Effects of propane-2, 2-diphosphonate (PDP) on matrix-induced ectopic bone formation in comparison to ethane-1-hydroxy-1, 1-diphosphonate (EHDP).

The most prominent effect of propane-2, 2-diphosphonate (PDP) and ethane-1-hydroxy-1, 1-diphosphonate (EHDP) on matrix-induced ectopic bone in the rat was a dose-dependent inhibition of osteogenesis in the early phases of development. The delay was seen as a consequence of osteoprogenitor cell inhibition. Additionally, later phases of bone maturation were disturbed by interference with the mineralization and remodeling processes. However, direct effects on the calcium phosphates of bone are only of minor additional value, which remains of lesser importance in comparison to the cellular impairment. After withdrawal of diphosphonates, the effects were nearly completely remitted. Neither PDP nor EHDP, even given in high doses, resulted in a lasting reduction in ectopic mass. The remission may be referred to the recovery of cell activities, whereas the mineral impregnation of osteoidosis was, if at all, of little importance. For treatment of ectopic osteogenesis PDP proved inefficient.

Immunohistochemical observations on bone morphogenetic protein in normal and abnormal conditions.

Localization of bone morphogenetic protein (BMP) in human tissues and cells is important for investigating the mechanism of bone induction. A stable cell line secreting monoclonal antibody against bovine BMP (bBMP-McAb) was obtained by the hybridoma technique. The result of immunohistochemical staining (ABC method) showed that BMP is distributed along collagen fibers of normal bone, in periosteal cells, and in mesenchymal cells of marrow stroma. Little BMP can be found in bone cells of lamellar bone or in calcified bone matrix. BMP may be abundant in human tooth anlagen such as predentin, cells of the outer and inner enamel epithelium, and cells of dental sac generating bone. BMP is found in the cytoplasm of tumor cells of osteosarcoma and chondrosarcoma. Immunohistochemical staining showed that BMP plays a role in bone fracture healing. The ability of BMP-McAb to detect BMP and to inhibit the generation of new bone also makes it potentially useful in diagnosing, treating, and providing a prognosis for osteosarcoma and other bone diseases.

The radiotoxicology of Radithor. Analysis of an early case of iatrogenic poisoning by a radioactive patent medicine.

Radithor was a radioactive patent medicine that was touted as a metabolic stimulant and aphrodisiac. We have obtained several original samples of Radithor and have used these historical specimens and a computer-based calculation model to perform a retrospective analysis of a famous case of Radithor-related radium poisoning. Our data suggest that the victim's cumulative skeletal radiation dose may have exceeded 350 Sv by the time he died. This figure far exceeds most current estimates of what radiation exposure level would constitute a rapidly lethal dose if given acutely. The physiological response to longterm internal radiation exposure and the highly localized nature of alpha particle irradiation may require the development of new models for the assessment of risk in cases of internal alpha particle irradiation.

Aluminium deposition in bone after contamination of drinking water supply.

Two healthy individuals who drank water accidentally contaminated at source with aluminium sulphate solution were investigated 6-7 months later. Bone biopsy specimens showed discrete lines of positive staining for aluminium, the distribution being compatible with acute exposure some months previously. These findings show that under certain conditions normal individuals can absorb aluminium via the gut, and that such aluminium can be deposited in bone.

A critical crosslink region in human-bone-derived collagen type I. Specific cleavage site at residue Leu95.

Collagen was extracted from human adult bone by limited pepsin digestion and collagen types were purified by consecutive salt precipitation first under neutral and then under acid conditions. In SDS/PAGE, all collagen type I preparations showed a protein band [alpha 1s(I)] migrating between alpha 1(I) and alpha 2(I) as well as a band [alpha 2s(I)] migrating in front of alpha 2(I). The collagenous nature of the pepsin-stable alpha 1s(I) protein was clearly demonstrated by digestion with human-leucocyte-derived collagenase, immunoblotting with antibodies against collagen type I and amino acid analysis. Partial amino acid sequencing of alpha 1(I) and alpha 1s(I) identified alpha 1s(I) as a shortened alpha 1(I) chain due to a specific cleavage site between residues Leu95 and Asp96 which is in close vicinity to the hydroxylysine-derived crosslink at position 87. In circular dichroism, the proportion of thermally labile collagen molecules was proportional to the amount of shortened alpha 1(I) and alpha 2(I) chains, respectively. The melting temperature was found to be 36 +/- 0.5 degrees C as judged from circular dichroism and susceptibility to proteolysis. Our data provide clear evidence that a shortened alpha 1-derived collagen chain can be extracted from human adult bone whereas it is hardly found in human skin. The unique cleavage site might provide important information about the collagen I molecule embedded in the calcified matrix of human bone.

Regional patterns of bone loss and altered bone remodeling in response to calcium deprivation in laboratory rabbits.

This study explores the effects of a calcium-deficient diet on patterns of bone remodeling, and examines regional differences in the amount of bone lost. Skeletally mature female rabbits (n = 6) were fed a calcium-deficient diet (0.10% Ca2+ and 0.50% P) for 14 weeks. A separate group of rabbits (n = 4) were fed a maintenance diet (1.2% Ca2+ and 0.45% P). Bone mineral content, serum calcium, and serum phosphorus were measured each week during the experimental period. Following sacrifice, the L3 vertebra, femoral head, proximal tibial metaphysis, and tibial midshaft were analyzed histomorphometrically. Rabbits had 20% less vertebral bone after only 14 weeks of a calcium-deficient diet. As in human postmenopausal osteoporosis, bone loss in calcium-deficient rabbits occurs in the trabecular bone of the lumbar spine before that in the trabecular bone of the lower extremity. Calcium-deficient diets alone do not lead to increased osteoid volume or thickness. Because bone loss is relatively rapid and because the pattern of loss is similar in some respects to that found in humans, adult rabbits may provide an attractive model of calcium deficiency osteoporosis in a skeletally mature mammal in which remodeling is predominant over modeling.

Characterization of interfaces in human calcified tissue.

Thermostimulated current spectroscopy has been applied to the investigation of molecular mobility in human calcified tissue. A comparative study of extracts at various stages of demineralization is presented. The response of an organic-mineral complex interphase has been identified.

[Morphological aspects of the mineral phase of the bone]. / Morfologicheskaia kharakteristika mineral'noi fazy kosti.

By means of light, scanning and transmission electron microscopy structure of the mineral phase of human compact bone have been studied. Mechanical properties of deorganified bone samples have been determined. High compressive strength may be in connection with interactions of plate-like mineral particles at the level of hydrate shell of the bone mineral crystallites.

[Bone mineral content and quantitative computerized tomography]. / Contenuto minerale osseo e tomografia computerizzata quantitativa.

Many methods are used to determine bone mineral content (BMC). Quantitative computed tomography (QCT) appears to be the most reliable method also because it allows the trabecular and the cortical bone to be measured separately. QCT is usually performed on the first four lumbar vertebral bodies. BMC is expressed in mg/ml and a mean value is calculated. Three hundred and fifteen subjects were studied (281 women and 34 men). The patients affected with Paget's disease or malignancies, with or without bone metastases, were not included in this study. The measurements were performed by means of a General Electric 9800 tomograph with software and calibration phantom (QCT-Bone program by Image Analysis). Fractured vertebrae were not included when calculating the mean value since an increased density is caused by fracture. The BMC of any studied vertebral body is considered in comparison with the BMC of the other lumbar vertebrae of the same subject. When the BMC of a given vertebral body exceeds the others by 25 mg/ml or more, nodules and/or stripes are observed during multiple-slice scanning of the bone. Lytic areas or angiomas are observed when the BMC of a given vertebral body is -25 mg/ml or higher. In calculating the mean vertebral BMC, vertebrae with both +25 and -25 must be excluded. In this way the method reliability increases.

[Environmental history as an anthropologic topic. Contribution to "chemical anthropology"]. / Umweltgeschichte als anthropologische Fragestellung. Beitrag einer "Chemical Anthropology".

Human population history is firmly connected with temporal and regional changes of the environment. Whether natural or anthropogene, alteration of environmental features lead to changes of human life-style and to the development of adaptive strategies. The demand of resources for his subsistence has led man to diverse impacts on his environment since ever. Thus, environmental history is a scientific topic for anthropologists. The research potential of trace element studies of excavated human skeletons for the reconstruction of natural and socio-cultural environments as well as for distribution patterns of hazardous substances is outlined for the European Middle Ages. The scientific value of unravelling past man/environment-interrelationships for both the historical and applied sciences and the place of any "chemical anthropology" within this context is discussed.

Effects of low molecular weight heparin and unfragmented heparin on induction of osteoporosis in rats.

A comparison between the effect of low molecular weight heparin (LMWH) and unfragmented heparin (UH) on induction of osteoporosis was made in 60 rats treated with either UH (2 IU/g bw), LMWH in 2 doses (2 XaI U/g or 0.4 XaI U/g) or placebo (saline) for 34 days. Studied variables were: bone mineral mass in femora; fragility of humera; zinc and calcium levels in serum and bone ash and albumin in plasma. A significant reduction in bone mineral mass was found in all heparin-treated rats. There was no difference between UH and LMWH in this respect. The effect was dose-dependent in LMWH-treated animals. The zinc contents in bone ash were decreased in all heparin-treated rats as compared with controls. No recognizable pattern was seen in alterations of zinc or calcium in serum. The fragility of the humera, tested as breaking strength did not differ between treatment groups and controls. In conclusion, if dosed according to similar factor Xa inhibitory activities, LMWH induces osteoporosis to the same extent as UH and in a dose-dependent manner. The zinc content in bone ash was decreased after heparin treatment, irrespective of type of heparin given.

A biophysical study of posttraumatic ectopic ossification. A case report.

Sites of ectopic ossification (EO) due to sports injury in a long-distance runner were biopsied and analyzed. The mineral content was compared to that of normal adult bone and to paraosteoarthropathy (POA) using biophysical methods. The degree of mineralization of bone tissue (DMBT) was employed as an evaluation of maturity of calcified tissue. Fluoride and carbonate content were determined. The crystal lattice dimensions expressed by the a and c parameters, crystal size, and/or lattice perfection were obtained with x-ray diffraction. All values were near those found in nonpathologic, newly deposited human compact bone. DMBT, crystal size, and/or lattice perfection were near those observed in a 24- to 30-month-old POA. The data suggest that posttraumatic EO represents otherwise normal young bone.

Limitations of ZAF correction factors in the determination of calcium/phosphorus ratios: important forensic science considerations relevant to the analysis of bone fragments using scanning electron microscopy and energy-dispersive x-ray microanalysis.

A series of calcium phosphate standards having calcium/phosphorus (Ca/P) molar ratios of 0.50, 1.00, 1.50, and 1.67, respectively, was prepared for bulk specimen analysis using scanning electron microscopy (SEM) and energy-dispersive X-ray microanalysis (EDXA). The standards were mounted on carbon planchettes as either pure crystals or crystals embedded in epoxy resin. Ten different samples of each embedded and non-embedded standard were analyzed in a JEOL 100 CX electron microscope interfaced with a Kevex 8000 EDXA system using a lithium-drifted silicon detector and a multichannel analyzer. The Ca/P ratios were determined by calculating both net peak intensities without matrix corrections and atomic kappa-ratios using the MAGIC V computer program with ZAF correction factors for quantitative analysis. There was such extensive absorption of phosphorus X-rays in standards embedded in an epoxy matrix that the observed Ca/P ratios were statistically compatible with four different standards ranging in theoretical Ca/P ratios from 1.0 to 1.67. Although the non-embedded crystals showed a greater separation in the Ca/P ratios, both methods of preparation produced serious flaws in analysis. Direct application of the discovery of this caveat to the identification of suspected bone fragments for forensic science purposes is discussed.

A colorimetric assay specific for gamma-carboxyglutamic acid-containing proteins: its utility in protein purification procedures.

A colorimetric method for the detection of gamma-carboxyglutamic acid (Gla)-containing proteins after reaction with 4-diazobenzenesulfonic acid is presented. Proteins can be visualized after electroblotting from polyacrylamide gels onto membrane supports, after dot-blotting onto membranes, or in solution as a red colored product with an absorbance maximum at 530 nm. The method is specific since other proteins without gamma-carboxyglutamic acid do not form a red color. The presence of other proteins does not inhibit or affect color production by gamma-carboxyglutamic acid-containing proteins. Application of the method for staining a Western blot of a crude extract of bone resulted in staining of only the gamma-carboxyglutamic acid-containing proteins. The usefulness of the method was verified when a second gamma-carboxyglutamic acid-containing protein, prothrombin, also resulted in red color production. A linear color response is seen up to 17 microM for the gamma-carboxyglutamic acid-containing protein bone Gla protein and up to 27 microM for the amino acid. The detection limit is down to 1 microgram of bone Gla protein or 0.17 nmol of the protein on electroblots or dot blots. The simplicity of the method allows rapid screening for gamma-carboxyglutamic acid-containing proteins or allows monitoring of purifications of these proteins in chromatographic or electrophoretic separations.

Concentration of ciprofloxacin in bone tissue after single parenteral administration to patients older than 70 years.

The concentrations of ciprofloxacin produced in bone, cartilage and menisci after a single administration of 200 mg were determined at different intervals in a group of patients with an average age of 80 years. Concentrations of 0.11 to 0.94 mg/kg bone tissue were measured after 0.5 to 5 hours. In the cartilage a concentration of active substance was measureable only once (4.18 mg/kg). In the presence of marked circulatory disorders the active substance concentrations reached in the bone were above those found in the seriously damaged muscle. Although the concentrations reached in the bone are effective, no risk should be taken in osteomyelitis. Ciprofloxacin should therefore be used at high dosage and possibly be combined with another substance. Given for therapeutic purposes, a single dose of ciprofloxacin is naturally not effective enough, and given for prophylactic purposes, not safe enough to prevent a post-traumatic osteitis.

Parathyroid glands in chronic aluminum intoxication.

In chronic renal failure, aluminum overload may influence parathyroid function. In a study of possible aluminum-induced parathyroid abnormalities, parathyroid glands from nine parathyroidectomized patients on hemodialysis were examined by light and electron microscopy and by X-ray microanalysis. Aluminum overload was assessed by the presence of stainable aluminum (aluminum surface, 23.3% +/- 11% of total surface) in bone biopsy specimens. The mean plasma aluminum concentration was 7.7 +/- 1.9 mumol/L. All patients but one had elevated plasma concentrations of immunoreactive parathyroid hormone as well as osteitis fibrosa. The aluminum concentrations in bone and parathyroid gland from these patients were significantly higher than those in tissue from patients on hemodialysis without stainable bone aluminum. Abundant aluminum deposits were present in parathyroid chief cell cytoplasm in lipoid bodies, lipofuscin granules, and mitochondria. These cells exhibited features of active hormonal synthesis and contained numerous secretory granules. The data show that in the parathyroid glands of these aluminum-intoxicated patients the presence of aluminum deposits neither induced cellular damage or chief cell necrosis nor interfered with the production of parathyroid hormone.

Serum bone Gla protein as an indicator of parathyroidectomy in patients with secondary hyperparathyroidism.

Bone Gla protein (BGP) is a vitamin K-dependent protein which is a marker of bone turnover. To determine whether serum BGP is a useful indicator for parathyroidectomy in patients with secondary hyperparathyroidism, we measured serum BGP levels. Thirty-seven patients with secondary hyperparathyroidism who were followed up for more than 1 year after parathyroidectomy were studied. All patients underwent total parathyroidectomy and autotransplantation. Controls were 46 patients who were treated by chronic hemodialysis for more than 3 years. Serum BGP levels (normal: less than 6.5 ng/ml) were markedly increased in 37 patients with parathyroidectomy, ranging from 4.2 ng/ml to 645 ng/ml, with a mean value of 278.8 +/- 159.8 ng/ml (mean +/- standard deviation) versus 65.0 +/- 85.2 ng/ml in the 46 controls (p less than 0.001). Patients with a high BGP level had severe bone and joint pain. Serum BGP in patients with parathyroidectomy was significantly correlated with serum alkaline phosphatase and mPTH (p less than 0.001 for both). The total weight of resected parathyroid tumors was 2,152 +/- 1,368 mg, and tumor weights ranged from 200 mg to 5,600 mg. There was a highly significant correlation between BGP level and tumor weight (r = 0.656, p less than 0.001). The 2 patients who showed BGP levels below 10 ng/ml had tumor weights of only 470 mg and 240 mg, respectively, and revealed no improvement of pain postoperatively, although their mPTH levels were increased. These results suggested that BGP measurement is a sensitive method for detecting increased bone turnover and is possibly useful as an indicator for parathyroidectomy in patients with secondary hyperparathyroidism.

Prospective evaluation of aluminum loading from formula in infants with uremia.

Assessment of potential aluminum loading from regular ingestion of a commercial infant formula (Similac PM 60/40), as the only milk substitute, was made in 14 infants aged 9.6 +/- 4.4 months who were also undergoing continuous cycling peritoneal dialysis. Tissue aluminum accumulation was assessed by serial measurements of plasma aluminum levels, from the increment in plasma aluminum after a standardized deferoxamine infusion, and from quantitative histomorphometry of bone and measurements of total bone aluminum content. Initial mean plasma aluminum levels were 0.61 +/- 0.32 mumol/L, (normal 0.30 +/- 0.04 mumol/L), and values were less than 0.92 mumol/L during the follow-up of 20 +/- 8 months. Plasma aluminum levels increased from 0.59 +/- 0.18 to 0.88 +/- 0.22 mumol/L after a single dose of deferoxamine. The histochemical stain for bone aluminum was negative for all patients, and the bone aluminum content was 0.27 +/- 0.22 mmol/kg dry weight (normal 0.08 +/- 0.04 mmol/kg dry weight). Thus the infant formula Similac PM 60/40 can be safely used in infants with chronic renal failure.

Effect of aluminum on performance and mineral metabolism in young chicks and laying hens.

This study was performed to determine the long-term effects of dietary aluminum on egg production and reproductive parameters in the mature laying hen and on growth rate and feed efficiency in young chicks. The diets used in these studies were adequate in phosphorus and other essential nutrients. Aluminum added to constitute 0.30% of the diet severely depressed growth and reduced feed efficiency, bone ash and plasma phosphorus in male Ross x Leghorn chicks. At the same time, 0.15% added aluminum mildly depressed growth, feed efficiency and bone ash but had no effect on plasma phosphorus levels. The reduction in bone ash was relatively mild, and no clinical signs of rickets were observed. In laying hens, diets containing 0.15% added aluminum did not affect egg production, but 0.30% added aluminum reduced production significantly. Long-term exposure to aluminum increased percent shell in both groups receiving aluminum, whereas egg weight remained similar to that in controls. There were no changes in hatchability or bone ash associated with dietary aluminum. Although dietary aluminum influenced bone aluminum content, egg aluminum content was not affected. These studies indicate that dietary aluminum interferes with systems in addition to phosphorus metabolism.