Search

The osteocyte is often considered to be a cell that is hardly active, yet it turns out to have a central role in bone remodeling. It functions as a mechanoreceptor that translates mechanical stimuli into biochemical signals. The expression of PGE2 by loaded osteocytes and the consequent activation of the cAMP/PKA pathway, together with stabilization of β-catenin, may permit cross talk with the canonical Wnt/β-catenin pathway, the activation of which is increased by the load-dependent reduction of the expression of DKK1 and of sclerostin (Sost, the secretion product of old osteocytes). These signal pathways, as well as TGF-β and BMP expression, stimulate the differentiation and activity of osteoblasts, reduce their apoptosis, and enhance bone formation. The expressions of MEPE, FGF-23 and DMP-1 further contribute to the osteocytic control of bone metabolism. Periosteocytic osteolysis, if definitively demonstrated, would constitute another fundamental function of osteocytes. A thorough understanding of the osteocyte’s activities could open up new strategies in the therapy of metabolic bone diseases.

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.

Osteoprotegerin (OPG) and the receptor activator of nuclear factor (NF)-kB ligand (RANKL) are key regulators of osteoclastogenesis. The present study had the main aim of showing the localization of OPG and RANKL mRNA and protein in serial sections of the rat femurs and tibiae by immunohistochemistry (IHC) and in situ hybridization (ISH). The main results were: (1) OPG and RANKL mRNA and protein were co-localized in the same cell types, (2) maturative/hypertrophic chondrocytes, osteoblasts, lining cells, periosteal cells and early osteocytes were stained by both IHC and ISH, (3) OPG and RANKL proteins were mainly located in Golgi areas, and the ISH reaction was especially visible in active osteoblasts, (4) immunolabeling was often concentrated into cytoplasmic vacuoles of otherwise negative proliferative chondrocytes; IHC and ISH labeling increased from proliferative to maturative/hypertrophic chondrocytes, (5) the newly laid down bone matrix, cartilage-bone interfaces, cement lines, and trabecular borders showed light OPG and RANKL immunolabeling, (6) about 70% of secondary metaphyseal bone osteocytes showed OPG and RANKL protein expression; most of them were ISH-negative, (7) osteoclasts were mostly unstained by IHC and variably labeled by ISH. The co-expression of OPG and RANKL in the same bone cell types confirms their strictly coupled action in the regulation of bone metabolism.

Porous, non-resorbable, granular hydroxyapatite (pHA), either pure or mixed with fibrin, or soaked in solutions of the growth factors PDGF, TGF, IGF, or TNF, has been implanted in rabbits under the derma and periosteum of the skull, in contact with the calvarial bone. The degree of osteogenesis in the implant was evaluated by histological analysis and histomorphometry 20 and 60 days after the implant. The results showed that new bone formation occurred in all the implants, most evidently in those containing TGF, TNF or IGF. The histomorphometric results, however, showed no significant differences between the groups, perhaps because of technical artifacts and the small number of animals. On the other hand, the formation of new bone was due to osteoconduction rather than osteoinduction, as shown by the observation that osteogenesis decreased with the distance of pHA granules from the calvarium. The earliest evidence of osteogenesis was the presence of dense borders on the surface of pHA: some of them were vacuolated and contained glycoproteins, others resembled osteoid borders. The bone formed at 20 days was of woven type, whereas that present at 60 days was of both woven and lamellar type, suggesting that some degree of remodeling takes place as the implant ages.

The hormone leptin is considered to have a role in the prevention of osteoporosis and probably acts on bone tissue through inhibition of osteoclasia. Its action has been attributed to interference in osteoprotegerin (OPG)/OPG-ligand equilibrium. Contradictory data also have been reported, casting doubts on the positive effect on bone mass of the hormone, at least in males. To date, the relation between serum leptin levels of dialysis patients and renal osteodystrophy, defined by histomorphometric and histodynamic parameters of bone, has not been studied.The study included 46 hemodialysis patients (32 men, 14 women; age, 57.2 +/- 11.4 years). A transiliac bone biopsy after double-tetracycline labeling was performed for histological, histomorphometric, and histodynamic studies. Blood samples were drawn for leptin, intact parathyroid hormone (PTH), whole PTH (PTH1-84), OPG, bone alkaline phosphatase, calcium, phosphate, 25-hydroxycholecalciferol, and calcitriol. Serum leptin was measured by means of a radioimmunoassay.Eighteen patients had mixed osteodystrophy (MO); 17 patients, hyperparathyroidism; 9 patients, adynamic bone disease (ABD); and 2 patients, osteomalacia. Aluminum histochemistry results were positive in 1 patient with ABD and 1 patient with MO. A sex difference was found in serum leptin levels (48.9 +/- 38 ng/mL in women and 12.2 +/- 13.2 ng/mL in men; P0.0002). In the entire population, lnleptin correlated significantly with body mass index (BMI; P0.01). SD score (SDS) leptin (adjusted for BMI, sex, and age) correlated inversely with PTH1-84 level and osteoclastic surface (OcS/BS; P0.05) and had a borderline correlation with bone formation rate. Correlations between leptin levels and other parameters were enhanced in men. SDS leptin correlated inversely with OcS/BS (P0.01), osteoclastic number (P0.01), and mineral apposition rate (P0.01). In addition, SDS leptin had a borderline inverse correlation with osteoblast surface (P0.06) and significant correlation with OPG level (P0.05). No difference was found in serum leptin levels between histological groups.The reported data confirm the finding of a positive relation between serum leptin level and BMI and greater levels in women compared with men. Serum leptin level is connected to bone resorption and also bone formation, both inversely related to serum leptin levels. The decrease in osteoclasia that accompanies increasing serum leptin levels does not seem to be related to an enhanced OPG effect because it was accompanied by decreased OPG levels. Low-turnover bone disease does not appear to be caused by increased serum leptin levels. The nature of the interrelation between serum leptin and PTH1-84 levels requires further study.

The secondary localization of neoplastic cells in bone is a severe event which often brings the life of patients suffering from cancer to a dramatic end. It mostly occurs in carcinomas of the prostate, breast, lung, thyroid and kidney, but its real frequency is hard to determine, because statistics are spoiled by diverging criteria for patient selection, evaluation methods, bone types, cancer cell phenotypes, and other variables. Its clinical interest is very high, because bone metastases not only testify to an advanced, often irreversible stage of neoplastic disease, but may also be responsible for severely distressing and painful, potentially lethal changes such as fractures, nerve compression syndromes, and hypercalcemia. These changes also pose very interesting biological problems. For all these reasons, bone metastases have been and continue to be at the center of a number of investigations which, however, have not completely solved the many physiopathological problems connected with their development and with the changes they induce in bone remodeling. The present paper, based on personal experience and on data recorded in the literature, reviews the main aspects of these problems. Emphasis is laid on the long and complicated route neoplastic cells must follow to reach bone marrow spaces and grow within them — on the way they spread through the stroma at the primary site, on how they reach the capillary vessels and penetrate into them by degrading the vascular basal lamina and the endothelium, how they migrate along the bloodstream and escape the surveillance of immunocompetent cells, so reaching the bone marrow sinusoids, where they grow, how they again degrade the wall of the capillary vessels, penetrate into the bone marrow spaces and grow in them, and how they modify the bone remodeling process, so giving rise to clinically manifest skeletal metastases. Each of these steps is mediated and controlled not only by the direct relationship between neoplastic cells and normal bone cells and structures, but also by diffusible, autocrine and/or paracrine substances such as growth factors, cytokines and enzymes which can be expressed by neoplastic and host cells, or be stored in the calcified matrix and then be released and activated during osteoclastic bone resorption. In these processes, a primary role is assigned to metalloproteinases and their inhibitors as regulators of the spread of neoplastic cells from the primary site into the stroma, their penetration through the vessel wall, their extravasation, their localization in bone marrow spaces, and their induction of angiogenesis. The role of the RANKL-RANK-OPG system, as well as that of PTH, PTHrP, 1,25(OH)2D3, cytokines and other factors (M-CSF, TNF-α), is discussed in connection with the process of bone resorption and the regulation of osteoclast activity, together with the role of growth factors, especially of TGF-β, BMP, IGF-I, and is considered in connection with bone formation and osteoblast activity. The alterations induced by neoplastic cells in the functional phases of the basic multicellular unit, and the resulting osteoclast-osteoblast uncoupling and modifications of bone remodeling, are discussed. The histopathology, ultrastructure and immunohistochemistry of predominantly osteolytic, predominantly osteoblastic and mixed bone metastases, and of the neoplastic cells present in them, are described and illustrated. On the whole, this review shows that, although several types of data are still not available, an impressive amount of information has been gathered on the physiopathology of bone metastases and their relationship with bone and bone marrow. This not only attests a keen scientific interest in bone metastases, but, more importantly, justifies the hope that the knowledge we need to achieve their prevention and therapy will become accessible in the near future.

Background: The intact parathyroid hormone (PTH) assay evaluates levels of serum 1-84 PTH and other N-terminally truncated PTH fragments, mainly PTH "7-84." This PTH molecule has been found experimentally to interfere with biological activity of PTH 1-84, perhaps through its binding to the PTH receptor complex. Therefore, assuming that high levels of PTH 7-84 are a cause of bone resistance to PTH, it has been hypothesized that a decreased 1-84 to 7-84 PTH ratio caused by a relative increase in PTH 7-84 level might help in the noninvasive diagnosis of low-turnover osteodystrophy (LTO). Methods: This study was performed in 35 patients with chronic renal failure on hemodialysis therapy who underwent bone biopsy for a histological, histomorphometric, and histodynamic study. In addition, blood samples were obtained for intact PTH, 1-84 PTH, and total PTH assays. PTH 7-84 level was obtained from the difference between total and 1-84 PTH assay results. Results: Nine patients had LTO (8 patients, adynamic bone disease; 1 patient, osteomalacia), 12 patients had hyperparathyroidism (HP), and 14 patients had mixed osteodystrophy (MO). On average, 1-84 PTH levels were approximately 60% of mean values for intact PTH. The two assays were strictly correlated. Average 1-84 to 7-84 PTH ratios were 1.57 ± 0.85, 1.73 ± 1.31, and 1.95 ± 2.1 in the three histological groups (LTO, HP, and MO, respectively), with no significant difference. Conclusion: Contrary to previous expectations, results do not favor the hypothesis of a role of 7-84 PTH in bone resistance in renal osteodystrophy. The 1-84 to 7-84 PTH ratio is not a marker of LTO and is of no use in noninvasive histological diagnosis. © 2002 by the National Kidney Foundation, Inc.

Background. Numerous growth factors and cytokines are known to modulate bone turnover. An important, recently discovered complex involved in osteoclastogenesis is the osteoprotegerinuosteoprotegerin-ligand (OPGuOPGL) cytokine complex, which is produced by osteoblasts. Many factors, including parathyroid hormone (PTH), appear to affect bone turnover through this pathway. In this disorder, the role of the OPGu OPGL system in the pathogenesis of renal osteodystrophy, a disease with either low or high bone turnover, has not been investigated so far. Methods. Thirty-nine chronic haemodialysis patients had bone biopsies, including histomorphometric and histodynamic examinations. In addition, the following serum biochemistry parameters were measured: serum OPG, intact PTH, PTH 1–84, total PTH, osteocalcin, total and bone alkaline phosphatases, 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol. Results. On average, serum OPG levels were above the normal range. They were lower in adynamic bone disease (ABD) patients, than in patients with predominant hyperparathyroidism (HP) or mixed osteodystrophy (MO). Significant negative correlations were found between serum OPG and PTH levels, and between serum OPG and parameters of bone resorption (ESuBS) and bone formation (ObSuBS and BFRuBS) in HP and MO patients with PTH values O1000 pguml. For intact PTH levels O300 pguml, serum OPG was significantly lower in the group with ABD than in those with HP or MO (P-0.05). Conclusion. In renal osteodystrophy the OPGuOPGL system is involved in the regulation of bone turnover induced by PTH. The determination of serum OPG levels could be of use in the diagnosis of low turnover bone disease, at least in association with PTH levels O300 pguml.

Background: Comparison of renal osteodystrophy in predialysis and hemodialysis has been rarely reported. Distinct patterns of renal osteodystrophy could be found in these conditions. In addition the use of parathyroid hormone (PTH) and other markers for noninvasive diagnosis may result in different predictive values in predialysis and hemodialysis patients. Methods: 79 consecutive patients with conservative chronic renal failure and 107 patients on hemodialysis were studied. All patients were subjected to bone biopsy for histological and histomorphometric evaluation. The patients had no exposure to aluminium before dialysis and relatively low exposure while on hemodialysis. Results: In the predialysis patients, bone biopsies showed 9 cases of adynamic bone disease (ABD) and 8 cases of osteomalacia (OM), 50 patients with mixed osteodystrophy and 2 cases of hyperparathyroidism. Among the hemodialysis patients 12 cases had ABD, 3 cases OM, 30 mixed osteodystrophy, and 61 patients hyperparathyroidism. In the predialysis patients with chronic renal failure, bone aluminium was on average 4.5 mg/kg dry weight, while in dialysis patients the average value was 35.4 mg/kg dry weight. Discriminant analysis of low turnover osteodystrophy (ABD and OM) by intact PTH showed higher accuracy in dialysis than in predialysis patients. Correlation studies of intact PTH versus bone formation rate, osteoblast surface/bone surface and osteoclast surface/bone surface showed significantly steeper slopes in dialysis than in predialysis patients, which indicates that bone resistance to PTH is more marked in predialysis patients. Conclusions: The prevalence of ABD and OM in the geographic area investigated is lower than in other reports. Aluminium exposure does not seem to be the cause of low turnover osteodystrophy in the present population. The predictive value of intact PTH in the noninvasive diagnosis of renal bone disease is higher in hemodialysis patients than in predialysis patients. Predialysis chronic renal failure, when compared to the dialysis stage, seems to be characterized by resistance of bone tissue to PTH.

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.

During the maturation stage of amelogenesis, the loss of matrix proteins combined with an accentuated but regulated influx of calcium and phosphate ions into the enamel layer results in the “hardest” tissue of the body. The aim of the present investigation was to examine the effects of chronic hypocalcemia on the maturation of enamel. Twenty-one-day old male Wistar rats were given a calcium-free diet and deionized water for 28 days, while control animals received a normal chow. The rats were perfused with aldehyde and the mandibular incisors were processed for histochemical and ultrastructural analyses and for postembedding colloidal gold immunolabeling with antibodies to amelogenin, ameloblastin, and albumin. The maturation stage enamel organ in hypocalcemic rats exhibited areas with an apparent increase in cell number and the presence of cyst-like structures. In both cases the cells expressed signals for ameloblastin and amelogenin. The content of the cysts was periodic acid–Schiff- and periodic acid–silver nitrate–methanamine-positive and immunolabeled for amelogenin, ameloblastin, and albumin. Masses of a similar material were also found at the enamel surface in depressions of the amelo-blast layer. In addition, there were accumulations of glycoproteinaceous matrix at the interface between ameloblasts and enamel. In decalcified specimens, the superficial portion of the enamel matrix sometimes exhibited the presence of tubular crystal “ghosts.” The basal lamina, normally separating ameloblasts and enamel during the maturation stage, was missing in some areas. Enamel crystals extended within membrane invaginations at the apical surface of ameloblasts in these areas. Immunolabeling for amelogenin, ameloblastin, and albumin over enamel was variable and showed a heterogeneous distribution. In contrast, enamel in control rats exhibited a homogeneous labeling for amelogenin, a concentration of ameloblastin at the surface, and weak reactivity for albumin. These results suggest that diet-induced chronic hypocalcemia interferes with both cellular and extracellular events during enamel maturation. (J Histochem Cytochem 48:1043–1057, 2000)

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. (J Histochem Cytochem 48:1059–1077, 2000)

Reconstruction of skeletal defects can be carried out by implantation of autologous bone removed from the iliac crest by surgery. To avoid this operation, and to achieve more favourable conditions of implantation, autologous bone can be replaced by biomaterials. For this reason, a morphological investigation has been carried out on the ossification process promoted by implantation in cranio-facial bones of granular, porous, non-resorbable hydroxyapatite mixed with fibrin glue. The results show that this material promotes ossification through the initial formation of fibrillar connective tissue, whose collagen fibrils form osteoid-like borders on the surface of hydroxyapatite. Deposition of calcium salts on these borders leads to formation of bone tissue which is completed in about 9 months, remains practically unchanged during many years, and is compact and hard enough to allow prosthesis application.

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.

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 (P0.05). 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.

Bone formation in hydroxyapatite-fibrin implants has been reported several times. However, available studies refer to experimental animals, or are limited to short periods after implantation. We report the results of histological, histochemical and ultrastructural studies carried out 2.5-8 yr after implantation of non-resorbable, porous hydroxyapatite (HA) and fibrin glue in human maxillofacial bones. Prominent ossification was found in all cases, with the presence of normally structured spongy bone. HA granules were embedded in the calcified bone matrix. They had not elicited inflammatory reactions and did not induce bone resorption. Ossification was preceded by the appearance of alkaline phosphatase activity on fibroblast-like cells, and by the formation of dense collagenous layers, similar to osteoid borders, on the surface of HA granules. The early phases of the calcification process occurred in these borders, with the appearance of calcification nodules adjacent to alkaline phosphatase-positive osteoblast-like cells. A remodeling process similar to that occurring in normal bones was found in the newly formed bone. These results justify the conclusion that HA-fibrin implants lead to the formation of long-lasting bone that does not differ from that of the normal maxillofacial skeleton. Mixing the HA granules with fibrin has the advantage of creating an easily mouldable material which can be adapted to any skeletal surface and stays in place after surgery.

Summary The bone tissue formed in orthotopic or heterotopic implants of granular, porous hydroxyapatite and fibrin glue was examined several (2½–8) years after implantation. The results showed distinct ossification in all cases and the functional situation and external appearance were also satisfactory. The reconstituted spongy and compact bone underwent a remodelling process similar to that of normal bone. Most of the hydroxyapatite granules were embedded in the bone tissue; a few were very close to the fibrillar connective tissue of the intertrabecular spaces and were either covered by osteoid-like collagenous borders or were in contact with osteoclast-like giant cells. Even if the results refer only to a few patients, and were obtained from a cross-sectional study, they allow the conclusion to be reached that the implantation of porous hydroxyapatite and fibrin glue leads to the formation of long-lasting bone whose hardness is equal to, or greater than, that of normal bone.

Osteoporosis is a very common skeletal disorder characterized by reduced bone mass and altered trabecular microarchitecture that leads to bone fragility and fractures. Such disease is due to alterations of the remodeling process that occurs in the basic multicellular units that are transitory cellular complexes including an osteoclastic phase (osteoclast activation and resorption of microscopic portions of bone), a reversion phase (osteoclast replacement by so-called postosteoclastic cells), and an osteoblastic phase (osteoblastic reconstruction of the resorbed bone matrix till the initial volume is regained). Bone remodeling is regulated by a number of systemic and local factors; among the former, besides physical activity and mechanical stresses, a primary role is played by hormones such as parathyroid hormone, vitamin D metabolites, estrogens, calcitonin, and glucocorticoids; among the latter, several growth factors (macrophage colony-stimulating factor, transforming growth factor β, platelet-derived growth factor, fibroblast growth factor 1, bone morphogenetic protein, and insulin-like growth factor 1), as well as the osteoprotegerin-receptor activator of nuclear factor-κ B ligand system and the sclerostin, play a primary function. The remodeling phases can be evaluated by static and dynamic histomorphometry. Their abnormalities may lead to several osteopathies, the most common of which is osteoporosis (above all senile and postmenopausal), a rather elusive disease chiefly due to its slow development. The use of animal models in its study is emphasized.

Bone is one of the many biological tissues characterized by the still poorly defined process of mineralization, that is, by the deposition of inorganic substance in their organic matrix, and is one that, chiefly due to its important biological functions, but also because of its wide‐ spread presence in primates and its easy availability, has been investigated for hundred of years by thousand of investigators (see reviews by [1-3]). Its study, however, is a demanding task, because of the complexity and variability of the tissue – factors that derive mainly from differences in its histological types, biochemical composition, phases of maturation, and de‐ gree of mineralization (the terms ‘mineralization’, ‘biomineralization’, and ‘calcification’ are treated as equivalents in this paper; see Bonucci [2]). These differences, although recently emphasized [4-5], have not always received due recognition. In this connection, revealing indicative examples include, for instance, the differences between the compact, lamellar bone and spongy, woven bone [4], or between the dense rostrum bone of toothed whales [6] and the medullary bone of birds [7]. These differences may lead to incorrect interpretations of apparently contrasting results, but may also lead to new insights if they are recognized to derive from distinctive traits of tissues belonging to the same broad type, which is what they are. The concepts that follow, which chiefly focus on the local mechanism of matrix calcifica‐ tion, mainly refer to woven bone, which raises fewer technical issues than compact bone, but they are valid for, and can be extended to, all other types of bone and hard tissue.

The role of phospholipids in biological mineralization has been hypothesized but not fully elucidated. In order to identify phospholipids at the ultrastructural level in the mineralizing extracellular matrix, rat epiphyseal cartilage and metaphyseal bone have been labeled with the phospholipase A(2) (PLA(2))-gold method. The specificity and the efficiency of phospholipid detection have been evaluated by postembedding labeling of sections from epoxy- or hydrophilic resin-embedded samples, and by preembedding labeling of cryosectioned samples. The efficiency of the labeling was higher in cryosections than in hydrophilic resin-embedded specimens, while lower efficiency was found in epoxy resin-embedded samples. A 2- to 6-fold increase of the labeling density in calcified with respect to uncalcified areas of cartilage and bone has been found, depending on the specimen preparation used. The labeling intensity was significantly higher, at the periphery of the calcifying nodules in the epiphyseal cartilage matrix and in the calcifying osteoid, while the fully calcified bone matrix presented a weak labeling. Matrix vesicles, which are considered a possible source of extracellular phospholipids, appeared labeled in cryosections and in epoxy resin-embedded samples after a preincubation with PLA(2), which also increased the labeling of the intracellular membranes. The localization of phospholipids in the areas of initial mineralization suggests some hypotheses on the possible involvement of these molecules in the mineral phase deposition process.

The morphological distribution of noncollagenous proteins in bone matrix is poorly known, chiefly because their amount changes with the type of bone, and they are not easily recognizable under the electron microscope. The aim of this study was to obtain further ultrastructural data on these proteins and on their relationships with collagen fibrils and the calcification process. Specimens of woven bone from the upper metaphysis of the tibia of chick embryos were examined after en bloc treatment with acetone solution of KMn04, or aqueous or alcoholic solutions of phosphotungstic acid or uranyl acetate. Untreated specimens were used as controls. Untreated, decalcified, and decalcified and stained sections were examined. As expected, the results showed that the woven bone matrix of the chick embryo consists of irregularly oriented collagen fibrils which outline wide interfibrillary spaces. These contain calcification nodules at the calcification front and are full of irregularly oriented crystals in completely calcified matrix. These spaces contain greater than expected amounts of filamentous or granular organic structures (probably corresponding to noncollagenous proteins), which are in a close relationship with needle- and filament-like crystals. The collagen fibrils are calcified to a lower degree than the interfibrillary spaces, and often appear uncalcified, or decalcified, when the interfibrillary spaces do not. This suggests that either the former calcify after the latter, or the respective mineral phases differ in their degree of solubility, so that the decalcification of collagen fibrils precedes that of interfibrillary spaces. The results confirm that woven bone contains an amount of interfibrillar noncollagenous material greater than lamellar bone, and show that woven bone calcification occurs not only in and on collagen fibrils, but chiefly between them, in relationship with noncollagenous filamentous material.

Bone sialoprotein was immunolocalized at the EM level in thin Lowicryl K4M sections of rat bone. Because of the unconventional EM morphology of the bone matrix seen in thin demineralized acrylate sections, the pattern of immunolabeling was compared with detailed structural images of demineralized bone obtained using an en bloc treatment of tissue samples with the cationic electron 'dye,' Malachite Green (MG), which provides stabilization and retention of anionic material throughout specimen processing. A system of structures corresponding to the sites of bone sialoprotein (BSP) immunoreactivity, as seen in Lowicryl K4M this sections, could be readily identified in the MG-treated, epoxy thing sections. This system includes the cement lines, and aggregates of similar material within mineralized bone and mineralizing osteoid. The virtual identity of BSP distribution with the arrangement of the MG-visualized material indicates that a BSP-enriched, noncollagenous phase can be demonstrated using different, unrelated tissue preparation and imaging protocols for EM. Besides improving our understanding of the distribution of bone sialoprotein in bone, these data assign a previously unrecognized structural dimension to noncollagenous material in the bone matrix.

Alkaline phosphatase (ALP) activity is a new histomorphometric index of the extent of osteoblastic surfaces involved in mineralization. To assess its validity in the evaluation of bone formation, we carried out a comparative study between histomorphometric values obtained on the basis of the extent of tetracycline labeling and of the length of ALP-positive endosteal surfaces. The following variables were compared (indicated by ALP when based on the extent of ALP positivity): trabecular mineralizing surface (MS/BS vs. ALP.SBS); osteoid mineralizing surface (MS/OS vs. ALPS/OS); bone formation rate (BFR/BS vs. ALP.BFR/BS); and adjusted appositional rate (Aj.AR vs. ALP.Aj.AR). Bone biopsies from 39 patients with chronic renal failure and different types of renal osteodystrophy were considered (48 ± 12 years of age; 19 men and 20 women). Patients were double labeled with tetracycline and biopsies were embedded in glycol-methacrylate at +4°C. Patients showed various types of renal osteodystrophy and were assigned to different groups of pathologies. Although it differed in incidence according to the different groups, ALP activity was found in typical plump osteoblasts bordering osteoid seams and in flat cells, either in contact with osteoid or along the quiescent surfaces of bone in continuity with it. Tetracycline codistributed with all these features to variable extents, according to groups. In all patients, however, ALPS/BS and ALPS/OS respectively exceeded MS/BS and MS/OS. In consequence of this, ALP.BFR/BS and ALP.Aj.AR were greater than BFR/BS and Aj.AR, respectively. For each of the variable considered, differences among groups of patients with different types of renal osteodystrophy were highly significant. Good correlations were found between the variables measured with the two methods. Correlations of ALP.BFR/BS and ALP.Aj.AR with serum indices of bone metabolism (calcium, phosphate, osteocalcin, ALP, and C-terminal PTH) were comparable with those of BFR/BS and Aj.AR, respectively. On the basis of the results obtained in our population study, the length of bone surfaces covered by ALP-positive osteoblastic cells greatly overestimates the extent of mineralizing surface as assessed by tetracycline labeling and cannot be assumed to be a reliable indicator of the true extent of the mineralizing surface. Histodynamic variables evaluated by considering bone surfaces positive to ALP to be mineralizing surfaces do not give more accurate information on bone metabolism compared with those derived by taking into account the extent of tetracycline labeling.

Evidence of healing of secondary hyperparathyroidism in chronically hemodialyzed uremic patients treated with long-term intravenous calcitriol. The aim of this study was to assess the effect of a long-term course of high-dose i.v. pulses of calcitriol (CLT) on hyperparathyroid bone disease (HBD) and functional mass of parathyroid glands of chronically hemodialyzed uremic (CHU) patients. We prospectively studied nine CHU patients treated with CLT, 30 ng/kg/body wt, i.v., thrice weekly over a period of eight months. Plasma concentrations of intact parathyroid hormone (iPTH), bone GLA protein (bGLA) and bone isoenzyme of alkaline phosphatase (biALP) were sampled throughout. Transiliac bone biopsies were made before and after the start of CLT therapy. Double scanning scintigraphy of the neck with 201Tl-99Tc was made before, during and eight months after the start of the treatment. All patients but one, who later responded to higher than planned CLT doses, had significant decreases of plasma iPTH (F = 76; P < 0.0001; ANOVA). The mean pretreatment value of PTH was 966 ±160 (mean ± SE) pg/ml and it had decreased significantly by the first week (T = 2.4, P < 0.04), and had fallen an average of 80% by the 35th week. Ionized plasma calcium concentration was 1.19 ± .01 mmol/liter which rose significantly (F = 13.5; P < 0.0001) by the 14th week to maximal peak levels, averaging 1.34 ± .02 mmol/liter. Changes in biALP were parallel to those of iPTH, while bGLA tended to increase immediately after the start of the therapy and to significantly decrease thereafter (T = 3.2; P < 0.01). There were significant decreases in all histomorphometric indices of HBD: the osteoid volume from 12 ± 1.8 to 4.8 ± 0.5% (P < 0.001), the osteoid surface from 45.7 ± 3.2 to 27 ± 5.2% (P < 0.001), the osteoblastic surface from 11.8 ± 1.7 to 4.8 ± 1.08% (P < 0.001), the eroded surface from 6.6 ± 1.2 to 4.3 ± 0.9 (P < 0.002), and the osteoclast surface from 2.2 ± 0.3 to 1.2 ± 0.3% (P < 0.009); there was even renormalization in some individual patients. The bone formation rate was measured in six of the nine patients and was found to be lowered in all subjects who had had significant PTH decreases. The median of intensity of 201Tl uptake before therapy scored 7.8 (range 4 to 9.2) arbitrary visual units and decreased significantly (P < 0.001; Wilcoxon test) to 0.4 (0 to 8.25) after eight months of CLT treatment, suggesting significant decreases of the mass of the parathyroid glands. Thus, this study demonstrates that long-term therapy with high-dose i.v. pulses of CLT in CHU with proven HBD is able to consistently decrease their circulating iPTH to levels comparable with those of subjects with mild or no HBD and also to decrease the functional mass of their parathyroid glands. Further studies will ascertain what dose of CLT is required to maintain this state of euparathyroidism once the hyperparathyroidism has been reversed.

We defined two distinct maturational compartments (proliferative and secretory) of osteogenic cells in vivo on the basis of ALP activity, BrdU incorporation, cell shape, and BSP production. BSP immunoreactivity was found to mark cells in the secretory but not in the proliferative compartment. We established the phenotypic similarity of primitive marrow stromal cells with proliferating perichondral cells (fibroblast-like, ALP+, BrdU+, BSP-). This suggests the potential functional equivalence of the two cell types as committed non-secretory osteogenic cells and points to the duality of osteogenic cell compartments as a generalized feature of bone formation. We further showed that although BSP secretion is a hallmark of the onset of osteogenesis, BSP antigenicity is lost both in osteoid and in a large proportion of mature osteoblasts during subsequent phases of bone deposition. This suggests that bone formation may not be a uniform event, as bone cells actually deposit antigenically, and likely biochemically, distinct matrices at specific times.

Bone sialoprotein (BSP), a bone matrix-enriched glycoprotein containing the Arg-Gly-Asp (RGD) motif and endowed with cell binding properties, was localized in osteoblasts and early bone matrix of developing rat bone at the ultrastructural level. Preliminary light microscopic observations indicated that intracellular labelling was restricted to a paranuclear dot corresponding to the "negative Golgi image" of classical histology. The same pattern was observed whether antisera against the fully glycosylated protein or a peptide antiserum to a stretch of amino acids in human BSP sequence were employed. At the EM level, we obtained labeling over the Golgi area of osteoblasts but not over the rER. The labeling was concentrated over distensions of the trans Golgi and over pro-secretory granules. In the matrix, BSP was distributed in a non-random manner. The label was concentrated over spherical aggregates of finely fibrillar material corresponding to the sites of early mineral deposition (so-called "mineralization nodules"). Such BSP-positive foci were seen both close to and away from the cell surface. The predominant association of BSP with Golgi and post-Golgi secretory structures and its absence from rER, as well as the reproducibility of the same pattern of localization with different antisera, might indicate a slow transit of the protein through the Golgi, not necessarily associated with protein glycosylation.

The effects of ipriflavone on bone cells were studied in vitro on pre-osteoclastic (FLG 29.1) and osteoblast-like (Saos-2) cells grown for 48 h either separately or in co-cultures, with or without the addition of PTH. Histological, ultrastructural and histochemical (TRAP-activity demonstration) methods were used. The main results show that ipriflavone reduces replication of FLG 29.1 cells and inhibits TRAP production by these cells both in controls and in co-cultures treated with PTH. Moreover, it has a moderate stimulatory effect on proliferation of osteoblast-like cells and reduces the PTH-induced degenerative changes of Saos-2 cells. These results suggest that the inhibitory effect of ipriflavone on FLG 29.1 cells might be indirect and might be mediated by the osteoblast-like cells.

Nuclear inclusions, identical to those characteristic of Paget's disease of bone, were observed in giant cells in four of eight cases of primary oxalosis. The giant cells containing nuclear inclusions were directly involved in phagocytosis of large oxalate crystals in the context of typical foreign body granulomas in the bone marrow. Cytochemically, all of them exhibited strong tartrate-resistant acid phosphatase activity, and a proportion of them also tartrate-resistant acid ATPase. The inclusions consisted of typical arrays of filamentous material as described in Paget's disease, admixed with variable proportions of electron-dense material closely reminiscent of nucleolar pars fibrillaris and fibrillary centres. These data indicate: (a) the occurrence of Paget-like inclusions in a bone disease unrelated to Paget's disease, not causally related to viral infection, and resulting from an inborn metabolic derangement; and (b) the occurrence of Paget-like inclusions in foreign body giant cells as opposed to osteoclasts. We suggest that the occurrence of paramyxovirus-like nuclear inclusions in either osteoclasts or giant cells may represent an epiphenomenon of cell fusion and giant cell formation whenever appropriate stimuli act on latently infected precursor cells. Furthermore, our data suggest that nucleoli may represent the specific site of virus-like inclusion formation.

This report presents a case of osteopetrosis in a 25-year-old male, which was complicated by the development of osteomyelitis in the maxilla and mandible following traumatic injury and tooth extractions. The osteomyelitis in the mandible was refractory to marginal resection and antibiotic therapy. Partial resection with mandible reconstruction was then carried out. Light and backscattered electron scanning microscopy revealed sclerosis of spongy bone and variations in mineral density of the bone matrix. There was also a prominent periosteal bone formation in regions affected by osteomyelitis. An 18-month follow-up showed absence of active infections in the face and oral structures, with a focal area of bone exposure in the right parasymphysis. However, development of anemia and bone marrow deficiency will likely affect prognosis. The importance of preventive oral health care and dental/periodontal managements in osteopetrosis is emphasized.

Sebbene l’osso appaia un tessuto strutturalmente omogeneo, in quanto essenzialmente rappresentato da una matrice calcificata, presenta in realta aspetti morfologici molto diversi che riflettono generalmente attivita funzionali differenti. Tali aspetti sono da tempo differenziati in quattro ordini di strutture, che dal macroscopico al microscopico comprendono: I ordine: ripartizione dell’osso compatto e spugnoso; II ordine: sistemi osteonici, sistemi interstiziali; III ordine: strutture microscopiche, come le cellule e le fibre collagene; IV ordine: strutture submicroscopiche, come i cristallini di idrossiapatite.

Matrix Gla protein (MGP) and fetuin-A are inhibitors of arterial calcifications. In blood of rats, calcium–phosphate–fetuin–MGP complexes, produced in bone, have been identified. Indeed, an association between bone resorption, release of such complexes, and arterial calcifications has been reported. We have investigated the synthesis and localization of fetuin-A and MGP in bone of hemodialysis patients and the possible contribution of bone cells in arterial calcifications. Bone biopsies from 11 hemodialysis patients were used for histology, in situ hybridization of fetuin-A and MGP messenger RNA (mRNA), immunohistochemistry of fetuin-A, and total, carboxylated, and non-carboxylated MGP proteins. Patients showed various types of renal osteodystrophy, or normal bone. MGP was synthesized and expressed (total and carboxylated) by osteoblasts, osteocytes, and most osteoclasts, while fetuin-A by osteoblasts and osteocytes. Fetuin-A and carboxylated MGP proteins were positive in the calcified matrix, while total MGP was negative. Osteoid seams were negative to fetuin-A, lightly positive to carboxylated MGP, and occasionally positive to total MGP. Undercarboxylated MGP was mostly undetectable. In adult humans, fetuin-A is produced also by osteoblasts, and not only by hepatocytes, as previously believed. MGP, essentially carboxylated, is synthesized by osteoblasts and most osteoclasts. Increased bone turnover can be an important contributor to arterial calcifications.

Sclerostin, encoded by the SOST gene, is a recently identified protein which seems to affect bone remodeling by inhibiting bone formation via Wnt pathways. A previous study on OPG and RANKL, two cytokines involved in the control of osteoclastogenesis, showed that the anabolic effect produced by intermittent treatment with parathyroid hormone was characterized by an increase in OPG/RANKL mRNA ratio in the primary spongiosa of metaphyseal bone of rat femur, and by its falling in the secondary spongiosa, in comparison to controls (Silvestrini et al. (2007a)). Considering that Wnt pathway components seem to regulate osteoclast formation and bone resorption by repression of RANKL transcription and by positive regulation of OPG gene in osteoblastic cells, we have evaluated, in the same rats, whether and how SOST mRNA and protein in the primary and secondary metaphyseal bone are affected by PTH. SOST mRNA and protein significantly fell in both primary and secondary spongiosa where only a few osteocytes were positive to sclerostin. These data show that in the two metaphyseal areas no relationship does exist between the trends of OPG and RANKL mRNA and that of SOST, suggesting that there are no direct links between the effects induced by PTH on these molecules, at least in terms of gene expression.

Effects of main sources of bone biopsy sample variability have been examined. Variability was assessed in double iliac crest biopsies of 12 subjects with normal or pathological bone. Components of variance were estimated as follows: two biopsies per patient; three specimens at different distances from compact bone; three sections per specimen; three microscopic fields per section. The following indicators were measured: bone volume (BV/TV); osteoid volume (OV/BV); osteoid surface (OS/BS); osteoblast surface (Ob.S/BS); eroded surface (ES/BS); osteoclast surface (Oc.S/BS); osteoid thickness (O.Th). Sources of variability were assessed by ANOVA for random effects. On the basis of the results, between fields variation gave the main contribution to the error of single measures (BV/TV, 50%; O.Th, 70%; OV/BV, Ob.S/BS, OS/BS, Oc.S/BS, ES/BS more than 80%). Distance from compact bone affected mostly the BV/TV (40%) and the O.Th (10%) error. When bone specimens at intermediate distance from cortical bone are examined, variations due to different biopsies, different section and different microscopic field are largely reduced by measuring 12 microscopic fields for BV/TV (14%) and 48 microscopic fields for the other indicators (O.Th 16%; OV/BV, Ob.S/BS, OS/BS, Oc.S/BS, ES/BS more than 30%). The precision can be only slightly improved by further increasing the number of the microscopic fields.

We have studied the effects of the treatment with corticosterone (CORT), parathyroid hormone (PTH), or both (CORT + PTH), and of their withdrawal (CORT-rec and CORT + PTH-rec), on the osteoprotegerin (OPG) and receptor activator of nuclear factor-kB ligand (RANKL) localization and expression and on histomorphometric parameters in primary and secondary spongiosa of rat femur and tibia metaphyses. In the secondary spongiosa of the CORT group, the bone remodeling and the OPG/RANKL mRNA ratio decreased. In the PTH group, the bone turnover and the structural and connectivity indices increased, and the OPG/RANKL mRNA ratio fell; this ratio rose, however, in the primary spongiosa. In the CORT + PTH group, remodeling values intermediate between those of the CORT and PTH groups, were detected in the secondary spongiosa, where OPG and RANKL mRNA rose. Return towards control values was found in the recovery groups. The Cartilage Growth Plate Width was reduced in the CORT and CORT + PTH groups and returned to normal values in the recovery groups, while it was not affected by PTH. Independently of treatments, both OPG and RANKL mRNA and proteins were co-localized in the same cartilage and bone cells and in several bone marrow cells. In conclusion, the catabolic effects induced by CORT treatment occur together with an OPG fall and a RANKL rise. In the PTH group in which the bone turnover increase, the OPG and RANKL mRNA expressions differ in the primary and secondary spongiosa, confirming that the bone tissue in these sites can have different metabolic trends.

Fetuin-A of hepatic origin circulates in large amounts in serum, but also is expressed in bone, where it is an inhibitor of transforming growth factor beta (TGF-beta)/bone morphogenetic protein (BMP) proteins. Together with matrix GLA protein (MGP), fetuin-A is able to make up a complex with calcium and phosphate that is more soluble than calcium and phosphate alone, preventing its deposition in extraskeletal tissues. Experimental results suggested that this complex is made at bone tissue level. The aim of this study is to evaluate whether serum fetuin-A and MGP are influenced by type of renal osteodystrophy, they correlate with bone histomorphometric and histodynamic parameters, and/or serum levels may influence bone turnover.Thirty-eight hemodialysis patients who volunteered to undergo a bone biopsy were studied. Patients (27 men, 11 women) had a mean age of 55.2 +/- 11.8 years and dialysis vintage of 75.7 +/- 57.4 months. They were not administered vitamin D or drugs connected with mineral metabolism. They underwent transiliac bone biopsy after tetracycline labeling. Biopsies were performed for histological, histomorphometric, and histodynamic evaluation and aluminum histochemistry. Serum fetuin-A and MGP were measured by using enzyme-linked immunosorbent assay kits.Serum fetuin-A levels were significantly less than normal, whereas MGP levels were less than the normal average. Fetuin-A levels in patients with hyperparathyroidism, mixed osteodystrophy, and low-turnover osteodystrophy were 0.219 +/- 0.1, 0.27 +/- 0.1, and 0.197 +/- 0.1 ng/mL, respectively (P = not significant). Fetuin-A level significantly correlated inversely with values for several histomorphometric parameters, such as osteoid volume (OV/BV), osteoblastic surface (Ob.S/BS), osteoid surface (OS/BS), and osteoclastic surface (Oc.S/BS). Logistic regression showed odds ratios of 5.3 and 4.9 for the association of high fetuin-A levels with low values for OS/BS and Ob.S/BS, respectively. Results of multiple regression analysis with intact parathyroid hormone and fetuin-A levels as independent variables and OV/BV and Ob.S/BS as dependent variables showed that independent variables correlated significantly with dependent variables, positively for intact parathyroid hormone levels and inversely for fetuin-A levels. MGP levels in patients with hyperparathyroidism, mixed osteodystrophy, and low-turnover osteodystrophy were not significantly different (3.94 +/- 0.86, 3.40 +/- 0.99, and 5.64 +/- 2.4 nmol/L, respectively). By dividing MGP serum values into tertiles, mean values for OV/BV were different (analysis of variance, P0.04), with a greater value in the higher MGP tertile. By exclusion of 3 extravariant cases (3 SDs greater than the mean), 1 case for each type of osteodystrophy, a significant correlation between bone formation rate and MGP serum level was found (P0.05). In addition, a significant correlation was found between MGP level and trabecular thickness.Fetuin-A and MGP levels correlated with bone formation parameters. This association could be caused by an effect of these proteins on bone formation, presumably mediated by the TGF-beta/BMP system. Fetuin-A, as opposed to MGP, is known to inhibit the TGF-beta/BMP complex, a protein-cytokine system that appears to be an important regulator of bone formation and probably a factor with an important role in renal osteodystrophy.

It has long been recognized that histochemistry and cytochemistry offer the only ways of gathering information about the biochemical composition of tissues and cells without disrupting their microscopic architecture. A variety of methods have been put forward for studying nuclei acids, proteins, carbohydrates, lipids, enzymes and other components of intact tissues and cells. By now, many of these have only a historical interest. Some do, however, survive in microscopic and ultramicroscopic applications, and have become incorporated in the most refined and precise techniques that are currently available. Histochemical reactions range from the classic procedures carried out on histological sections to yield final stained products recognizable under the light microscope (Figure 1), to those which are applied on ultrathin sections, using heavy metals or other electron-dense compounds to reveal specific components under the electron microscope (Figure 2A); others range from procedures based on the antigen-antibody reaction that are capable of revealing the presence of specific biological molecules (Figure 2B), to the biophysical techniques which permit the qualitative and quantitative analysis of elements (Figure 3); lastly, there are the recently proposed ultra-high resolution methods that allow nanoparticles to be recognized. This brief review, which is based on personal experience and on the data in the literature, will discuss the most important methods now being used.

25-hydroxyvitamin D levels and bone histomorphometry in hemodialysis renal osteodystrophy.BackgroundThe importance of 25-hydroxyvitamin D (25-OHD) serum levels in hemodialysis chronic renal failure has not been so far histologically evaluated. Information still lacking relate to the effect of 25-OHD deficiency on serum parathyroid hormone (PTH) levels and on bone and its relationship with calcitriol levels.MethodsThis retrospective study has been performed on a cohort of 104 patients on hemodialysis from more than 12 months, subjected to transiliac bone biopsy for histologic, histomorphometric, and histodynamic evaluation. The patients, 61 males and 43 females, mean age 52.9 ± 11.7 years, hemodialysis length 97.4 ± 61.4 months, were treated with standard hemodialysis and did not receive any vitamin D supplementation. Treatment with calcitriol was not underway at the time of the biopsy. Transiliac bone biopsies were performed after double tetracycline labels. In addition, serum intact PTH (iPTH), alkaline phosphatase, and 25-OHD were measured. Calcitriol serum levels was also measured in a subset of patients (N = 53). The patients were divided according to serum 25-OHD levels in three groups: (1) 0 to 15 (15 patients), (2) 15 to 30 (38 patients), and (3) >30 ng/mL (51 patients).ResultsThere was no significant difference in average age, hemodialysis age, serum PTH [490 ± 494, 670 ± 627, and 489 ± 436 pg/mL, respectively (mean ± SD)], alkaline phosphatase, and calcitriol between the three groups. The parameters double-labeled surface, trabecular mineralizing surface, and bone formation rate were significantly lower in group 1 than in the other groups (P < 0.03, < 0.03, and < 0.02, respectively). Osteoblast surface and adjusted apposition rate were borderline significantly lower in group 1 (P < 0.06 and < 0.10). There was no statistical difference in the biochemical and bone parameters between groups 2 and 3. A positive significant correlation was found between several bone static and dynamic parameters and 25-OHD levels in the range 0 to 30 ng/mL, showing a vitamin D dependence of bone turnover at these serum levels. However, actual evidence of an effect on bone of 25-OHD deficiency was found at serum levels below 20 ng/mL. With increasing 25-OHD levels beyond 40 ng/mL, a downslope of parameters of bone turnover was also observed.ConclusionSince PTH serum levels are equally elevated in low and high 25-OHD patients, while calcitriol levels are constantly low, an effect of 25-OHD deficiency (group 1) on bone, consisting of a mineralization and bone formation defect, can be hypothesized. The effect of vitamin D deficiency or bone turnover is found below 20 ng/mL. The optimal level of 25-OHD appears to be in the order of 20 to 40 ng/mL. Levels of the D metabolite higher than 40 ng/mL are accompanied by a reduction of bone turnover.

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.