Showing papers in "Calcified Tissue International in 1982"
TL;DR: Tartrate-resistant acid phosphatases of bone may be suitable biochemical probes for osteoclast function, but it will be necessary to achieve further purification in order to develop analytical methods with sufficient sensitivity and specificity to ensure precise localization and quantitation.
Abstract: Organ cultures of newborn mouse calvaria were used to test the hypothesis that tartrate-resistant acid phosphatase might serve as a biochemical marker for osteoclast function. When bone resorption was stimulated in vitro with either parathyroid hormone or 1,25(OH)2D3, there was a significant increase in both tartrate-resistant and tartrate-sensitivity acid phosphatase activity in the medium relative to cultured controls. Tartrate-resistant activity was localized histochemically primarily over the osteoclast and appeared as three distinct activity bands when electrophoresed on polyacrylamide gels. The tartrate-sensitive activity was found primarily associated with bone cells other than the osteoclast using histochemical techniques, and was resolved into five bands on polyacrylamide gels. The results obtained from biochemical assays, histochemical observations, and polyacrylamide gel electrophoresis suggest that bone resorption in vitro results in the release of tartrate-resistant acid phosphatase from osteoclasts and tartrate-sensitive acid phosphatase from other bone cells as well as osteoclasts. Tartrate-resistant acid phosphatases of bone may be suitable biochemical probes for osteoclasts function, but it will be necessary to achieve further purification in order to develop analytical methods with sufficient sensitivity and specificity (e.g., immunochemical) to ensure precise localization and quantitation.
961 citations
TL;DR: Periostea consisting of the osteogenic layer and the fibrous layer of the periosteum were dissected from 17-day-old embryonic chick calvariae, and physiologic mineralization of osteoid produced in vitro did occur in this system by the addition of the alkaline phosphatase substrateβ-glycerophosphate.
Abstract: Periostea consisting of the osteogenic layer and the fibrous layer of the periosteum were dissected from 17-day-old embryonic chick calvariae, leaving the osteoblasts behind on bone. The dissected periostea were folded with the osteogenic cells in apposition. The explants were cultured on plasma clots for up to 6 days, during which time osteodifferentiation was observed followed by osteoid formation in between the two layers. These cultures consistently mineralized in the presence of 5 or 10 mMβ-glycerophosphate. The mineralization and osteoid formation displayed many characteristics identical with those seen in vivo. Specifically, the osteoid formed was birefringent under polarized light, the central zone of osteoid became mineralized within 24 h of formation in vitro, and a clear border between mineralized and non-mineralized osteoid suggestive of a mineralization front was present. The unmineralized osteoid at the periphery was surrounded by osteoblasts. These data suggest that physiologic mineralization of osteoid produced in vitro did occur in this system by the addition of the alkaline phosphatase substrateβ-glycerophosphate.
215 citations
Journal Article•
TL;DR: It is demonstrated that carbonate exists in two crystallographically distinct sites in the apatite crystal structure and is consistent with a carbonate-for-phosphate substitution mechanism, where four carbonate groups replace three phosphate groups.
Abstract: A range of synthetic carbonated apatites were characterized using infrared and Raman spectroscopy, X-ray diffraction, and chemical analysis and compared to stoichiometric hydroxyapatite and dental enamel. Synthetic apatites were prepared by aqueous precipitation and by solid-state reaction at high temperatures. A method is described for the carbonate determination of apatite samples using gas chromatography. This study demonstrates that carbonate exists in two crystallographically distinct sites in the apatite crystal structure and is consistent with a carbonate-for-phosphate substitution mechanism, where four carbonate groups replace three phosphate groups. Sodium and hydroxyl ions are shown to be involved with the substitution of carbonate ions in the apatite structure. Observed sodium/carbonate and calcium/phosphorus molar ratios confirm this mechanism. Substitution of this type may explain the predominant mode of carbonate substitution in biological apatites.
174 citations
TL;DR: The osteoblastic properties of the ROS 17/2 osteosarcoma cell line are documented, and it is suggested that PTH inhibition of alkaline phosphatase represents a physiological response to the hormone in these cells, and cyclic AMP as a mediator of this PTH effect.
Abstract: The effect of parathyroid hormone (PTH 1–34 bovine) on alkaline phosphatase activity was investigated in an osteoblast-like clonal cell line derived from rat osteosarcoma (ROS 17/2). ROS 17/2 alkaline phosphatase resembled the bone enzyme in levamisole sensitivity and electrophoretic mobility but differed in heat stability. The specific activity of ROS 17/2 alkaline phosphatase increased with time in culture. This increase was inhibited by PTH (1–34) and (-)-isoproterenol in a dose-dependent manner starting at near-physiological hormone concentrations. The ID50 values were 0.02 nM for PTH (1–34) and 1.7 nM for isoproterenol. The two hormones stimulated ROS 17/2 adenylate cyclase, albeit at higher concentrations: Km values were 13 nM for PTH (1–34) and 16 nM for isoproterenol. The rise in alkaline phosphatase was also inhibited by dibutyryl cyclic AMP and 8-bromocyclic AMP (0.1 mM). These findings further document the osteoblastic properties of the ROS 17/2 osteosarcoma cell line, suggest that PTH inhibition of alkaline phosphatase represents a physiological response to the hormone in these cells, and implicate cyclic AMP as a mediator of this PTH effect.
162 citations
TL;DR: The measurement of plasma BGP appears to provide a specific index of bone metabolism that may in some circumstances be more sensitive than serum alkaline phosphatase measurement, however, further studies are necessary to establish the clinical value of Plasma BGP measurement by RIA in the management of patients with bone diseases.
Abstract: Bone Gla protein (BGP) was measured in the plasma by radioimmunoassay (RIA) during treatment of 59 patients with bone diseases including Paget's disease (N=9), primary hyperparathyroidism (N=25), chronic renal failure (N=20), and cancer involving bone (N=5). Plasma BGP was increased above normal in all patients. BGP decreased in the patients with Paget's disease following the acute and chronic administration of salmon calcitonin. Plasma BGP was higher in women then in men with primary hyperparathyroidism. Following parathyroidectomy, BGP decreased in both sexes but the decrease was significant in women only. Plasma BGP was increased in patients with renal osteodystrophy and did not change after hemodialysis. In the patients with bone cancer, plasma BGP decreased during treatment of the attendant hypercalcemia with salmon calcitonin. Although plasma BGP and serum alkaline phosphatase (AP) levels were generally correlated in these studies, there were examples of dissociation between the two. The measurement of plasma BGP appears to provide a specific index of bone metabolism that may in some circumstances be more sensitive than serum alkaline phosphatase measurement. However, further studies are necessary to establish the clinical value of plasma BGP measurement by RIA in the management of patients with bone diseases.
154 citations
TL;DR: A new semiautomatic technique combining advantages of the manual and fully automatic methods is described for obtaining quantitative static and dynamic histologic data of bone, showing that static andynamic parameters of bone are obtained with a degree of error well within the acceptable range for biologic measurements.
Abstract: A new semiautomatic technique combining advantages of the manual and fully automatic methods is described for obtaining quantitative static and dynamic histologic data of bone The hardware consists of a photomicroscope, digitizing platen, digitizer, plotter/printer, floppy disc drive, and computer The microscope is equipped with a drawing tube through which the image of the digitizing platen is projected over the optical field The investigator selects and traces all histologic structures to be measured by moving a cursor on the digitizing platen which is visible by its projection over the histologic field The results on accuracy and static and dynamic precision of this method show that static and dynamic parameters of bone are obtained with a degree of error (less than 20%) well within the acceptable range for biologic measurements Comparison of this method with the grid technique according to Merz and Schenck showed that for almost all micromorphometric parameters comparable absolute data are obtained Due to the higher precision of our method, however, the number of optical fields evaluated in obtaining these comparable data could be reduced to 25% of the number of fields evaluated by the Merz and Schenck technique The time requirements for quantitative evaluation of a histologic slide of bone by our technique are 40-50 min; 20-25 min is needed for quantitative evaluation of osteocytes
145 citations
TL;DR: During the past 20 years, great strides have been made in defining bioelectric phenomena in the skeletal system and a rapidly expanding base of fundamental data suggest that many important advances can be made as new research endeavors characterize effects of weak pulsing electric currents on cell behavior.
Abstract: During the past 20 years, great strides have been made in defining bioelectric phenomena in the skeletal system. As a result, it is possible to treat, clinically, disordered function in these tissues. The success of these efforts and a rapidly expanding base of fundamental data suggest that many important advances can be made as new research endeavors characterize effects of weak pulsing electric currents on cell behavior. Future endeavors will involve investigators versed in biochemical, biophysical , developmental ; endocrinological , physical-chemical, and physiological aspects of the skeletal and other systems. Unfortunately, few of these individuals are equipped by past experience or training to deal easily with the principles and techniques of electricity or electromagnetism. In such an environment, confusion of concepts and terminology is an ever present and real danger. An example of the magnitude of the problem is evident in an edi tor ial , ent i t led \" E l e c t r i c a l Osteogenesis--Pro and Con,\" that appeared a short while ago in Calcif ied Tissue Research [1]. The author, a pioneer in bioelectric research, dealt with the two main electrical methods for inducing ununited fractures to heal, namely, direct-current (D.C) electrodes and pulsing electromagnetic fields (PEMFs). In the process, differences between the two methods were not delineated and the uninitiated reader could have assumed that, since both employed electricity, they were basically the same. This assumption is quite natural when there is a paucity of information, and it is made frequently now by neophytes, as it was during the beginning of the au thor ' s invest igat ions . For example , in 1967-1968 it was thought that development of weak
130 citations
TL;DR: The results show that histomorphometric data of bone cannot be easily compared when different techniques are employed for obtaining bone samples, and sampling variations are kept smaller when bone specimens are obtained in a vertical manner.
Abstract: Quantitative bone histology was done in undecalcified sections of iliac crest bone specimens obtained from 84 normal American individuals Samples were obtained within 12 h after death in a vertical and horizontal manner from both the right and left iliac crests In addition to the determination of normal values of micromorphometric parameters of bone in these healthy American subjects, the following studies were carried out: (a) comparison of variance of micromorphometric parameters of bone obtained from the right versus left iliac bone (40 pairs), (b) comparison of micromorphometric parameters of bone obtained in a vertical versus horizontal manner (12 pairs), (c) evaluation of variance with increasing distance from the compact zone in bone samples obtained in a vertical manner (44 pairs), (d) analysis of variation between bone samples obtained more anteriorly versus posteriorly along the iliac crest (N=40), (e) comparison of differences in micromorphometric parameters obtained from age-matched men versus premenopausal women (N=12), and (f) plotting of histograms for assessment of distribution of micromorphometric parameters The results show that histomorphometric data of bone cannot be easily compared when different techniques are employed for obtaining bone samples Sampling variations are kept smaller when bone specimens are obtained in a vertical manner Anterior/posterior variation does not cause major sampling error If ranges of variation are taken into account, quantitative bone histology is a valuable tool for assessment of bone structure and bone cells
123 citations
TL;DR: Implantation of mineral-containing bone fragments into calvarial defects in rats initiates a rapid and reproducible resorption of the bone matrix, and a dense tissue develops with mononucleated as well as multinucleated cells surrounding and between the bone fragments.
Abstract: Implantation of mineral-containing bone fragments into calvarial defects in rats initiates a rapid and reproducible resorption of the bone matrix. After 7 days, a dense tissue develops with mononucleated as well as multinucleated cells surrounding and between the bone fragments. Electron microscopy revealed that these cells belong to the mononuclear phagocytic system: they were identified as macrophages, epithelioid cells, foreign body giant cells, and Langerhans cells. In addition to the common ultrastructural characteristics, these cells had electron-dense, focal specializations along their cell membrane with a coating on the exterior, corresponding to subplasmalemmal linear densities. Small, unidentified cells with electron-dense ground cytoplasm were often seen in close proximity to more differentiated cells. No halisteresis had occurred on the surfaces of the bone fragments. Indentations resembling Howship's lacunae were frequent; these contained mononucleated as well as multinucleated cells. Some surfaces were frayed and collagen fibers were exposed, but the cells apposed to these surfaces did not have ruffled borders as are seen in osteoclasts. Some bone fragments were broken up and cell processes had penetrated deep into the cracks, separating pieces of matrix. Small matrix particles were phagocytosed by macrophages, but not by epithelioid cells or giant cells. It appears that enzymes capable of degrading bone matrix components were secreted by the more differentiated cells of the mononuclear phagocytic system. They eroded the bone surface in a way reminiscent of osteoclastic bone resorption. They also entered the canaliculi to act from within the bone fragment, a process possible only in dead bone. We suggest a possible relationship of these cells with osteoclasts.
96 citations
TL;DR: The bone mineral status of 17 girls with Turner's syndrome was evaluated by single photon absorptiometry and bones of girls who received estrogen replacement therapy were less demineralized than those of the others.
Abstract: The bone mineral status of 17 girls with Turner's syndrome was evaluated by single photon absorptiometry. Bone mineral content (BMC) was 25.4% below that predicted by normalization for age, sex, height, weight, and bone width. Only 25% of this demineralization could be attributed to delayed skeletal maturation. Bones of girls who received estrogen replacement therapy were less demineralized than those of the others. The bone mineral deficit became less pronounced with advancing age. It could not be determined if the apparent effect of estrogens was related to age or if the apparent improvement with age was really due to an effect of estrogen treatment. For 8 subjects followed longitudinally there was no significant change in the BMC deficit.
82 citations
TL;DR: The data confirm the osteoblast-like nature of the isolated cell populations and demonstrate that these cells retain their differentiated function in culture.
Abstract: Osteoblast-like and osteoclast-like cells freed from neonatal calvaria by sequential enzymatic digestion after 6-7 days in culture were placed in diffusion chambers and implanted in the peritoneal cavities of CD-1 mice. About half of the chambers also contained a dead calvarium to test for the need of an "inducer." After 20 days, 11 of 18 chambers containing to osteoblast-like cells formed large foci of mineralized bone that corresponded to alkaline phosphatase activity throughout the chambers. Moreover, only type I (i.e., bone) collagen was formed. Occasional deposits of bone were found in only 3 of 22 chambers containing the osteoclast-like cells. The presence of dead bone did not affect any of the results. These data confirm the osteoblast-like nature of the isolated cell populations and demonstrate that these cells retain their differentiated function in culture.
TL;DR: For a compound to inhibit potently the transformation of amorphous calcium phosphate into hydroxyapatite, it is suggested that the minimum structural requirement is a phosphate group and, at some other position, either another phosphate group preferably or a carboxylic moiety.
Abstract: For a compound to inhibit potently the transformation of amorphous calcium phosphate into hydroxyapatite, it is suggested that the minimum structural requirement is a phosphate group and, at some other position, either another phosphate group preferably or a carboxylic moiety. Primary amino groups abolish inhibitor potential. Inhibitor potency is modified by various secondary factors, including the number and proximity of active groups, their stereochemistry, steric factors, the lability of the molecule, and in special instances its lipophilicity. Parameters used to monitor the transformation indicate that inhibitors can be grouped into two classes, and it is suggested that this is because one class acts as a hydroxyapatite crystal growth inhibitor. The close proximity of two phosphate groups or of a phosphate and multiple carboxylic groups is proposed to determine in part whether or not a compound acts as a crystal growth inhibitor. Further, bulky side groups render a molecule inactive as a crystal growth poison, although it will still inhibit by other mechanisms.
TL;DR: It is suggested that the action exerted by osteocytes on the surrounding calcified matrix, whatever the function of these cells, is not uniform throughout the skeleton and is to some extent correlated with the activity of the other bone cells—osteoblasts and osteoclasts.
Abstract: Size and density of osteocyte lacunae were evaluated at different levels of long bones to investigate whether or not the proportion of bone tissue occupied by osteocytes changes in skeletal regions, characterized by clear-cut differences in bone turnover rates. Statistical analysis of the results shows that the mean cross-sectional area of osteocyte lacunae (C) is lowest in compact bone of diaphysis and metaphysis, highest in spongy bone of metaphysis and epiphysis. On the contrary, the mean surface of bone tissue surrounding each osteocyte (T=bidimensional osteocyte territory, indirectly calculated from the number of lacunae/mm2 of bone) is largest in compact bone of diaphysis, smallest in metaphyseal spongiosa, and shows intermediate values in the cortex of metaphysis and in epiphyseal spongiosa. The proportion of bone tissue occupied by osteocyte lacunae (%C/T) appears to follow at different levels of long bones, the same pattern recorded for the data of bone turnover rate, by the tetracycline labeling technique: it is lowest in mid-diaphyses, highest in metaphyses, and intermediate in epiphyses. On the basis of these findings, it is suggested that the action exerted by osteocytes on the surrounding calcified matrix, whatever the function of these cells, is not uniform throughout the skeleton and is to some extent correlated with the activity of the other bone cells—osteoblasts and osteoclasts.
TL;DR: It is proposed that subjects with XLH have serum 1,25(OH)2D levels within appropriate age- and growth-rate-matched normal ranges, suggesting the presence of a relative deficiency of this active vitamin D metabolite.
Abstract: In order to determine whether a defect in vitamin D metabolism might play a role in the pathogenesis of X-linked hypophosphatemic rickets and osteomalacia (XLH), we compared the serum 1,25-dihydroxyvitamin D [1,25(OH)2D] level in 52 normal subjects and 37 patients with XLH In untreated patients, adults were found to have values similar to age-matched controls, while youths had values similar to growth-rate-matched controls but significantly lower than the levels of age-matched controls who were growing at a normal rate In contrast, treated XLH patients of all ages had serum levels significantly lower than both controls and untreated XLH patients Further, the serum levels of 1,25(OH)2D in these treated patients had a significant inverse linear correlation with serum 25-(OH)D concentrations We propose that subjects with XLH have serum 1,25(OH)2D levels within appropriate age- and growth-rate-matched normal ranges However, in the presence of hypophosphatemia, we would have anticipated elevated levels of 1,25(OH)2D; viewed in this light the serum 1,25(OH)2D levels are inadequate, suggesting the presence of a relative deficiency of this active vitamin D metabolite
TL;DR: It is indicated that stimulated salivary secretions are supersaturated with respect to dicalcium phosphate dihydrate; this is a substantially greater degree of supersaturation than previously reported.
Abstract: Previous ultrafiltration studies indicated that up to one-half of the calcium and two-thirds of the phosphate in human salivary secretions may be bound by salivary proteins. Since this binding is an important variable in determining the extent of salivary supersaturation with respect to calcium phosphate salts, and since the amount of binding reported is surprisingly large, calcium and phosphate ion-binding by salivary macromolecules has been reexamined. From experiments using equilibrium dialysis, it was found that (1) the fraction of salivary calcium involved in macromolecular complexes ranges from a few percent for unstimulated secretions, to no more than about 10% for stimulated glandular salivas, and (2) salivary proteins do not bind phosphate ions to any significant extent. These findings, and experiments using an improved ultrafiltration membrane, indicate that the earlier results were artifacts of the ultrafiltration technique. Fractionation of salivary proteins, followed by equilibrium dialysis measurements, showed that the anionic proline-rich proteins and a basic proline-rich glycoprotein are responsible for most of the calcium binding now observed. The finding that macromolecular complexes of salivary calcium and phosphate have been overestimated in the past, leads to the conclusion that salivary calcium and phosphate ion activities in stimulated salivary secretions may be up to 50 to 100% higher than previously thought. Revised values were therefore used to recalculate the degree of salivary supersaturation with respect to calcium phosphate salts. The results indicate that stimulated salivary secretions are supersaturated with respect to dicalcium phosphate dihydrate; this is a substantially greater degree of supersaturation than previously reported.
TL;DR: It is demonstrated that physiological amounts of glucocorticoids reduce bone resorption, normalize serum calcium, and restore the production of 1,25-dihydroxyvitamin D3.
Abstract: A case is reported of a hypercalcemic patient with primary Addison's disease. A combination of increased calcium input into the extracellular space and reduced calcium removal by the kidney accounted for the hypercalcemia. The mechanisms responsible for the reduction in calcium removal were decreased glomerular filtration and increased tubular calcium reabsorption. Both renal factors were secondary to volume depletion and improved rapidly during rehydration with saline infusion. The enhanced calcium mobilization was probably of skeletal origin. It persisted irrespective of volume status until hydrocortisone treatment was instituted. Serum 1,25-dihydroxyvitamin D3 levels were below 10 pg/ml, even after normalization of the glomerular filtration rate, but returned slowly to the normal range during corticosteroid substitution. Serum 25-hydroxyvitamin D3 and parathyroid hormone levels were within the normal range. Our case report therefore demonstrates that physiological amounts of glucocorticoids reduce bone resorption, normalize serum calcium, and restore the production of 1,25-dihydroxyvitamin D3.
TL;DR: The uronate and DNA contents are reduced in formalin-fixed bone while hydroxyproline content is elevated and mineral parameters and lipid parameters are not affected.
Abstract: Formalin fixation of hard tissues alters the levels of some, but not all, of the parameters routinely measured by bone researchers. Although mineral parameters (ash weight, particle size (beta 1/2 002) and Ca/PO4 ratio) and lipid parameters (total lipid content and composition, extractability, and Ca-acidic phospholipid phosphate content) are not affected by formalin fixation, the uronate and DNA contents are reduced in formalin-fixed bone while hydroxyproline content is elevated.
TL;DR: The results emphasize the similarity of the structural requirements for the inhibition of matrix synthesis and the stimulation of resorption by active vitamin D metabolites in bone and support the importance of the 1-hydroxyl function to the biologic activity of vitamin D in the skeleton.
Abstract: The effects of selected vitamin D3 metabolites and analogs on bone collagen synthesis in vitro were examined in organ cultures of neonatal mouse calvarial bone. The incorporation of [3H]proline into the collagenase-digestible fraction of newly synthesized protein was progressively inhibited by 1α,25-dihydroxyvitamin D3 (1α,25-(OH)2D3) (10−12 M to 10−7 M) in 24-h cultures, and incorporation into noncollagen protein was also blunted at the higher doses employed. The synthetic analog 1α-hydroxyvitamin D3 (1α-OHD3) was almost 300-fold less potent an inhibitor of collagen synthesis than was 1α,25(OH)2D3, and the natural metabolites 25-hydroxyvitamin D3 (25OHD3) and 24R,25-dihydroxyvitamin D3 (24R,25(OH)2D3), 1000-fold less potent, although the dose-response curve for each of these compounds was not parallel with that for 1α,25(OH)2D3. The 24S,25(OH)2D3 enantiomer was four-fold less potent than 24R,25-(OH)2D3 or 25OHD3, and vitamin D3 showed less than 2% the activity of 25OHD3. The responses were unaffected by the substitution of 0.4% bovine albumin for 5% horse serum in the medium, and no stimulation of collagen synthesis was observed in response to 25-hydroxylated metabolites between 2×10−14 and 2×10−6 M or in cultures treated for up to 96 h with 24R,25(OH)2D3 (2×10−10M). The overall results emphasize the similarity of the structural requirements for the inhibition of matrix synthesis and the stimulation of resorption by active vitamin D metabolites in bone. In addition, these studies support the importance of the 1-hydroxyl function to the biologic activity of vitamin D in the skeleton.
Journal Article•
TL;DR: The results are consistent with the proposition that phosphoproteins have a regulatory role in mineralization processes and could provide a mechanism by which dietary and salivary phosphoprotein exert an anticariogenic effect.
Abstract: A chromatography column containing hydroxyapatite beads was used to study the effect of different proteins on the rate of hydroxyapatite dissolution. The four phosphoproteins tested (phosvitin, alpha sl-casein, beta-casein and kappa-casein) markedly reduced the rate of hydroxyapatite dissolution. Three nonphosphorylated proteins had a relatively smaller effect. The effect of the protein in reducing the hydroxyapatite dissolution rate has been attributed to protein binding to the surface of hydroxyapatite. The reduction in dissolution rate, expressed as the change in nmol calcium released per min per nmol of phosphoprotein bound to hydroxyapatite, increased with increasing number of phosphoserine residues of the protein. The results are consistent with the proposition that phosphoproteins have a regulatory role in mineralization processes and could provide a mechanism by which dietary and salivary phosphoproteins exert an anticariogenic effect.
TL;DR: In 12 hypercalcemic patients with osteolytic bone lesions and in the 20 normocalcemic patients, the mean serum calcium decreased to final levels that were subnormal and significantly lower than those obtained after treatment of 8 patients with primary hyperparathyroidism.
Abstract: For 2 weeks 27 patients with hypercalcemia received a standard oral treatment with (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (APD) as the sole agent. Results were grouped according to causes of hypercalcemia and compared with effects of APD in 13 normocalcemic patients with Paget's disease of bone and 7 with osteoporosis. In 12 hypercalcemic patients with osteolytic bone lesions and in the 20 normocalcemic patients, the mean serum calcium decreased to final levels that were subnormal and significantly lower than those obtained after treatment of 8 patients with primary hyperparathyroidism. In 3 patients with myeloma and in 4 tumor patients without bone lesions, serum calcium did not always decrease to the normal range. Implications of these observations for the mechanism of hypercalcemia are discussed.
Journal Article•
TL;DR: Hydroxyapatite synthesized by various "standard" ways exhibits marked differences with preparation method, and deuterizability was used as an indicator of ease of diffusion along the X-ion channels, a property that may be related to dissolution kinetics.
Abstract: Hydroxyapatite synthesized by various "standard" ways exhibits marked differences with preparation method. Specimens were prepared with two precipitation methods, a reflux method, a hydrothermal method, a high-temperature (1000 degrees C) solid-state reaction method, and by conversion of chlorapatite at 1000 degrees C. They were compared in detail by use of several techniques, the major ones being x-ray diffraction including Rietveld structure refinements, quantitative i.r. analyses, and deuteration kinetics studies. At least some of the specimens differed with respect to each of the approximately 14 properties measured. The major lattice parameter differences could be largely accounted for by structurally incorporated H2O, CO2-3, and + O2- for 2(OH)-. Deuterizability was used as an indicator of ease of diffusion along the X-ion channels, a property that may be related to dissolution kinetics. The differently prepared specimens differed in deuterizability by at least two orders of magnitude. The high-temperature preparations, which were monoclinic, deuterated little at 110 degrees C, even in 1000 h. The precipitated and reflux specimens deuterated readily. There were general indications of correlation between ease of diffusion and features providing passing sites for the diffusing species, e.g., OH- disorder, vacancies, and distortions in the walls of the X-ion channels (mostly by CO3 for PO4), and possibly OH- vacancies. Correlation of structural H2O, present in the aqueous preparations, with ease of diffusion is still ambiguous.
TL;DR: The results show that, when compared to the value found in half calvaria incubated in a control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with Al as well as with bPTH, 1,25-, or 24,25(OH)2D3 and sharply decreased with higher Al concentrations.
Abstract: The present study was undertaken to test the in vitro action of aluminum on bone phosphatase activities and the possible interaction of this metal with parathyroid hormone (bPTH) or vitamin D3 dihydroxymetabolites [1,25- and 24,25(OH)2D3). Three-day-old rat calvaria were incubated for 24 h with one of the following: bPTH at 5×10−8M, 1,25-or 24,25(OH)2D3 at 2.5×10−9M, Al at concentrations ranging from 3×10−11M to 6×10−6M, or their corresponding solvents. Al effects were also investigated when the medium phosphate or calcium concentrations were modified. In some experiments, Al was added simultaneously with bPTH or one of the vitamin D3 metabolites at the beginning of the 24 h incubation. At the end of all incubations, acid and alkaline phosphatase activities were measured in bone cytoplasmic extract. The results show that: (a) When compared to the value found in half calvaria incubated in a control medium, the bone acid and alkaline phosphatase content is significantly higher in paired halves incubated with Al (3×10−11M to 1.5×10−6M) as well as with bPTH, 1,25-, or 24,25(OH)2D3 and sharply decreased with higher Al concentrations (6×10−6M). (b) The Al effect on phosphatase activities is modified in a free phosphate or a free calcium medium. (c) The presence of Al at 1.5×10−6M or 6×10−6M significantly decreases the bPTH or 1,25(OH)2D3-induced stimulation of bone phosphatase activities. (d) A similar interaction could not be found between Al and 24,25-(OH)2D3.
TL;DR: It was found that the principal chromatographic fractions showed similarities of both size and amino acid composition among species and it is suggested that these characteristic proteins arise during the degradation of the matrix which accompanied mineralization.
Abstract: The matrix proteins of the developing dental enamel of five mammalian species were isolated and subjected to chromatographic, electrophoretic, and amino acid analyses. It was found that the principal chromatographic fractions showed similarities of both size and amino acid composition among species. The major amelogenin protein of the cow, hamster, human, and sheep was of about 30,000 daltons and of the pig enamel matrix about 20,000 daltons. In each species a higher molecular weight fraction, greater than 40,000 daltons, was detected. In the lower molecular weight range an amelogenin polypeptide enriched in leucine, a fraction rich in tyrosine, and a fraction of intermediate size (Bovine matrix "Component-14") were identified in each case. It is suggested that these characteristic proteins arise during the degradation of the matrix which accompanied mineralization.
TL;DR: The results indicate that the mobilization of calcium from bone to maintain pregnancy and lactation occurs by a mechanism independent of vitamin D.
Abstract: The effect of vitamin D on bone changes during the reproductive cycle in female rats has been investigated. One group of female rats was maintained on a vitamin D-deficient diet and another group of a vitamin D-replete diet from weaning. Both groups were mated with normal males and changes in their bones were determined histomorphometrically during pregnancy, lactation, and after weaning. All vitamin D-deficient rats had bone changes typical of rickets. Pregnancy caused significant reductions in mineralized tissue of trabecular and cortical bone in the vitamin D-deficient rats. Lactation caused further significant reductions in mineralized tissues of cortical and trabecular bone in both the vitamin D-deficient and vitamin D-replete animals, with the greatest changes seen at weaning. Some restoration of mineralized tissues occurred following weaning. There was an increase in tetracycline-labeled bone surface in the vitamin D-replete animals during lactation, likely due to an increase in bone formation rates. In the vitamin D-deficient animals during lactation, there was a decrease in tetracycline-labeled bone surface, likely due to severely depressed bone mineralization. These results indicate that the mobilization of calcium from bone to maintain pregnancy and lactation occurs by a mechanism independent of vitamin D.
TL;DR: Observations lend further support to the hypothesis that the ruffle-ended cells are very active in mineralizing enamel and that the smooth- ended cells are in a passive, restorative phase.
Abstract: Rats were injected with45Ca and horseradish peroxidase to determine the patterns of45Ca incorporation into incisor enamel and the morphological types of the overlying maturation ameloblasts.45Ca autoradiography showed no differences in the patterns of incorporation into enamel between routinely embedded and freeze-dried specimens. Enamel overlaid by ruffle-ended ameloblasts was much more heavily labeled while that overlaid by smooth-ended ameloblasts showed only moderate labeling. The observations lend further support to the hypothesis that the ruffle-ended cells are very active in mineralizing enamel and that the smooth-ended cells are in a passive, restorative phase.
TL;DR: It is concluded that the factor(s) responsible for osteoclast differentiation resides specifically within bone matrix and is intimately associated with the mineral phase and can occur ectopically, outside of the developing vertebrate body.
Abstract: The present report describes the first in a series of studies designed to identify the factor or factors responsible for eliciting osteoclast differentiation. Particles of mineralized and demineralized bone, hydroxyapatite (HA), and eggshell were grafted onto the chorioallantoic membranes (CAMs) of chick embryos. After 3 or 6 days, portions of CAMs with associated grafts were harvested, processed for light and electron microscopy, and examined for the presence of multinucleated giant cells with the morphological characteristics of osteoclasts. Light microscopic examination revealed that, within only 3 days, many particles of mineralized materials had become surrounded or engulfed by multinucleated giant cells. Ultrastructurally, all such cells possessed a vacuolated and mitochondriaenriched cytoplasm, but they differed in the nature of the contacts formed at the cell-particle interface. With eggshell, the cells developed filopodia but lacked clear zones and ruffled membranes. With HA, clear zones were evident but cytoplasmic extensions and membrane ruffling were absent. Implants of mineralized bone, however, elicited the formation of giant cells with prominent clear zones and ruffling of the plasma membrane like that observed in bonafide osteoclasts. In contrast, grafts of demineralized bone did not evoke giant cell formation but rather recruited two cell types morphologically akin to either fibroblasts or macrophages. We conclude that the factor(s) responsible for osteoclast differentiation resides specifically within bone matrix and is intimately associated with the mineral phase. Further, in response to such a factor(s), osteoclast differentiation can occur ectopically, outside of the developing vertebrate body.
TL;DR: It is suggested that the tendency to hypercalcemia in ovarian-insufficient females given 1-hydroxylated vitamin D compounds may be related to a diminished metabolism of 1,25-dihydroxyvitamin D3.
Abstract: The effect of ovarian insufficiency on calcium metabolism has been thought to involve an increased bone resorptive effect of parathyroid hormone and possible impaired synthesis of 1,25-dihydroxyvitamin D3. In the present study a rat model allowing for controlled serum levels of parathyroid hormone and 1,25-dihydroxyvitamin D3 was used. Oophorectomy in this species is associated with increased serum levels of 1,25-dihydroxyvitamin D3 and decreased bone mass. Although thyroparathyroidectomy increased bone mass, an increased sensitivity of bone to parathyroid hormone in oophorectomized rats was not observed. Thus the development of the osteopenia did not seem to be related to increased parathyroid hormone sensitivity or to reduced levels of 1,25-dihydroxyvitamin D3. Exogenous 1,25-dihydroxyvitamin D3 increased bone mass in oophorectomized as well as intact rats. Intestinal calcium transport was increased by moderate doses of 1,25-dihydroxyvitamin D3. Intestinal calcium transport was also reduced by thyroparathyroidectomy and increased by the administration of parathyroid extract. A tendency for increased accumulation of 1,25-dihydroxyvitamin D3 in blood in oophorectomized rats has been noted. It is suggested that the tendency to hypercalcemia in ovarian-insufficient females given 1-hydroxylated vitamin D compounds may be related to a diminished metabolism of 1,25-dihydroxyvitamin D3.
TL;DR: Measurements of serum vitamin D metabolites, calcium and phosphorus as well as measurements of the equilibrium dissociation constant for duodenal 1,25(OH)2D3 receptor in 15-, 18, 19, and 20-day chick embryos in comparison to that in 1- and 118-day-old chicks and to vitamin D-deficient chicks showed that levels rise from 15 and 18 to days 19 and 20 of embryonic development while serum phosphate levels are stable.
Abstract: This study presents measurements of serum vitamin D metabolites, calcium and phosphorus as well as measurements of the equilibrium dissociation constant for duodenal 1,25(OH)2D3 receptor in 15-, 18-, 19-, and 20-day chick embryos in comparison to that in 1- and 118-day-old chicks and to vitamin D-deficient chicks. The present results showed that: (a) serum 1,25(OH)2D and 24,25(OH)2D levels rise from 15 and 18 to days 19 and 20 of embryonic development while serum phosphate levels are stable; (b) serum calcium levels rise at hatching to adult levels; (c) the duodenal 1,25(OH)2D3 receptor is detectable in 15-day-old embryo and has a Kd similar to that of 118-day-old vitamin D-replete chicks; and (d) the activity of 1,25(OH)2D3 receptor in chick duodenal cytosol is maximal at hatching.
Journal Article•
TL;DR: A significant difference was found between the concentrations of Gla and Ser(P) with respect to the calcium content of the tissue, which may reflect the fact that the two classes of proteins have different biological functions and are concentrated in the tissue by different physical chemical mechanisms.
Abstract: The contents of two classes of calcium binding proteins of bone matrix, osteocalcin and phosphoprotein, were measured as a function of age of the animal and of the mineral content of chicken bone powder, fractionated according to particle density by differential centrifugation in bromoform-toluene solutions. Whole bone and separated fractions were analyzed for gamma-carboxyglutamic acid (Gla) and for O-phosphoserine [Ser(P)] as indications of their osteocalcin and phosphoprotein contents, respectively. Except for the Ser(P)/collagen ratio of 2-year-old chicken bone, the concentrations of Gla and Ser(P) in the organic protein matrix of the bone increased with increasing mineral content of the tissue, calculated on the basis of the total protein content, the noncollagenous protein content, and the collagen content of the tissue sample. However, a significant difference was found between the concentrations of Gla and Ser(P) with respect to the calcium content of the tissue. The Gla concentration remained relatively constant, whereas the Ser(P) concentration decreased markedly with increasing calcium content. This may reflect the fact that the two classes of proteins have different biological functions and are concentrated in the tissue by different physical chemical mechanisms.
TL;DR: The fall in serum calcium and phosphate was associated with a rise in the serum concentration of PTH and 1,25(OH)2D3, coupled with increases in net calcium absorption and calcium balance, showing that this vitamin D metabolite contributes as a hormone to calcium homeostasis.
Abstract: In 10 patients with Paget's disease of bone and 2 patients with osteoporosis, we studied the effects of hypocalcemia and hypophosphatemia induced by disodium-(3-amino-1-hydroxypropylidene)-1,-bisphosphonate (APD) treatment on the serum concentration of PTH and 1,25-dihydroxyvitamin D [1,25(OH)2D3] and on calcium absorption and balance. The fall in serum calcium and phosphate was associated with a rise in the serum concentration of PTH and 1,25(OH)2D3, coupled with increases in net calcium absorption and calcium balance. The concentration of 1,25(OH)2D3 was significantly related (P<0.001) to the serum calcium (r=0.66), the serum phosphate (r=0.78), and the serum PTH (r=0.71), confirming the interrelated control of these parameters on 1,25(OH)2D3 production. Moreover, the rise in 1,25(OH)2D3 caused an appropriate rise in calcium absorption (r=0.74) and calcium balance (r=0.86), showing that this vitamin D metabolite contributes as a hormone to calcium homeostasis.