Effect of treadmill gait on bone markers and bone mineral density of quadriplegic subjects

Quadriplegic subjects present extensive muscle mass paralysis which is responsible for the dramatic decrease in bone mass, increasing the risk of bone fractures. There has been much effort to find an efficient treatment to prevent or reverse this significant bone loss. We used 21 male subjects, mean age 31.95 ± 8.01 years, with chronic quadriplegia, between C4 and C8, to evaluate the effect of treadmill gait training using neuromuscular electrical stimulation, with 30-50% weight relief, on bone mass, comparing individual dual-energy X-ray absorptiometry responses and biochemical markers of bone metabolism. Subjects were divided into gait (N = 11) and control (N = 10) groups. The gait group underwent gait training for 6 months, twice a week, for 20 min, while the control group did not perform gait. Bone mineral density (BMD) of lumbar spine, femoral neck, trochanteric area, and total femur, and biochemical markers (osteocalcin, bone alkaline phosphatase, pyridinoline, and deoxypyridinoline) were measured at the beginning of the study and 6 months later. In the gait group, 81.8% of the subjects presented a significant increase in bone formation and 66.7% also presented a significant decrease of bone resorption markers, whereas 30% of the controls did not present any change in markers and 20% presented an increase in bone formation. Marker results did not always agree with BMD data. Indeed, many individuals with increased bone formation presented a decrease in BMD. Most individuals in the gait group presented an increase in bone formation markers and a decrease in bone resorption markers, suggesting that gait training, even with 30-50% body weight support, was efficient in improving the bone mass of chronic quadriplegics.

Effects of hyperprolactinemia and ovariectomy on the tibial epiphyseal growth plate and bone formation in mice

PURPOSE: To evaluate the effects of ovariectomy and the hyperprolactinemia procedure in the tibial epiphyseal growth plate of female mice.METHODS: In this study, the epiphyseal growth plate of ovariectomized (OVX) and/or rendered hyperprolactinemic female mice by 50 days of treatment with 200 μg metoclopramide (M) was evaluated morphologically, morphometrically and immuno-histochemically. Forty female and adult mice were divided into four groups according to treatment: V group - animals treated with saline solution; H group - hyperprolactinemic animals; Ovx/V group - ovariectomized animals and treated with saline solution; Ovx/H group - hyperprolactinemic and ovariectomized animals. After the treatment period, the animals were sacrificed, tibia was removed and fixed in 10% buffered formalin and decalcified in 10% formic acid. The material was immersed in paraffin and subjected to histological processing in paraffin. The sections were stained with Masson's trichrome and immunohistochemistry was carried out for the pro-apoptotic protein BCL-2. The images for the morphological and morphometric study were analyzed with the imaging program AxioVision 4.8 (Carl-Zeiss(r), Germany).RESULTS: The combination of hyperprolactinemia and the ovariectomy procedure decreased the number of resting chondrocytes 1.5-fold, the number of proliferative chondrocytes 1.8-fold; the percentage of resting cartilage 2.4-fold and the percentage of trabecular bone 2.1-fold, compared with respective control animals.CONCLUSION: The procedure of ovariectomy combined with the metoclopramide-induced hyperprolactinemia in female mice has showed marked bone degeneration due to significant decrease of cell proliferation in the epiphyseal growth plate and bone formation.

Contribution of matrix vesicles and alkaline phosphatase to ectopic bone formation

Endochondral calcification involves the participation of matrix vesicles (MVs), but it remains unclear whether calcification ectopically induced by implants of demineralized bone matrix also proceeds via MVs. Ectopic bone formation was induced by implanting rat demineralized diaphyseal bone matrix into the dorsal subcutaneous tissue of Wistar rats and was examined histologically and biochemically. Budding of MVs from chondrocytes was observed to serve as nucleation sites for mineralization during induced ectopic osteogenesis, presenting a diameter with Gaussian distribution with a median of 306 ± 103 nm. While the role of tissue-nonspecific alkaline phosphatase (TNAP) during mineralization involves hydrolysis of inorganic pyrophosphate (PPi), it is unclear how the microenvironment of MV may affect the ability of TNAP to hydrolyze the variety of substrates present at sites of mineralization. We show that the implants contain high levels of TNAP capable of hydrolyzing p-nitrophenylphosphate (pNPP), ATP and PPi. The catalytic properties of glycosyl phosphatidylinositol-anchored, polidocanol-solubilized and phosphatidylinositol-specific phospholipase C-released TNAP were compared using pNPP, ATP and PPi as substrates. While the enzymatic efficiency (k cat/Km) remained comparable between polidocanol-solubilized and membrane-bound TNAP for all three substrates, the k cat/Km for the phosphatidylinositol-specific phospholipase C-solubilized enzyme increased approximately 108-, 56-, and 556-fold for pNPP, ATP and PPi, respectively, compared to the membrane-bound enzyme. Our data are consistent with the involvement of MVs during ectopic calcification and also suggest that the location of TNAP on the membrane of MVs may play a role in determining substrate selectivity in this micro-compartment.

Building bone tissue: matrices and scaffolds in physiology and biotechnology

Deposition of bone in physiology involves timed secretion, deposition and removal of a complex array of extracellular matrix proteins which appear in a defined temporal and spatial sequence. Mineralization itself plays a role in dictating and spatially orienting the deposition of matrix. Many aspects of the physiological process are recapitulated in systems of autologous or xenogeneic transplantation of osteogenic precursor cells developed for tissue engineering or modeling. For example, deposition of bone sialoprotein, a member of the small integrin-binding ligand, N-linked glycoprotein family, represents the first step of bone formation in ectopic transplantation systems in vivo. The use of mineralized scaffolds for guiding bone tissue engineering has revealed unexpected manners in which the scaffold and cells interact with each other, so that a complex interplay of integration and disintegration of the scaffold ultimately results in efficient and desirable, although unpredictable, effects. Likewise, the manner in which biomaterial scaffolds are "resorbed" by osteoclasts in vitro and in vivo highlights more complex scenarios than predicted from knowledge of physiological bone resorption per se. Investigation of novel biomaterials for bone engineering represents an essential area for the design of tissue engineering strategies.

Association between indoxyl sulfate and bone histomorphometry in pre-dialysis chronic kidney disease patients

Introduction: Experimental studies have suggested that indoxyl sulfate (IS), a protein-bound uremic toxin, may be involved in the development of renal osteodystrophy. Objective: evaluate the association between IS levels and biochemical parameters related to mineral metabolism and bone histomorphometry in a cohort of pre-dialysis chronic kidney disease (CKD) patients. Methods: This is a post-hoc analysis of an observational study evaluating the association between coronary calcification and bone biopsy findings in 49 patients (age: 52 ± 10 years; 67% male; estimated glomerular filtration rate: 36 ± 17 ml/min). Serum levels of IS were measured. Results: Patients at CKD stages 2 and 3 presented remarkably low bone formation rate. Patients at CKD stages 4 and 5 presented significantly higher osteoid volume, osteoblast and osteoclast surface, bone fibrosis volume and bone formation rate and a lower mineralization lag time than CKD stage 2 and 3 patients. We observed a positive association between IS levels on one hand and the bone formation rate, osteoid volume, osteoblast surface and bone fibrosis volume on the other. Multivariate regression models confirmed that the associations between IS levels and osteoblast surface and bone fibrosis volume were both independent of demographic and biochemical characteristics of the study population. A similar trend was observed for the bone formation rate. Conclusion: Our findings demonstrated that IS is positively associated with bone formation rate in pre-dialysis CKD patients.

Congenital hyperthyroidism: autopsy report

We report the autopsy of a stillborn fetus with congenital hyperthyroidism born to a mother with untreated Graves' disease, whose cause of death was congestive heart failure. The major findings concerned the skull, thyroid, heart, and placenta. The cranial sutures were closed, with overlapping skull bones. The thyroid was increased in volume and had intense blood congestion. Histological examination showed hyperactive follicles. The heart was enlarged and softened, with dilated cavities and hemorrhagic suffusions in the epicardium. The placenta had infarctions that involved at least 20% of its surface, and the vessels of the umbilical cord were fully exposed due to a decrease in Wharton 's jelly. Hyperthyroidism was confirmed by the maternal clinical data, the fetal findings of exophthalmia, craniosynostosis, and goiter with signs of follicular hyperactivity. Craniosynostosis is caused by the anabolic action of thyroid hormones in bone formation during the initial stages of development. The delayed initiation of treatment in the present case contributed to the severity of fetal hyperthyroidism and consequent fetal death.

Insulin impairs the maturation of chondrocytes in vitro

The precise nature of hormones and growth factors directly responsible for cartilage maturation is still largely unclear. Since longitudinal bone growth occurs through endochondral bone formation, excess or deficiency of most hormones and growth factors strongly influences final adult height. The structure and composition of the cartilaginous extracellular matrix have a critical role in regulating the behavior of growth plate chondrocytes. Therefore, the maintenance of the three-dimensional cell-matrix interaction is necessary to study the influence of individual signaling molecules on chondrogenesis, cartilage maturation and calcification. To investigate the effects of insulin on both proliferation and induction of hypertrophy in chondrocytes in vitro we used high-density micromass cultures of chick embryonic limb mesenchymal cells. Culture medium was supplemented with 1% FCS + 60 ng/ml (0.01 µM) insulin and cultures were harvested at regular time points for later analysis. Proliferating cell nuclear antigen immunoreactivity was widely detected in insulin-treated cultures and persisted until day 21 and [³H]-thymidine uptake was highest on day 14. While apoptosis increased in control cultures as a function of culture time, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-labeled cells were markedly reduced in the presence of insulin. Type II collagen production, alkaline phosphatase activity and cell size were also lower in insulin-treated cultures. Our results indicate that under the influence of 60 ng/ml insulin, chick chondrocytes maintain their proliferative potential but do not become hypertrophic, suggesting that insulin can affect the regulation of chondrocyte maturation and hypertrophy, possibly through an antiapoptotic effect.

Body fat and cholecalciferol supplementation in elderly homebound individuals

Vitamin D deficiency, observed mainly in the geriatric population, is responsible for loss of bone mass and increased risk of bone fractures. Currently, recommended doses of cholecalciferol are advised, but since there are few studies evaluating the factors that influence the serum levels of 25-hydroxyvitamin D (25(OH)D) following supplementation, we analyzed the relationship between the increase in serum 25(OH)D after supplementation and body fat. We studied a group of 42 homebound elderly subjects over 65 years old (31 women) in order to assess whether there is a need for adjustment of the doses of cholecalciferol administered to this group according to their adipose mass. Baseline measurements of 25(OH)D, intact parathyroid hormone and bone remodeling markers (osteocalcin and carboxy-terminal fraction of type 1 collagen) were performed. Percent body fat was measured by dual-energy X-ray absorptiometry. The patients were divided into three groups according to their percent body fat index and were treated with cholecalciferol, 7,000 IU a week, for 12 weeks. The increases in serum levels of 25(OH)D were similar for all groups, averaging 7.46 ng/mL (P < 0.05). It is noteworthy that this increase only shifted these patients from the insufficiency category to hypovitaminosis. Peak levels of 25(OH)D were attained after only 6 weeks of treatment. This study demonstrated that adipose tissue mass does not influence the elevation of 25(OH)D levels following vitamin D supplementation, suggesting that there is no need to adjust vitamin D dose according to body fat in elderly homebound individuals.

Proteoliposomes as matrix vesicles' biomimetics to study the initiation of skeletal mineralization

During the process of endochondral bone formation, chondrocytes and osteoblasts mineralize their extracellular matrix by promoting the formation of hydroxyapatite (HA) seed crystals in the sheltered interior of membrane-limited matrix vesicles (MVs). Ion transporters control the availability of phosphate and calcium needed for HA deposition. The lipidic microenvironment in which MV-associated enzymes and transporters function plays a crucial physiological role and must be taken into account when attempting to elucidate their interplay during the initiation of biomineralization. In this short mini-review, we discuss the potential use of proteoliposome systems as chondrocyte- and osteoblast-derived MVs biomimetics, as a means of reconstituting a phospholipid microenvironment in a manner that recapitulates the native functional MV microenvironment. Such a system can be used to elucidate the interplay of MV enzymes during catalysis of biomineralization substrates and in modulating in vitro calcification. As such, the enzymatic defects associated with disease-causing mutations in MV enzymes could be studied in an artificial vesicular environment that better mimics their in vivo biological milieu. These artificial systems could also be used for the screening of small molecule compounds able to modulate the activity of MV enzymes for potential therapeutic uses. Such a nanovesicular system could also prove useful for the repair/treatment of craniofacial and other skeletal defects and to facilitate the mineralization of titanium-based tooth implants.

Experimental model of heterotopic ossification in Wistar rats

Heterotopic ossification (HO) is a metaplastic biological process in which there is newly formed bone in soft tissues adjacent to large joints, resulting in joint mobility deficit. In order to determine which treatment techniques are more appropriate for such condition, experimental models of induced heterotopic bone formation have been proposed using heterologous demineralized bone matrix implants and bone morphogenetic protein and other tissues. The objective of the present experimental study was to identify a reliable protocol to induce HO in Wistar rats, based on autologous bone marrow (BM) implantation, comparing 3 different BM volumes and based on literature evidence of this HO induction model in larger laboratory animals. Twelve male Wistar albino rats weighing 350/390 g were used. The animals were anesthetized for blood sampling before HO induction in order to quantify serum alkaline phosphatase (ALP). HO was induced by BM implantation in both quadriceps muscles of these animals, experimental group (EG). Thirty-five days after the induction, another blood sample was collected for ALP determination. The results showed a weight gain in the EG and no significant difference in ALP levels when comparing the periods before and after induction. Qualitative histological analysis confirmed the occurrence of heterotopic ossification in all 12 EG rats. In conclusion, the HO induction model was effective when 0.35 mL autologous BM was applied to the quadriceps of Wistar rats.

An animal experimental study of porous magnesium scaffold degradation and osteogenesis

Our objective was to observe the biodegradable and osteogenic properties of magnesium scaffolding under in vivo conditions. Twelve 6-month-old male New Zealand white rabbits were randomly divided into two groups. The chosen operation site was the femoral condyle on the right side. The experimental group was implanted with porous magnesium scaffolds, while the control group was implanted with hydroxyapatite scaffolds. X-ray and blood tests, which included serum magnesium, alanine aminotransferase (ALT), creatinine (CREA), and blood urea nitrogen (BUN) were performed serially at 1, 2, and 3 weeks, and 1, 2, and 3 months. All rabbits were killed 3 months postoperatively, and the heart, kidney, spleen, and liver were analyzed with hematoxylin and eosin (HE) staining. The bone samples were subjected to microcomputed tomography scanning (micro-CT) and hard tissue biopsy. SPSS 13.0 (USA) was used for data analysis, and values of P<0.05 were considered to be significant. Bubbles appeared in the X-ray of the experimental group after 2 weeks, whereas there was no gas in the control group. There were no statistical differences for the serum magnesium concentrations, ALT, BUN, and CREA between the two groups (P>0.05). All HE-stained slices were normal, which suggested good biocompatibility of the scaffold. Micro-CT showed that magnesium scaffolds degraded mainly from the outside to inside, and new bone was ingrown following the degradation of magnesium scaffolds. The hydroxyapatite scaffold was not degraded and had fewer osteoblasts scattered on its surface. There was a significant difference in the new bone formation and scaffold bioabsorption between the two groups (9.29±1.27 vs 1.40±0.49 and 7.80±0.50 vs 0.00±0.00 mm3, respectively; P<0.05). The magnesium scaffold performed well in degradation and osteogenesis, and is a promising material for orthopedics.

Serum 25-hydroxyvitamin D and biochemical markers of bone metabolism in patients with juvenile idiopathic arthritis

Our objective was to evaluate the concentrations of serum 25-hydroxyvitamin D [25(OH)D], serum calcium, serum phosphorus, alkaline phosphatase, and parathormone (PTH) in patients with polyarticular juvenile idiopathic arthritis (JIA) and to associate them with disease duration and activity, bone mineral density and use of medications. In a cross-sectional and controlled study, 30 patients with polyarticular JIA were evaluated and compared to 30 healthy individuals matched for age and gender. Clinical status, anthropometry, laboratory markers in both patients and controls, and bone mineral density, only in the patients, were measured. Of the 30 patients included in the study, 23 (76.7%) were female and 16 (53.3%) non-Caucasian; mean age was 14 years (range = 4 to 20 years). Mean disease duration was 5 years (range = 1 to 12 years). The mean concentrations of serum albumin-corrected calcium (9.04 ± 0.41 mg/dL) and alkaline phosphatase (153.3 ± 100.1 IU) were significantly lower in patients with JIA than in controls (P < 0.0001 and P = 0.001, respectively). No differences in 25(OH)D, PTH or serum phosphorus were observed between JIA and control subjects. Regarding 25(OH)D concentration, 8 patients (26.7%) and 5 controls (16.7%) had 25(OH)D concentrations compatible with deficiency (lower than 20 ng/mL) and 14 patients (46.7%) and 18 controls (60%) had concentrations compatible with insufficiency (20-32 ng/mL). These values were not associated with disease activity, use of medications or bone mineral density. We observed a high frequency of 25(OH)D insufficiency and deficiency in the study sample. The compromised bone metabolism emphasizes the importance of follow-up of JIA patients.

Bone marrow contribution to eosinophilic inflammation

Allergen-induced bone marrow responses are observable in human allergic asthmatics, involving specific increases in eosinophil-basophil progenitors (Eo/B-CFU), measured either by hemopoietic assays or by flow cytometric analyses of CD34-positive, IL-3Ralpha-positive, and/or IL-5-responsive cell populations. The results are consistent with the upregulation of an IL-5-sensitive population of progenitors in allergen-induced late phase asthmatic responses. Studies in vitro on the phenotype of developing eosinophils and basophils suggest that the early acquisition of IL-5Ralpha, as well as the capacity to produce cytokines such as GM-CSF and IL-5, are features of the differentiation process. These observations are consistent with findings in animal models, indicating that allergen-induced increases in bone marrow progenitor formation depend on hemopoietic factor(s) released post-allergen. The possibility that there is constitutive marrow upregulation of eosinophilopoiesis in allergic airways disease is also an area for future investigation.

Red blood cells derived from peripheral blood and bone marrow CD34+ human haematopoietic stem cells are permissive to Plasmodium parasites infection

The production of fully functional human red cells in vitro from haematopoietic stem cells (hHSCs) has been successfully achieved. Recently, the use of hHSCs from cord blood represented a major improvement to develop the continuous culture system for Plasmodium vivax. Here, we demonstrated that CD34+hHSCs from peripheral blood and bone marrow can be expanded and differentiated to reticulocytes using a novel stromal cell. Moreover, these reticulocytes and mature red blood cells express surface markers for entrance of malaria parasites contain adult haemoglobin and are also permissive to invasion by P. vivax and Plasmodium falciparum parasites.

Genetic diversity in soybean germplasm identified by SSR and EST-SSR markers

The objectives of this work were to investigate the genetic variation in 79 soybean (Glycine max) accessions from different regions of the world, to cluster the accessions based on their similarity, and to test the correlation between the two types of markers used. Simple sequence repeat markers present in genomic (SSR) and in expressed regions (EST-SSR) were used. Thirty SSR primer-pairs were selected (20 genomic and 10 EST-SSR) based on their distribution on the 20 genetic linkage groups of soybean, on their trinucleotide repetition unit and on their polymorphism information content. All analyzed loci were polymorphic, and 259 alleles were found. The number of alleles per locus varied from 2-21, with an average of 8.63. The accessions exhibit a significant number of rare alleles, with genotypes 19, 35, 63 and 65 carrying the greater number of exclusive alleles. Accessions 75 and 79 were the most similar and accessions 31 and 35, and 40 and 78, were the most divergent ones. A low correlation between SSR and EST-SSR data was observed, thus genomic and expressed microsatellite markers are required for an appropriate analysis of genetic diversity in soybean. The genetic diversity observed was high and allowed the formation of five groups and several subgroups. A moderate relationship between genetic divergence and geographic origin of accessions was observed.