A Controlled Study of the Effects of Alendronate in a Growing Mouse Model of Osteogenesis Imperfecta

Recent studies have reported that bisphosphonates reduce fracture incidence and improve bone density in children with osteogenesis imperfecta (OI). However, questions still persist concerning the effect of these drugs on bone properties such as ultrastructure and quality, particularly in the growing patient. To address these issues, the third-generation bisphosphonate alendronate was evaluated in the growing oim/oim mouse, an animal model of moderate-to-severe OI. Alendronate was administered to 6-week-old mice during a period of active growth at a dosage of 73 microg alendronate/kg/day for the first 4 weeks and 26 microg alendronate/kg/day for the next 4 weeks. Positive treatment effects included a reduction in the number of fractures sustained by the alendronate-treated oim/oim mice compared with untreated oim/oim mice (2.1+/-2.0 vs 3.2+/-1.6 fractures per mouse), increased femoral metaphyseal density (0.111+/-0.02 vs 0.034+/-0.04 g/cm2), a tendency towards reduced tibial bowing (4.0+/-3.7 vs 6.1+/-5.8 degrees), and towards increased femoral diameter (1.22+/-0.12 vs 1.15+/-0.11 mm). Potential negative effects included a persistence of calcified cartilage in the treated oim/oim metaphyses compared with treated wildtype (+/+) (33.8+/-11.1 vs 22.1+/-10.2%), and significantly shorter femora compared with nontreated oim/oim mice (14.8+/-0.67 vs 15.3+/-0.37 mm). This preclinical study demonstrates that alendronate is effective in reducing fractures in a growing mouse model of OI, and is also an important indicator of potential positive and negative outcomes of third-generation bisphosphonate therapy in children with OI.     

Alendronate Treatment of the Brtl Osteogenesis Imperfecta Mouse Improves Femoral Geometry and Load Response Before Fracture but Decreases Predicted Material Properties and Has Detrimental Effects on Osteoblasts and Bone Formation

Long courses of bisphosphonates are widely administered to children with osteogenesis imperfecta (OI), although bisphosphonates do not block mutant collagen secretion and may affect bone matrix composition or structure. The Brtl mouse has a glycine substitution in col1a1 and is ideal for modeling the effects of bisphosphonate in classical OI. We treated Brtl and wildtype mice with alendronate (Aln; 0.219 mg/kg/wk, SC) for 6 or 12 wk and compared treated and untreated femora of both genotypes. Mutant and wildtype bone had similar responses to Aln treatment. Femoral areal BMD and cortical volumetric BMD increased significantly after 12 wk, but femoral length and growth curves were unaltered. Aln improved Brtl diaphyseal cortical thickness and trabecular number after 6 wk and cross‐sectional shape after 12 wk. Mechanically, Aln significantly increased stiffness in wildtype femora and load to fracture in both genotypes after 12 wk. However, predicted material strength and elastic modulus were negatively impacted by 12 wk of Aln in both genotypes, and metaphyseal remnants of mineralized cartilage also increased. Brtl femoral brittleness was unimproved. Brtl osteoclast and osteoblast surface were unchanged by treatment. However, decreased mineral apposition rate and bone formation rate/bone surface and the flattened morphology of Brtl osteoblasts suggested that Aln impaired osteoblast function and matrix synthesis. We conclude that Aln treatment improves Brtl femoral geometry and load to fracture but decreases bone matrix synthesis and predicted material modulus and strength, with striking retention of mineralized cartilage. Beneficial and detrimental changes appear concomitantly. Limiting cumulative bisphosphonate exposure of OI bone will minimize detrimental effects.     

Strontium Ranelate Reduces the Fracture Incidence in a Growing Mouse Model of Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a genetic bone dysplasia characterized by brittle bones with increased fracture risk. Although current treatment options to improve bone strength in OI focus on antiresorptive bisphosphonates, controlled clinical trials suggest they have an equivocal effect on reducing fracture risk. Strontium ranelate (SrR) is a promising therapy with a dual mode of action that is capable of simultaneously maintaining bone formation and reducing bone resorption, and may be beneficial for the treatment of OI. In this study, SrR therapy was investigated to assess its effects on fracture frequency and bone mass and strength in an animal model of OI, the oim/oim mouse. Three‐week‐old oim/oim and wt/wt mice were treated with either SrR or vehicle (Veh) for 11 weeks. After treatment, the average number of fractures sustained by SrR‐treated oim/oim mice was significantly reduced compared to Veh‐treated oim/oim mice. Micro–computed tomographic (μCT) analyses of femurs showed that both trabecular and cortical bone mass were significantly improved with SrR treatment in both genotypes. SrR significantly inhibited bone resorption, whereas bone formation indices were maintained. Biomechanical testing revealed improved bone structural properties in both oim/oim and wild‐type (wt/wt) mice under the treatment, whereas no significant effects on bone brittleness and material quality were observed. In conclusion, SrR was able to effectively reduce fractures in oim/oim mice by improving bone mass and strength and thus represents a potential therapy for the treatment of pediatric OI. © 2015 American Society for Bone and Mineral Research.     

Dysfunction of Caveolae-Mediated Endocytic TβRI Degradation Results in Hypersensitivity of TGF-β/Smad Signaling in Osteogenesis Imperfecta

Osteogenesis imperfecta (OI) is a genetic disorder caused by mutations of type I collagen-related genes, and excessive transforming growth factor-beta (TGF-β) signaling is a common mechanism. TGF-β/Smad signaling has inhibitory effects on osteoblast differentiation and maturation and is mainly transduced and regulated by the internalization of a tetrameric receptor complex comprising types I and II TGF-β receptors (TβRI and TβRII). During internalization, clathrin-mediated endocytosis enhances TGF-β/Smad signaling via Smad2/3 phosphorylation and receptors recycling, while caveolae-mediated endocytosis turns off TGF-β/Smad signaling by promoting receptor ubiquitination and degradation. In this study, using an animal model of OI (Colla2^oim, osteogenesis imperfecta murine [oim]/oim mouse), we found that osteoblastic cells of oim/oim mice were more sensitive to the inhibitory effects of TGF-β on osteoblast differentiation and maturation and had much higher cell membrane protein levels of TGF-β receptors than those of wild-type (wt)/wt mice. Further results showed that clathrin-mediated endocytosis of TβRI was enhanced, whereas caveolae-mediated TβRI endocytic degradation was reduced in oim/oim mice, combined with reduced caveolin-1 (Cav-1) phosphorylation. In addition, type I collagen downregulated TβRI via focal adhesion kinase (FAK) and Src activation-dependent Cav-1 phosphorylation. To further examine this mechanism, 4-week-old oim/oim and wt/wt mice were treated with either TβRI kinase inhibitor (SD-208) or vehicle for 8 weeks. SD-208 treatment significantly reduced the fracture incidence in oim/oim mice. Micro–computed tomography and biomechanical testing showed that femoral bone mass and strength were significantly improved with SD-208 treatment in both genotypes. Additionally, SD-208 significantly promoted osteoblast differentiation and bone formation and inhibited bone resorption. In conclusion, dysfunction of caveolae-mediated endocytic TβRI degradation is a possible mechanism for the enhanced TGF-β/Smad signaling in OI. Targeting this mechanism using a TβRI kinase inhibitor effectively reduced fractures and improved bone mass and strength in OI model and, thus, may offer a new strategy for the treatment of OI. © 2022 American Society for Bone and Mineral Research (ASBMR).     

Transient disturbance in physeal morphology is associated with long-term effects of nitrogen-containing bisphosphonates in growing rabbits.

Bisphosphonates have clinical benefit in children with severe osteogenesis imperfecta or osteoporosis and potential benefit in children with Perthes disease or undergoing distraction osteogenesis. However, there is concern about the effects of bisphosphonates on the physis and bone length. In 44 growing rabbits, zoledronic acid caused a transient disruption of physeal morphology, retention of cartilaginous matrix in trabeculae and cortical bone of the metaphysis, and a minor decrement in tibial bone length at maturity. INTRODUCTION: Data from growing animal models suggest that bisphosphonates cause retention of longitudinal cartilaginous septa at the chondro-osseous junction, extension of trabeculae to the metaphyseal-diaphyseal junction, and varying dose-dependent effects on longitudinal growth. However, there is a lack of data regarding effects of intermittent use of nitrogen-containing bisphosphonates on the physis and on tibial length in models reaching maturity. MATERIALS AND METHODS: Contralateral tibias of juvenile rabbits were examined after right tibial distraction osteogenesis from two previous studies. Animals were randomized to receive 0.1 mg/kg zoledronic acid (ZA) IV at 8 weeks of age (ZA*1) or 8 and 10 weeks of age (ZA*2) or saline. Body mass was analyzed from 5 to 44 weeks of age; tibial length and proximal physeal-metaphyseal histology and histomorphometry were analyzed at 8-52 weeks of age. RESULTS: Tibial length was 3% less at 14 weeks of age in the ZA*2-treated versus saline group (p<0.05) in both studies, and this difference persisted at maturity in the long-term study group (26 weeks of age, p<0.05). Total body mass gain from 5 to 26 weeks of age was 14% less in ZA*2-treated than saline animals (p<0.05). Rate of weight gain from 8 to 10 weeks of age was 76% less in ZA*2 compared with saline animals (p<0.05). Radiographs showed radiodense lines in the metaphyses of ZA-treated bones, corresponding to the number of doses. Histologically, lines resulting from the first dose of ZA contained longitudinal cartilaginous matrix cores surrounded by bone, whereas those from the second dose contained spherical cores of matrix caused by transient disruption of physeal morphology after the first dose of ZA. Resorption of these lines at later times was radiographically and histologically evident, but remnants of cartilaginous matrix remained in the cortical bone of ZA-treated animals. CONCLUSIONS: ZA treatment within the final 13.5% of the rabbit tibial growth period caused a transient disruption in physeal morphology and resorption associated with retention of cartilaginous matrix and coinciding with a persistent 3% decrement in tibial length. Disruption of physeal morphology and potential loss of bone length should be considered when administering nitrogen-containing bisphosphonates to children before closure of the major physes.     

Differential effects of alendronate treatment on bone from growing osteogenesis imperfecta and wild-type mouse

Bisphosphonates have been reported to decrease the number of fractures in children with osteogenesis imperfecta (OI). The current study sought to further explore bisphosphonate-associated bone changes in OI by investigating the effects of alendronate (ALN) treatment on bone mechanical and material properties in osteogenesis imperfecta (oim/oim) and wild-type (+/+) mice treated with 26-73 microg kg(-1) day(-1) of ALN for 8 weeks via subcutaneously implanted pumps. Femoral three-point bend tests to evaluate cortical bone were combined with geometric and material density analysis. Cortical and trabecular architecture of metaphyseal bone were histomorphometrically evaluated and material density assessed by quantitative backscattered electron imaging (qBEI). For the cortical oim/oim bone, which revealed principally inferior biomechanical properties compared to +/+ bone, ALN neither improved cortical strength or any other mechanical property, nor affected cortical width (Ct.Wi.) or material density. In contrast, for the +/+ mice, bone strength was enhanced (+22%, P < 0.05) though coupled with increased brittleness (+28%, P < 0.05). This mechanical improvement was associated with an increase in Ct.Wi. (+17.3%, P = 0.02) and a reduction in heterogeneity of cortical mineralization (Ca(Width), -4%, P = 0.04). In the metaphysis, ALN raised cancellous bone volume (BV/TV) significantly in oim/oim as well as in +/+ mice (+97%, P = 0.008 and +200%, P < 0.0001, respectively). This occurred without any change in either material density or trabecular thickness (Tb.Th.) in the oim/oim mice, while in the +/+ mice, material density increased slightly but significantly (+3%, P = 0.004), and Tb.Th. increased by 77% (P < 0.0001). Taken together, these results illustrate the differential effects of ALN on oim/oim vs. +/+ bone, as well as on specific skeletal sites, i.e., cortical vs. trabecular bone. ALN augmented the mechanical, geometrical, and material properties of +/+ cortical and trabecular bone, while the only observable improvement to the oim/oim bone was increased cancellous bone volume. This suggests that in this mouse model of OI, the previously demonstrated bisphosphonate-associated reduction in fractures is primarily attributable to increased metaphyseal bone mass.     

Aminobisphosphonates Cause Osteoblast Apoptosis and Inhibit Bone Nodule Formation In Vitro

Bisphosphonates are widely used for the treatment of bone diseases associated with increased osteoclastic bone resorption. Bisphosphonates are known to inhibit biochemical markers of bone formation in vivo, but it is unclear to what extent this is a consequence of osteoclast inhibition or a direct inhibitory effect on cells of the osteoblast lineage. In order to investigate this issue, we studied the effects of various bisphosphonates on osteoblast growth and differentiation in vitro. The aminobisphosphonates pamidronate and alendronate inhibited osteoblast growth, caused osteoblast apoptosis, and inhibited protein prenylation in osteoblasts in a dose-dependent manner over the concentration range 20-100 microM. Further studies showed that alendronate in a dose of 0.1 mg/kg inhibited protein prenylation in calvarial osteoblasts in vivo, indicating that alendronate can be taken up by osteoblasts in sufficient amounts to inhibit protein prenylation at clinically relevant doses. Pamidronate and alendronate inhibited bone nodule formation at concentrations 10-fold lower than those required to inhibit osteoblast growth. These effects were not observed with non-nitrogen-containing bisphosphonates or with other inhibitors of protein prenylation and were only partially reversed by cotreatment with a fourfold molar excess of ss-glycerol phosphate. We conclude that aminobisphosphonates cause osteoblast apoptosis in vitro at micromolar concentrations and inhibit osteoblast differentiation at nanomolar concentrations by mechanisms that are independent of effects on protein prenylation and may be due in part to inhibition of mineralization. While these results need to be interpreted with caution because of uncertainty about the concentrations of bisphosphonates that osteoblasts are exposed to in vivo, our studies clearly demonstrate that bisphosphonates exert strong inhibitory effects on cells of the osteoblast lineage at similar concentrations to those that cause osteoclast inhibition. This raises the possibility that inhibition of bone formation by bisphosphonates may be due in part to a direct inhibitory effect on cells of the osteoblast lineage.     

Long‐Term Protective Effects of Zoledronic Acid on Cancellous and Cortical Bone in the Ovariectomized Rat

Current bisphosphonate therapies effectively prevent bone loss in postmenopausal women. We studied the effect of a single intravenous dose of ZOL in ovariectomized rats. Protection from bone loss was dose dependent, lasting for up to 32 weeks, supporting the rationale for an annual intravenous dosing regimen of ZOL for treatment of postmenopausal osteoporosis. Introduction : Once‐yearly dosing with zoledronic acid (ZOL) 5 mg can increase BMD and reduce fracture rate in postmenopausal women with low BMD. The primary objective of this study was to determine the duration of bone protective effects of a single dose of ZOL in ovariectomized rats, an animal model of postmenopausal osteopenia. Secondary objectives were to determine the effects on bone turnover and mechanical properties. Materials and Methods : Female Wistar rats (10 per group) received single intravenous doses of ZOL 0.8, 4, 20, 100, or 500 μg/kg, alendronate 200 μg/kg, or isotonic saline 4 days before bilateral ovariectomy. Sham‐operated controls were pretreated with saline. Mass and density of cancellous and cortical bone (pQCT) were measured at 4‐wk intervals for 32 wk. Bone architecture (μCT), bone formation dynamics (fluorochrome label‐based histomorphometry), and biomechanical strength in compression testing were also assessed at 32 wk. Results : Ovariectomy‐associated BMD loss was significantly attenuated for 32 wk by ZOL ≥4 μg/kg for total BMD, ZOL ≥20 μg/kg for cortical BMD, and ZOL ≥4 μg/kg for cancellous BMD (p < 0.01 versus ovariectomized controls). Alendronate 200 μg/kg was of equivalent potency to ZOL 20 μg/kg. Ovariectomy‐associated decreases in trabecular architectural parameters were dose‐dependently attenuated by ZOL. Alendronate 200 μg/kg was equivalent to ZOL 20 μg/kg. The bone resorption marker TRACP5b indicated transient suppression of elevated osteoclast activity by ZOL relative to OVX‐rats even at the lowest dose of 0.8 μg/kg, whereas at 100–500 μg/kg, the effect was significant relative to the OVX control for the entire duration of the study of 32 wk. Bone formation parameters were not significantly affected by ZOL 20 μg/kg but were significantly reduced by ZOL 100–500 μg/kg. Alendronate 200 μg/kg was equivalent to ZOL 100 μg/kg. ZOL produced dose‐related improvements in bone strength parameters after ovariectomy. Alendronate 200 μg/kg was of similar potency to ZOL 20 μg/kg. Conclusions : The duration and magnitude of the bone‐protecting effect of a single intravenous dose of ZOL in ovariectomized rats is dose dependent and lasts for up to 32 wk. Compared with alendronate, ZOL shows 10‐fold higher potency in preventing bone loss. These data support the use of an annual intravenous ZOL dosing regimen for the treatment of osteoporosis.     

Effects of alendronate and pamidronate on cultured rat metatarsal bones: failure to prevent dexamethasone-induced growth retardation

Bisphosphonates are widely used anti-resorptive drugs in the adult population. In children, their use has mainly been limited to patients with osteogenesis imperfecta. However, the powerful effects of bisphosphonates on bone turnover have raised concern about their long-term effects on the growing skeleton. We aimed to study the effects of two commonly used bisphosphonates, alendronate (Aln) and pamidronate (Pam) on normal bone growth as well as their potential to prevent glucocorticoid-induced growth retardation. Effects on bone growth were studied in fetal rat metatarsal bones (day E20) that were cultured for 5-47 days and measured every 2-7 days. Cellular mechanisms were investigated in metatarsal bones and also in the human chondrocytic cell line HCS-2/8. Chondrocyte viability (WST-1), proliferation (BrdU incorporation), differentiation (collagen type X immunohistochemistry) and apoptosis (TUNEL and Cell Death ELISA) were determined. At a clinically relevant concentration of bisphosphonates (1 microM), metatarsal bone growth was stimulated by both Aln (p<0.001 for length and p<0.05 for width) and Pam (p<0.05 for both length and width) from day 19 of culture. The growth-stimulatory effect was associated with increased chondrocyte proliferation (+21% with Aln and +24% with Pam), while cell differentiation and apoptosis were not affected. Despite the finding that both Aln and Pam (1 muM) rescued HCS-2/8 cells from undergoing dexamethasone-induced apoptosis, neither of them was able to prevent dexamethasone-induced growth retardation of fetal rat metatarsal bones. Aln and Pam have the capacity to stimulate the growth of cultured fetal rat metatarsal bones; an effect associated with increased proliferation of growth plate chondrocytes. Our experimental data suggest that bisphosphonates are ineffective in preventing glucocorticoid-induced growth retardation. Nevertheless, based on our in vitro data, both Aln and Pam appear safe to use in growing children, at least with regard to their effects on linear bone growth.     

Inhibition of bone resorption by alendronate and risedronate does not require osteoclast apoptosis

Bisphosphonate inhibition of bone resorption was proposed to be due to osteoclast apoptosis. We tested this hypothesis for both the N-containing bisphosphonates alendronate and risedronate, which inhibit farnesyldiphosphate synthase and thus protein isoprenylation, and for clodronate and etidronate, which are metabolized to adenosine triphosphate (ATP) analogs. We found, in dose-response studies, that alendronate and risedronate inhibit bone resorption (in pit assays) at doses tenfold lower than those reducing osteoclast number. At an N-bisphosphonate dose that inhibited resorption and induced apoptosis, the antiapoptotic caspase inhibitor, Z-VAD-FMK, maintained osteoclast (Oc) number but did not prevent inhibition of resorption. Furthermore, when cells were treated with either alendronate alone or in combination with Z-VAD-FMK for 24 or 48 h, subsequent addition of geranylgeraniol, which restores geranylgeranylation, returned bone resorption to control levels. On the other hand, Z-VAD-FMK did block etidronate and clodronate inhibition of resorption. Moreover, in cells treated with etidronate, but not alendronate or risedronate, Z-VAD-FMK also prevented actin disruption, an early sign of osteoclast inhibition by bisphosphonates. These observations indicate that, whereas induction of apoptosis plays a major role in etidronate and clodronate inhibition of resorption, alendronate and risedronate suppression of bone resorption is independent of their effects on apoptosis.     

阿仑膦酸钠抑制骨巨细胞瘤细胞生长和诱导其凋亡的研究

[目的]骨巨细胞瘤是一种潜在的恶性病变，具有手术后易复发的特点。二膦酸盐是抗骨质疏松药，可以抑制破骨细胞性骨吸收，近来发现其还有抗肿瘤作用。本研究是探讨第3代二膦酸盐——阿仑膦酸钠是否能够抑制骨巨细胞瘤细胞生长，诱导骨巨细胞瘤细胞凋亡，探讨应用二膦酸盐能否成为一个防止骨巨细胞瘤复发的方法。[方法]在体外培养骨巨细胞瘤细胞，给予不同浓度的阿仑膦酸钠，作用不同时间后，应用M1Tr法检测骨巨细胞瘤细胞的活性是否受到抑制，TUNEL染色法观察骨巨细胞瘤细胞经药物作用后是否发生凋亡，流式细胞术检测凋亡率，观察药物作用后凋亡蛋白Caspase-3活性的表达是否增加。[结果]经阿仑膦酸钠作用后瘤细胞活性减低，可以发现阿仑膦酸钠抑制骨巨细胞瘤细胞生长的作用可以随时间和浓度的增加而增高。TUNEL法观察到瘤细胞凋亡染色阳性，流式细胞仪检测阿仑膦酸钠作用后骨巨细胞瘤细胞的凋亡率也随着时间和浓度的增加而增高。进一步检测随着阿仑膦酸钠浓度的提高，骨巨细胞瘤细胞的Caspase-3活性表达也增加。[结论]阿仑膦酸钠对于体外培养的骨巨细胞瘤细胞的活性有抑制作用，可以抑制其生长并诱导肿瘤细胞内的Caspase-3活性表达，促其凋亡，阿仑膦酸钠可能成为治疗和预防骨巨细胞瘤复发的一个治疗方法，但还需要进一步的体内实验研究。     

Fourier Transform Infrared Imaging Spectroscopy (FT-IRIS) of Mineralization in Bisphosphonate-treated oim/oim Mice

Fourier transform infrared microscopy (FT-IRM) and imaging spectroscopy (FT-IRIS) are increasingly used to analyze the molecular components of mineralized tissues. A primary advantage of these techniques is the capability to simultaneously image the quantity and quality of multiple components in histological sections at 7 µm spatial resolution. In the current study, FT-IRM and FT-IRIS were used to characterize bone mineralization in a mouse model of osteogenesis imperfecta (OI) after treatment with the bisphosphonate alendronate (ALN). This application is currently relevant since recent studies have demonstrated great promise for the treatment of children with OI with bisphosphonates, but have not identified bisphosphonate-associated bone quality changes. Growing oim/oim mice, a model of moderate-to-severe OI, were treated with ALN (73 µg ALN/kg/day for 4 weeks followed by 26 µg/kg/day for 4 weeks) or saline from 6 to 14 weeks of age, and mineralization was evaluated in femoral cortical and metaphyseal bone. Infrared vibrations of the mineral (a carbonated apatite) and the matrix phases were monitored. The relative amounts of mineral and matrix present (min:matrix), the relative amount of carbonate present in the mineral (carb:min), and the crystallinity of the mineral phase were calculated. In untreated oim/oim bone, the min:matrix was greater and the crystallinity was reduced (indicative of less mature mineral) in the primary versus the secondary spongiosa, most likely due to the presence of calcified cartilage. With ALN treatment, the oim/oim mm:matrix increased in the secondary spongiosa, but the mineral crystallinity was not changed. In the cortical bone, no changes were evident with ALN treatment. These data demonstrate that in this mouse model, ALN treatment results in increased metaphyseal bone mineralization, but does not improve mineral maturity.     

The critical role of the epidermal growth factor receptor in endochondral ossification

Loss of epidermal growth factor receptor (EGFR) activity in mice alters growth plate development, impairs endochondral ossification, and retards growth. However, the detailed mechanism by which EGFR regulates endochondral bone formation is unknown. Here, we show that administration of an EGFR-specific small-molecule inhibitor, gefitinib, into 1-month-old rats for 7 days produced profound defects in long bone growth plate cartilage characterized by epiphyseal growth plate thickening and massive accumulation of hypertrophic chondrocytes. Immunostaining demonstrated that growth plate chondrocytes express EGFR, but endothelial cells and osteoclasts show little to no expression. Gefitinib did not alter chondrocyte proliferation or differentiation and vascular invasion into the hypertrophic cartilage. However, osteoclast recruitment and differentiation at the chondro-osseous junction were attenuated owing to decreased RANKL expression in the growth plate. Moreover, gefitinib treatment inhibited the expression of matrix metalloproteinases (MMP-9, -13, and -14), increased the amount of collagen fibrils, and decreased degraded extracellular matrix products in the growth plate. In vitro, the EGFR ligand transforming growth factor α (TGF-α) strongly stimulated RANKL and MMPs expression and suppressed osteoprotegerin (OPG) expression in primary chondrocytes. In addition, a mouse model of cartilage-specific EGFR inactivation exhibited a similar phenotype of hypertrophic cartilage enlargement. Together our data demonstrate that EGFR signaling supports osteoclastogenesis at the chondro-osseous junction and promotes chondrogenic expression of MMPs in the growth plate. Therefore, we conclude that EGFR signaling plays an essential role in the remodeling of growth plate cartilage extracellular matrix into bone during endochondral ossification.     

Alendronate administration and skeletal response during chronic alcohol intake in the adolescent male rat.

Alendronate is an aminobisphosphonate that inhibits bone resorption in osteoporotic humans and rats but does not induce osteomalacia. Several bisphosphonates, including alendronate, also have direct positive actions on osteoblasts, bone formation, and mineralization. We studied the effects of alendronate on skeletal development in adolescent male rats during chronic alcohol intake. Four groups of age- and weight-matched male Sprague-Dawley rats (35 days of age) were fed the Lieber-DeCarli diet containing 36% of calories as EtOH (E), the EtOH diet plus 60 mg/kg alendronate (EA) every other day intraperitoneally (ip), an isocaloric diet (I), or the isocaloric diet plus 60 mg/kg alendronate (IA) every other day ip. Body weight, femur length, serum levels of osteocalcin (OC), insulin-like growth factor 1 (IGF-1), testosterone, and luteinizing hormone (LH); femur distal metaphyseal and middiaphyseal bone mineral density (BMD) and tibial metaphyseal gene expression for alpha-1-type I collagen (Col I), OC, and bone alkaline phosphatase (AP); and femur strength by four-point bending to failure were measured after 28 days of feeding and alendronate injections. Serum alcohol levels at death were 156 +/- 13 mg/dl (E) and 203 +/- 40 mg/dl (EA). Alendronate given to alcohol-fed rats increased metaphyseal BMD by more than 3-fold over rats fed alcohol alone. Alendronate given to isocaloric pair-fed rats increased metaphyseal BMD by more than 2.5-fold over rats fed the isocaloric diet alone. Cortical BMD was reduced by alcohol but was increased by alendronate. Alcohol consumption reduced serum IGF-1 levels, and alendronate increased IGF-1 levels in alcohol-fed rats. Serum OC, testosterone, and LH were unaffected by alcohol and alendronate. Quantitative dot blot hybridization using rat complementary DNA (cDNA) probes and normalization against 18S subunit ribosomal RNA (rRNA) levels revealed no changes in tibial metaphyseal gene expression for type I collagen, osteocalcin, or alkaline phosphatase. Alcohol significantly reduced the biomechanical properties of the femurs that were partially compensated by alendronate. Chronic alcohol consumption uncouples formation from ongoing resorption, and resorption is inhibited by alendronate. However, alendronate's positive effects on osteoblast-mediated mineralization during chronic alcohol consumption point to the potential use of bisphosphonates in the treatment of decreased bone formation secondary to alcohol-induced diminished osteoblast function.     

Bone healing in children with osteogenesis imperfecta treated with bisphosphonates

The long-term effects of bisphosphonate treatment in children with osteogenesis imperfecta (OI) are unknown. The aim of this study was to evaluate whether treatment with bisphosphonates interferes with the healing of fractures in a group of children with OI. Seven subjects (6 boys), aged 11.4 +/- 5.95 years, were followed for 2.5 +/- 0.84 years after the start of treatment with intravenous pamidronate (9 mg/kg/y) and/or oral alendronate (5 or 10 mg/d). Orthopaedic surgery of 24 bones was performed after 2.33 +/- 4.14 months of treatment, with 1.6 +/- 0.84 osteotomies per bone. Ambulation was started after 26.1 +/- 32.28 days. Reoperation was required in 8% of the bones due to fracture below primary fixation. Pseudoarthrosis was seen in one fracture, an osteotomy of the proximal femur (14% of the patients, as expected in an OI population). These results suggest that treatment with bisphosphonates at the administered doses does not interfere with fracture healing. Larger and longer studies are warranted.     

Follow-Up of Six Patients with Neurofibromatosis 1-Related Osteoporosis Treated with Alendronate for 23 Months

This is the first prospective follow-up study to describe the effects of oral alendronate medication on neurofibromatosis 1 (NF1)-related osteoporosis. NF1 is a neurocutaneous skeletal syndrome associated with increased fracture risk and high frequency of osteopenia and osteoporosis. Alendronate is a bisphosphonate drug which inhibits the function of bone-resorbing osteoclasts, ultimately leading to an increase in bone mineral density (BMD) and reduction in fracture risk. However, in vitro studies have shown that NF1 osteoclasts display insensitivity to apoptotic signals caused by bisphosphonates. Our aim was to monitor the effects of alendronate medication in patients with NF1. Five men and one woman, aged 28–76 years, with NF1-related osteoporosis were enrolled to the study. Study participants did not have other conditions and were not taking any medication known to affect bone. The medication included a weekly dose of 70 mg alendronate and a daily 20 μg vitamin D supplementation. After 23 months of follow-up, BMD was increased in five out of six patients, but the increase was not statistically significant. Serum levels of the bone turnover markers CTX and PINP were reduced, suggesting slower bone remodeling, as expected. An unexpected result was that serum levels of the osteoclast activity marker TRAP5b did not change during the follow-up. One new stress fracture of the tibia was documented during the alendronate therapy. Even though the study group was small, the findings of the current study (one new fracture and one patient with decreased BMD) call for a larger study to assess the efficacy of bisphosphonates in NF1-related osteoporosis.     

Effect of alendronate therapy in children with osteogenesis imperfecta

OBJECTIVE: To evaluate the effect of orally administered alendronate in children with osteogenesis imperfecta. METHODS: Thirty children (16 girls and 14 boys; mean age at baseline 10.7 +/- 6.0 years; range 4-16 years) with osteogenesis imperfecta type I (n = 22), III (n = 2), or IV (n = 6) were treated with alendronate (5 mg/day in patients aged 4-10 years and 10 mg/day in children >10 years of age) for 3 years. RESULTS: After 1 year of alendronate therapy we observed a significant increase in areal and volumetric bone mineral density Z-scores (from -2.03 +/- 1.51 to -1.04 +/- 1.20, and from -1.91 +/- 1.38 to -1.33 +/- 1.30, respectively, P < 0.001), together with a significant drop in fracture rate (from 3.77 +/- 1.57 to 0.13 +/- 0.57, P < 0.000001), relief of chronic pain (from 3.83 +/- 1.44 days of pain/week to 0.73 +/- 0.77, P < 0.000001) and improvement in ambulation/mobility (P < 0.00002). After additional 2 years of therapy there were no further significant changes in these parameters, however the improvement was still remarkable in comparison to the pretreatment values (P < 0.003, P < 0.004, P < 0.000001, P < 0.000001 and P < 0.00001, respectively). A significant drop in markers of bone turnover (urinary deoxypyridinoline and serum osteocalcin) occurred after 3 years of therapy (P < 0.003 and 0.004, respectively). No adverse reactions were observed throughout the treatment. CONCLUSIONS: Alendronate has positively influenced quality of life in paediatric patients with osteogenesis imperfecta. Bisphosphonate therapy should be used only in the context of a well-defined protocol.     

The effect of alendronate doped calcium phosphates on bone cells activity

This study demonstrates that octacalcium phosphate (OCP) is a suitable substrate for alendronate local action towards bone cells. The results of the structural, spectroscopic, and microscopic investigation show that soaking OCP into alendronate solutions provoked the deposition of long crystalline rod-shaped formations, most likely a calcium alendronate complex, onto the calcium phosphate. The amount of alendronate loaded onto OCP increased as a function of the bisphosphonate concentration in solution. Osteoblast and osteoclast response was tested in single and in co-cultures on OCP containing 6.4 wt.% AL (OCP-AL), and for comparison on hydroxyapatite (HA) containing a similar amount (5.9 wt.%) of AL (HA-AL), as well as on pure OCP and HA as reference materials. Alendronate loaded materials displayed a beneficial effect on osteoblast activity and differentiation, whereas they inhibited osteoclast proliferation and differentiation. Crosstalking between osteoblast-like MG63 cells and human osteoclasts enhanced their response to alendronate. Moreover, OCP displayed a greater stimulating effect than HA on osteoblast differentiation, and AL promotion of osteoblast differentiation and mineralization was enhanced in OCP-AL with respect to HA-AL.     

A randomized,controlled dose‐ranging study of risedronate in children with moderate and severe osteogenesis imperfecta

Moderate to severe osteogenesis imperfecta is associated with multiple fractures in childhood. There are no published data regarding the effects of third‐generation bisphosphonates in these children. This randomized study investigated which of three different doses of risedronate was most effective in reducing fracture incidence. We randomly assigned 53 children with moderate to severe osteogenesis imperfecta to receive 0.2, 1, or 2 mg/kg per week of risedronate. We assessed safety, fracture incidence, and bone measurement outcomes at 3, 6, 12, 18, and 24 months. At 24 months, 69% of children assigned 0.2 mg/kg per week had had new fractures compared with 44% receiving 1 mg/kg per week and 75% receiving 2 mg/kg per week. Poisson regression with age and prior fracture as covariates showed that there was no difference in incident nonvertebral fracture between groups. Fracture rate diminished in each group during the trial compared with previous the 2 years (p = .005). Lumbar spine bone mineral density increased significantly (p = .009) only in the 2 mg/kg per week group. Long bone bowing deformities reduced more in children receiving 1 or 2 mg/kg per week of risedronate [odds ratio (OR) 0.67, 95% confidence interval (CI) 0.48–0.93 per unit increase in risedronate dose, p = .015]. There were no serious adverse events. Bone mass increased and bowing deformities reduced with increasing risedronate dose. Children suffered fewer fractures irrespective of risedronate dose. The most appropriate dose of risedronate for children with moderate to severe osteogenesis imperfecta in this study was 2 mg/kg per week. © 2010 American Society for Bone and Mineral Research     

Age- and genotype-dependence of bone material properties in the osteogenesis imperfecta murine model (oim).

Cortical mineralization of long bones was studied in collagen alpha2(I)-deficient mice (oim) used as a model for human osteogenesis imperfecta. Aspects of the age development of the mice were characterized by combining nanometer- to micrometer-scale structural analysis with microhardness measurements. Bone structure was determined from homozygous (oim/oim) and heterozygous (oim/+) mice and their normal (+/+) littermates as a function of animal age by small-angle X-ray scattering (SAXS) and quantitative backscattered electron imaging (qBEI) measurements. SAXS studies found anomalies in the size and arrangement of bone mineral crystals in both homozygous and heterozygous mice aged 1-14 months. Generally, the crystals were smaller in thickness and less well aligned in these mice compared with control animals. An increase in the mean crystal thickness of the bone was found within all three genotypes up to an age of 3 months. Vicker's hardness measurements were significantly enhanced for oim bone (homozygotes and heterozygotes) compared with controls. The microhardness values were correlated directly with increased mineral content of homozygous and heterozygous compared with control bone, as determined by qBEI analysis. There was also a significant increase of mineral content with age. Two possibilities for collagen-mineral association are discussed for explaining the increased hardness and mineral content of oim/oim bone, together with its decreased toughness and thinner mineral crystals. As a consequence of the present measurements, one model for oim bone could incorporate small and densely packed mineral crystals. A second model for possible collagen-mineral association in oim material would consist of two families of mineral crystals, one being smaller and the other being much larger than the crystals found in normal mouse long bones.