Local bone injections of LPS and M-CSF increase bone resorption by different pathways in vivo in rats.

We investigated the local in vivo action of lipopolysaccharide (LPS), a potent monocyte activator, and of macrophage colony-stimulating factor (M-CSF), a hemopoietic growth factor influencing monocyte differentiation, on bone resorption in normal female 8-wk-old rats. LPS (2 injections of 0.5 microgram), M-CSF (2 injections of either 12.5, 25, 100, or 500 ng), or vehicle was injected into bone marrow space through a thin catheter implanted, under hydrochloride anesthesia, in the distal end of the right femur. Histomorphometry was performed after staining of the tartrate-resistant acid phosphatase (TRAP). The number of osteoclasts and of TRAP-positive marrow cells (considered as osteoclast precursors) were counted in the secondary spongiosa. LPS caused a 3-fold increase in osteoclast surface, a 4.5-fold increase in the number of osteoclasts, but no change in the number of TRAP-positive marrow cells. M-CSF induced a striking dose-dependent biphasic effect on the number of TRAP-positive marrow cells and on bone resorption (no change with the lowest or with the highest concentrations, although the two intermediate doses significantly increased resorption surfaces and the number of osteoclasts). Our results demonstrate a local in vivo effect of LPS and of M-CSF on bone resorption and suggest that these substances act at different stages of osteoclast development and function.

Influence of calcium and vitamin D deficient diet on calcitonin gene expression in the ultimobranchial cells of the developing chicken.

Recently hatched chickens were fed a vitamin D and calcium deficient diet for 4 weeks. Calcitonin (CT) biosynthesis in the ultimobranchial glands (UBG) was studied during the treatment by means of in situ hybridization of specific CT mRNAs and immunocytochemical detection of the CT intracellular stores. Circulating CT levels were measured by radioimmunoassay. Within 1 week of the start of treatment, the deficient animals had significantly lowered plasma calcium concentrations and a dramatic fall of plasma CT levels, but the UBGs were not much affected. From week 2 to week 4, the UBG underwent a gradual atrophy. The tissue became lacunar due to the presence of an abnormally developed cystic component. Although calcemia returned to normal at week 4, the cellular endocrine cords were dramatically reduced, corresponding to the undetectable circulating CT levels. However, the UB glands always contained persistent CT-secreting cells, mainly at the periphery of the tissue or in contact with enlarged parathyroid tissue inclusions. These endocrine UB cells contained large amounts of hybridizable CT mRNA and immunodetectable stores of the mature hormone, and their ultrastructural features were quite unaffected compared to normal ones. Thus, we conclude that, in the chicken, severe calcium malnutrition led to a striking reduction of CT biosynthesis in the UB glands by decreasing the number of secretory cells and not by triggering modifications of the biosynthetic activity of the UB endocrine cells.

Effects of transforming growth factor-beta on long-term human cord blood monocyte cultures.

Transforming growth factor-beta (TGF-beta) modulates growth and differentiation in many cell types and is abundant in bone matrix. We recently showed that human cord blood monocytes cultured in the presence of 1,25(OH)2D3 acquire some features of osteoclast precursors. Since TGF-beta has been shown to influence bone resorption in organ culture, we have studied the effect of TGF-beta (1-1,000 pg/ml) on cord blood monocyte cultures. These cells were cultured on plastic substrate during 3 weeks in the presence of 20% horse serum and 10(-9) M 1,25(OH)2D3. TGF-beta, from a concentration of 10 pg/ml in the culture medium, decreased in a dose dependent manner the formation of multinucleated cells. At a concentration of TGF-beta of 1 ng/ml, the multinucleated cells were reduced to 2.1% +/- 0.3%, compared to 19.3% +/- 1.5% in control cultures. TGF-beta inhibited in a dose-dependent manner the proliferation of cord blood monocytes as assessed by 3H-thymidine incorporation at 7 and 14 days of culture. The fusion index was also decreased by 3 weeks of treatment with TGF-beta. Indomethacin did not reverse the inhibitory effects of TGF-beta. The expression of the osteoclastic phenotype was assessed using two different antibodies: 23C6, a monoclonal antibody directed against the vitronectin receptor, which is highly expressed by osteoclasts but not by adult monocytes, and an antibody to HLA-DR, which is not present on osteoclast. TGF-beta decreased the expression of HLA-DR and increased in a dose-dependent manner the proportion of 23C6-labeled cells; these results suggest that TGF-beta could modulate a differentiation effect to the osteoclastic phenotype. However, when cord blood monocytes were cultured on devitalized rat calvariae prelabeled with 45Ca, TGF-beta did not induce any 45Ca release from bone cultured with monocytes, suggesting that full osteoclastic differentiation was not achieved. These results emphasize the complex role of TGF-beta in the local regulation of bone cell differentiation and in bone remodeling.

Formation of multinucleated cells with osteoclast precursor features in human cord monocytes cultures.

A common lineage between monocytes and osteoclasts has been suggested but not yet proved, and an osteoclast precursor might be an immature cell of the monocyte-macrophage family. We therefore compared the ability of cord blood and adult monocytes in long-term culture to differentiate toward osteoclasts. Both adult and cord monocytes were cultured for 3 weeks in the presence of 20% horse serum. The proportion of multinucleated cells formed was influenced by 1,25(OH)2D3 in cord, but not in adult monocyte cultures: 10(-9)M 1,25(OH)2D3 increased multinucleated cells from 13 +/- 2 to 26 +/- 1% of total cells in cord monocyte cultures. The formation of multinucleated cells in cord monocyte cultures, in the presence of 10(-9) M 1,25(OH)2D, was decreased by salmon calcitonin (dose dependently from 10(-8) to 10(-6) M) and increased by 1-34 parathormone (100 ng/ml). None of these hormones induced any modification of the proportion of multinucleated cells formed in adult monocytes culture. Specific antigens on the membrane of the cells obtained after 3 weeks culture in the presence of 10(-9) M 1,25(OH)2D3 were assessed by immunocytochemistry. The respective proportion of adult and cord labeled cells was 64 +/- 11 vs. 63 +/- 6% with Leu M5 (specific for monocyte) and 68 +/- 7 vs. 30 +/- 10% (P less than 0.05) with the anti-HLA DR antibody. The monoclonal antibody 23C6 is specific to the vitronectin receptor, which is highly expressed by osteoclasts--41 +/- 2% of the cells in cord monocyte cultures--but none in the adult monocytes culture were labeled with 23C6 at the end of the culture period.(ABSTRACT TRUNCATED AT 250 WORDS)

Deferoxamine test and bone disease in dialysis patients with mild aluminum accumulation.

Aluminum bone disease is a frequent complication of dialysis patients. The deferoxamine (DFO) test has been advocated as a noninvasive procedure for the diagnosis of AI bone lesion. However most of these studies have been performed in symptomatic patients with significant AI bone disease. Whether this test may provide similar data at an earlier stage of AI toxicity is not known. The present study evaluates prospectively 28 patients with mild AI load. Patients studied ranged in age from 21 to 65 years; duration of dialysis was 5.6 +/- 3.2 years; deferoxamine, 40 mg/kg body weight, was infused at the end of dialysis. Serum AI was measured before DFO administration and before the next dialysis treatment. Bone biopsies were performed in all patients. Cortical bone AI was determined biochemically; trabecular and cortical bone AI were also determined histochemically. Mean basal serum AI (43.2 +/- 30.8 micrograms/L) and cortical bone AI (25.7 +/- 35.2 micrograms/g) were moderately increased. Basal serum AI correlated (r = 0.77) with the increment in serum AI after DFO infusion. After DFO, stainable trabecular and cortical bone AI correlated in a similar manner with both basal serum AI and increment in serum AI. Only biochemically determined cortical bone AI was not significantly related to basal serum AI. Nineteen of the 28 patients had evidence of osteitis fibrosa on bone biopsy. Stained AI surfaces but not trabecular AI were different in patients with low and patients with high bone formation rates. The bone findings, assessed as bone formation rates and resorption surfaces, did not correlate with biochemically or histochemically determined bone AI.(ABSTRACT TRUNCATED AT 250 WORDS)

Cyclosporin A induces in vivo inhibition of resorption and stimulation of formation in rat bone.

We investigated a possible "in vivo" effect of cyclosporin A, an immunosuppressive agent, on normal rat bone remodeling. At an oral daily dose of 7 mg/kg for 14 days, the blood level of cyclosporin A was in the usual effective range and no change in renal function or magnesium metabolism was observed. Treated rats had decreased bone resorption: urinary hydroxyproline, plasma acid phosphatase, and the number of osteoclasts in caudal vertebrae were significantly reduced. By contrast, bone formation assessed by dynamic histomorphometry after double tetracycline labeling was increased. No modification of calciotropic hormones (vitamin D metabolites and parathyroid hormone as assessed by urinary cyclic AMP) was observed at the end of the treatment. These results suggest that in vivo cyclosporin A treatment induces bone remodeling modifications related to either a direct or a lymphokine-mediated effect on bone cells.

Influence of blood calcium on calcitonin receptors in isolated chick osteoclasts.

Isolated osteoclasts obtained from young chickens fed a normal (+Ca) or deficient (-Ca) calcium and vitamin D diet for 3 weeks, were studied for their ability to bind salmon calcitonin (sCT). Osteoclasts obtained from -Ca chickens, when incubated with 0.1 mumol sCT/l, doubled cyclic (c)AMP production and retracted from a glass support, as observed by scanning electron microscopy. The presence of receptors was also demonstrated by autoradiography and competition analysis of 125I-labelled sCT binding. The number of receptors per cell was 0.9 +/- 0.1 x 10(4). In contrast, osteoclasts obtained from +Ca chickens did not increase cAMP production and did not retract in the presence of 0.1 mumol sCT/l. No specific binding of 125I-labelled sCT could be demonstrated on these osteoclasts. Plasma levels of calcium and calcitonin were measured in +Ca and -Ca chickens. The plasma concentration of calcium was markedly lower at 3 weeks in -Ca than in +Ca chickens. The plasma concentration of calcitonin was decreased in -Ca chickens compared with +Ca chickens at the first week and kept decreasing during the 3 weeks. These results strongly support the hypothesis that calcium and vitamin D intake regulate plasma calcitonin levels in chickens, and that calcitonin receptors can be detected on chicken osteoclasts only when blood calcium is decreased by a diet deficient in calcium and vitamin D.

Different schedules of administration of (3 amino-1-hydroxypropylidene)-1, 1 bisphosphonate induce different changes in pig bone remodeling.

Intermittent administration of antiosteoclastic agents has been proposed in order to increase trabecular bone volume (TBV). We evaluated the effect of two different intermittent schedules of administration of (3 amino-1-hydroxypropylidene)-1, 1 bisphosphonate (AHPrBP) on pig bone remodeling for a period of 60 days. AHPrBP (1.6 mumol/kg/injection) was given subcutaneously daily (group A1), or 5 consecutive days out of 21 days (group A2), or 1 out of every fourth day (group A3). Compared to control animals, group A1 significantly increased trabecular bone volume (TBV) (+62%) with a marked decrease in bone resorption assessed by interstitial bone thickness. Bone formation assessed by mean wall thickness (MWT) was also decreased due to a decrease in the number and activity of osteoblasts. There was not a delay in the coupling mechanism as assessed by the reversal surfaces. The two groups receiving intermittent schedules had markedly different results. Group A2 had very similar changes to group A1 despite receiving four time less drug. Compared to group A1 and A2, group A3 had smaller decrease in resorption and higher bone formation rate with identical MWT. These differences between group A2 and A3 were associated with similar levels of parathyroid hormone and vitamin D metabolites. Different bone concentrations induced by the two different schedules of AHPrBP may explain the greater effect on bone resorption and osteoblast recruitment in group A2 and thus a milder effect of the AHPrBP administration once every fourth day.

Bone histological heterogeneity in postmenopausal osteoporosis: a sequential histomorphometric study.

We studied sequential bone biopsies performed at 6 to 24 month intervals from 14 untreated osteoporotic women (64 +/- 7). Subgroups were defined, respectively, by increased osteoclastic resorption surfaces and decreased osteoblastic surfaces +/- 2 S.D. Normal values were obtained from bone biopsy of 23 normal women (61 +/- 8). When patients were divided into subgroups according to the above criteria the first biopsy showed that 3 out of the 14 patients had high resorption surfaces and 6 had low osteoblastic surfaces. Eight patients spontaneously changed during the study. In 2 patients there was a change in resorption surfaces, in 3 in osteoblastic surfaces and in 3 a change in both osteoblastic and resorption surfaces was observed. Considering the first or second bone biopsy results the patient variance was higher than the control subject's variance; however the variance between the first and second bone biopsy of one patient was not different from the variance inside the group of patients. The average intraindividual variation of the parameters on sequential biopsies was of the same order as the one we previously observed on simultaneous bone biopsies of normal and hemodialyzed patients. We concluded that if osteoporosis is a heterogeneous disorder, subgroups cannot be definitively defined on the basis of cellular parameters of bone remodelling assessed on bone biopsies.

Increased bone aluminum deposition after subtotal parathyroidectomy in dialyzed patients.

Ten dialyzed patients underwent a systematic bone biopsy before and 19 +/- 9 months after subtotal parathyroidectomy (PTX). At the end of the follow-up period all the patients, except two, who complained of proximal myalgia, were asymptomatic. Compared to the bone biopsy specimen obtained prior surgery, decreased bone formation without mineralization impairment was observed after PTX. Despite an average decrease in aluminum gels intake after PTX, an increase in stained aluminum was observed (0.69 +/- 0.79 versus 1.20 +/- 0.95 mm/mm2, P less than 0.050). Aluminum accumulation depended on the pre-PTX bone aluminum load: pre- and post-PTX bone aluminum loads were correlated (r = 0.78, P less than 0.01). Bone aluminum accumulation was not related to the amount of aluminum gel intake after PTX; however, only two patients free of both bone aluminum deposit prior to PTX and aluminum gel intake after PTX had no stainable aluminum on the second bone biopsy after PTX. The only patient who had no decrease in bone formation after PTX had no increase in bone aluminum. Assuming that the patients had no aluminum deposit prior to dialysis, we measured the rate of bone aluminum accumulation. It rose from 0.11 +/- 0.09 mm/mm2/year prior to PTX to 0.40 +/- 0.25 mm/mm2/year after PTX (P less than 0.05) in the six patients who were maintained on phosphate binders and who had a decrease in bone formation after PTX. These six patients had unchanged aluminum gel intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Histomorphometric evidence of deleterious effect of aluminum on osteoblasts.

Bone histomorphometry was performed in 26 hemodialyzed patients to study the relation between the dynamic parameters of bone formation and aluminum deposition. Patients were divided into two groups according to whether bone formation rate at tissue level (Svft) was above or below normal: 0.089 mu 3/mu 2 per day. The 12 patients who constituted group II, defined by a Svft less than 0.089 mu 3/mu 2 per day, had markedly decreased extent of double-labeled surfaces (m = 1.3 +/- 6.5%), and these were absent in 8 of 12 patients. Osteomalacia, defined by decreased formation with increased mean osteoid thickness (greater than 15 micron), was present in only 3 of 12 patients in group II. The 14 patients who constituted group I, defined by a Svft greater than 0.089 mu 3/mu 2 per day, had both increased total labeled surfaces and mineralization rate. Osteomalacia was present in none of the group I patients. In trabecular bone, group II patients had increased stainable aluminum deposition, compared to group I patients, whether estimated as total stainable aluminum (2.16 +/- 1.34 vs 0.17 +/- 0.28 mm/mm2) or stainable percent of trabecular surfaces (42 +/- 19 vs 4 +/- 5%). This last parameter was inversely related to osteoblastic surfaces (r = -0.49, n = 26, P less than 0.01) and total labeled surfaces (r = -0.72, n = 26, P less than 0.01). Therefore, massive aluminum deposition was not invariably associated with impaired mineralization but with decreased formation due to decreased extent of active formation surfaces. In the group I patients, moderate aluminum deposition was not associated with the mineralization arrest observed in these patients.(ABSTRACT TRUNCATED AT 250 WORDS)

Multifactorial low remodeling bone disease during cyclic total parenteral nutrition.

The physiopathology of metabolic bone disease described during long term total parenteral nutrition is poorly understood. We therefore prospectively assessed bone status of seven adult patients [mean age, 42 +/- 16 (SD) yr] treated with cyclic total parenteral nutrition for a period of 7 +/- 2 (SD) months. All patients had hypercalciuria (381 +/- 96 mg/day) associated with negative calcium balance in six of seven patients (-49 +/- 120 mg/day). A correlation was found (r = +0.74, P less than 0.01) between protein intake and calciuria. Two patients developed slight transient hypercalcemia. Serum magnesium and phosphate levels remained within the normal range. A high aluminum load due to the added phosphate solution (253 +/- 84 micrograms/day) was associated with increased serum aluminum levels (52 +/- 38 micrograms/liter). Normal serum levels of 25 hydroxyvitamin D (12 +/- 7 ng/ml) and low normal 1,25 dihydroxyvitamin D levels (21 +/- 8 pg/ml) were found. Serum PTH was normal in five and increased in two of the seven patients. However, in these two patients skeletal unresponsiveness to the action of PTH was found. A new histomorphometric picture of bone was observed; it consisted of a markedly reduced bone formation with subnormal osteoclastic activity leading to a low trabecular bone volume. No osteomalacia was found. The aluminum load may have played a role in these bone defects. The hypercalciuria with negative calcium balance was attributed to the cyclic amino-acid delivery during TPN.

Effects of iron overload on bone remodeling in pigs.

For study of the effects of an iron overload on bone remodeling, 5 control pigs were compared with 5 pigs given a total dose of 10.8 g of parenteral iron in 36 days. Treated pigs developed an iron tissue overload demonstrated by a marked increase in bone and liver iron. Except for a modest increase in SGOT, there was no biochemical or histologic sign of liver damage. Serum levels of 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D were unchanged in the treated pigs. There was no accumulation of iron in the parathyroid glands and the serum immunoreactive parathyroid hormone level was unchanged in the treated animals. Bone histomorphometry after double tetracycline labeling showed that in the treated pigs osteoblast cell surfaces, double and total labeled surfaces, appositional rate, and formation at tissue level were significantly decreased, and reversal surfaces were increased. Mineralization was not impaired because the osteoid thickness was unchanged. From the morphometric measurements it was concluded that osteoblast recruitment and the collagen synthesis rate were decreased. Mean wall thickness, which indicates the amount of bone synthesized, was also lowered. In contrast, the osteoclastic resorption surfaces and the depth of lacunae resulting from osteoclast resorption were unchanged by treatment. Despite this imbalance between formation and resorption, trabecular bone mass estimated on trabecular bone volume and bone ash was unchanged after 36 days' treatment. Perls' stain revealed that iron deposits were present in osteoblast and osteoclast cells and also inside the bone matrix, because there was a linear deposit along the trabecular surfaces, cement line, and osteoid-mineralized bone interface. Therefore, because treatment induced no modification of the major humoral regulators of bone metabolism, it is suggested that iron, which was present in bone cells and matrix, could play a role in bone remodeling.

Evidence for defective osteoblastic function. A role for alcohol and tobacco consumption in osteoporosis in middle-aged men.

In a group of 11 men ranging in age from 35 to 50 years with idiopathic osteoporosis, most were mild alcoholics and heavy smokers. Two had absorptive hypercalciuria. Histomorphometry showed that the patients had low trabecular bone volume and mean trabecular thickness when compared with age-matched control subjects. Mean wall thickness was also markedly reduced in patients as compared with control subjects. The quantity of resorbed bone was extrapolated from the calculated mean interstitial bone thickness. Resorption was not significantly different in patients and control subjects. Consequently, in this group of patients with severe osteoporosis, the pathogenesis was characterized by markedly decreased bone formation.

Calcium phosphate metabolism and bone disease in patients with homozygous thalassemia.

Calcium and phosphate metabolism were studied in 22 patients with homozygous thalassemia. The overall results showed no significant difference for serum calcium, phosphorus, alkaline phosphatase, immunoreactive parathyroid hormone, or 25-hydroxyvitamin D between thalassemic and control children. However, during the winter, serum 25-hydroxycholecalciferol levels were very significantly decreased in thalassemic children. A study of the hands showed thin metacarpal cortices related to increased resorption. Histomorphometric study of four iliac bone biopsies showed normal osteoclastic resorption and decreased bone formation. Prussian blue staining and x-ray electron microprobe analysis showed iron deposits inside the bone. Whether this finding is critical in the pathogenesis of the bone disease in unknown.

Bone histomorphometry in hemodialysed patients.

We performed bone histomorphometry in thirty hemodialysed patients. Ten patients had a double iliac bone biopsy to estimate bone histomorphometry reproductibility. There was no difference between the mean results for each of the 10 patients at each site. However, there was an intra-individual variation which was small for the parameters of formation and particularly osteoid thickness and mineralizing rate and greater for resorption parameters. Mineralization rate appeared the most reliable and discriminant parameter. These 30 patients were separated in two groups according to their mineralizing rate (MR); patients with an MR greater than 0.3 mu/day were in group I and had severe hyperparathyroidism without major impairment of bone mineralization and high formation rate. They also had high serum alkaline phosphatases and high serum parathyroid levels measured with a COOH terminal antibody (iPTH). Patients with a low MR less than 0.3 mu/day (group II) had a severe mineralization defect with low formation rate, normal alkaline phosphatase and significantly lower levels of iPTH than in group I. This last type of histological bone lesion could not be due to aluminum intoxication since the level of serum aluminum was the same in the two groups. The mineralizing defect appeared to be inversely correlated with the percent of osteoid surfaces covered by osteoblast and with the iPTH level. These data suggest that during the course of renal osteodystrophy PTH stimulates not only bone resorption but also bone mineralization by increasing osteoblastic number.