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1.  Extracellular ATP Released by Osteoblasts Is A Key Local Inhibitor of Bone Mineralisation 
PLoS ONE  2013;8(7):e69057.
Previous studies have shown that exogenous ATP (>1µM) prevents bone formation in vitro by blocking mineralisation of the collagenous matrix. This effect is thought to be mediated via both P2 receptor-dependent pathways and a receptor-independent mechanism (hydrolysis of ATP to produce the mineralisation inhibitor pyrophosphate, PPi). Osteoblasts are also known to release ATP constitutively. To determine whether this endogenous ATP might exert significant biological effects, bone-forming primary rat osteoblasts were cultured with 0.5-2.5U/ml apyrase (which sequentially hydrolyses ATP to ADP to AMP + 2Pi). Addition of 0.5U/ml apyrase to osteoblast culture medium degraded extracellular ATP to <1% of control levels within 2 minutes; continuous exposure to apyrase maintained this inhibition for up to 14 days. Apyrase treatment for the first 72 hours of culture caused small decreases (≤25%) in osteoblast number, suggesting a role for endogenous ATP in stimulating cell proliferation. Continuous apyrase treatment for 14 days (≥0.5U/ml) increased mineralisation of bone nodules by up to 3-fold. Increases in bone mineralisation were also seen when osteoblasts were cultured with the ATP release inhibitors, NEM and brefeldin A, as well as with P2X1 and P2X7 receptor antagonists. Apyrase decreased alkaline phosphatase (TNAP) activity by up to 60%, whilst increasing the activity of the PPi-generating ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) up to 2.7-fold. Both collagen production and adipocyte formation were unaffected. These data suggest that nucleotides released by osteoblasts in bone could act locally, via multiple mechanisms, to limit mineralisation.
doi:10.1371/journal.pone.0069057
PMCID: PMC3706437  PMID: 23874866
2.  Inorganic pyrophosphatase induces type I collagen in osteoblasts 
Bone  2009;46(1):81.
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.
doi:10.1016/j.bone.2009.08.055
PMCID: PMC2818162  PMID: 19733704
PPi; Pi; Tissue-Nonspecific Alkaline Phosphatase; Calcification; Enpp1
3.  The Role of Phosphatases in the Initiation of Skeletal Mineralization 
Calcified tissue international  2012;93(4):299-306.
Endochondral ossification is a carefully orchestrated process mediated by promoters and inhibitors of mineralization. Phosphatases are implicated, but their identities and functions remain unclear. Mutations in the tissue-nonspecific alkaline phosphatase (TNAP) gene cause hypophosphatasia, a heritable form of rickets and osteomalacia, caused by an arrest in the propagation of hydroxyapatite (HA) crystals onto the collagenous extracellular matrix due to accumulation of extracellular inorganic pyrophosphate (PPi), a physiological TNAP substrate and a potent calcification inhibitor. However, TNAP knockout (Alpl−/−) mice are born with a mineralized skeleton and have HA crystals in their chondrocyte- and osteoblast-derived matrix vesicles (MVs). We have shown that PHOSPHO1, a soluble phosphatase with specificity for two molecules present in MVs, phosphoethanolamine and phosphocholine, is responsible for initiating HA crystal formation inside MVs and that PHOSPHO1 and TNAP have nonredundant functional roles during endochondral ossification. Double ablation of PHOSPHO1 and TNAP function leads to the complete absence of skeletal mineralization and perinatal lethality, despite normal systemic phosphate and calcium levels. This strongly suggests that the Pi needed for initiation of MV-mediated mineralization is produced locally in the perivesicular space. As both TNAP and nucleoside pyrophosphohydrolase-1 (NPP1) behave as potent ATPases and pyrophosphatases in the MV compartment, our current model of the mechanisms of skeletal mineralization implicate intravesicular PHOS-PHO1 function and Pi influx into MVs in the initiation of mineralization and the functions of TNAP and NPP1 in the extravesicular progression of mineralization.
doi:10.1007/s00223-012-9672-8
PMCID: PMC3594124  PMID: 23183786
Biomineralization; Bone and cartilage development; Metabolic bone disease; Animal model
4.  Optimisation of the differing conditions required for bone formation in vitro by primary osteoblasts from mice and rats 
The in vitro culture of calvarial osteoblasts from neonatal rodents remains an important method for studying the regulation of bone formation. The widespread use of transgenic mice has created a particular need for a reliable, simple method that allows the differentiation and bone-forming activity of murine osteoblasts to be studied. In the present study, we established such a method and identified key differences in optimal culture conditions between mouse and rat osteoblasts. Cells isolated from neonatal rodent calvariae by collagenase digestion were cultured for 14–28 days before staining for tissue non-specific alkaline phosphatase (TNAP) and bone mineralisation (alizarin red). The reliable differentiation of mouse osteoblasts, resulting in abundant TNAP expression and the formation of mineralised ‘trabecular-shaped’ bone nodules, occurred only following culture in α minimum essential medium (αMEM) and took 21–28 days. Dexamethasone (10 nM) inhibited bone mineralisation in the mouse osteoblasts. By contrast, TNAP expression and bone formation by rat osteoblasts were observed following culture in both αMEM and Dulbecco’s modified Eagle’s medium (DMEM) after approximately 14 days (although ~3-fold more effectively in αMEM) and was strongly dependent on dexamethasone. Both the mouse and rat osteoblasts required ascorbate (50 μg/ml) for osteogenic differentiation and β-glycerophosphate (2 mM) for mineralisation. The rat and mouse osteoblasts showed similar sensitivity to the well-established inhibitors of mineralisation, inorganic pyrophosphate (PPi) and adenosine triphosphate (ATP; 1–100 μM). The high efficiency of osteogenic differentiation observed following culture in αMEM, compared with culture in DMEM possibly reflects the richer formulation of the former. These findings offer a reliable technique for inducing mouse osteoblasts to form bone in vitro and a more effective method for culturing bone-forming rat osteoblasts.
doi:10.3892/ijmm.2014.1926
PMCID: PMC4199408  PMID: 25200658
osteoblast; bone formation; alkaline phosphatase; mineralisation
5.  Mineralisation of reconstituted collagen using polyvinylphosphonic acid/polyacrylic acid templating matrix protein analogues in the presence of calcium, phosphate and hydroxyl ions 
Biomaterials  2010;31(25):6618-6627.
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.
doi:10.1016/j.biomaterials.2010.04.060
PMCID: PMC2904357  PMID: 20621767
extrafibrillar mineralisation; intrafibrillar mineralisation; matrix protein analogues; reconstituted collagen fibrils; tissue engineering materials
6.  Hierarchical and non-hierarchical mineralisation of collagen 
Biomaterials  2010;32(5):1291-1300.
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.
doi:10.1016/j.biomaterials.2010.10.018
PMCID: PMC3003335  PMID: 21040969
7.  Effects of pH on the Production of Phosphate and Pyrophosphate by Matrix Vesicles' Biomimetics 
Calcified tissue international  2013;93(3):222-232.
During endochondral bone formation, chondrocytes and osteoblasts synthesize and mineralize the extracellular matrix through a process that initiates within matrix vesicles (MVs) and ends with bone mineral propagation onto the collagenous scaffold. pH gradients have been identified in the growth plate of long bones, but how pH changes affect the initiation of skeletal mineralization is not known. Tissue-nonspecific alkaline phosphatase (TNAP) degrades extracellular inorganic pyrophosphate (ePPi), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/ phosphodiesterase-1 (NPP1), while contributing Pi from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine (DPPC) liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP and PPi was studied at pH 8 and 9 and compared to the data determined at pH 7.4. While catalytic efficiencies in general were higher at alkaline pH, PPi hydrolysis was maximal at pH 8 and indicated a preferential utilization of PPi over ATP, at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph (SCL) containing 1 mM ATP as substrate and amorphous calcium phosphate (ACP) or calciumphosphate- phosphatidylserine complexes (PS-CPLX) as nucleators. Propagation of mineralization was significantly more efficient at pHs 7.5 and 8 than at pH 9. Since a slight pH elevation from 7.4 to 8 promotes considerably more hydrolysis of ATP, ADP and AMP primarily by TNAP, this small pH change facilitates mineralization, especially via upregulated PPi hydrolysis by both NPP1 and TNAP, further elevating the Pi/PPi ratio, thus enhancing bone mineralization.
doi:10.1007/s00223-013-9745-3
PMCID: PMC3752608  PMID: 23942722
biomineralization; alkaline pH; microenvironment; proteoliposomes; pyrophosphate; ATP
8.  Loss of Skeletal Mineralization by the Simultaneous Ablation of PHOSPHO1 and Alkaline Phosphatase Function: A Unified Model of the Mechanisms of Initiation of Skeletal Calcification 
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.
doi:10.1002/jbmr.195
PMCID: PMC3179344  PMID: 20684022
OSTEOMALACIA; OSTEOIDOSIS; SCOLIOSIS; CALCIFICATION; BIOMINERALIZATION; HYPOPHOSPHATASIA; AKP2; TNAP
9.  Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation 
British Journal of Cancer  2012;106(3):525-537.
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.
doi:10.1038/bjc.2011.583
PMCID: PMC3273345  PMID: 22233923
microcalcifications; breast cancer; mineralisation; calcifications; hydroxyapatite; mammography
10.  Extracellular Matrix Mineralization Promotes E11/gp38 Glycoprotein Expression and Drives Osteocytic Differentiation 
PLoS ONE  2012;7(5):e36786.
Osteocytes are terminally differentiated osteoblasts which reside in a mineralized extracellular matrix (ECM). The factors that regulate this differentiation process are unknown. We have investigated whether ECM mineralization could promote osteocyte formation. To do this we have utilised MLO-A5 pre-osteocyte-like cells and western blotting and comparative RT-PCR to examine whether the expression of osteocyte-selective markers is elevated concurrently with the onset of ECM mineralization. Secondly, if mineralization of the ECM is indeed a driver of osteocyte formation, we reasoned that impairment of ECM mineralization would result in a reversible inhibition of osteocyte formation. Supplementation of MLO-A5 cell cultures with ascorbic acid and phosphate promoted progressive ECM mineralization as well as temporally associated increases in expression of the osteocyte-selective markers, E11/gp38 glycoprotein and sclerostin. Consistent with a primary role for ECM mineralization in osteocyte formation, we also found that inhibition of ECM mineralization, by omitting phosphate or adding sodium pyrophosphate, a recognized inhibitor of hydroxyapatite formation, resulted in a 15-fold decrease in mineral deposition that was closely accompanied by lower expression of E11 and other osteocyte markers such as Dmp1, Cd44 and Sost whilst expression of osteoblast markers Ocn and Col1a increased. To rule out the possibility that such restriction of ECM mineralization may produce an irreversible modification in osteoblast behaviour to limit E11 expression and osteocytogenesis, we also measured the capacity of MLO-A5 cells to re-enter the osteocyte differentiation programme. We found that the mineralisation process was re-initiated and closely allied to increased expression of E11 protein after re-administration of phosphate or omission of sodium pyrophosphate, indicating an ECM mineralization-induced restoration in osteocyte formation. These results emphasise the importance of cell-ECM interactions in regulating osteoblast behaviour and, more importantly, suggest that ECM mineralization exerts pivotal control during terminal osteoblast differentiation and acquisition of the osteocyte phenotype.
doi:10.1371/journal.pone.0036786
PMCID: PMC3346717  PMID: 22586496
11.  Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures 
Bone  2013;53(2):478-486.
Studies on various compounds of inorganic phosphate, as well as on organic phosphate added by post-translational phosphorylation of proteins, all demonstrate a central role for phosphate in biomineralization processes. Inorganic polyphosphates are chains of orthophosphates linked by phosphoanhydride bonds that can be up to hundreds of orthophosphates in length. The role of polyphosphates in mammalian systems, where they are ubiquitous in cells, tissues and bodily fluids, and are at particularly high levels in osteoblasts, is not well understood. In cell-free systems, polyphosphates inhibit hydroxyapatite nucleation, crystal formation and growth, and solubility. In animal studies, polyphosphate injections inhibit induced ectopic calcification. While recent work has proposed an integrated view of polyphosphate function in bone, little experimental data for bone are available. Here we show in osteoblast cultures producing an abundant collagenous matrix that normally shows robust mineralization, that two polyphosphates (PolyP5 and PolyP65, polyphosphates of 5 and 65 phosphate residues in length) are potent mineralization inhibitors. Twelve-day MC3T3-E1 osteoblast cultures with added ascorbic acid (for collagen matrix assembly) and β-glycerophosphate (a source of phosphate for mineralization) were treated with either PolyP5 or PolyP65. Von Kossa staining and calcium quantification revealed that mineralization was inhibited in a dose-dependent manner by both polyphosphates, with complete mineralization inhibition at 10 μM PolyP. Cell proliferation and collagen assembly were unaffected by polyphosphate treatment, indicating that polyphosphate inhibition of mineralization results not from cell and matrix effects but from direct inhibition of mineralization. This was confirmed by showing that PolyP5 and PolyP65 bound to synthetic hydroxyapatite in a concentration-dependent manner. Tissue-nonspecific alkaline phosphatase (TNAP, ALPL) efficiently hydrolyzed the two PolyPs as measured by Pi release. Importantly, at the concentrations of polyphosphates used in this study which inhibited bone cell culture mineralization, the polyphosphates competitively saturated TNAP, thus potentially interfering with its ability to hydrolyze mineralization-inhibiting pyrophosphate (PPi) and mineralizing-promoting β-glycerophosphate (in cell culture). In the biological setting, TNAP may regulate mineralization by shielding the essential inhibitory substrate pyrophosphate from TNAP degradation, and in the same process, delay the release of phosphate from this source. In conclusion, the inhibition of mineralization by polyphosphates is shown to occur via direct binding to apatitic mineral and by mixed inhibition of TNAP.
doi:10.1016/j.bone.2013.01.020
PMCID: PMC3712787  PMID: 23337041
polyphosphates; phosphate; bone; biomineralization; extracellular matrix; osteoblasts
12.  Chondrogenic ATDC5 cells: An optimised model for rapid and physiological matrix mineralisation 
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.
doi:10.3892/ijmm.2012.1114
PMCID: PMC3573767  PMID: 22941229
ATDC5; chondrocyte; mineralisation; β-glycerophosphate; endochondral ossification; growth plate
13.  An Overview of Bone Cells and their Regulating Factors of Differentiation 
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.
PMCID: PMC3341892  PMID: 22589609
Bone cells; osteoblasts; osteoclasts; regulations
14.  The Phosphatonins: New Hormones the Cause of Numerous Congenital Bone Disorders 
Phosphorous (Pi) ions play a very important role in the regulation of numerous biological processes; at skeletal level, Pi is essential in the formation of hydroxyapatite crystals. Pi is also an element present in membrane phospholipids and in the nucleotides needed for the synthesis of DNA and RNA, and it is crucial in the activation and deactivation of a great many molecules, generally proteins, through processes of phosphorylation and dephosphorylation, the mechanism by which cells control their activity. A prolonged deficiency of Pi in the organism gives rise to major biological problems, such as skeletal demineralisation leading to rickets and/or osteomalacia, alterations of erythrocyte, leukocyte, and platelet function, and altered membrane integrity.
Parathormone (PTH) and vitamin D (1–25 OH2D3) have long been recognised as important regulators of Pi homeostasis. In recent years, there have emerged new Pi homeostasis-regulating molecules called “phosphatonins”. The phosphatonins are: fibroblast growth factor 23 (FGF23) and secreted frizzled related protein-4 (sFRP-4), which are inhibitors of reabsorption of Pi and of vitamin D by the renal tubule [1–25(OH)2D3], and FGF7 and matrix extracellular phosphoglycoprotein (MEPE), which inhibit Pi reabsorption by the renal tubule but have no effect on vitamin D. The term phospatonin was introduced to describe the factor(s) responsible for the inhibition of renal Pi reabsorption and for the alteration of 1-α hydroxylase activity. In 1994 Cai et al. described a case of osteogenic osteomalacia characterised by hypophosphaturia, hyperphosphataemia and reduced 1–25 OH2D3. Removal of the tumour led to the disappearance of these biochemical alterations and remission of the osteomalacic picture. Animal studies showed that transplantation of tumour cells into nude mice was able to reproduce the picture of hyperphosphataemia and hypophosphaturia. It was thus clear that factors produced by tumour cells were responsible for the clinical picture and the phoshatonins were identified as these factors. The most well-known phoshatonin is FGF23, a protein synthesised mainly by osteoblasts and osteocytes but also expressed in parathyroid and thyroid, cardiac, hepatic, and intestinal tissues. FGF23 acts on membrane receptors of the FGFr family, together with a co-factor named Klotho. At cellular level, glycosylation of FGF23 by the enzyme GALNT3 is fundamental to avoid its rapid degradation. FGF23 acts at renal level in a manner similar to PTH. Like PTH it inhibits tubular re-absorption of Pi by reducing the synthesis and increasing the degradation of the Pi transporters (NPTIIa). Unlike PTH, FGF23 inhibits 1-α hydroxylase activity, decreasing the production of 1–25 OH2D3. At bone level, FGF23 controls the mineralisation process and its synthesis and activity are controlled by factors such as MEPE and DMP1. As well as tumour-induced osteomalacia (TIO) in which there is overproduction of phosphatonin, activating mutations in the FGF23, FGFr and Klotho genes are responsible for disorders having the same biochemical and skeletal profile. Knockout (KO) mice for GALNT3, FGF23 or Klotho, important for understanding the mechanism of action of these molecules, have a phenotype opposite to that of hyperphosphataemic disorders, being characterised by hyperphosphataemia and increased renal tubular transport of Pi. These animals present ectopic and vascular calcifications. A human model analogous to the phenotype of the KO mice is that of tumoural calcinosis (CT). CT is a genetic disorder due to activating mutations in the GALNT3, FGF23, FGFr and Klotho genes. In the past, a distinction was made between familial forms and hyperphosphataemic/hyperostotic forms. The most recent data suggest that these variants are different clinical expressions of the same clinical picture. CT is characterised by extraskeletal and vascular calcifications with hyperphosphataemia and hypophosphaturia. Despite the considerable body of available data, many aspects of phsophatonin activity are still being studied. The use of cell models to evaluate the function of these molecules could be very important both for the genetic diagnosis of phosphatonin-related diseases and for laying the foundations for future targeted therapies.
PMCID: PMC3213798
15.  A review of the biology of calcium phosphate sequestration with special reference to milk 
In milk, a stable fluid is formed in which sequestered nanoclusters of calcium phosphate are substructures in casein micelles. As a result, calcium and phosphate concentrations in milk can be far in excess of their solubility. Variations of calcium, phosphate and casein concentrations in milks, both within and among species, are mainly due to the formation of the nanocluster complexes. Caseins evolved from tooth and bone proteins well before the evolution of lactation. It has therefore been suggested that the role of caseins in milk is an adaptation of an antecedent function in the control of some aspect of biomineralisation. There is new evidence that nanocluster-type complexes are also present in blood serum and, by implication, in many other closely related biofluids. Because such fluids are stable but nevertheless supersaturated with respect to the bone and tooth mineral hydroxyapatite, they allow soft and mineralised tissues to co-exist in the same organism with relative ease. An appreciable concentration of nanocluster complexes exists in fresh saliva. Such saliva may stabilise tooth mineral and help to repair demineralised lesions. In the extracellular matrix of bone, nanocluster complexes may be involved in directing the amorphous calcium phosphate to intrafibrillar spaces in collagen where they can mature into oriented apatite crystals. Thus, evidence is accumulating that calcium phosphate sequestration by phosphopeptides to form equilibrium complexes, first observed in milk, is more generally important in the control of physiological calcification.
doi:10.1007/s13594-014-0177-2
PMCID: PMC4302223  PMID: 25632319
Amorphous calcium phosphate; Blood; Saliva; Milk; Urine; Bone; Tooth
16.  Central Role of Pyrophosphate in Acellular Cementum Formation 
PLoS ONE  2012;7(6):e38393.
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.
doi:10.1371/journal.pone.0038393
PMCID: PMC3366957  PMID: 22675556
17.  Mineralisation of collagen rich soft tissues and osteocyte lacunae in Enpp1−/− mice 
Bone  2014;69:139-147.
Ecto-nucleotide pyrophosphatase/phosphodiesterases (NPPs) hydrolyse nucleotide triphosphates to the corresponding nucleotide monophosphates and the mineralisation inhibitor, pyrophosphate (PPi). This study examined the role of NPP1 in osteocytes, osteoclasts and cortical bone, using a mouse model lacking NPP1 (Enpp1−/−). We used microcomputed tomography (μCT) to investigate how NPP1 deletion affects cortical bone structure; excised humerus bones from 8, 15 and 22-week old mice were scanned at 0.9 μm. Although no changes were evident in the cortical bone of 8-week old Enpp1−/− mice, significant differences were observed in older animals. Cortical bone volume was decreased 28% in 22-week Enpp1−/− mice, whilst cortical porosity was reduced 30% and 60% at 15 and 22-weeks, respectively. This was accompanied by up to a 15% decrease in closed pore diameter and a 55% reduction in the number of pores. Cortical thickness was reduced up to 35% in 15 and 22-week Enpp1−/− animals and the endosteal diameter was increased up to 23%. Thus, the cortical bone from Enpp1−/− mice was thinner and less porous, with a larger marrow space. Scanning electron microscopy (SEM) revealed a decrease in the size and number of blood vessel channels in the cortical bone as well as a 40% reduction in the mean plan area of osteocyte lacunae. We noted that the number of viable osteocytes isolated from the long bones of Enpp1−/− mice was decreased ≤ 50%. In contrast, osteoclast formation and resorptive activity were unaffected by NPP1 deletion. μCT and histological analysis of Enpp1−/− mice also revealed calcification of the joints and vertebrae as well as soft tissues including the whisker follicles, ear pinna and trachea. This calcification worsened as the animals aged. Together, these data highlight the key role of NPP1 in regulating calcification of both soft and skeletal tissues.
Highlights
•We examine the role of NPP1 in osteocytes, osteoclasts and cortical bone.•Osteocyte lacunae are reduced in size and number in NPP1 knockout mice.•Sclerostin levels are increased in NPP1 knockout mice.•Osteoclast formation and resorptive activity are unaffected by NPP1 deletion.•Mice lacking NPP1 display widespread calcification of collagen rich soft tissues.
doi:10.1016/j.bone.2014.09.016
PMCID: PMC4228085  PMID: 25260930
NPP1; Osteocytes; Osteoclasts; Soft tissue mineralisation; Pyrophosphate
18.  The Role of Pyrophosphate/Phosphate Homeostasis in Terminal Differentiation and Apoptosis of Growth Plate Chondrocytes 
Bone  2010;47(3):657-665.
Extracellular inorganic phosphate (Pi) concentrations are the highest in the growth plate just before the onset of mineralization. The study reported here demonstrates that Pi not only is required for hydroxyapatite mineral formation but also modulates terminal differentiation and apoptosis of growth plate chondrocytes. Extracellular Pi stimulated terminal differentiation marker gene expression, including the progressive ankylosis gene (ank), alkaline phosphatase (APase), matrix metalloproteinase-13 (MMP-13), osteocalcin, and runx2, mineralization, and apoptosis of growth plate chondrocytes. The stimulatory effect of extracellular Pi on terminal differentiation and apoptosis events of growth plate chondrocytes was dependent on the concentration, the expression levels of type III Na+/Pi co-transporters, and ultimately Pi uptake. A high extracellular Pi concentration was required for the stimulation of apoptosis, whereas lower Pi concentrations were required for the most effective stimulation of terminal differentiation events, including terminal differentiation marker gene expression and mineralization. Suppression of Pit-1 was sufficient to inhibit the stimulatory effects of extracellular Pi on terminal differentiation events. On the other hand, increasing the local extracellular Pi concentration by overexpressing ANK, a protein transporting intracellular PPi to the extracellular milieu where it is hydrolyzed to Pi in the presence of APase, resulted in marked increases of hypertrophic and early terminal differentiation marker mRNA levels, including APase, runx2 and type X collagen, and slight increase of MMP-13 mRNA levels, but decreased osteocalcin mRNA level, a late terminal differentiation markers. In the presence of levamisole, a specific APase inhibitor to prevent hydrolysis of extracellular PPi to Pi, ANK overexpression of growth plate chondrocytes resulted in decreased mRNA levels of hypertrophic and terminal differentiation markers but increased MMP-13 mRNA levels. In conclusion, with extracellular PPi inhibiting and extracellular Pi stimulating hypertrophic and terminal differentiation events, a precise regulation of PPi/Pi homeostasis is required for the spatial and temporal control of terminal differentiation events of growth plate chondrocytes.
doi:10.1016/j.bone.2010.06.018
PMCID: PMC2926124  PMID: 20601283
Apoptosis; progressive ankylosis protein (ANK); alkaline phosphatase; growth plate chondrocytes; phosphate; pyrophosphate
19.  Purinergic signalling in the musculoskeletal system 
Purinergic Signalling  2013;9(4):541-572.
It is now widely recognised that extracellular nucleotides, signalling via purinergic receptors, participate in numerous biological processes in most tissues. It has become evident that extracellular nucleotides have significant regulatory effects in the musculoskeletal system. In early development, ATP released from motor nerves along with acetylcholine acts as a cotransmitter in neuromuscular transmission; in mature animals, ATP functions as a neuromodulator. Purinergic receptors expressed by skeletal muscle and satellite cells play important pathophysiological roles in their development or repair. In many cell types, expression of purinergic receptors is often dependent on differentiation. For example, sequential expression of P2X5, P2Y1 and P2X2 receptors occurs during muscle regeneration in the mdx model of muscular dystrophy. In bone and cartilage cells, the functional effects of purinergic signalling appear to be largely negative. ATP stimulates the formation and activation of osteoclasts, the bone-destroying cells. Another role appears to be as a potent local inhibitor of mineralisation. In osteoblasts, the bone-forming cells, ATP acts via P2 receptors to limit bone mineralisation by inhibiting alkaline phosphatase expression and activity. Extracellular ATP additionally exerts significant effects on mineralisation via its hydrolysis product, pyrophosphate. Evidence now suggests that purinergic signalling is potentially important in several bone and joint disorders including osteoporosis, rheumatoid arthritis and cancers. Strategies for future musculoskeletal therapies might involve modulation of purinergic receptor function or of the ecto-nucleotidases responsible for ATP breakdown or ATP transport inhibitors.
doi:10.1007/s11302-013-9381-4
PMCID: PMC3889393  PMID: 23943493
Bone; Cartilage; Joints; Arthritis; Muscular dystrophy; Cancer
20.  Proteomic analysis of mineralising osteoblasts identifies novel genes related to bone matrix mineralisation 
International Orthopaedics  2010;35(3):447-451.
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.
doi:10.1007/s00264-010-1076-7
PMCID: PMC3047647  PMID: 20556378
21.  Altered Bone Development and an Increase in FGF-23 Expression in Enpp1−/− Mice 
PLoS ONE  2012;7(2):e32177.
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.
doi:10.1371/journal.pone.0032177
PMCID: PMC3281127  PMID: 22359666
22.  miRNA-221 and miRNA-222 synergistically function to promote vascular calcification 
Cell Biochemistry and Function  2013;32(2):209-216.
Vascular calcification shares many similarities with skeletal mineralisation and involves the phenotypic trans-differentiation of vascular smooth muscle cells (VSMCs) to osteoblastic cells within a calcified environment. Various microRNAs (miRs) are known to regulate cell differentiation; however, their role in mediating VSMC calcification is not fully understood. miR-microarray analysis revealed the significant down-regulation of a range of miRs following nine days in culture, including miR-199b, miR-29a, miR-221, miR-222 and miR-31 (p < 0.05). Subsequent studies investigated the specific role of the miR-221/222 family in VSMC calcification. Real-time quantitative polymerase chain reaction data confirmed the down-regulation of miR-221 (32.4%; p < 0.01) and miR-222 (15.7%; p < 0.05). VSMCs were transfected with mimics of miR-221 and miR-222, individually and in combination. Increased calcium deposition was observed in the combined treatment (two-fold; p < 0.05) but not in individual treatments. Runx2 and Msx2 expression was increased during calcification, but no difference in expression was observed following transfection with miR mimics. Interestingly, miR-221 and miR-222 mimics induced significant changes in ectonucleotide phosphodiesterase 1 (Enpp1) and Pit-1 expression, suggesting that these miRs may modulate VSMC calcification through cellular inorganic phosphate and pyrophosphate levels. © 2013 The Authors. Cell Biochemistry and Function published by John Wiley & Sons, Ltd.
doi:10.1002/cbf.3005
PMCID: PMC4158883  PMID: 24604335
calcification; Enpp1; microarray; miRNA; vascular smooth muscle cell
23.  Disorders of Phosphate Homeostasis and Tissue Mineralisation 
Endocrine development  2009;16:10.1159/000223693.
Phosphate is absorbed from the diet in the gut, stored as hydroxyapatite in the skeleton, and excreted with the urine. The balance between these compartments determines the circulating phosphate concentration. Fibroblast growth factor 23 (FGF23) has recently been discovered and is part of a previously unrecognised hormonal bone-kidney axis. Phosphate-regulating gene with homologies to endopeptidases on the X chromosome, and dentin matrix protein 1 regulate the expression of FGF23 in osteocytes, which then is O-glycosylated by UDP-N-acetyl-alpha-d-galactosamine: poly-peptide N-acetylgalactosaminyl-transferase 3 and secreted into the circulation. FGF23 binds with high affinity to fibroblast growth factor receptor 1c in the presence of its co-receptor Klotho. It inhibits, either directly or indirectly, reabsorption of phosphate and the synthesis of 1,25-dihydroxy-vita-min-D by the renal proximal tubule and the secretion of parathyroid hormone by the parathyroid glands. Acquired or inborn errors affecting this newly discovered hormonal system can lead to abnormal phosphate homeostasis and/or tissue mineralisation. This chapter will provide an update on the current knowledge of the pathophysiology, the clinical presentation, diagnostic evaluation and therapy of the disorders of phosphate homeostasis and tissue mineralisation.
doi:10.1159/000223693
PMCID: PMC3810012  PMID: 19494665
24.  Physiologic and pathologic functions of the NPP nucleotide pyrophosphatase/phosphodiesterase family focusing on NPP1 in calcification 
Purinergic Signalling  2006;2(2):371-377.
The catabolism of ATP and other nucleotides participates partly in the important function of nucleotide salvage by activated cells and also in removal or de novo generation of compounds including ATP, ADP, and adenosine that stimulate purinergic signaling. Seven nucleotide pyrophosphatase/phosphodiesterase NPP family members have been identified to date. These isoenzymes, related by up conservation of catalytic domains and certain other modular domains, exert generally non-redundant functions via distinctions in substrates and/or cellular localization. But they share the capacity to hydrolyze phosphodiester or pyrophosphate bonds, though generally acting on distinct substrates that include nucleoside triphosphates, lysophospholipids and choline phosphate esters. PPi generation from nucleoside triphosphates, catalyzed by NPP1 in tissues including cartilage, bone, and artery media smooth muscle cells, supports normal tissue extracellular PPi levels. Balance in PPi generation relative to PPi degradation by pyrophosphatases holds extracellular PPi levels in check. Moreover, physiologic levels of extracellular PPi suppress hydroxyapatite crystal growth, but concurrently providing a reservoir for generation of pro-mineralizing Pi. Extracellular PPi levels must be supported by cells in mineralization-competent tissues to prevent pathologic calcification. This support mechanism becomes dysregulated in aging cartilage, where extracellular PPi excess, mediated in part by upregulated NPP1 expression stimulates calcification. PPi generated by NPP1modulates not only hydroxyapatite crystal growth but also chondrogenesis and expression of the mineralization regulator osteopontin. This review pays particular attention to the role of NPP1-catalyzed PPi generation in the pathogenesis of certain disorders associated with pathologic calcification.
doi:10.1007/s11302-005-5304-3
PMCID: PMC2254483  PMID: 18404477
ANK; ANKH; cartilage; inorganic phosphate; inorganic pyrophosphate; osteopontin
25.  Asfotase-α improves bone growth, mineralization and strength in mouse models of neurofibromatosis type-1 
Nature medicine  2014;20(8):904-910.
Mineralization of the skeleton depends on the balance between levels of pyrophosphate (PPi), an inhibitor of hydroxyapatite formation, and phosphate generated from PPi breakdown by alkaline phosphatase (ALP). We report here that ablation of Nf1, encoding the RAS/GTPase–activating protein neurofibromin, in bone–forming cells leads to supraphysiologic PPi accumulation, caused by a chronic ERK–dependent increase in genes promoting PPi synthesis and extracellular transport, namely Enpp1 and Ank. It also prevents BMP2–induced osteoprogenitor differentiation and, consequently, expression of ALP and PPi breakdown, further contributing to PPi accumulation. The short stature, impaired bone mineralization and strength in mice lacking Nf1 in osteochondroprogenitors or osteoblasts could be corrected by enzyme therapy aimed at reducing PPi concentration. These results establish neurofibromin as an essential regulator of bone mineralization, suggest that altered PPi homeostasis contributes to the skeletal dysplasiae associated with neurofibromatosis type-1 (NF1), and that some of the NF1 skeletal conditions might be preventable pharmacologically.
doi:10.1038/nm.3583
PMCID: PMC4126855  PMID: 24997609
bone mineralization; neurofibromin; Neurofibromatosis type I; osteoblast; mesenchymal stem cell; pyrophosphate; Ank; Enpp1/PC1

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