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Mineralizing osteoblasts are regularly used to study osteogenesis and model in vivo bone formation. Thus, it is important to verify that the mineral and matrix being formed in situ are comparable to those found in vivo. However, it has been shown that histochemical techniques alone are not sufficient for identifying calcium phosphate-containing mineral. The goal of the present study was to demonstrate the use of Fourier transform infrared imaging (FTIRI) as a tool for characterizing the spatial distribution and colocalization of the collagen matrix and the mineral phase during the mineralization process of osteoblasts in situ. MC3T3-E1 mouse osteoblasts were mineralized in culture for 28 days and FTIRI was used to evaluate the collagen content, collagen cross-linking, mineralization level and speciation, and mineral crystallinity in a spatially resolved fashion as a function of time. To test whether FTIRI could detect subtle changes in the mineralization process, cells were treated with risedronate (RIS). Results showed that collagen deposition and mineralization progressed over time and that the apatite mineral was associated with a collagenous matrix rather than ectopic mineral. The process was temporarily slowed by RIS, where the inhibition of osteoblast function caused slowed collagen production and cross-linking, leading to decreased mineralization. This study demonstrates that FTIRI is a complementary tool to histochemistry for spatially correlating the collagen matrix distribution and the nature of the resultant mineral during the process of osteoblast mineralization. It can further be used to detect small perturbations in the osteoid and mineral deposition process.

Young mice overexpressing Runx2 specifically in cells of the osteoblastic lineage failed to gain bone mass and exhibited a dramatic increase in bone resorption, leading to severe osteopenia and spontaneous vertebral fractures. The objective of the current study was to determine whether treatment with a bisphosphonate (risedronate, Ris), which reduces fractures in postmenopausal as well as in juvenile osteoporosis, was able to improve bone quality and reduce vertebral fractures in mice overexpressing Runx2. Four-week-old female Runx2 mice received Ris at 2 and 10 μg/kg subcutaneously twice a week for 12 weeks. Runx2 and wild-type mice received vehicle (Veh) as control. We measured the number of new fractures by X-ray and bone mineral density (BMD) by DEXA. We evaluated bone quality by histomorphometry, micro-CT, and Fourier transform infrared imaging (FTIRI). Ris at 20 μg/kg weekly significantly reduced the average number of new vertebral fractures compared to controls. This was accompanied by significantly increased BMD, increased trabecular bone volume, and reduced bone remodeling (seen in indices of bone resorption and formation) in the vertebrae and femoral metaphysis compared to Runx2 Veh. At the femur, Ris also increased cortical thickness. Changes in collagen cross-linking seen on FTIRI confirmed that Runx2 mice have accelerated bone turnover and showed that Ris affects the collagen cross-link ratio at both forming and resorbing sites. In conclusion, young mice overexpressing Runx2 have high bone turnover-induced osteopenia and spontaneous fractures. Ris at 20 μg/kg weekly induced an increase in bone mass, changes in bone microarchitecture, and decreased vertebral fractures.

Bisphosphonates (BPs) slow bone loss by reducing initiation of new basic multicellular units (BMUs). Whether or not BPs simply prevent osteoclasts from initiating new BMUs that resorb bone or also reduce the amount of bone they resorb at the BMU level is not clear. The goal of this study was to determine the effects of BPs on three morphological parameters of individual BMUs, resorption depth (Rs.De), area (Rs.Ar), and width (Rs.Wi). After 1 year of treatment with vehicle (VEH), alendronate (ALN; 0.10, 0.20, or 1.00 mg/kg/day), or risedronate (RIS; 0.05, 0.10, or 0.50 mg/kg/day), resorption cavity morphology was assessed in vertebral trabecular bone of beagle dogs by histology. Animals treated with ALN or RIS at the doses representing those used to treat postmenopausal osteoporosis (0.20 and 0.10 mg/kg/day, respectively) had significantly lower Rs.Ar (-27%) and Rs.Wi (-17%), with no difference in Rs.De, compared to VEH-treated controls. Low doses of ALN and RIS did not affect any parameters, whereas higher doses resulted in similar changes to those of the clinical dose. There were no significant differences in the resorption cavity measures between RIS and ALN at any of the dose equivalents. These results highlight the importance of examining parameters beyond erosion depth for assessment of resorption parameters. Furthermore, these results suggest that in addition to the well-known effects of BPs on reducing the number of active BMUs, these drugs also reduce the activity of osteoclasts at the individual BMU level at doses at and above those used clinically for the treatment of postmenopausal osteoporosis.

The purpose of the present study was to examine the effects of vitamin K(2) and risedronate on bone formation and resorption, the osteocyte lacunar system, and porosity in the cortical bone of glucocorticoid (GC)-treated rats. Forty-nine female Sprague-Dawley rats, 3 months of age, were randomized into five groups according to the following treatment schedule: age-matched control, GC administration, and GC administration with concomitant administration of vitamin K(2), risedronate, or vitamin K(2) + risedronate. At the end of the 8-week experiment, classical bone histomorphometric analysis was performed, and the osteocyte lacunar system and porosity were evaluated on the cortical bone of the tibial diaphysis. GC administration decreased percent cortical bone area and increased percent marrow area as a result of decreased periosteal bone formation, and increased endocortical bone erosion, and increased cortical porosity. Vitamin K(2) prevented a reduction in periosteal bone formation but did not affect percent cortical bone and marrow areas. Risedronate prevented a reduction in periosteal bone formation and an increase in endocortical bone erosion, resulting in prevention of alterations in percent cortical bone and marrow areas. Both vitamin K(2) and risedronate increased osteocyte density and lacunar occupancy and prevented a GC-induced increase in cortical porosity. Vitamin K(2) and risedronate had additive effects on osteocyte density and lacunar occupancy and a synergistic effect on cortical porosity. The present study showed the efficacy of vitamin K(2) and risedronate for bone formation and resorption, the osteocyte lacunar system, and porosity in the cortical bone of GC-treated rats.

Risedronate is used in osteoporosis treatment. Postmenopausal women enrolled in the Vertebral Efficacy with Risedronate Therapy trial received either risedronate (5 mg/day) or placebo for 3 years. Subjects received calcium and vitamin D supplementation if deficient at baseline. Lumbar spine bone mineral density (BMD) was measured at baseline and at 3 years. Quantitative back-scattered electron imaging (qBEI) was performed on paired iliac crest biopsies (risedronate, n = 18; placebo, n = 13) before and after treatment, and the mineral volume fraction in the trabecular bone was calculated. Combining dual-energy X-ray absorptiometric values with the mineral volume fraction for the same patients allowed us to calculate the relative change in trabecular bone volume with treatment. This showed that the effect on BMD was likely to be due partly to changes in matrix mineralization and partly due to changes in bone volume. After treatment, trabecular bone volume in the lumbar spine tended to increase in the risedronate group (+2.4%, nonsignificant) but there was a significant decrease (-3.7%, P < 0.05) in the placebo group. Calcium supplementation with adequate levels of vitamin D led to an approximately 3.3% increase in mineral content in the bone material independently of risedronate treatment. This increase was larger in patients with lower matrix mineralization at baseline and likely resulted from correction of calcium/vitamin D deficiency as well as from reduced bone remodeling. Combining BMD and bone mineralization density distribution data show that in postmenopausal osteoporosis 3-year treatment with risedronate preserves or may increase trabecular bone volume, unlike placebo. This analysis also allows, for the first time, separation of the contributions of bone volume and matrix mineralization to the increase in BMD.

Antiremodeling agents reduce bone loss in part through direct actions on osteoclasts. Their effects on osteoblasts and bone formation activity are less clear and may differ at sites undergoing modeling vs. remodeling. Skeletally mature intact beagles, 1-2 years old at the start of the study, were treated daily with clinically relevant doses of alendronate (0.10 or 0.20 mg/kg), risedronate (0.05 or 0.10 mg/kg), raloxifene (0.50 mg/kg), or vehicle (1 mL/kg). Dynamic bone formation parameters were histologically assessed on periosteal, endocortical/trabecular, and intracortical bone envelopes of the rib. Raloxifene significantly increased periosteal surface mineral apposition rate (MAR), a measure of osteoblast activity, compared to all other treatments (+108 to +175%, P < 0.02), while having no significant effect on MAR at either the endocortical/trabecular or intracortical envelope. Alendronate (both 0.10 and 0.20 doses) and risedronate (only the 0.10 dose) significantly (P < or = 0.05) suppressed MAR on the endocortical/trabecular envelope, while none of the bisphosphonate doses significantly altered MAR at either the periosteal or intracortical envelopes compared to vehicle. Based on these results, we conclude that (1) at clinically relevant doses the two classes of antiremodeling agents, bisphosphonates and selective estrogen receptor modulators, exert differential effects on osteoblast activity in the canine rib and (2) this effect depends on whether modeling or remodeling is the predominant mechanism of bone formation.

We previously demonstrated that suppression of bone remodeling allows microdamage to accumulate, thereby leading to reduced bone toughness in dog bone. In this study we evaluated the relationships between bone remodeling at the iliac crest and skeletal activation frequency, microdamage accumulation, or biomechanical properties of lumbar vertebrae using the same dogs to determine whether bone remodeling at the iliac crest can predict damage accumulation and mechanical parameters of the lumbar spine following treatment with antiresorptive agents. Thirty-six female beagles, 1 to 2 years old, were divided into three groups. The control group was treated daily for 12 months with saline vehicle. The remaining two groups were treated daily with oral risedronate at a dose of 0.5 mg/kg/day, or alendronate at 1.0 mg/kg/day orally. The doses of these bisphosphonates were 5 to 6 times the clinical doses approved for treatment of osteoporosis in humans. After sacrifice, the right ilium and L2 vertebra were assigned to histomorphometry. The left ilium and L3 vertebra were used for microdamage analysis. The L4 vertebra was mechanically tested to failure in compression, and bone toughness calculated from the stress-strain curve. There was a strong positive relationship for activation frequency (Ac.f) between ilium and lumbar vertebrae (r2 = 0.82; P < 0.0001). Iliac crest Ac.f underestimates Ac.f in L2, but L2 Ac.f reaches a minimum threshold and does not decline further when iliac crest Ac.f is below 0.10/yr. Microdamage (Cr.S.Dn) accumulation at the ilium was significantly associated with increased microdamage accumulation in the L3 lumbar vertebra (r2 = 0.43, P < 0.0001). The data also show that bisphosphonate treatment increased Cr.S.Dn at a faster rate in L3 than in the iliac crest. Although bisphosphonate treatment decreased bone toughness in L4, this decrease demonstrated no relationship to decreased Ac.f in the ilium. These results clearly indicate that bone remodeling data obtained from iliac crest biopsy could be used to estimate the activation frequency and microdamage burden in the vertebral column.

Among the bisphosphonates (BPs), the aminobisphosphonates (aminoBPs) have much stronger bone-resorption-inhibitory activities (BRIAs) than nonaminobisphosphonates (nonaminoBPs). However, aminoBPs have inflammatory effects. We previously reported that in mice: (i) all aminoBPs tested (10-40 micromol/kg) induced various inflammatory reactions (including induction of histidine decarboxylase), whereas clodronate (a non-aminoBP) (10-160 micromol/kg) inhibited these reactions; and (ii) a clear sclerotic line (tentatively called the BP line) was detectable in the tibia by radiography a few weeks after a single injection of either alendronate (a typical aminoBP) (1.6 micromol/kg) or clodronate (160 micromol/kg), and this BP-line formation (a marker for the BRIAs of BPs) was not reduced in mice given both alendronate and clodronate. In this study, using this murine model, we compared clodronate, etidronate (another typical non-aminoBP), alendronate, etidronate + alendronate, and clodronate + alendronate in terms of their inflammatory effects and/or BP-line formation. For BP-line formation, 480 micromol/kg etidronate was needed (single injection). At 160 micromol/kg, etidronate inhibited the histidine decarboxylase induction, but not the other inflammatory reactions induced by alendronate. However, etidronate (unlike clodronate) also inhibited alendronate-induced BP-line formation (even at 40 micromol/kg). Etidronate (160 micromol/kg) also inhibited the physicochemical changes in the tibia induced by six, weekly injections of alendronate. Therefore, depending on the dose, etidronate can inhibit alendronate's inflammatory actions and its BRIA. These results, together with those reported previously, suggest that a strategy utilizing clodronate (but not etidronate) plus an aminoBP might prevent or reduce the inflammatory side effects induced by aminoBPs while preserving their powerful BRIAs. We discuss the mechanisms underlying the antagonism between aminoBPs and non-aminoBPs.

We examined the effects of one-year high-dose bisphosphonates (risedronate 0.5 mg/kg/day or alendronate 1.0 mg/kg/day) on the three-dimensional (3-D) microstructural and mechanical properties of canine cancellous bone. A high-resolution micro-CT scanner was used to scan cubic specimens produced from the first lumbar vertebrae. Microstructural properties of the specimens were calculated directly from the 3-D datasets and the mechanical properties of the specimens were determined. Our data demonstrate significant microarchitectural changes in the bisphosphonate-treated cancellous bone that was typically plate-like, denser, with thicker and more trabeculae compared with those of the controls. Consistent with architectural changes, the Young's moduli of cancellous bone increased in all three directions with the greatest increase in primary axial loading (cephalo-caudal) direction after treatment. Our results suggest a bone remodeling-adaptation mechanism stimulated by bisphosphonates that increases bone volume fraction, thickens trabeculae, changes trabeculae towards more plate-like, and increases mechanical properties. The secondary degree of anisotropy contributed significantly to the explained variance in bone strength, and the primary or tertiary degree of anisotropy improved the explanation of variances for Young's moduli, i.e., 79% of strength variances or 74-83% of modulus variances could be explained by the combined anisotropy and bone volume fraction. These significant improvements of cancellous bone architecture provide a rationale for the clinical observation that fracture risk decreased by 50% in the first year of bisphosphonate therapy with only a 5% increase in bone mineral density. We conclude that bisphosphonates enhance mechanical properties and reduce fracture risk by improving architectural anisotropy of cancellous bone 3-D microarchitecture.

Bisphosphonates exert a potent inhibitory effect on bone resorption. Several studies have been performed, with contradictory results, to ascertain whether the effect of bisphosphonates on osteoclasts could be produced, at least in part, by modulation of the synthesis of resorption-promoting factors by osteoblasts. The aim of this study was to evaluate the effect of etidronate (10-4-10-9 M) and alendronate (10-7-10-12 M) on the production of IL-6 and IL-11 using human osteoblast cultures. Cytokines were quantified by ELISA, and mRNA expression was tested. Treatment with alendronate and etidronate had no effect on the synthesis of IL-6 or IL-11, and IL-6 and IL-11 mRNA levels. These results were obtained both in nonstimulated cultures and in cultures stimulated by means of TNF-a, IL-1b, and TNF-a+IL-1b, with or without FCS. In conclusion, a possible indirect osteoclast-mediated effect of alendronate and etidronate on bone resorption would not be exerted through reduction in osteoblastic synthesis of IL-6 and IL-11.

The effects of risedronate, alendronate, and etidronate were assessed in calcifying fibroblastic colony-forming unit (CFU-f) cultures of rat bone marrow cells in vitro. Biphasic effects on the formation of bone-like colonies were observed. Treatment with high concentrations (10(-5)-10(-4)M) of alendronate and risedronate caused a total inhibition of colony formation whereas etidronate had relatively little effect. It was also found that intermediate concentrations (10(-6)M) of alendronate and risedronate decreased the formation of colonies displaying osteoblastic characteristics such as alkaline phosphatase expression, collagen accumulation, and calcification. At lower concentrations (10(-9)-10(-7)M), risedronate and alendronate increased the formation of fibroblastic colonies, suggesting a mild anabolic effect, however, the formation of colonies with osteoblastic properties was not affected. Treating the cells with a combination of bisphosphonate and 1 mM geranylgeraniol could to some extent abrogate the cytotoxic effects of alendronate or risedronate, suggesting the involvement of the mevalonate pathway. The colony-stimulating activity of these bisphosphonates was, however, unaffected.

Two kinds of remodeling, stochastic and targeted, have been proposed based on the observation that microdamage in bone can initiate the remodeling process. Bisphosphonates are known to suppress stochastic bone remodeling. It has been hypothesized that bisphosphonates allow microdamage to accumulate, suggesting that they also suppress targeted remodeling. This study investigated whether suppression of remodeling using bisphosphonates inhibits remodeling targeted to repair microdamage, or whether the suppression of stochastic remodeling alone can account for the observed increase in damage accumulation. Beagle dogs were divided into three groups: control (CNT), risedronate-treated (RIS), and alendronate (ALN)-treated groups. The doses of both bisphosphonates were 6 times higher than the clinical doses. After 1-year treatment, animals were sacrificed and the right 9th rib was assigned to microdamage analysis. There were 3.06 times more associations between cracks and resorption spaces in CNT than expected (P < 0.005), indicating that remodeling normally targets cracks for repair, i.e., cracks can initiate a new remodeling event. However, although there was increased microdamage accumulation in RIS and ALN compared with CNT, fewer cracks than expected were associated with resorption spaces. The observation in RIS and ALN that there were fewer associations between cracks and resorption spaces than expected indicates that both targeted and non-targeted remodeling are suppressed in these groups. These data further suggest that the complete suppression of targeted remodeling could account for the increased microdamage burden.

We have compared the measurements obtained by different methods: dual energy X-ray absorptiometry (DXA), histomorphometry, ash weight, and two morphometric indices (robusticity and bone weight/bone length index) in the orchidectomized (ORX) rat model of male osteoporosis. We examined 144 male wistar rats: 48 sham-operated, 48 ORX, and 48 ORX-treated with a bisphosphonate (risedronate) 2 or 10 mg/kg/day, 5 days per week. Rats were sacrificed at 2, 4, 8, or 16 weeks after the beginning of the study. DXA was performed on a Hologic QDR 2000 on the whole body, whole tibia, and tibial metaphysis. Bone volumes (C.BV/C.TV, and BV/TV) were measured by histomorphometry on the proximal tibial. A significant correlation was obtained between weight measured by DXA and scale (r = 0.993, P <0.000001). However, DXA underestimated weight by 0.3%. This discrepancy was dependent on the rat's weight. The weight bone length (WL) index was linearly correlated with BMD (r = 0.86), BMC (r = 0.96), and ash weight (r = 0.97). Correlation with robusticity was lower than with the WL index. A significant correlation was found between BMC of the metaphyseal region and the bone volumes but this explained only 27% of the variance; correlation with BMD was poorer (r = 0.40). BMC and ash weight were highly correlated (r = 0.992, P <0.000001). However, DXA overestimated BMC by 11% and the overestimation was found to be clearly dependent on the net mineral content of the bone.

Bisphosphonates, therapeutic reagents against tumoral bone diseases (Paget's disease or osteoporosis), are potent inhibitors of bone resorption. The mechanisms by which they directly act on mature osteoclasts remain unclear. Using a recently developed technique for isolation of highly purified mammalian mature osteoclasts, we demonstrated that etidronate [ethane-1-hydroxy-1,1-diphosphonate (EHDP), 1-hydroxy-1,1-ethylidenebisphosphonate], inhibited directly osteoclastic bone-resorbing activity by pit assay. In addition, EHDP also directly induced apoptosis and disrupted actin rings in osteoclasts. The data support previous data on non-purified osteoclasts and results in vivo.

Calcitriol has been widely used in the management of osteoporosis, but its efficiency is a matter of controversy. It is not known whether combinations of calcitriol and antiresorptive agents such as etidronate and calcitonin are superior to calcitriol alone in the treatment of postmenopausal osteoporosis. To make this determination, 30 Turkish women with postmenopausal osteoporosis between 45 and 68 years of age were randomized to receive either intermittent cyclical etidronate (400 mg/day, for 14 days) followed by 60 days of cyclical calcitriol therapy 0.25 microg twice daily (group 1; n = 10), or calcitriol 0.25 microg twice daily (group 2; n = 10), or calcitriol 0.25 microg/day in combination with 100 IU intranasal salmon calcitonin taken every other day (group 3; n = 10) through a 1-year period. Bone mineral density (BMD) of lumbar spine (L2 to L4) was determined for each patient by dual-photon absorptiometry (153Gd) at baseline, after 6 months, and at the end of the study. There was no significant difference among groups with respect to mean spinal BMD at baseline, after 6, and after 12 months. No significant spinal BMD changes occurred in any group from baseline, after 6 months, and after 12 months. Four patients in groups 1 and 2 and five patients in group 3 developed hypercalcemia at least once during therapy. Hypercalciuria occurred at least once in 9, 10, and 7 patients in groups 1, 2, and 3, respectively. One patient in group 2 developed a renal stone at the end of the study. Mean urine hydroxyproline levels did not change significantly in any group with respect to baseline. The data suggest that one-year treatment with calcitriol, given either alone or in combination with antiresorptive agents, does not improve spinal BMD in Turkish women with postmenopausal osteoporosis, and is associated with a high rate of adverse events.

A computer model of trabecular bone turnover has been developed, based on concepts of Jonathan Reeve [1]. This model predicts changes in bone volume by summing bone resorption and formation over a large number of remodeling sites. Clinical data [histomorphometry and bone mineral content (BMC)] from two clinical studies using an antiresorptive drug (etidronate disodium, EHDP) in postmenopausal osteoporosis were used to test the model. The results for BMC obtained from the EHDP and placebo groups in each study at 60 and 120 weeks were correctly predicted by the model from the histomorphometric data obtained from baseline and week 60 biopsies. The parameter in this model having the greatest influence on predicted changes in bone volume was found by sensitivity analysis to be activation frequency. These results suggest that the contribution of bone turnover to BMC can be predicted solely by considering the cell kinetics of the basic multicellular unit (BMU), and that, in the case of antiresorptive drugs, maximal effects on bone volume may be achieved by pharmacological reduction of activation frequency. The results also suggest that the present model may be useful in predicting in clinical studies the effects of EHDP and similar drugs on bone turnover.

The study was designed to determine the skeletal effects of withdrawal of estrogen and diphosphonate treatment in the estrogen-deplete state. Groups of ovariectomized (OVX) rats were treated with vehicle alone, estrogen, or the diphosphonates etidronate or risedronate for a 180-day period. A group of sham-operated control rats was treated for 180 days with vehicle alone. All treatments were then terminated, followed by sequential sacrifice of rats at 0, 35, 90, 180, and 360 days after withdrawal of treatment. The proximal tibia from each animal was processed undecalcified for quantitative bone histomorphometry. At the end of the treatment period, vehicle-treated OVX rats were characterized by cancellous osteopenia and increased bone turnover relative to vehicle-treated control rats. Treatment of OVX rats with estrogen or diphosphonates depressed bone turnover and protected against cancellous osteopenia. During the withdrawal period, OVX rats previously treated with estrogen exhibited rapid bone loss associated with increased bone turnover. The bone protective effect of the hormone in OVX rats was nearly completely lost by 90 days of withdrawal. In contrast, OVX rats maintained low levels of bone turnover and normal cancellous bone mass at 180 days of withdrawal from diphosphonate treatment. The results suggest that estrogen-deplete women who are withdrawn from estrogen replacement are at high risk for subsequent bone loss. They further suggest that widely spaced periods of intermittent diphosphonate treatment may be sufficient to prevent the development of osteopenia in postmenopausal and oophorectomized women.

A single, high dose of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) results in three different types of lesions along the enamel surface of the rat incisor, one of which is seen as a "bright band" crossing the final enamel surface in the scanning electron microscope (SEM). The present study presents the structural surface features of final enamel formation and its subsequent maturation in normal and HEBP-exposed rats. The position of the bright band is examined in relation to where the Tomes processes pits disappear (DTPP), where the boundary between "light" and "dark" enamel (LDB) as seen by SEM is located, and in particular, where the so-called opaque boundary (OB) is positioned. Groups of rats were given a subcutaneous dose of 0, 5, or 10 mg P/kg body wt of HEBP and killed at intervals of either 12 hours or 2 or 9 days. The mandibular incisors were processed for SEM after enzymatic digestion of enamel organ remains. Enamel surface nodules, 100-300 nm in diameter and composed of smaller units, were evident at the start of final enamel formation which was defined as the area from DTPP to LDB. With increasing maturation, the nodules merged to form a smooth surface. In HEBP-treated animals, growth and merging of these surface nodules became arrested at the time of injection resulting in an irreversible "porous" stage corresponding to that part of the surface enamel. This area--the bright band--developed corresponding to the start of the area of final enamel formation and was subsequently carried incisally during the eruption of the incisor.(ABSTRACT TRUNCATED AT 250 WORDS)

Hypercalcemia of malignancy is a commonly encountered serious clinical problem that often requires aggressive therapy. In order to combine the rapid hypocalcemic effects of calcitonin with the more delayed effect of a bisphosphonate, we administered etidronate, 7.5 mg/kg/day intravenously and salmon calcitonin, 100 IU subcutaneously, every 12 hours for 3 days in 9 patients with hypercalcemia associated with malignancy. The mean serum calcium concentration fell from 3.33 +/- 0.1 mmol/liter (mean +/- SEM) to 2.88 +/- 0.1 mmol/liter within 24 hours (P less than 0.001). All patients had a fall in the serum calcium concentration of greater than 0.5 mmol/liter and it returned to normal in 7 of the 9 patients. We conclude that the combination of salmon calcitonin with etidronate more effectively lowers the serum calcium concentration in patients with hypercalcemia of malignancy then the use of either agent alone.

Bisphosphonates are potent inhibitors of bone resorption. In previous studies, we have shown that ovariectomy accelerates bone resorption and 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP) inhibits ovariectomy-accelerated bone resorption in female Wistar adult rats. As interleukin 1 (IL-1) stimulates bone resorption in vitro and in vivo, we have investigated the effects of ovariectomy and HEBP administered in vivo on IL-1 secretion from peritoneal macrophages in adult rats. Ovariectomy or sham surgery were performed in female Wistar rats at 40 weeks of age. Ovariectomized and sham-operated rats were administered with HEBP (10 mg) or saline, 10 times in total, from 43 to 46 weeks of age. Paraffin oil-induced peritoneal macrophages at 46 weeks of age were cultured for 24 hours. Lipopolysaccharide (LPS)-stimulated peritoneal macrophages from ovariectomized rats secreted more IL-1 than sham-operated rats. HEBP decreased LPS-stimulated IL-1 secretion from peritoneal macrophages in ovariectomized rats, but not in sham-operated rats. In vivo administration of HEBP decreased IL-1 secretion only in postovariectomy hyperresorptive states. These results suggest that alterations in LPS-stimulated IL-1 secretion from oil-induced peritoneal macrophages may be responsible, at least in part, for the postovariectomy acceleration in bone resorption and its inhibition by HEBP.

Calvaria taken from rats (normal or thyroparathyroidectomized) given a diphosphonate (EHDP 10 mg/kg daily for 7 days) were placed in special Ussing chambers for the measurement of phosphate fluxes to and from bone. When compared with appropriate controls, the experimental calvaria showed a marked decrease in influx, K, a significant increase in the projected equilibrium concentration, E/K, where E is the efflux and a significant increase in the calcium concentration in the medium. It is concluded that this large increase in passive "solubility" is caused by the diphosphonate's ability to stabilize the small amount of regulator phase (brushite or brushite-apatite mixture) present in bone.

Chicks chronically prelabelled with 45Ca, 3H-tetracycline, and 3H-proline were used to measure the weekly effect of EHDP (5 mg P/kg for 28 days) on bone turnover at the whole bone level in vivo. Direct measurements were made of cortical bone resorption (loss of 3H-tetracycline and 3H-collagen from whole femur, blood-bone ratio of 45Ca), skeletal collagen formation and bone mineralization (collagen and calcium mass per whole femur, respectively). Chicks were sacrificed after 5, 14, 21 and 28 days of EHDP administration. By five days of treatment, EHDP caused a greater inhibition of bone mineralization (86%) than bone resorption (30-39%) without affecting skeletal collagen mass. The blood-bone ratio of 45Ca decreased 34%, which was similar in degree to the inhibition of 3H-tetracycline loss (30%) and 3H-collagen loss (39%). Almost complete inhibition of bone resorption and bone mineralization occurred by 14 days of treatment without effects on skeletal collagen mass. No additional effect was seen at 21 and 28 days of EHDP treatment. In chicks, EHDP inhibits almost completely bone mineralization (bone formation) and cortical bone resorption without affecting skeletal collagen mass.

Stimulated by the rather sparse information in the literature on cellular changes induced by EHDP, we carried out electron microscopic investigations on young bone tissue and on de novo bone formation. Cellular changes could be observed during continuous administration of EHDP. The osteoblasts demonstrated temporary storing of crystalloid structures in the mitochondria, and atypical osteocytes showed persistent changes indicative of hyperactivity. The osteoclasts exhibited varying ultrastructural features with respect to the number and appearance of nuclei, Golgi, RER, and lysosomes. These changes under the influence of EHDP could be an indication of altered activity of the osteoclast. The possible interference of EHDP with bone cell metabolism is discussed.

It has been hypothesized that phagocytosis of a layer of mineralized material which is sometimes deposited along the root surface following trauma is an important factor in the initiation of the resorption of teeth. To test this hypothesis, freezing of the mouse incisor periodontium was used as a model where deposition of mineral was prevented by the systemic administration of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP, 10 mg P/kg b.w.) for time periods varying from 2 to 31 days. The animals were killed either 24 hours or 1 month after the last injection and their mandibles were processed for light microscopy. In the HEBP-treated animals, killed 24 hours after the last injection, no mineralized material had been deposited along the root surface, and very little resorption was seen. Large amounts of bone matrix were deposited in the intraperiodontal space (often causing localized ankylosis). In HEBP-treated animals killed 1 month after the last injection, again no layer of mineralized material along the root surface was seen, but now root resorption had become manifest, occupying up to 40% of the cement surface. This occurred irrespective of the duration of HEBP administration. In the saline-treated animals, extensive resorption of the incisor was noted (up to 90% of its surface). It is concluded that the layer of mineralized material deposited after the periodontium is frozen does not play a decisive part in the onset of root resorption. HEBP treatment slows down root resorption but does not prevent it.

For a compound to inhibit potently the transformation of amorphous calcium phosphate into hydroxyapatite, it is suggested that the minimum structural requirement is a phosphate group and, at some other position, either another phosphate group preferably or a carboxylic moiety. Primary amino groups abolish inhibitor potential. Inhibitor potency is modified by various secondary factors, including the number and proximity of active groups, their stereochemistry, steric factors, the lability of the molecule, and in special instances its lipophilicity. Parameters used to monitor the transformation indicate that inhibitors can be grouped into two classes, and it is suggested that this is because one class acts as a hydroxyapatite crystal growth inhibitor. The close proximity of two phosphate groups or of a phosphate and multiple carboxylic groups is proposed to determine in part whether or not a compound acts as a crystal growth inhibitor. Further, bulky side groups render a molecule inactive as a crystal growth poison, although it will still inhibit by other mechanisms.

The diphosphonates dichloromethanediphosphonate (Cl2MDP) and to a minor degree 1-hydroxyethane-1,1-diphosphonate (EDHP) increased the glycogen content in cultured cartilage cells of rabbit ear. This effect was not due to different concentrations of glucose in the medium.

The intracellular effects of different diphosphonates on splenic macrophages were compared after intravenous injection of liposomes with encapsulated dichloromethylene diphosphonate (C12MDP), 3-amino-1-hydroxypropane-1,1-diphosphonate (APD), or 1 hydroxyethane-1,1-diphosphonate (EHDP) in similar concentrations. Intracellular C12MDP was by far the most toxic compound for macrophages in the spleen. APD encapsulated in liposomes in the highest possible concentration eliminated marginal zone macrophages only and EHDP did not affect any of the macrophage subpopulations in the spleen.

Both the intracellular compartment and bone mineral are supposed to play a role in acid-base balance by contributing to the extrarenal acid buffering capacity. Bisphosphonates could affect extrarenal acid buffering capacity by interfering with the formation and/or dissolution of bone mineral. In the present study, rats were pretreated with either 1-hydroxyethylidene-1, 1-bisphosphonate (HEBP, 10 mg/kg.day sc), with prevailing inhibitory action on bone mineral formation, or dichloromethylene bisphosphonate (Cl2MBP, 10 mg p/kg.day sc) with prevailing action on bone resorption, or NaCl injections (controls) for 7 days. In intact rats, blood acid-base variables were influenced by neither HEBP, nor Cl2MBP. Two hours after nephrectomy and before acute acid loading, HEBP-but not Cl2MBP-pretreated rats displayed a significant increase in both blood HCO3- and PCO2. After HCl infusion (2.5 mEq/kg), the relative decrement in blood HCO3- (difference in blood HCO3- before and after acid loading) was transiently more important in the two bisphosphonate pretreated groups than in controls. After a 24 hour fasting period, nephrectomized animals pretreated with Cl2MBP displayed significantly lower blood HCO3- and pH values than controls or HEBP-pretreated rats. These results suggest that bisphosphonates influence extrarenal buffering capacity according to their prevailing inhibitory action on either bone mineral formation and/or dissolution. These compounds could interfere with the release rate of bone proton buffers. However, in the presence of normal renal function, this effect does not disturb the blood acid-base equilibrium.

Administration of large quantities of ethane-1-hydroxy-1,1-diphosphonate to growing chicks resulted in a decrease in percent bone ash and an increase in percent osteoid. The degree of inhibition of bone mineral accumulation was a function of both duration and quantity of ethane-1-hydroxy-1,1-diphosphonic acid administration. The inhibition of bone mineral accumulation could be partially corrected with administration of 1,25-dihydroxyvitamin D3. Administration of high levels of ethane-1-hydroxy-1,1-diphosphonate also resulted in inhibition of intestinal calcium absorption. This could be reversed or prevented by the administration of 1,25-dihydroxyvitamin D3.

The effects on serum uric acid (SUA) of two diphosphonates (EHDP at 5 and 20 mg/kg/day and Cl2MDP at 400 and 1600 mg/day) were studied in 49 pagetic patients treated for 6 months. Patients were divided into two groups: group I, initially normouricemic (SUA less than 385 mumol/l); group II, initially hyperuricemic (SUA greater than or equal to 385 mumol/l). SUA was significantly decreased (P less than 0.01) after 6 months of diphosphonate therapy in all group II patients. However, 3 months after withdrawal of therapy, SUA returned to values not significantly different from those initially recorded in this group. SUA did not change during or after treatment in the group I patients. Groups I and II could not be differentiated on the basis of initial serum alkaline phosphatase or urinary hydroxyproline values. In response to therapy, both groups showed the same reduction in these parameters. These results suggest that diphosphonates have no effect at a single level in uric acid metabolism. They certainly reduce the part of the urate pool coming from the nucleic acids of the increased bone cell population by reducing the number of osteoclasts and osteoblasts, which is extremely high in pagetic bone. They also must act on uric acid metabolism through other mechanisms which need to be investigated in further studies.

Effects of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) on the dentinogenesis of the rat maxillary incisor were microradiographically examined 96 hours after a single subcutaneous injection. HEBP produced hypomineralized incremental bands in the enamel (HPOe) and in the dentine (outer HPO1 and inner HPO2) in a dose-dependent manner. In the labial dentine, both HPO1 and HPO2 did not extend apically beyond the apical limit of the HPOe and were thus located in the circumpulpal dentine. The two lesions in the labial dentine fully developed with a dosage of 10 mg/ P/kg or larger, and with 15 mg P/kg or larger, gross hypoplastic lesions developed. Therefore, histological analysis at shorter postinjection intervals was carried out on the labial dentine with 10 mg P/kg of HEBP. The HPO1 appeared along the dentine-predentine junction by 24 hours and was characterized by reduced numbers of inorganic crystals and 10 nm particles which invested the collagen fibrils. Therefore, the HPO1 was judged to be produced by the disturbance of transformation of the predentine to dentine. The precursor lesion of HPO2 appeared in the proximal predentine by 4 hours, and was characterized by reduced number of collagen fibrils and unusually coarse interfibrillar stippled material. This lesion migrated to the dentine by 96 hours and became the HPO2, which was characterized by reduced number of collagen fibrils. Individual collagen fibrils in the HPO2 were invested by as many inorganic crystals and 10 nm particles as in the normal dentine. The HPO2 is considered to be produced by the disturbance of organic matrix formation.