Amanda Garces

Ibandronate for prevention of femoral head deformity after ischemic necrosis of the capital femoral epiphysis in immature pigs.

BACKGROUND Femoral head deformity is the most serious sequela of ischemic necrosis of the immature femoral head. The purpose of this study was to determine if a highly potent antiresorptive agent, ibandronate, can inhibit bone resorption during the repair of the infarcted femoral head and thus alter the repair process. We hypothesized that preservation of the trabecular framework by inhibiting osteoclastic bone resorption would minimize the development of deformity in a piglet model of ischemic necrosis. The effect of ibandronate on long-bone growth was also assessed. METHODS Ischemic necrosis of the right femoral head was produced in twenty-four piglets by placing a ligature tightly around the femoral neck. The animals were divided into three groups according to whether they received saline solution, prophylactic treatment, or post-ischemia treatment. The contralateral, untreated femoral heads from the animals that had received saline solution served as the normal control group. At eight weeks, the femoral heads were assessed for deformity with radiography and for trabecular bone indices with histomorphometry. Also, the length of femur from the untreated side was measured on the radiographs and compared among the groups. RESULTS Radiographic assessment showed that the epiphyseal quotient, determined by dividing the maximum height of the osseous epiphysis by the maximum diameter, was better preserved in the prophylactic (p < 0.001) and post-ischemia (p = 0.02) treatment groups than in the group treated with saline solution. Histomorphometric assessment also showed that the trabecular bone indices were better preserved in the prophylactic and the post-ischemia treatment groups than in the group treated with saline solution (p < 0.01). The mean femoral length on the untreated side of the animals treated with ibandronate was reduced compared with the length on the untreated side of the animals that had received saline solution (p </= 0.01). CONCLUSIONS Ibandronate preserves the trabecular structure of the osseous epiphysis and prevents femoral head deformity during the early phase of repair of ischemic necrosis in the piglet model.

Novel therapeutic approach for cancer using four cardiovascular hormones.

Background  The atrial natriuretic peptide (ANP) gene synthesizes four cardiovascular hormones, i.e. vessel dilator, long‐acting natriuretic peptide, kaliuretic peptide and ANP, which decrease the number of human pancreatic adenocarcinoma cells in culture by 65%, 47%, 37%, and 34%, respectively.

Increased VEGF expression in the epiphyseal cartilage after ischemic necrosis of the capital femoral epiphysis.

Ischemic injury to the immature femoral head produces epiphyseal cartilage damage and cessation of endochondral ossification. This study suggests that VEGF facilitates the repair of the necrotic epiphyseal cartilage, which is essential for restoration of endochondral ossification and re‐establishment of the growth of the immature femoral head after ischemic necrosis.

Local administration of ibandronate and bone morphogenetic protein-2 after ischemic osteonecrosis of the immature femoral head: a combined therapy that stimulates bone formation and decreases femoral head deformity.

BACKGROUND Bisphosphonate therapy has been shown to preserve the osteonecrotic femoral head in experimental and short-term clinical studies. However, a lack of new bone formation within the preserved femoral head due to the inhibition of bone remodeling is a concern. The purpose of this investigation was to determine if combined therapy consisting of ibandronate and bone morphogenetic protein-2 (BMP-2) can preserve the shape of the femoral head and stimulate new bone formation in an immature animal model of ischemic osteonecrosis. METHODS Ischemic osteonecrosis was surgically induced in immature pigs. Four groups were studied: normal, treated with saline solution, treated with ibandronate, and treated with both ibandronate and BMP-2 (the ibandronate + BMP-2 group). The animals were killed eight weeks after surgery. Radiographic, histological, and histomorphometric assessments were performed. RESULTS Radiographic assessment showed better preservation of the femoral head shape-i.e., a 54% (CI [95% confidence interval]: 22%, 86%) higher mean epiphyseal quotient-in the ibandronate + BMP-2 group than in the saline group. Histological assessment showed increased trabecular bone in the ibandronate + BMP-2 group as compared with that in the saline group. The mean values for trabecular bone volume, thickness, and number and for osteoblast surface were an average of 400% (CI: 242%, 558%), 212% (CI: 166%, 259%), 71% (CI: 6%, 137%), and 2402% (CI: 2113%, 2693%) higher, respectively, in the ibandronate + BMP-2 group than in the saline group. The osteoclast number was significantly reduced in the ibandronate + BMP-2 group compared with that in the saline group (-59% [CI: -75%, -42%]). The mean osteoblast surface value in the ibandronate + BMP-2 group was significantly higher (2567% [CI: 2258%, 2877%]) than that in the ibandronate group. Heterotopic ossifications were present in the capsule of the hip joint in the ibandronate + BMP-2 group. CONCLUSIONS A combination of ibandronate and BMP-2 decreased femoral head deformity while stimulating bone formation in an immature animal model of ischemic osteonecrosis.

Hypoxia and HIF-1α expression in the epiphyseal cartilage following ischemic injury to the immature femoral head

UNLABELLED HIF-1alpha has been shown to be a central mediator of cellular response to hypoxia. The role it plays after ischemic injury to the immature femoral head is unknown. The purpose of this study was to determine the region of the femoral head affected by hypoxia following ischemic injury to the immature femoral head and to determine the site of HIF-1alpha activation and revascularization. We hypothesize that the epiphyseal cartilage, rather than the bony epiphysis, is the site of HIF-1alpha activation following ischemic osteonecrosis and that the epiphyseal cartilage plays an important role in the revascularization process. MATERIALS AND METHODS Femoral head osteonecrosis was surgically induced in 56 immature pigs. Hypoxyprobe staining, cell viability assay, HIF-1alpha western blot, RT-qPCR of HIF-1alpha, VEGF, VEGFR2, and PECAM, and micro-CT assessments of microfil-infused femoral heads were performed. RESULTS Severe hypoxia was present in the bony epiphysis and the lower part of the epiphyseal cartilage following ischemia. In the bony epiphysis, extensive cell death and tissue necrosis was observed with degradation of proteins and RNAs which precluded further analysis. In the epiphyseal cartilage, the loss of cell viability was limited to its deep layer with the remainder of the cartilage remaining viable. Furthermore, the cartilage from the ischemic side showed a significant increase in HIF-1alpha protein level and HIF-1alpha expression. VEGF expression in the cartilage was dramatically and significantly increased at 24 h, 2 and 4 weeks (p<0.05 for all) with 5 to 10 fold increase being observed on the ischemic side compared to the normal side. PECAM and VEGFR2 expressions in the cartilage were both significantly decreased at 24 h but returned to the normal levels by 2 and 4 weeks, respectively. Micro-CT showed revascularization of the cartilage on the ischemic side with the vessel volume/total volume equaling the normal side by 4 weeks. CONCLUSIONS Acute ischemic injury to the immature femoral head induced severe hypoxia and cell death in the bony epiphysis and the deep layer of the epiphyseal cartilage. Viable chondrocytes in the superficial layer of the epiphyseal cartilage showed HIF-1alpha activation and VEGF upregulation with subsequent revascularization occurring in the cartilage.

In vivo cerebrovascular actions of amyloid β-peptides and the protective effect of conjugated estrogens

Vascular dysfunction and inflammatory processes may be early events in the pathology of Alzheimers disease (AD). Even though amyloid beta-peptides (Abeta) play a prominent role in the initiation and progression of cellular dysfunction in AD, the precise in vivo actions of various Abeta-peptides has not been established. The cerebrovascular actions of the major Abeta-peptides (1-40) and (1-42) in live animals were investigated using an open cranial window technique. We show here that the Abeta-peptides cause vascular lesions, especially in the arterioles. In one set of experiments, leukocytes and platelets were tagged with Rhodamine 6G, soluble Abeta(1-40) infused intravenously for 2 minutes, and the vasculature video recorded for 90 minutes. In a second set of experiments, soluble Abeta(1-40) infusion was followed 30 minutes later by an infusion of soluble Abeta(1-42) and the vasculature recorded for 90 minutes. Fluorescent and transmission electron microscopic examinations demonstrated the following cerebrovascular action of Abeta-peptides: endothelial cell damage, leukocyte adhesion, platelet activation, thrombus formation, impeded blood flow, and smooth muscle cell damage. The vascular disruption observed were similar to those observed in the brains of some AD patients and may represent the initial phase of a vascular inflammatory response associated with cerebral amyloid angiopathy. The combination of Abeta(1-40) and (1-42) produced significantly more vascular disruption than Abeta(1-40) alone. Oral administration of conjugated estrogens in ovariectomized female rats protected them from the deleterious actions of Abeta-peptides. The reported protective effect of estrogen against AD may be mediated in part through prevention of cerebrovascular Abeta toxicity.

Immunocytochemical Localization of Atrial Natriuretic Peptide, Vessel Dilator, Long-acting Natriuretic Peptide, and Kaliuretic Peptide in Human Pancreatic Adenocarcinomas

We recently found that four peptide hormones synthesized by the same gene completely inhibit the growth of human pancreatic adenocarcinomas in athymic mice. The present immunocytochemical investigation was designed to determine where in the adenocarcinomas these peptide hormones localize. Atrial natriuretic peptide, vessel dilator, long-acting natriuretic peptide, and kaliuretic peptide localized to the cytoplasm and nucleus of the human pancreatic adenocarcinomas, which is consistent with their ability to decrease DNA synthesis in the nucleus of this cancer. In this first investigation of where these peptide hormones with anticancer effects localize in any cancer, these peptide hormones also localized to the endothelium of capillaries and fibroblasts within these cancers. This is the first demonstration of growth-inhibiting peptide hormones localizing to the nucleus, where they inhibit DNA synthesis and may interact with growth-promoting hormones that localize there as the etiology of their ability to inhibit the growth of adenocarcinomas both in vitro and in vivo.

Ischaemic injury to femoral head induces apoptotic and oncotic cell death

Aims: The mechanism of cell death in ischaemic osteonecrosis of the femoral head is not clear. Therefore, this study was designed to clarify the mode of cell death following ischaemic osteonecrosis of the femoral head in an established pig model. Methods: Morphological assessment, terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labelling (TUNEL) assay, detection of DNA laddering and transmission electron microscopy studies were performed to determine whether apoptosis is one of the pathways of cell death following ischaemic osteonecrosis of the femoral head. Results: Mode of cell death was investigated from 2 to 14 days following the surgical induction of ischaemia. Ischaemic femoral heads showed morphological evidence of cell death by oncotic and apoptotic pathways in earlier stages of osteonecrosis. TUNEL positive cells were seen from 2 to 14 days following the induction of ischaemia. DNA samples obtained from ischaemic femoral heads following the induction of ischaemia showed nucleosomal ladders indicating apoptotic cell death. Electron micrographs also showed morphological changes associated with apoptosis. Conclusions: This study demonstrates that oncosis is not the sole mechanism of cell death following ischaemic injury of the femoral head. Both apoptosis and oncosis are involved as a result of ischaemic injury to the femoral head.

Bisphosphonate-modified gold nanoparticles: a useful vehicle to study the treatment of osteonecrosis of the femoral head

Legg-Calvé-Perthes disease (LCPD) is a juvenile form of osteonecrosis of the femoral head that presents in children aged 2-14 years. To date, there is no effective medical therapy for treating LCPD largely due to an inability to modulate the repair process, including the predominance of bone resorption. This investigation aims to evaluate the feasibility of using gold nanoparticles (GNPs) that are surface modified with a bisphosphonate compound for the treatment of osteonecrosis at the cellular level. Studies have found osteoclast-mediated resorption to be a process that contributes significantly to the pathogenesis of femoral head deformities arising from Perthes disease. Our in vitro model was designed to elucidate the effect of alendronate-(a bisphosphonate) modified GNPs, on osteoclastogenesis and osteoclast function. RAW 264.7 macrophage cells were cultured with recombinant mouse receptor activator of NF-κB ligand (RANKL), which stimulates osteoclastogenesis, and were then treated with alendronate-modified GNPs for 24, 48, and 72 h. Cell proliferation, osteoclast function, and osteoclast morphology were evaluated by trypan blue dye exclusion assay, tartrate-resistant acid phosphatase (TRAP) staining, and transmission electron microscopy (TEM) imaging. Comparative studies were performed with GNPs that were only stabilized with citrate ions and with alendronate alone. Neither osteoclastogenesis nor osteoclast function were adversely affected by the presence of the citrate-GNP. Alendronate-modified GNPs had an enhanced effect on inducing osteoclast apoptosis and impairing osteoclast function when compared to unbound alendronate populations.

A comparison of the anti-inflammatory activities of conjugated estrogens and 17-β estradiol

AbstractObjective and design: Unregulated chronic inflammatory process partly due to an estrogen deficiency may render postmenopausal women vulnerable to degenerative conditions such as arthritis, osteoporosis, atherosclerosis, and Alzheimer’s disease. Current confusion regarding therapeutic efficacy of estrogen replacement therapy may be due to different estrogen formulations used, short term therapy, as well as advanced stage of the disease. Materials and methods: We compared anti-inflammatory activities of two major estrogen preparations, conjugated equine estrogen (CEE) and 17-β estradiol, using an animal model (rat mesentery) of in vivo inflammatory reaction to intravenously infused amyloid-β, examined by video recording and subsequently analyzed by transmission electron microscopy. Cellular markers of inflammation were monitored: leukocyte migration, platelet activation, mast cell activation/degranulation, and endothelial disruption. Results: Low doses of CEE (0.3 mg/kg for 3 weeks) demonstrated significant anti-inflammatory activity, whereas even at high doses (2.0 mg) 17-β estradiol had only minimal activity. Conclusion: CEE, a mixture of several compounds, may have some component(s) with significant anti-inflammatory activity. The anti-inflammatory activity of CEE may have a role in prevention of several degenerative diseases associated with menopause.