Phosphate Ions in Bone: Identification of a Calcium–Organic Phosphate Complex by 31P Solid-State NMR Spectroscopy at Early Stages of Mineralization

Previous ³¹P cross-polarization and differential cross-polarization magic angle spinning (CP/MAS and DCP/MAS) solid-state NMR spectroscopy studies of native bone and of the isolated crystals of the calcified matrix synthesized by osteoblasts in cell culture identified and characterized the major PO₄^–3 phosphate components of the mineral phase. The isotropic and anisotropic chemical shift parameters of the minor HPO₄^–2 component in bone mineral and in mineral deposited in osteoblast cell cultures were found to differ significantly from those of brushite, octacalcium phosphate, and other synthetic calcium phosphates. However, because of in vivo and in vitro evidence that phosphoproteins may play a significant role in the nucleation of the solid mineral phase of calcium phosphate in bone and other vertebrate calcified tissues, the focus of the current solid-state ³¹P NMR experiments was to detect the possible presence of and characterize the phosphoryl groups of phosphoproteins in bone at the very earliest stages of bone mineralization, as well as the possible presence of calcium-phosphoprotein complexes. The present study demonstrates that by far the major phosphate components identified by solid-state ³¹P NMR in the very earliest stages of mineralization are protein phosphoryl groups which are not complexed with calcium. However, very small amounts of calcium-complexed protein phosphoryl groups as well as even smaller, trace amounts of apatite crystals were also present at the earliest phases of mineralization. These data support the hypothesis that phosphoproteins complexed with calcium play a significant role in the initiation of bone calcification.     

The Enamel Protein Amelotin Is a Promoter of Hydroxyapatite Mineralization

Amelotin (AMTN) is a recently discovered protein that is specifically expressed during the maturation stage of dental enamel formation. It is localized at the interface between the enamel surface and the apical surface of ameloblasts. AMTN knock‐out mice have hypomineralized enamel, whereas transgenic mice overexpressing AMTN have a compact but disorganized enamel hydroxyapatite (HA) microstructure, indicating a possible involvement of AMTN in regulating HA mineralization directly. In this study, we demonstrated that recombinant human (rh) AMTN dissolved in a metastable buffer system, based on light scattering measurements, promotes HA precipitation. The mineral precipitates were characterized by scanning and transmission electron microscopy and electron diffraction. Colloidal gold immunolabeling of AMTN in the mineral deposits showed that protein molecules were associated with HA crystals. The binding affinity of rh‐AMTN to HA was found to be comparable to that of amelogenin, the major protein of the forming enamel matrix. Overexpression of AMTN in mouse calvaria cells also increased the formation of calcium deposits in the culture medium. Overexpression of AMTN during the secretory stage of enamel formation in vivo resulted in rapid and uncontrolled enamel mineralization. Site‐specific mutagenesis of the potential serine phosphorylation motif SSEEL reduced the in vitro mineral precipitation to less than 25%, revealing that this motif is important for the HA mineralizing function of the protein. A synthetic short peptide containing the SSEEL motif was only able to facilitate mineralization in its phosphorylated form (^PS^PSEEL), indicating that this motif is necessary but not sufficient for the mineralizing properties of AMTN. These findings demonstrate that AMTN has a direct influence on biomineralization by promoting HA mineralization and suggest a critical role for AMTN in the formation of the compact aprismatic enamel surface layer during the maturation stage of amelogenesis. © 2015 American Society for Bone and Mineral Research.     

Characterization of very young mineral phases of bone by solid state 31Phosphorus magic angle sample spinning nuclear magnetic resonance and X-ray diffraction

Summary The properties of bone mineral change with age and maturation. Several investigators have suggested the presence of an initial or precursor calcium phosphate phase to help explain these differences. We have used solid state ³¹P magic angle sample spinning (MASS) nuclear magnetic resonance (NMR) and X-ray radial distribution function (RDF) analyses to characterize 11-and 17-day-old embryonic chick bone and fractions obtained from them by density fractionation. Density fractionation provides samples of bone containing Ca-P solid-phase deposits even younger and more homogeneous with respect to the age of mineral than the calcium phosphate (Ca-P) deposits in the whole bone samples. The analytical techniques yield no evidence for any distinct phase other than the poorly crystal-line hydroxyapatite phase characteristic of mature bone mineral. In particular, there is no detectable crystalline brushite [DCPD, CaHPO₄ 2H₂O< 1%] or amorphous calcium phosphate (< 8–10%) in the most recently formed bone mineral. A sizeable portion of the phosphate groups exist as HPO₄ ^2– in a brushite (DCPD)-like configuration. These acid phosphate moieties are apparently incorporated into the apatitic lattice. The most likely site for the brushite-like configuration is probably on the surface of the crystals.     

Importance of Phosphorylation for Osteopontin Regulation of Biomineralization

Previous in vitro and in vivo studies demonstrated that osteopontin (OPN) is an inhibitor of the formation and growth of hydroxyapatite (HA) and other biominerals. The present study tests the hypotheses that the interaction of OPN with HA is determined by the extent of protein phosphorylation and that this interaction regulates the mineralization process. Bone OPN as previously reported inhibited HA formation and HA-seeded growth in a gelatin-gel system. A transglutaminase-linked OPN polymer had similar effects. Recombinant, nonphosphorylated OPN and chemically dephosphorylated OPN, had no effect on HA formation or growth in this system. In contrast, highly phosphorylated milk OPN (mOPN) promoted HA formation. The mOPN stabilized the conversion of amorphous calcium phosphate (a noncrystalline constituent of milk) to HA, whereas bone OPN had a lesser effect on this conversion. Mixtures of OPN and osteocalcin known to form a complex in vitro, unexpectedly promoted HA formation. To test the hypothesis that small alterations in protein conformation caused by phosphorylation account for the differences in the observed ability of OPN to interact with HA, the conformation of bone OPN and mOPN in the presence and absence of crystalline HA was determined by attenuated total reflection (ATR) infrared (IR) spectroscopy. Both proteins exhibited a predominantly random coil structure, which was unaffected by the addition of Ca²⁺. Binding to HA did not alter the secondary structure of bone OPN, but induced a small increase of β-sheet (few percent) in mOPN. These data taken together suggest that the phosphorylation of OPN is an important factor in regulating the OPN-mediated mineralization process.     

Effect of matrix bound phosphate and fluoride on mineralization of dentin

Completely demineralized bovine dentin mineralized to a high degree, but only after extraction of about half of its organic phosphate. Initiation of mineral formation depended on the concentrations of Ca²⁺, P _i , HCO ₃ ^? and H⁺ ions in the incubation solution. After nucleation, mineral accretion by the dentin continued in solutions that did not, themselves, initiate mineralization. The nucleation phase, but not the subsequent accretion of mineral, was sensitive to small changes in temperature. Inclusion of 0.05 mM NaF in the solution markedly reduced the amount of mineral deposited during the nucleation phase but apparently enhanced subsequent mineralization.     

Formation and structure of Ca-deficient hydroxyapatite

Summary When amorphous calcium phosphate (ACP) was transformed to crystalline hydroxyapatite (HA) in a series of aqueous slurry concentrations ranging from low to high, the higher slurry concentrations produced more Ca-deficient HA as measured by Ca/P ratio and heat-produced pyrophosphate. We feel that the excess solution phosphate produced in the higher slurry transformations results in lower Ca/P ratio HA. It has been suggested that an ACP is the precursor to bone apatite. Regulation of the in vivo ACP slurry concentration could then control the stoichiometry and, therefore, the metabolic activity of bone apatite. X-ray radial distribution function (RDF) analyses showed that CO ₃ ²⁻ substitution in HA creates far greater structural distortions than do Ca deficiencies. The latter, however, do produce small, but observable, structural distortions when compared to stoichiometric HA. It now seems clear that the RDF of bone apatite can be modeled by a synthetic, Ca-deficient, CO ₃ ²⁻ -containing HA.     

Fourier transform infrared microscopy of calcified turkey leg tendon

Fourier transform infrared microscopy (FT-IRMS) was used to monitor spatial variations in the quality and quantity of the mineral phase in calcified turkey tendon. Spectral maps were generated by analysis of 50 μm ×~ 50 μm areas within different regions of the tendon. Spectra of the transitional region, where nonmineralized matrix ends and mineralized matrix begins, revealed marked changes in the spectrally determined mineral-to-matrix ratio, whereas regions deeper into the mineralization front showed a relatively constant ratio. Since spectra of EDTA-demineralized matrix were similar to those of nonmineralized matrix, the nonmineralized regions of the tendon were used for spectral subtraction. The broad, relatively featureless contour of the mineral ν₁,ν₃ phosphate region (900–1200 cm⁻¹) showed only subtle changes at different stages of mineralization. Second derivatives of these spectra were calculated and compared with those of synthetic, poorly crystalline hydroxyapatite (HA). The peak positions seen in second-derivative spectra of the mineral near the transitional region were within ±2 cm⁻¹ of the least mature synthetic HAs whereas spectra of the mineral deeper into the mineralization front were within ±2 cm⁻¹ of the most mature synthetic HAs. Spectra from cross- and longitudinal sections at equivalent positions in the tendon, and polarized FT-IRMS data were analyzed to determine the effect of mineral orientation on the parameters used to characterize the mineral. Spectra of cross- and longitudinal sections of the tendon showed no major differences in either the ν₁,ν₃ phosphate region or the amide I, II, or III components (1200–1800 cm⁻¹). However, polarized FT-IR spectra revealed dramatic differences in both of these regions. Despite these differences, second-derivative analysis of the ν₁,ν₃ regions revealed no significant changes in the positions of the underlying bands used to characterize the environments of the phosphate ion in poorly crystalline HA. The results of this study demonstrate the power of FT-IRMS to monitor spatial variations of the mineral phase in calcified tissue. Also, the incorporation of polarized radiation provides a method capable of assessing the molecular orientation of the mineral phase relative to the collagen matrix. Received: 17 January 1995 / Accepted: 26 May 1995     

Persistence of complexed acidic phospholipids in rapidly mineralizing tissues is due to affinity for mineral and resistance to hydrolytic attack:In Vitro data

Acidic phospholipids, complexed with calcium and inorganic phosphate, are components of extracellular matrix vesicles. Both the complexed acidic phospholipids and matrix vesicles have previously been shown to serve as hydroxyapatite (HA) nucleators in solution and when implanted in a muscle pouch. The present study supplies evidence that complexed acidic phospholipids can persist in mineralizing tissues both because of their affinity for HA and because of their resistance to hydrolysis by phospholipase A₂. Calcium-phosphatidylserine-phosphate complex (CPLX-PS) synthesized with ¹⁴C-labeled phosphatidylserine (PS) was used to measure CPLX-PS affinity for HA using a Langmuir adsorption isotherm model. The affinity was shown to be higher and more specific than that of PS itself (K = 8.66 ml/μmol; N, the number of binding sites = 20.4 μmol/m² as compared with previously reported values for PS of K = 3.33 ml/μmol, and N = 4.87 μmol/m²). Incorporated into synthetic liposomes and incubated in a calcium phosphate solution in which mineralization is induced by an ionophore, CPLX-PS showed behavior distinct from free PS. As previously reported, PS in these liposomes totally blocked HA formation. On the other hand, CPLX-PS in similar concentrations had a varied response, having no effect, slightly inhibiting, or actually promoting HA formation. CPLX-PS was also shown to be a poorer substrate for phospholipase A₂ than PS, with Km = 4.63 mM for CPLX-PS and Km = 0.27 mM for PS; and V_max= 0.029 ml/minute for CPLX-PS and V_max= 0.066 ml/minute for PS. These data explain how complexed acidic phospholipids may persist in the growth plate and facilitate initial mineral deposition. Received: 8 June 1995 / Accepted: 9 August 1995     

Calcium phosphate formation in aqueous suspensions of multilamellar liposomes

Summary The present study examined calcium phosphate precipitation in aqueous suspensions of multilamellar liposomes as a possiblein vitro model for matrix vesicle mineralization. Liposomes were prepared by dispersing CHCl₃-evaporated thin films of 7:2:1 and 7:1:1 molar mixtures of phosphatidylcholine, dicetyl phosphate, and cholesterol in aqueous solutions containing 0, 25, or 50 mM PO₄ and 0 or 0.8 mM Mg. After removal of unencapsulated PO₄ by gel filtration, the liposomes were suspended in 1.33 mM Ca/0.8 mM Mg solutions and made permeable to these cations by the addition of the ionophore X-537A. All experiments were carried out at pH 7.4, 22°C, and 240 mOsm. In the absence of entrapped PO₄, Ca²⁺ taken up by the liposomes was largely bound to inner membrane surfaces. With PO₄ present, Ca²⁺ uptake increased as much as sixfold with maximum accumulations well above values sufficient for solid formation. Precipitated solids appeared to be located predominantly in the aqueous intermembranous spaces of the liposomes. Amorphous calcium phosphate (ACP) precipitated initially in the presence of entrapped Mg²⁺, then subsequently converted to apatite intermixed with some octacalcium phosphate. The stability of the liposomal ACP was somewhat greater than that observed in bulk solutions under comparable conditions of pH, temperature, and electrolyte makeup. In time, the mineral deposits caused entrapped PO₄ to leak from the liposomes. These findings suggest that the precipitation within liposomes is similar to that which occurs in macro-volume synthetic systems but that the precipitated solid eventually impairs the integrity of the surrounding intermembranous space. These observations may offer a partial explanation for the ability of matrix vesiclesin vivo to act as nidi for extracellular mineralization.     

The effect of dietary xylitol on recalcifying and newly formed cortical long bone in rats

Summary Thirty-six 3-week-old male Wistar rats were labeled with a single intraperitoneal tetracycline injection. Twenty-four of them were then fed a Ca-deficient basal diet for 3 weeks, while the control group received the basal diet supplemented with CaCO₃ (12 g/kg). The tetracycline labeling was then repeated and six animals in each group were decapitated. The diet of the remaining formerly Ca-deficient animals was returned to normal, and half the test rats also received xylitol supplementation (50 g/kg). After 4 weeks of rehabilitation the labeling was repeated and the animals were decapitated and their tibias were prepared.The tibias were measured in terms of weight and density and cross sections were prepared for the examination of mineral content. Bone element analysis was performed by scanning electron microscopy with electron-probe microanalysis, examining separately the bone areas formed during the various dietary periods. Areas of the former Ca-deficient and newly formed cortical bone were identified by tetracycline fluorescence under ultraviolet light, and the amount of cortical bone in each group was measured.The mineralization-promoting effect of dietary xylitol as compared with CaCO₃ supplementation alone was seen more clearly in the newly formed periosteal bone than in remineralization of the formerly Ca-deficient bone, the concentrations of Ca and P being significantly elevated (P < 0.05), as also was the total mineral content (P < 0.01). The cortical bone volume was similar following the CaCO₃ and CaCO₃ + xylitol supplementations, suggesting unaltered formation of the organic matrix.The results show that the effect of xylitol on bone during dietary Ca rehabilitation particularly concerns newly formed bone mineral. This may be due to the slow process of remodeling in the former mineralized bone or to xylitol induced effects on osteoblast and/or osteoclast metabolism.     

Complementary Information on In Vitro Conversion of Amorphous (Precursor) Calcium Phosphate to Hydroxyapatite from Raman Microspectroscopy and Wide-Angle X-Ray Scattering

In addition to mechanical functions, bones have an essential role in metabolic activity as mineral reservoirs that are able to absorb and release ions. Bioapatite, considered the major component in the mineralized part of mammalian bones, is a calcium phosphate mineral with a structure that closely resembles hydroxyapatite (HA, Ca₁₀[PO₄]₆[OH]₂) with variable chemical substitutions. It is important to note that it continues to be chemically active long after it has been initially deposited. Detailed understanding of changes in the mineral phase as HA matures is essential for understanding how normal bone achieves its remarkable mechanical performance, how it is altered in disease, as well as the effects of therapeutic interventions. A model system for investigation of the in vivo maturation of HA is available, namely, the in vitro conversion of amorphous calcium phosphate (ACP) to HA in a supersaturated solution of calcium and phosphate ions. In the present study, this system was employed to correlate with the changes in chemistry and poorly crystalline HAP crystal size, shape, and habit. The results of the X-ray diffraction as well as Raman analyses showed that as the crystallites mature in the 002 and 310 directions both the full width at half-height and wavelength at maximum of the Raman peaks change as a function of reaction extent and crystallite maturation, size, and shape. Moreover, such analyses can be performed in intact bone specimens through Raman microspectroscopic and imaging analyses with a spatial resolution of 0.6–1 μ, by far superior to the one offered by other microspectroscopic techniques, thus potentially yielding important new information on the organization and mineral quality of normal and fragile bone.     

Resolution-enhanced fourier transform infrared spectroscopy study of the environment of phosphate ion in the early deposits of a solid phase of calcium phosphate in bone and enamel and their evolution with age: 2. Investigations in thev 3 PO4 domain

Summary Resolution-enhanced Fourier Transform Infrared (FTIR) spectra of early mineral deposits in enamel and bone show bands at 1020, 1100, 1110, 1125, and 1145 cm⁻¹ in thev ₃PO₄ domain which do not belong to well crystallized stoichiometric hydroxyapatite. Bands at 1020 and 1100 cm⁻¹ have been shown to occur in nonstoichiometric apatites containing HPO ₄ ²⁻ ions. Though the bands at 1110 and 1125 cm⁻¹ have not been found in any well crystallized apatite, they are present in newly precipitated apatite. These latter bands disappear progressively during maturation in biological as well as synthetic samples, and partial dissolution of synthetic apatites shows that they belong to species that exhibit an inhomogeneous distribution in the mineral, and that are the first to be solubilized. Comparison of the FTIR spectra of biological apatites with those of synthetic, nonapatitic-containing phosphate minerals shows that the presence of these bands does not arise from nonapatitic, well-defined phases; they are due to the local environment of phosphate ions which may possibly be loosely related or perhaps unrelated to the phosphate groups present in the well-crystallized nonapatitic calcium phosphates. Resolution-enhanced FTIR affords a very precise characterization of the mineral phases which may be very useful in characterizing pathological deposits of Ca−P mineral phases.     

<Superscript>1</Superscript>H MAS and <Superscript>1</Superscript>H → <Superscript>31</Superscript>P CP/MAS NMR Study of Human Bone Mineral

Chemical structure of human bone mineral was studied by solid-state nuclear magnetic resonance (NMR) with magic-angle spinning (MAS). Trabecular and cortical bone samples from adult subjects were compared with mineral standards: hydroxyapatite (HA), hydrated and calcined, carbonatoapatite of type B with 9 wt% of CO ₃ ^2– (CHA-B), brushite (BRU) and mixtures of HA with BRU. Proton spectra were acquired with excellent spectral resolution provided by ultra-high speed MAS at 40 kHz. 2D ¹H-³¹P NMR heteronuclear correlation was achieved by cross-polarization (CP) under fast MAS at 12 kHz. ³¹P NMR was applied with CP from protons under slow MAS at 1 kHz. Appearance of ³¹P rotational sidebands together with their CP kinetics were analyzed. It was suggested that the sidebands of CP spectra are particularly suitable for monitoring the state of apatite crystal surfaces. The bone samples appeared to be deficient in structural hydroxyl groups analogous to those in HA. We found no direct evidence that the HPO ₄ ^2– brushite-like ions are present in bone mineral. The latter problem is extensively discussed in the literature. The study proves there is a similarity between CHA-B and bone mineral expressed by their similar NMR behavior.     

The Role of Phosphatases in the Initiation of Skeletal Mineralization

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 (PP_i), 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 P_i 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 PHOSPHO1 function and P_i influx into MVs in the initiation of mineralization and the functions of TNAP and NPP1 in the extravesicular progression of mineralization.     

The Role of Fetuin-A in Physiological and Pathological Mineralization

Mineralization in higher vertebrates is restricted to bones and teeth. Pathological calcification is mostly known in vasculature but can basically affect all soft tissues. Simply put, tissue mineralization occurs through the interplay of three key determinants: extracellular matrix suitable for mineralization, extracellular levels of inorganic phosphate and calcium, and the levels of mineralization inhibitors that may be expressed systemically or locally. In this article we describe the role of a prototypic systemic inhibitor protein of mineralization, the hepatic plasma protein α₂-Heremans-Schmid glycoprotein/fetuin-A. Fetuin-A mediates the formation of stable colloidal mineral–protein complexes called calciprotein particles (CPPs). Thus, fetuin-A is important in the stabilization and clearance of amorphous mineral precursor phases. Efficient clearance of CPPs and, thus, of excess mineral from circulation prevents local buildup of mineral and calcification of soft tissue. Besides calcium phosphate binding, fetuin-A also acts as a carrier for lipids, which may influence calcification, inflammation, and apoptosis. Fetuin-A-deficient (Ahsg ^?/?) mice show impaired growth of their long bones and premature growth plate closure. We posit that the absence of fetuin-A in the growth plate causes simultaneous lack of calcification inhibition and excess lipid hormone signaling, leading to premature growth plate mineralization and shortened long bones. This suggests that fetuin-A regulates endochondral ossification through mineralization inhibition and lipid (hormone) binding.     

Do laser/LED phototherapies influence the outcome of the repair of surgical bone defects grafted with biphasic synthetic microgranular HA + β-tricalcium phosphate? A Raman spectroscopy study

The treatment of bone loss is difficult. Many techniques are proposed to improve repair, including biomaterials and, recently, phototherapies. This work studied bone mineralization by Raman spectroscopy assessing intensities of Raman peaks of both inorganic (～960, ～1,070 cm^?1) and organic (～1,454 cm^?1) contents in animal model. Six groups were studied: clot, laser, light-emitting diode (LED), biomaterial (HA + β-tricalcium phosphate), laser + biomaterial, and LED + biomaterial. Defects at right tibia were performed with a drill. When indicated, defects were further irradiated at a 48-h interval during 2 weeks. At the 15th and 30th days, the tibias were withdrawn and analyzed. The ～960-cm^?1 peak was significantly affected by phototherapy on both clot- and biomaterial-filled defects. The ～1,070-cm^?1 peak was affected by both time and the use of the LED light on clot-filled defects. On biomaterial-filled defects, only the use of the laser light significantly influenced the outcome. No significant influence of either the time or the use of the light was detected on clot-filled defects as regards the ～1,454-cm^?1 peak. Raman intensities of both mineral and matrix components indicated that the use of laser and LED phototherapies improved the repair of bone defects grafted or not with biphasic synthetic microgranular HA + β-tricalcium phosphate.     

Effects of Lanthanum on Composition, Crystal Size, and Lattice Structure of Femur Bone Mineral of Wistar Rats

The application of lanthanum (La) in industry, medicine, and agriculture may cause accumulation of the element in human body. This article examines the effects of La on the femur bone mineral of male Wistar rats after administration of La(NO₃)₃ by gavage at the dose of 2.0 mg La(NO₃)₃ · kg⁻¹ · day⁻¹ over a 6-month period. Chemical analysis confirmed La accumulation in bone and loss in bone mineral. Thermogravimetric analysis showed a decrease in the mineral-to-matrix ratio and an increase in carbonate content. Fourier-transform infrared spectrometry revealed elevation in the contents of labile carbonate and acidic phosphate. The synchrotron radiation small-angle X-ray scattering study presented a smaller mean thickness of the mineral crystals in the bone of La-treated rats. The synchrotron radiation-extended X-ray absorption fine structure analysis indicated that the La treatment resulted in a lowered disorder in the crystals. The smaller size, more adsorbed labile carbonate, and more acidic phosphate made the bone mineral easier to dissolve, as revealed in the kinetic measurement of bone demineralization. These findings suggest that La retards bone maturation of rats.     

Alkaline phosphatase inhibition by levamisole prevents 1,25-dihydroxyvitamin D3-stimulated bone mineralization in the mouse

Summary To determine the relationship between alkaline phosphatase (AP), 1,25(OD)₂D₃ and bone formationin vivo, we have examined the effects of levamisole, a stereospecific inhibitor of AP on bone formation and on 1,25(OH)₂D₃-stimulated bone mineralization in the mouse. Normal mice were injected daily with levamisole at doses of 40 and 80 mg/kg/b.w. The compound was given alone or in combination with 1,25(OH)₂D₃ infusion (0.05 μg/kg/d) for 7 days. Treatment with levamisole alone inhibited the serum AP activity (mainly of skeletal origin in mice) by 18.4 and 61.3% for the low and high dose respectively. No deleterious effect on body growth, tibia length, and bone cells population was detected. The moderate inhibition of AP activity produced by the lower dose of levamisole alone (18.4%) or in combination with 1,25(OH)₂D₃ (37.9%) was associated with a reduced endosteal matrix apposition rate (MaAR) determined by double³H-proline labeling method. This effect was related to a levamisole-induced fall in serum phosphate. Despite the moderate inhibition of AP activity, the mineral apposition rate (MiAR) determined by the double tetracycline labeling method remained normal. Moreover, 1,25(OH)₂D₃ infusion still resulted in increased MiAR which was stimulated to the same extent as in the absence of levamisole. By contrast, the more severe inhibition of AP activity induced by 80 mg/kg of levamisole alone (61.3%) or in combination with 1,25(OH)₂D₃ (45.8%) inhibited both the MaAR and the MiAR and prevented the stimulatory effect of 1,25(OH)₂D₃ on bone mineralization. The data show that AP activity affects the bone matrix and mineral apposition ratesin vivo and that severe inhibition of AP activity inhibits the 1,25(OH)₂D₃-induced stimulation of bone mineralization in the mouse.     

Influence of estrogen and progesterone on matrix-induced endochondral bone formation

Summary The influence of estradiol and progesterone, alone or in combination, on the discrete phases of matrix-induced endochondral bone formation was investigated. Administration of estradiol and progesterone in combination increased mesenchymal cell proliferation, as indicated by [³H] thymidine incorporaton into acid precipitable material. However, ornithine decarboxylase (ODC) activity was significantly suppressed by the combination of estradiol and progesterone. Also, this treatment did not influence the³⁵SO₄ incorporation into proteoglycans on day 7. Mineralization of newly induced bone was quantitated by alkaline phosphatase,⁴⁵Ca incorporation into bone mineral and calcium content, and was found to be significantly increased by progesterone alone and in combination with estradiol in both matrix-induced plaques and tibial metaphysis. These results demonstrated the stimulatory role of progesterone in combination with estradiol in bone formation and mineralization.     

Quantitative computed tomography: Comparison of two calibration phantoms

Vertebral trabecular mineral density of healthy Japanese (91 females and 67 males) was measured using the quantitative computed tomography (QCT) technique in a cross-sectional study. We compared estimates of vertebral bone density using two calibration phantoms: the Genant K₂HPO₄ phantom developed by Genant and a solid CaCO₃ phantom developed by Chugai Pharmaceutical. Using the CaCO₃ phantom, the rate of decrease of spinal trabecular mineral with age in control females averaged 1.1% per year from age 20 to 80, with an accelerated loss demonstrated at menopause (1.8%). Trabecular bone mass in male controls declined an average 0.9% per year. There was a highly significant correlation between the results obtained with each phantom (r=0.980, p<0.001). This relation was linear over the range of bone mass (60 –172 mg K₂HPO₄/cm³), and was expressed by the equation y(CaCO₃)=1.26×(K₂HPO₄)+12.3 There was, however, some dispersion of the data around the regression. The calibration phantom used for the measurement of the vertebral trabecular bone should need to be more consistent in longitudinal studies. Apart from this consideration, a solid Chugai phantom can be said to be useful having practical advantages in its flexibility.