E. Bonucci

Bone loss in response to long-term glucocorticoid therapy

A number of studies have shown that an excess of glucocorticoids induces osteoporosis, but the mechanism(s) and the time course of the reduction of bone mass remain uncertain. In order to clarify this issue we carried out a longitudinal clinical and histomorphometric study of patients requiring long-term glucocorticoid treatment. In 23 patients (9 men, 10 post- and 4 premenopausal women) biochemical and bone histomorphometric investigations were carried out before and during treatment with 10-25 mg/day of prednisone. Histomorphometric analysis of bone biopsies of the iliac crest showed that the decrease of TBV (up to -27%, P less than 0.001) occurs predominantly within the first 5-7 months of treatment; during the subsequent stages, which include observations after 12 months of treatment, only minor changes were observed. Therefore trabecular bone loss can be satisfactorily described by a negative exponential function. None of the other histomorphometric parameters (osteoid surfaces, resorption surfaces, etc.) showed significant changes. However, the histological features of the bone biopsies during steroid therapy, showing a virtual lack of osteoblastic activity, ruled out an increase of bone resorption. Moreover, the dynamic study of the bone formation by double tetracycline labelling showed, in a small subgroup of patients, a decrease of the apposition rates (from 0.763 +/- 0.053 to 0.305 +/- 0.074 microns/day (mean +/- SE) after treatment). No significant changes, at any time during steroid treatment, were observed in serum alkaline phosphatase, 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D, 1,25-dihydroxyvitamin D, parathyroid hormone or urinary calcium excretion. Serum calcium increased significantly within the first 1-2 months of therapy and then it returned to baseline. Urinary hydroxyproline excretion decreased significantly within the first 1-2 months and continued to fall throughout the treatment. Thus, both biochemical and histological findings suggest that long-term glucocorticoid therapy causes a reduction of bone turnover, that the bone loss occurs predominantly within the first 6 months of treatment and that patients with lower bone mass have a lower rate of bone loss.

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.

Renal bone disease in 76 patients with varying degrees of predialysis chronic renal failure: a cross-sectional study

BACKGROUND Renal osteodystrophy has been studied less extensively in predialysis than in dialysis patients. Different types or histological patterns in their natural evolution from moderate to advanced severity of renal insufficiency are only partially known, with special regard to adynamic bone disease and its relationship with osteomalacia. METHODS We conducted a cross-sectional retrospective study on 76 unselected patients with chronic renal failure undergoing conservative treatment, with a wide range of severity of renal insufficiency. All the patients were subjected to bone biopsy for histological and histomorphometric evaluation. The patients, 44 males and 32 females ranging in age from 18 to 72 years and with serum creatinine 1.2-11.4 mg/dl, had not been exposed to aluminium-containing drugs and had never been treated with vitamin D or calcitriol. RESULTS Ten patients had normal bone, nine were diagnosed with adynamic bone disease, 26 with mild mixed osteodystrophy, seven with predominant osteomalacia, 22 with advance mixed osteodystrophy, and two with predominant hyperparathyroidism. Patients with adynamic bone disease had less severe chronic renal failure than the other pathological subgroups, intact PTH above the upper limit of normal, normocalcaemia, and reduced serum osteocalcin in line with a significantly lower ObS/BS. Osteomalacia was found in a more advanced stage of chronic renal failure with relative hypocalcaemia and more severe metabolic acidosis. A creatinine clearance of 20 ml/min served as a clear demarcation between this histological group and adynamic bone disease. CONCLUSIONS It is postulated that adynamic bone disease is a form of renal osteodystrophy, separate from osteomalacia, appearing when bone resistance to PTH develops, probably a transient stage to more hyperparathyroid histological classes with increasing severity of chronic renal failure.

Bone sialoprotein (BSP) secretion and osteoblast differentiation: relationship to bromodeoxyuridine incorporation, alkaline phosphatase, and matrix deposition.

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.

Renal Osteodystrophy in Predialysis and Hemodialysis Patients: Comparison of Histologic Patterns and Diagnostic Predictivity of Intact PTH

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.

Tartrate-Resistant Acid Phosphatase Activity in Rat Osteoblasts and Osteocytes

SummaryTartrate-resistant acid phosphatase activity, generally known as a distinctive cytochemical feature of osteoclasts, has been demonstrated in osteoblasts and osteocytes in low-temperature glycol methacrylate-embedded rat bone specimens.

Culture and differentiation of chondrocytes entrapped in alginate gels

SummaryWe studied the response to culture conditions and the differentiative ability in suspension culture in alginate gels of resting chondrocytes from the preosseous cartilage of adult pig scapula. It was found that the maximum rate of chondrocyte duplication is reached at the fourth day in culture whereas the rate of proteoglycan synthesis and alkaline phosphatase expression do not gain a maximum value before the seventh day. During the culture time, the chondrocytes undergo differentiation as it is demonstrated by the alkaline phosphatase specific activity increase and by morphological criteria (hypertrophy, increase of the number of mitochondria per cell, increased endoplasmic reticulum, matrix vesicle production). The alginate gels can be easily dissolved to obtain cell populations in which the variation of cytosolic calcium concentration following a proliferative stimulus can be conveniently observed using the conventional procedure of Fura 2.

The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption.

The organic-inorganic relationships in bone matrix undergoing osteoclastic resorption have been studied in rat tibial diaphyses using electron microscope techniques in an attempt to identify the steps of the resorption process. Results suggest that bone resorption occurs in two phases: the first, an extracellular phase, leads to bone matrix fragmentation and partial dissolution, and the second, an intracellular phase, to complete digestion of the breakdown products of the bone matrix. The first component of the bone matrix to be attacked by the osteoclast is the ground substance. This induces the release of the crystals lying between, and on, the collagen fibrils; any crystals lying within fibrils are released later, when the fibrils break up. As this stage proceeds, the collagen fibrils retain their normal intrinsic texture, but gradually loose their lateral aggregation, appearing as individual fibrils (some of them uncovered by crystals), mixed with fragments of fibrils and many free crystals. The loosened but otherwise structurally normal collagen fibrils, and their fragments, are strongly argyrophilic. Complete dissolution of the disaggregated fibrils occurs outside the cell, both in the resorption zone and in the initial portion of the channels of the ruffled border. The free crystals present in the resorption zone and those phagocytosed in cytoplasmic vacuoles are organic-inorganic structures, whose organic component (the crystal ghost) is, at least in part, of proteoglycan nature. Dissolution of inorganic material occurs within the cytoplasmic vacuoles of the osteoclasts. Results are viewed in relation to the process of bone resorption and, as far as crystal ghosts are concerned, to that of bone calcification. A tentative summary of the various steps involved in the mechanism of bone resorption is given.

Immunohistochemical localization of osteonectin in developing human and calf bone using monoclonal antibodies.

SummaryOsteonectin was immunolocalized in human fetal and calf neonatal developing bone using newly developed monoclonal antibodies. The protein was localized to the cytoplasm of osteoblasts and young osteocytes. In bone matrix, strong reactivity was found in newly laid down osteoid. Bone matrix immunoreactivity was enhanced by pretreatment of sections with proteases, possibly because of an unmasking of epitopes engaged in protein-protein interactions. Osteonectin immuno-reactivity was also found in preosteoblasts in all types of human fetal osteogenesis (membranous, endochondral, subperiosteal, and mantellar (Meckels cartilage) ossification), and in some chondrocytes of metaphyseal growth plate, posibly modulating towards an osteoblastic phenotype.

Zinc mapping in bone tissues by histochemistry and synchrotron radiation-induced X-ray emission: correlation with the distribution of alkaline phosphatase.

Zinc distribution in osteons was mapped by synchrotron radiation-induced X-ray emission analysis in both human and porcine adult bone, as well as in porcine bone by histochemistry using Timms method. Both procedures showed that zinc is not uniformly distributed, being in its highest concentration on haversian bone surfaces. When Timms method was applied in conjunction with a procedure leading to partial zinc extraction, three zinc pools were specifically detected: a loose one, found in the mineralizable osteoid; a mineral one, bound to the bone mineral; and a tenacious one, firmly bound to an organic component located in the osteoid and mineralizing organic matrix. The alkaline phosphatase distribution was also mapped in porcine adult bone by histochemistry and immunohistochemistry and it was found codistributed with tenacious zinc mainly at the calcification front. The data suggest that alkaline phosphatase is buried as a bone matrix protein during initial mineralization.