Deposition Of Calcium Phosphate Minerals Research Articles

STIM1 a Calcium Sensor Promotes the Assembly of an ECM that Contains Extracellular Vesicles and Factors that Modulate Mineralization

Bone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. Several proteins work in coordination and contribute to this tightly regulated process. STIM1 (Stromal interaction molecule 1) is one such protein that has been recently identified to function in bone and enamel mineralization. STIM1 is a calcium sensor localized on the ER membrane and is well recognized for its physiological role in maintaining calcium homeostasis. We have demonstrated earlier that DMP1 stimulation of preosteoblasts and preodontoblasts stimulate calcium release from internal Ca2+ stores and this store depletion is sensed by STIM1. Store-operated calcium entry (SOCE) is one of the major Ca2+ influx mechanisms following store depletion in the ER. To demonstrate a role for STIM1 in dentin matrix mineralization, we generated transgenic DPSCs in which STIM1 was either overexpressed or silenced. These cells were characterized for their differentiation potential by gene expression analysis, Alizarin Red staining and the topology of the matrix examined by Field Emission Scanning Electron Microscopy (FESEM). Results suggest upregulation of genes involved in mineralization and increased calcium deposition with STIM1 overexpression. FESEM results demonstrate that STIM1 overexpression resulted in release of large amount of extracellular microvesicles and promoted matrix mineralization. Interestingly, knockdown of STIM1 resulted in release of fewer microvesicles and less mineral deposits in the ECM. Reduced dentin thickness, malformed and highly porous alveolar bone of STIM-1 null mice confirmed the role of STIM1 in the formation of calcified tissues. Overall, STIM1 is a crucial molecule in biomineralization as STIM1 can influence intracellular Ca2+ oscillations and thus provide a signal for activation of upstream and downstream effectors to promote precursor cell differentiation and matrix mineralization.

Differences in mineral composition and morphology between men and women in aortic valve calcification

Aortic valve calcification leads to the deposition of calcium phosphate minerals in the extracellular matrix of the aortic valve leaflets. The mineral deposits can severely narrow the opening of the aortic valve, leading to aortic stenosis. There are no therapies to halt or slow down disease progression and the mechanisms governing aortic valve calcification are still poorly understood. Recently, several studies have shown that for the same aortic stenosis severity, women present significantly lower calcification loads than men. The cause of this sex-related difference is unknown. To understand this difference, we analyzed mineral deposits from surgically excised calcified human aortic valves with different material characterization techniques. We find profound differences in mineral composition and morphology between sexes, which strongly suggest that minerals form slower in women than in men and follow a different mineralization pathway. This finding paves the way for new approaches specifically geared towards men or women in the diagnosis and treatment of aortic valve calcification. Statement of significanceAortic valve calcification is a health disorder with increasing prevalence and high morbidity and mortality. Currently there is no approved effective treatment; the only available therapeutic option is invasive valve replacement, to which not all patients are suited. The main reason for such lack of treatment options is our lack of understanding of the calcification mechanism. In this study, we show profound differences in mineral composition and morphology between sexes, suggesting that aortic valve calcification follows different mineralization pathways in men and women. These findings pave the way for new approaches specifically geared towards men or women in the diagnosis and treatment of aortic valve calcification.

Effect of the Ionic Concentration of Simulated Body Fluid on the Minerals Formed on Cross-Linked Elastin-Like Polypeptide Membranes.

Deposition of calcium phosphate minerals on the elastin-rich medial layers of arteries can cause severe cardiovascular complications. There are no available treatments for medial calcification, and the mechanism of mineral formation on elastin layers is still unknown. We recently developed an in vitro model of medial calcification using cross-linked elastin-like polypeptide (ELP) membranes immersed in simulated body fluid (SBF). While mineral phase evolution matched that observed in a mouse model of medial calcification, the long incubation required was a practical limitation of this model. Using higher SBF ion concentrations could be a solution to speed up mineral deposition, but its effect on the mineralization process is still not well understood. Here we analyze mineral formation and phase transformation on ELP membranes immersed in high concentration SBF. We show that while mineral deposition is significantly accelerated in these conditions, the chemistry and morphology of the minerals deposited on the ELP membranes and the overall mineralization process are strongly affected. Overall, this work suggests that while the use of low concentration SBF in this in vitro model is more appropriate to study medial calcification associated with the loss of calcification inhibitors, higher SBF ion concentration may be more relevant to study medial calcification in patients with life-threatening diseases such as chronic kidney disease.

Differing calcification processes in cultured vascular smooth muscle cells and osteoblasts

Arterial medial calcification (AMC) is the deposition of calcium phosphate mineral, often as hydroxyapatite, in the medial layer of the arteries. AMC shares some similarities to skeletal mineralisation and has been associated with the transdifferentiation of vascular smooth muscle cells (VSMCs) towards an osteoblast-like phenotype. This study used primary mouse VSMCs and calvarial osteoblasts to directly compare the established and widely used in vitro models of AMC and bone formation. Significant differences were identified between osteoblasts and calcifying VSMCs. First, osteoblasts formed large mineralised bone nodules that were associated with widespread deposition of an extracellular collagenous matrix. In contrast, VSMCs formed small discrete regions of calcification that were not associated with collagen deposition and did not resemble bone. Second, calcifying VSMCs displayed a progressive reduction in cell viability over time (≤7-fold), with a 50% increase in apoptosis, whereas osteoblast and control VSMCs viability remained unchanged. Third, osteoblasts expressed high levels of alkaline phosphatase (TNAP) activity and TNAP inhibition reduced bone formation by to 90%. TNAP activity in calcifying VSMCs was ∼100-fold lower than that of bone-forming osteoblasts and cultures treated with β-glycerophosphate, a TNAP substrate, did not calcify. Furthermore, TNAP inhibition had no effect on VSMC calcification. Although, VSMC calcification was associated with increased mRNA expression of osteoblast-related genes (e.g. Runx2, osterix, osteocalcin, osteopontin), the relative expression of these genes was up to 40-fold lower in calcifying VSMCs versus bone-forming osteoblasts. In summary, calcifying VSMCs in vitro display some limited osteoblast-like characteristics but also differ in several key respects: 1) their inability to form collagen-containing bone; 2) their lack of reliance on TNAP to promote mineral deposition; and, 3) the deleterious effect of calcification on their viability.

Hierarchical surface morphology on Ti6Al4V via patterning and hydrothermal treatment towards improving cellular response

Titanium implants are widely used in load bearing applications. Failure analysis for several decades revealed poor interfacial bonding at implant-tissue interface is the major cause. Diverse morphological and chemical modifications are carried out to overcome to this issue. In this study, we developed a simple, user friendly combinatorial approach of embossing followed by hydrothermal treatment for generating nano/micro/macro hierarchical architecture on Ti6Al4V surface with deposition of calcium phosphate minerals for improving osseointegration property. The hydrothermally treated groove/pillar patterned sample had uniformly distributed nanorods with 65 ± 3 nm diameter, surface roughness ~118 nm and ~532 nm height, respectively. Presence of phosphate and calcium were confirmed through EDS and XRD with Ca/P ratio ~1.66 which is similar to apatite like deposition. Calcium phosphate apatite formation was also confirmed on the surface by XPS. The hydrothermal treated samples demonstrated improved hMSC cell adhesion, migration, proliferation and differentiation as compared to untreated ones. Therefore, this method has potential to minimize interfacial failure through morphological fixation via tissue ingrowth as well as ongrowth.

Placental Vascular Calcification and Cardiovascular Health: It Is Time to Determine How Much of Maternal and Offspring Health Is Written in Stone.

Vascular calcification is the deposition of calcium phosphate minerals in vascular tissue. Vascular calcification occurs by both active and passive processes. Extent and tissue-specific patterns of vascular calcification are predictors of cardiovascular morbidity and mortality. The placenta is a highly vascularized organ with specialized vasculature that mediates communication between two circulatory systems. At delivery the placenta often contains calcified tissue and calcification can be considered a marker of viral infection, but the mechanisms, histoanatomical specificity, and pathophysiological significance of placental calcification are poorly understood. In this review, we outline the current understanding of vascular calcification mechanisms, biomedical consequences, and therapeutic interventions in the context of histoanatomical types. We summarize available placental calcification data and clinical grading systems for placental calcification. We report on studies that have examined the association between placental calcification and acute adverse maternal and fetal outcomes. We then review the intersection between placental dysfunction and long-term cardiovascular health, including subsequent occurrence of maternal vascular calcification. Possible maternal phenotypes and trigger mechanisms that may predispose for calcification and cardiovascular disease are discussed. We go on to highlight the potential diagnostic value of placental calcification. Finally, we suggest avenues of research to evaluate placental calcification as a research model for investigating the relationship between placental dysfunction and cardiovascular health, as well as a biomarker for placental dysfunction, adverse clinical outcomes, and increased risk of subsequent maternal and offspring cardiovascular events.

The ER Ca2+ sensor STIM1 can activate osteoblast and odontoblast differentiation in mineralized tissues

ABSTRACTBone and dentin development requires temporal and spatial deposition of calcium phosphate mineral. A host of proteins works in concert to contribute to this tightly regulated process while malfunction in this scheme often leads to pathological defects. We have reported earlier that DMP1 stimulation of preosteoblasts leads to calcium release from internal Ca2+ stores and this store depletion is sensed by the ER Ca2+ sensor STIM1 (stromal interaction molecule 1). In this study, we first assessed the temporal and spatial localization of STIM1 protein during the development of bone and dentin by immunohistochemical methods. We further analyzed the function of STIM1 by establishing a stable MC3T3-E1 cell-line by overexpressing STIM1 (MC3T3-E1/STIM1 OE). Under mineralizing conditions, STIM1 overexpressing cells showed increased calcium deposits with higher expression of key osteogenic markers, such as Runx2 and type I collagen, BMP4 when compared with the control cells. Our results demonstrate that during mineralized matrix formation STIM1, the key ER sensor protein, can promote cellular differentiation in the presence of extracellular calcium.

Human Alternative Macrophages Populate Calcified Areas of Atherosclerotic Lesions and Display Impaired RANKL-Induced Osteoclastic Bone Resorption Activity.

Vascular calcification is a process similar to bone formation leading to an inappropriate deposition of calcium phosphate minerals in advanced atherosclerotic plaques. Monocyte-derived macrophages, located in atherosclerotic lesions and presenting heterogeneous phenotypes, from classical proinflammatory M1 to alternative anti-inflammatory M2 macrophages, could potentially display osteoclast-like functions. To characterize the phenotype of macrophages located in areas surrounding the calcium deposits in human atherosclerotic plaques. Macrophages near calcium deposits display an alternative phenotype being both CD68 and mannose receptor-positive, expressing carbonic anhydrase type II, but relatively low levels of cathepsin K. In vitro interleukin-4-polarization of human primary monocytes into macrophages results in lower expression and activity of cathepsin K compared with resting unpolarized macrophages. Moreover, interleukin-4 polarization lowers expression levels of the osteoclast transcriptional activator nuclear factor of activated T cells type c-1, associated with increased gene promoter levels of the transcriptional repression mark H3K27me3 (histone 3 lysine 27 trimethylation). Despite higher expression of the receptor activator of nuclear factor κB receptor, receptor activator of nuclear factor κB ligand/macrophage colony-stimulating factor induction of nuclear factor of activated T cells type c-1 and cathepsin K expression is defective in these macrophages because of reduced Erk/c-fos-mediated downstream signaling resulting in impaired bone resorption capacity. These results indicate that macrophages surrounding calcium deposits in human atherosclerotic plaques are phenotypically defective being unable to resorb calcification.

Biomineralization Studies on Cellulose Membrane Exposed to Biological Fluids of Anodonta cygnea

The present work proposes to analyse the results obtained under in vitro conditions where cellulose artificial membranes were incubated with biological fluids from the freshwater bivalve Anodonta cygnea. The membranes were mounted between two half 'Ussing chambers' with different composition solutions in order to simulate epithelial surfaces separating organic fluid compartments. The membrane surfaces were submitted to two synthetic calcium and phosphate solutions on opposite sides, at pH 6.0, 7.0 or 9.0 during a period of 6 hours. Additional assays were accomplished mixing these solutions with haemolymph or extrapallial fluid from A. cygnea, only on the calcium side. A selective ion movement, mainly dependent on the membrane pore size and/or cationic affinity, occurred with higher permeability for calcium ions to the opposite phosphate chamber supported by calcium diffusion forces across the cellulose membrane. In general, this promoted a more intense mineral precipitation on the phosphate membrane surface. A strong deposition of calcium phosphate mineral was observed at pH 9.0 as a primary layer with a homogeneous microstructure, being totally absent at pH 6.0. The membrane showed an additional crystal phase at pH 7.0 exhibiting a very particular hexagonal or cuttlebone shape, mainly on the phosphate surface. When organic fluids of A. cygnea were included, these crystal forms presented a high tendency to aggregate under rosaceous shapes, also predominantly in the phosphate side. The cellulose membrane was permeable to small organic molecules that diffused from the calcium towards the phosphate side. In the calcium side, very few similar crystals were observed. The presence of organic matrix from A. cygnea fluids induced a preliminary apatite-brushite crystal polymorphism. So, the present results suggest that cellulose membranes can be used as surrogates of biological epithelia with preferential ionic diffusion from the calcium to the phosphate side where the main mineral precipitation events occurred. Additionally, the organic fluids from freshwater bivalves should be also thoroughly researched in the applied biomedical field, as mineral nucleators and crystal modulators on biosynthetic systems.

Physicochemical and cell biological characterization of PMMA bone cements modified with additives to increase bioactivity

Polymethylmethacrylate (PMMA) bone cement is the most widely used material in surgery to fix joint replacements in the bone. In this study, we propose a new approach to generate bioactive PMMA surfaces directly at the site of implantation by adding the amphiphilic molecule phosphorylated 2-hydroxyethylmethacrylate (HEMA-P) to commercial PMMA bone cement, both with or without addition of 1-5% soluble calcium and carbonate salts. The setting behavior as well as the mechanical properties, the bonding quality at the metal/cement interface, mineral deposition, and cellular response for different cement modifications were investigated in vitro. The addition of HEMA-P resulted in entirely positive effects with respect to proliferation and differentiation of osteoblast-like cells (SaOs-2) and a very tight contact at the metal/cement interface. No detrimental changes of other properties were detected. The additional incorporation of salts provoked an increased deposition of calcium phosphate minerals but no further improvement in SaOs-2 cell differentiation. A significant decrease in polarization resistance for cements with high salt content (5%) was attributed to debonding between metal and cement. The results suggest an improved clinical performance of PMMA/HEMA-P composites, which might be further enhanced by small amounts of the soluble salts.

The Role of Cellular Senescence During Vascular Calcification: A Key Paradigm in Aging Research

Vascular calcification has severe clinical consequences and is considered an accurate predictor of future adverse cardiovascular events. Vascular calcification refers to the deposition of calcium phosphate mineral, most often hydroxyapatite, in arteries. Extensive calcification of the vascular system is a key characteristic of aging. In this article, we outline the mechanisms governing vascular calcification and highlight its association with cellular senescence. This review discusses the molecular mechanisms of cellular senescence and its affect on calcification of vascular cells, the relevance of phosphate regulation and the function of FGF23 and Klotho proteins. The association of vascular calcification and cellular senescence with the rare human aging disorder Hutchison-Gilford Progeria Syndrome (HGPS) is highlighted and the mouse models used to try to determine the underlying pathways are discussed. By understanding the pathways involved in these processes novel drug targets may be elucidated in an effort to reduce the effects of cellular aging as a risk factor in cardiovascular disease.

Biomimetic deposition of calcium phosphate minerals on the surface of partially demineralized dentine modified with phosphorylated chitosan

The aim of this study is to remineralize partially demineralized dentine sections using phosphorylated chitosan (P-chi) based on mimicking the nucleating role of the phosphorylated noncollagenous proteins in the biomineralization of dentine. The surface of partially demineralized dentine sections, mainly composed of type I collagen, were modified by covalent immobilization of P-chi on the collagen surface. The dentine sections were subsequently put into remineralizing solution for remineralization, and then the remineralizing effect was investigated. The remineralization effect of this methodology was compared with that of fluoride. Moreover, the influence of surface properties of samples on remineralizing effect was investigated. The experimental results indicated that the effect of fluoride on remineralization of dentine was limited when residual crystals were lacking on the surface of partially demineralized dentine, whereas the covalent immobilization of P-chi can significantly induce deposition of calcium phosphate minerals on the surface of the partially demineralized dentine. This biomimetic methodology resulted in favorable surface properties (i.e., highly negative charge and low interfacial free energy between substrate and remineralizing medium) for crystal nucleation. Thus, P-chi can facilitate surface remineralization of dentine and thereby could find application in the minimally invasive management of dentine caries and dentine hypersensitivity.

Chemical and topographical modification of PHBV surface to promote osteoblast alignment and confinement

Proper cell attachment and distribution, and thus stronger association in vivo between a bone implant and native tissue will improve the success of the implant. In this study, the aim was to achieve promotion of attachment and uniform distribution of rat mesenchymal stem cell-derived osteoblasts by introducing chemical and topographical cues on poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) film surfaces. As the chemical cues, either alkaline phosphatase was covalently immobilized on the film surface to induce deposition of calcium phosphate minerals or fibrinogen was adsorbed to improve cell adhesion. Microgrooves and micropits were introduced on the film surface by negative replication of micropatterned Si wafers. Both chemical cues improved cell attachment and even distribution on the PHBV films, but Fb was more effective especially when combined with the micropatterns. Cell alignment (<10 degrees deviation angle) parallel to chemically modified microgrooves (1, 3, or 8 microm groove width) and on 10 microm-thick Fb lines printed on the unpatterned films was achieved. The cells on unpatterned and 5 microm-deep micropitted films were distributed and oriented randomly. Results of this study proved that microtopographies on PHBV can improve osseointegration when combined with chemical cues, and that microgrooves and cell adhesive protein lines on PHBV can guide selective osteoblast adhesion and alignment.

A composite coating by electrolysis-induced collagen self-assembly and calcium phosphate mineralization

A composite coating that is composed of collagen protein and calcium phosphate minerals is considered to be bioactive and may enhance bone growth and fixation of metallic orthopedic implants. In this study, we have successfully developed a uniform collagen fibril/octacalcium phosphate composite coating on silicon substrate by electrolytic deposition (ELD). The coating deposition was done through applying a constant potential to the cathode in a three-electrode electrochemistry cell that contain a mild acidic (pH 4.8–5.3) aqueous solution of collagen molecules, calcium and phosphate ions. The coating process involved self-assembly of collagen fibrils and the deposition of calcium phosphate minerals as a result of cathode reaction and local pH increase. The two steps could be synchronized to form a bone-like composite at nanometer scale through proper adjustment of the solution and deposition parameters. Coating morphology, crystal structure and compositions were analyzed by optical and fluorescence microscopy, scanning and transmission electron microscopy, energy dispersive X-ray analysis, inductively coupled argon plasma optical emission spectrophotometry, and Fourier-transformed infrared spectroscopy. Under typical deposition conditions, the cathode (Si) surface formed a thin (100nm) layer of calcium phosphate coating, on top of which a thick (∼100μm) composite layer formed. The porous composite layer consists of a collagen fibril network on which clusters of octacalcium phosphate crystals nucleate and grow. By combining photolithography and ELD, we were also able to pattern the composite coating into regular arrays of squares. Preliminary results by nanoindentation tests showed that properly prepared composite coating may have higher elastic modulus and scratch resistance than monolithic porous calcium phosphate coating. The results not only provide a novel bioactive coating for biomedical implants, but also establish a new experimental protocol for studying biomineralization mechanisms of collagen based biological tissues.

Biodegradation Study of Amorphous and Crystalline Calcium Metaphosphate in the SBF and Tris-Buffer Solution

Two types of pure and Na{sub 2}O added calcium metaphosphate (CMP), amorphous and crystalline, were prepared by the quenching from the melt and the crystallization of the amorphous CMP, respectively. Two specimens were soaked into tris-buffer solution and SBF for 1 day to 3 weeks. The weight decrease of the amorphous specimens was significantly higher than that of the crystalline specimens with the soaking time in the both solutions. The addition of Na{sub 2}O in CMP effects the weight change in the amorphous CMP, but not significant in crystalline CMP. And the addition of Na{sub 2}O in CMP also changes the deposition of calcium phosphate minerals, which is related to the dissolution of the CMP materials. The degradation and deposition was faster in the SBF than the tris-buffer solution. (orig.)

Compartmental control of mineral formation: adaptation of a biomineralization strategy for biomedical use

A common biomineralization strategy is the use of lipid vesicle compartments to sequester ions, control ion transport, and to control the shape and size of inorganic mineral particles deposited within developing tissues. In this paper, we describe an adaptation of this bioinspired strategy for inducing rapid formation of calcium phosphates for potential medical and dental use. Calcium and inorganic phosphate salts were separately loaded into temperature-sensitive liposomes composed of 90% 1,2-bis(palmitoyl)- sn-glycero-3-phosphocholine and 10% 1,2-bis(myristoyl)- sn-glycero-3-phosphocholine. Leakage of calcium and inorganic phosphate from liposomes stored at room temperature was negligible over a period of several weeks. Calcium-loaded liposomes released entrapped calcium when heated to 36–37°C, while phosphate-loaded liposomes released entrapped inorganic phosphate (P i) at slightly lower temperatures (33–34°C). Release of entrapped ions at this temperature was due to increased permeability of phospholipid bilayers at the lipid chain melting temperature. Aqueous suspensions containing mixtures of calcium and P i-loaded liposomes formed calcium phosphate mineral when heated to 37°C, as indicated by a rapid drop in suspension pH from 7 to approximately 5. This result was consistent with diffusion of both calcium and phosphate ions out of the liposomes and their reaction to form calcium phosphate mineral. Such a strategy could be used in medical and dental procedures to induce rapid inorganic mineral formation from an injectable liposome-containing fluid. This was illustrated by applying a liposome suspension to warm human dentin and enamel surfaces, which resulted in deposition of calcium phosphate minerals onto the tissue substrates.