Accentuated osteoclastic response to parathyroid hormone undermines bone mass acquisition in osteonectin-null mice

Matricellular proteins play a unique role in the skeleton as regulators of bone remodeling, and the matricellular protein osteonectin (SPARC, BM-40) is the most abundant non-collagenous protein in bone In. the absence of osteonectin, mice develop progressive low turnover osteopenia, particularly affecting trabecular bone. Polymorphisms in a regulatory region of the osteonectin gene are associated with bone mass in a subset of idiopathic osteoporosis patients, and these polymorphisms likely regulate osteonectin expression. Thus it is important to determine how osteonectin gene dosage affects skeletal function. Moreover, intermittent administration of parathyroid hormone (PTH) (1-34) is the only anabolic therapy approved for the treatment of osteoporosis, and it is critical to understand how modulators of bone remodeling, such as osteonectin, affect skeletal response to anabolic agents. In this study, 10 week old female wild type, osteonectin-haploinsufficient, and osteonectin-null mice (C57Bl/6 genetic background) were given 80 mu g/kg body weight/day PTH(1-34) for 4 weeks. Osteonectin gene dosage had a profound effect on bone microarchitecture. The connectivity density of trabecular bone in osteonectin-haploinsufficient mice was substantially decreased compared with that of wild type mice, suggesting compromised mechanical properties. Whereas mice of each genotype had a similar osteoblastic response to PTH treatment, the osteoclastic response was accentuated in osteonectin-haploinsufficient and osteonectin-null mice. Eroded surface and osteoclast number were significantly higher in PTH-treated osteonectin-null mice, as was endosteal area. In vitro studies confirmed that PTH induced the formation of more osteoclast-like cells in marrow from osteonectin-null mice compared with wild type. PTH treated osteonectin-null bone marrow cells expressed more RANKL mRNA compared with wild type. However, the ratio of RANKL:OPG mRNA was somewhat lower in PTH treated osteonectin-null cultures. Increased expression of RANKL in response to PTH could contribute to the accentuated osteoclastic response in osteonectin(-/-) mice, but other mechanisms are also likely to be involved. The molecular mechanisms by which PTH elicits bone anabolic vs. bone catabolic effects remain poorly understood. Our results imply that osteonectin levels may play a role in modulating the balance of bone formation and resorption in response to PTH. (c) 2008 Elsevier Inc. All rights reserved.

Phosphate Binder Impact on Bone Remodeling and Coronary Calcification - Results from the BRiC Study

Background and Aims: Calcium-containing phosphate binders have been shown to increase the progression of vascular calcification in hemodialysis patients. This is a prospective study that compares the effects of calcium acetate and sevelamer on coronary calcification (CAC) and bone histology. Methods: 101 hemodialysis patients were randomized for each phosphate binder and submitted to multislice coronary tomographies and bone biopsies at entry and 12 months. Results: The 71 patients who concluded the study had similar baseline characteristics. On follow-up, the sevelamer group had higher levels of intact parathyroid hormone (498 +/- 352 vs. 326 +/- 236 pg/ml, p = 0.017), bone alkaline phosphatase (38 +/- 24 vs. 28 +/- 15 U/l, p = 0.03) and deoxypyridinoline (135 +/- 107 vs. 89 +/- 71 nmol/l, p = 0.03) and lower LDL cholesterol (74 +/- 21 vs. 91 +/- 28 mg/dl, p = 0.015). Phosphorus (5.8 +/- 1.0 vs. 6 +/- 1.0 mg/dl, p = 0.47) and calcium (1.27 +/- 0.07 vs. 1.23 +/- 0.08 mmol/l, p = 0.68) levels did not differ between groups. CAC progression (35 vs. 24%, p = 0.94) and bone histological diagnosis at baseline and 12 months were similar in both groups. Patients of the sevelamer group with a high turnover at baseline had an increase in bone resorption (eroded surface, ES/BS = 9.0 +/- 5.9 vs. 13.1 +/- 9.5%, p = 0.05), whereas patients of both groups with low turnover at baseline had an improvement in bone formation rate (BFR/BS = 0.015 +/- 0.016 vs. 0.062 +/- 0.078, p = 0.003 for calcium and 0.017 +/- 0.016 vs. 0.071 +/- 0.084 mu m(3)/mu m(2)/day, p = 0.010 for sevelamer). Conclusions: There was no difference in CAC progression or changes in bone remodeling between the calcium and the sevelamer groups. Copyright (C) 2008 S. Karger AG, Basel

The effect of oestrogen and alendronate therapies on postmenopausal bone loss around osseointegrated titanium implants

Objectives This study evaluated the influence of oestrogen deficiency and its therapies on bone tissue around osseointegrated implants. Methods Implants were placed in 66 female rats tibiae. The animals were assigned into five groups: control (CTL), sham, ovariectomy (OVX), oestrogen (EST), and alendronate (ALE). While CTL was sacrificed 60 days after implant placement, other groups were subjected to ovariectomy or sham surgery according to group and euthanized after 90 days. Blood and urine samples were collected at sacrifice day for osteocalcin (OCN) and deoxypyridinoline (DPD) quantification. Densitometry of femur and lumbar vertebrae was performed in order to evaluate rats` skeletal impairment. Non-decalcified sections were referred to fluorescent and light microscopy for analyses of mineral apposition rate (MAR), eroded and osteoclastic surfaces, bone-to-implant contact (BIC), and bone area fraction occupancy (BAFO). Results Results from the OVX group showed significantly lower bone mineral density (BMD), BIC, BAFO, and MAR, while OCN, deoxipiridinoline, eroded surface and ostecoclastic surface were increased compared with the other groups of the study. ALE reduced OCN and DPD concentrations, MAR, osteoclastic and eroded surfaces, and no difference was in BIC and BAFO relative to SHAM. EST and CTL showed similar results to SHAM for measurements. Conclusions Oestrogen deficiency exerted a negative influence on bone tissue around implants, while oestrogen replacement therapy and alendronate were effective against its effects. Although alendronate therapy maintained the quantity of bone around implants, studies evaluating bone turnover kinetics are warranted. To cite this article:Giro G, Coelho PG, Pereira RMR, Jorgetti V, Marcantonio E Jr, Orrico SRP. The effect of oestrogen and alendronate therapies on postmenopausal bone loss around osseointegrated titanium implants.Clin. Oral Impl. Res. 22, 2011; 259-264.doi: 10.1111/j.1600-0501.2010.01989.x.

Fibroblast Growth Factor 23 in Hemodialysis Patients: Effects of Phosphate Binder, Calcitriol and Calcium Concentration in the Dialysate

Background: Fibroblast growth factor 23 (FGF23) concentrations increase early in chronic kidney disease (CKD), and the influence of current CKD-mineral and bone disorder (MBD) therapies on serum FGF23 levels is still under investigation. Methods: In this post-hoc analysis of a randomized clinical trial, phosphate binders and calcitriol were washed out of 72 hemodialysis patients who were then submitted to bone biopsy, coronary tomography and biochemical measures, including FGF23. They were randomized to receive sevelamer or calcium acetate for 1 year and the prescription of calcitriol and the calcium concentration in the dialysate were adjusted according to serum calcium, phosphate and PTH and bone biopsy diagnosis. Results: At baseline, bone biopsy showed that 58.3% had low-turnover bone disease, whereas 38.9% had high-turnover bone disease, with no significant differences between them with regard to FGF23. Median baseline FGF23 serum levels were elevated and correlated positively with serum phosphate. After 1 year, serum FGF23 decreased significantly. Repeated measures ANOVA analysis showed that the use of a 3.5-mEq/l calcium concentration in the dialysate, as well as the administration of calcitriol and a calcium-based phosphate binder were associated with higher final serum FGF23 levels. Conclusions: Taken together, our results confirm that the current CKD-MBD therapies have an effect on serum levels of FGF23. Since FGF23 is emerging as a potential treatment target, our findings should be taken into account in the decision on how to manage CKD-MBD therapy. Copyright (C) 2010 S. Karger AG, Basel

Binding and internalization of the melanocyte stimulating hormone receptor ligand [Nle(4), D-Phe(7)]alpha-MSH in B16 melanoma cells

The current study aims to ascertain the fate of the melanocyte stimulating hormone (MSH) receptor and its ligand [Nle(4), D-Phe(7)]alpha-MsH (NDP-MSH) following binding to murine B16 melanoma cells. Cells were incubated with [I-125]-NDP-MSH for up to 180 min and binding, internalization and degradation determined. Intracellular trafficking of the radiolabel was assessed !using Percoll density gradient centrifugation of homogenized cells. Receptor down-regulation and receptor mRNA levels were also measured over 96 hr after exposure to 1 mu M ligand. NDP-MSH accumulation increased with time in a temperature-dependent manner and was inhibited by excess peptide. The ligand was rapidly internalized and translocated to the lysosomal compartment where it was degraded. Internalization was accompanied by a loss or down-regulation of cell surface receptors, suggesting internalization of the NDP-MSH-receptor complex. No recycling of the receptors between the plasma membrane and intracellular compartments could be detected in this cell-hue. Approximately 15% of the surface receptors were resistant to down-regulation, possibly indicating receptor heterogeneity. Down-regulation persisted ibr up to 96 hr and was accompanied by a decrease in MSH receptor mRNA levels 48 hr after treatment. However, before this time, transcript levels were the same in treated and control cells. In contrast to what was seen with NDP-MSH, cell surface receptors removed with trypsin wc:re rapidly replaced. These results show that NDP-MSH not only induced MSH receptor :internalization but also inhibited receptor turnover, resulting in a prolonged down-regulation. It is concluded that, in B16 cells, the MSH receptor undergoes ligand-dependent internalization, resulting in a prolonged down-regulation. Copyright (C) 1996 Elsevier Science Ltd

Influence of Estrogen Deficiency on Bone Around Osseointegrated Dental Implants: An Experimental Study in the Rat Jaw Model

Purpose: The aim of this study was to evaluate the influence of estrogen deficiency on bone around osseointegrated dental implants in a rat jaw model. Materials and Methods: This study used 16 female rats that had the first molars bilaterally extracted and were allowed to heal for 30 days before implant placement. Sixty days after implant placement, the animals were randomly subjected to sham surgery or ovariectomy (OVX). The animals were euthanized 90 days after OVX. Bone-to-implant contact, bone area fraction occupancy between implant threads, mineral density, turnover markers, and cells positive for tartrate-resistant acid phosphatase were assessed for the 2 groups. Results: The results showed that OVX group presented a decrease of systemic bone density, alterations in bone turnover markers, and an increase of cells positive for tartrate-resistant acid phosphatase compared with the sham-surgery group. However, no difference relative to bone-to-implant contact and bone area fraction occupancy was observed between groups. Conclusions: The findings of this study demonstrate that estrogen deficiency may not be considered a risk factor for osseointegrated implant failure in jaw bone. (C) 2011 American Association of Oral and Maxillofacial Surgeons J Oral Maxillofac Surg 69:1911-1918, 2011

Serum from children with polyarticular juvenile idiopathic arthritis (pJIA) inhibits differentiation, mineralization and may increase apoptosis of human osteoblasts ""in vitro""

We examined the effects of polyarticular juvenile idiopathic arthritis (pJIA) serum on proliferation, differentiation, mineralization, and apoptosis of human osteoblast cells (hOb) in culture. The hOb were cultured with 10% serum from active pJIA and healthy controls (CT) and were tested for DNA synthesis, alkaline phosphatase (AP) activity, osteocalcin (OC) secretion, calcium levels, caspase 3 activity, and DNA fragmentation. None of the patients had used glucocorticoids for at least 1 month before the study, or any other drug that can affect bone mineral metabolism. Human inflammatory cytokine levels (IL-6, IL-8, IL-10, IL-1 beta, TNF-alpha, and IL-12p70) were measured in pJIA and CT sera. Low levels of AP activity was observed in pJIA cultures compared with CT cultures (67.16 +/- 53.35 vs 100.11 +/- 50.64 mu mol p-nitrophenol/h(-1) mg(-1) protein, P=0.008). There was also a significant decrease in OC secretion (9.23 +/- 5.63 vs 12.82 +/- 7.02 ng/mg protein, P=0.012) and calcium levels (0.475 +/- 0.197 vs 0.717 +/- 0.366 mmol/l, P=0.05) in pJIA hOb cultures. No difference was observed in cell proliferation (323.56 +/- 108.23 vs 328.91 +/- 88.03 dpm/mg protein, P=0.788). Osteoblasts cultured with JIA sera showed lower levels of DNA and increased fragmentation than osteoblasts cultured with CT sera. pJIA sera showed higher IL-6 values than CT (21.44 +/- 9.31 vs 3.58 +/- 2.38 pg/ml, P<0.001), but no difference was observed related to IL-8, IL-10, IL-1 beta, TNF-alpha, and IL-12p70 between pJIA and controls. This study suggests that serum from children with pJIA inhibits differentiation, mineralization and may increase apoptosis of hOb cultures, and inflammatory cytokines such as IL-6 might be a mechanism in this find. These results may represent an alternative therapeutic target for prevention and treatment of bone loss in JIA.

Vitamin D insufficiency: A risk factor to vertebral fractures in community-dwelling elderly women

Objective:To determine the risk factors for the presence of moderate/severe vertebral fracture, specifically 25-hydroxyvitamin D (25-OHD). Study design: Cross-sectional study conducted for 2 years in the city of Sao Paulo, Brazil including community-dwelling elderly women. Methods: Bone mineral density (BMD), serum 25-OHD, intact parathyroid hormone (iPTH), calcium and estimated glomerular filtration rate (eGFR) were examined in 226 women without vertebral fractures (NO FRACTURE group) and 189 women with at least one moderate/severe vertebral fracture (FRACTURE group). Vertebral fracture assessment (VFA) was evaluated using both the Genant semiquantitative (SQ) approach and morphometry. Results: Patients in the NO FRACTURE group had lower age, increased height, higher calcium intake, and higher BMD compared to those patients in the FRACTURE group (p < 0.05). Of interest, serum levels of 25-OHD in the NO FRACTURE group were higher than those observed in the FRACTURE group (51.73 nmol/L vs. 42.31 nmol/L, p < 0.001). Reinforcing this finding, vitamin D insufficiency (25-OHD < 75 nmol/L) was observed less in the NO FRACTURE group (82.3% vs. 93.65%, p = 0.001). After adjustment for significant variables within the patient population (age, height, race, calcium intake, 25-OHD, eGFR and sites BMD), the logistic-regression analyses revealed that age (OR = 1.09, 95% Cl 1.04-1.14, p < 0.001) femoral neck BMD (OR = 0.7, 95% CI 0.6-0.82, p < 0.001) and 25-OHD <75 nmol/L (OR = 2.38, 95% CI 1.17-4.8, p = 0.016) remains a significant factor for vertebral fracture. Conclusion: Vitamin D insufficiency is a contributing factor for moderate/severe vertebral fractures. This result emphasizes the importance of including this modifiable risk factor in the evaluation of elderly women. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Dog culling and replacement in an area endemic for visceral leishmaniasis in Brazil

Measures employed to control visceral leishmaniasis in Brazil have focused on vector control by residual insecticide spraying and diagnosis of infection with elimination of positive dogs. We describe dog culling and replacement in a Brazilian endemic area (the Alvorada District, Aracatuba, SP) in order to better understand dog population dynamics when elimination of the dog reservoir is adopted as the main control measure. From August 2002 to July 2004, 60.9% of the estimated dog population for the area was culled with a mean age of 34 months old. The presence of anti-Leishmania sp. antibodies was recorded for only 26.7% of the euthanized canines. Replacement was observed in 38.8% of the cases, some of them by 2 or more dogs and in a mean time of 4 months. Dogs were replaced mostly by puppies of both sexes with a mean age of 6.8 months. From August 2002 to April 2005 we were able to follow-up 116 of these dogs, during a mean time of 8.7 months. Canine visceral leishmaniasis seropositivity by ELISA was observed in 42.2% of the followed dogs, 30.6% of which were already positive at the first evaluation. By the end of the follow-up period 37% of the dogs were submitted to euthanasia, with a mean age of 18.3 months. In the studied CVL endemic area of Brazil, euthanasia and the subsequent replacement ratio were high, increasing the dog population turnover and leading to a younger population that might be more susceptible to a variety of other infectious diseases in addition to CVL. Dog culling as a control strategy for VL should be reassessed. (C) 2008 Elsevier B.V. All rights reserved.

Flow-through peritoneal dialysis in neonatal enema-induced hyperphosphatemia

Fleet enemas are hypertonic solutions with an osmotic action and a high concentration of phosphate. When retained in the human body they have a great toxic potential, causing severe hydro-electrolyte disorders in children, especially in newborns. We report the case of a previously healthy 8-day-old newborn who needed neonatal intensive care treatment after the inadvertent administration of an osmotically active hypertonic phosphate enema. Taking into account that phosphate removal by peritoneal dialysis (PD) strongly depends on total dialysate turnover, we chose continuous flow PD (CFPD) as the treatment option, with a successful outcome. Clinical experience with this dialytic modality is limited to a few case reports in pediatric and adult patients. To the best of our knowledge, we report here the first description of CFPD in the setting of acute phosphate nephropathy in the neonatal period. The modality of PD described here has potential as an alternative management option as it is a highly efficient, methodologically simple, and low-cost method without any need for sophisticated equipment. Physicians and parents should be aware of the adverse effects of a hypertonic phosphate enema and should never use these medications in infants and newborns.

Effects of bone remodelling on calcium mass transfer during haemodialysis

Background. During haemodialysis, calcium balance can affect, or be affected by, mineral metabolism. However, when dialysate calcium concentration (d[Ca]) is chosen or kinetic models are employed to calculate calcium balance, bone remodelling is rarely considered. In this study, we examined whether bone remodelling affects calcium mass transfer during haemodialysis. Methods. We dialysed 23 patients using a d[Ca] of 1.0, 1.25, 1.5 or 1.75 mmol/L. Calcium mass transfer was measured and associated with remodelling bone factors. Results. Calcium balance varied widely depending on the d[Ca]. Calcium removal was -578 +/- 389, -468 +/- 563, +46 +/- 400 and +405 +/- 413 mg when a d[Ca] of 1.0, 1.25, 1.5 or 1.75 mmol/L was used, respectively (1.0 and 1.25 VS 1.5 and 1.75 mmol/L, P<0.001; 1.5 vs 1.75 mmol/L, P<0.05). Univariate analysis showed that calcium balance correlated with calcium gradient, parathyroid hormone (PTH), osteocalcin and dialysis vintage. Multivariate analysis revealed that calcium balance was dependent on calcium gradient, PTH and osteocalcin. Conclusions. These results suggest that bone remodelling could affect calcium mass transfer during haemodialysis.

Strength Training Preserves the Bone Mineral Density of Postmenopausal Women Without Hormone Replacement Therapy

Objective: The study was designed to evaluate the effects of strength training (ST) on the bone mineral density (BMD) of postmenopausal women without hormone replacement therapy. Method: Subjects were randomized into untrained (UN) or trained (TR) groups. The TR group exercised three ST sessions per week for 24 weeks, and body composition, muscular strength, and BMD of the lumbar spine and femur neck were evaluated. Results: Body weight, mass index, and fat percentage were lower after 24 weeks only in the TR group (p < .05). SR also improved the one repetition maximum test in 46% and 39% of upper and lower limbs, respectively. The percentage of demineralization was higher in the UN group than in the TR group at the lumbar spine and femoral neck (p < .05). Discussion: Results indicated that 24 weeks of ST improved body composition parameters, increased muscular strength, and preserved BMD in postmenopausal women.

Low protein diet changes the energetic balance and sympathetic activity in brown adipose tissue of growing rats

Objective: The aim of this study was to assess the effects of protein restriction in growing rats. Methods: Rats (approximate weight, 100 g) were maintained with low-protein (LP; 6%) or normo-proteic (control; 17%) diets, and at the end of the 15th day, hormonal and biochemistry parameters and energetic balance were evaluated. Data were analyzed using Student`s t test (with statistical significance set at P <= .05). Results: LP animals were hyperphagic and showed increased energetic gain (24%) and energy expenditure (EE) compared with controls. The increase in EE was followed by increased sympathetic activity in brown adipose tissue, evidenced by increased norepinephrine turnover, suggesting increased thermogenesis. In spite of hyperphagia, protein ingestion in LP animals was lower than that of controls (P < 0.01). The LP diet impaired body growth and caused deep alterations in body chemical composition, with an increase in carcass lipid content (64%) and reductions of protein and water. In LP animals, postprandial glycemia was unchanged, and insulinemia was lower than in controls (P <= .01). Reduction in fasting glycemia without changes in insulinemia also was detected (P < .01), suggesting increased insulin sensitivity. The LP diet caused a 100% increase in serum leptin (P < .01). Conclusions: Protein restriction led to an increase in EE, with probable activation of thermogenesis in brown adipose tissue, evidenced by an increase in catecholamines levels. Despite the higher EE, energetic gain and lipids increased. The high level of leptin associated with hyperphagia led to the supposition that these animals are leptin resistant, and the increase in insulin sensitivity, suggested by the relation between insulin and glycemia in fasting and fed animals, might contribute to lipid accumulation. (C) 2009 Elsevier Inc. All rights reserved.

Fatty acid synthesis and generation of glycerol-3-phosphate in brown adipose tissue from rats fed a cafeteria diet

In vivo fatty acid synthesis and the pathways of glycerol-3-phosphate (G3P) production were investigated in brown adipose tissue (BAT) from rats fed a cafeteria diet for 3 weeks. In spite of BAT activation, the diet promoted an increase in the carcass fatty acid content. Plasma insulin levels were markedly increased in cafeteria diet-fed rats. Two insulin-sensitive processes, in vivo fatty acid synthesis and in vivo glucose uptake (which was used to evaluate G3P generation via glycolysis) were increased in BAT from rats fed the cafeteria diet. Direct glycerol phosphorylation, evaluated by glycerokinase (GyK) activity and incorporation of [U-(14)C]glycerol into triacylglycerol (TAG)-glycerol, was also markedly increased in BAT from these rats. In contrast, the cafeteria diet induced a marked reduction of BAT glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase-C activity and incorporation of [1-(14)C]pyruvate into TAG-glycerol. BAT denervation resulted in an approximately 50% reduction of GyK activity, but did not significantly affect BAT in vivo fatty acid synthesis, in vivo glucose uptake, or glyceroneogenesis. The data suggest that the supply of G3P for BAT TAG synthesis can be adjusted independently from the sympathetic nervous system and solely by reciprocal changes in the generation of G3P via glycolysis and via glyceroneogenesis, with no participation of direct phosphorylation of glycerol by GyK.

Morphologic evaluation and expression of matrix metalloproteinases-2 and 9 and nitric oxide during experimental periodontal disease in rat

The immunopathologic and inflammatory mechanisms involved in periodontal disease (PD) include the participation of host resident, inflammatory cells and chemical mediators. Metalloproteinases (MMPs) and nitric oxide (NO) play essential role in extracellular matrix turnover of periodontal tissue destruction. In this study, by means of RT-PCR through semi-quantitative densitometric scanning methods, the expression of MMPs -2 and -9 and inducible NO synthase (iNOS) was temporally and spatially investigated during the destructive mechanisms of experimentally induced PD in rats. Samples from different periods were microscopically analyzed and compared with the contralateral side (control). Our results showed significant expression of MMP-9 and iNOS in tissues affected by PD, as compared with controls, three days after PD induction, simultaneously with the beginning of alveolar bone loss. At 7 days post induction, only the MMP-9 mRNA presented a significantly higher expression, as compared with the respective controls. Thus, in the rat ligature-induced PD, MMP-9 and iNOS might importantly participate in the early stages of the disease, including inflammatory cell migration, tissue destruction and alveolar bone resorption. Also, we may suggest that the exuberant presence of PMNs may be related to the important expression of iNOS and MMP-9 found at 3 days post induction.