Showing papers in "Calcified Tissue International in 1989"

The carbonate environment in bone mineral: a resolution-enhanced Fourier Transform Infrared Spectroscopy Study.

Abstract: The environment of carbonate ions in bones of different species (rat, rabbit, chicken, cow, human) was investigated by Fourier Transform Infrared Spectroscopy (FTIR) associated with a self-deconvolution technique. The carbonate bands in thev 2 CO 3 2− domain show three components which were identified by using synthetic standards and different properties of the apatitic structure (ionic affinity for crystallographic locations, ionic exchange). The major component at 871 cm−1 is due to carbonate ions located in PO 4 3− sites (type B carbonate). A band at 878 cm−1 was exclusively assigned to carbonate ions substituting for OH− ions in the apatitic structure (type A carbonate). A band at 866 cm−1 not previously observed was shown to correspond to a labile carbonate environment. The intensity ratio of type A to type B carbonate appears remarkably constant in all bone samples. The 866 cm−1 carbonate band varies in its relative intensity in different species.

Long-term effects of ovariectomy and aging on the rat skeleton

Abstract: The long-term skeletal effects of ovariectomy and aging were studied in female Sprague-Dawley rats sacrificed at 270, 370, and 540 days after bilateral ovariectomy (OVX) or sham surgery at 90 days of age. The proximal tibia was processed undecalcified for quantitative bone histomorphometry. For continuity, data from these late time points were combined with previously published data from earlier time points (0-180 days). A biphasic pattern of cancellous bone loss was detected in the proximal tibial metaphysis of OVX rats. An initial, rapid phase of bone loss out to 100 days was followed by an intermediate period of relative stabilization of cancellous bone volume at the markedly osteopenic level of 5-7%. After 270 days, a slow phase of bone loss occurred during which cancellous bone volume declined to 1-2%. Both the initial, rapid phase and the late, slow phase of bone loss in OVX rats were associated with increased bone turnover. In control rats, cancellous bone volume remained constant at 25-30% out to 270 days (12 months of age), then decreased to approximately 10% by 540 days (21 months of age). This age-related bone loss was also associated with increased bone turnover. It is interesting to note that the proximal tibial growth plates were closed in approximately a quarter of the control rats by 15-21 months of age. Our data indicate that a slow rate of bone loss and increased bone turnover persist in OVX rats during the later stages of estrogen deficiency. Therefore, the development of osteopenia is coincident with increased bone turnover in OVX rats as well as in aged, control rats.

Performance evaluation of a dual-energy X-ray bone densitometer

Abstract: We tested a dual-energy bone densitometer (LUNAR DPX) that uses a stable x-ray generator and a K-edge filter to achieve the two energy levels. A conventional scintillation detector in pulse-counting mode was used together with a gain stabilizer. The densitometer normally performs spine and femur scans in about 6 minutes and 3 minutes, respectively, with adequate spatial resolution (1.2×1.2mm). Total body scans take either 10 minutes or 20 minutes. The long-term (6 months, n=195) precision of repeat measurement on an 18-cm thick spine phantom was 0.6% at the medium speed. Precision errorin vivo was about 0.6, 0.9 and 1.5% for spine scans (L2-L4) at slow, medium and fast speeds, while the error was 1.2 and 1.5 to 2.0%, respectively, for femur scans at slow and medium speed. The precision of total body bone density was 0.5%in vitro andin vivo. The response to increasing amounts of calcium hydroxyapatite was linear (r=0.99). The densitometer accurately indicated (within 1%) the actual amount of hydroxyapatite after correction for physiological amounts of marrow fat. The measured area corresponded exactly (within 0.5%) to that of known annuli and to the radiographic area of spine phantoms. There was no significant effect of tissue thickness on mass, area, or areal density (BMD) between 10 and 24cm of water. The BMD values for both spine and femurin vivo correlated highly (r=0.98, SEE=.03 g/cm2) with those obtained using conventional153Gd DPA. Similarly, total body BMD correlated highly (r=0.96, SEE=.02g/cm2) with DPA results.

Predicting fractures in women by using forearm bone densitometry.

Abstract: In 1,076 women the forearm bone mineral content (BMC) had been measured with single photon gamma absorptiometry (SPA) 10-16 years ago. The incidence of fractures incurred in the ensuing years (1975-1985) was recorded. The BMC had been significantly less in those women who were to have the types of fractures that are related to bone fragility but only in women measured during the first post- menopausal decades. From age 50-69, diminished BMC at the distal forearm site was associated with a threefold increase of relative risk whereas at the proximal site there was about a sixfold increase in relative risk. This association was not evident in the population over 70 years of age because of a high frequency of fractures (mostly of the spine) in subjects with relatively high BMC values. Body weight and grip strength were significantly lower in the fracture group over 70 years of age.

Increasing age-adjusted risk of fragility fractures: a sign of increasing osteoporosis in successive generations?

Abstract: The age-adjusted incidence of fragility fractures is increasing. This is the outcome of almost all epidemiologic studies that have been done throughout the world during the last 40 years. However, a few studies that have shown an unchanged incidence of hip fractures are as often cited in recent literature, which motivates a discussion of the issue. There are signs that favor the fact that the increasing incidence of fractures in the elderly is evidence of a deterioration of the skeleton in successive generations. Its possible reasons are discussed. The epidemiology of fractures has been studied more thoroughly in Malmr, Sweden than in any other place. The favorable conditions for such investigations in our city have been used in many studies since the beginning of this century [1, 2], up to the present, where all the fractures from those occurring at birth [3] to the fractures sustained by already hospitalized elderly [4] have been investigated. These investigations together with all other studies on fracture epidemiology (to our knowledge) are reviewed in the first part of this article. In the second, possible reasons for the increasing ageadjusted incidence of fragility fractures are discussed.

Mineral induction by immobilized polyanionic proteins.

Abstract: The purpose of this study was to investigate the mineral induction capacity in vitro of polyanionic proteins covalently bound to a surface. Rat dentin gamma-carboxyglutamate-containing protein of the osteocalcin type (Gla-protein), proteoglycan (PG), and phosphoprotein (PP-H), as well as phosvitin (PhV) and bovine serum albumin (BSA), were covalently linked to agarose beads. There were incubated at 37 degrees C in solutions with a Ca/P molar ratio of 1.67, [Ca][P] molar products in the range 1.0-1.8 mM2, and an ionic strength of 0.165. The incubations were performed at constant pH and composition conditions; no spontaneous precipitation occurred under these conditions. Mineral formation, as monitored by scanning electron microscopy (SEM), was induced by all immobilized polyanions, including enzymatically dephosphorylated PP-H and PhV. No mineral was induced by BSA. The mineral inductive capacity of immobilized polyanionic proteins, as judged by the SEM after identical incubations, was found to differ between the different ligands. The mineral induced by PP-H and PG was shown by X-ray diffraction to be apatitic. It was concluded that, although polyanionic proteins in solution may inhibit mineral induction and growth, very minute quantities of such molecules, when immobilized on a surface, induce mineral at physiological concentrations of calcium and phosphate ions. The data presented may be taken to suggest that PP-H and PG, and perhaps other polyanions, may possibly be responsible for mineral nucleation in dentin and bone. The results, however, also point to the rather limited specificity in this type of reaction.

The effects of muscle-building exercise on bone mineral density of the radius, spine, and hip in young men.

Abstract: We previously demonstrated that muscle-building exercise is associated with increases in serum Gla-protein, serum 1,25(OH)2D, and urinary cyclic AMP. These studies were interpreted to mean that this form of exercise increases bone formation and modifies the vitamin D-endocrine system to provide more calcium for bone. The present investigation was carried out in normal young adult white men to determine the effects of exercise on bone mineral density at weight-bearing and nonweight-bearing sites. Twelve men who had regularly engaged in muscle-building exercises (use of weights, exercise machines, or both) for at least 1 year and 50 age-matched controls (aged 19-40 years) were studied. The body weights of the two groups were not different from each other (78 +/- 2 vs. 74 +/- 1 kg, NS). Bone mineral density (BMD) of the lumbar spine, trochanter, and femoral neck was measured by dual-photon absorptiometry, and BMD of the midradius was measured by single photon absorptiometry. It was found that muscle-building exercise was associated with increased BMD at the lumbar spine (1.35 +/- 0.03 vs. 1.22 +/- 0.02 g/cm2, P less than 0.01), trochanter (0.99 +/- 0.04 vs. 0.86 +/- 0.02 g/cm2, P less than 0.01), and femoral neck (1.18 +/- 0.03 vs. 1.02 +/- 0.02 g/cm2, P less than 0.001) but not at the midradius (0.77 +/- 0.02 vs. 0.77 +/- 0.01 g/cm2, NS). These studies provide additional evidence that muscle-building exercise is associated with increases in BMD at weight-bearing sites but not at nonweight-bearing sites.

Deterring bone loss by exercise intervention in premenopausal and postmenopausal women.

Abstract: This study investigated the efficacy of 4 years of exercise intervention in deterring bone loss in middle-aged women, and is a correction and extension of previously published data. Sixty-two control subjects (mean age 50.8) and 80 exercise subjects (mean age 50.1) completed a 4-year study. Subjects exercised three times a week, 45 minutes per session. Bilateral radius, ulna, and humerus bone mineral content (BMC) and width (W) were measured on each subject 11 times over the 4-year period. The two groups did not differ initially in age, height, or weight, but the control group had a greater maximum VO2 (ml/kg/min) than the exercise group. Slopes and intercepts of the bone variables vs. time were determined for each subject, and these values were used for between-group comparisons of loss. The control group BMC and BMC/W declined significantly in all three bones in both arms. The exercise group rate of decline was significantly less than that of the control group for 12 of the 18 bone variables. The greatest effect of the exercise intervention was on the ulna and radius. Exercise subjects lost significantly less than control subjects in left and right ulna and radius BMC and BMC/W, and left ulna and radius W. Lesser differences between groups were observed in the humerus. BMC and W loss rates of the left humerus were reduced in the exercise group, with no difference between exercise and control subjects in the other humerus variables. To determine if menopausal status influenced the response to exercise, we analyzed the difference between groups for premenopausal and postmenopausal subjects separately. Regardless of menopausal status, exercise subjects had lower bone loss rates than control subjects. In both premenopausal and postmenopausal subjects, exercise reduced bone loss significantly for 10 of the 18 bone variables. It can be concluded that physical activity significantly reduces bone loss in the arms of middle-aged women.

Crystal dissolution of biological and ceramic apatites.

Abstract: High resolution transmission electron microscopy (Hr TEM) studies on biological and synthetic calcium phosphate have provided information on the dissolution process at the crystal level. The purpose of this study was to investigate the dissolution of ceramic hydroxyapatite (HA) after implantation using Hr TEM. Recovered HA ceramic implanted in bony and nonbony sites in animals and in periodontal pockets in humans were used for the study. For comparison, sections of human fluorotic enamel with caries and sections of shark enameloid previously exposed to 0.1 HCl were similarly investigated. Hr TEM studies demonstrated that in both the biological and ceramic apatites, the lattice and atomic defects were the starting points in the dissolution process. However, significant differences in the process of dissolution were observed: (1) biological apatite crystals showed preferential core dissolution whereas ceramic apatite crystals showed nonspecific dissolution at the cores and at the surfaces; (2) the dissolution of biological apatites appeared to consistently extend along the crystal's c-axis whereas dissolution of the ceramic HA did not appear to be correlated with the crystal's c-axis. The observed differences in crystal dissolution between biological and ceramic apatites may be attributed to the following: (1) the unique crystal/protein interaction present with biological apatites but absent in ceramic HA; (2) differences in defect distribution between biological and ceramic apatites which are due to the differences in the original of these defects; and (3) the longer morphological c-axis of biological apatites compared with that of ceramic apatites.(ABSTRACT TRUNCATED AT 250 WORDS)

Indomethacin modulation of load-related stimulation of new bone formation in vivo.

Abstract: The capacity of bone to organize and reorganize its structure in response to changing mechanical demands is well recognized. However, the mechanism by which the changing mechanical environment is detected, and the means by which this information is translated into a stimulus for structural modification, are not understood. A group of substances suggested to be involved in the initial transduction of strain information are the prostaglandins. In this experiment we used a single period of dynamic loading to stimulate an adaptive osteogenic responsein vivo. Loading was performed in the presence and absence of indomethacin. Measurements of the periosteum 5 days after loading showed that the presence of indomethacin at the time of loading reduced the osteogenic response. Though consistent with the hypothesis that prostaglandins are involved in the initial transduction of tissue strain into a biochemical response, this result is not sufficient to demonstrate this conclusively because reduced prostaglandin levels during the 24 hours immediately after the period of loading may affect many other points in the cascade of events between strain transduction and adaptive new bone formation. Furthermore, indomethacin at the relatively high levels we used (40 mg/kg) may have effects other than those on prostaglandin synthesis.

Prediction of vertebral strength by dual photon absorptiometry and quantitative computed tomography

Abstract: We measured the lumbar bone mineral of 19 cadavers (10 women, 9 men) by dual photon absorptiometry (DPA) and quantitative computed tomography (QCT). In addition, we determined the ultimate load and stress of each vertebra, and finally ash content and volumetric ash density of the vertebral body. We found that single energy QCT was inferior to DPA and dual energy QCT in the prediction of the ultimate load or stress of vertebrae (P<0.001). The ultimate stress of predicted by using the dual energy QCT results (r=0.71; SEE=36.3 N/cm2) whereas the ultimate vertebral load was best predicted by using the DPA (BMC) results (r=0.80; SEE=740 N). If the QCT finding was multiplied with the surface area of the vertebral body it could be used to predict the ultimate load with good accuracy (r=0.74; SEE=841 N). All the above correlations were higher in women than in men. The frequency of vertebral compression fractures in the material was wel correlated with the bone mineral findings. A nonlinear (third degree) relationship between mineral content and mechanical characteristics is proposed but within the area of measurement used in clinical practice a linear (first degree) equation is preferred.

Familial resemblance of radial bone mass between premenopausal mothers and their college-age daughters

Abstract: The influences of heredity and environmental factors on radial bone mass were evaluated in 84 premenopausal mothers with their biological daughters (ages 18–22). Mid- and distal radial bone mineral content (BMC) and density (BMD) were assessed using single-photon absorptiometry. As a group, the daughters (mean age 18.6 years) had 5–10% less bone mass at both the distal and midradial sites than their mothers (mean age 44.2 years). Familial resemblance estimates showed significant relationships between mothers and daughters for mid-and distal BMC and BMD after considering the influence of body mass index (BMI). Daughters with a maternal family history of osteoporosis had 6–7% lower but nonsignificant values of mid- (P=0.086) and distal BMC (P=0.075) compared to values of women with a negative family history, whereas mothers with a positive family history had 3–4% lower (NS) values of distal and mid-BMC compared to those of mothers with a negative family history after adjustment for BMI. Multiple regression analyses showed BMI to be the most important determinant of the bone values of the mothers, and both BMI and dietary calcium intake were found to be significant for the daughters. The findings of this study suggest that hereditary contributions from the mothers play an overwhelmingly critical role in the accrual of bone mass by their daughters by ages 18–22, but that environmental influences on bone consolidation during the premenopausal decades may be more important in promoting optimal (peak) bone mass and thereby may help to delay the postmenopausal onset of osteoporotic fractures.

Microdamage in response to repetitive torsional loading in the rat tibia.

Abstract: Tibiae from 60 male Wistar rats, aged 13 ±1 weeks, were divided into six groups for mechanical and histological testing. Bones were loaded repetitively in torsion at 90 deg.s−1. Group 1 was subjected to 5,000 loading cycles at a twist angle of 3.6°, groups 2–5 to 10,000 cycles at 3.6, 5.4, 7.2, and 9.0°, respectively, and group 6 was tested to failure. Six transverse sections from the middiaphysis were then cut, bulk-stained in basic fuchsin, and hand ground to 30–50 μm to examine the presence of microcracks. Cracks were classified as running parallel to lamellae, crossing lamellae, crossing the full thickness of the cortex, or invading vascular canals. Results for fatigue testing showed that the tibiae exhibited a gradual decrease in torque (P<0.05), average stress (P<0.01), stiffness (P<0.01) and energy absorbed (P<0.01) from the initial loading cycle. Analysis of microdamage showed an increase in the variety of cracks from groups, 1–5. Analysis of deviance demonstrated a strong dependence of crack probability on the level of loading for all crack types (P<0.05) except those crossing lamellae. This study reinforces the evidence that yielding of bone observed during repetitive loading is caused by diffuse structural damage such as microcracking or debonding.

Efficacy of wheat germ lectin-precipitated alkaline phosphatase in serum as an estimator of bone mineralization rate: comparison to serum total alkaline phosphatase and serum bone Gla-protein.

Abstract: Serum levels of total alkaline phosphatase activity (S-T-AP), wheat germ lectin-precipitated alkaline phosphatase activity (S-L-AP), and bone Gla-protein immunoreactivity (S-BGP) were measured in 26 patients (23 females and 3 males) aged 35–73 years (mean 59 years) with primary hyperparathyroidism (n=7), hyperthyroidism (n=9), and hypothyroidism (n=10) in whom the bone mineralization rate (m) was determined by47Ca-kinetics (continuously expanding calcium pool model). A weak positive correlation (r=0.42,P 0.50). The highest correlation coefficient (r=0.81,P<0.001) was found between S-BGP and m which could be predicted with an error of 3.4 mmol Ca/day. S-BGP showed a closer correlation to S-L-AP (r=0.71,P<0.001) than to S-T-AP (r=0.58,P<0.01). We concluded that S-L-AP predicts bone mineralization at organ level better than S-T-AP in selected metabolic bone disorders and that the supernatant activity shows no relation to bone turnover. We find the assay easy to handle and suitable for large-scale use in the diagnosis and monitoring of metabolic bone disease.

Increase of vertebral density by combination therapy with pulsatile 1-38hPTH and sequential addition of calcitonin nasal spray in osteoporotic patients.

Abstract: Combination therapy with the biologically active (1–38) human parathyroid hormone peptide and calcitonin using pulsatile and sequential activation of the skeleton for 14 months in patients with low-turnover osteoporosis resulted in an increase in trabecular bone mass. These favorable responses were observed without any significant changes in cortical (forearm) bone mass content.

The effect of aging on fracture healing in the rat.

Abstract: The effect of age on the biomechanical properties of healing tibial fractures was studied by comparing the fracture healing in 2-year-old male Wistar rats with the fracture healing in 3-month-old male Wistar rats after 40 and 80 days of healing. There were no significant differences in the mechanical parameters after 40 days of healing, but after 80 days, a considerable delay in the fracture healing process was noted in the old rats compared with the young adult rats when evaluated by maximum load, maximum stress, stiffness, and energy absorption in a three-point bending procedure. In the contralateral, nonfractured bones, the tibiae from the old animals sustained higher loads and had higher stiffness than the bones from the young adult animals, but stress values, elastic modulus, and capacity for energy absorption was much lower in the old animals.

Suggestion of a Deficient Osteoblastic Function in Diabetes Mellitus: The Possible Cause of Osteopenia in Diabetics

Abstract: The mechanism underlying diabetic osteopenia is still unclear and may involve osteoblastic activity and/or the deficit of insulin's anabolic action. Bone gla protein (BGP) is synthesized by the osteoblast and its synthesis increases with 1,25(OH)2D3 and fluoride. Because 1,25(OH)2D3 also stimulates insulin secretion, sodium fluoride administration can be used to investigate deficient osteoblastic activity in diabetics, as reflected by BGP levels. BGP was determined before and after administering sodium fluoride at a dosage of 50 mg/day/15 days to three groups: 14 patients with insulin-dependent diabetes, 16 diabetics on oral antidiabetic treatment, and 25 controls, all of similar age, sex, and characteristics. Basal BGP values (mean±SD) were low in diabetics on insulin treatment (4.3±1.1 ng/ml) and in diabetics on oral antidiabetics (5.8±1.2 ng/ml) as compared with controls (6.5±0.7 ng/ml) (P<0.001 and <0.05, respectively). After giving fluoride, BGP values did not change in the two diabetic groups but did vary in controls (8.1±0.6 ng/ml,P<0.001). These results suggest that deficient osteoblast function could be responsible for osteopenia in diabetics.

The predictive value of fracture, disease, and falling tendency for fragility fractures in women

Abstract: In 1,076 women who had been interviewed from 1970 to 1976 with regard to previous fracture and disease, all fragility fractures that occurred from 1975 to 1985 were recorded. Hip and vertebral fractures were good predictors of future fragility fractures but mainly in younger women (less than 70 years) and this was also the case for fracture of the distal end of the forearm in the age group 40-59. Later in life, falling tendency became the important predictor.

Bone-resorbing cytokines enhance release of macrophage colony-stimulating activity by the osteoblastic cell MC3T3-E1.

Abstract: It has been observed that bone resorption in response to interleukin 1 (IL 1) or tumor necrosis factor (TNF) is accompanied by an increase in osteoclast number. Because the osteoclast is of hemopoietic lineage, recruitment could be regulated by colony-stimulating factors, one of which may be macrophage colony-stimulating factor (MCSF). In this study, we show that the constitutive release of M-CSF activity by the osteoblastic cell MC3T3-E1 is enhanced by the presence of recombinant IL 1α, recombinant TNFα, or by the concurrent presence of purified transforming growth factorβ (TGFβ) and epidermal growth factor (EGF). Increased release of CSF by the osteoblast in response to these agents may provide a signal for the growth and maturation of osteoclast precursors leading to subsequent bone resorption.

The dual role of polyelectrolytes and proteins as mineralization promoters and inhibitors of calcium oxalate monohydrate

Abstract: Polyelectrolytes and protein molecules appear to be able to act not only as crystallization inhibitors when present in solution, but also as promoters of crystal growth when immobilized onto surfaces. Because this is especially relevant for systems in which heterogeneous nucleation can occur, the influence of poly-L-glutamic (PGlu) acid, poly-L-aspartic (PAsp) acid, and human serum albumin (HSA) on the nucleation and growth inhibition of calcium oxalate monohydrate (COM) was studied using the Constant Composition (CC) kinetics technique. The overgrowth of COM on hydroxyapatite (HAP) seed crystals pretreated with HSA was also investigated. Pronounced differences in inhibiting and nucleating potential were found for the various additives. HSA, a relatively poor growth inhibitor when present in solution, was found to nucleate very regular, hexagonal COM crystals when immobilized on a surface and to enhance the overgrowth of COM when adsorbed on HAP surfaces.

Developmental expression of genes in chick growth cartilage detected by in situ hybridization.

Abstract: We have usedin situ hybridization to examine expression of collagen type I, II, and X mRNA and osteonectin mRNA in the chick epiphysis. Tissue samples from the proximal tibial growth cartilage were fixed in modified Carnoy's solution, dehydrated in ethanol, and embedded in paraffin. Longitudinal and transverse sections were dimineralized with HCl and digested with hyaluronidase and proteinase K.In situ hybridization was carried out using biotinylated cDNA probes; the hybridized probe was detected using a streptavidin-biotinylated alkaline phosphatase conjugate. This procedure permitted detection of the corresponding mRNAs in cartilage with high sensitivity and low background. Osteonectin mRNA was detected in proliferating cartilage; lower levels of osteonectin mRNA were seen in the mid-hypertrophic region. This mRNA species was also expressed in cells that border the vascular canals in the premineralized region of the epiphysis. Collagen type X mRNA was detected throughout the hypertrophic zone. As localizated of collagen type X mRNA coresponded to the site of maximal synthesis of the protein, reported in other studies, our results would further support the suggestion that this protein is associated with mineralization of cartilage. Collagen type II mRNA was seen in both the proliferating and the hypertrophic regions of the cartilage. Highest levels of expression were observed in the proliferative region. The results suggest that the transcriptional control of collagen type II and X by cells of the proliferating and hypertrophic regions of the growth cartilage may be related.

The dentino-enamel junction: a structural and microanalytical study of early mineralization.

Abstract: The spatial localization of enamel and dentin apatite crystals of the rat tooth has been studied by electron microscopic methods--bright field, selected-area dark field, and electron spectroscopic imaging. The sequential events of dentin calcification followed by the formation and growth of enamel crystals were determined and compared to previous studies. In dentin, initial sites of mineral deposition occur in areas subjacent to the dentino-enamel junction (DEJ). The subsequent expansion of these deposits progresses towards the DEJ to the terminal ends of dentin collagen fibrils. Concomitantly, an electron-dense enamel matrix is released by ameloblasts; with the presence of this matrix, the growth of enamel crystals occurs from the underlying calcified dentin. Enamel crystal growth continues to within close proximity of the plasma membrane of ameloblasts. A close spatial relationship between enamel and the crystals of calcified dentin collagen fibrils was observed by selected-area dark field imaging. Such areas of crystal intimacy show a co-localization of calcium and phosphorus extending from calcified collagen fibrils to enamel sheaths which encase enamel crystals. A working model of the spatial relationship between crystals of dentin and enamel is presented and discussed in light of mechanisms by which calcified dentin may promote the formation of enamel crystals.

Excretion of pyridinium cross-links of collagen in ovariectomized rats as urinary markers for increased bone resorption.

Abstract: Groups of 19-day-old rats were ovariectomized or were given sham operations Measurements in urine of the pyridinium cross-links of collagen, pyridinoline and deoxypyridinoline, 7 weeks after surgery showed significantly higher amounts of cross-links relative to creatinine in the ovariectomized groups compared with the controls Analyses before and after acid hydrolysis of the urine revealed that the increased excretion was only as free cross-link with no change in the concentrations of the bound forms The loss of trabecular bone in the ovariectomized group was confirmed by immunocytochemical staining with antibodies to type I collagen There were no differences between the ovariectomized and control groups in the concentrations of cross-links in the tibial bone or the articular cartilage Measurements of free pyridinoline and deoxypyridinoline in urine therefore appear to provide a good index of the increased bone resorption induced by estrogen deficiency

Chemical changes in hydroxyapatite biomaterial under in vivo and in vitro biological conditions.

Abstract: The introduction of a synthetic calcium phosphate into a biological environment is likely to result in surface-mediated chemical events. On the basis of such an assessment, we studied the chemical changes occurring in the mineral after exposure of a synthetic hydroxyapatite ceramic to bothin vivo (implantation in human) andin vitro (cell culture) conditions. A small amount of the material was phagocytized but the major remaining part behaved as a secondary nucleator as evidenced by the appearance of a newly formed mineral. Morphologically, the newly formed mineral appeared as tiny crystals precipitated and grown from the surface of the initial synthetic crystals. The density of the additional mineral increased from the periphery to the core of each biomaterial aggregate. Chemically, it was identified by IR spectroscopy as a carbonated apatitic mineral. We propose that the adsorption of biomolecules could inhibit precipitation, accounting for the increasing amount of precipitate from the periphery to the core of the aggregates.

Fixation and demineralization of bone tissue for immunohistochemical staining of neuropeptides

Abstract: The present study in the rat demonstrates the feasibility of applying immunohistochemical staining techniques on bone tissue for studies of substances such as neuropeptides contained in nerve fibers. Two fixation procedures, as well as the influence of demineralization on neuropeptide antigenicity, were studied in bone and for comparison in small intestine.In vivo perfusion with paraformaldehyde and picric acid, followed by demineralization in a solution of either EDTA-cacodylate or buffered EDTA-sucrose, proved to be the most appropriate with respect to preserved substances were tested. In the bone tissue, immunoreactivity was found to four neuropeptides: substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide, and neuropeptide Y, and also to the catecolamine-synthesizing enzyme tyrosine hydroxylase. The described method for identifying intraosseal neuropeptides offers a new means of studying skeletal innervation and bioactive substances in bone tissue.

Effect of carbonate content on the ESR spectrum near g = 2 of carbonated calciumapatites synthesized from aqueous media.

Abstract: The ESR spectrum of X-irradiated carbonated apatites synthesized at low temperature was studied as a function of their carbonate content. Using13C-enriched samples, four different carbonate-derived radicals and a surface O− ion could be identified. Isotropic CO 3 − and CO 2 − ions are present at a B site in the apatite lattice, and anisotropic CO 3 − and CO 2 − radicals are located at the surface of the crystallites. Only the isotropic ESR signals increase with increasing carbonate content. The anisotropic signal ascribed to a surface CO 2 − radical is mainly responsible for the so-called asymmetric ESR signal near g=2. It is argued that this surface signal may still be composite and caused by several very similar CO 2 − ions. The consequences for phenomenological ESR studies of calcified tissues are discussed.

Transient-steady state phenomena in microdamage physiology: A proposed algorithm for lamellar bone

Abstract: This algorithm suggests that in steady states the momentary burden of unrepaired microdamage (MDx) in lamellar bone equals the rate of creation of new MDx multiplied by the time taken to repair a locus of MDx completely in the biomechanical sense. That "repair period" equals about 0.6 years in healthy human adults. When MDx production suddenly increases, the momentary MDx burden begins to increase too, and does so for a time equal to the repair period and in proportion to the increased MDx production. After the repair period elapses, the momentary MDx would tend to reach and stay at a maximum value as long as increased MDx production continued. Prolonging the repair period, preventing the creation of new remodeling units to repair MDx, or delaying mineralization of the new bone made by those units would also increase MDx burdens. Reducing MDx production or the repair period, or accelerating the creation of new modeling units would have the opposite effects on the momentary MDx burden but would also go through a transient phase before developing the new steady state conditions. Exploiting these relationships quantitatively and experimentally requires expressing them mathematically and using for the terms in any equations things one can define logically and measure practically. Accordingly, the article suggests a special definition of a unit amount of microdamage, how to measure it, and simple algebra and equations for calculating some effects of microdamage on the biologic system.

Time course of calcium absorption in humans: evidence for a colonic component.

Abstract: We examined the time course of calcium absorption (CaAbs) in 155 studies, using a double isotope technique. The subjects were 118 healthy peri-menopausal women (mean age 53.3 years), studied as impatients under metabolic balance conditions. We measured the ratio of radiolabeled calcium (oral:IV) in serum and urine for 144 hours after the oral dose, and generated a composite CaAbs curve for all 155 studies using normalized data. Although CaAbs was 80.9% complete at 3 hours, it was still only 95.8% complete at 7 hours; the remaining 4.2% was absorbed in a slower late component, and did not reach completion until about 26 hours. The rapid initial component probably represents mainly small intestinal absorption and the late component, colonic. At the dietary intakes of our subjects, we estimate the size of the late component at about 6.8 mg/day. For fully accurate measurements of CaAbs, it is necessary to allow for this small late component.

Osteocalcin levels in diabetic subjects

Abstract: Because a series of reports suggests the existence of altered bone and mineral metabolism in diabetes mellitus, we studied 106 diabetic subjects (42 insulin-dependent (IDD) and 64 noninsulin dependent (NIDD)) to determine whether a difference in bone turnover (evaluated by serum osteocalcin (OC)) could be found in comparison with normal controls. OC levels in diabetic subjects were lower than the age- and sex-specific predicted values. The reduction was especially evident in male and female NIDD (Z-score: −1.12±0.92, t=8.4,P P>0.1), was not significantly different from normal. Total serum calcium (Ca) and calcitonin (CT) showed an apposite pattern, being higher in all the diabetic subgroups (with the exception of Ca in female IDD), whereas parathyroid hormone (PTH) was lower than expected in each diabetic subset. By multiple regression analysis, the reduction of OC was related to PTH and CT levels and to the type of treatment. Subjects controlled with diet showed differences of greater magnitude from the expected normal values than those treated with oral hypoglycemic agents or insulin (Z-score: −1.28±1.05 vs. −0.85±0.90 and −0.63±0.97, respectively;P=0.05). However, the variance explained by these three factors was small, suggesting that other variables (possibly α,25(OH)2D) exerted important influences on OC levels.

FT-IR microscopy of endochondral ossification at 20μ spatial resolution

Abstract: A Fourier transform infrared spectrometer has been coupled with an optical microscope to study the distribution and characteristics of the mineral phase in calcifying tissues at 20μ spatial resolution. This represents the first biophysical application of this technique. High quality spectra were obtained in a relatively short scan time (1–2 minutes) from thin longitudinal sections of normal and rachitic rat femurs. Substantial spatial variations in the extent and structure of the mineral phase were observed as a function of spatial position both within and beyond the growth plates, as judged by the phosphate vibrations in the 900–1200 cm−1 spectral region. The current experiments reveal the utility of FT-IR micrscopy in identification of sites where mineralization has occurred. In addition to vibrations from the inorganic components, the Amide I and Amide II motions of the protein constituents are readily observed and may be useful as a probe of protein/mineral interactions.