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Biomineralisation of collagen involves functional motifs incorporated in extracellular matrix protein molecules to accomplish the objectives of stabilising amorphous calcium phosphate into nanoprecursors and directing the nucleation and growth of apatite within collagen fibrils. Here we report the use of small inorganic polyphosphate molecules to template hierarchical intrafibrillar apatite assembly in reconstituted collagen in the presence of polyacrylic acid to sequester calcium and phosphate into transient amorphous nanophases. The use of polyphosphate without a sequestration analogue resulted only in randomly-oriented extrafibrillar precipitations along the fibrillar surface. Conversely, the use of polyacrylic acid without a templating analogue resulted only in non-hierarchical intrafibrillar mineralisation with continuous apatite strands instead of discrete crystallites. The ability of using simple non-protein molecules to recapitulate different levels of structural hierarchy in mineralised collagen signifies the ultimate simplicity in Nature’s biomineralisation design principles and challenges the need for using more complex recombinant matrix proteins in bioengineering applications.

The complex morphologies of mineralised collagen fibrils are regulated through interactions between the collagen matrix and non-collagenous extracellular proteins. In the present study, polyvinylphosphonic acid, a biomimetic analogue of matrix phosphoproteins, was synthesised and confirmed with FTIR and NMR. Biomimetic mineralisation of reconstituted collagen fibrils devoid of natural non-collagenous proteins was demonstrated with TEM using a Portland cement-containing resin composite and a phosphate-containing fluid in the presence of polyacrylic acid as sequestration, and polyvinylphosphonic acid as templating matrix protein analogues. In the presence of these dual biomimetic analogues in the mineralisation medium, intrafibrillar and extrafibrillar mineralisation via bottom-up nanoparticle assembly based on the nonclassical crystallisation pathway could be identified. Conversely, only large mineral spheres with no preferred association with collagen fibrils were observed in the absence of biomimetic analogues in the medium. Mineral phases were evident within the collagen fibrils as early as 4 hours after the initially-formed amorphous calcium phosphate nanoprecursors were transformed into apatite nanocrystals. Selected area electron diffraction patterns of highly mineralised collagen fibrils were nearly identical to those of natural bone, with apatite crystallites preferentially aligned along the collagen fibril axes.

Introduction

The physiologic selectivity of calcification in bone tissue reflects selective co-expression by osteoblasts of fibrillar collagen I and of tissue nonspecific alkaline phosphatase (TNAP), which hydrolyzes the calcification inhibitor pyrophosphate (PPi) and generates phosphate (Pi). Humans and mice deficient in the PPi-generating ecto-enzyme NPP1 demonstrate soft tissue calcification, occurring at sites of extracellular matrix expansion. Significantly, the function in osteoblasts of cytosolic inorganic pyrophosphatase (abbreviated iPPiase), which generates Pi via PPi hydrolysis with neutral pH optimum, remains unknown. We assessed iPPiase in Enpp1−/− and wild type (WT) mouse osteoblasts and we tested the hypothesis that iPPiase regulates collagen I expression.

Methods

We treated mouse calvarial osteoblasts with ascorbate and β-glycerol phosphate to promote calcification, and we assessed cytosolic Pi and PPi levels, sodium-dependent Pi uptake, Pit-1 Pi co-transporter expression, and iPPiase and TNAP activity and expression. We also assessed the function of transfected Ppa1 in osteoblasts.

Results

Inorganic pyrophosphatase but not TNAP was elevated in Enpp1−/− calvariae in situ. Cultured primary Enpp1−/− calvarial osteoblasts demonstrated increased calcification despite flat TNAP activity rather than physiologic TNAP up-regulation seen in WT osteoblasts. Despite decreased cytosolic PPi in early culture, Enpp1−/− osteoblasts maintained cytosolic Pi levels comparable to WT osteoblasts, in association with increased iPPiase, enhanced sodium-dependent Pi uptake and expression of Pit-1, and markedly increased collagen I synthesis. Suppression of collagen synthesis in Enpp1−/− osteoblasts using 3,4-dehydroproline markedly suppressed calcification. Last, transfection of Ppa1 in WT osteoblasts increased cytosolic Pi and decreased cytosolic but not extracellular PPi, and induced both collagen I synthesis and calcification.

Conclusions

Increased iPPiase is associated with “Pi hunger” and increased calcification by NPP1-deficient osteoblasts. Furthermore, iPPiase induces collagen I at the levels of mRNA expression and synthesis and, unlike TNAP, stimulates calcification by osteoblasts without reducing the extracellular concentration of the hydroxyapatite crystal inhibitor PPi.

Background:

Mammographic microcalcifications represent one of the most reliable features of nonpalpable breast cancer yet remain largely unexplored and poorly understood.

Methods:

We report a novel model to investigate the in vitro mineralisation potential of a panel of mammary cell lines. Primary mammary tumours were produced by implanting tumourigenic cells into the mammary fat pads of female BALB/c mice.

Results:

Hydroxyapatite (HA) was deposited only by the tumourigenic cell lines, indicating mineralisation potential may be associated with cell phenotype in this in vitro model. We propose a mechanism for mammary mineralisation, which suggests that the balance between enhancers and inhibitors of physiological mineralisation are disrupted. Inhibition of alkaline phosphatase and phosphate transport prevented mineralisation, demonstrating that mineralisation is an active cell-mediated process. Hydroxyapatite was found to enhance in vitro tumour cell migration, while calcium oxalate had no effect, highlighting potential consequences of calcium deposition. In addition, HA was also deposited in primary mammary tumours produced by implanting the tumourigenic cells into the mammary fat pads of female BALB/c mice.

Conclusion:

This work indicates that formation of mammary HA is a cell-specific regulated process, which creates an osteomimetic niche potentially enhancing breast tumour progression. Our findings point to the cells mineralisation potential and the microenvironment regulating it, as a significant feature of breast tumour development.

Bone matrix mineralisation plays a critical role in the determination of the overall biomechanical competence of bone. However, the molecular mechanisms of bone matrix mineralisation have not been fully elucidated. We used a proteomic approach to identify proteins and genes that may play a role in osteoblast matrix mineralisation. Proteomic differential display revealed a protein band that appeared only in mineralising mouse 7F2 osteoblasts. In-gel protein digestion and mass spectrometry proteomic analysis of this protein band identified 16 proteins. Furthermore, their corresponding transcripts were upregulated. This identification of proteins that may be associated with bone matrix mineralisation presents important new information toward a better understanding of the precise mechanisms of this process.

Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Alkaline phosphatase (TNAP) plays a crucial role promoting mineralization of the extracellular matrix by restricting the concentration of the calcification inhibitor inorganic pyrophosphate (PPi). Mutations in the TNAP gene cause hypophosphatasia, a heritable form of rickets and osteomalacia. Here we show that PHOSPHO1, a phosphatase with specificity for phosphoethanolamine and phosphocholine, plays a functional role in the initiation of calcification and that ablation of PHOSPHO1 and TNAP function prevents skeletal mineralization. Phospho1−/− mice display growth plate abnormalities, spontaneous fractures, bowed long bones, osteomalacia, and scoliosis in early life. Primary cultures of Phospho1−/− tibial growth plate chondrocytes and chondrocyte-derived matrix vesicles (MVs) show reduced mineralizing ability, and plasma samples from Phospho1−/− mice show reduced levels of TNAP and elevated plasma PPi concentrations. However, transgenic overexpression of TNAP does not correct the bone phenotype in Phospho1−/− mice despite normalization of their plasma PPi levels. In contrast, double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality. We conclude that PHOSPHO1 has a nonredundant functional role during endochondral ossification, and based on these data and a review of the current literature, we propose an inclusive model of skeletal calcification that involves intravesicular PHOSPHO1 function and Pi influx into MVs in the initiation of mineralization and the functions of TNAP, nucleotide pyrophosphatase phosphodiesterase-1, and collagen in the extravesicular progression of mineralization. © 2011 American Society for Bone and Mineral Research.

Bone is a specialised connective tissue and together with cartilage forms the strong and rigid endoskeleton. These tissues serve three main functions: scaffold for muscle attachment for locomotion, protection for vital organs and soft tissues and reservoir of ions for the entire organism especially calcium and phosphate. One of the most unique and important properties of bone is its ability to constantly undergo remodelling even after growth and modelling of the skeleton have been completed. Remodelling processes enable the bone to respond and adapt to changing functional situations. Bone is composed of various types of cells and collagenous extracellular organic matrix, which is predominantly type I collagen (85–95%) called osteoid that becomes mineralised by the deposition of calcium hydroxyapatite. The non-collagenous constituents are composed of proteins and proteoglycans, which are specific to bone and the dental hard connective tissues. Maintenance of appropriate bone mass depends upon the precise balance of bone formation and bone resorption which is facilitated by the ability of osteoblastic cells to regulate the rate of both differentiation and activity of osteoclasts as well as to form new bone. An overview of genetics and molecular mechanisms that involved in the differentiation of osteoblast and osteoclast is discussed.

Background

Inorganic pyrophosphate (PPi) is a physiologic inhibitor of hydroxyapatite mineral precipitation involved in regulating mineralized tissue development and pathologic calcification. Local levels of PPi are controlled by antagonistic functions of factors that decrease PPi and promote mineralization (tissue-nonspecific alkaline phosphatase, Alpl/TNAP), and those that increase local PPi and restrict mineralization (progressive ankylosis protein, ANK; ectonucleotide pyrophosphatase phosphodiesterase-1, NPP1). The cementum enveloping the tooth root is essential for tooth function by providing attachment to the surrounding bone via the nonmineralized periodontal ligament. At present, the developmental regulation of cementum remains poorly understood, hampering efforts for regeneration. To elucidate the role of PPi in cementum formation, we analyzed root development in knock-out (−/−) mice featuring PPi dysregulation.

Results

Excess PPi in the Alpl−/− mouse inhibited cementum formation, causing root detachment consistent with premature tooth loss in the human condition hypophosphatasia, though cementoblast phenotype was unperturbed. Deficient PPi in both Ank and Enpp1−/− mice significantly increased cementum apposition and overall thickness more than 12-fold vs. controls, while dentin and cellular cementum were unaltered. Though PPi regulators are widely expressed, cementoblasts selectively expressed greater ANK and NPP1 along the root surface, and dramatically increased ANK or NPP1 in models of reduced PPi output, in compensatory fashion. In vitro mechanistic studies confirmed that under low PPi mineralizing conditions, cementoblasts increased Ank (5-fold) and Enpp1 (20-fold), while increasing PPi inhibited mineralization and associated increases in Ank and Enpp1 mRNA.

Conclusions

Results from these studies demonstrate a novel developmental regulation of acellular cementum, wherein cementoblasts tune cementogenesis by modulating local levels of PPi, directing and regulating mineral apposition. These findings underscore developmental differences in acellular versus cellular cementum, and suggest new approaches for cementum regeneration.

Endochondral bone formation requires a cartilage template, known as the growth plate, and vascular invasion, bringing osteoblasts and osteoclasts. Endochondral chondrocytes undergo sequences of cell division, matrix secretion, cell hypertrophy, apoptosis, and matrix calcification/mineralisation. In this study, two critical steps of endochondral bone formation, the deposition of collagen X-rich matrix and blood vessel attraction/invasion, were investigated by immunohistochemistry. Fourteen multiple osteochondromas and six secondary peripheral chondrosarcomas occurring in patients with multiple osteochondromas were studied and compared to epiphyseal growth plate samples. Mutation analysis showed all studied patients (expect one) to harbour a germ-line mutations in either EXT1 or EXT2. Here, we described that homozygous mutations in EXT1/EXT2, which are causative for osteochondroma formation, are likely to affect terminal chondrocyte differentiation and vascularisation in the osteocartilaginous interface. Contrastingly, terminal chondrocyte differentiation and vascularisation seem to be unaffected in secondary peripheral chondrosarcoma. In addition, osteochondromas with high vascular density displayed a higher proliferation rate. A similar apoptotic rate was observed in osteochondromas and secondary peripheral chondrosarcomas. Recently, it has been shown that cells with functional EXT1 and EXT2 are outnumbering EXT1/EXT2 mutated cells in secondary peripheral chondrosarcomas. This might explain the increased type X collagen production and blood vessel attraction in these malignant tumours.

Objective

Extracellular inorganic pyrophosphate (ePPi) plays a key role in the regulation of normal and pathologic mineralization. The purpose of this work was to evaluate the role of P1 and P2 purine receptors in modulating ePPi production by articular chondrocytes.

Methods

Porcine cartilage explants and chondrocyte monolayers were cultured in the presence of P1 agonists, or a P2 agonist or antagonist and inhibitors of P2 signaling. Ambient media ePPi concentrations were measured after 48 to 96 hours.

Results

The P1 agonists NECA and CGS 21680 significantly decreased ePPi concentrations surrounding chondrocytes and cartilage explants. The P2 agonist, ADP, increased ePPi levels, and the P2 antagonist, suramin, decreased ePPi concentrations. Thapsigargin and BAPTA, which dampen Ca2+-related P2 signaling, suppressed the response to ADP.

Conclusions

Purine receptors are important regulators of ePPi production by chondrocytes. P1 receptor stimulation diminishes and P2 receptor stimulation enhances ePPi production. Alterations in receptor signaling or aberrations of extracellular purine nucleotide metabolism resulting in abnormal quantities or proportions of P1 and P2 receptor ligands could foster changes in ePPi production that in turn affect mineralization. We propose a homeostatic role for extracellular purine nucleotides and purine receptors in stabilizing ePPi concentrations.

Biocalcification of collagen matrices with calcium phosphate and biosilicification of diatom frustules with amorphous silica are two discrete processes that have intrigued biologists and materials scientists for decades. Recent advancements in the understanding of the mechanisms involved in these two biomineralisation processes have resulted in the use of biomimetic strategies to replicate these processes separately using polyanionic, polycationic or zwitterionic analogues of extracellular matrix proteins to stabilise amorphous mineral precursor phases. To date, there is a lack of a universal model that enables the subtleties of these two apparently dissimilar biomineralisation processes to be studied together. Here, we utilise the eggshell membrane as a universal model for differential biomimetic calcification and silicification. By manipulating the eggshell membrane to render it permeable to stabilised mineral precursors, it is possible to introduce nanostructured calcium phosphate or silica into eggshell membrane fibre cores or mantles. We provide a model for infiltrating the two compartmental niches of a biopolymer membrane with different intrafibre minerals to obtain materials with potentially improved structure-property relationships.

The development of chondrogenic cell lines has led to major advances in the understanding of how chondrocyte differentiation is regulated, and has uncovered many signalling pathways and gene regulatory mechanisms required to maintain normal function. ATDC5 cells are a well established in vitro model of endochondral ossification; however, current methods are limited for mineralisation studies. In this study we demonstrate that culturing cells in the presence of ascorbic acid and 10 mM β-glycerophosphate (βGP) significantly increases the rate of extracellular matrix (ECM) synthesis and reduces the time required for mineral deposition to occur to 15 days of culture. Furthermore, the specific expression patterns of Col2a1 and Col10a1 are indicative of ATDC5 chondrogenic differentiation. Fourier transform-infrared spectroscopy analysis and transmission electron microscopy (TEM) showed that the mineral formed by ATDC5 cultures is similar to physiological hydroxyapatite. Additionally, we demonstrated that in cultures with βGP, the presence of alkaline phosphatase (ALP) is required for this mineralisation to occur, further indicating that chondrogenic differentiation is required for ECM mineralisation. Together, these results demonstrate that when cultured in the presence of ascorbic acid and 10 mM βGP, ATDC5 cells undergo chondrogenic differentiation and produce a physiological mineralised ECM from Day 15 of culture onwards. The rapid and novel method for ATDC5 culture described in this study is a major improvement compared with currently published methods and this will prove vital in the pursuit of underpinning the molecular mechanisms responsible for poor linear bone growth observed in a number of chronic diseases such as cystic fibrosis, chronic kidney disease, rheumatological conditions and inflammatory bowel disease.

Introduction:

Biphosphonate-induced osteonecrosis of the jaws (ONJ) was first described in the literature in 2003. Biphosphonates, which are structural analogues of inorganic pyrophosphate, inhibit the formation, aggregation and dissolution of calcium phosphate crystals; they have a high affinity for the mineralised bone matrix and can inhibit osteoclast-mediated osseous resorption. They are divided into non-aminobiphosphonates (etidronate, clodronate, tiludronate) and aminobiphosphonates (alendronate, ibandronate, neridronate, risedronate, pamidronate, zoledronate), which have a more powerful inhibitory effect on osteoclast-mediated osseous resorption. In 2009 the AAOMS estimated the incidence of i.v. biphosphonate-induced ONJ to be 0.8–12%, and that of ONJ induced by oral biphosphonates to be 0.01–0.1%. In recent years, preventive measures have been introduced, to be adopted in all patients about to begin biphosphonate treatment, as well as behavioural measures to be adopted in patients under bisphosphonate therapy requiring dental treatment to prevent ONJ.

Aims:

To establish the incidence of the disease over the years, and whether this incidence fell in 2009; to investigate correlations between ONJ and gender, age, underlying disease, type of biphosphonate taken and duration of biphosphonate treatment; to investigate treatments instituted.

Materials and methods:

We collected data on all cases of ONJ diagnosed at the Careggi Hospital in Florence up to December 2009: the sample was made up of 59 patients (24 men, 35 women). We performed a statistical analysis of the data in our possession. No patient was excluded from the study.

Results:

The incidence of the disease was higher in the women (59.32% versus 40.68%) and this difference was statistically significant. The mean age at diagnosis was 66 years, 8 months, with a difference emerging between the women (67 yrs 6 mths) and the men (65 yrs 3 mths). The drug responsible for the highest number of cases of ONJ was zoledronate (86.44%), followed by alendronate and pamidronate (both 5.08%), and risendronate (3.40%). The most frequent of the underlying diseases requiring the use of biphosphonates was multiple myeloma (32.20%), followed by breast cancer (25.42%) and osteoporosis (10.17%). These were followed, in order of frequency, by prostate cancer, lung cancer, lymphomas and leukaemia. The oldest diagnoses dated back to 2004. The year with the smallest number of diagnosed cases was 2009, in which 4 cases of ONJ were detected (6.78% of the total). Of these, 3 patients had taken alendronate (in 2 cases for osteoporosis) and 1 zometa. The duration of treatment varied, ranging from 3 months to 11 years (mean 30.4 months).

The appearance of the lesions was due to: tooth extractions (77.97%), trauma caused by dental prostheses (15.25%), endodontics (1.69%), implantology (1.69%), no dental intervention (1.69%). In most cases the lesions were mandibular (57.6%), in 27.1% maxillary and in 15.2% of cases present at both levels.

At diagnosis, 56 patients were at stage 2 (95%) and 3 at stage 3 (5%) of the AAOMS staging system. The stage 3 patients were treated with surgical mandibular resection and vascularised graft, while the stage 2 patients received pharmacological therapy, in some cases associated with minor surgery and hyperbaric oxygen therapy (HBOT).

Conclusions:

From the data gathered in recent years on patients diagnosed with ONJ at our service, we can conclude that zoledronate caused most of the cases of ONJ. It was thus confirmed as the drug with the greatest potency and affinity, as has been shown by the literature. It can be noted that osteoporosis was found to be the third most frequent underlying disease and that the incidence of ONJ fell in 2009, presumably due to increased understanding of the condition, but that, contrary to the overall mean, in 2009 half of the cases were osteoporosis patients who had taken alendronate.

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Here, we have studied phosphosubstrate catalysis by osteoblast-derived MVs at physiologic pH, analyzing the hydrolysis of ATP, ADP, and PPi by isolated wild-type (WT) as well as TNAP-, NPP1- and PHOSPHO1-deficient MVs. Comparison of the catalytic efficiencies identified ATP as the main substrate hydrolyzed by WT MVs. The lack of TNAP had the most pronounced effect on the hydrolysis of all physiologic substrates. The lack of PHOSPHO1 affected ATP hydrolysis via a secondary reduction in the levels of TNAP in PHOSPHO1-deficient MVs. The lack of NPP1 did not significantly affect the kinetic parameters of hydrolysis when compared with WT MVs for any of the substrates. We conclude that TNAP is the enzyme that hydrolyzes both ATP and PPi in the MV compartment. NPP1 does not have a major role in PPi generation from ATP at the level of MVs, in contrast to its accepted role on the surface of the osteoblasts and chondrocytes, but rather acts as a phosphatase in the absence of TNAP. © 2010 American Society for Bone and Mineral Research.

ANK is a multipass transmembrane protein transporter thought to play a role in the export of intracellular inorganic pyrophosphate and so to contribute to the pathophysiology of chondrocalcinosis. As transforming growth factor-beta-1 (TGF-β1) was shown to favor calcium pyrophosphate dihydrate deposition, we investigated the contribution of ANK to the production of extracellular inorganic pyrophosphate (ePPi) by chondrocytes and the signaling pathways involved in the regulation of Ank expression by TGF-β1. Chondrocytes were exposed to 10 ng/mL of TGF-β1, and Ank expression was measured by quantitative polymerase chain reaction and Western blot. ePPi was quantified in cell supernatants. RNA silencing was used to define the respective roles of Ank and PC-1 in TGF-β1-induced ePPi generation. Finally, selective kinase inhibitors and dominant-negative/overexpression plasmid strategies were used to explore the contribution of several signaling pathways to Ank induction by TGF-β1. TGF-β1 strongly increased Ank expression at the mRNA and protein levels, as well as ePPi production. Using small interfering RNA technology, we showed that Ank contributed approximately 60% and PC-1 nearly 20% to TGF-β1-induced ePPi generation. Induction of Ank by TGF-β1 required activation of the extracellular signal-regulated kinase (ERK) pathway but not of p38-mitogen-activated protein kinase or of protein kinase A. In line with the general protein kinase C (PKC) inhibitor calphostin C, Gö6976 (a Ca2+-dependent PKC inhibitor) diminished TGF-β1-induced Ank expression by 60%, whereas a 10% inhibition was observed with rottlerin (a PKCδ inhibitor). These data suggest a regulatory role for calcium in TGF-β1-induced Ank expression. Finally, we demonstrated that the stimulatory effect of TGF-β1 on Ank expression was inhibited by the suppression of the Ras/Raf-1 pathway, while being enhanced by their constitutive activation. Transient overexpression of Smad 7, an inhibitory Smad, failed to affect the inducing effect of TGF-β1 on Ank mRNA level. These data show that TGF-β1 increases ePPi levels, mainly by the induction of the Ank gene, which requires activation of Ras, Raf-1, ERK, and Ca2+-dependent PKC pathways in chondrocytes.

Nucleotide pyrophosphatase phosphodiesterase 1 (NPP1) is required for the conversion of extracellular ATP into inorganic pyrophosphate (PPi), a recognised inhibitor of hydroxyapatite (HA) crystal formation. A detailed phenotypic assessment of a mouse model lacking NPP1 (Enpp1−/−) was completed to determine the role of NPP1 in skeletal and soft tissue mineralization in juvenile and adult mice. Histopathological assessment of Enpp1−/− mice at 22 weeks of age revealed calcification in the aorta and kidney and ectopic cartilage formation in the joints and spine. Radiographic assessment of the hind-limb showed hyper-mineralization in the talocrural joint and hypo-mineralization in the femur and tibia. MicroCT analysis of the tibia and femur disclosed altered trabecular architecture and bone geometry at 6 and 22 weeks of age in Enpp1−/− mice. Trabecular number, trabecular bone volume, structure model index, trabecular and cortical thickness were all significantly reduced in tibiae and femurs from Enpp1−/− mice (P<0.05). Bone stiffness as determined by 3-point bending was significantly reduced in Enpp1−/− tibiae and femurs from 22-week-old mice (P<0.05). Circulating phosphate and calcium levels were reduced (P<0.05) in the Enpp1−/− null mice. Plasma levels of osteocalcin were significantly decreased at 6 weeks of age (P<0.05) in Enpp1−/− mice, with no differences noted at 22 weeks of age. Plasma levels of CTx (Ratlaps™) and the phosphaturic hormone FGF-23 were significantly increased in the Enpp1−/− mice at 22 weeks of age (P<0.05). Fgf-23 messenger RNA expression in cavarial osteoblasts was increased 12-fold in Enpp1−/− mice compared to controls. These results indicate that Enpp1−/− mice are characterized by severe disruption to the architecture and mineralization of long-bones, dysregulation of calcium/phosphate homeostasis and changes in Fgf-23 expression. We conclude that NPP1 is essential for normal bone development and control of physiological bone mineralization.

Mineralization of growth plate cartilage is a critical event during endochondral bone formation, which allows replacement of cartilage by bone. Ankylosis protein (Ank), which transports intracellular inorganic pyrophosphate (PPi) to the extracellular milieu, is expressed by hypertrophic and, especially highly, by terminally differentiated mineralizing growth plate chondrocytes. Blocking Ank transport activity or ank expression in terminally differentiated mineralizing growth plate chondrocytes led to increases of intra- and extracellular PPi concentrations, decreases of alkaline phosphatase (APase) expression and activity, and inhibition of mineralization, whereas treatment of these cells with the APase inhibitor levamisole led to an increase of extracellular PPi concentration and inhibition of mineralization. Ank-overexpressing hypertrophic nonmineralizing growth plate chondrocytes showed decreased intra- and extracellular PPi levels; increased mineralization-related gene expression of APase, type I collagen, and osteocalcin; increased APase activity; and mineralization. Treatment of Ank-expressing growth plate chondrocytes with a phosphate transport blocker (phosphonoformic acid [PFA]) inhibited uptake of inorganic phosphate (Pi) and gene expression of the type III Na+/Pi cotransporters Pit-1 and Pit-2. Furthermore, PFA or levamisole treatment of Ank-overexpressing hypertrophic chondrocytes inhibited APase expression and activity and subsequent mineralization. In conclusion, increased Ank activity results in elevated intracellular PPi transport to the extracellular milieu, initial hydrolysis of PPi to Pi, Pi-mediated upregulation of APase gene expression and activity, further hydrolysis and removal of the mineralization inhibitor PPi, and subsequent mineralization.

Background/Aims

Ectopic osteochondral differentiation, driven by ENPP1-catalyzed generation of the chondrogenesis and calcification inhibitor inorganic pyrophosphate (PPi), promotes generalized arterial calcification of infancy. The multiligand receptor for advanced glycation end-products (RAGE), which promotes atherosclerosis and diabetic cardiovascular and renal complications, also mediates chondrocyte differentiation in response to RAGE ligand calgranulins such as S100A11. Here, we tested RAGE involvement in ENPP1 deficiency-associated arterial calcification.

Methods

Because ectopic artery calcification in Enpp1–/– mice is Pi-dependent and mediated by PPi deficiency, in vitro studies on effects of S100A11 and RAGE on mouse aortic explants were conducted using exogenous Pi, as well as alkaline phosphatase to hydrolyze ambient PPi.

Results

S100A11 induced cartilage-specific collagen IX/XI expression and calcification dependent on RAGE in mouse aortic explants that was inhibited by the endogenous RAGE signaling inhibitor soluble RAGE (sRAGE). Enpp1–/– aortic explants demonstrated decreased Pi-stimulated release of sRAGE, and increased calcification and type IX/XI collagen expression that were suppressed by exogenous sRAGE and by Rage knockout. Last, Rage knockout suppressed spontaneous aortic calcification in situ in Enpp1–/– mice.

Conclusion

Cultured Enpp1–/– aortic explants have decreased Pi-stimulated release of sRAGE, and RAGE promotes ectopic chondrogenic differentiation and arterial calcification in Enpp1–/– mice.

Generalized arterial calcification of infancy (GACI) is a life-threatening disorder in young infants. Cardiovascular symptoms are usually apparent within the first month of life. The symptoms are caused by calcification of large and medium-sized arteries, including the aorta, coronary arteries, and renal arteries. Most of the patients die by 6 months of age because of heart failure. Recently, homozygous or compound heterozygous mutations for the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene were reported as causative for the disorder. ENPP1 regulates extracellular inorganic pyrophosphate (PPi), a major inhibitor of extracellular matrix calcification. A newborn was diagnosed with GACI. The infant died at the age of 7 weeks of cardiac failure and the parents were referred to Molecular Biology and Cytogenetic lab for further workup. Cytogenetics analysis was performed on the parents, which showed normal karyotypes and mutational analysis for the ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) gene was also performed. The mutational analysis showed that both father and mother of the deceased infant were heterozygous carriers of the mutation c.749C>T (p.P250L) in exon 7 of ENPP1 and it was likely, that the deceased child carried the same mutation homozygous on both alleles and died of GACI resulting from this ENPP1 mutation. The couple was counseled and monitored for the second pregnancy. Amniocentesis was performed at 15 weeks of gestation for mutational analysis of the same gene in the second pregnancy. The analysis was negative for the parental mutations. One month after the birth of a healthy infant, peripheral blood was collected from the baby and sent for reconfirmation. The results again were negative for the mutation and the baby was on 6 months follow up and no major symptoms were seen. The parents of the child benefited enormously by learning about the disease much in advance and also its risk of recurrence. The main aim of this study is to emphasize on two aspects: (i) the importance of modern molecular techniques in diagnosis such a syndrome and (2) the difficulties faced by the physician to provide appropriate diagnosis and the adequate genetic counseling to the family without molecular facilities.

Objective

Ecto-Nucleotide Pyrophosphatase/Phosphodiesterase 1 (NPP1) generates inorganic pyrophosphate (PPi), a physiologic inhibitor of hydroxyapatite deposition. In a previous study, we found NPP1 expression to be inversely correlated with the degree of atherosclerotic plaque calcification. Moreover, function-impairing mutations of ENPP1, the gene encoding for NPP1, are associated with severe, artery tunica media calcification and myointimal hyperplasia with infantile onset in humans. NPP1 and PPi have the potential to modulate atherogenesis by regulating arterial smooth muscle cell (SMC) differentiation and function, including increase of pro-atherogenic osteopontin (OPN) expression. Hence, this study tested the hypothesis that NPP1 deficiency modulates both atherogenesis and atherosclerotic intimal plaque calcification.

Methods and Results

Npp1/ApoE double deficient mice were generated by crossing mice bearing the ttw allele of Enpp1 (that encodes a truncation mutation) with ApoE null mice and fed with high fat/high cholesterol atherogenic diet. Atherosclerotic lesion area and calcification were examined at 13, 18, 23 and 28 weeks of age. The aortic SMCs isolated from both ttw/ttw ApoE−/− and ttw/+ ApoE−/− mice demonstrated decreased Opn expression. The 28 weeks old ttw/ttw ApoE−/− and ttw/+ ApoE−/− had significantly smaller atherosclerotic lesions compared with wild type congenic ApoE−/− mice. Only ttw/ttw but not ttw/+ mice developed artery media calcification. Furthermore in ttw/+ mice, there was a tendency towards increased plaque calcification compared to ApoE−/− mice without Npp1 deficiency.

Conclusion

We conclude that Npp1 promotes atherosclerosis, potentially mediated by Opn expression in ApoE knockout mice.

Aim

To report a potential adverse effect of intensified treatment with sodium hyaluronate artificial tears.

Methods

Five cases of deep calcium deposition in the cornea associated with ocular surface disease and frequent use of hyaluronic acid artificial tears are described. All patients used one formulation of phosphate buffered hyaluronate eye drops when rapid calcification developed. All eyes required corneal graft surgery for visual rehabilitation. Specimens at keratoplasty were available for light microscopy and investigation by dispersive x ray analysis. The phosphate concentration in the medication used for topical treatment was measured and compared to alternative hyaluronate preparations.

Results

Light microscopy showed dense mineralisation of the entire stroma. The crystalline deposits consisted of hydroxyapatite, Ca5(PO4)3OH. A 50‐fold higher concentration of phosphate was measured in the sodium hyaluronate eye drops used for treatment (50.9 mmol/l) when compared with normal serum. The other hyaluronate formulations showed phosphate concentrations from <0.1 mmol/l to 10.9 mmol/l.

Conclusions

The hyaluronate artificial tear formulation “Hylo‐Comod” favours the formation of insoluble crystalline calcium phosphate deposits in presence of epithelial keratopathy. This is because of its high phosphate concentration and typically frequent instillation. Manufacturers and prescribers should be aware that topical preparations may contain considerable amounts of phosphate which may lead to sight threatening corneal complications.

BACKGROUND: Osteoblast phenotypic abnormality, namely the expression of collagen type III, has been shown previously in fracture non-union woven bone. AIMS: To investigate osteoblasts from fracture non-unions for evidence of gene expression of non-collagenous bone matrix proteins that have been implicated in mineralisation, namely matrix gla protein (MGP), osteonectin, osteopontin, and osteocalcin. MGP is a consistent component of bone matrix, but there are no reports of osteoblasts in the skeleton expressing the gene for MGP, and the site of synthesis of skeletal MGP (perhaps the liver) has yet to be determined. METHODS: Biopsies from normally healing human fractures and non-unions were examined by means of in situ hybridisation, using 35S labelled probes and autoradiography to disclose levels of gene expression. RESULTS: In normally healing fractures, mature osteoblasts on woven bone were negative for MGP mRNA, but positive for osteonectin, osteopontin, and osteocalcin mRNA molecules. In non-unions, osteoblasts displayed a novel phenotype: they were positive for MGP mRNA, in addition to osteonectin, osteopontin, and osteocalcin mRNA molecules. CONCLUSIONS: Mature osteoblasts in slowly healing fractures have an unusual phenotype: they express the gene encoding MGP, which indicates that control of osteoblast gene expression in non-unions is likely to be abnormal. This might be of importance in the pathogenesis of non-uniting human fractures, and is of current interest given the emerging status of MGP as an inhibitor of mineralisation.

Alkaline phosphatase (ALP) promotes bone formation by degrading inorganic pyrophosphate (PPi), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (Pi), an inducer of hydroxyapatite formation. Pi is a crucial molecule in differentiation and mineralization of osteoblasts. In this study, a method to immobilize ALP on fibrin scaffolds with tightly controllable pore size and pore interconnection was developed, and the biological properties of these scaffolds were characterized both in vitro and in vivo. Microporous, nanofibrous fibrin scaffolds (FS) were fabricated using a sphere-templating method. ALP was covalently immobilized on the fibrin scaffolds using 1-ethyl-3-(dimethylaminopropyl)carbodiimide hydrochloride (EDC). Scanning electron microscopic observation (SEM) showed that mineral was deposited on immobilized alkaline phosphatase fibrin scaffolds (immobilized ALP/FS) when incubated in medium supplemented with β-glycerophosphate, suggesting that the immobilized ALP was active. Primary calvarial cells attached, spread and formed multiple layers on the surface of the scaffolds. Mineral deposition was also observed when calvarial cells were seeded on immobilized ALP/FS. Furthermore, cells seeded on immobilized ALP/FS exhibited higher osteoblast marker gene expression compared to control FS. Upon implantation in mouse calvarial defects, both the immobilized ALP/FS and FS alone treated group had higher bone volume in the defect compared to the empty defect control. Furthermore, bone formation in the immobilized ALP/FS treated group was statistically significant compared to FS alone group. However, the response was not robust.

Bone undergoes continuous remodelling throughout adult life, and the equilibrium between bone formation by osteoblasts and bone resorption by osteoclasts defines the final bone mass. Here we show that Snail1 regulates this balance by controlling osteoblast differentiation. Snail1 is necessary for the early steps of osteoblast development, and it must be downregulated for their final differentiation. At the molecular level, Snail1 controls bone mass by repressing the transcription of both the osteoblast differentiation factor Runx2 and the vitamin D receptor (VDR) genes in osteoblasts. Sustained activation of Snail1 in transgenic mice provokes deficient osteoblast differentiation, which, together with the loss of vitamin D signalling in the bone, also impairs osteoclastogenesis. Indeed, the mineralisation of the bone matrix is severely affected, leading to hypocalcemia-independent osteomalacia. Our data show that the impact of Snail1 activity on the osteoblast population regulates the course of bone cells differentiation and ensures normal bone remodelling.

Inorganic pyrophosphate (PPi) is generated by ATP hydrolysis in the cells and also present in extracellular matrix, cartilage and bodily fluids. Fueling an alternative pathway for energy production in cells, PPi is hydrolyzed by inorganic pyrophosphatase (PPA1) in a highly exergonic reaction that can under certain conditions substitute for ATP-derived energy. Recombinant PPA1 is used for energy-regeneration in the cell-free systems used to study the zymology of ATP-dependent ubiquitin-proteasome system, including the role of sperm-borne proteasomes in mammalian fertilization. Inspired by an observation of reduced in vitro fertilization (IVF) rates in the presence of external, recombinant PPA1, this study reveals, for the first time, the presence of PPi, PPA1 and PPi transporter, progressive ankylosis protein ANKH in mammalian spermatozoa. Addition of PPi during porcine IVF increased fertilization rates significantly and in a dose-dependent manner. Fluorometric assay detected high levels of PPi in porcine seminal plasma, oviductal fluid and spermatozoa. Immunofluorescence detected PPA1 in the postacrosomal sheath (PAS) and connecting piece of boar spermatozoa; ANKH was present in the sperm head PAS and equatorial segment. Both ANKH and PPA1 were also detected in human and mouse spermatozoa, and in porcine spermatids. Higher proteasomal-proteolytic activity, indispensable for fertilization, was measured in spermatozoa preserved with PPi. The identification of an alternative, PPi dependent pathway for ATP production in spermatozoa elevates our understanding of sperm physiology and sets the stage for the improvement of semen extenders, storage media and IVF media for animal biotechnology and human assisted reproductive therapies.