Studies of the mechanism of biological calcification

Ultrastructural changes duringin vitro calcification of a collagen-containing matrix derived from beef tendon were correlated with the time of calcification and with the effect of calcification inhibitors. The early stage of calcification is characterized by a molar Ca/P ratio of 1.0–1.2 and by the formation of intrafibrillar electron dense particles. These particles were 50 to 100 Å in diameter and produced no sharp fringes when examined by selected area electron diffraction. During later stages, the electron dense particles appear to coalesce and serve as centers for the growth of hydroxyapatite crystals. At concentration that inhibit hydroxyapatite formation, methylenediphosphonate allows uptake of a small amount of Ca²⁺ and HPO₄ ^2? with a molar Ca/P of 1.0–1.2 and the formation of the non-crystalline electron dense particles but prevents subsequent growth of hydroxyapatite. Phosphonoacetate depresses Ca²⁺ and HPO₄ ^2? uptake further and prevents formation of both noncrystalline and crystalline mineral. These findings are consistent with the multistep model of matrix calcification proposed earlier and indicate that the formation of a non-crystalline precursor of hydroxyapatite is induced by the matrix from a soluble phase that is comparable to physiological fluids.     

Matrix vesicles produced by osteoblast-like cells in culture become significantly enriched in proteoglycan-degrading metalloproteinases after addition of β-Glycerophosphate and ascorbic acid

Matrix vesicles, media vesicles, and plasma membranes from three well-characterized, osteoblast-like cells (ROS 17/2.8, MG-63, and MC-3T3-E1) were evaluated for their content of enzymes capable of processing the extracellular matrix. Matrix vesicles were enriched in alkaline phosphatase specific activity over the plasma membrane and contained fully active neutral, but not acid, metalloproteinases capable of digesting proteoglycans, potential inhibitors of matrix calcification. Matrix vesicle enrichment in neutral metalloproteinase varied with the cell line, whereas collagenase, lysozyme, hyaluronidase, and tissue inhibitor of metalloproteinases (TIMP) were not found in any of the membrane fractions examined. MC-3T3-E1 cells were cultured for 32 days in the presence of ascorbic acid (100 g/ml), -glycerophosphate (5 mM), or a combination of the two, to assess changes in matrix vesicle enzymes during calcification. Ascorbate or -glycerophosphate alone had no effect, but in combination produced significant increases in both active and total neutral metalloproteinase in matrix vesicles and plasma membranes, with the change seen in matrix vesicles being the most dramatic. This correlated with an increase in the formation of von Kossa-positive nodules. The results of the present study indicate that osteoblast-like cells produce matrix vesicles enriched in proteoglycan-degrading metalloproteinases. In addition, the observation that matrix vesicles contain significantly increased metalloproteinases under conditions favorable for mineralization in vitro lends support to the hypothesis that matrix vesicles play an important role in extracellular matrix processing and calcification in bone.     

Chronic Mineral Dysregulation Promotes Vascular Smooth Muscle Cell Adaptation and Extracellular Matrix Calcification

In chronic kidney disease (CKD) vascular calcification occurs in response to deranged calcium and phosphate metabolism and is characterized by vascular smooth muscle cell (VSMC) damage and attrition. To gain mechanistic insights into how calcium and phosphate mediate calcification, we used an ex vivo model of human vessel culture. Vessel rings from healthy control subjects did not accumulate calcium with long-term exposure to elevated calcium and/or phosphate. In contrast, vessel rings from patients with CKD accumulated calcium; calcium induced calcification more potently than phosphate (at equivalent calcium-phosphate product). Elevated phosphate increased alkaline phosphatase activity in CKD vessels, but inhibition of alkaline phosphatase with levamisole did not block calcification. Instead, calcification in CKD vessels most strongly associated with VSMC death resulting from calcium- and phosphate-induced apoptosis; treatment with a pan-caspase inhibitor ZVAD ameliorated calcification. Calcification in CKD vessels was also associated with increased deposition of VSMC-derived vesicles. Electron microscopy confirmed increased deposition of vesicles containing crystalline calcium and phosphate in the extracellular matrix of dialysis vessel rings. In contrast, vesicle deposition and calcification did not occur in normal vessel rings, but we observed extensive intracellular mitochondrial damage. Taken together, these data provide evidence that VSMCs undergo adaptive changes, including vesicle release, in response to dysregulated mineral metabolism. These adaptations may initially promote survival but ultimately culminate in VSMC apoptosis and overt calcification, especially with continued exposure to elevated calcium.Cardiovascular disease accounts for more than half of all deaths in patients with chronic kidney disease (CKD).¹ Vascular calcification is an important contributor to this cardiovascular mortality,^1,2 beginning in the first decade of life in children who are on dialysis.³ Calcification can occur in the tunica intima or media in patients with CKD but, at least in its early stages in young patients with CKD, is typically medial.⁴ Epidemiologic studies have highlighted the impact of dysregulated mineral metabolism and linked both elevated phosphate (P) and calcium (Ca) to accelerated vascular calcification.^5,6 In vitro studies using human vascular smooth muscle cells (VSMCs) have provided mechanistic insights into the role of Ca and P in the initiation and progression of calcification and have shown that in response to raised extracellular Ca and P levels, VSMCs undergo specific phenotypic changes, including osteo/chondrocytic differentiation and vesicle release.^7,8 Recent work has shown that mineral dysregulation in CKD leads to remarkably similar processes of VSMC phenotypic modulation in vivo⁴ beginning before dialysis and culminating in VSMC apoptosis and calcification in dialysis. This suggests that prolonged exposure to worsening mineral dysregulation, as well as other factors specific to the dialysis milieu, accelerate VSMC damage, reducing their inhibitory capacity and promoting calcification.To date, studies into the mechanisms that drive calcification in CKD vessels have been hampered by the lack of an appropriate in vitro model; explanted VSMCs lack the matrix and architecture of a normal vessel and undergo substantial phenotypic changes so that they may no longer be representative of contractile VSMCs in vivo.^9–11 To address this, we used an ex vivo model of intact human arteries from children and tested the response of CKD vessels (from predialysis and dialysis patients) and age-matched healthy subjects to exposure to high Ca and P levels. We show that VSMCs in different vessel types have fundamentally different responses to high Ca and P as a result of phenotypic changes induced during long-term exposure to dysregulated mineral metabolism in vivo. We hypothesize that these are adaptive changes that may provide survival benefits in predialysis but are overwhelmed in dialysis, leading to VSMC apoptosis and accelerated calcification.     

Synthesis of noncollagenous extracellular matrix proteins during development of mineralized nodules by rat periodontal ligament cells In vitro

To characterize the mineralized nodules produced by rat periodontal ligament (PDL) cells in vitro, we have studied the synthesis and distribution of mineralized tissue proteins at various stages of nodule formation. PDL cells were obtained from coagulum in the socket at 2 days after tooth extraction and cultured in Dulbecco's Modified Eagles Medium (DMEM) containing 10% fetal bovine serum and antibiotics. Confluent cells were grown in the presence of ascorbic acid (50 g/ml), dexamethasone (5 M), and -glycerophosphate (10 mM) for 3 weeks. Four stages showing distinct morphological characteristics during development of mineralized nodules were identified. Protein synthesis and deposition of proteins into the matrix were studied during these stages by metabolic labeling with [³⁵S]methionine for 24 hours. Large quantities of SPARC (secreted protein, acidic and rich in cysteine) were synthesized by confluent cells but decreased during the progress of mineralized nodule formation. Two forms of osteopontin (OPN) (67 kDa and 61 kDa) were synthesized in comparable quantities by confluent cells; OPN and bone sialoprotein (BSP) were induced by dexamethasone and represented the major proteins in the mineralized matrix. The 67 kDa form of OPN was the predominant species in the mineralized matrix. Both OPN and BSP were localized by immunogold electron microscopy on globular as well as fused electron-dense structures at sites of tissue mineralization.     

Bone remodeling in pathologic conditions

Summary Bone remodeling in pathologic conditions was studied with the scanning electron microscope (SEM). Benign and malignant ossification were examined in cases of myositis ossificans, ossifying fibroma, osteoid osteoma, and osteosarcoma, Resorption of bone due to invasion by non-ossifying tumors was found in cases of squamous cell carcinoma, adenocarcinoma, ameloblastoma, and multiple myeloma. Bone formation due to excessive production of growth hormone was studied in a case of acromegaly. Resorption of bone due to pathologic processes resembled the pattern found in surfaces which were undergoing resorption by osteoclasts. Lamelar-cortical bone formation in acromegaly was similar in nature to normal bone. The deformities were related to the excessive continuous osteogenesis that occurs in these instances. Neoplastic ossification was characterized by calcifying globules, the diameters of which ranged from 1 to 3 m. The surfaces of these globules were constructed of minute calcospherites with diameters ranging from 0.1 to 0.3 m. It is suggested that the pattern of globular calcification is similar to the type that was found with the SEM in fetal bone and cartilage, during healing of fractured bone, and also with the TEM in normal and pathologic calcification.     

Fetuin-A Protects against Atherosclerotic Calcification in CKD

Reduced serum levels of the calcification inhibitor fetuin-A associate with increased cardiovascular mortality in dialysis patients. Fetuin-A–deficient mice display calcification of various tissues but notably not of the vasculature. This absence of vascular calcification may result from the protection of an intact endothelium, which becomes severely compromised in the setting of atherosclerosis. To test this hypothesis, we generated fetuin-A/apolipoprotein E (ApoE)-deficient mice and compared them with ApoE-deficient and wild-type mice with regard to atheroma formation and extraosseous calcification. We assigned mice to three treatment groups for 9 wk: (1) Standard diet, (2) high-phosphate diet, or (3) unilateral nephrectomy (causing chronic kidney disease [CKD]) plus high-phosphate diet. Serum urea, phosphate, and parathyroid hormone levels were similar in all genotypes after the interventions. Fetuin-A deficiency did not affect the extent of aortic lipid deposition, neointima formation, and coronary sclerosis observed with ApoE deficiency, but the combination of fetuin-A deficiency, hyperphosphatemia, and CKD led to a 15-fold increase in vascular calcification in this model of atherosclerosis. Fetuin-A deficiency almost exclusively promoted intimal rather than medial calcification of atheromatous lesions. High-phosphate diet and CKD also led to an increase in valvular calcification and aorta-associated apoptosis, with wild-type mice having the least, ApoE-deficient mice intermediate, and fetuin-A/ApoE-deficient mice the most. In addition, the combination of fetuin-A deficiency, high-phosphate diet, and CKD in ApoE-deficient mice greatly enhanced myocardial calcification, whereas the absence of fetuin-A did not affect the incidence of renal calcification. In conclusion, fetuin-A inhibits pathologic calcification in both the soft tissue and vasculature, even in the setting of atherosclerosis.Hemodialysis (HD) patients experience a cardiovascular mortality of up to 20% per year, and vascular calcification is a strong independent risk factor of cardiovascular death.^1,2 Pathologic calcification is driven both by an elevated serum calcium phosphate product and by differentiation of vascular or mesenchymal cells into osteoblast-like cells, becoming mineralization competent.Serum is a metastable solution with respect to calcium phosphate precipitation. Once started, calcification proceeds rapidly in the presence of calcifiable templates such as collagen, elastin, and cell debris.^3–5 Fetuin-A accounts for approximately 50% of the capacity of serum to inhibit the spontaneous apatite formation from solutions supersaturated in calcium and phosphate.⁶ The inhibition is achieved by rapid formation of soluble colloidal fetuin-A calcium phosphate complexes, termed calciprotein particles (CPPs).^7–9We previously showed that HD and calciphylaxis patients have depressed fetuin-A serum levels accompanied by a reduced capacity of their serum to inhibit calcium phosphate precipitation.⁵ In cross-sectional studies in HD patients, fetuin-A deficiency was identified as an inflammation-related predictor of cardiovascular and all-cause mortality, respectively.^10,11 In patients without chronic kidney disease (CKD), fetuin-A levels correlated inversely with advanced coronary calcification.^12,13 Fetuin-A–deficient (Ahsg−/−) mice maintained on the DBA/2 background exhibit a fully penetrating phenotype with extensive soft tissue calcification, whereas C57BL/6 Ahsg−/− mice represent “borderline calcifying” mice whereby rapid calcification can be induced by additional metabolic challenges or induction of CKD.^5,14Calcification of the aorta or larger vessels is conspicuously absent in Ahsg−/− mice; therefore, the role for fetuin-A as an inhibitor of vascular calcification was uncertain.^15,16 Absent vascular calcification in Ahsg−/− mice may be related to the protective mechanisms by an intact endothelium, which is severely compromised in humans with atherosclerosis and thus may serve as a nidus for subsequent calcification. To test this hypothesis, we created Ahsg−/−/apolipoprotein E double-deficient (ApoE−/−) mice maintained on the C57BL/6 genetic background to dissect the contribution of fetuin-A, CKD, and an elevated calcium-phosphorus product (Ca × P) to the development of atheroma formation and vascular calcification in an established murine model of atherosclerosis.     

Odontoblast-like differentiation and mineral formation of pulpsphere derived cells on human root canal dentin in vitro

Background

The revitalization or regeneration of the dental pulp is a preferable goal in current endodontic research. In this study, human dental pulp cell (DPC) spheres were applied to human root canal samples to evaluate their potential adoption for physiological tissue-like regeneration of the dental root canal by odontoblastic differentiation as well as cell-induced mineral formation.

Methods

DPC were cultivated into three-dimensional cell spheres and seeded on human root canal specimens. The evaluation of sphere formation, tissue-like behavior and differentiation as well as mineral formation of the cells was carried out with the aid of optical light microscopy, immunohistochemical staining and scanning electron microscopy (SEM).

Results

Spheres and cells migrated out of the spheres showed an intense cell-cell- and cell-dentin-contact with the formation of extra cellular matrix. In addition, the ingrowth of cell processes into dentinal tubules and the interaction of cell processes with the tubule walls were detected by SEM-imaging. Immunohistochemical staining of the odontoblast specific matrix proteins, dentin matrix protein-1, and dentin sialoprotein revealed an odontoblast-like cell differentiation in contact with the dentin surface. This differentiation was confirmed by SEM-imaging of cells with an odontoblast specific phenotype and cell induced mineral formation.

Conclusions

The results of the present study reveal the high potential of pulp cells organized in spheres for dental tissue engineering. The odontoblast-like differentiation and the cell induced mineral formation display the possibility of a complete or partial “dentinal filling” of the root canal and the opportunity to combine this method with other current strategies.

     

Ultrastructural Analysis of Vascular Calcifications in Uremia

Accelerated intimal and medial calcification and sclerosis accompany the increased cardiovascular mortality of dialysis patients, but the pathomechanisms initiating microcalcifications of the media are largely unknown. In this study, we systematically investigated the ultrastructural properties of medial calcifications from patients with uremia. We collected iliac artery segments from 30 dialysis patients before kidney transplantation and studied them by radiography, microcomputed tomography, light microscopy, and transmission electron microscopy including electron energy loss spectrometry, energy dispersive spectroscopy, and electron diffraction. In addition, we performed synchrotron x-ray analyses and immunogold labeling to detect inhibitors of calcification. Von Kossa staining revealed calcification of 53% of the arteries. The diameter of these microcalcifications ranged from 20 to 500 nm, with a core-shell structure consisting of up to three layers (subshells). Many of the calcifications consisted of 2- to 10-nm nanocrystals and showed a hydroxyapatite and whitlockite crystalline structure and mineral phase. Immunogold labeling of calcification foci revealed the calcification inhibitors fetuin-A, osteopontin, and matrix gla protein. These observations suggest that uremic microcalcifications originate from nanocrystals, are chemically diverse, and intimately associate with proteinaceous inhibitors of calcification. Furthermore, considering the core-shell structure of the calcifications, apoptotic bodies or matrix vesicles may serve as a calcification nidus.Cardiovascular mortality in patients with ESRD is dramatically increased when compared with the general population.¹ Cardiovascular calcifications are major predictors of mortality in patients with ESRD.^2,3 Ectopic calcification is a tightly regulated process that results from an imbalance between proteinaceous and small-molecule inhibitors (e.g., fetuin, matrix gla protein, pyrophosphate) and inducers of mineralization (e.g., an elevated serum calcium-phosphate product).^4,5 In cell culture models, hyperphosphatemia leads to a transformation of vascular smooth muscle cells to osteoblast-like cells, which then express bone-specific proteins.⁶ Thus, vascular wall calcifications in particular do not seem to result from passive precipitation of calcium and phosphate but rather involve active cellular processes.So far, it is generally assumed that the mineral deposited in the vascular wall has the physicochemical properties of hydroxyapatite, the main mineral phase of bone; however, for the ultrastructure and systematic characterization of uremic cardiovascular calcifications, limited information is available in the literature. These previous studies used different tissues and yielded different results: Hydroxyapatite [Ca₁₀(PO₄)₆OH₂]—as seen in the skeleton—was described as the sole mineral phase of uremic arterial calcifications in two studies,^7,8 whereas another study found both brushite and hydroxyapatite in calcifications of stenotic arteriovenous fistulas.⁹ In a rodent study of uremic calcification, whitlockite, a magnesium-containing crystal [(Ca,Mg)₃(PO₄)₂], was detected in addition to hydroxyapatite.¹⁰Different mineral phases arising during uremic medial calcification may potentially be the result of different pathomechanisms ultimately affecting dissolution of the calcifications and the choice of therapy. We therefore performed a systematic ultrastructural analysis of uremic media calcifications in patients with ESRD.     

Serum Calcification Propensity Predicts All-Cause Mortality in Predialysis CKD

Medial arterial calcification is accelerated in patients with CKD and strongly associated with increased arterial rigidity and cardiovascular mortality. Recently, a novel in vitro blood test that provides an overall measure of calcification propensity by monitoring the maturation time (T₅₀) of calciprotein particles in serum was described. We used this test to measure serum T₅₀ in a prospective cohort of 184 patients with stages 3 and 4 CKD, with a median of 5.3 years of follow-up. At baseline, the major determinants of serum calcification propensity included higher serum phosphate, ionized calcium, increased bone osteoclastic activity, and lower free fetuin-A, plasma pyrophosphate, and albumin concentrations, which accounted for 49% of the variation in this parameter. Increased serum calcification propensity at baseline independently associated with aortic pulse wave velocity in the complete cohort and progressive aortic stiffening over 30 months in a subgroup of 93 patients. After adjustment for demographic, renal, cardiovascular, and biochemical covariates, including serum phosphate, risk of death among patients in the lowest T₅₀ tertile was more than two times the risk among patients in the highest T₅₀ tertile (adjusted hazard ratio, 2.2; 95% confidence interval, 1.1 to 5.4; P=0.04). This effect was lost, however, after additional adjustment for aortic stiffness, suggesting a shared causal pathway. Longitudinally, serum calcification propensity measurements remained temporally stable (intraclass correlation=0.81). These results suggest that serum T₅₀ may be helpful as a biomarker in designing methods to improve defenses against vascular calcification.The extent of vascular calcification is an established predictor of cardiovascular events and mortality risk in patients with CKD.¹ Mineral deposition within the medial layer of the large- and medium-sized muscular arteries is commonly seen with normal aging,² but it is markedly accelerated in patients with CKD, representing a key element of the premature vascular aging seen in this population.³ Calcification of the tunica media leads to stiffening of the arterial wall,⁴ which is associated with increased pulse pressure, pulse wave velocity (PWV), and wave reflection.⁵ These hemodynamic changes can result in increased cardiac afterload, left ventricular hypertrophy and fibrosis, reduced diastolic coronary blood flow and subendocardial ischemia, abnormal endothelial function, and damage to the microcirculation of the kidney and brain.⁶Exposure of the vasculature to the uremic milieu triggers cellular processes closely resembling physiologic bone formation within the vascular wall and cardiac valves.⁷ A key event in this phenomenon is the transdifferentiation of contractile vascular smooth muscle cells to an osteochondrocytic mineralizing phenotype.⁸ A number of systemic and paracrine factors important in regulating mineralization in bone have also been identified as potentiators of this process in the arterial wall. Less attention, however, has been paid to the common final pathway of the mineralization process itself—the formation of hydroxyapatite, which may occur as a cell-dependent or purely physiochemical process. In uremic serum, crystalline hydroxyapatite is present in spindle-shaped high molecular weight protein–mineral complexes termed secondary calciprotein particles (CPP), which are derived from spherical, amorphous calcium phosphate-containing primary CPP.⁹The main protein component in both primary and secondary CPP is fetuin-A (Fet-A), a liver-derived regulator of extracellular matrix mineralization.¹⁰ Serum CPP levels can be estimated indirectly by calculating the difference between Fet-A concentrations before and after sedimentation of the high molecular weight species with high-speed centrifugation, leaving only monomeric or free Fet-A in solution.¹¹ Higher serum Fet-A reduction ratios (CPP Fet-A) have been associated with declining renal function, increased systemic inflammation (high-sensitivity C-reactive protein [hsCRP]), and procalcific cytokine production in addition to increased coronary calcification scores and aortic stiffness.^11,12 However, although CPP Fet-A shows promise as a marker of extraosseous mineral stress, there is currently no data linking higher CPP Fet-A levels to patient outcome.Recently, a novel test was developed that measures the overall calcification propensity of serum.¹³ This test is based on timing the transformation of amorphous calcium phosphate–containing primary CPP to crystalline hydroxyapatite–containing secondary CPP. Primary and secondary CPP are formed sequentially in vitro on the addition of supraphysiologic concentrations of buffered calcium and phosphate solutions to patient serum. The balance of potentiating and inhibitory factors present in each serum sample governs the transformation time (serum calcium phosphate precipitation time [T₅₀]).In this study, we provide the first analysis of the clinical and biochemical determinants of serum calcification propensity (T₅₀) in a well described prospective cohort of patients with stages 3 and 4 CKD. We examined the relationship of T₅₀ with longitudinal changes in aortic stiffness and its association with all-cause mortality in this population. We hypothesized that increased serum calcification propensity (i.e., reduced serum T₅₀) would be associated with progressive aortic stiffening and predict poor survival.     

Studies of the mechanism of biological calcification

Uncalcified matrix derived from beef tendon will bind approximately 2 μmoles Sr²⁺/100 mg matrix. Sr²⁺ uptake is inhibited by Mg²⁺, Ca²⁺ and urea but is not influenced by inorganic phosphate, phosphoacetate, nor methylenediphosphonate. Sr²⁺ inhibits net calcification and this inhibition is reversed by Ca²⁺ but not by HPO ₄ ^2? . Although net strontium apatite formation is not induced by this matrix, an elevated Sr²⁺ uptake by previously calcified matrix was observed. The latter does not require added phosphate, is accompanied by a nearly equimolar release of Ca²⁺ to the soluble phase and is inhibited by compounds that also inhibit the⁴⁵Ca²⁺ and³²P-HPO ₄ ^2? exchange reactions. These results are interpreted to indicate that Sr²⁺ can bind to the matrix by a Mg²⁺ and urea-sensitive process as well as by incorporation into the matrix-bound mineral phase. It is proposed that the latter occurs by a heteroionic exchange mechanism that is not identical to the isoionic exchange which requires added phosphate. The possible relationship of these observations to a multi-step calcification process is discussed.     

A comparative study of alkaline phosphatase in calcifying cartilage,odontoblasts and the enamel organ

The enzyme alkaline phosphatase (AP) (EC 3.1.3.1) in three different calcification areas was studied by means of a spectrophotometric micro method using p-nitrophenylphosphate as a substrate. Rat maxillary incisor odontoblasts and enamel organ from the zones of matrix formation and maturation and tissue from rabbit metatarsal cartilage were allowed to react with the substrate in glycine-NaOH buffer at room temperature. The reaction was found to be linear for a minimum of 20 min. The pH optima for AP from these tissues were in the pH range of 10.0–10.3. In order to compare AP from the four calcification areas different parameters were studied. Heating at 56°C or 60°C for varying times revealed that the enzymes were almost completely inactivated after 10 min. Mg²⁺ ions activated the enzymes by about 25% at concentrations of 2.5 mM (enamel organ 1.25 mM); while only higher concentrations of Mg²⁺ had an inactivating effect, Ca²⁺ and PO ₄ ^3– ions were inactivating at varying concentrations. F^– ions show no effect on AP activity at concentrations below 250 mM (enamel organ 125 mM) but caused inactivation of the enzymes at about 50% at 1 M. EDTA was found to be a very effective AP inactivator at concentrations above 0.06 mM, whereas urea did not noticeably affect the enzyme reactions at concentrations below 1 M. At higher concentrations, inactivation was observed. In order to determine AP localization in the epiphyseal plate successive 40-m-thick, freeze-sectioned slices were analyzed. The activity was highest nearest the zone of cartilage calcification and decreased towards the reserve cell zone.It was concluded that the same AP isoenzyme is present in these quite different calcification loci.     

Studies of enamel proteins during maturation

Summary The solubility characteristics, amino acid composition and molecular size, and aggregation of the protein components of the organic matrix of bovine and rat enamel have been studied as a function of progressive stages in the maturation of the tissue. The age of the animal, type of tooth and location of the enamel on the crown of the tooth and the amount of calcification were used as indices of enamel maturation in the bovine species, whereas location of the anemel on the tooth crown was used as an index of maturation in the rat.The most immature enamel contains about 15–20% protein by weight, whereas the most mature enamel contains 0.1% protein or less. The proteins in the organic matrix of immature enamel are relatively insoluble in near neutral solutions of 0.5 M EDTA and are characterized by their high concentrations of proline, glutamic acid, leucine, and histidine (immature protein components). Conversely the proteins and peptides of fully mature enamel are soluble in 0.5 M EDTA and are characterized by their relatively high concentrations of aspartic and glutamic acids, serine and glycine (mature protein and peptide components). The very marked decrease in the protein content of maturing and mature enamel, and the differences in amino acid composition of mature enamel compared with immature enamel, suggest that maturation of enamel is characterized biochemically by the selective loss of certain peptides and the retention of certain other peptides, the latter possibly due to their interaction with the mineral ion constituents.The biochemical processes accompanying maturation appear to begin early in the development of the enamel at a time when the organic matrix and its protein constituents are immature as judged by their insolubility in EDTA and by their overall amino acid composition. In consists of the selective loss and the selective retention of certain components from the enamel which tend to decrease the proline and histidine contents and increase the serine, aspartic acid and glycine contents of the immature, EDTA-insoluble matrix, the latter components also becoming diffusible and suggesting protein and/or peptide degradation or depolymerization. Despite the changes in the overall composition of the immature, EDTA-insoluble enamel matrix which occur as a function of progressive stages in the maturation process, they all contain soluble, nondiffusible components of similar composition which are rich in proline, histidine and leucine.At stages of development intermediate between the most immature and the fully mature states, the enamel matrix contains a mixture of immature and mature proteins, the relative proportions of which are principally responsible for its overall amino acid composition. However, since the progressive biochemical changes of maturation are first observed in the immature, EDTA-insoluble enamel matrix, these changes also contribute to the overall differences in the amino acid composition.There is no evidence that the overall compositional changes in the enamel at any stage of development is due principally to compositional changes in the individual protein and peptide components per se. It is proposed that if peptide bond hydrolysis occurs during enamel maturation, it is catalysed in part by the enamel crystals per se.     

Progression of aortic valve stenosis: TGF-β1 is present in calcified aortic valve cusps and promotes aortic valve interstitial cell calcification via apoptosis

Background

Aortic valve stenosis characteristically progresses due to cuspal calcification, often necessitating valve replacement surgery. The present study investigated the hypothesis that TGF-β1, a cytokine that causes calcification of vascular smooth muscle cells in culture, initiates apoptosis of valvular interstitial cells as a mechanistic event in cuspal calcification.

Methods

Noncalcified and calcified human aortic valve cusps were obtained at autopsy or at the time of cardiac surgery. The distributions within cusps of TGF-β1, latent-TGF-β1-associated peptide, and TGF-β receptors were studied using immunohistochemistry. The effects of TGF-β1 on mechanistic events contributing to aortic valve calcification were also investigated using sheep aortic valve interstitial cell (SAVIC) cultures.

Results

Immunohistochemistry studies revealed that calcific aortic stenosis cusps characteristically contained within the extracellular matrix qualitatively higher levels of TGF-β1 than noncalcified cusps. Noncalcified normal valves demonstrated only focal intracellular TGF-β1. Addition of TGF-β1 to SAVIC cultures led to a cascade of events, including: cellular migration, aggregation, formation of apoptotic-alkaline phosphatase enriched nodules, and calcification of these nodules. The time course of these events in the SAVIC culture system was rapid with nodule formation with apoptosis by 72 hours, and calcification after 7 days. Furthermore, ZVAD-FMK, an antiapoptosis agent (caspase inhibitor), significantly inhibited calcification and apoptosis induced by TGF-β1, but had no effect on nodule formation. However, cytochalasin D, an actin-depolymerizing agent, inhibited nodule formation, but not calcification.

Conclusions

TGF-β1 is characteristically present within calcific aortic stenosis cusps, and mediates the calcification of aortic valve interstitial cells in culture through mechanisms involving apoptosis.     

Action of Estradiol on Epiphyseal Growth Plate Chondrocytes

Estrogen plays an important role in the human growth plate by accelerating growth and promoting epiphyseal fusion in both sexes. Nevertheless, the precise mechanisms responsible for these effects are poorly understood. In the present study, we examined the role of 17-estradiol (E2) on cell proliferation and viability, type X collagen synthesis, alkaline phosphatase activity, and matrix calcification in primary cultures of resting, proliferating, and prehypertrophic chondrocytes derived from explants of the bovine fetal epiphyseal growth plate. Growth plate chondrocytes were isolated and separated into maturationally distinct subpopulations, which were cultured for 7–21 days to high density in either (1) serum-free medium, (2) 1 nM thyroid hormone (T₃), (3) E2 concentrations ranging from 10^–13 M to 10^–7 M, or (4) a combination of T₃ and E2. To compare E2 effects in both sexes, chondrocytes were harvested from 8 fetuses of both sexes. After hormone treatment, cell cultures were analyzed for cell number and viability, collagen type X, alkaline phosphatase (ALP), and matrix calcification. Neither DNA content nor cell viability were affected by the duration or type of hormone treatment. By itself, E2 stimulated maturation of all subpopulations only in pharmacologic doses (10^–7 M). Physiologic E2 concentrations were no different than negative controls treated with ITS (insulin, transferrin, and selenite). Regardless of E2 concentrations, the addition of E2 to 1 nM T₃ did not appreciably affect the response to T₃ alone, which stimulates maturation of the phenotype. All effects were comparable in both male and female chondrocytes, in all cell subpopulations (maturation stages) and fetuses of varying gestational age. These findings indicate that at physiologic concentrations, the effects of E2 on fetal bovine growth plate chondrocyte appear to be indirect and independent of T₃, suggesting that, in vivo, E2 acts in concert with other factors or hormones to induce fusion of the growth plate.     

Abdominal aorta and pelvic artery calcifications on plain radiographs may predict mortality in chronic kidney disease,hemodialysis and renal transplantation

Purpose

Vascular calcification is common in chronic kidney disease (CKD) and predicts poor patient outcomes. While computed tomography is the gold standard for evaluation of vascular calcification, plain radiograph offers a simpler and less costly alternative. The calcification of abdominal aorta, iliac and femoral arteries has been evaluated by plain radiograph, but the data on their outcome predictabilities are still limited. The present study investigated the role of abdominal aortic calcification (AAC) and pelvic arterial calcification (PAC) in predicting overall morality in non-dialysis CKD stages 2–5 (CKD 2–5), maintenance hemodialysis (HD) and long-term kidney transplant (KT) patients.

Methods

Four hundred and nineteen patients were included. Lateral abdominal and pelvic radiographs were obtained. The degree of AAC and PAC was evaluated according to the methods described previously by Kaupplia et al. and Adragao et al. Patients were followed prospectively for 5 years.

Results

AAC and PAC scores correlated well with the correlation coefficients of 0.442 for CKD 2–5, 0.438 for HD and 0.586 for KT (p < 0.001). Patients with AAC score > 6 or PAC score > 1 were older, showed higher prevalence of DM and had higher serum phosphate and PTH but lower serum albumin and eGFR. A more severe degree of AAC was associated with an increase in KT duration, whereas a more severe degree of PAC was associated with worsening kidney function and prolonged dialysis vintage. Kaplan–Meier survival curves revealed AAC score > 6 as a significant predictor of all-cause mortality in CKD 2–5 but not in HD or KT, whereas PAC score > 1 was a significant predictor of all-cause mortality in all three populations. After adjusting for age, the predictability of AAC was lost, whereas PAC remained an independent predictor of mortality in all three populations. Adjustments for cardiovascular and CKD risk factors including age, gender, BMI, DM, serum albumin, calcium and phosphate attenuated the predictability of PAC in HD but not in CKD 2–5 or KT patients.

Conclusion

PAC was better than AAC in predicting mortality in CKD, HD and KT patients.

     

Evaluation of four animal models of intrarenal calcium deposition and assessment of the influence of dietary supplementation with essential fatty acids on calcification

Firstly, to determine a satisfactory animal model for induction of intrarenal calcification, a study of four previously described animal models of intrarenal calcification was carried out which showed that intraperitoneal injection of 10% calcium gluconate into female Sprague-Dawley rats was most effective. We then investigated the hypothesis that dietary supplementation with essential fatty acids could reduce the intrarenal calcification developing as a result of intraperitoneal calcium injection. Using a combination of fish oil and evening primrose, oil, we demonstrated a significant difference in renal parenchymal calcification, which was 940±240 g Ca/g dry weight renal parenchyma in unsupplemented animals and 320–370 ±55–65 g Ca/g dry weight renal parenchyma in supplemented animals (means±SEM, P<0.005). It was also demonstrated that there was synergism between eicosapentaenoic acid (EPA) and -linolenic acid (GLA): dietary supplementation with a combined oil preparation containing 27 mg/ml EPA and 67 mg/ml GLA mixed as 2% with food was as effective as oils containing either 400 mg/ml EPA or 80 mg/ml GLA mixed as 4% of food.     

Association between AST-120 and abdominal aortic calcification in predialysis patients with chronic kidney disease

Background

Vascular calcification is associated with mortality and cardiovascular events in patients with chronic kidney disease. AST-120, which adsorbs uremic toxins, is reported to reduce the risk of cardiovascular disease and death in chronic kidney disease patients. The aim of the current study was to investigate the association between abdominal aortic calcification and the use of AST-120 in predialysis chronic kidney disease patients.

Methods

A retrospective analysis was conducted including 199 predialysis chronic kidney disease patients (stages 4 and 5) who underwent abdominal plain computed tomography in our institution between 2005 and 2010. Abdominal aortic calcification was assessed by aortic calcification index (ACI). Patients were divided into two groups based on whether or not AST-120 was administered for at least six months, and ACI was compared between the two groups.

Results

The aortic calcification index was significantly lower in patients taking AST-120 [12.2 (2.5–30.3) vs. 25.7 (13.4–45.3) %, P < 0.001]. According to multivariate linear regression analysis, the use of AST-120 was independently and significantly correlated with ACI after adjusting for confounding factors.

Conclusions

The use of AST-120 was independently associated with less aortic calcification in predialysis chronic kidney disease patients.     

Correlation of cell necrosis and tissue calcification with ischemia/reperfusion injury after liver transplantation

Background

The cellular events following liver ischemia/reperfusion (I/R) during transplantation are largely unknown. The spectrum of I/R damage to the liver can be clinically revealed by the development of primary graft dysfunction or nonfunction. Because viral-induced liver necrosis has been associated with the development of calcifications in an animal model, we investigated the spectrum of I/R changes identified at an ultrastructural level among livers after liver transplant (LT).

Materials and methods

Random liver biopsies from five recipients with different degrees of liver dysfunction (LD) were processed for light (LM) and electron (EM) microscopic examination. The degree of calcification was estimated as mild-moderate or severe. The degree of cell vacuolization, used as a surrogate marker of cell necrosis, was reported as mild-moderate or severe.

Results

Two patients with severe LD had obvious calcifications by LM and EM examinations. Both showed significant vacuole formation, suggesting a severe degree of cell necrosis, and both succumbed to the sequelae of their LD. One patient showed evidence of mild calcifications at EM (but not LM) examination, with mild vacuole formation. The remaining two patients displayed no microcalcifications. Both presented only mild vacuole formation. Both patients recovered from LD and are currently alive.

Conclusion

In this preliminary report, we conclude that the clinically observed degree of LD after orthotopic liver transplant (OLT)correlates well with ultrastructural modifications. These include calcification and vacuole formation. We believe that both findings can be used as surrogate markers of a clinically significant hepatic I/R injury.     

Relationship between glucose exposure via peritoneal dialysis solutions and coronary artery calcification in non-diabetic peritoneal dialysis patients

Introduction

Vascular calcification is frequent in dialysis patients and is associated with increased mortality. Impaired glucose metabolism is proposed as a contributing factor for vascular calcification. We investigated whether glucose exposure via dialysate may have a role in vascular calcification in non-diabetic peritoneal dialysis patients.

Method

We measured coronary artery calcification by multi-slice computerized tomography in 50 prevalent non-diabetic peritoneal dialysis patients and assessed its relations with fasting blood glucose, homeostasis model assessment of insulin resistance (HOMA-IR), and glucose exposure from peritoneal dialysis fluid.

Results

Twenty-four patients (48%) had no coronary calcification. When patients were grouped according to the presence or absence of calcification, patients with calcification were mostly men and had higher burden of cardiovascular disease history, vitamin D dose intake, serum calcium, total glucose exposure from dialysis solution, and lower total weekly Kt/V _urea. In multivariate analysis, dialysate glucose exposure was an independent predictor of coronary artery calcification score, besides serum calcium and Kt/V _urea.

Conclusion

These data suggest that high glucose exposure from dialysis solution, which is potentially correctable, is a risk factor for vascular calcification in non-diabetic PD patients.     

Cross-linked polypentapeptide of elastin as a calcifiable matrix: Molecular weight dependence

Summary The polypentapeptide, (L·Val¹-L·Pro²-Gly³-L·Val⁴-Gly⁵) _n , when cross-linked by γ-irradiation was shown to calcify when exposed to dialyzates of calcium and phosphate augmented fetal bovine sera and the molecular weight dependence of this calcification is investigated. Five molecular weight fractions, labeled I to V in order of increasing polymer size from under 12,000 dalton (I), that is, < 30, to over 100,000 daltons (V), that is, > 240, were γ-irradiation cross-linked at 10–12 MRAD to form matrices I–V. Calcium-45 was used to follow the time course and relative amount of calcium uptake from the sera. Scanning electron microscopy and electron probe microanalysis were used to characterize the extent of matrix calcification. All matrices took up calcium-45 from the sera; however, only matrices formed from polypentapeptide with greater than 100 calcified, that is, matrices III, IV, and V. Matrix V with > 240 calcified massively and in a manner comparable to chemically cross-linked polypentapeptide with ∼ 40 using nonaugmented sera. Presumably, γ-irradiation results in chain breakage. The γ-irradiation cross-linked matrices with values of ranging from under 30 to greater than 240 establish the molecular weight dependence of matrix calcification.