Showing papers in "Calcified Tissue International in 1975"

The absorption and scattering of light in bovine and human dental enamel

Abstract: The reflectance and transmission of thin slabs of dental enamel has been measured at all wavelengths between 220 and 700 nm by means of an integrating sphere. From the results the true scattering and absorption coefficients have been computed. The theoretical model used is an extended two-flux model, which is presented and discussed. The absorption spectrum of the dissolved organic component of enamel was also determined. An absorption peak at 270 nm is common to all the spectra. This peak in the bovine enamel spectrum is about three times as high as in the spectrum of human enamel. The peak of the dissolved material is about as high as the peak of the corresponding enamel. Hence it is concluded that the organic component, presumably aromatic amino acids, is responsible for most or all of the observed optical absorption.

Osteogenesis by chondrocytes from growth cartilage of rat rib.

Abstract: Chondrocytes were isolated from growth cartilage and resting cartilage of rat rib and cultivatedin vitro. The cultivated chondrocytes were placed in Millipore diffusion chambers, which were then implanted into the abdominal cavities of rats for several weeks and prepared for histological analysis. The results indicate that growth cartilage cells have a remarkable osteogenic potential, even after cultivationin vitro, whereas resting cartilage cells show no osteogenic activity. However, growth cartilage cells alone do not form new bone but require the participation of certain host cells to initiate osteogenic differentiation.

Effect of carbonate and biological macromolecules on formation and properties of hydroxyapatite.

Abstract: Amorphous calcium phosphate (ACP) was transformed at 25 degrees to hydroxyapatite (HA) in horse and bovine serum; solutions of serum-protein fractions in tris-HC1 buffer (pH 7.4), and pH 7.4 buffers containing from 0.1 to 10 times physiological CO3(2-) concentration. The ACP-to-HA transformation was slower in whole serum and serum fractions than in control buffer solution. The observed adsorption of serum proteins on ACP and HA probably inhibits both the dissolution of the ACP particles and the growth of HA crystals. After 72 h all transformations were complete as determined by X-ray diffraction. The HA crystal dimensions decreased with increasing C03(2-) but the shape, as shown by X-ray linewidths, was relatively constant up to about 4% CO3(2-). At 15% CO3(2-) the crystals were more equiaxial and less needle-like in habit. The radial distribution function (RDF) of HA with 3.7% CO3(2-) is less well resolved than the RDF of HA with ambient CO3(2-) (1.1%). The peaks are less sharp and their amplitude falls more rapidly with increasing atomic separation than for low CO3(2-)-HA. These effects show that CO3(2-) decreases the regularity of the atomic arrangement when incorporated in HA. The rapid decrease, with increasing CO3(2-) content, of the IR splitting of the P-O bending mode of CO3(2-)-HA is attributed to reduced crystal size and possibly to a perturbation of the crystal field due to CO3(2-)-induced lattice distortion. Finally, for bone mineral, it is probable that the poor resolution of the X-ray and IR patterns is due, in large part, to small crystal size and internal disorder caused by CO3(2-).

Variations in the composition of developing rat incisor enamel.

Abstract: The developing enamel of rat incisors was dissected into a series of samples extending from the newly-formed partially-mineralised matrix to the mature enamel. Chemical analysis showed that, on a dry weight basis, the tissue achieved the composition of mature enamel well before the completion of mineral uptake. The enamel at this stage was porous and relatively soft. As more mineral was acquired, its hardness increased. Throughout the developing region, the Ca:P ratio remained fairly constant, but the CO2:P and Mg:P ratios both decreased due, apparently, to dilution by an influx of relatively CO2- and Mg-free mineral.

Hpo-4(2-) content in enamel and artificial carious lesions

Abstract: The HPO 4 2− and CO 3 2− content was determined in sound enamel and in material collected from artificially produced carious lesions. The method described shows that the HPO 4 2− content can be determined from the 875 cm−1 infrared absorption band if correction for the CO 3 2− contribution are made. The I. R. spectra show that the HPO 4 2− content in sound human or bovine enamel is about 5% by weight. In artificially produced carious lesions (pH=4.0), the HPO 4 2− content is in the order of 15 wt%. Most likely, the HPO 4 2− ions in sound and carious enamel have a different environment.

Ethane-1-hydroxy-1, 1-diphosphonate (EHDP) effects on growth and modeling of the rat tibia

Abstract: One hundred gram male Sprague-Dawley rats were divided into groups which were injected daily for 10 or 30 days with vehicle (control group), 02, 04, 20, or 100 mg ethane-1-hydroxy-1,1-diphosphonate (EHDP)/kg/day The proximal tibial metaphysis and epiphysis were assayed for changes in percentage of hard tissue and bone formation factors Knowing these, information about hard tissue resorption was deduced

Extraction of a calcium-phospholipid-phosphate complex from bone.

Abstract: A calcium-phospholipid-phosphate complex with a constant 1:1 calcium to total phosphate molar ratio is shown to exist in rabbit and calf bone. This complex, which may be involved in the transport and deposition of bone mineral, appears to constitute a significantly greater proportion of the lipids of younger bone than of more mature bone. The comples was isolated by a modified Folch extraction employing ultrasonic disruption of cellular material. Evidence is presented to show that the complex is a natural constituent of bone rather than one created artifactually during extraction.

Quantitative histological evaluation of early fracture healing of cortical bones immobilized by stainless steel and composite plates.

Abstract: Internal fixation devices of less bending stiffness than conventional plates made of stainless steel or vitallium were compared with conventional plates in a study of fracture healing. The material for this investigation was a fine graphite fiber reinforced methyl methacrylate resin composite with a modulus of elasticity approximately ten times less than that of stainless steel. Osteotomies were performed on canine radii. Internal fixation was accomplished by means of a composite plate on the left side, and a stainless steel plate on the right. Clinical assessment, as well as biomechanical and quantitative histological techniques, were used to compare osteotomy healing of the two sides. At four months, all osteotomies had healed and the bioengineering tests showed radii from the two sides had equivalent strength. However, significantly less cortical porosity was found in the side with the composite plate (6.8%), as compared to that of the stainless steel plated side (14%). These results suggest that a less stiff fixation plate may have some advantage in the treatment of long bone fracture if there is no implant failure, and if union rates are equivalent.

Aging effects on osteon remodeling.

Abstract: Analysis of partial cross sections of 101 human tibiae indicated that osteon remodeling in the outer cortex is affected by age. The frequency of resorption spaces remained constant throughout life suggesting no loss of osteoclast function with age. However, the frequency of both forming osteons and osteons which were structurally complete but not completely mineralized increased with age. This suggests that protein matrix synthesis by osteoblasts slows with age and that initial mineralization, possibly mediated by osteoblasts, and final mineralization, possibly mediated by osteocytes, becomes increasingly deficient with increasing age. The frequency of osteons which have dense (sclerotic) inner lamellae decreases with age. This supports a hypothesis that such lamellae are functional, perhaps representing a specialized, labile, mineral phase and that osteons having this feature become less frequent as part of the general degenerative changes associated with aging.

Effect of phospholipids on the transformation of amorphous calcium phosphate to hydroxyapatitein vitro

Abstract: The conversion of amorphous calcium phosphate (ACP) to crystalline hydroxyapatite (HA) was studiedin vitro in the presence or absence of phosphatidyl serine (PS) and other phospholipids. ACP transformation and HA crystal growth were monitored by electron microscopy, selected-area electron diffraction and X-ray diffraction, and by measuring supernatant calcium and phosphate. PS, and other acidic phospholipids, had a significant stabilizing effect on ACP. With preformed ACP at ratios of only 1 lipid molecule per 30–50 Ca atoms, PS markedly delayed HA crystal formation. When PS was present during ACP precipitation, inhibition of conversion to HA was less pronounced, but crystal habit and aggregation were greatly altered resulting in stacks of thin, membrane-like sheets approximately 38–42 A thick. PS appeared to be most effective in blocking ACP to HA conversion when oriented primarily on the surface; it most affected subsequent crystal formation when distributed throughout the amorphous precursor. Phospholipids possessing anionic, and hence Ca-binding properties, were effective in stabilizing ACP; neutral zwitterion lipids, which have amphipathic properties but do not bind Ca, were not. In view of the presence of anionic lipids in matrix vesicles and their association with early mineral deposits, the current findings add further evidence that lipids may play a role in the control of normal mineralizationin vivo.

Precipitation of calcium phosphates from electrolyte solutions

Abstract: Precipitation and precipitate transformation in the system sodium phosphate (pre-adjusted to pH 7.4)—calcium chloride (25°) was studied by means of radiometric analysis using45Ca and32P as tracers. Changes in the pH and the total concentrations of calcium and phosphate were followed during solid phase formation and the data were used to calculate composition changes of the precipitates and their supernatants. In all investigated systems two-step precipitation was observed, the precursor being more basic than the secondary precipitate. The composition of the latter was mostly within the range of the composition of octacalcium phosphate. The course of further chemical changes was dependent on the pH established during secondary precipitation. The heterogeneous exchange of the radionuclides between the solid phase and their supernatant solutions was also followed as a function of time. The results indicate that recrystallization through the mother liquid accompanied by composition changes is the dominant mechanism of equilibration of the solid phases.

A quantitative method for the application of compressive forces to bone in tissue culture.

Abstract: A quantitative method for the application of compressive forces to long bones in culture is described. Using this method it was found that a physiological pressure of 80 g/cm sq. applied to tibiae of 16-day-old chick embryos reduced glucose consumption to 50% of controls. Twenty four hours after the release of pressure glucose utilization again increased, approaching control levels. A pressure of 80 g/cm sq. also stimulated thymidine incorporation into DNA. The same pressure decreased the size of the extracellular fluid pool by 8%, but had no effect on the rate of equilibration of this pool with22Na.

The effect of a low-calcium diet in lactating rats; observations on the rapid development and repair of osteoporosis.

Abstract: Female rats were given a low-calcium diet (0.05%) during the last three weeks of the lactating period, followed by a normal diet (1.03% calcium) during the first three weeks after lactation. The resulting bone loss and its recovery were studied by means of microradiography, tetracycline-uptake, quantitative estimation of the cortical area of cross-sections from the femoral midshaft, and estimation of total body calcium. The cortical area in the femoral midshaft fell to 46% of its original value during depletion, and then rose to 78% during the first three weeks after weaning. Total body calcium fell from 1.12% to 0.60% of body weight and then increased to 0.89%. Removal of bone occurred mainly in the spongiosa and on the endosteal side of the cortex. Subsequently, new bone was laid down on the endosteal side, but also to some extent on the periosteal side of the cortex. The mineral density of this new bone was low. During the recovery phase resorption cavities within the cortex were filled in a concentric manner as in Haversian remodeling. Neither this feature nor the low mineral density of bone are normally present in the rat.

Chondrocyte metabolism as affected by vitamin A.

Abstract: Chondrocytes from 13-day-old embryonic chick sterna were cultured for 6 days in the presence of vitamin A (10 I.U./ml). Vitamin A treated chondrocytes became flattened and stellate within the first 24 hours of culture. After 6 days of culture, the treated cells contained 59% less DNA than the untreated controls. Sulfated glycosaminoglycan synthesis was inhibited 84%, and a greater percentage of GAG was secreted into the medium (90%) than in control cultures (78%). Vitamin A inhibited cell proliferation, and to varying degrees, RNA and protein synthesis, and these effects are dose dependent.

Prostaglandins: Bone resorption stimulating factors released from monkey gingiva

Abstract: Gingival fragments from monkeys have been found to release a factorin vitro into incubation medium which stimulates bone resorption in organ culture. Indomethacin effectively blocks the occurrence of this stimulatory factor in the gingival incubation medium. All of this bone resorptive activity can be accounted for by prostaglandin-like material. The prostaglandins contributing to the bone resorptive activity have been found to be prostaglandins E1 and E2.

Cellular localization of cyclic AMP in periodontal tissues during experimental tooth movement in cats.

Abstract: Using an immune-histochemical method, cyclic AMP was localized in cells of periodontal tissues in orthodontically-treated cats. Sixteen cats were treated for periods ranging from 1 hour to 4 weeks. Fresh, frozen, undecalcified 6 μ sections of the tissues were incubated with rabbit anti-cyclic AMP antibodies, followed by sequential incubations with sheep anti-rabbit IgG, rabbit anti-peroxidase IgG and horseradish peroxidase. In the final step, the peroxidase was demonstrated by the diaminobenzidine (DAB) method. It was found that the number of intensely stained cells increased within a short time in areas in which bone resorption or apposition occurred later. However, differences in the pattern of cellular activation were found to exist between areas of compression and tension. The alveolar osteocytes appeared to be affected to only a slight degree by the applications of mechanical forces. These results indicate: (a) that our immune-histochemical method was useful in following the cellular distribution of cyclic AMP during bone remodeling; and (b) that mechanical forces may affect only a small part of the bone cell population and therefore cannot be regarded as an efficient means to bring about extensive bone remodeling.

Evidence for the presence of secondary calcium phosphate in bone and its stabilization by acid production.

Abstract: Calvaria from three 4-day-old chicks were incubated in a variety of buffers to study the parameters controlling the equilibration of bone with its supporting fluid environment. Strong inferential chemical evidence was obtained for the presence in viable bone of some calcium phosphate phase of high solubility apparently governed by the Ksp of CaHPO4·2H2O. In dead bone, this phase underwent spontaneous conversion to a higher Ca/P ratio. In viable bone the soluble phase appeared to be stabilized by the metabolic production of acid (lactate) as revealed by the effects of selective inhibitors.

Preliminary Trial of Low Doses of Human Parathyroid Hormone 1–34 Peptide in Treatment of Osteoporosis

Abstract: It has recently been suggested that parathyroid hormone (PTH), administered chronically in low doses so as to induce a positive calcium balance, may be effective in the treatment of osteoporosis (11). This hypothesis was based in part on the relative sensitivity of the multiple effects of the parathyroids on calcium metabolism (12) and in part on the known anabolic effects of PTH in low doses on young rat bone (17, 5, 21). In the adult dog, which provides a better model than the rat of adult human calcium metabolism, Parsons and Reit demonstrated that chronic low-dose infusions of bovine PTH (0.025 to 0.1 µg/kg/h) resulted in increased calcium absorption without a concomitant rise in urinary calcium, in spite of moderate hypercalcaemia at the higher dose levels. These results provided the justification for a preliminary trial of low doses of a synthetic amino-terminal fragment of human PTH (hPTH 1–34 (8, 20)) in the treatment of primary osteoporosis.

Effect of weightlessness on mineral metabolism; metabolic studies on Skylab orbital space flights.

Abstract: After eight years of preparation, which took place before and during the Apollo-Moon space-flight series, the National Aeronautics and Space Administration in May 1973 launched into orbit a cylinder 22 feet in diameter and 48 feet in length called the Skylab Orbital Workshop The living quarters and storage area of this cylinder contained nearly all of the life support and scientific experimentation equipment and supplies for three separately launched three-man astronaut crews The first crew remained in orbit for 28 days, the second for 60 days, and the last crew for 84 days

Frozen thin-sections of rapidly forming bone: Bone cell ultrastructure

Abstract: Frozen thin-sections of fresh medullary bone, which had been calcifying for 2 to 4 days, were prepared and examined in the electron microscope. The bone was obtained from male Japanese quail treated with estradiol valerate. Large numbers of 200–800 A electron dense granules, which consisted of 50–75 A subparticles, were seen within mitochondria. A population of electron-dense particles slightly smaller than ribosomes was also observed. Most granules were not present after conventional fixation, dehydration, embedding, sectioning and staining. Other structures which were visible, also on the basis of their intrinsic electron density, were nuclei with regions which resembled condensed chromatin, ribosomes and rough endoplasmic reticulum. By the use of osmium tetroxide vapor staining, inner and outer membranes and cristae of mitochondria were delineated. Other membranous components were not detected. Since very little loss or dislocation of cell components can occur during the preparation of frozen thin-sections, the micrographs obtained may be a more accurate representation of cell ultrastructure.

Effect of long-term administration of fluoride on physico-chemical properties of the rat incisor enamel.

Abstract: Rats were maintained on drinking water containing different amounts of fluoride (0, 9, 23 45, 68, and 113 ppm) for 70 days. Physico-chemical properties of the incisor enamel were examined after fluoride administration, using contact microradiography, histochemistry, and microhardness tests. The tooth enamel formed during high fluoride exposure showed marked hypocalcification. Much of organic substance in the enamel seemed to have been retained. In addition, the microhardness of enamel showed a marked decrease. These changes were most prominent in the outer region of enamel and were proportional to the concentration of fluoride administered. Such changes following fluoride administration indicated inhibition of enamel maturation,i.e., an inhibition of the progressive deposition of minerals and/or an inhibition of organic matrix withdrawal by ameloblasts. Enamel seemed more affected by fluoride than dentine.

The extraction of phosphoproteins from bovine dentin

Abstract: The phosphoprotein obtained by the neutral pH tris buffer extraction of acetic acid demineralized bovine dentin has been compared with the phosphoprotein extracted directly during the neutral pH EDTA demineralization process. The phosphoproteins isolated by DEAE-cellulose chromatography from the neutral pH EDTA demineralization extract are not identical to those isolated by the same procedure from the dentin which had been subjected to acid demineralization. The two demineralization procedures yield phosphoproteins different in amino acid content and in presence of 260 nm UV absorbing moiety. Even after sequential acid demineralization, trisbuffer extraction and EDTA extraction, the residual dentin contains bound phosphoprotein. A peptide fragment containing both collagen and phosphoprotein moieties has been isolated following digestion and cleavage of the insoluble dentin collagen with cyanogen bromide. The acid demineralization process appears to be accompanied by degradation which removes both protein and non-protein components from the phosphoprotein.

The metabolism of labeled parathyroid hormone. VII Attempts to modify hormone deposition.

Abstract: Using a125I-labeled parathyroid hormone with apparently full biological activity, a series of experiments was performed on rats to determine the effects of several physiological parameters on the deposition and metabolism of the hormone in the target tissues, liver, kidney and bone.

Calcium absorption in the elderly.

Abstract: Calcium absorption can be measured in at least three ways — by the balance procedure, by intubation of the small intestine with a double or triple lumen tube and by the oral administration of radioactive calcium.

Morphology and size-distribution of sound and acid-treated enamel crystallites

Abstract: Citric acid dissolves crystallites of enamel by initially etching out approximately hexagonal holes in the core of the crystallites, parallel to their long axis. Such acid-treatment influences the crystallite diameter only slightly since the distribution of the diameters of crystallites with a hollow core isnot essentially different from those found in sound enamel. In both cases, the average diameter is 37 nm. Crystallites having a central defect and an outer diameter of about 40 nm are split into two parts of approximately 15 nm in diameter following acid treatment. The central defect is caused exclusively by the acid and not by damage from the electron beam, nor by a combination of acid treatment and electron beam damage.

Transfer of 45Ca during amelogenesis studied by autoradiography and electron microscopy

Abstract: 45Ca transfer through the stratum intermedium and the secreting ameloblasts towards enamel has been studied by quantitative electron microscopical autoradiography in tooth-germs of newborn cats following intravenous injection of the isotope. Two transfer pathways were demonstrated. The relatively more important direct path passed through the stratum intermedium and ameloblast intercellular spaces and reached the enamel directly. The second pathway consisted in an intracellular transfer through the ameloblasts. 45Ca penetrated the cell through its basal pole. The mitochondria were the most highly labeled organelles at the different experimental time intervals studied. A maximum of radioactivity was respectively noticed at 30 min and 1 h in the endoplasmic reticulum and the Golgi apparatus. A total absence of silver grains was noted over the secretory ameloblastic bodies. At 6 h, the highest labeling was observed over enamel and especailly over the inner enamel along the enameldentin junction.

Amorphous calcium precipitations in human aortic valve.

Abstract: Spheroidal, granular, and fibrillar particles were observed in human aortic valve calcifications. Spheroids were similar to amorphous calcium phosphate shown previously in synthetic preparations and in bone. The frequent coexistence of needle shaped hydroxyapatite crystals with granular and fibrillar particles suggests that the latter are also amorphous calcium phosphate and may be precursors of hydroxyapatite. All three types of particles had a tendency to form laminated and spherular secondary structures and failed to give a crystalline pattern by electron diffraction.

Transmembrane bone matrix gelatin-induced differentiation of bone

Abstract: In response to chemically-defined bone matrix gelatin (BMG) inside a diffusion chamber implanted in a muscle pouch, mesenchymal cells migrate directionally, aggregate and differentiate into new bone, on theoutside of the chamber. BMG diffuses through double membranes 275 to 300 μm in thickness. The inner membrane of pore size is 0.025 μm and the outer membrane of pore size is 0.45 μm. The inner membrane is 1/20 the pore size and the combination is twice the thickness of membranes previously reported to transfer osteoinductive activity of living cells. Autoradiographs show35S-cysteine-labelled BMG produces very high transmembrane grain counts while3H-proline labelled BMG produces very low transmembrane grain counts. Electron micrographs demonstrate that gelatin-derived, uranyl-acetate-stained fine granules interspersed with ruthenium red-staining coarse granules, diffuse through the membrane of 0.025 μm pore size from the inside out. Solitary pale-staining collagen fibrils, possibly formed in interstitial fluid by renaturation of BMG are found in the interior of the chamber and in the interior of the outer 0.45 μm but not the inner 0.025 μm pore size membrane. Densely-stained new bone collagen fiber bundles cover the outer membrane, fill the 0.45 μm subsurface pores for a depth of 0.20 to 30 μm, and thereby attach the new cartilage and bone deposits to the outer surface of the chamber. BMG powders solubilize rapidly in diffusion chambers and produce high yields of new bone. The relationship between denatured collagen and renatured gelatin fibrils in the process of transfer of the bone morphogen from BMG to mesenchymal cell receptors is an intriguing subject for further investigation.

The metabolism of labeled parathyroid hormone. V. Collected biological studies.

Abstract: Biologically active 125I-labeled parathyroid hormone (125I-PTH) was used in a series of studies in dogs and chickens designed to confirm and augment earlier studies in rats. As in rats, a three exponential equation was required to describe disappearance of 125I-PTH from the blood in the dog. The first two "half-lives" (1.8 and 7 min) accounted for the bulk of the dose. Also as in rats, deposition of apparently intact hormone took place rapidly in kidney, liver and bone in both the dog and the chicken. Degradation occurred very rapidly in all three target organs. Three labeled hormones of different biological activities were compared in the rat. Inactive, oxidized hormone was rejected by the liver but showed markedly increased deposition in kidney and the higher the purity of the hormone the higher was its uptake by liver. Exploration of a wide range of dosages revealed few effects on distribution (smaller deposition in liver and kidney at highest dosages, 65 mug/rat). Fresh sera did not degrade hormone rapidly or extensively. There was no deposition of hormone in intestinal mucosa, marrow, and red cells. Nephrectomy increased deposition in liver and bone. Finally, the perfused liver was capable of extensive degradation of the hormone.