Bone material properties in actively bone-forming trabeculae in postmenopausal women with osteoporosis after three years of treatment with once-yearly Zoledronic acid.

Zoledronic acid (ZOL), a third-generation aminobisphosphonate, showed pronounced antifracture efficacy in a phase III clinical trial [Health Outcomes and Reduced Incidence with Zoledronic Acid Once Yearly-Pivotal Fracture Trial (HORIZON-PFT)] when administered yearly (5-mg infusions of ZOL), producing significant reductions in morphometric vertebral, clinical vertebral, hip, and nonvertebral fractures by 70%, 77%, 41%, and 25%, respectively, over a 3-year period. The purpose of this study was to analyze the biopsies obtained during the HORIZON clinical trial (152 patients, 82 ZOL and 70 placebo) by means of Raman microspectroscopy (a vibrational spectroscopic technique capable of analyzing undecalcified bone tissue with a spatial resolution of approximately 0.6 µm) to determine the effect of ZOL therapy on bone material properties (in particular mineral/matrix ratio, lamellar organization, carbonate and proteoglycan (based on spectral identification of glycosaminoglycan) content, and mineral maturity/crystallinity) at similar tissue age (based on the presence of tetracycline double labels). The results indicated that while ZOL administration increased the mineral/matrix ratio compared with placebo, it also resulted in mineral crystallites with a quality profile (based on carbonate content and maturity/crystallinity characteristics) of younger (with respect to tissue age) bone. Since the comparisons between ZOL- and placebo-treated patients were performed at similar tissue age at actively forming bone surfaces, these results suggest that ZOL may be exerting an effect on bone matrix formation in addition to its well-established antiresorptive effect, thereby contributing to its antifracture efficacy.

Circulating fibronectin affects bone matrix, whereas osteoblast fibronectin modulates osteoblast function.

The bone matrix is composed mostly of collagen, but the initial and continuous presence of fibronectin was found to be crucial for collagen matrix integrity in vitro. It has been assumed that osteoblasts produce the fibronectin required for bone matrix formation. Using transgenic mice, we conditionally deleted fibronectin in the osteoblasts and in the liver using the cre-loxP system. We also used mice with mutated fibronectin and conditionally deleted beta(1)-integrin in osteoblasts to identify the receptor involved in fibronectin effects on osteoblasts. Conditional deletion of fibronectin in the differentiating osteoblasts [using the 2.3 kb collagen-alpha1(I) promoter] failed to show a decrease in fibronectin amount in the bone matrix despite evidence of successful deletion. Using these mice we established that osteoblast-derived fibronectin solely affects osteoblast function. This effect was not mediated by integrins that bind to the RGD motif. Conditional deletion of fibronectin in the liver showed a marked decrease in fibronectin content in the matrix associated with decreased mineral-to-matrix ratio and changed biomechanical properties but had no effect on osteoblasts or osteoclasts. In conclusion, osteoblast fibronectin affects osteoblasts function. This does not seem to be mediated by the RGD motif on fibronectin. In contrast, liver-derived fibronectin affects bone matrix properties without affecting osteoblast or osteoclast function. A novel role for liver-derived circulating fibronectin thus was defined and delineated from that of locally produced fibronectin.

Effects of intravenous zoledronic acid once yearly on bone remodeling and bone structure.

UNLABELLED: In a substudy of the HORIZON pivotal fracture trial, in which yearly intravenous zoledronic acid 5 mg was found to significantly reduce risk of various fracture types in patients with postmenopausal osteoporosis, 152 patients underwent bone biopsy. Zoledronic acid reduced bone turnover by 63% and preserved bone structure and volume, with evidence of ongoing bone remodeling in 99% of biopsies obtained. INTRODUCTION: In the HORIZON pivotal fracture trial (PFT), enrolling 7,736 women with postmenopausal osteoporosis, three annual intravenous infusions of the bisphosphonate zoledronic acid (5 mg) significantly reduced morphometric vertebral, clinical vertebral, hip, and nonvertebral fractures by 70%, 77%, 41%, and 25%, respectively. Whereas 79% of patients received zoledronic acid/placebo only (stratum I, n = 6,113), 21% received concomitant treatment with other antiresorptive drugs, excluding other bisphosphonates, PTH, and strontium (stratum II, n = 1,652). MATERIALS AND METHODS: To determine effects on bone remodeling and bone architecture, iliac crest bone biopsies were obtained in 152 patients on active treatment or placebo at 3 yr after double tetracycline labeling. In five patients, only qualitative histology was performed, leaving 147 biopsy cores (79 on active treatment and 68 on placebo) for microCT analysis and histomorphometry. RESULTS: Analysis of bone structure by microCT revealed higher trabecular bone volume (BV/TV) in the zoledronic acid group (median, 16.6% versus 12.8%; p = 0.020). In addition, patients treated with zoledronic acid exhibited higher trabecular numbers (p = 0.008), decreased trabecular separation (p = 0.011), and a trend toward improvement in connectivity density (p = 0.062), all indicating better preservation of trabecular structure after treatment with zoledronic acid. Qualitative analysis revealed presence of tetracycline label in 81 of 82 biopsies from patients on zoledronic acid and all 70 biopsies from placebo patients, indicative of continued bone remodeling. No bone pathology was observed. Zoledronic acid induced a 63% median (71% mean) reduction of the activation frequency (Ac.f; p < 0.0001) and reduced mineralizing surface (MS/BS; p < 0.0001) and volume referent bone formation rate (BFR/BV) versus placebo, indicating reduced bone turnover. Mineral appositional rate was higher in the zoledronic acid group (p = 0.0002), suggesting improved osteoblast function compared with placebo. Mineralization lag time was similar in the two groups, whereas osteoid volume (OV/BV; p < 0.0001) and osteoid thickness (O.Th; p = 0.0094) were lower in zoledronic acid-treated patients, indicating normal osteoid formation and mineralization of newly formed bone. Concomitant administration of other antiresorptive osteoporosis therapies (e.g., raloxifene, tamoxifen, tibolone, ipriflavone) did not significantly alter the tissue level response to zoledronic acid. CONCLUSIONS: Annual dosing for 3 yr with zoledronic acid 5 mg intravenously resulted in a median 63% (mean, 71%) reduction of bone turnover and preservation of bone structure and mass without any signs of adynamic bone. Concomitant treatment with other osteoporosis therapies did not significantly affect the bone response to zoledronic acid.

TNF-induced structural joint damage is mediated by IL-1.

Blocking TNF effectively inhibits inflammation and structural damage in human rheumatoid arthritis (RA). However, so far it is unclear whether the effect of TNF is a direct one or indirect on up-regulation of other mediators. IL-1 may be one of these candidates because it has a central role in animal models of arthritis, and inhibition of IL-1 is used as a therapy of human RA. We removed the effects of IL-1 from a TNF-mediated inflammatory joint disease by crossing IL-1alpha and beta-deficient mice (IL-1-/-) with arthritic human TNF-transgenic (hTNFtg) mice. Development of synovial inflammation was almost unaffected on IL-1 deficiency, but bone erosion and osteoclast formation were significantly reduced in IL-1-/-hTNFtg mice, compared with hTNFtg mice based on an intrinsic differentiation defect of IL-1-deficient monocytes. Most dramatically, however, cartilage damage was absent in IL-1-/-hTNFtg mice. Chimera studies revealed that protection of cartilage is based on the loss of IL-1 on hematopoietic, but not mesenchymal, cells, leading to decreased expression of ADAMTS-5 and MMP-3. These data show that TNF-mediated cartilage damage is completely and TNF-mediated bone damage is partially dependent on IL-1, suggesting that IL-1 is a crucial mediator for inflammatory cartilage and bone degradation.

Antitumor effects of clinical dosing regimens of bisphosphonates in experimental breast cancer bone metastasis.

BACKGROUND: Bisphosphonates exhibit direct antitumor activity in animal models, but only at high doses that are incompatible with the clinical dosing regimens approved for the treatment of cancer patients with skeletal metastases. We compared the antitumor effects of clinical dosing regimens of the bisphosphonates zoledronic acid and clodronate in a mouse model of bone metastasis. METHODS: Mice (n = 6-10 per group) were treated with zoledronic acid, clodronate, or vehicle starting before (preventive protocols) or after (treatment protocols) intravenous injection with human B02/GFP.2 breast cancer cells, which express green fluorescent protein (GFP) and luciferase and metastasize to bone. Zoledronic acid was given as daily, weekly, or single doses at a cumulative dose of 98-100 microg/kg body weight, equivalent to the 4-mg intravenous dose given to patients. Clodronate was given as a daily dose (530 microg/kg/day), equivalent to the daily 1600-mg oral clinical dose given to patients. Bone destruction was measured by radiography, x-ray absorptiometry or tomography, and histomorphometry (as the ratio of bone volume to tissue volume). Skeletal tumor burden was measured by histomorphometry (as the ratio of tumor burden to soft tissue volume [TB/STV]) and luciferase activity. All statistical tests were two-sided. RESULTS: In treatment protocols, daily clodronate was less effective at decreasing the TB/STV ratio than daily (53% versus 87%, difference = 34%, 95% confidence interval [CI] = 16% to 44%, P < .001) or weekly (53% versus 90%, difference = 37%, 95% CI = 19% to 46%, P < .001) zoledronic acid-dosing regimens. Compared with vehicle, a single dose of zoledronic acid decreased tumor burden by only 16% (95% CI = 9% to 22%, P < .001). In preventive protocols, daily clodronate and daily or weekly zoledronic acid decreased the TB/STV ratio by 49% (95% CI = 40% to 57%, P = .006), 83% (95% CI = 68% to 98%, P < .001), and 66% (95% CI = 47% to 84%, P < .001), respectively, compared with vehicle, whereas a single dose of zoledronic acid decreased tumor burden by only 13% (95% CI = -2% to 28%, P = .84). Mice treated with a daily preventive regimen of clodronate or with a daily or weekly preventive regimen of zoledronic acid showed a decreased B02/GFP.2 cell tumor burden compared with vehicle, whereas a single preventive dose of zoledronic acid had no effect. CONCLUSION: Daily or repeated intermittent therapy with clinical doses of bisphosphonates inhibits skeletal tumor growth in a mouse model.

The molecular scaffold Gab2 is a crucial component of RANK signaling and osteoclastogenesis.

Morphogenesis and remodeling of bone involve synthesis of bone matrix by osteoblasts and coordinate resorption of bone by osteoclasts. Defective bone remodeling caused by altered osteoclast activity underlies a multitude of osteopenic disorders. Receptor activator of NF-kappaB (RANK) and its ligand RANKL have been identified as essential factors involved in osteoclast development and bone remodeling, but their mechanism and interacting factors have not been fully characterized. Here we report that the molecular adapter Grb-2-associated binder-2 (Gab2) associates with RANK and mediates RANK-induced activation of NF-kappaB, Akt and Jnk. Inactivation of the gene encoding Gab2 in mice results in osteopetrosis and decreased bone resorption as a result of defective osteoclast differentiation. We also show that Gab2 has a crucial role in the differentiation of human progenitor cells into osteoclasts. We have thus identified a new, key regulatory scaffold molecule, Gab2, that controls select RANK signaling pathways and is essential for osteoclastogenesis and bone homeostasis.