Effects of 3 years treatment with once-yearly zoledronic acid on the kinetics of bone matrix maturation in osteoporotic patients.

UNLABELLED: Once-yearly administration of intravenous zoledronic acid for 3 years in humans affects the kinetics of matrix filling in by mineral, independent of bone turnover. INTRODUCTION: Yearly 5-mg infusions of zoledronic acid (ZOL) for 3 years have shown pronounced antifracture efficacy. The purpose of the present study was to test whether ZOL affects the kinetics of forming bone material properties maturation. METHODS: Iliac crest biopsies from the HORIZON-PFT clinical trial were analyzed by Raman microspectroscopy in actively bone-forming surfaces as a function of tissue age in trabecular and osteonal bone, to determine ZOL's effect on bone material quality indices maturation kinetics. RESULTS: Mineral/matrix ratio increased in both groups as a function of tissue age, at both osteonal- and trabecular-forming surfaces; ZOL exhibiting the greatest increase in the trabecular surfaces only. The proteoglycan content showed a dependency on tissue age in both trabecular and osteonal surfaces, with ZOL exhibiting lower values in the tissue age 8-22 days in the trabecular surfaces. Mineral crystallinity (crystallite length and thickness) showed a dependence on tissue age, with ZOL exhibiting lower crystallite length compared with placebo only in the 8- to 22-day-old tissue at trabecular surfaces, while crystal thickness was lower in the 1- to 5-day-old tissue at both osteonal and trabecular surfaces. CONCLUSIONS: The results of the present study suggest that once-yearly administration of intravenous ZOL for 3 years in humans does not exert any adverse effects on the evolution of bone material properties at actively forming osteonal and trabecular surfaces, while it may have a beneficial effect on the progression of the mineral-to-matrix ratio and mineral maturity bone quality indices.

Altered cellular signalling and decreased platelet sensitivity to adenosine in insulin-dependent diabetic patients with proliferative retinopathy.

Platelets from patients with insulin-dependent diabetes with proliferative retinopathy showed the same reactivity to ADP as those from control subjects. Responsiveness of platelets to the aggregation inhibitor adenosine and to the analogue N-ethylcarboxamidoadenosine was decreased in diabetes. In contrast, responsiveness to the anti-aggregatory effects of prostaglandin I2 was not significantly altered in diabetes. Platelets from diabetic patients exhibited decreased formation of cyclic AMP in response to N-ethylcarboxamidoadenosine compared with those from control subjects. In contrast, when adenylyl cyclase was stimulated by prostaglandin I2 or by forskolin, no differences in cyclic AMP formation were observed between control and diabetic platelets. Diabetes was associated with an apparent loss of high-affinity binding of [3H]N-ethylcarboxamidoadenosine to platelet membranes. Possible mechanisms that could contribute to this diabetes-induced change in signalling through the platelet A2 adenosine receptor are discussed.

Protein malnutrition attenuates bone anabolic response to PTH in female rats.

PTH is indicated for the treatment of severe osteoporosis. Elderly osteoporotic patients frequently suffer from protein malnutrition, which may contribute to bone loss. It is unknown whether this malnutrition may affect the response to PTH. Therefore, the aim of the present study was to assess whether an isocaloric low-protein (LP) diet may influence the bone anabolic response to intermittent PTH in 6-month-old female rats. Six-month-old female rats were either pair fed an isocaloric LP diet (2.5% casein) or a normal-protein (NP) diet (15% casein) for 2 weeks. The rats continued on their respective diet while being treated with 5- or 40-µg/kg recombinant human PTH amino-terminal fragment 1-34 (PTH-[1-34]) daily, or with vehicle for 4 weeks. At the end of this period, areal bone mineral density, bone mineral content, microstructure, and bone strength in axial compression of proximal tibia or 3-point bending for midshaft tibia tests were measured. Blood was collected for the determination of IGF-I and osteocalcin. After 4 weeks of PTH-(1-34), the dose-dependent increase of proximal tibia bone mineral density, trabecular microstructure variables, and bone strength was attenuated in rats fed a LP diet as compared with rats on a NP intake. At the level of midshaft tibia cortical bone, PTH-(1-34) exerted an anabolic effect only in the NP but not in the LP diet group. Protein malnutrition was associated with lower IGF-I levels. Protein malnutrition attenuates the bone anabolic effects of PTH-(1-34) in rats. These results suggest that a sufficient protein intake should be recommended for osteoporotic patients undergoing PTH therapy.

Assessing bone quantity by pQCT.

We have tested the ability of the XCT960A to detect bone loss in OVX-rats, as well as bone gain in the proximal tibial metaphysis of healthy rats treated with hPTH(1-34). The results demonstrated that high precision can be achieved, with CV's for most measurement parameters in the range of 1.6 to 5.9% being obtained in vivo with repositioning of animals. Significant changes in bone parameters in the tibia were observed already at 2 weeks following OVX or PTH-therapy, while whole bone mass measured in the tibia by DEXA ex vivo did not change significantly for up to 24 weeks. For the proximal rat tibia at location 5mm distal to the knee joint was identified as an optimal site. At this location, cortices are fairly parallel thus reducing the partial volume effect, the area is relatively rich in cancellous bone increasing the magnitude of bone gain or loss, and the site (2mm below the growth plate) is relevant for comparisons with histomorphometric measurements. The results demonstrate that pQCT can be adapted for use in small animals such as rats, and that it is a sensitive, reproducible, non-invasive method available to monitor changes in bone mass, bone density, and geometric properties. Future studies should help to establish whether the moment of inertia, moment of resistance and the newly added bone strength index provided by the machine are predictive in any way for bone strength as obtained from biomechanical testing procedures. Peripheral QCT in small animals is an important addition for drug evaluation because it is more sensitive than DEXA and allows for shorter duration of experiments. This non-invasive method can reliably measure changes in cancellous and cortical bone mass over time following ovariectomy or administration of the bone anabolic hormone hPTH(1-34). pQCT should be viewed as a complimentary technique to static and dynamic histomorphometry, which does not replace either of these methods. Its value in the field of basic research should be evaluated.

Bone tissue and its mineralization in aged estrogen-depleted rats after long-term intermittent treatment with parathyroid hormone (PTH) analog SDZ PTS 893 or human PTH(1-34).

Intermittently administered parathyroid hormone (PTH) is a potent bone anabolic agent. We aimed to determine the impact of long-term treatment with PTH on bone structure, dynamics, and mineralization. We ovariectomized (ovx) 1-year-old rats with the exception of a baseline and a sham-operated group. Twelve weeks later, a 36 week treatment with PTH analog SDZ PTS 893 (12.5, 25, 50, 100 microg/kg), human PTH(1-34) (25, 50, 100 microg/kg), or vehicle (ovx, sham) was initiated. Bone dynamics, structure, and mineralization were evaluated in the lumbar spine and in the femoral diaphysis. Cancellous bone turnover was elevated 12 weeks postovariectomy in estrogen-deficient, vehicle-treated animals, but returned to the level of the sham group by 48 weeks. The animals experienced substantial cancellous bone loss associated with a reduction of trabecular number and presented with a partly rod-like trabecular network. After 36 weeks of treatment with SDZ PTS 893 or human PTH(1-34), cancellous bone formation rates and turnover were raised in all treated groups compared with age-matched controls. The mineral apposition rate was increasing with dose. This amplified matrix synthesis led to trabecular thickening, but not to an increase in trabecular number, resulting in a crude, plate-like cancellous network with a high bone volume fraction. Fluorochrome label-based cortical bone dynamics demonstrated that a thick ring of new bone was formed at the endocortex by activation of modeling drifts during treatment. Treatment-induced cortical bone formation was increased with dose at the subperiosteal and endocortical envelopes, but substantially higher at the latter. Intracortical bone turnover was elevated near the endocortex. Bone mineralization was undisturbed in all compartments. The average degree of mineralization was lowered slightly, reflecting the increased portion of new bone formed during treatment. In summary, the main anabolic effect was mediated for both peptides by an increase in bone apposition with dose, persisting throughout treatment that lasted more than one third of the lifespan of the rats, and direct activation of bone-forming surfaces. As a result, a substantial amount of new bone, maintained at elevated turnover and adequate mineralization levels, formed predominantly at compartments exposed to bone marrow.

Long-term therapy of ovariectomy-induced osteopenia with parathyroid hormone analog SDZ PTS 893 and bone maintenance in retired breeder rats.

The aim of the study was to assess the long-term anabolic effect of the parathyroid hormone (PTH) analog SDZ PTS 893 in a dose-response manner, and to determine the ability of the antiresorptive agents estradiol and alendronate to maintain bone mass after withdrawal of SDZ PTS 893. One hundred thirty retired breeder Wistar rats were distributed into 13 groups with 10 rats in each group: 1 baseline group, 2 sham groups, and 10 ovariectomized groups. Treatment was initiated 12 weeks after ovariectomy. SDZ PTS 893 treatment was administered daily subcutaneously (Monday to Friday) for 36 weeks. Treatment regimens were as follows: (1) baseline (-12 weeks); (2) ovariectomy (ovx) (0 weeks); (3) sham (36 weeks); (4) ovx (36 weeks); (5) SDZ PTS 893 12.5 microg/kg/day (36 weeks); (6) SDZ PTS 893 25 microg/kg/day (36 weeks); (7) SDZ PTS 893 50 microg/kg/day (36 weeks); (8) SDZ PTS 893 100 microg/kg/day (36 weeks); for the maintenance part of the study: (9) sham (48 weeks); ovx animals treated with SDZ PTS 893, 50 microg/kg/day for 36 weeks followed by 12 weeks of treatment regimens: (10) placebo; (11) SDZ PTS 893 50 microg/kg/day; (12) estradiol 10 microg/kg/day; or (13) alendronate 28 microg/kg (2 injections/week). The effects of ovx, SDZ PTS 893 treatment, and maintenance regimens were measured at four skeletal sites: lumbar vertebra; femoral diaphysis; distal femoral metaphysis; and proximal femoral metaphysis (femoral neck). At these sites, bone density and bone strength were measured as treatment endpoints. Furthermore, bone dimensions were measured at the midpoint of the femur. The results showed that SDZ PTS 893 increased bone strength in a dose-dependent manner at all skeletal sites tested. At the vertebral body and distal femoral metaphysis, apparent ash density increased in a similar way. There was a slight decrease in cortical density at the mid-diaphyseal site. Static histomorphometry showed increased bone area due to a decreased marrow area (endosteal net bone gain) but also due to increased tissue area (periosteal net bone gain). For maintenance, continuous SDZ PTS 893 therapy was most efficient, followed by alendronate and estradiol treatment with regard to preservation of bone mass and strength. It is concluded that the new PTH analog SDZ PTS 893 has a highly anabolic, dose- and time-dependent effect on all skeletal sites tested. Bone formation is induced at both endosteal and periosteal surfaces.

The effect of enterocystoplasty on bone strength assessed at four different skeletal sites in a rat model.

The objective of the study was to investigate bone strength at four different skeletal sites in a chronic animal model of urinary diversion. Young male Wistar rats (120) were allocated randomly to four groups undergoing ileocystoplasty; ileocystoplasty and resection of the ileocecal segment; colocystoplasty; or sham operation (controls). After 8 months the lumbar vertebrae, femora, and tibiae were harvested at necropsy. Bone strength was assessed biomechanically at four different skeletal sites: vertebra L3, femoral middiaphysis, femoral neck, and distal femoral metaphysis. Bone mass and architecture were assessed using standard static histomorphometry of the proximal tibial metaphysis (trabecular bone volume [BV/TV]; trabecular number [Tb.N]) and ash weight. Statistically significant differences of biomechanical parameters between groups were observed at three skeletal sites with corresponding changes in tibial histomorphometry. Isolated ileocystoplasty resulted in decreased maximum load values of L3 (-16.4%; p < 0.0035) and a substantial reduction in tibial BV/TV (-34.7%; p < 0.05). Ileocystoplasty combined with resection of the ileocecal segment led to a significant loss of bone strength of L3 (-32.4%; p < 0.0015) and a dramatic reduction of tibial BV/TV (-45.9%; p < 0.01). Loss of tibial metaphyseal bone mass was predominantly caused by a decrease in Tb.N. (p < 0.01). Colonic augmentation had no significant effect on bone strength or histomorphometric values. In conclusion, this is the first experimental study to demonstrate the relevance of histomorphometrically proven bone loss after enterocystoplasty in terms of biomechanical variables.

Influence of a low calcium and phosphorus diet on the anabolic effect of human parathyroid hormone (1-38) in female rats.

Parathyroid hormone (PTH) or synthetic N-terminal PTH fragments administered intermittently have been established as anabolic agents in animal and human bones. In the present study, the influence of a low calcium diet on the anabolic effect of human PTH(1-38) [hPTH(1-38)] was investigated. Forty-eight 10-week-old female Sprague-Dawley rats were randomly assigned to a diet with a low calcium content (LCa) or a diet with the recommended amount of calcium (RCa). After an adaptation period of 15 days, the rats were randomly assigned to hPTH(1-38) treatment (+LCa/+RCa) or vehicle only (-LCa/-RCa) for an additional 14 days. Total bone mineral density (BMD) values of several bones were determined using quantitative computed tomography and from ratios of ash weight to volume. Biomechanical competence of the fourth lumbar vertebrae and of the right femora was assessed. An anabolic effect could be detected in both PTH-treated groups. However, the bones of the +LCa group showed significantly lower BMD and also a diminished increase in maximal breaking force compared with those of the +RCa group. The study demonstrates that the anabolic effect of hPTH(1-38) is blunted by the LCa diet. This suggests that, during PTH treatment, dietary calcium intake is critical.

PTH and interactions with bisphosphonates.

We report that a therapeutic dose of the antiresorptive bisphosphonate alendronate administered to skeletally mature rats for the duration of 16 weeks significantly blunted the anabolic response to a high dose SDZ PTS 893 in the tibia and femur but not in lumbar vertebra. Effects were seen at the level of bone mass (DEXA, pQCT) as well as in biomechanical tests. In one arm of this study, rats were switched to vehicle injections after 8 weeks on alendronate for another 8 weeks before being challenged with the anabolic stimulus (washout). This recovery period was insufficient for full recovery and the response to SDZ PTS 893 was still greatly reduced after this procedure. Serial pQCT-measurements suggest that part of the interaction happened during the first two weeks of PTH treatment when bone-lining cells are activated by the anabolic drug. In addition bisphosphonate pretreated rats failed to catch up with the vehicle control at all time points suggesting a second level of drug interaction. The failure of the 'washout' period to restore the normal response to PTH is suggestive of a physico-chemical interaction on the level of the matrix embedded bisphosphonate with the overlaying bone lining cells, rather than of direct effects of the drug on osteoblasts or their precursor cells. Overall the data raises the possibility, that bisphosphonate treated patients respond to PTH and SDZ PTS 893 with a delay which could affect the shorter bone mass measurements carried out at 6 months to 1 year. Additionally, bisphosphonate pre-treated rats did not develop the full anabolic response over time. Clinical investigators studying anabolic drugs such as PTH should be aware of potential long-term interactions of bisphosphonates when assessing the outcome of their experiments. However, the beneficial effect of bisphosphonates like alendronate on PTH-induced bone remodeling, as well as its potent action in the protection of bone loss after cessation of anabolic therapy might outweigh the worries about a small delay in the bone response to parathyroid hormone.

Multi-lineage potential of human mesenchymal stem cells following clonal expansion.

Bone marrow contains mesenchymal cells that can be isolated and grown in vitro. Using appropriate treatment protocols such cultures can be induced to differentiate to yield osteoblasts, adipocytes, and chondrocytes. However, previous experiments had not addressed the question whether single pluripotent stem cells exist and can give rise to these different cell lineages or whether bone marrow mesenchymal cell preparations represent a mixture of committed precursors. We have used human adult bone marrow-derived mesenchymal cells obtained from iliac crest biopsies to demonstrate clonal outgrowth after limiting dilution and we show that some clones can be expanded over more than 20 cumulative population doublings and differentiated to osteoblasts, adipocytes, and chondrocytes. Our data provide direct experimental evidence that cultures of bone marrow-derived mesenchymal cells contain individual cells that fulfil two essential stem cell criteria: (i) extensive self-renewal capacity and (ii) multi-lineage potential.

Bone degeneration and recovery after early and late bisphosphonate treatment of ovariectomized wistar rats assessed by in vivo micro-computed tomography.

Bisphosphonates are antiresorptive drugs commonly used to treat osteoporosis. It is not clear, however, what the influence of the time point of treatment is. Recently developed in vivo micro-computed tomographic (CT) scanners offer the possibility to study such effects on bone microstructure in rats. The aim of this study was to determine the influence of early and late zoledronic acid treatment on bone in ovariectomized rats, using in vivo micro-CT. Twenty-nine female Wistar rats were divided into the following groups: ovariectomy (OVX, n = 5), OVX and zoledronic acid (ZOL) at week 0 (n = 8), OVX and ZOL at week 8 (n = 7), and sham (n = 9). CT scans were made of the proximal tibia at weeks 0, 2, 4, 8, 12, and 16; and bone structural parameters were determined in the metaphysis. Two fluorescent labels were administered to calculate dynamic histomorphometric parameters. At week 16, all groups were significantly different from each other in bone volume fraction (BV/TV), connectivity density, and trabecular number (Tb.N), except for the early ZOL and control groups which were not significantly different for any structural parameter. After ZOL treatment at week 8, BV/TV, structure model index, Tb.N, and trabecular thickness significantly improved in the late ZOL group. The OVX and ZOL groups showed, respectively, higher and lower bone formation rates than the control group. Early ZOL treatment inhibited all bone microstructural changes seen after OVX. Late ZOL treatment significantly improved bone microstructure, although the structure did not recover to original levels. Early ZOL treatment resulted in a significantly better microstructure than late treatment. However, late treatment was still significantly better than no treatment.

Comparison of the effects of carvedilol, propranolol, and verapamil on in vitro platelet function in healthy volunteers.

The effects of the two beta-blockers carvedilol and propranolol and the calcium antagonist verapamil on platelet aggregation induced by ADP, epinephrine, or collagen was examined in the serum of eight healthy volunteers. The inhibitory potential of the three drugs on the arachidonic acid-prostaglandin pathway was measured by the assessment of the formation of the stable metabolite thromboxane B2. Furthermore, the influence of drugs on intracellular cAMP levels was measured in platelets aggregated with ADP, epinephrine, or collagen. All three drugs were able to inhibit platelet aggregation induced by a threshold concentration of ADP, epinephrine (10 microM), or collagen (1 micrograms/ml) in a concentration-dependent manner, preventing aggregation completely in the high micromolar range. Propranolol is 1.6 and 2.3 times more active than verapamil and carvedilol in inhibiting ADP-induced platelet aggregation. Although the two beta-blockers were marginally more potent than verapamil in inhibiting platelet aggregation induced by collagen, this was not statistically significant. Both propranolol and verapamil were slightly more active than carvedilol in inhibiting epinephrine-induced platelet aggregation, a trend consistent with the IC50 values. Intracellular cAMP levels decreased in platelets aggregated with ADP, epinephrine, and collagen. The inhibition of platelet aggregation with the three drugs resulted in a return of intracellular cAMP levels to basal values. Carvedilol and propranolol were of similar potency and around 1.5-3 times as potent as verapamil for all agonists. The formation of thromboxane B2, an end product of the arachidonic acid pathway, could be completely inhibited by the three drugs. Propranolol was 2.4 and 2.1 times more potent than carvedilol and verapamil when platelets were aggregated with ADP.(ABSTRACT TRUNCATED AT 250 WORDS)

Lipids and thrombosis.

There is extensive evidence of important interactions between plasma lipoproteins and platelet function. Some population groups, particularly hypercholesterolaemic patients, have strong evidence of abnormal platelet function which is mediated by the binding of lipoproteins, especially oxidized LDL, to surface receptors. Additionally, abnormal plasma lipid levels precipitate membrane composition changes by increasing the cholesterol:phospholipid ratio. The resulting changes in microviscosity seem to affect transmembrane signalling and might in some cases influence receptor binding. This not only has important therapeutic implications with regard to lipid-lowering drug therapy but also with regard to the potential beneficial effects of dietary therapy.

Comparison of the healing and revascularization of onlayed autologous and lyophilized allogeneic rib grafts to the edentulous maxilla.

The healing and revascularization of onlayed autologous and lyophilized allogeneic rib grafts to the edentulous maxilla in the Macaca fascicularis monkey were studied using clinical, histologic, and microangiographic methods at varying intervals of up to eight months. Results indicated that healing and revascularization were similar but resorption of the allografts occurred approximately three months later than resorption of the autografts. Both grafting systems appeared to have minimal osteogenic potential. Osteoinduction and the final bony augmentation obtained were less than were seen with comparable autologous and allogeneic interpositional grafts.

Decreased sensitivity to adenosine in platelets from patients with familial hypercholesterolaemia--a change reversed by cholestyramine treatment.

Platelet-rich plasma was obtained from patients with untreated heterozygous familial hypercholesterolaemia (FH), from FH patients treated with cholestyramine and from control subjects. Responsiveness of platelets to the aggregation inhibitors adenosine, its analogue N-ethylcarboxamidoadenosine (NECA) and prostaglandin I2 was decreased in FH. Patients on cholestyramine therapy showed normal responsiveness to adenosine and NECA. There were only minor changes in the binding of [3H]NECA to high-affinity binding sites on platelet membranes from untreated FH or cholestyramine-treated FH patients. The initial rate of cyclic AMP formation in response to a high concentration of NECA was severely decreased in platelets from FH patients. By contrast, the rate of cyclic AMP formation in response to forskolin or a high concentration of prostaglandin I2 was unchanged. These data point to a defect in the coupling of the platelet A2 adenosine receptor to adenylyl cyclase in untreated FH patients.

Systemic administration of a novel octapeptide, amylin-(1---8), increases bone volume in male mice.

Amylin increases bone mass when administered systemically to mice. However, because of its size, the full peptide is not an ideal candidate for the therapy of osteoporosis. The fragment, amylin-(1---8), stimulates osteoblast proliferation in vitro, although it is without effect on carbohydrate metabolism. The present study assessed the effects of daily administration of this peptide on sexually mature male mice for 4 wk. Amylin-(1---8) almost doubled histomorphometric indices of osteoblast activity but did not change measures of bone resorption. Trabecular bone volume increased by 36% as a result of increases in both trabecular number and trabecular thickness, and tibial cortical width increased by 8%. On three-point bending tests of bone strength, displacement to fracture was increased by amylin-(1---8), from 0.302 +/- 0.013 to 0.351 +/- 0. 017 mm (P = 0.02). In a separate experiment using dynamic histomorphometry with bone-seeking fluorochrome labels, amylin-(1---8) was administered by local injection over the calvariae of female mice. Amylin-(1---8) (40 nM) increased the double-labeled surface threefold. The effect was dose dependent from 0.4 to 40 nM and was greater than that of an equimolar dose of human parathyroid hormone-(1---34) [hPTH-(1---34)]. Mineral apposition rate was increased by 40 nM amylin-(1---8) but not by hPTH-(1---34). Amylin-(1---8) thus has significant anabolic effects in vivo, suggesting that this peptide or analogs of it should be further evaluated as potential therapies for osteoporosis.

Skeletal growth and long-term bone turnover after enterocystoplasty in a chronic rat model.

OBJECTIVE: To investigate skeletal growth and bone metabolism in a chronic animal model of urinary diversion. MATERIALS AND METHODS: Young male Wistar rats (120) were allocated randomly to four groups undergoing: ileocystoplasty, ileocystoplasty and resection of the ileocaecal segment, colocystoplasty, and controls. All animals received antibiotics for 1 week after surgery; half of each group remained on oral antibiotics. Bone-related biochemistry was measured in serum and urine. Dual-energy X-ray absorptiometry and peripheral quantitative computed tomography (pQCT) were used to determine bone mass ex vivo. RESULTS: Most (90%) of the rats survived the study period (8 months); six rats died from bowel obstruction at the level of the entero-anastomosis and four had to be killed because of persistent severe diarrhoea. Vital intestinal mucosa was found in all augmented bladders. There were no differences in bone length and volume. Loss of bone mass was almost exclusively in rats with ileocystoplasty and resection of the ileocaecal segment (-37.5%, pQCT, P < 0.01). There was no hyperchloraemic metabolic acidosis or gross impairment of renal function. Hypomagnesaemia, hypocalcaemia and decreased insulin-like growth factor-binding protein 3 were the only significant findings on blood analysis. Deoxypyridinoline crosslinks in urine were higher in rats with an enterocystoplasty than in controls. CONCLUSIONS: Enterocystoplasty in rats neither impairs skeletal growth nor bone quantity, but leads to significant loss of bone mass when combined with resection of the ileocaecal segment. Rarefaction of the trabecular network is confined to the metabolically highly active cancellous compartment, most likely as a consequence of intestinal malabsorption.