Inhibition of bone matrix apposition by (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) in the mouse

To elucidate the mechanism of action of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP, formerly APD) on bone metabolism, we have studied the influence of low doses of AHPrBP on bone resorption and formation in the mouse. Thirty-five-day-old mice were given daily injections of 0.16, 1.6, or 16 mumol/kg BW per day of AHPrBP for 10 days. At sacrifice biochemical parameters were measured in serum and bone ash, and histomorphometric parameters of bone formation and resorption were determined on undecalcified sections of caudal vertebrae after double 3H-proline and double tetracycline labelings. Serum calcium and 1,25-dihydroxyvitamin D levels remained normal at all dosage levels. Compared to controls, AHPrBP at doses of 1.6 and 16 mumol/kg per day increased the number of osteoclasts and the number of nuclei per osteoclast but markedly decreased the number of acid phosphatase-stained osteoclasts. Thus, AHPrBP appears to inhibit osteoclastic activity in vivo in part through reduction of acid phosphatase activity. At doses of 1.6 and 16 mumol/kg per day AHPrBP reduced serum alkaline phosphatase and the osteoblastic surface and decreased the endosteal osteoid surface and thickness. Both the matrix apposition rate and the mineral apposition rate were progressively reduced at the endosteal level, although they were not significantly changed at the periosteal level. Greater inhibition of bone resorption than bone formation resulted in increased endosteal bone density and bone mineral content. AHPrBP at a dose of 0.16 mumol/kg per day did not alter either the osteoclastic bone resorption or the mineral and matrix apposition rates.     

Influence of magnesium supplementation on bone turnover in the normal young mouse

The effect of magnesium (Mg) supplementation on bone metabolism has been studied in the normal young mouse. Weanling male mice were given Mg-supplemented drinking water (5 mM or 32 mM Mg) for 4 weeks. Mineral and skeletal changes were assessed by biochemical methods and by histomorphometric analysis of endosteal bone formation and resorption parameters evaluated on tetracycline double-labeled, undecalcified caudal vertebrae. Magnesium supplementation increased serum and urinary Mg concentrations and bone Mg content. Both the calcification rate and the extent of tetracycline double-labeled osteoid surface increased progressively in Mg-treated mice, whereas the mineralization lag time was shortened. The osteoblastic surface was reduced, leading to a fall in osteoid surface. Stimulation of bone mineralization was associated with a rise in extracellular calcium (Ca) and phosphorus (P) concentrations whereas serum 25-OHD and 1,25(OH)2D levels remained normal. The Mg supplementation increased the number of acid phosphatase stained chondroclasts and osteoclasts and the extent of resorbing surface showing histochemically stained osteoclasts. Although urinary OH-proline increased above normal, Ca, P, and cyclic adenylic acid (cAMP) excretion and phosphate concentration (TmP/GFR) remained normal, suggesting that parathyroid hormone (PTH) secretion was not altered. The trabecular bone volume remained normal, showing that the increased bone resorption was balanced by the stimulated bone mineralization. The results show that Mg supplementation influenced both bone formation and resorption in the young mouse, and that the stimulation of bone mineralization was the result of increased extracellular mineral availability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of prostaglandins E1, E2, and F2 alpha on osteoclast formation in mouse bone marrow cultures.

Prostaglandins (PG) act as direct inhibitors of mature osteoclasts, but although resorption-inhibition is also observed initially PG increase bone resorption in organ culture. This suggests that PG influence bone resorption in organ culture through actions on cell types other than mature osteoclasts. We have therefore tested the effects of PG E1, E2, and F2 alpha on the differentiation of osteoclastic phenotype in mouse bone marrow cultures using bone resorption and calcitonin receptors (CTR) as markers of osteoclastic differentiation. We found that PGE2 (10(-6)-10(-9) M) and PGE1 (10(-6)-10(-7) M) induced a significant increase in CTR-positive cell numbers, to levels five to eight times those seen in controls and similar to the number induced by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. Bone resorption was increased (10(-7) M PGE2 and 10(-6) M PGE1) in association with the increased CTR-positive cell numbers, suggesting that the PG also induced resorptive function. 1,25-(OH)2D3 increased both the number of CTR-positive cells and the extent of resorption per cell; the additional presence of PG did not affect the number of CTR-positive cells but did reduce bone resorption compared with 1,25-(OH)2D3 alone. PGF2 alpha had no significant effect on CTR-positive cell induction or bone resorption. The results suggest that PGE1 and E2 induce osteoclastic differentiation in mouse bone marrow cultures and inhibit the function of the osteoclasts thus formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histogenesis of hyperosteoidosis in 1,25(OH)2D3-treated rats fed high levels of dietary calcium

The purpose of this investigation was to determine by sequential quantitative morphometry the histogenesis of metaphyseal changes induced in rats fed high levels of dietary calcium and treated with pharmacologic doses of 1,25(OH)2D3. Young adult female rats were placed on a diet containing 2.5% calcium and 0.3% phosphorus and administered either ethanol or 135 ng (5 units) 1,25(OH)2D3 in ethanol IP daily for 10 days. Rats were terminated at Days 1, 2, 3, 4, 6, 8, and 10. At Day 1 the proximal tibias from rats treated with 1,25(OH)2D3 had a dramatic increase in osteoclasts/mm total trabecular surface perimeter compared with placebo-treated rats. Osteoclast numbers decreased in 1,25(OH)2D3-treated rats to the levels in placebo-treated rats by Days 3 and 4 and decreased significantly below placebo-treated levels at Days 6, 8, and 10. Active resorbing surface was significantly increased at Days 1 and 2 and decreased at Days 8 and 10 in 1,25(OH)2D3-treated rats compared with placebo-treated rats. From Day 4 through Day 10 in 1,25(OH)2D3-treated rats, there was a progressive increase in osteoblasts/mm total trabecular surface perimeter, osteoid surface, active osteoid surface, and metaphyseal osteoid. Metaphyseal osteoid increased markedly at Days 8 and 10 in 1,25(OH)2D3-treated rats and caused a significant increase in the amount of osseous tissue in the metaphysis. Metaphyseal mineralized bone, however, was not consistently affected by 1,25(OH)2D3 treatment. Serum calcium and phosphorus were elevated in 1,25(OH)2D3-treated rats at more time periods. In rats fed high levels of dietary calcium, repeated supraphysiologic doses of 1,25(OH)2D3 result in a net increase in metaphyseal osseous tissue, predominantly osteoid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Bone resorption induced by 1 alpha,25 dihydroxyvitamin D(3) in vivo is not altered by inactivation of the plasminogen activator inhibitor 1

One of the proteolytic systems produced by bone cells is the plasminogen activator/plasmin pathway, which involves the two plasminogen activators and the type 1 plasminogen activator inhibitor (PAI-1) and results in plasmin generation. We have recently demonstrated that this pathway plays a specific role in the degradation of the nonmineralized matrix of bone in vitro. To evaluate whether PAI-1 is required during bone resorption in vivo, we studied the effects of PAI-1 inactivation on bone metabolism using systemic administration of 1alpha,25 dihydroxyvitamin D(3) [1, 25(OH)(2)D(3)] as model. PAI-1-deficient (PAI-1-/-) and wild-type (WT) mice were injected intraperitoneally with 1,25(OH)(2)D(3) (2 microg/kg) or vehicle every other day during 4 weeks and analyzed using biochemical parameters of bone turnover, histomorphometric analysis of the proximal tibial metaphysis, and pQCT analysis of the distal femoral metaphysis. PAI-1 inactivation did not affect bone metabolism in vehicle-treated mice. Treatment with 1,25(OH)(2)D(3) induced bone resorption similarly in PAI-1-/- and WT mice, as assessed by the increase in the urinary excretion of calcium (2. 2-fold and 2.3-fold, respectively) and of pyridinoline crosslinks (by 24% and 22%, respectively). In addition, a comparable reduction in bone mass was observed in PAI-1-/- and WT mice after treatment with 1,25(OH)(2)D(3), as evidenced by the decrease in the femoral calcium content (by 25% and 32%, respectively), in the trabecular bone volume (by 50% and 40%, respectively), in the trabecular mineral content (by 17% and 15%, respectively), and in the cortical mineral content (by 45% and 52%, respectively). The parameters of bone turnover also increased after 1,25(OH)(2)D(3) treatment. Serum osteocalcin was, respectively, 25% and 28% higher in PAI-1-/- and WT mice treated with 1,25(OH)(2)D(3) compared with the mice injected with vehicle. Similarly, the osteoid surface increased in 1, 25(OH)(2)D(3)-treated PAI-1-/- and WT mice by 40% and 51%, respectively, the mineral apposition rate increased by 15% and 8%, respectively, and the bone formation rate by 54% and 48%, respectively. These data indicate that PAI-1 is not critical during bone resorption induced by 1,25(OH)(2)D(3) in vivo.     

Bone response to phosphate and vitamin D metabolites in the hypophosphatemic male mouse

The hypophosphatemic male mouse (Hyp/y), the proposed model for human vitamin D-resistant rickets (VDRR), is characterized by chronic hypophosphatemia, dwarfism, and rachitic and osteomalacic bone lesions. We have reported that treatment of Hyp/y mice with phosphate salts (Pi) heals rickets but does not correct the defective endosteal bone mineralization. In an attempt to cure osteomalacia, mutant male animals were treated with Pi combined with 25-hydroxyvitamin D3 (25OHD3, 1 microgram/kg/day), 24,25-dihydroxyvitamin D3 [24,25(OH)2D3, 0.5 microgram/kg/day], or 1,25-dihydroxyvitamin D3 [1,25(OH)2D3, 0.05--0.25 microgram/kg/day] infused constantly for 3 weeks. The biochemical and skeletal effects of treatment were assessed by analytical methods and bone histomorphometry. The results show that only 1,25(OH)2D3 produced a dose-dependent elevation of serum calcium and phosphorus, and greatly improved bone mineralization at doses high enough to increase serum calcium and phosphorus concentrations within or above the normal range. Better improvement of bone mineralization was obtained when Pi was combined to 1,25(OH)2D3. In conjunction with the correction of hypocalcemia, Pi + 1,25(OH)2D3 suppressed the stimulation of bone turnover induced by Pi supplementation. The results show that, as in VDRR children, 1,25(OH)2D3 produces beneficial effects on bone lesions in Hyp/y mice, mainly through enhancement of mineral availability. However, the persistence of osteomalacia despite correction of serum mineral concentrations suggests that there is a specific bone cell resistance to mineral and/or hormonal influences in Hyp/y mice.     

Treatment of congenital osteopetrosis in the rabbit with high-dose 1,25-dihydroxyvitamin D

Osteopetrosis is a congenital metabolic bone disease characterized by skeletal sclerosis resulting from defective osteoclast-mediated bone resorption. Osteopetrosis has been described in several animal species (mouse, rat, and rabbit) and in children. Bone marrow transplantation, originally shown to reverse the skeletal sclerosis in some animal mutations, has been effective in curing osteopetrosis in some children. Unfortunately, not all children with osteopetrosis are candidates for or respond to bone marrow transplantation. Recent studies have shown that several animal mutations and some children inheriting osteopetrosis have significantly elevated serum levels of 1,25-(OH)2D. Based on the possibility that there may be a resistance to 1,25-(OH)2D, high-dose calcitriol therapy has been used to treat some children and stimulated some parameters of resorption. In this study, we have examined the effects of high-dose calcitriol therapy on various serum and skeletal parameters in the osteopetrotic rabbit. Mutant rabbits and normal littermates were given continuous infusions of calcitriol via subcutaneously implanted osmotic minipumps for 2 weeks at a dose of 0.5, 2.5, or 25 micrograms/kg/per day. Untreated mutant rabbits are hypocalcemic and hypophosphatemic in the presence of elevated serum 1,25-(OH)2 levels in comparison with their normal littermates. Calcitriol infusions resulted in dose-dependent increases in circulating 1,25-(OH)2D levels in both normal and mutant rabbits. However, evaluation of other serum parameters and the skeletal response demonstrated significant differences between osteopetrotic and normal rabbits. At the highest dose, normal animals rapidly became hypercalcemic and osteoporotic, accompanied by weight loss and a failure to thrive; mutants remained hypocalcemic and osteopetrotic but did not exhibit the deleterious physical effects seen in treated normal littermates. Although the number of osteoclasts increased in both mutants and normals, osteoclast phenotype in the former remained abnormal. These data indicate that although very high levels of circulating 1,25-(OH)2D were achieved in osteopetrotic mutants, activation of osteoclast-mediated bone resorption with subsequent improvement of skeletal sclerosis was not observed.     

Inhibition by aminohydroxypropylidene bisphosphonate (AHPrBP) of 1,25(OH)2 vitamin D3-induced stimulated bone turnover in the mouse

Summary The purpose of this study was to evaluate whether the 1,25(OH)₂D₃-induced increased bone mineralization in the mouse occurs in response to stimulation of bone resorption. In order to inhibit bone resorption, 35-day-old mice were given 16 μmol/kg/day of (3-amino-1-hydroxypropylidene)-1,1-bisphosphonate (AHPrBP) for 10 days, the first injection occurring 3 days prior to the continuous infusion of 0.06, 0.13, or 0.20 μg/kg/day of 1,25(OH)₂D₃ for 7 days. Two groups of mice were treated with AHPrBP or 1,25(OH)₂D₃ alone. The skeletal changes were assessed by histomorphometric study of caudal vertebrae after double³H-proline and double tetracycline labelings for evaluation of the matrix apposition rate (MaAR) and mineral apposition rate (MiAR), respectively. Treatment with AHPrBP alone or combined to 1,25(OH)₂D₃ decreased the number of acid phosphatase-stained osteoclasts and reduced the endosteal MaAR and MiAR and the amount of osteoid. When given alone, 1,25(OH)₂D₃ increased serum calcium above normal, enhanced the number of histochemically active osteoclasts, and stimulated the endosteal MiAR. Pretreatment with AHPrBP blocked both the increase in serum calcium and the stimulation of the MiAR induced by 1,25(OH)₂D₃ infusion though serum 1,25(OH)₂D₃ levels rose according to the dose given. The results show that 1) the serum calcium and the bone resorbing responses to 1,25(OH)₂D₃ infusion are prevented by pretreatment with AHPrBP, and 2) the stimulatory effect of 1,25(OH)₂D₃ on the mineralization rate is blocked when bone resorption is inhibited. The data indicate that 1,25(OH)₂D₃ promotes bone mineralization in the mouse mainly in response to stimulation of bone resorption.     

Antisense oligodeoxynucleotide evidence that a unique osteoclastic protein-tyrosine phosphatase is essential for osteoclastic resorption.

This study tested the hypothesis that a unique osteoclastic transmembrane protein tyrosine phosphatase (PTP-oc) is involved in osteoclastic resorption by determining whether suppression of PTP-oc expression with a specific phosphorothioated 20-mer PTP-oc antisense oligodeoxynucleotide (oligo) would inhibit basal, 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]-stimulated, and PTH-stimulated osteoclastic resorption. Treatment of rabbit osteoclasts with 1 microM of the antisense oligo for up to 4 days showed a time-dependent reduction in PTP-oc protein level, indicating that this PTP-oc antisense oligo was effective. To assess the effect of PTP-oc antisense oligo on osteoclastic resorption, rabbit osteoclasts were pretreated for 3 days with 1 microM of the antisense, a scramble oligo, or vehicle, respectively, followed by a 3-day treatment with vehicle, 10 nM of 1,25(OH)2D3, or 10 nM of parathyroid hormone (PTH). 1,25(OH)2D3 and PTH each alone increased PTP-oc cellular level and stimulated resorptive activity of rabbit osteoclasts. The antisense oligo treatment, but not the scramble oligo, decreased the basal and the stimulated resorption activity and reduced the PTP-oc protein level. Treatment with the PTP-oc antisense oligo, but not the scramble oligo, also markedly increased the Y527 phosphorylation level of c-src in rabbit osteoclasts. In conclusion, these results provide the first antisense oligo evidence that PTP-oc plays an essential role in osteoclastic resorption.     

Henoch-Schoenlein syndrome: a clinical and morphological study of renal biopsies

In 36 patients with incipient and advanced renal failure (CCr 80--30 ml/min X 1.73 m2), serum chemistry including ionized Ca, serum PTH and fractional intestinal absorption of Ca (whole body counter; two-dose-technique; 47Ca p.o. and i.v. to correct for urinary and endogenous fecal loss were measured. Quantitative bone histology after in vivo tetracycline double labeling was evaluated from undecalcified sections before and 18 months after therapy with vitamin D3 or 5,6-trans-25-OH-CC in a dose sufficient to raise intestinal absorption and/or urinary excretion of Ca. Intestinal absorption of Ca was impaired in some patients at a GFR of 60 ml/min/1.73 m2. After up to 10000 U/d 5,6-trans-25-OH-CC and 8000 IU/d vitamin D3, respectively, fractional intestinal absorption of Ca rose and was normalized in all patients. There was a concomitant rise in urinary Ca. Serum PTH fell, but did not always return into the normal range. Ionized Ca rose in all patients. Bone histology was evaluated in 17 of these 36 patients after informed consent was obtained. The mass of mineralized bone (Vv) rose in 7/17 patients, pointing to a positive calcium balance. Volumetric density of osteoid (Vvos) and surface density of osteoid (Svos) fell in 10/17 patients concomitant with an increase in the fraction of mineralizing seams and a decrease in the number of lamellae in osteoid seams. Osteoclastic resorption (OCl) fell as did the fraction of woven osteoid seams. However, woven osteoid failed to disappear completely and osteoclastic resorption stayed elevated in some patients. 5,6-trans-25-OH-CC and vitamin D3, in doses that normalized intestinal absorption of Ca, failed to restore completely bone histology to normal although mineralization and collagen texture of osteoid were consistently improved. The dose response characteristics to vitamin D of different abnormalities of Ca metabolism appear to be non-uniform.     

Increased bone volume and reduced bone turnover in vitamin D-replete rabbits by the administration of 24R,25-dihydroxyvitamin D3.

To study the effect of a large dose of 24R,25(OH)2D3 on bone metabolism, we treated vitamin D-replete rabbits with the agent for eight weeks. Fifteen rabbits 20 weeks of age were divided into three groups of five animals each. Group I received only the vehicle; groups II and III were given the agent at doses of 10 micrograms/kg/d, and 100 micrograms/kg/d, respectively. Through the dosing period, serum calcium, phosphorus, alkaline phosphatase, and creatinine levels were not altered. By the end of the experiment, serum 1,25(OH)2D or serum 25(OH)D levels did not change, nor did the PTH level. Serum 24,25(OH)2D levels for groups I, II, and III were 5.25 +/- 3.40, 76.16 +/- 19.90 (p less than .01), and 199.0 +/- 30.90 (p less than .01) ng/ml, respectively. The bone mineral content (BMC) significantly increased in group III. The percentages of BMC increase in group III over group I were 14.5% on the femur, 34.1% (p less than .01) on the sixth lumbar vertebra, and 23.3% (p less than .05) on the seventh lumbar vertebra. A marked increase of bone mineral densities in the cancellous bone-rich regions was seen in group III. Bone histomorphometry on the seventh lumbar vertebra demonstrated that both the eroded surface and the osteoclast number were reduced and the surfaces indicating bone formation such as the osteoid surface and the tetracycline double labeled surface were also reduced. However, both the osteoid thickness and the mineral apposition rate increased and the mineral formation rate at the tissue level remained approximately equal to that in the control.(ABSTRACT TRUNCATED AT 250 WORDS)     

Histological observations on the failure of rachitic rat bones to respond to 1,25/OH)2D3

Rachitic rats, maintained on diets with low or normal P contents, were given daily intraperitoneal doses of 1,25(OH)2D3 or 25OHD3 at levels of 100 or 200 ng. Plasma chemistry was measured and the ash content and histological appearance of the bones investigated. Using labeled material it was shown that the dosing levels of 1,25(OH)2D3 employed ensured a higher than normal plasma concentration of that metabolite over the period between doses. 1,25(OH)2D3 was not effective as 25OHD3 in raising bone ash or reducing the amount of osteoid. The difference between the effects of the metabolites was evident at both dietary P levels, but more marked at the higher P level. In contrast, the metabolites reduced the width of the epiphyseal plate to an approximately similar degree, and this is possibly the reason why there are discrepancies between previous reports of the effectiveness of 1,25(OH)2D3 compared with 25OHD3 or vitamin D3. Dosing with 1,25(OH)2D3 failed to maintain a constant plasma Pi value over the period between doses in animals fed the low P diet.     

Parathyroid hormone sensitizes long bones to the stimulation of bone resorption by 1,25-dihydroxyvitamin D3.

In response to hypocalcemia the serum PTH level increases rapidly followed by a PTH-induced rise in 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] production. Therefore, bone is first exposed to increased PTH levels before increased 1,25-(OH)2D3 levels. In the present study the effect of pretreatment with PTH on 1,25-(OH)2D3-induced bone resorption was examined. Bone resorption was measured as release of prelabeled 45Ca during culture from 17-day-old fetal mice radii/ulnae and metatarsals. Radii/ulnae and metatarsals are characterized by differences in development. In radii/ulnae mature osteoclasts are present, whereas in metatarsals only different stages of preosteoclasts can be found. Preincubation for 24 h but not 4 h with PTH increases the stimulation of bone resorption by 1,25-(OH)2D3 in fetal radii/ulnae but not in metatarsals. Coincubation of PTH and 1,25-(OH)2D3 did not result in a significant change in bone resorption compared to 1,25-(OH)2D3 alone. The observed difference in the effect of pretreatment with PTH between radii/ulnae and metatarsals indicates that PTH does not stimulate the development of early osteoclast precursors but that a certain level of differentiation of the osteoclast precursor is required. Pretreatment with prostaglandin E2 resulted in an effect similar to that of PTH. Inhibition of prostaglandin synthesis by indomethacin prevented the potentiation of 1,25-(OH)2D3-induced bone resorption by pretreatment with PTH. Thus, the present study demonstrates that PTH sensitizes responses to 1,25-(OH)2D3. PTH must be present before 1,25-(OH)2D3 to observe a potentiation of 1,25-(OH)2D3-induced bone resorption.(ABSTRACT TRUNCATED AT 250 WORDS)     

1,25-Dihydroxyvitamin D3-induced calcium efflux from calvaria is mediated by protein kinase C.

1,25-dihydroxyvitamin D3 (1,25(OH)2D3) is an important regulator of bone metabolism involved in both formation and resorption. Traditionally it was assumed that vitamin D receptors are intracellular. Recent data indicate that vitamin D may also act through a membrane receptor, specifically raising intracellular calcium and inositol 1,4,5 triphosphate. The present study was undertaken to explore further the mechanism(s) of vitamin D-induced bone resorption in cultured bone. 1,25(OH)2D3 induced a dose-dependent increase of calcium efflux from cultured bone. This increase was completely obliterated by inhibition of protein kinase C (PKC) with either staurosporine or calphostin C. In cultured rat calvariae, 1,25(OH)2D3 also induced a dose-dependent translocation of PKC from cytosol to membrane. The activation of PKC by 1, 25(OH)2D3 occurred following a 30-s incubation, peaked at 1 minute, and disappeared by 5 minutes. 1,25(OH)2D3 did not increase cAMP production in similarly cultured calvaria. These results suggest that the action of 1,25(OH)2D3 on calcium flux from cultured bone is mediated, in part, via activation of PKC.     

Ultrastructural development of hyperosteoidosis in 1,25(OH)2D3-treated rats fed high levels of dietary calcium

To evaluate the sequential ultrastructural pathogenesis of the increase in osseous tissue and hyperosteoidosis previously demonstrated in rats administered supraphysiologic doses of 1,25(OH)2D3 and fed high levels of dietary calcium, young adult female rats were placed on a 2.5% calcium and 0.3% phosphorus diet, administered ethanol or 135 ng (5 units) 1,25(OH)2D3 IP daily, and killed after 2, 4, 6, 8, and 10 days. Metaphyseal trabeculae from 1,25(OH)2D3 and placebo-treated rats were examined. Osteoblast hypertrophy characterized by increased cytoplasmic area, abundant rough endoplasmic reticulum, and prominent Golgi apparatus was evident in 1,25(OH)2D3-treated rats at Day 4. These osteoblasts were interpreted to be active in matrix synthesis. Widened osteoid seams were present at Day 6. Osteoblast hypertrophy and widened osteoid seams persisted through Day 10 in 1,25(OH)2D3-treated rats. The unmineralized bone matrix in 1,25(OH)2D3-treated rats contained more numerous cytoplasmic processes from adjacent osteoblasts than did control animals and loosely arranged collagen fibrils, which failed to aggregate in regions adjacent to the osteoid-mineralized bone interface as in placebo-treated rats. Osteoid seams in 1,25(OH)2D3-treated rats contained irregular electron-dense foci, which were often concentrated around embedded cytoplasmic processes. Osteocytic hypertrophy characterized by increased cytoplasmic area, developed rough endoplasmic reticulum, and increased numbers of mitochondria was evident at Day 2 and was sustained through Day 10 in 1,25(OH)2D3-treated rats. Variable-sized aggregates of electron-dense deposits similar to those concentrated around osteoblast cytoplasmic processes were observed in the pericellular space and on and immediately adjacent to the plasma membranes of osteocytes and embedding osteoblasts in 1,25(OH)2D3-treated rats as early as Day 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vitamin D deficiency in guinea pigs: exacerbation of bone phenotype during pregnancy and disturbed fetal mineralization,with recovery by 1,25(OH)2D3 infusion or dietary calcium-phosphate supplementation

Vitamin D (D) deficiency during human pregnancy appears to disturb fetal growth and mineralization, but fetal development is normal in D-deficient rats and vitamin D receptor gene-ablated mice. We used the guinea pig model to investigate maternal and fetal effects of D deficiency. Pregnant (Pr) and nonpregnant (NPr) animals were fed a D-replete (+D) or D-deficient diet (-D) for 8 weeks. We further studied whether the effects of a -D diet are reversed by continuous 1,25(OH)2D3 infusion (-D+1,25) and/or by a lactose-, Ca- and P-enriched D-deficient diet (-D+Ca/P). Bone analyses included histomorphometry of the proximal tibiae, dual-energy X-ray absorptiometry (DXA), and quantitative computed tomography (QCT) of the femora. Depletion of 25(OH)D3 and 1,25(OH)2D3 levels and the D-deficiency syndrome were more severe in pregnant animals. Indeed, Pr/-D but not NPr/-D guinea pigs were hypophosphatemic, and showed robust increases in growth plate width and osteoid surface and thickness; in addition, bone mineral density on DXA was lower in Pr/-D animals only, which was exclusively in cortical bone on QCT. Bone phenotype was partly normalized in Pr/-D+1,25 and Pr/-D+Ca/P animals. Compared with +D fetuses, -D fetuses had very low or undetectable 25(OH)D3 and 1,25(OH)2D3, were hypercalcemic and hypophosphatemic, and had lower osteocalcin levels. In addition, body weight and total body bone mineral content were 10-15% lower; histomorphometry showed hypertrophic chondrocyte zone expansion and hyperosteoidosis. 1,25(OH)2D3 levels were restored in -D+1,25 fetuses, and the phenotype was partially corrected. Similarly, the fetal +D phenotype was rescued in large part in -D+Ca/P fetuses, despite undetectable circulating 25(OH)D3 and 1,25(OH)2D3. We conclude that pregnancy markedly exacerbates D deficiency, and that augmenting Ca and P intake overrides the deleterious effects of D deficiency on fetal development.     

Derivation of osteoclasts from hematopoietic colony-forming cells in culture.

The osteoclast is known to be derived from the hematopoietic stem cell, but its lineage remains controversial. There is evidence that osteoclastic differentiation is induced through a contact-dependent interaction between bone marrow stromal cells and hematopoietic precursors. To analyze osteoclastic lineage, colonies were generated in semisolid medium from mouse spleen cells in the presence of Wehi-conditioned medium, interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF), or macrophage colony-stimulating factor (M-CSF) with or without erythropoietin (epo). After 5-8 days colonies were picked and phenotyped and incubated with 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] on bone slices or coverslips with bone marrow-derived cell lines (ts8 or ST2) that induce osteoclastic differentiation. Cells of osteoclastic phenotype [as judged by calcitonin receptor (CTR) expression or bone resorption] were observed only in multilineage colonies. The ability of cells that generate macrophage colonies (CFU-M) to generate osteoclasts was tested by incubating alveolar or peritoneal macrophages on ts8 or ST2 cells. Despite colony formation, no osteoclastic differentiation was detectable. Last, individual cells from blast cell colonies were incubated (1 cell per culture well) on ts8 or ST2 cells in the presence of 1,25-(OH)2D3 and epo (to expose the lineage potential of the plated cell). We found CTR-positive (CTRP) cells in 6 of 66 macrophage colonies, 7 of 12 granulocyte-macrophage (GM) colonies, and 49 of 50 colonies containing multiple lineages other than GM colonies. No single-lineage CTRP colonies were observed. Although most macrophage colonies did not contain CTRP, no CTRP were observed in colonies from which macrophages were absent. These results suggest that osteoclasts are derived from a multilineage precursor rather than from CFU-M.     

Cyclosporine A inhibits calcemic hormone-induced bone resorption in vitro

We have investigated the in vitro effects of cyclosporine (CsA), a potent immunosuppressive agent, on bone resorption induced by calcemic hormones. CsA inhibited parathyroid hormone (PTH), prostaglandin E2, 1,25-dihydroxy vitamin D3 (1,25(OH)2D3), and osteoclast-activating factor induced resorption of fetal rat limb bones in a dose-dependent manner. Established ongoing resorptive activity in bone was also inhibited by CsA. The CsA inhibition of bone resorption could be partially surmounted by higher concentrations of PTH and 1,25(OH)2D3. The inhibitory effects of CsA on limb bone resorption were reversible. Neither protein nor DNA synthesis were inhibited by treatment of limb bones with CsA. Thus, the inhibitory effect of this agent on bone resorption is not a cytotoxic one. These data could suggest that the induction of bone resorption by the calcemic hormones involves an immune cell derived mediator such as a lymphokine.     

The effect of hemodialysis, vitamin D metabolites and renal transplantation on the skeletal demineralization associated with renal osteodystrophy: a computerized histomorphometric analysis

Twenty-three patients with end-stage renal failure treated by hemodialysis or transplantation were followed for up to 10 years. Sequential full thickness iliac crest bone biopsies were obtained to assess the effects on bone disease of hemodialysis, treatment with 1,25-dihydroxycholecalciferol [1,25-(OH)2D3] and 24,25-dihydroxycholecalciferol [24,25-(OH)2D3] and renal transplantation. The biopsies were analyzed by a computerized histomorphometric technique which allowed accurate measurements of calcified bone and osteoid areas. Serum aluminum and parathyroid hormone concentrations were also monitored. Hemodialysis was associated with a loss of calcified bone and an increase in osteoid areas. The progressive bone loss was arrested but not reversed following treatment with either 1,25-(OH)2D3 or 24,25-(OH)2D3. Osteoid area was unchanged or reduced following treatment with 1,25-(OH)2D3 in all but three patients who had serum aluminum concentrations in excess of 5 mumol/l. 24,25-(OH)2D3 was not effective in reducing osteoid area, and combined treatment with 1,25 and 24,25-(OH)2D3 had no effect beyond that expected with 1,25-(OH)2D3 alone. Bone biopsies showed loss of calcified bone and an increase in osteoid areas one year and more after successful renal transplantation in five patients. Nineteen of the 23 patients developed serum aluminum concentrations greater than 3 mumol/l, probably because of the use of oral aluminum hydroxide as a phosphate binding agent. In these patients serum parathyroid hormone concentrations greater than 600 pg/ml appeared to prevent the development of osteopenia.