The nuclear factor of activated T cells (NFAT) transcription factor NFATp (NFATc2) is a repressor of chondrogenesis.

Nuclear factor of activated T cells (NFAT) transcription factors regulate gene expression in lymphocytes and control cardiac valve formation. Here, we report that NFATp regulates chondrogenesis in the adult animal. In mice lacking NFATp, resident cells in the extraarticular connective tissues spontaneously differentiate to cartilage. These cartilage cells progressively differentiate and the tissue undergoes endochondral ossification, recapitulating the development of endochondral bone. Proliferation of already existing articular cartilage cells also occurs in some older animals. At both sites, neoplastic changes in the cartilage cells occur. Consistent with these data, NFATp expression is regulated in mesenchymal stem cells induced to differentiate along a chondrogenic pathway. Lack of NFATp in articular cartilage cells results in increased expression of cartilage markers, whereas overexpression of NFATp in cartilage cell lines extinguishes the cartilage phenotype. Thus, NFATp is a repressor of cartilage cell growth and differentiation and also has the properties of a tumor suppressor.

The effects of alendronate in the treatment of experimental osteonecrosis of the hip in adult rabbits.

OBJECTIVE: Characterize the effects of alendronate (ALN) on the repair process of the osteonecrotic femoral head as well as the development of secondary osteoarthritis in the ipsilateral hip in an established experimental model of osteonecrosis. METHODS: Osteonecrosis of the femoral head was induced surgically in 60 adult, male New Zealand white rabbits. Animals were randomized in two placebo- (saline) and two treatment-groups (ALN 150 microg/kg/day S.C., 3x per wk) and were euthanized at 6 and 12 months post-operatively. Contralateral hip was used as control. Micro-Quantitative-CT (microQCT) analysis as well as histological assessment was performed in the femoral head and the acetabulum. Mankin Score was used to assess cartilage degeneration in the acetabulum. RESULTS: Repair in the osteonecrotic femoral head in the placebo group led to a significantly increased bone volume fraction (BVF) and volumetric bone mineral density (vBMD) in the trabecular region and to an increase in porosity in the cortical and subchondral region when compared to the normal femoral head on the contralateral side. ALN treatment significantly further increased BVF and vBMD in the trabecular region, and significantly reduced porosity and increased vBMD in the necrotic subchondral and cortical bone when compared to placebo. ALN led to a significant increase in vBMD in the subchondral region of the osteoarthritic acetabulum as well as to a significant reduction in articular cartilage degeneration. CONCLUSION: Inhibition of bone resorption by ALN treatment during repair of the osteonecrotic femoral head significantly increased bone mass in the trabecular region of the femoral head, inhibited subchondral resorption and reduced cartilage degeneration in the acetabulum.

Radioautographic visualization and biochemical identification of O-phosphoserine- and O-phosphothreonine-containing phosphoproteins in mineralizing embryonic chick bone.

We injected NaH2(33)PO4 into normal 14-d-old embryonic chicks and examined the long bones by both radioautography and biochemical analyses from 10 to 240 min after the injection was completed. At 30 min, determination of the radiographic grain density revealed that 33P was concentrated principally in fibroblasts, preosteoblasts, and osteoblasts. With time, there was a progressive increase in the density of silver grains located over both the osteogenic cells and the regions of uncalcified (osteoid) and calcified extracellular organic matrices. Biochemical analyses identified 33P-O-phosphoserine as the major 33P component in glutaraldehyde-treated whole demineralized bone tissue and in EDTA-soluble, nondiffusible proteins extracted from the bones, both at the same time periods that 33P-induced silver grains were visualized by radioautography. 33P-O-phosphothreonine was also identified in experiments using a dosage of 10 mCi per embryo. The results provide the first combined direct biochemical and radioautographic identification that phosphoproteins are synthesized in bone and are located morphologically at the sites of mineralization. The data provide further evidence that phosphoproteins play a critical role in the biological calcification of vertebrate tissues.

Receptor-ligand interaction between CD44 and osteopontin (Eta-1).

The CD44 family of surface receptors regulates adhesion, movement, and activation of normal and neoplastic cells. The cytokine osteopontin (Eta-1), which regulates similar cellular functions, was found to be a protein ligand of CD44. Osteopontin induces cellular chemotaxis but not homotypic aggregation, whereas the inverse is true for the interaction between CD44 and a carbohydrate ligand, hyaluronate. The different responses of cells after CD44 ligation by either osteopontin or hyaluronate may account for the independent effects of CD44 on cell migration and growth. This mechanism may also be exploited by tumor cells to promote metastasis formation.

Noncollagenous nature of the proteins of shark enamel.

The proteins, soluble and insoluble at neutral pH, of relatively mature enamel of two different species of sharks were distinctly different from both collagen and embryonic enamel proteins and similar to the proteins isolated from mature human and bovine enamel.

Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity.

Cell-mediated (type-1) immunity is necessary for immune protection against most intracellular pathogens and, when excessive, can mediate organ-specific autoimmune destruction. Mice deficient in Eta-1 (also called osteopontin) gene expression have severely impaired type-1 immunity to viral infection [herpes simplex virus-type 1 (KOS strain)] and bacterial infection (Listeria monocytogenes) and do not develop sarcoid-type granulomas. Interleukin-12 (IL-12) and interferon-gamma production is diminished, and IL-10 production is increased. A phosphorylation-dependent interaction between the amino-terminal portion of Eta-1 and its integrin receptor stimulated IL-12 expression, whereas a phosphorylation-independent interaction with CD44 inhibited IL-10 expression. These findings identify Eta-1 as a key cytokine that sets the stage for efficient type-1 immune responses through differential regulation of macrophage IL-12 and IL-10 cytokine expression.

The presence of protein-bound gamma-carboxyglutamic acid in calcium-containing renal calculi.

The amino acid gamma-carboxyglutamic acid (Gla) is found in four blood-clotting proteins, in a bone protein, in kidney protein, and in the protein present in various ectopic calcifications. This paper reports the presence of Gla in the EDTA-soluble, nondialyzable proteins of calcium-containing renal calculi including calcium oxalate, hydroxyapatite, and mixed stores of apatite and struvite (MgNH4PO4). Calculi composed of pure struvite and those composed of only uric acid or cystine do not contain Gla. From calcium oxalate and hydroxyapatite stontes, a protein of about 17,000 daltons was obtained which contained about 40 residues of Gla per 1,000 amino acids. The amino acid composition of this protein had no apparent relationship to the Gla-containing bone protein or to the similarly-sized F1 fragment of prothrombin which contains about 64 residues of Gla per 1,000 amino acid residues. The Gla-rich protein in calcium-containing renal stones thus may be a different Gla-containing protein. These data as well as other studies demonstrating the presence of Gla in pathologically calcified tissues not normally containing Gla suggest that the Gla-containing proteins may be of considerable pathophysiological significance.

Isolation, characterization and partial amino acid sequence of a phosphorylated polypeptide (E4) from bovine embryonic dental enamel.

A phosphorylated polypeptide (E4) of molecular weight 5000-6000, has been isolated from bovine embryonic enamel by Bio-Gel P-10 gel filtration and DE-52 ion-exchange chromatography. The peptide contains three serine residues all of which are phosphorylated. All three O-phosphoserine residues are in glutamic acid-O-phosphoserine-tyrosine sequences that are distributed relatively evenly along the polypeptide chain. Although it was not possible to sequence the entire polypeptide chain directly by automatic peptide sequencing, a partial sequence and peptide map was constructed on the basis of the sequence and composition of peptides derived by cyanogen bromide, trypsin and chymotrypsin digestion. The presence of glutamic acid, tyrosine and leucine adjacent to and near the O-phosphoserine residues may be important in calcium binding and in mineralization.

Studies on the interaction between heparin and mouse bone collagenase.

Mouse bone collagenase was found to be tightly bound to a heparin-substituted gel at low ionic strength. The bond was reversible, however, and the collagenase could be elutted at high ionic strength. In addition to providing a method for purifying the enzyme with high yield, the results suggest that the strong ionic bond between heparin and collagenase may partially explain the mechanism wherein heparin enhances the activity of mouse bone collagenase.

Three-dimensional spatial relationship between the collagen fibrils and the inorganic calcium phosphate crystals of pickerel (Americanus americanus) and herring (Clupea harengus) bone.

High-voltage (1.0 MV) electron microscopy and stereomicroscopy, electron probe microanalysis, electron diffraction and three-dimensional computer reconstruction, have been used to examine the spatial relationship between the inorganic crystals of calcium phosphate and the collagen fibrils of pickerel and herring bone. High-voltage stereo electron-micrographs were obtained of cross-sections of the cylinder-shaped intramuscular bones in uncalcified regions, in regions where only one or only several crystals had been deposited in some of the fibrils, and in successive sections containing progressively more mineral crystals until the stage of full mineralization was reached. High-resolution electron probe microanalysis confirmed that the electron-dense particles contained calcium and phosphorus. In the earliest stages of mineralization and progressing throughout the mineralization process, the crystals are located only within the collagen fibrils; crystals are not observed free in the extracellular spaces between collagen fibrils. The progressive increase in the mass of mineral deposited in the bone tissue with time occurs, essentially, completely within the collagen fibrils including the stage of full mineralization. At this stage, cross-sectional profiles of collagen fibrils are completely obliterated by mineral. A small number of crystals that are located on or close to the surface of the fibrils appear to extend a very short distance into the spaces between the fibrils. These ultrastructural observations of the very onset of calcification in which nucleation of the calcium phosphate crystals is clearly shown to begin within specific volumes of collagen fibrils, and of the subsequent temporal and spatial sequences of this phenomenon, which shows that calcification continues wholly within the collagen fibrils until maximum calcification is achieved, add important information on the basic physical chemical mechanism of the calcification and the structural elements that are involved. The spatial and temporal independence of the sites where mineralization is initiated establishes that such ultrastructural locations within individual collagen fibrils represent independent, physical chemical nucleation loci. The findings are totally inconsistent with the proposal that crystals must first be deposited in matrix vesicles, or other components such as mitochondria, and subsequently released and propagated in the interfibrillar space, until they eventually reach and impregnate the hole zone regions of the collagen fibrils. Three-dimensional computer reconstruction of serial transverse and longitudinal sections demonstrates periodic swellings along the collagen fibrils, corresponding to the hole zone region of their axial period as mineralization proceeds.(ABSTRACT TRUNCATED AT 400 WORDS)

A unique protonated phosphate group in bone mineral not present in synthetic calcium phosphates. Identification by phosphorus-31 solid state NMR spectroscopy.

The detailed chemical composition and microstructure of freshly deposited bone mineral, and how these properties change with maturation of the mineral, have been studied intensively and still remain controversial. For example, current analytical technology is inadequate for the unambiguous characterization of the monohydrogen phosphate ions in bone mineral. Using a differential cross polarization/magic angle spinning solid state nuclear magnetic resonance spectroscopy technique, we suppress the dominant orthophosphate (PO4-3) signal to reveal the spectra of the minor phosphate constituents. This method depends upon differences in the cross polarization time constants for phosphorus-31 nuclei in protonated and non-protonated phosphate ions. It is now possible for the first time to directly measure both the proportion of acid phosphate (HPO4-2) as well as the parameters which characterize its isotropic and anisotropic chemical shift. In bone from three species at several developmental stages, we have found a single type of acid phosphate species, identical in all of the specimens examined. The phosphorus-31 isotropic chemical shift of this acid phosphate group in bone mineral corresponds precisely with that of acid phosphate in octacalcium phosphate, and not with that of brushite. In contrast, the bone acid phosphate anisotropic chemical shift parameters are close to those of brushite, and differ significantly from those of octacalcium phosphate. The orthophosphate resonances of bone mineral, synthetic hydroxyapatite and synthetic octacalcium phosphate share identical chemical isotropic shifts, and similar chemical shift anisotropies. The implication of these results is that the intimate structure of the acid phosphate group in bone mineral is unique, and that none of the common synthetic calcium phosphates accounts well for all of the observed solid state phosphorus-31 NMR properties of bone mineral.

On the problem of covalent linkages between phosphoproteins and collagen in bovine dentin and bone.

The majority of phosphoproteins in bovine bone and dentin are insoluble in EDTA and guanidine hydrochloride (Gu.HCl) at 2 degrees C. After removal of EDTA and Gu.HCl-soluble proteins at 2 degrees C, collagen alpha-chains and alpha-chain polymers were extracted from bovine bone and dentin in Gu.HCl at elevated temperatures and purified by several chromatographic techniques and SDS-PAGE. Small amounts of O-phosphoserine were found in all collagen components. In contrast, O-phosphoserine was not detected in the purified collagen components soluble in EDTA or Gu.HCl at 2 degrees C nor was hydroxyproline detected in the EDTA-soluble phosphoproteins. In contrast, although the vast majority of EDTA-insoluble collagen and phosphoprotein molecules can be readily dissociated by a variety of molecular sieving and ion-exchange chromatographic procedures, a small number are very strongly associated or covalently cross-linked. These results are consistent with the findings that both hydroxyproline and hydroxylysine are present in purified phosphoprotein components released from the EDTA-insoluble tissue by bacterial collagenase. The hydroxylysine/100 hydroxyproline ratios in the phosphoprotein-collagen complexes are much higher than those in dentin or bone collagens.

Isolation of calcium-phosphate crystals of bone by non-aqueous methods at low temperature.

We have developed low temperature nonaqueous solution methods as well as low power plasma ashing for the degradation of the organic matrix of bone power which have permitted us to obtain bone crystals essentially free of organic matrix constituents without any significant change in their composition, overall structure, or internal short-range order. We have also been able to disperse the crystals, which has made it possible to examine the isolated crystals by X-ray diffraction and resolution-enhanced Fourier transform infrared (FTIR) spectroscopy and isolated single crystals by high resolution transmission electron microscopy (TEM) and electron diffraction. TEM of isolated single crystals of fish, chicken, mouse and bovine bone have clearly demonstrated that the crystals are very thin plates. No rod or needle-like crystals were observed in any of the bone samples in the four species studied including the earliest crystals deposited. Although there were variations in the size distribution of the crystals in the different species studied, in general the average crystal dimensions were very similar.

Structural studies of the mineral phase of calcifying cartilage.

The calcified cartilage of the epiphyseal growth plate of young calves has been studied by x-ray diffraction. Fourier transform infrared spectroscopy, magic angle 31P nuclear magnetic resonance spectroscopy, and chemical composition. The powdered tissue was separated by density centrifugation as a function of mineral content and thus qualitatively of the age of the calcium-phosphorus mineral phase. The individual density centrifugation fractions were examined separately. X-ray diffraction of the samples, especially of the lowest density fractions, revealed very poorly crystalline apatite. Fourier transform infrared spectroscopy and 31P nuclear magnetic resonance spectroscopy revealed the presence of significant amounts of nonapatitic phosphate ions. The concentration of such nonapatitic phosphates increases during the early stages of mineralization but then decreases as the mineral content steadily rises until full mineralization is achieved. The total concentration of carbonate ions was found to be much lower in calcified cartilage than in bone from the same organ (scapula). The carbonate ions are located in both A sites (OH-) and B sites (PO4(3-)), with a distribution similar to that found in bone mineral. However, discrepancies between infrared resolution factors of phosphate and carbonate bands are consistent with a heterogeneous distribution of carbonate ions in poorly organized domains of the solid phase of calcium phosphate. These initial studies permit one to characterize the calcium phosphate mineral phase as a very poorly crystalline, immature calcium phosphate apatite, rich in labile nonapatitic phosphate ions, with a low concentration of carbonate ions compared with bone mineral of the same animal, indeed from the bone of the same organ (scapula).

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.

Structural and chemical characteristics and maturation of the calcium-phosphate crystals formed during the calcification of the organic matrix synthesized by chicken osteoblasts in cell culture.

The calcium-phosphate (CA-P) crystals formed in the extracellular organic matrix synthesized by chicken osteoblasts in cell culture were examined after 30, 40, and 60 days of culture by a number of physical and chemical techniques including chemical analyses, X-ray diffraction, transmission electron microscopy of isolated crystals, and resolution-enhanced Fourier transform infrared spectroscopy. The data reveal that the solid inorganic calcium-phosphate phase consists of a very poorly crystalline apatite, having a low carbonate content and containing acid phosphate groups. The chemical and structural characteristics are generally similar to the apatite crystals found in young newly synthesized bone but there were small but significant differences found. The major significant differences noted were the rate at which maturational changes occurred in the crystals formed in cell culture compared with those noted in vivo and in synthetic carbonate apatite crystals equilibrated with the same cell culture medium, and the persistence of labile groups, especially HPO4(-2) ions during a relatively long period of incubation. Despite extensive chemical efforts to degrade the organic constituents and to disperse the individual crystals isolated from the organic matrix constituents, a large proportion of the crystals were found to be organized in both loosely and densely packed relatively large roughly spherical aggregates. A few of the aggregates were organized in the form of fibrils with the crystals oriented with their c-axes roughly parallel to the long axes of the crystal aggregate. With briefer periods of chemical treatment, larger aggregates of crystals were occasionally observed in which there was a distinct axial periodicity of approximately 70 nm. In such collagen-crystal fragments, the crystals were well-oriented with their c-axis roughly parallel to the long axes of the aggregate similar to the organization and relationships between crystals and collagen fibrils in native bone. Isolated crystals were in the shape of thin plates. At the end of 30 days of culture, many of the crystals were clearly larger than those observed in native chick bone, except for those in the very youngest (7- to 8-day-old) embryos. At the end of 40 and 60 days of culture, the crystal habit remained as thin plates but the crystals were predominantly smaller, similar to those found in older embryo and postnatal chicken bone. The marked tendency of the crystals to form relatively large aggregates that resist dispersion by techniques that readily disperse the crystals of bone, and the presence of a significant number of larger crystals has also been observed in studies of calcified cartilage. Resolution enhanced FTIR spectroscopy revealed the presence of a high concentration of labile phosphate groups, especially after 30 days of culture and just after the plateau of mineralization is reached.

Protein kinases of cultured osteoblasts: selectivity for the extracellular matrix proteins of bone and their catalytic competence for osteopontin.

The enzyme activities of the major kinases found within the cytosolic and microsomal fractions of embryonic avian calvaria osteoblasts were assayed for their specificity for various noncollagenous extracellular matrix (ECM) proteins of bone. At least 6 proteins with M(r)'s of 66, 58, 50, 36, 30, and 22 kD out of more than 30 of the noncollagenous proteins of the bone ECM were phosphorylated by the kinase(s) found in both osteoblast cellular fractions. The purification and N-terminal sequence analysis of three of the above proteins, M(r)'s 66 and 58 kD (+50 kD), identified them as chicken bone sialoprotein (BSP) and osteopontin (OPN), respectively. Heparin, a specific inhibitor of factor-independent protein kinase (FIPK) activity, blocked the phosphorylation of all six ECM proteins by the microsomal kinase(s) but only inhibited the phosphorylation of the 66, 50, and 36 kD by the cytosolic enzyme(s). Casein kinase II (a known FIPK) showed a similar phosphorylation pattern of the same bone ECM proteins as the FIPK(s) found in osteoblast cell extracts, while purified cyclic adenosine monophosphate (cAMP)-dependent protein kinase did not phosphorylate any of the ECM proteins. Use of dephosphorylated casein showed that in comparison with casein kinase II, casein was a poor substrate for the FIPK found in the osteoblast cellular extracts. Further studies, using FIPK(s) of osteoblasts and purified chicken OPN or bacterially produced recombinant murine OPN as a substrate, showed that both species of OPN were excellent substrates for the FIPK(s) found in osteoblasts. The phosphorylation of the purified chicken and recombinant mouse OPNs were evaluated by quantitative analysis using commercially available protein kinases. cAMP-dependent kinase showed no phosphorylation of either protein, and cyclic guanodine monophosphate (cGMP)-dependent kinase and protein kinase C incorporated 1.2 and 0.5 mol phosphate/mol OPN, respectively. However, both chicken and mouse OPNs were significantly phosphorylated by casein kinase II (9.3 and 9.0 mol of phosphate/mol of OPN, respectively). These results demonstrate that the noncollagenous proteins of the bone ECM, and in particular OPN, are predominantly phosphorylated by FIPK(s), and this class of kinase is the major enzyme found within the microsomal fraction of osteoblasts.

Effects of fixation and demineralization on the retention of bone phosphoprotein and other matrix components as evaluated by biochemical analyses and quantitative immunocytochemistry.

Aqueous tissue processing and demineralization procedures may adversely affect the inorganic mineral phase of a calcified sample and, where mineral and organic constituents interact, may consequently also indirectly alter organic matrix ultrastructure and distribution. In the present work, the effects of demineralization have been investigated on the retention in chicken bone of two phosphoamino acids, O-phosphoserine and O-phosphothreonine, found in bone phosphoproteins proposed to be important in vertebrate mineralization and, more specifically, on the retention and distribution of a 66 kD bone phosphoprotein (66 kD BPP, osteopontin) also implicated in the calcification process. In tibiae fixed initially with 1% glutaraldehyde and then demineralized in 0.5 N HCl, 0.5 N acetic acid, or 0.1 M EDTA (all containing 1% glutaraldehyde), amino acid analyses and quantitative immunocytochemistry revealed that the phosphoamino acid content and the distribution of the 66 kD BPP were essentially the same as in fixed undemineralized controls. However, demineralization slightly altered the ultrastructural appearance of immunolabeled, electron-dense patches of organic material in the bone matrix. In unfixed bone demineralized with any of these acids, there was a substantial loss of phosphoamino acids and the 66 kD BPP from the bone matrix. The relative ability of these acids to extract phosphoproteins from unfixed bone was found to decrease in the order EDTA greater than HCl greater than acetic acid. These results emphasize the differential effects on structural components of various demineralization and extraction procedures for biochemical and immunocytochemical studies of biologic tissues. Furthermore, they demonstrate that initial fixation with glutaraldehyde retains phosphoproteins in bone, with or without demineralization, while being adequate for immunocytochemical localization of certain bone matrix proteins and that an understanding of the action of specimen preparation on organic constituents (as well as inorganic components) is essential for accurately describing ultrastructural matrix-mineral relationships.

Structural and composition studies on the mineral of newly formed dental enamel: a chemical, x-ray diffraction, and 31P and proton nuclear magnetic resonance study.

The present report describes a study of the development and maturation of the mineral component of dental enamel. We prepared porcine enamel of different stages of maturation, from the very immature enamel of unerupted teeth, with a mineral content of 45%, to fully mature enamel, with a mineral content of approximately 99%. We fractionated the less mature enamel by density centrifugation and examined the enamel density fractions and unfractionated enamel by a variety of chemical and physical techniques, including conventional and radial distribution function x-ray diffraction analysis, conventional and Fourier transform infrared spectroscopy, 31P and 1H nuclear magnetic resonance spectroscopy, and chemical analysis. The three most immature preparations, from unerupted teeth, had mineral contents of 45, 67, and 91 and Ca/P molar ratios of 1.41, 1.44, and 1.47. Density distribution histograms of the three fractions show that the early maturation of dental enamel mineral is accompanied by an increase in tissue density, reflecting the increase in mineral content. The density distribution in each sample is relatively narrow, indicating that the maturation process occurs at a fairly homogeneous rate, with all enamel in an anatomically defined zone mineralizing to about the same extent. X-ray diffraction studies indicate that even the least mature, least mineralized of these immature samples is considerably more crystalline than the most mature bone mineral studied and that crystalline perfection of the enamel crystals crystals increases further with maturation. Both the a and c axes of the mineral unit cell expand significantly during early stages of maturation. Solid-state 31P nuclear magnetic resonance spectroscopy studies indicate that dental enamel contains a DCPD-like HPO4 component in an apatitic lattice, similar to the component previously observed in bone and some synthetic calcium phosphates. The proportion of this DCPD-like component decreases with maturation but is readily detectable even in fully mature enamel. The infrared spectroscopic studies indicate that the 3570 cm-1 band ascribed to the OH- group of the hydroxyapatite crystals is absent in the least mature enamel but can be detected and becomes progressively stronger as the enamel becomes more mature. The increase in the content of the OH- groups of the apatite crystals is concomitant with the observed increase in unit cell parameters. Similar studies on very young and very old mature bone of four different species failed to detect the presence of OH- groups.

Structure, composition, and maturation of newly deposited calcium-phosphate crystals in chicken osteoblast cell cultures.

Characterization of the very early calcium phosphate (CaP) crystals deposited in bone or in osteoblast cell cultures has been hampered by the overwhelming presence of organic matrix components and cells that obscure spectral analyses. We have overcome this problem using isolated protein-free crystals and have obtained new data including 31P nuclear magnetic resonance (NMR) spectra for the first time from mineral crystals deposited during osteoblast calcification in culture. Crystals were isolated from cultures at two time points: (a) at first calcium accumulation (day 8-10) and (b) after 60 days of culture, to assess maturational changes. The analyses show that the chemical composition overall and short range order of the early and mature crystals are characteristic of the apatite crystals found in young embryonic chick bone in vivo. No mineral phase other than apatite was detected by any of the methods used. 31P NMR spectroscopy identified the HPO4 groups as those present in bone apatite. Similar to bone apatites, no OH groups were detected by Fourier transform infrared (FTIR) spectroscopy. The temporal maturational changes in composition and structure of the mineral phase were difficult to assess because of the continuous deposition of crystals throughout culturing. The pathway of the maturational changes observed were similar to those occurring in chick bone in vivo and synthetic apatite crystals in vitro although to a much smaller extent.