted and 22 after 11 years of implantation in the aortic and mitral valve position in humans. In areas of calcification vizualized by Von Kossa staining and microradiography, immunostaining using polyclonal antibodies against calcium-bindingproteins showed osteopontin positive staining and no staining for osteocalcin, bone sialoprotein or osteonectin. In uncalcified areas and in non-implanted valves, staining for osteopontin or other calcium-binding proteins was negative. Western blot analysis of macroscopically calcified and uncalcified areas showed that several proteins were adsorbed in implanted valves and confirmed the presence of osteopontin in the calcified areas, while no immunolabelling was found in non-calcified areas, in uncalcified valves and in non-implanted valves. Thus the presence of osteopontin in the calcified areas of bioprosthetic heart valves implanted in human indicates that this protein is associated with bioprosthetic valvular calcification. Since these valves are made of non-living connective tissue, and no cell immunostained for osteopontin was found around the calcified area, this study suggests that a non-cellular mediated mechanism involving protein adsorption may play a role in bioprosthetic valvular calcification.     

A ferric chloride pre-treatment to prevent calcification of Nafion membrane used for implantable biosensors.

Since the perfluorosulfonated ionomer Nafion, commonly used for the protection of biosensors, experiences calcification in a biological environment, we evaluated the efficacy of preincubating Nafion membranes in a FeCl3 solution to reduce the number of nucleation sites responsible for the growth of the calcium phosphate crystals. Nafion membranes were prepared and divided into two groups. In the first group, the Nafion membranes were pre-incubated in 0.1 M FeCl3 for a 24 h period. In the second group, no pre-incubation took place. All membranes were placed in a culture medium for a period of up to 4 weeks. All membranes were then examined for changes in: (1) their surface topography (using scanning electron microscopy (SEM)); (2) their near surface chemical properties (using energy dispersive X-ray (EDX)); and (3) their permeability to glucose. The membranes that were not pre-incubated in FeCl3 showed significant cracking of the Nafion surface, extensive calcium phosphate deposits and a resulting decrease in permeability. In contrast, the membranes treated with FeCl3 showed almost no cracking, very little calcium phosphate deposits and no change in permeability to glucose. This study demonstrated that FeCl3 significantly reduces calcification of Nafion and thus should help in preserving the in vivo function of implantable biosensors that utilize Nafion in their design.     

Elevated extracellular calcium stimulates secretion of bone morphogenetic protein 2 by a macrophage cell line

It has been suggested that macrophages and multinucleated giant cells are responsible for phagocytosis of resorbable calcium phosphate (CaP) compounds implanted in bone defects. However, function of macrophages around the CaP, if continuously exposed to various concentration of extracellular calcium ions ([Ca(2+)](o)), is still unknown. The present study showed that when resorbable octacalcium phosphate was implanted in mouse calvaria, macrophage-like cells were observed around the implant during bone formation. Then, experiments were designed to investigate whether secretion of bone morphogenetic protein 2 (BMP-2) is enhanced by [Ca(2+)](o) in a macrophage cell line (J774A.1) in vitro. The mRNA expression and the secretion of BMP-2 in J774A.1 cells were significantly increased when incubated in the medium with [Ca(2+)](o) up to 14mM. The promotion of mRNA expression was maintained even when incubated with a small amount of minute CaP crystals. The present results suggest that [Ca(2+)](o) above physiological concentration may stimulate macrophages to induce osteogenic cytokine, such as BMP-2, for bone formation by osteoblast.     

Impression cytology changes and corneoconjunctival calcification in patients with chronic renal failure

OBJECTIVE: To evaluate the relationship between corneoconjunctival calcification and conjunctival squamous metaplasia in patients with chronic renal failure (CRP). STUDY DESIGN: We studied impression cytology in 45 CRF patients on regular hemodialysis and 30 age- and sex-matched controls. Specimens were obtainedfrom the temporal bulbar conjunctiva using cellulose acetate filter paper. The samples were fixed in a mixture of 70% ethyl alcohol, 37% formaldehyde and glacial acetic acid and then stained with a combination of periodic acid- Schiff and Gill's modified Papanicolaou stain and graded by a masked observer. Corneoconjunctival calcification was graded by the Porter-Crombie classification. RESULTS: Of 45 study patients, 4 (9%) disclosed grade 0, 23 (51%) grade 1, 14 (31%) grade 2 and 4 grade 3 (9%) impression cytology changes. There was a statistically significant difference between the patient and control groups (p < 0.001). Calcium deposits were more frequent and extensive in CRF patients than in controls (p = 0.01). There was no correlation between impression cytology and calcium deposit grades (p = 0.128). However the presence of conjunctival inflammation correlated with the existence of extensive squamous metaplasia (p < 0.001). CONCLUSION: The severity of conjunctival changes in CRF patients on regular hemodialysis are not related to calcium deposition but to acute conjunctival inflammation.