Efficacy assessment of methylcellulose-based thermoresponsive hydrogels loaded with gallium acetylacetonate in osteoclastic bone resorption.

Homeostatic imbalance involving progressive stimulation of osteoclast (OC) differentiation and function will lead to an increased risk of fragility fractures. In this regard, we investigated gallium acetylacetonate (GaAcAc) as a possible treatment for osteoclastic bone resorption. Further, the extent to which suitable delivery systems can enhance the therapeutic potential of GaAcAc was evaluated. GaAcAc solution (10-50 µg/mL) suppressed OC differentiation using murine monocytic RAW 264.7 or hematopoietic stem cells. Methylcellulose-based hydrogels were fabricated and characterized based on biocompatibility with bone cells, GaAcAc loading, and thermoresponsive behavior using storage (G') and loss (G″) moduli parameters. Compared to GaAcAc solution, hydrogels loaded with GaAcAc (GaMH) were more effective in suppressing OC differentiation and function. The number and extent of bone resorption pits from ex vivo studies were markedly reduced with GaMH treatment. Mechanistic assessment of GaMH efficacy showed superiority, compared to GaAcAc solution, in downregulating the expression of key markers involved in mediating OC differentiation (such as NFAT2, cFos, TRAF6, and TRAP) as well as in bone resorption by OCs (cathepsin K or CTSK). Additional studies (in vitro and in vivo) suggested that the performance of GaMH could be ascribed to controlled release of GaAcAc and the ability to achieve prolonged bio-retention after injection in BALB/c mice, which plausibly maximized the therapeutic impact of GaAcAc. Overall, the work demonstrated, for the first time, the therapeutic efficacy of GaAcAc and the therapeutic potential of GaMH delivery systems in osteoclastic bone resorption.

Histochemical assessment on osteoclasts in long bones of toll-like receptor 2 (TLR2) deficient mice.

OBJECTIVE: Toll-like receptor 2 (TLR2), recognizes a wide variety of pathogen-associated molecular patterns such as lipopolysaccharides, peptidoglycans, and lipopeptides, and is generally believed to be present in monocytes, macrophages, dendritic cells, and vascular endothelial cells. However, no histological examination of osteoclasts, which differentiate from precursors common to macrophages/monocytes, has been performed in a non-infected state of TLR2 deficiency. The objective of this study was to examine the histological properties and function of osteoclasts in the long bones of 8-week-old male TLR2 deficient (TLR2-/-) mice to gain insight into TLR2 function in biological circumstances without microbial infection. METHODS: Eight-week-old male wild-type and TLR2-/- mice were fixed with paraformaldehyde solution, and their tibiae and femora were used for micro-CT analysis, immunohistochemistry, transmission electron microscopy, and real-time PCR analysis. RESULTS: TLR2-/- tibiae and femora exhibited increased bone volume of metaphyseal trabeculae and elevated numbers of TRAP-positive osteoclasts. However, the number of multinucleated TRAP-positive osteoclasts was reduced, whereas mononuclear TRAP-positive cells increased, despite the high expression levels of Dc-Stamp and Oc-Stamp. Although TRAP-positive multinucleated and mononuclear osteoclasts showed the immunoreactivity and elevated expression of RANK and siglec-15, they revealed weak cathepsin K-positivity and less incorporation of the mineralized bone matrix, and often missing ruffled borders. It seemed likely that, despite the increased numbers, TLR2-/- osteoclasts reduced cell fusion and bone resorption activity. CONCLUSION: It seems likely that even without bacterial infection, TLR2 might participate in cell fusion and subsequent bone resorption of osteoclasts.

Assessment of Biochemical Bone Turnover Markers and Bone Mineral Density in Thin and Normal-Weight Children.

Objective There is scant research examining the prevalence of thinness in early childhood, despite its potential negative consequences for health and development across the life course. The objective of this study was to assess bone status through measurement of bone mineral density and biochemical bone turnover markers, with special attention paid to carboxylated (c-OC) as well as undercarboxylated (uc-OC) forms of osteocalcin, in the groups of thin and normal-weight children. Design The study included 80 healthy prepubertal children (median age 7.0 years), who were divided (according to Cole's international cutoffs) into 2 subgroups: thin children ( n = 40, body mass index [BMI] = 13.5 kg/m2) and normal-weight children ( n = 40, BMI = 16.1 kg/m2). Bone mineral density (BMD) and bone mineral content (BMC) were assessed by dual-energy x-ray absorptiometry method. Serum concentrations of C-terminal telopeptide of collagen type I (CTX), total osteocalcin (OC), and c-OC, and uc-OC forms of osteocalcin were determined using enzyme-linked immunosorbent assays. Results In thin children, we observed higher levels of bone resorption marker CTX compared with normal-weight peers. Total osteocalcin concentrations were comparable in both groups of children; however, in thin children we observed higher median values of uc-OC (34.40 vs. 29.30 ng/mL, P < 0.05) and similar c-OC levels (25.65 vs. 28.80 ng/mL). The ratio of c-OC to uc-OC was significantly lower ( P < 0.05) in thin than in normal-weight children. Total BMD and BMC were significantly decreased ( P < 0.0001) in thin children compared with normal-weight peers (0.724 ± 0.092 vs. 0.815 ± 0.060 g/cm2 and 602.7 ± 159.2 vs. 818.2 ± 220.1 g, respectively). Conclusion Increased concentrations of CTX and uc-OC might lead to disturbances in bone turnover and a decrease in bone mineral density in thin children.

Histochemical assessment for osteoblastic activity coupled with dysfunctional osteoclasts in c-src deficient mice.

Since osteoblastic activities are believed to be coupled with osteoclasts, we have attempted to histologically verify which of the distinct cellular circumstances, the presence of osteoclasts themselves or bone resorption by osteoclasts, is essential for coupled osteoblastic activity, by examining c-fos-/- or c-src-/- mice. Osteopetrotic c-fos deficient (c-fos-/-) mice have no osteoclasts, while c-src deficient (c-src-/-) mice, another osteopetrotic model, develop dysfunctional osteoclasts due to a lack of ruffled borders. c-fos-/- mice possessed no tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts, and showed very weak tissue nonspecific alkaline phosphatase (TNALPase)-reactive mature osteoblasts. In contrast, c-src-/- mice had many TNALPase-positive osteoblasts and TRAPase-reactive osteoclasts. Interestingly, the parallel layers of TRAPase-reactive/osteopontin-positive cement lines were observed in the superficial region of c-src-/- bone matrix. This indicates the possibility that in c-src-/- mice, osteoblasts were activated to deposit new bone matrices on the surfaces that osteoclasts previously passed along, even without bone resorption. Transmission electron microscopy demonstrated cell-to-cell contacts between mature osteoblasts and neighboring ruffled border-less osteoclasts, and osteoid including many mineralized nodules in c-src-/- mice. Thus, it seems likely that osteoblastic activities would be maintained in the presence of osteoclasts, even if they are dysfunctional.

Quantifying the Balance Between Total Bone Formation and Total Bone Resorption: An Index of Net Bone Formation.

CONTEXT: Bone gain vs loss across the skeleton loss depends on the balance between total bone formation and total bone resorption. OBJECTIVE: The objective of the study was to determine whether resorption and formation markers can be combined to gauge net bone formation across the skeleton. DESIGN: The study included a cohort followed up across menopause transition (Study of Women's Health Across the Nation). SETTING AND PARTICIPANTS: Community-dwelling women, 42-52 years old, premenopausal or early perimenopausal at baseline, participated in the study. OUTCOME: The study included the following measures: 1) bone balance index (BBI) created by estimating the relationship between resorption (urinary N-telopeptide) and formation (osteocalcin) markers when the total formation equals the total resorption in 685 women with stable bone mineral density (BMD) (>5 y before the final menstrual period [FMP]) and applying this relationship to measured bone turnover markers in 216 women beginning to lose bone (≤2 y from FMP); and 2) annualized percentage declines over the following 3-4 years in the lumbar spine (LS) and femoral neck (FN) BMD. RESULTS: Adjusted for covariates, the BBI was greater (more favorable) in women with a greater body mass index (P = .03) and lower (less favorable) in women closer to the FMP (P = .007). Each SD decrement in BBI was associated with 0.27%/y faster LS BMD decline (P 0.04) and a 38% higher odds of faster-than-average loss of LS bone mass (P = .008, c-statistic 0.76). BBI was not associated with decline in FN BMD. Urinary N-telopeptide alone was not associated with either LS or FN BMD decline. CONCLUSIONS: An index that quantifies net bone formation vs resorption can be created from bone turnover markers and may help identify individuals at high risk for LS bone loss.

[Assessment of selected bone metabolism marker concentrations in children with cystic fibrosis]. / Analiza stezen wybranych parametrów metabolizmu kostnego u dxieci chorych na mukowiscydoze.

AIM: The aim of this study was to assess bone formation and resorption processes and bone metabolism regulators, such as osteoprotegerin and fetuin-A in children with cystic fibrosis. MATERIAL AND METHODS: We examined 45 children with cystic fibrosis aged 5-13 years treated at the Institute of Mother and Child in Warsaw. The control group consisted of 35 healthy children in the same synage range without any diseases which may influence bone metabolism. We determined serum calcium and phosphate levels by colorimetric methods, vitamin D3 by the chemiluminiscence method and bone metabolism markers (osteocalcin, 5b isoenzyme of tartrate-resistant acid phosphatase, osteoprotegerin, fetuin-A) by immunoenzymatic methods. RESULTS: Mean serum concentrations of calcium and phosphate in the studied children were within the reference ranges. However, the level of 25-hydroxyvitamin D3 was significantly lower in patients with cystic fibrosis compared to the controls (19.3±7.6 vs 25.2±8.9 ng/ml, p<0.01). In cystic fibrosis children we observed a statistically significant lower concentration of osteocalcin (81.9±28.9 vs 97.9±28.6 ng/ ml, p<0.01) and similar activity of 5b isoenzyme of tartrate-resistant acid phosphatase (12.5±2.9 vs 13.4±3.5 U/L) as compared to healthy peers. Mean serum concentration of osteoprotegerin in patients with CF was significantly lower than in the control children (4.1±0.98 vs 4.59±0.86 pmol/l, p<0.05). Serum concentration of fetuin-A was comparable in both groups of children. CONCLUSIONS: In children with cystic fibrosis changes in the profile of bone metabolism markers were observed. Even patients with CF who are clinically stable and supplemented with vitamins are at risk of osteopenia and osteoporosis in their later life. Therefore, they should be under a comprehensive medical and nutritional care in order to achieve their optimal peak bone mass.

Causal assessment of dietary acid load and bone disease: a systematic review & meta-analysis applying Hill's epidemiologic criteria for causality.

BACKGROUND: Modern diets have been suggested to increase systemic acid load and net acid excretion. In response, alkaline diets and products are marketed to avoid or counteract this acid, help the body regulate its pH to prevent and cure disease. The objective of this systematic review was to evaluate causal relationships between dietary acid load and osteoporosis using Hill's criteria. METHODS: Systematic review and meta-analysis. We systematically searched published literature for randomized intervention trials, prospective cohort studies, and meta-analyses of the acid-ash or acid-base diet hypothesis with bone-related outcomes, in which the diet acid load was altered, or an alkaline diet or alkaline salts were provided, to healthy human adults. Cellular mechanism studies were also systematically examined. RESULTS: Fifty-five of 238 studies met the inclusion criteria: 22 randomized interventions, 2 meta-analyses, and 11 prospective observational studies of bone health outcomes including: urine calcium excretion, calcium balance or retention, changes of bone mineral density, or fractures, among healthy adults in which acid and/or alkaline intakes were manipulated or observed through foods or supplements; and 19 in vitro cell studies which examined the hypothesized mechanism. Urine calcium excretion rates were consistent with osteoporosis development; however calcium balance studies did not demonstrate loss of whole body calcium with higher net acid excretion. Several weaknesses regarding the acid-ash hypothesis were uncovered: No intervention studies provided direct evidence of osteoporosis progression (fragility fractures, or bone strength as measured using biopsy). The supporting prospective cohort studies were not controlled regarding important osteoporosis risk factors including: weight loss during follow-up, family history of osteoporosis, baseline bone mineral density, and estrogen status. No study revealed a biologic mechanism functioning at physiological pH. Finally, randomized studies did not provide evidence for an adverse role of phosphate, milk, and grain foods in osteoporosis. CONCLUSIONS: A causal association between dietary acid load and osteoporotic bone disease is not supported by evidence and there is no evidence that an alkaline diet is protective of bone health.

Molecular and structural assessment of alveolar bone during tooth eruption and function in the miniature pig, sus scrofa.

The development of alveolar bone adjacent to the tooth root during tooth eruption is not well understood. This study tested the hypothesis that predominantly woven bone forms adjacent to tooth roots during tooth eruption, but that this immature structure transitions to lamellar bone when the tooth comes into function. Additionally, bone resorption was predicted to play a key role in transitioning immature bone to more mature, load-bearing tissue. Miniature pigs were compared at two occlusal stages, 13 weeks (n = 3), corresponding with the mucosal penetration stage of M(1) tooth eruption, and 23 weeks (n = 3), corresponding with early occlusion of M(1) /M(1) . Bone samples for RNA extraction and qRT-PCR analysis were harvested from the diastema and adjacent to M(1) roots on one side. Following euthanasia, bone samples for haematoxylin and eosin and TRAP staining were harvested from these regions on the other side. In contrast to expectations, both erupting and functioning molars had reticular fibrolamellar structure in alveolar bone adjacent to M(1) . However, the woven bone matrix in older pigs was thicker and had denser primary osteons. Gene expression data and osteoclast cell counts showed a tendency for more bone resorptive activity near the molars than at distant sites, but no differences between eruptive stages. Thus, although resorption does occur, it is not a primary mechanism in the transition in alveolar bone from eruption to function. Incremental growth of existing woven bone and filling in of primary osteons within the mineralized scaffold generated the fortification necessary to support an erupted and functioning tooth.

The pilot study of DXA assessment in chinese HIV-infected men with clinical lipodystrophy.

The aim of this study was to evaluate human immunodeficiency virus (HIV)-infected patient's body composition changes by dual-energy X-ray absorptiometry (DXA) and to analyze factors associated with lipodystrophy (LD). Total-body composition was measured by DXA in HIV-infected men and healthy men. HIV-infected men were divided into LD patients and non-LD patients according to whether they were complicated with LD. Healthy men were selected as controls. Fat mass (FM) of HIV-infected patients correlated negatively with the duration of HIV infection and with the duration of highly active antiretroviral therapy regimen (r(s)=-0.448 and -0.563; p=0.032 and 0.000, respectively). Multiple linear regression results showed that FM had positive correlation with weight and bone mineral content (BMC) and had negative correlation with lean mass (LM). Total body and regional FMs were found to be significantly different among LD patients, non-LD patients, and controls-the lowest in LD patients and the highest in controls (p<0.05). Total body, trunk, and leg BMCs of LD patients were lower than those of controls (p<0.05). Lumbar bone mineral density of LD patients was lower than that of non-LD patients and controls (p=0.04 and 0.007). LM of LD patients was higher than that of non-LD patients, and trunk LM had statistical difference between the 2 groups (p=0.003). Applying DXA to assess HIV-infected patient's body composition changes could provide objective information for physicians to prevent LD and osteoporosis.

Morphological assessment of basic multicellular unit resorption parameters in dogs shows additional mechanisms of bisphosphonate effects on bone.

Bisphosphonates (BPs) slow bone loss by reducing initiation of new basic multicellular units (BMUs). Whether or not BPs simply prevent osteoclasts from initiating new BMUs that resorb bone or also reduce the amount of bone they resorb at the BMU level is not clear. The goal of this study was to determine the effects of BPs on three morphological parameters of individual BMUs, resorption depth (Rs.De), area (Rs.Ar), and width (Rs.Wi). After 1 year of treatment with vehicle (VEH), alendronate (ALN; 0.10, 0.20, or 1.00 mg/kg/day), or risedronate (RIS; 0.05, 0.10, or 0.50 mg/kg/day), resorption cavity morphology was assessed in vertebral trabecular bone of beagle dogs by histology. Animals treated with ALN or RIS at the doses representing those used to treat postmenopausal osteoporosis (0.20 and 0.10 mg/kg/day, respectively) had significantly lower Rs.Ar (-27%) and Rs.Wi (-17%), with no difference in Rs.De, compared to VEH-treated controls. Low doses of ALN and RIS did not affect any parameters, whereas higher doses resulted in similar changes to those of the clinical dose. There were no significant differences in the resorption cavity measures between RIS and ALN at any of the dose equivalents. These results highlight the importance of examining parameters beyond erosion depth for assessment of resorption parameters. Furthermore, these results suggest that in addition to the well-known effects of BPs on reducing the number of active BMUs, these drugs also reduce the activity of osteoclasts at the individual BMU level at doses at and above those used clinically for the treatment of postmenopausal osteoporosis.

Short-term safety assessment in the use of intravenous zoledronic acid in children.

The clinical side effects of the potent new bisphosphonate zoledronic acid in children are unknown. In this study of 34 children with various bone disorders, the frequency of postinfusion flu-like illness, hypocalcemia, and hypophosphatemia was 85%, 74%, and 82%, respectively. No renal side effects were detected after up to 3 consecutive infusions.

An assessment of ADAMs in bone cells: absence of TACE activity prevents osteoclast recruitment and the formation of the marrow cavity in developing long bones.

ADAMs (A Disintegrin And Metalloprotease domain) are metalloprotease-disintegrin proteins that have been implicated in cell adhesion, protein ectodomain shedding, matrix protein degradation and cell fusion. Since such events are critical for bone resorption and osteoclast recruitment, we investigated whether they require ADAMs. We report here which ADAMs we have identified in bone cells, as well as our analysis of the generation, migration and resorptive activity of osteoclasts in developing metatarsals of mouse embryos lacking catalytically active ADAM 17 [TNFalpha converting enzyme (TACE)]. The absence of TACE activity still allowed the generation of cells showing an osteoclastic phenotype, but prevented their migration into the core of the diaphysis and the subsequent formation of marrow cavity. This suggests a role of TACE in the recruitment of osteoclasts to future resorption sites.

Avaliação do efeito do risedronato sódico na consolidação das fraturas: estudo experimental em ratos / Assessment of the effect of sodium risedronate in fracture healing: experimental essay in rats

Estudo experimental em 40 ratos para avaliar a influência do risedronato sódico no tratamento de fraturas em animais nutridos e desnutridos. Os animais foram aleatoriamente distribuídos em quatro grupos submetidos ou não a dieta aprotéica, e recebendo ou não o risedronato, por gavagem. Os animais foram submetidos à fratura da tíbia direita por osteoclasia manual, sob anestesia, no dia 15 do experimento e sacrificados 28 dias depois. As variáveis analisadas foram a evolução ponderal, avaliações radiográfica, densitométrica e histomorfométrica do calo ósseo, e dosagens séricas do cálcio, fósforo, fosfatase alcalina, proteínas totais, albumina e osteocalcina. O risedronato interferiu positivamente no processo de consolidação de fraturas /An experimental study was carried out in 40 rats to assess the influence of sodium risedronate in fracture healing in normal and undernourished rats. The animals were distributed in four groups, at random, which received or not risedronate by gavage. The animals underwent fractures of the right tibia by...

Non-invasive assessment of equine bone: an update.

This paper summarizes traditional and current methods of non-invasive assessment of bone in the horse. The description and potential clinical utility of two non-invasive technologies with major development in the last decade are presented, namely, (1) serum biochemical markers for bone turnover and (2) quantitative ultrasound. Serum biochemical markers of bone formation valid in horses are osteocalcin, carboxy-terminal peptide of type I procollagen and bone-specific alkaline phosphatase. The cross-linked carboxy-terminal telopeptide of type 1 collagen c-telopeptides of type I collagen and total deoxypyridinoline are the serum markers for bone degradation. These markers respond more rapidly to skeletal changes than other bone assessment techniques, but ideally each horse needs to be compared with itself. Quantitative ultrasound is radiation free and is a well-tolerated technique for measuring bone properties in horses. This device allows bone speed of sound measurements at various sites using the axial transmission mode along the cortex and gives information about stiffness, architecture, porosity and bone mass.A combination of different non-invasive assessment techniques is recommended for the evaluation of bone biphasic modelling-remodelling activity and the mineral phase with its architecture. The potential clinical and research use of these techniques is considered.

Biochemical markers in the assessment of bone disease.

As the mean age of our population increases, increasing attention has been paid to the diseases associated with aging, including diseases of the skeleton such as osteoporosis. Effective means of treating and possibly preventing such skeletal disorders are emerging, making their early recognition an important goal for the primary care physician. Although bone density measurements and skeletal imaging studies remain of primary diagnostic importance in this regard, a large number of assays for biochemical markers of bone formation and resorption are being developed that promise to complement the densitometry measurements and imaging studies, providing an assessment of the rates of bone turnover and an earlier evaluation of the effects of therapy. In this review, emphasizing the recent literature, the major biochemical markers currently in use or under active investigation are described, and their application in a number of diseases of the skeleton including osteoporosis is evaluated.

Recent advances in the ultrastructural assessment of osteoclastic resorptive functions.

Ultrastructural enzyme and immunocytochemical studies have made great contributions to clarifying intriguing questions as to the actual role of osteoclastic ruffled borders in bone resorption. In the present study, vacuolar-type H(+)-ATPase and cysteine-proteinase (cathepsin) were localized in osteoclasts by means of light and electron microscopic immunocytochemistry. The specific immunoreactivity of vacuolar-type H(+)-ATPase was detected along the ruffled border membranes, associated pale vacuoles, and cisterns of the rough-surfaced endoplasmic reticulum of osteoclasts. Anti-cathepsin B immunoreaction occurred in Golgi vesicles, lysosomes, pale vesicles and vacuoles, and the extracellular canals of ruffled borders of osteoclasts. The resorbing bone surfaces were also immunoreactive for anti-cathepsin B. In a coculture system of osteoclasts with devitalized dentine slices, a specific H(+)-ATPase inhibitor (bafilomycin A1) markedly reduced both demineralized areas and resorption lacuna formation on the dentine slices. On the other hand, the cathepsin inhibitor, E-64, inhibited only resorption lacuna formation but had no effect on demineralization of the dentine slices. These results suggest that H(+)-ATPase and cathepsins in osteoclasts are involved, respectively, in the extracellular solubilization of apatite crystals and subsequent degradation of bone matrix and that the ruffled border-clear zone complex of osteoclasts is the main site of cell-matrix interactions during bone resorption processes.

Simultaneous assessment of bone collagen synthesis and degradation in patients with different malignant tumours. Comparison with the results of 99mTc-methylene bisphosphonate bone scintigraphy.

We report on the diagnostic validity of the serum concentrations of the C-terminal propeptide of type I procollagen (a marker of bone formation) and of the urinary excretion of deoxypyridinoline (a marker of bone resorption) in a consecutive series of 89 tumour patients who were routinely examined by 99mTc-methylene bisphosphonate bone scintigraphy for detection of bone metastases. Z score analysis reveals that the discriminating power of deoxypyridinoline is superior to that of calcium excretion whereas the discriminating power of the C-terminal propeptide concentrations is inferior to that of bone alkaline phosphatase values. Accuracy (as assessed by the area under the receiver-operating characteristic curve) was 0.75 for deoxypyridinoline and 0.82 for the C-terminal propeptide. Combination of both markers did not yield an increase of accuracy (0.82) compared with the determination of the C-terminal propeptide concentrations alone. There was a correlation (r = +0.398; p < 0.0001) between C-terminal propeptide concentrations and deoxypyridinoline excretion values in the group of 89 patients examined. Further studies should be done to elucidate whether the determination of bone collagen turnover is suitable as a screening procedure for detecting bone metastases.