Effect of pyrophosphate and two diphosphonates on 45Ca and 32Pi uptake and mineralization by matrix vesicle-enriched fractions and by hydroxyapatite

Inorganic pyrophosphate (PPi) and two diphosphonates, ethane-1-hydroxy-1, 1-diphosphonate (EHDP) and dichloromethylene diphosphonate (Cl2MDP), were found to inhibit in vitro mineralization induced by matrix vesicle-enriched fractions from chicken epiphyseal cartilage. Inhibitor concentrations from 0.20 to 20 microM caused a dosage-dependent decrease in 45Ca and 32Pi uptake by the vesicle fraction. These inhibitors were also tested in a hydroxyapatite (HA)-seeded system to help distinguish between effects on the mineral vs nonmineral portions of the vesicle fraction. The order of inhibition of the HA-seeded system was EHDP greater than PPi greater than Cl2MDP, except for inhibitor concentrations of 0.20 microM where EHDP was the least inhibitory. This variation may be due to differences in the binding of the inhibitors to HA crystals. In general, inhibition of HA mineralization was greatest during later time periods, whereas vesicle ion uptake was affected more during early stages of incubation. The differential effects of the three inhibitors were most obvious at the 2.0 microM concentration. With PPi substantial inhibition of HA-seeded mineralization was observed even in late stages of the study; in contrast, with time the vesicle fraction overcame this inhibition. This suggests that alkaline phosphatase, an enzyme notably enriched in matrix vesicles, catalyzed the hydrolysis of PPi, reducing its concentration to a level where mineralization could proceed. Our findings show that matrix vesicle-induced mineralization differs significantly from apatite-induced mineralization. The data support the concept that vesicle alkaline phosphatase acts, at least in part, to remove physiological crystal growth inhibitors.     

Matrix mineralization in MC3T3-E1 cell cultures initiated by β-glycerophosphate pulse

MC3T3-E1 cells, grown in the presence of serum and ascorbate, express alkaline phosphatase and produce an extensive collagenous extracellular matrix that can be mineralized by the addition of β-glycerophosphate (β-GP). In the present work, we study the influence of concentration and duration of β-GP treatment on the mineralization pattern in 4-week-old cell cultures. Amount and structure of mineral deposition were monitored by von Kossa staining, light, and electron microscopy, as well as small-angle X-ray scattering (SAXS) of unstained specimens. SAXS measures the total surface of the mineral phase and is therefore preferentially sensitive to very small crystals (typically <50 nm). It was used to determine the ratio (M) of small crystals to collagen matrix. A variety of mineralization patterns was observed to occur simultaneously, some associated with collagen within nodules or in deeper layers of the cultures and some independent of it. At a β-GP concentration of 10 mmol, mineralization was initiated after about 24 h and continued to increase, irrespective of whether the high level of β-GP was maintained or reduced to 2 mmol. With shorter pulses (<24 h), no significant mineralization was observed in the week following β-GP pulse. With continuous treatment at 5 mmol β-GP, the first signs of mineralization were detected 14 days after the beginning of treatment in the 4-week-old cultures, but no mineralization at all occurred at lower β-GP concentrations. When cells were grown without ascorbic acid for 4 weeks, only two cell layers without collagen matrix were found. In these cultures, no mineralization detectable by SAXS could be induced with β-GP. These data indicate that, in viable cells, high doses of β-GP are essential for the nucleation of mineral crystals, but not for the progression of mineralization once crystals had been nucleated. In contrast, when 4-week-old cell cultures were devitalized, M was found to increase immediately, even at 2 mmol β-GP. These results suggest that, in MC3T3-E1 cell cultures, cell viability is essential for prevention of spontaneous mineralization of the extracellular matrix.     

The deposition of calcium pyrophosphate and phosphate by matrix vesicles isolated from fetal bovine epiphyseal cartilage

Summary Since calcium (Ca) deposition by isolated fetal bovine matrix vesicles is selectively supported by nucleoside triphosphate, and since the Ca deposits appear to be amorphous by transmission electron microscopy [1], attempts were made to study further the nature of these Ca deposits. Calcification of isolated matrix vesicles was allowed to occur in a calcifying medium in which either inorganic phosphate (Pi) or [γ-P]ATP was labeled with³²P.³²P in Ca P (pyrophosphate) deposits were analyzed by a Dowex 1×10 anion exchange chromatography. The results of the analysis indicate that the (³²P) radioactivity was mainly associated with Pi when Pi in the calcifying media was labeled with³²P. In contrast,³²P was found to be associated with inorganic pyrophosphate (PPi) when [γ-³²P]ATP was used. Using a specific enzyme coupling assay for PPi, the presence of PPi in the Ca deposits was demonstrated. The amounts of Pi and PPi in the Ca deposits initiated by fetal calf matrix vesicles were found to be approximately equal. To exclude the possibility that the major part of PPi of Ca P deposit existed as adsorbed form, the deposition was performed under the conditions in which Pi was omitted from calcifying medium. The results of these experiments showed that substantial amount of PPi and Ca deposits remained the same and was not correlated to the amount of Pi in these deposits. In contrast, Pi of CaP was decreased if Pi was omitted from the calcifying medium. Thus, it appears that the major portion of PPi exists as mineral rather than adsorbed form. The moles of Ca deposited were approximately equal to the sum of moles Pi and PPi deposited. Levamisole at 10 mM inhibited 70% of ATPase (specific release of³²Pi from [γ-³²P]ATP at pH 7.6) activity, 18% of Ca deposition, and 34% of Pi deposition during 3 hours of incubation. Adenosine monophosphate (AMP) or adenosine diphosphate (ADP) at 1 mM exerted earlier and greater inhibition on Ca and P (Pi and PPi) deposition than did 10 mM levamisole. Adenosine (1 mM) had little effect on Ca P deposition. Prolonged incubations which allowed enzymatic degradation of ADP and AMP substantially reduced the ADP and AMP inhibition. Levamisole was able to potentiate the AMP and ADP inhibition since it prevents further breakdown of AMP or ADP by alkaline phosphatase. Thus, we concluded that alkaline phosphate is not the major factor in thein vitro Ca P deposition by fetal bovine matrix vesicles. Instead, it appears that nucleoside trisphosphate pyrophosphohydrolase is directly involved in the early stages of deposition since both the enzyme and Ca P deposition were inhibited by AMP or ADP. The possible involvement of ATP phyophosphohydrolase in chondrocalcinosis is suggested.     

Genetics of chondrocalcinosis

Rapid developments in genetic analysis have enabled the dissection of a variety of arthropathies that are inherited in a Mendelian manner. These disorders include calcium crystal arthropathies such as calcium pyrophosphate dihydrate deposition (CPPD) disease and hydroxyapatite deposition disease. In CPPD disease, mutations in a recently discovered gene, ANKH, have been demonstrated in five affected families and may also be associated with the idiopathic deposition of calcium pyrophosphate dihydrate crystals. The product of ANKH appears to be involved in cellular transport of inorganic pyrophosphate (PPi) and mutations in ANKH have been shown to have a significant impact on the regulation of intra- and extracellular levels of PPi. In families with hydroxyapatite deposition disease, no gene locus has yet been linked to the disorder.     

Procedure for the study of acidic calcium phosphate precursor phases in enamel mineral formation

Summary Considerable evidence suggests that an acidic calcium phosphate, such as octacalcium phosphate (OCP) or brushite, is involved as a precursor in enamel and other hard tissue formation. Additionally, there is in vitro evidence suggesting that fluoride accelerates and magnesium inhibits the hydrolysis of OCP to hydroxyapatite (OHAp). As the amount of OCP or brushite in enamel cannot be measured directly in the presence of an excess of hydroxyapatite, a procedure was developed that allows for their indirect in vivo quantification as pyrophosphate. This permits study of the effects of fluoride and magnesium ions on enamel mineral synthesis. Rat incisor calcium phosphate was labeled by intraperitoneal injection of NaH₂ ³²PO₄. The rats were then subjected to various fluoride and magnesium treatments with subcutaneous implanted osmotic pumps. They were then killed at predetermined intervals; the nascent sections of the incisors were collected, cleaned, and pyrolyzed at 500°C for 48 hours to convert acidic calcium phosphates to calcium pyrophosphate; the pyrophosphate was separated from orthophosphate by anion-exchange chromatography; and the resulting fractions were counted by liquid scintillation spectrometry. The activities of the pyro- and orthophosphate fractions were used to calculate the amount of acidic calcium phosphate present in the nascent mineral. The results demonstrated that the percentage of radioactive pyrophosphate in nascent incisors decreased with time, with increasing serum F^- concentration, and with decreasing serum magnesium content. The technique described here should prove to be a powerful new tool for studying the effects of various agents on biological mineral formation.     

Retinoic acid stimulates pyrophosphate elaboration by cartilage and chondrocytes

Abnormal metabolism of extracellular inorganic pyrophosphate (PPi) by articular cartilage contributes to calcium pyrophosphate dihydrate (CPPD) crystal formation and the resultant arthritis known as CPPD deposition disease. The factors causing excess PPi elaboration in affected cartilage remain poorly defined. Retinoic acid (RA), a naturally occurring vitamin A metabolite, promotes cartilage degeneration and mineralization, two correlates of CPPD crystal deposition. RA was examined as a potential modifier of cartilage PPi elaboration. All-trans RA (200–1000 nM) increased PPi levels in culture medium of normal porcine cartilage and chondrocytes 2–3-fold over control values at 96 hours of incubation (P < 0.01). IGF1 and anti-EGF antibody diminished the effects of RA on PPi elaboration. RA modestly increased activity of the PPi-generating ectoenzyme NTPPPH in culture medium (P < 0.01). As some RA effects are mediated through increased activity of TGF\, a known PPi stimulant, we examined the effect of anti-TGF\ antibody on RA-induced PPi elaboration. PPi levels in medium were reduced from 30 ± 7 fxM in cartilage cultures with 500 nM RA to 14 ± 4 ΜM PPi in cartilage cultures with RA and anti-TGF\. Anti-TGF\ antibody, however, had no significant effect on RA-induced PPi elaboration in chondrocyte cultures. Thus, RA, along with TGF\ and ascorbate, can now be included in the list of known PPi stimulants. All three of these factors promote mineralization in growth plate cartilage. These data support a central role for TGF\ in CPPD disease, and provide further evidence linking processes of normal and pathologic calcification in cartilage.     

Transforming growth factor beta-1 stimulates articular chondrocyte elaboration of matrix vesicles capable of greater calcium pyrophosphate precipitation

Objective To determine the role of transforming growth factor beta1 (TGFbeta) in early calcium pyrophosphate formation by measuring its effects on articular chondrocyte matrix vesicle (MV) formation, specific activity of the inorganic pyrophosphate(PPi)-generating enzyme nucleoside triphosphate pyrophospho-hydrolase (NTPPPH) and biomineralization capacity.Methods MV elaborated from mature porcine chondrocyte monolayers+/-TGFbeta were compared for protein content, NTPPPH activity, and ATP-dependent biomineralization. Precipitation of calcium pyrophosphate mineral phases by MV was determined by a radiometric assay and by Fourier transform infrared spectroscopy (FTIR).Results MV from monolayers exposed to TGFbeta were enriched in NTPPPH activity compared to MV from control monolayers (P< 0.01) and precipitated more calcium/mg MV protein than controls (P相似文献   

Calcium and orthophosphate deposits in vitro do not imply osteoblast-mediated mineralization: Mineralization by betaglycerophosphate in the absence of osteoblasts

It has been shown in several laboratories that addition of beta-glycerophosphate (βGP), a substrate for alkaline phosphatase (AP), to cultured osteoblast-like cells induces deposition of orthophosphate (Pi) and Ca within seven days. Even though this effect is regarded as an in vitro model of bone mineralization, it is not known whether it is specific for osteoblasts. We have, therefore, studied the amounts of Pi and Ca deposited after seven days with 10 mM βGP in culture wells containing confluent cultures of osteoblast-like cells (OB) derived from human trabecular bone expiants, human skin fibroblasts (SF), or culture medium alone (MED). Ox liver AP at an activity considerably greater than the endogenous AP activity of the cells, but comparable with that of other osteoblast models, was added to ensure a similar rate of Pi generation from βGP in all wells. βGP was converted quantitatively to Pi within seven days, leading to a nonphysiological 10-fold increase in the Pi concentration in the culture medium. After thorough rinsing on day seven, the OB and SF wells contained deposits of Pi and Ca, but the amounts were comparable for the two cell types. Smaller, but significant, amounts of Pi and Ca were also detectable even in rinsed MED wells. This suggests that the detection of such deposits in βGP experiments cannot necessarily be interpreted as a specific property of osteoblast cultures in vitro, and may simply reflect the presence of AP. It was also shown that, even though phosphatases in human plasma incubated for seven days in vitro with added 10 mM βGP can hydrolyze a large fraction of the βGP to Pi, endogenous organic phosphates in the plasma, which occur at a similar concentration, are hydrolyzed only slowly. This implies that no naturally occurring analogue of βGP is present at high concentrations in extracellular fluid in humans.     

The solid, calcium-phosphate mineral phases in embryonic chick bone characterized by high-voltage electron diffraction

The solid mineral phases of calcium-phosphate (Ca-P) in the long bones from embryonic chicks between the ages of 9 and 13 days have been examined by high voltage (1.0 MV) electron microscopy and electron microdiffraction. The study was undertaken to identify the chemical and crystallographic nature of the inorganic mineral phase(s) prepared under conditions which significantly reduce artifacts of specimen preparation and microscopic examination of the tissues. Electron microdiffraction patterns of solid mineral phase particles in the osteoid matrices of the subperiosteal region of tibiae were principally those of poorly crystalline hydroxyapatite. In rare instances (less than 1% of the estimated volume of the mineral phase present in the zone of early mineralization), relatively large single crystals were observed within clusters of hydroxyapatite. From calculations of both interplanar spacings and measurements of angular displacement of diffraction reflections from single crystal microdiffraction patterns, two distinct phases other than hydroxyapatite were identified: brushite and beta-tricalcium phosphate. A third phase, resembling an apatite, remains unidentified. The results suggest that very small amounts of nonapatitic phases of Ca-P exist in chicken bone tissue. No temporal relationship could be established between the nonapatitic and apatitic phases. There is at present no evidence from this study to support the concept that nonapatitic phases are precursors of a final apatitic phase in bone.     

Polyphosphates inhibit extracellular matrix mineralization in MC3T3-E1 osteoblast cultures

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 demonstrate in osteoblast cultures producing an abundant collagenous matrix that normally show 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. 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 P_i 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 (PP_i) and mineralizing-promoting β-glycerophosphate (in cell culture). In the biological setting, polyphosphates 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.     

Mixed-image longitudimetry: A new method of length measurement in microscopy

Summary The Ca²⁺-binding glycoprotein isolated from preosseous cartilage shows also alkaline phosphatase activity. The purification procedure indicates that the enzyme is inhibited in crude extract and conceivably in the intact tissue; the activity may be controlled by the proteoglycans present in the matrix. Other substrates are hydrolyzed by the purified enzyme in addition top-nitrophenylphosphate; the highest specific activity was measured with ATP and pyrophosphate (PPi) at pH 7.5 and 9.0 Mg²⁺ induces an activation of ATP and PPi hydrolysis; Ca²⁺ activates hydrolysis of ATP but inhibits that of PPi. The glycoprotein shows also transphosphorylase activity,l-serine being the best phosphate acceptor. The release or transfer of Pi catalyzed by the glycoprotein can be an important step in calcium phosphate precipitation.     

Effects of pH on the Production of Phosphate and Pyrophosphate by Matrix Vesicles’ Biomimetics

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 (PP_i), a mineralization inhibitor produced by ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1), while contributing P_i from ATP to initiate mineralization. TNAP and NPP1, alone or combined, were reconstituted in dipalmitoylphosphatidylcholine liposomes to mimic the microenvironment of MVs. The hydrolysis of ATP, ADP, AMP, and PP_i 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, PP_i hydrolysis was maximal at pH 8 and indicated a preferential utilization of PP_i over ATP at pH 8 versus 9. In addition, all proteoliposomes induced mineral formation when incubated in a synthetic cartilage lymph containing 1 mM ATP as substrate and amorphous calcium phosphate or calcium–phosphate–phosphatidylserine complexes as nucleators. Propagation of mineralization was significantly more efficient at pH 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 PP_i hydrolysis by both NPP1 and TNAP, further elevating the P_i/PP_i ratio, thus enhancing bone mineralization.     

Mechanism of action of β-glycerophosphate on bone cell mineralization

Summary Experiments were performed to determine whether -glycerophosphate (-GP) promoted mineralization in vitro by modulating bone cell metabolic activity and/or serving as a local source of inorganic phosphate ions (Pi). Using MC3T3-E1, ROS 17/2.8, and chick osteoblast-like cells in the presence of -GP or Pi, we examined mineral formation, lactate generation, alkaline phosphatase (AP) activity, and protein and phospholipid synthesis. Neither -GP nor Pi modulated any of the major biosynthetic activities of the bone cells. Thus, we found no change in the levels of phospholipids, and the total protein concentration remained constant. Measurement of lactate synthesis showed that -GP did not effect the rate of anaerobic glycolysis. Evaluation of medium Pi levels clearly indicated that -GP was hydrolyzed by bone cells; within 24 hours, almost 80% of 10 mM -GP was hydrolyzed. It is likely that this local increase in medium Pi concentration promoted rapid mineral deposition. Chemical, energy dispersive X-ray, and Fourier transform infrared analysis of the mineral formed in the presence of -GP showed that it was nonapatitic; moreover, mineral particles were also seen in the culture medium itself. Experiments performed with a cell-free system indicated that mineral particles formed spontaneously in the presence of AP and -GP and were deposited into a collagen matrix. We conclude that medium supplementation with -GP or Pi should not exceed 2 mM. If this value is exceeded, then there will be nonphysiological mineral deposition in the bone cell culture.     

Solid state31NMR studies of the conversion of amorphous tricalcium phosphate to apatitic tricalcium phosphate

Summary The hydrolytic conversion of a solid amorphous calcium phosphate of empirical formula Ca₉(PO₄)₆ to a poorly crystalline apatitic phase, under conditions where Ca²⁺ and PO₄ ³⁻ were conserved, was studied by means of solid-state magic-angle sample spinning³¹P-NMR (nuclear magnetic resonance). Results showed a gradual decrease in hydrated amorphous calcium phosphate and the formation of two new PO₄ ³⁻-containing components: an apatitic component similar to poorly crystalline hydroxyapatite and a protonated PO₄ ³⁻, probably HPO₄ ²⁻ in a dicalcium phosphate dihydrate (DCPD) brushite-like configuration. This latter component resembles the brushite-like HPO₄ ²⁻ component previously observed by³¹P-NMR in apatitic calcium phosphates of biological origin. Results were consistent with previous studies by Heughebaert and Montel [18] of the kinetics of the conversion of amorphous calcium phosphate to hydroxyapatite under the same conditions.     

The deposition of calcium pyrophosphate and phosphate by matrix vesicles isolated from fetal bovine epiphyseal cartilage

Since calcium (Ca) deposition by isolated fetal bovine matrix vesicles is selectively supported by nucleoside triphosphate, and since the Ca deposits appear to be amorphous by transmission electron microscopy, attempts were made to study further the nature of these Ca deposits. Calcification of isolated matrix vesicles was allowed to occur in a calcifying medium in which either inorganic phosphate (Pi) or [gamma-P]ATP was labeled with 32P. 32P in Ca P (pyrophosphate) deposits were analyzed by a Dowex 1 X 10 anion exchange chromatography. The results of the analysis indicate that the (32P) radioactivity was mainly associated with Pi when Pi in the calcifying media was labeled with 32P. In contrast, 32P was found to be associated with inorganic pyrophosphate (PPi) when [gamma-32P]ATP was used. Using a specific enzyme coupling assay for PPi, the presence of PPi in the Ca deposits was demonstrated. The amounts of Pi and PPi in the Ca deposits initiated by fetal calf matrix vesicles were found to be approximately equal. To exclude the possibility that the major part of PPi of Ca P deposit existed as adsorbed form, the deposition was performed under the conditions in which Pi was omitted from calcifying medium. The results of these experiments showed that substantial amount of PPi and Ca deposits remained the same and was not correlated to the amount of Pi in these deposits. In contrast, Pi of CaP was decreased if Pi was omitted from the calcifying medium. Thus, it appears that the major portion of PPi exists as mineral rather than adsorbed form.(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcific Periarthritis as the Only Clinical Manifestation of Hypophosphatasia in Middle‐Aged Sisters

Hypophosphatasia (HPP) is the inborn error of metabolism that features low serum alkaline phosphatase (ALP) activity caused by loss‐of‐function mutation(s) within the gene for the tissue nonspecific isoenzyme of ALP (TNSALP). In HPP, extracellular accumulation of inorganic pyrophosphate (PPi), a TNSALP substrate and inhibitor of mineralization, leads frequently to premature tooth loss and often to rickets or osteomalacia. In affected adults, the excess PPi sometimes also causes calcium pyrophosphate dihydrate (CPPD) deposition, PPi arthropathy, or pseudogout, or seemingly paradoxical deposition of hydroxyapatite crystals in ligaments or around joints when the condition is called calcific periarthritis (CP). We report three middle‐aged sisters with CP as the only clinical manifestation of HPP. Each presented during early adult life with recurrent episodes of pain principally around the shoulders, elbows, wrists, hips, or Achilles tendon. Otherwise, they were in good health, including no history of unusual dental disease, fractures, or pseudofractures. Calcific deposits were identified in symptomatic areas principally by ultrasonographic assessment but also confirmed radiographically. All three sisters had low serum levels of total and bone‐specific ALP, hyperphosphatemia, and increased serum concentrations of the TNSALP substrate pyridoxal 5′‐phosphate together characteristic of HPP. Mutation analysis revealed that each carried a single unique 18‐bp duplication within TNSALP (c.188_205dup18, p.Gly63_Thr68dup) as did two of their healthy sons and their mother, who was without signs of CPPD deposition or CP but had knee osteoarthritis. We find that CP can be the only complication of HPP in adults. Thus, multiple juxta‐articular deposits of hydroxyapatite causing CP may be a useful sign of HPP, especially when the CP is familial. © 2014 American Society for Bone and Mineral Research.     

The influence of hydroxyapatite and pyrophosphate on the formation product of calcium oxalate at different pHs

Summary The nucleating effect of hydroxyapatite (HAP) and the inhibitory effect of pyrophosphate (PPi) on calcium oxalate crystallization have been studied at different pH's in solution metastabely supersaturated with respect to calcium oxalate but saturated with respect to HAP. Crystallization was monitored by a decrease of calcium in the supernatant and formation products were calculated. At a pH above 6.0 already minimal HAP concentrations proved to be a suitable substrate for heterogeneous nucleation and growth of calcium oxalate. PPi showed a pronounced inhibitory effect on spontaneous as well as on HAP induced crystallization of calcium oxalate, this effect being highly pH dependent. HAP was found to neutralize the inhibitory effect of PPi in a molar ratio of 10:1.     

The Mineralization Regulator ANKH Mediates Cellular Efflux of ATP,Not Pyrophosphate

The plasma membrane protein ankylosis homologue (ANKH, mouse ortholog: Ank) prevents pathological mineralization of joints by controlling extracellular levels of the mineralization inhibitor pyrophosphate (PPi). It was long thought that ANKH acts by transporting PPi into the joints. We recently showed that when overproduced in HEK293 cells, ANKH mediates release of large amounts of nucleoside triphosphates (NTPs), predominantly ATP, into the culture medium. ATP is converted extracellularly into PPi and AMP by the ectoenzyme ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). We could not rule out, however, that cells also release PPi directly via ANKH. We now addressed the question of whether PPi leaves cells via ANKH using HEK293 cells that completely lack ENPP1. Introduction of ANKH in these ENPP1-deficient HEK293 cells resulted in robust cellular ATP release without the concomitant increase in extracellular PPi found in ENPP1-proficient cells. Ank activity was previously shown to be responsible for about 75% of the PPi found in mouse bones. However, bones of Enpp1^−/− mice contained <2.5% of the PPi found in bones of wild-type mice, showing that Enpp1 activity is also a prerequisite for Ank-dependent PPi incorporation into the mineralized bone matrix in vivo. Hence, ATP release precedes ENPP1-mediated PPi formation. We find that ANKH also provides about 25% of plasma PPi, whereas we have previously shown that 60% to 70% of plasma PPi is derived from the NTPs extruded by the ABC transporter, ABCC6. Both transporters that keep plasma PPi at sufficient levels to prevent pathological calcification therefore do so by extruding NTPs rather than PPi itself. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Inhibition of calcium pyrophosphate dihydrate crystal formation: Effects of carboxylate ions

Summary Proteoglycans are recognized to inhibit calcium pyrophosphate dihydrate (CPPD) crystal formation but the mechanisms are not known. To study the role of carboxylate (−CO₂ ⁻) ligands, the possible inhibitor effects of sodium acetate, sodium D-glucuronate, disodium malate, and trisodium citrate were studied using solution mixtures containing [Ca²⁺]=1.5 mM, [Mg²⁺]=0.5 mM, [PPi] =0.1 mM, [Pi]=0.1 mM, [Na⁺]=140 mM, 37°C, pH 7.4 with or without 9.5±0.5 mg CPPD (seed) crystals. These studies showed that monocarboxylates (acetate, glucuronate) have little inhibitive effect. Progressively greater inhibition was found with dicarboxylate (malate) and tricarboxylate (citrate) indicating that the arrangement of carboxylate (and sulfate) ligands on proteoglycan is more important than the inhibitory effect of individual ligands.     

Matrix vesicle plasma cell membrane glycoprotein-1 regulates mineralization by murine osteoblastic MC3T3 cells.

A naturally occurring nonsense truncation mutation of the inorganic pyrophosphate (PPi)-generating nucleoside triphosphate pyrophosphohydrolase (NTPPPH) PC-1 is associated with spinal and periarticular ligament hyperostosis and cartilage calcification in "tiptoe walking" (ttw) mice. Thus, we tested the hypothesis that PC-1 acts directly in the extracellular matrix to restrain mineralization. Cultured osteoblastic MC3T3 cells expressed PC-1 mRNA and produced hydroxyapatite deposits at 12-14 days. NTPPPH activity increased steadily over 14 days. Transforming growth factor-beta and 1,25-dihydroxyvitamin D3 increased PC-1 and NTPPPH in matrix vesicles (MVs). Because PC-1/NTPPPH was regulated in mineralizing MC3T3 cells, we stably transfected or infected cells with recombinant adenovirus, in order to express 2- to 6-fold more PC-1. PC-1/NTPPPH and PPi content increased severalfold in MVs derived from cells transfected with PC-1. Furthermore, MC3T3 cells transfected with PC-1 deposited approximately 80-90% less hydroxyapatite (by weight) than cells transfected with empty plasmid or enzymatically inactive PC-1. ATP-dependent 45Ca precipitation by MVs from cells overexpressing active PC-1 was comparably diminished. Thus, regulation of PC-1 controls the PPi content and function of osteoblast-derived MVs and matrix hydroxyapatite deposition. PC-1 may provide a novel therapeutic target in certain disorders of bone mineralization.