Medial artery calcification of uremic patients: a histological, histochemical and ultrastructural study.

Recent findings suggest that vascular calcification (VC) is an active process similar to bone mineralization, the vascular smooth muscle cells (VSMCs) undergoing phenotypic differentiation into osteoblastic cells and synthesizing calcification-regulating proteins found in bone. This study has investigated the VC process of uremic patients, with a morphologic approach. Epigastric artery samples from 49 uremic, non-diabetic patients were taken during kidney transplantation. Sections from paraffin-embedded samples were stained with hematoxylin/eosin and von Kossa. CD68 was immunohistochemically detected, and sections from frozen samples were stained with Oil Red O. Deeply calcified samples were stained with Picrosirius Red, PAS, and Alcian blue. Specimens from one patient with moderate and one with severe VC were examined under the electron microscope. None of the samples had atherosclerosis. Calcifications were found in the media of 38 patients. In 23, dot-like calcifications were irregularly scattered near the adventitia (light VC); in 11, granular calcifications formed concentric rings near the adventitia (moderate-advanced VC); in 4, zones of consolidated calcifications were found (severe VC). These zones were poor in collagen, glycoproteins and proteoglycans. In cases with moderate or severe VC, VSCMs showed necrotic changes. Matrix vesicles could be recognized in the extracellular spaces. In cases with severe VC, uncalcified or partially calcified membranous bodies were found, together with Liesegang rings. Patches of fibrin were also found. These findings point to a mainly degenerative mechanism of VC, which proceeds from the outer portion of the media. An active mechanism, however, cannot be excluded. A unifying hypothesis is suggested.

Bone turnover, osteopenia and vascular calcifications in hemodialysis patients. A histomorphometric and multislice CT study.

BACKGROUND: Several classical risk factors are at the base of vascular calcifications in hemodialysis patients. Among these, according to a general opinion, also bone turnover plays a role, which, however, requires a better definition. In addition, it has been suggested that there is a relationship between primary osteoporosis and vascular calcifications. This bone biopsy-based study on a hemodialysis patient cohort is a contribution to the evaluation of these alleged relations. METHODS: This study has been carried out on a cohort of 32 patients on maintenance hemodialysis, who were subjected to transiliac bone biopsy for histomorphometric, histodynamic and bone aluminum deposit evaluation. The patients were also examined with multislice computerized tomography for quantitation of heart and coronary calcifications. RESULTS: The patients were affected by renal osteodystrophy with a wide range of bone formation rate values. A significant negative correlation was found between the rate of bone turnover and log-transformed cardiac calcification score (p < 0.003). There were also negative significant correlations between the cardiac and coronary calcification score log and trabecular number (p < 0.02 and p < 0.05, respectively), while the correlations were positive with trabecular separation (p < 0.03 and p < 0.05, respectively). However, multiregression analysis, forward method, selected only age, hemodialysis age and serum Ca as predictive variables of cardiac and coronary calcification score log, while the histomorphometric and histodynamic variables were excluded. CONCLUSIONS: In this study, in spite of the suggestive findings of the univariate statistical approach, a further multivariate analysis was indicative of a spurious association between calcification scores and both bone turnover and histomorphometric parameters of trabecular mass and connectivity. Bone turnover and trabecular mass do not appear to be prominently connected with the extent of cardiac and coronary calcifications in hemodialysis patients.

Trabecular bone microarchitecture in mild primary hyperparathyroidism.

Primary hyperparathyroidism (PHPT) is a common endocrine disease. High levels of PTH cause demineralization of bone and increased risk of fracture. On the other hand, the effect of PHPT on bone structure is more ambiguous. The aim of this study was to evaluate the effect of PHPT on cancellous bone volume, structure, and microarchitecture. Thirteen transiliac biopsy specimens taken in untreated post-menopausal women aged 65+/-5 yr with primary hyperparathyroidism were compared with 13 biopsies taken in normal women aged 66+/-6 yr. None of the patients presented any other disorder affecting bone metabolism. In these samples we evaluated the direct and indirect histomorphometric parameters of bone microarchitecture using an image analysis system consisting of an epifluorescent microscope (Leica DMR) connected to an analogic 3 CCD camera (Sony DXC 390P) and a computer equipped with specific software for histomorphometric analyses. No significant differences between PHPT patients and controls in cancellous bone volume, trabecular thickness, and number were found. Two-dimensional parameters showed a preserved microarchitecture in PHPT patients. On the other hand, indirect parameters of microarchitecture [Marrow Star Volume (MSV) and Trabecular Bone Pattern Factor (TBPf)] showed a significant compromising of microarchitecture in these patients. PHPT patients have similar structural parameters to normal subjects. Concerning microarchitecture, indirect approach by MSV and TBPf shows a significant compromising of connectivity. These results can explain trabecular fragility observed in clinical studies on PHPT.

Effects of intermittent parathyroid hormone (PTH) administration on SOST mRNA and protein in rat bone.

Sclerostin, the secreted protein product of the SOST gene, which is mainly expressed by osteocytes, has recently been proposed as a negative regulator of bone osteoblastogenesis. Chronic elevation of PTH reduces SOST expression by osteocytes, while controversial results have been obtained by intermittent PTH administration. We have investigated the effects of intermittently administered PTH on SOST expression and sclerostin localization, comparing them with those of controls, as they appeared in three different bone segments of rat tibia: secondary trabecular metaphyseal and epiphyseal bone, and cortical diaphyseal bone. The histomorphometric results demonstrate that PTH enhances bone turnover through anabolic effects, as shown by the association of increased bone resorption variables with a significant rise in BV/TV, Tb.Th and Tb.N and a fall in Tb.Sp. PTH induces a SOST mRNA and protein fall in secondary metaphyseal trabeculae, diaphyseal bone and in epiphyseal trabeculae. Numbers of sclerostin immunopositive osteocytes/mm(2) show no change, compared with controls; there are fewer sclerostin-positive osteocytes in secondary metaphyseal trabeculae than in the other two bone areas, both in the control and PTH groups. The low numbers of sclerostin-positive osteocytes in the metaphyseal trabecular bone seem to be directly related to the fact that this area displays a high remodeling rate. The anabolic effects of PTH are in line with the fall of SOST mRNA and protein in all the three bone segments examined; the rise of bone turnover supports a negative role of SOST in bone formation.

Clinical, genetic, and cellular analysis of 49 osteopetrotic patients: implications for diagnosis and treatment.

BACKGROUND: Osteopetrosis, a genetic disease characterised by osteoclast failure, is classified into three forms: infantile malignant autosomal recessive osteopetrosis (ARO), intermediate autosomal recessive osteopetrosis (IRO), and autosomal dominant osteopetrosis (ADO). METHODS: We studied 49 patients, 21 with ARO, one with IRO, and 27 with type II ADO (ADO II). RESULTS: Most ARO patients bore known or novel (one case) ATP6i (TCIRG1) gene mutations. Six ADO II patients had no mutations in ClCN7, the only so far recognised gene implicated, suggesting involvement of yet unknown genes. Identical ClCN7 mutations produced differing phenotypes with variable degrees of severity. In ADO II, serum tartrate resistant acid phosphatase was always elevated. Bone alkaline phosphatase (BALP) was generally low, but osteocalcin was high, suggesting perturbed osteoblast differentiation or function. In contrast, BALP was high in ARO patients. Elevated osteoclast surface/bone surface was noted in biopsies from most ARO patients. Cases with high osteoclasts also showed increased osteoblast surface/bone surface. ARO osteoclasts were morphologically normal, with unaltered formation rates, intracellular pH handling, and response to acidification. Their resorption activity was greatly reduced, but not abolished. In control osteoclasts, all resorption activity was abolished by combined inhibition of proton pumping and sodium/proton antiport. CONCLUSIONS: These findings provide a rationale for novel therapies targeting pH handling mechanisms in osteoclasts and their microenvironment.

Aluminium hydroxide-induced granulomas in pigs.

The effect of intramuscular injection of 40 mg/2 ml aluminium hydroxide in the neck of pigs was examined in a number of ways. The investigation followed repeated slaughterhouse reports, according to which 64.8% of pigs from one particular farm were found at slaughter to have one or more nodules in the muscles of the neck (group slaughtered). The pigs had been injected with a vaccine containing 40 mg/2 ml dose of aluminium hydroxide as adjuvant. Research consisted of two phases: first, an epidemiological study was carried out, aimed at determining the risk factors for the granulomas. The results indicated that the vaccine was to be held responsible for the formation of granulomas. A clinical trial was then performed to further substantiate the initial hypothesis, by comparing pigs, which were aseptically inoculated twice with either the original vaccine or the adjuvant alone (groups vaccine and adjuvant) to pigs inoculated twice with apyrogenic bi-distilled water (group water) and to pigs inoculated once with the adjuvant and once with apyrogenic bi-distilled water (group adjuvant/water). Both studies agreed in their conclusions, which indicate that the high amount of aluminium hydroxide was the cause of the granulomas.

The osteoblastic phenotype in calcium-depleted and calcium-repleted rats: a structural and histomorphometric study.

In a previous report we showed that young rats fed a calcium-free diet for 28 days developed severe hypocalcaemia and showed a significant increase in serum alkaline phosphatase activity. The main histological and cytochemical changes exhibited by these animals in bone of the metaphyseal primary spongiosa were: (1) hyperplasia of osteoblasts, (2) an increase in the frequency of tartrate-resistant acid phosphatase (TRAP)-positive osteoblasts apposed to osteoid, and (3) an excessive amount of osteoid tissue. In addition to typical osteoblasts, there was a subpopulation of osteoblast-like cells with coated pits, lysosome-like bodies and large cytoplasmic processes. In the present study, we investigated how the above parameters change when calcium-depleted rats are placed on a normal diet for 7 days. Such a regimen normalized calcium concentration and alkaline phosphatase activity in the serum. The osteoid thickness returned to normal and, in some areas, was fully calcified. Most osteoblasts no longer showed TRAP activity and their ultrastructure was similar to that found in controls. Despite an intense alkaline phosphatase activity, some of them still exhibited a number of macrophagic characteristics. They were TRAP-positive, and showed electron-dense bodies in the cytoplasm facing bone, an abundance of coated pits, calcified spicules impinging on the cell membrane and large processes extending into the mineralized matrix. We concluded that calcium deficiency causes hyperplasia of osteoblasts in primary spongiosa and an increase in expression of TRAP. It also induces changes in their phenotype characterized by the acquisition of macrophagic cellular features. While TRAP activity is normalized by calcium repletion, macrophagic characteristics persist. These results suggest that the osteoblast can modulate its phenotype according to its physiological status.

Histomorphometric assessment of bone turnover in uraemic patients: comparison between activation frequency and bone formation rate.

AIMS: The histomorphometric assessment of bone formation rate (BFR/BS) in bone biopsies from uraemic patients is of crucial importance in differentiating low from high turnover types of renal osteodystrophy. However, since BFR/BS relies on osteoblasts, activation frequency (Ac.f), encompassing all remodelling phases, has recently been preferred to BFR/BS. This study was carried out to consider whether estimation of Ac.f is superior, in practical terms, to that of BFR/BS in distinguishing between different rates of bone turnover in uraemic patients. METHODS AND RESULTS: Bone biopsies from 27 patients in predialysis (20 men and seven women; mean age 53 +/- 12 years) and 37 in haemodialysis (22 men and 15 women; mean age 53 +/- 12 years) were examined. The types of renal osteodystrophy were classified on the basis of morphology. Bone formation rate and Ac.f were evaluated according to standardized procedures. The Ac.f was calculated both as a ratio between BFR/BS and wall thickness (W.Th) and as a reciprocal of erosion, formation and quiescent periods (EP, FP and QP). Patients were affected by renal osteodystrophy with predominant hyperparathyroidism (two predialysis and 16 dialysis), predominant osteomalacia (three predialysis and seven dialysis) or that of advanced (nine predialysis and five dialysis) or mild (seven predialysis and four dialysis) mixed type or adynamic type (six predialysis and five dialysis). Activation frequency, which with either formula requires the measurement of W.Th, i.e. the thickness of bone structural units (BSUs), was not calculated in three dialysis patients with severe hyperparathyroidism and in one predialysis and four dialysis patients with severe osteomalacia, because only incomplete BSUs were found. In dialysis, EP was higher in the adynamic than in the other types of osteodystrophy. During both predialysis and dialysis, FP was higher in osteomalacia than in the other forms of osteodystrophy, and in adynamic osteopathy than in hyperparathyroidism or in advanced and mild mixed osteodystrophy. During predialysis and dialysis, QP was higher in the adynamic than in the other forms of osteodystrophy. Correlations were found between BFR/BS and Ac.f, during predialysis (r=0.97) and dialysis (r=0.95). CONCLUSIONS: The superiority of Ac.f in assessing bone turnover, in comparison to BFR/BS, is conceptual rather than practical. The highest values for FP in osteomalacia and for QP in adynamic bone allow a clearer characterization of these low turnover conditions.

Bcl-2 and Bax expression in cartilage and bone cells after high-dose corticosterone treatment in rats.

The expression of Bcl-2 and Bax has been evaluated by immunohistochemistry in normal rats, and in rats after treatment with high-dose corticosterone (CORT). Proliferative (PC) and maturative/hypertrophic (MaHC) chondrocytes of the growth plate have been examined, as well as osteoblasts (Obs), osteocytes (Ots) and osteoclasts (Ocs) of the metaphyseal secondary spongiosa. For each cell type, the Bcl-2 and Bax immunopositive cells were expressed as a percentage of the total number of cells. Bcl-2 and Bax expression was considered to be enhanced when the percentage of positive cells rose. Bcl-2 and Bax were expressed in all cell types, and two main kinds of labeling distribution, both suggestive of association with intracellular organelles, were observed in the cytoplasm: scarce and spotty labeling (type 1) or abundant, granular and diffuse labeling (type 2). In some cases, nuclear membranes could also be seen to be positive. Positive PCs and Obs generally showed a labeling of type 1, MaHCs and Ocs of type 2, while Ots varied with labeling of type 1 or type 2. CORT administration induced a fall in the percentage of Bcl-2 immunopositive cells, and a rise in that of Bax immunopositive cells, in PCs and Ots. The same trend was observed in MaHCs, although the Bcl-2 decrease was not significant. The percentage of Bcl-2 and Bax immunopositive Obs rose, and their labeling distribution shifted from type 1- to type 2-labeled cells. Ocs showed the highest immunopositivity for both Bcl-2 and Bax, which did not change after CORT administration. These data suggest that CORT treatment, by lowering Bcl-2, and raising Bax expression, may promote the apoptotic process in PCs, MaHCs and Ots. Obs, however, do not undergo the same variations. This finding, together with the results of a previous study showing that CORT administration raises the frequency of apoptotic Obs, does not support a direct relationship between apoptosis and Bax overexpression, at least in Obs. The CORT effect might be related to cell types and their state of differentiation, so that Bcl-2 and Bax might regulate not only the machinery of cell death, but also cell proliferation and differentiation.

A histomorphometric, structural, and immunocytochemical study of the effects of diet-induced hypocalcemia on bone in growing rats.

Despite several studies on the effect of calcium deficiency on bone status, there is relatively little information on the ensuing histological alterations. To investigate bone changes during chronic hypocalcemia, weanling rats were kept on a calcium-free diet and deionized water for 28 days while control animals were fed normal chow. The epiphyseal-metaphyseal region of the tibiae were processed for histomorphometric, histochemical, and structural analyses. The distribution of bone sialoprotein (BSP), osteocalcin (OC), and osteopontin (OPN), three noncollagenous bone matrix proteins implicated in cell-matrix interactions and regulation of mineral deposition, was examined using postembedding colloidal gold immunocytochemistry. The experimental regimen resulted in serum calcium levels almost half those of control rats. Trabecular bone volume showed no change but osteoid exhibited a significant increase in all its variables. There were a multitude of mineralization foci in the widened osteoid seam, and intact matrix vesicles were observed in the forming bone. Many of the osteoblasts apposed to osteoid were tartrate-resistant acid phosphatase (TRAP)- and alkaline phosphatase-positive, whereas controls showed few such TRAP-reactive cells. Osteoclasts in hypocalcemic rats generally exhibited poorly developed ruffled borders and were inconsistently apposed to bony surfaces showing a lamina limitans. Sometimes osteoclasts were in contact with osteoid, suggesting that they may resorb uncalcified matrix. Cement lines at the bone-calcified cartilage interface in some cases were thickened but generally did not appear affected at bone-bone interfaces. As in controls, electron-dense portions of the mineralized matrix showed labeling for BSP, OC, and OPN but, in contrast, there was an abundance of immunoreactive mineralization foci in osteoid of hypocalcemic rats. These data suggest that chronic hypocalcemia affects both bone formation and resorption.

Changes in bone turnover after parathyroidectomy in dialysis patients: role of calcitriol administration.

BACKGROUND: Available data on changes in serum levels of bone markers after parathyroidectomy (PTx) in dialysis patients are not uniform. Changes are thought to be due to either a reduction in PTH activity per se or to a direct effect of vitamin D therapy on bone cells. We aimed to verify whether treatment with vitamin D modifies serum levels of markers of bone synthesis (alkaline phosphatase (AP), osteocalcin (BGP), procollagen type I C-terminal peptide (PICP)) and resorption (collagen type I C-terminal peptide (ICTP)) within a period of 15 days in haemodialysis patients with severe secondary hyperparathyroidism following PTx. METHODS: We randomized two groups (A, treatment and B, placebo, 10 patients each) with comparable basal PTH values and measured bone markers 3, 7 and 15 days after surgery. All patients were treated with calcium supplements (i.v. and p.o.), and group A also received calcitriol (2.4+/-1.0 microg/day, p.o.). RESULTS: In both groups, PTx induced significant changes in all the markers evaluated, except for BGP in group B. Compared to basal values, ICTP decreased from 481+/-152 ng/ml in group A and 277+/-126 ng/ml in group B to 267+/-94 and 185+/-71 ng/ml (M+/-SD) respectively, and PICP increased from 307+/-139 ng/ml in group A and 309+/-200 ng/ml in group B to 1129+/-725 and 1231+/-1267 ng/ml (M+/-SD) respectively, within 3 days of surgery. AP values increased after 15 days from 1115+/-734 mU/ml in group A and 1419+/-1225 mU/ml in group B to 1917+/-1225 and 1867+/-1295 mU/ml (M+/-SD) respectively. On the contrary, mean values of BGP were never different from basal levels after PTx in either group. In the two groups, the pattern of changes of all the bone markers after PTx was almost identical. Group A patients predictably required lower doses of oral calcium supplements to correct hypocalcaemia (16. 9+/-5.7 vs 22.1+/-5.0 g/10 days; M+/-SD, P<0.04). CONCLUSIONS: The opposite behaviour of serum PICP and ICTP after PTx, in both the treated and untreated groups suggests that quantitative uncoupling between bone synthesis and resorption is responsible for hypocalcaemia. This phenomenon, as reflected by the evaluated bone markers, is unaffected by calcitriol. Based on our data we conclude that immediately after parathyroid surgery, vitamin D therapy does not influence bone cell activity, but improves hypocalcaemia mainly through its known effect on intestinal calcium absorption.

Evaluation of apoptosis and the glucocorticoid receptor in the cartilage growth plate and metaphyseal bone cells of rats after high-dose treatment with corticosterone.

A connection has been suggested between glucocorticoid-induced osteopenia and an increase in the apoptosis of bone cells, and between the dimerization of the glucocorticoid receptor (GR) and the development of apoptosis. On this basis, a study has been carried out on the relationships between the occurrence of apoptotic cells and their detectable GR content, and between apoptosis frequency and changes in histomorphometric variables, in the growth plate and secondary spongiosa of rat long bones after the high-dose (10 mg/day) administration of corticosterone (CORT) and after recovery. The main results of the CORT treatment were: a significant increase in apoptotic osteoblasts, and a concomitant decrease in the histomorphometric variables of bone formation, with a reversal of both values during recovery; a nonsignificant increase in the apoptosis of osteoclasts, without changes in the histomorphometric variables of bone resorption; a significant increase in apoptotic terminal hypertrophic chondrocytes; the presence of GR in all types of skeletal cells in control rats, with different (cytoplasmic and/or nuclear) immunohistochemical detection in the same type of cell; a decrease in GR detection in proliferative chondrocytes and osteocytes in CORT and recovery groups, and in the maturative/hypertrophic chondrocytes of the recovery group; a fall in growth cartilage width, possibly due to the reduced proliferation of proliferative chondrocytes and increased apoptosis in terminal hypertrophic chondrocytes. In conclusion, pharmacological doses of CORT reduce bone formation by increasing osteoblast apoptosis; they reduce growth cartilage width, probably by inhibiting chondrocyte proliferation and increasing the apoptosis of terminal hypertrophic chondrocytes, and they reduce osteocyte GR. Although these effects appear to be mediated by the presence of GR in all skeletal cells, no precise correlation between GR immunohistochemical detection and apoptosis induction has been found.

Relationship between antral lymphocyte density and basal gastrin levels in patients with Helicobacter pylori infection.

BACKGROUND: The mechanism by which Helicobacter pylori causes hypergastrinaemia is not completely understood. AIM: To evaluate whether antral lymphocyte density could play a role in this alteration. METHODS: A total of 12 patients with active duodenal ulcer and 10 with non-ulcer dyspepsia were enrolled upon detection of Helicobacter pylori infection at endoscopy Enrolled as controls were 7 matched dyspeptic patients without Helicobacter pylori infection. Biopsy specimens were collected for Helicobacter pylori and histological assessments, and for antral lymphocyte density assessment by a histomorphometric method. A blood sample was obtained from each patient to determine basal gastrin levels. All patients were controlled by a further endoscopy 4 weeks after the end of Helicobacter pylori treatment. RESULTS: Antral lymphocyte density (5,464 +/- 1,328 and 5,635 +/- 1,186 vs 2,267 +/- 557 lymphocytes/mm2; p<0.001 and p<0.001, respectively) and gastrin levels (66.7 +/- 14.1 and 60.4 +/- 21.7 vs 40.7 +/- 7.8 pg/dl; p=0.004 and p=0.02, respectively) were higher in duodenal ulcer and non-ulcer dyspepsia patients than in controls, while no significant differences emerged between duodenal ulcer and non-ulcer dyspepsia patients. There was a significant direct correlation between antral lymphocyte density and gastrin levels both in duodenal ulcer (r=0.77; p=0.003) and in non-ulcer dyspepsia (r=0.75; p=0.03) patients, while no correlation was found in controls [r=0.12; p=0.8). After treatment, this correlation persisted in 10 eradication failure patients (r=0.68; p=0.027), but disappeared in those successfully cured. CONCLUSIONS: These data suggest that lymphocyte density in the antral mucosa could play a role in the impaired gastrin production occurring in patients with Helicobacter pylori infection.

Measurement variability and diagnostic sensitivity of gastric mucosal inflammatory cell morphometry.

OBJECTIVE: To assess the variability and sensitivity of morphometric measures of gastric mucosal lymphocyte and plasma cells to determine a systematic procedure for evaluating the density of these mononuclear inflammatory cells (MNC). STUDY DESIGN: Gastric biopsies of antrum (n = 3), incisura angularis (n = 2) and corpus (n = 3) from two controls and three patients with Helicobacter pylori-related gastritis (antral, diffuse or multifocal gastritis) were considered. In each biopsy, three fields from each of three sections were selected. In each field, stromal area was obtained by subtracting gland area (GA) from total area, and MNC were counted. Results were expressed as MNC/total mm2 and MNC/stromal mm2. Correlations with GA, coefficients of variation (CV), discriminant power analysis and analysis of variance were performed. RESULTS: Correlations always existed between GA and MNC/total mm2 and rarely between GA and MNC/stromal mm2. CV of MNC/stromal mm2 were lower (18%) than those of MNC/total mm2 (30%). High sensitivity (95.4%) and specificity (95.8%) were found for MNC/stromal mm2 but not for MNC/total mm2. Differences in MNC/stromal mm2 existed in all subjects (P < .0001). Highly significant differences in MNC/stromal mm2 were also found between normal and inflammatory states, gastric sites and sections. CONCLUSION: In contrast to MNC/total mm2, MNC/stromal mm2 is an unbiased estimate of MNC density. The following sampling procedure is proposed: two biopsies from each gastric site, two sections from each biopsy and two microscopic fields from each section.

Use of ultrasonography in the diagnosis of osteomalacia: preliminary results on experimental osteomalacia in the rat.

This study was performed to investigate the ability of ultrasonographic technique to distinguish osteomalacia from normal bone with the same mineral content. Ten rats with experimentally induced osteomalacia (group A) and 12 control rats having similar body size and weight (group B) were studied. Histomorphometric analysis confirmed the presence of osteomalacia in two rats from group A and showed normally mineralized bone in two rats from group B. Whole body bone mineral density, measured by dual-energy x-ray absorptiometry, was similar in the two groups (86 +/- 6 mg/cm2 in group A and 89 +/- 4 mg/cm2 in group B). The velocity of the ultrasound beam in bone was measured by densitometer at the first caudal vertebra of each rat. The velocity was measured when the first peak of the waveform reached a predetermined minimum amplitude value (amplitude-dependent speed of sound) as well as at the lowest point of this curve before it reaches the predetermined minimum amplitude (first minimum speed of sound). Although the amplitude-dependent speed of sound was similar in the two groups (1381.9 +/- 11.8 m/s in group A and 1390.9 +/- 17.8 m/s in group B), the first minimum speed of sound was clearly different (1446.1 +/- 8.9 m/s in group A and 1503.3 +/- 10.9 m/s in group B; P < 0.001). This study shows that ultrasonography could be used to identify alterations in bone quality, such as osteomalacia, but further studies need to be carried out before this method can be introduced into clinical practice.

Cytochemical demonstration of the glucocorticoid receptor in skeletal cells of the rat.

The in vivo localization of the glucocorticoid receptor (GR) was studied in cartilage and bone cells of femurs of young adult rats. Deparaffinized sections were treated with a polyclonal antibody raised against the amino-terminus of human GR; the immunoreaction was detected with the streptavidin-biotin amplification method. Histomorphometric, computer-assisted analysis of GR-positive cells was performed by counting the percentage of GR-immunostained cells in the proliferative and maturative/hypertrophic zone of the epiphyseal growth plate cartilage, and of the percentage of positive osteoblasts (OBs), osteoclasts (OCLS) and osteocytes (OCs) in the metaphyseal secondary ossification zone. Numbers of OBs and OCLs per mm of metaphyseal endosteal perimeter, and numbers of OCs per mm2 of trabecular area were also counted. Immunopositive cells were found both in cartilage and bone, with variable degree of nuclear and/or cytoplasmic immunostaining; immunonegative cells were present among the positive ones. Histomorphometry showed that about 54% of chondrocytes in the proliferative zone, and 55% of chondrocytes in the maturative/hypertrophic zone of the growth plate were labeled; in metaphyseal bone, 68% of OBs, 65% of OCs, and 98% of OCLs were GR-positive. The density of positive cells was 12.06 OBs/mm, 3.32 OCLs/mm, and 520.40 OCs/mm2. These results, for the first time obtained in vivo, show that GR is present in cartilage and bone cells, and that the degree of GR-immunostaining is variable in the same type of cell. This may be dependent on the cell cycle and stage of differentiation, and may reflect a variable cellular sensitivity to the stimulation of the glucocorticoid hormone.

Gastrectomy osteopenia in the rat: the role of vitamin B12 deficiency and the type of reconstruction of the digestive tract.

1. The mechanisms underlying gastrectomy osteopenia are not yet clear. The gastrectomy-associated cobalamin (vitamin B12) deficiency may favour osteopenia and skeletal fractures. Also, the exclusion of the duodenum from the food passage may contribute to gastrectomy osteopenia. To investigate this, rats were gastrectomized and the passage of nutrients restored either with the duodenum excluded (Roux Y) or included (Longmire). Sham-operated rats served as controls. In half of the rats in each gastrectomy group the serum B12 levels were normalized by parenteral administration of B12.2. Four months post operation, both gastrectomy groups showed a similar degree of osteopenia. There was normal bone mineralization; serum levels of parathyroid hormone were normal, but decreased for 25-hydroxyvitamin D, and elevated for 1,25-dihydroxyvitamin D; in urine there was decreased pH and excessive hyperphosphaturia.3.B12 therapy had no influence on any of the essential bone and mineral metabolic parameters.4. We conclude that osteopenia in the gastrectomized rat (i) is not due to B12 or folic acid deficiency, calcium deficiency or secondary hyperparathyroidism; (ii) is independent of the type of anatomic reconstruction of the digestive tract; (iii) appears to be related to disturbed vitamin D, phosphorus and acid-base metabolism.

Bone markers in the diagnosis of low turnover osteodystrophy in haemodialysis patients.

BACKGROUND: Renal osteodystrophy includes a number of low and high turnover bone histologic patterns which require a bone biopsy for their full identification. The role of intact PTH and several classical and more recent bone markers in the non-invasive diagnosis of renal bone disease in patients with CRF in HD requires further definition since available published data are limited. METHODS: In addition to intact PTH, alkaline phosphatase (AP) and osteocalcin (BGP), bone alkaline phosphatase isoenzyme (BALP), tartrate resistant acid phosphatase (TRAP), C-terminal cross-linked peptide of collagen type 1 (ICTP) and deoxypyridinoline (DPD) were measured in the serum of 41 patients on haemodialysis, subjected at the same time to transiliac bone biopsy for histomorphometric, histodynamic and aluminium histochemical examination. Histodynamic evaluation following double tetracycline label, was carried out in 37 patients. The patients had no evidence of active cytolytic and cholestatic liver disease and a history of very limited aluminium exposure. RESULTS: The patients had differing degrees of hyper-parathyroidism, with intact PTH ranging from normal to very elevated levels. Serum values of the markers BGP, ICTP and DPD, normally excreted through the kidneys, were on average very high. The correlation coefficients of the humoral parameters vs dynamic variables, such as BFR/BS, were high. The highest values were: intact PTH 0.798, AP 0.900, BALP 0.891, ICTP 0.807. The patients, grouped in low turnover osteodystrophy (LTO; 9), mixed osteodystrophy (MO; 9) and prevalent hyperparathyroidism (HP; 23), showed significant difference in the levels of most humoral and static and dynamic parameters (ANOVA). Bone aluminium histochemistry was negative in all cases. Discrimination of LTO patients from the other groups by humoral parameters, at the highest value of accuracy, showed 100% sensitivity and 93.7% specificity with a cut-off of 12.9 ng/ml for BALP; 88.9% sensitivity and 93.7% specificity with a cut-off of 21.5 ng/ml for DPD, and 88.9% sensitivity and 90.6% specificity with a cut-off of 79.7 pg/ml for intact PTH. The other markers had lower values. A standardized z-score approach for evaluation of all humoral parameters was also carried out. Using all variables, a correct classification of MO/HP and of LTO was possible in 93.8 and 88.9% of the cases, respectively. Predictive power was 96.8 and 80%, respectively for MO/HP and LTO. When the only variables used were intact PTH and BALP, a correct classification of MO/HP and LTO was possible in 90.6% and 88.9%, respectively. Predictive value of MO/HP was 96.7% and for LTO 72.7%. Predictive values using PTH and AP were 96.3% and 57.2%, respectively. CONCLUSION: Intact PTH and several relatively new bone markers are of certain value in the non-invasive diagnosis of renal osteodystrophy. However some of the humoral markers carry the same quality of information and the use of intact PTH and BALP may be adequate in the discrimination of bone histologic patterns. In cases exempt from liver disease, PTH and AP may be used as a less costly alternative. Bone biopsy could be chiefly limited to cases with borderline humoral values and to all those with a suspected aluminium overload.

A histomorphometric long-term longitudinal study of trabecular bone loss in glucocorticoid-treated patients: prednisone versus deflazacort.

Administration of a corticosteroid with minor osteopenic effects is considered an effective prevention of glucocorticoid osteoporosis. Deflazacort, an oxazolinic derivative of prednisolone, is reported to be less harmful to cancellous bone mass than other equally effective corticosteroids. However, comparative long-term studies, particularly on trabecular bone, are needed before a smaller detrimental effect on bone of deflazacort can be unequivocally confirmed. We conducted such a prospective long-term study using histomorphometric analysis of iliac bone. For the study, 18 pairs of nonimmobilized patients, matched for age, sex, menopausal state, corticosteroid dose, and type and severity of the disease, were randomly submitted to treatment with therapeutically equivalent doses of prednisone or deflazacort. Bone biopsies from iliac crest were taken before and at various times during treatment. In order to represent the time-related trabecular bone loss and find out possible differences between patients on prednisone or deflazacort, a previously described model of bone loss kinetics was applied. No significant differences in biochemical indices of bone turnover or in histomorphometric variables between prednisone- and deflazacort-treated patients were recorded before treatment. The mean duration of treatment at the final biopsy was similar for prednisone and deflazacort (15.8 and 15.2 months, respectively). Patients showed evident clinical improvement with both treatments. Osteoid and resorption surfaces showed no significant differences throughout the observation period in any of the 18 pairs. On the contrary, both steroids induced a significant decrease in trabecular bone, although the bone loss rate induced by prednisone was significantly higher than that induced by deflazacort (P < 0.05). The kinetics of bone loss and the different osteopenic effects of the two drugs are described by the negative exponential function fitted to data from patients never previously given glucocorticoids; the model yields highly significant difference (P approximately equal to 0.01) between the two drugs and allows estimation of the difference even 3 years after the beginning of treatment (-3.0%/year versus -1.1%/year for prednisone and deflazacort, respectively). This prospective long-term study confirms that an exponential model accurately describes the trabecular bone loss induced by long-term corticosteroid treatment and demonstrates that deflazacort, at therapeutically effective doses, induces less trabecular bone loss than prednisone.

In vivo incidence of apoptosis evaluated with the TdT FragEL DNA fragmentation detection kit in cartilage and bone cells of the rat tibia.

Previous studies have shown the occurrence of cell death by apoptosis in cartilage and bone cells, and have suggested a functional relationship between bone growth and remodelling on one hand, and numbers of apoptotic cells on the other. At present, no in vivo studies are available on the frequency of the apoptotic process measured at one time and in one place using the cartilage and bone cells of single specimens. The aim of the present investigation was to measure the in vivo incidence of apoptosis in cartilage and bone cells of the upper epiphysis and secondary ossification metaphyseal bone of the tibia in normal young adult rats. Apoptotic cells were visualized with the terminal deoxynucleotidyl transferase (TdT) FragEL DNA fragmentation detection kit, which is analogous to the TdT-mediated nick end-labelling (TUNEL) method. In the growth cartilage, only a few TUNEL-positive terminal hypertrophic chondrocytes were found; they were 1.32 +/- 0.70% of the total hypertrophic chondrocytes counted along the chondro-osseous junction. There were only a few apoptotic osteoblastic cells and osteocytes (0.22 +/- 0.22% and 0.15 +/- 0.16% of total osteoblasts and osteocytes respectively). TUNEL-positive osteoclasts were 1.03 +/- 0.57% of the total of osteoclastic cells; they usually showed only one or two apoptotic nuclei. The total number of TUNEL-positive bone marrow cells were also counted (56.78 +/- 10.29/mm2 of bone marrow spaces). Our results confirm that apoptosis does occur in hypertrophic chondrocytes and bone cells, and show that its frequency is very low. However, chiefly because of its short lifespan, the frequency of apoptosis in cartilage and bone may be higher than that shown by the TUNEL method. The static estimate that can be obtained with this method might lead to misleading conclusions on the physiological significance of such a dynamic, rapid and asynchronous process, whose precise importance in bone growth and remodelling remains to be determined.