Bone disease in children with homozygous β-thalassemia

The histological features of thalassemic bone are imperfectly known, and the roles of bone marrow hyperactivity, iron overload or vitamin D deficiency in the pathogenesis of the disease are not clearly identified. In this study we examined iliac crest biopsies from 17 transfusion-dependent children with homozygous β-thalassemia and severe radiological skeletal thalassemic changes, including widening of medullary spaces and osteoporosis. Rachitic lesions were not observed. Serum ferritin concentrations were increased in all but one subject. Iron deposits were histochemically detected in bone marrow, at the marrow-bone interface, along cement lines and mineralizing perimeters. Minor changes were present in trabecular bone, and osteomalacia was absent. By contrast, cortical bone exhibited severe changes including fissures and focal mineralization defects. Plasma 25-hydroxyvitamin D (25(OH)D) concentrations measured during the winter (December–May, 6.5 ± 4.9 ng/ml, mean ± SD, n = 6) and during the summer (June–November, 13.8 ± 8.4 ng/ml, n = 9) did not differ from those of age-matched children living in the same country. Seven patients had moderate hypocalcemia but no biological signs suggestive of vitamin D deficiency: all had normal alkaline phosphatase activity, normal or slightly elevated plasma phosphate, only two had low plasma 25(OH)D concentrations and two others supranormal values of plasma immunoreactive parathyroid hormone.These results show that iron overload and vitamin D deficiency do not seem to play an important role in the pathogenesis of thalassemic bone disease, which is characterized by cortical lesions probably related to marrow hyperactivity.     

Bone Mineral Density and Elemental Composition of Bone Tissues in “Red-Boned” Guishan Goats

Red-colored bones were first found in Guishan goats in the 1980s, and they were subsequently designated red-boned Guishan goats. However, the difference remains unclear between the bone mineral density (BMD) or elemental composition in bones between red-boned Guishan goats and common Guishan goats. Analysis of femoral bone samples by dual-energy X-ray absorptiometry and inductively coupled plasma optical emission spectrometry revealed an increase in bone mineral density in the femoral diaphysis and distal femur of red-boned Guishan goats at 18 and 36?months of age. The data revealed that BMD increased in both the red-boned and common Guishan goats from 18 to 36?months of age. The data also indicated that the ratio of the BMD values of red-boned to common Guishan goats was higher at 36?months of age than they were at 18?months of age. Furthermore, the levels of calcium, phosphorus, magnesium, barium, zinc, manganese, and aluminum were significantly higher in red-boned Guishan goats than common Guishan goats at 18 and 36?months of age. The results indicate that the red-boned Guishan goats were linked to the elevated levels of mineral salts observed in the bones and that this in turn may be linked to the elevated BMD levels encountered in red-boned Guishan goats. These reasons may be responsible for the red coloration in the bones of red-boned Guishan goats.     

Frontal Bone Insufficiency in Gsk3β Mutant Mice

The development of the mammalian skull is a complex process that requires multiple tissue interactions and a balance of growth and differentiation. Disrupting this balance can lead to changes in the shape and size of skull bones, which can have serious clinical implications. For example, insufficient ossification of the bony elements leads to enlarged anterior fontanelles and reduced mechanical protection of the brain. In this report, we find that loss of Gsk3β leads to a fully penetrant reduction of frontal bone size and subsequent enlarged frontal fontanelle. In the absence of Gsk3β the frontal bone primordium undergoes increased cell death and reduced proliferation with a concomitant increase in Fgfr2-IIIc and Twist1 expression. This leads to a smaller condensation and premature differentiation. This phenotype appears to be Wnt-independent and is not rescued by decreasing the genetic dose of β-catenin/Ctnnb1. Taken together, our work defines a novel role for Gsk3β in skull development.     

Status of Bone Allografting in Japan – Nation-Wide Survey of Bone Grafting Performed from 1995 through 1999

We report the status of bone allografting in Japan on the basis of the information obtained through questionnaires performed by the Japanese Orthopaedic Association (JOA). JOA performed a nation-wide survey in 2000, in order to clarify the current status of musculoskeletal tissue grafting in the orthopaedic practices in Japan. Conducted period was for 5 years from 1995 to 1999. As the results of this survey, it had been clarified that 92,984 bone graftings, which included autografts, allografts and synthetic bone substitutes, were performed during conducted 5 years. While the allografts were used only in 3,212 cases (3%), autograftings were performed in 64,193 cases (69%), synthetic bone substitutes were used in 25,576 cases (28%) in this series. The proportion of the number of operations for use bone substitutes increased every year, whereas that autografting decreased. The proportion of the number of allografting remained almost unaltered. Of the 706 institutions which answered to have experiences of tissue grafting, only 193 (27%) performed allograft.Since Kitasato University Hospital Bone Bank was developed in 1971, we have applied to clinical while doing basic research for preserved bone allograft. When extensive bone graft is required, allograft is very useful. In Japan, however, allograft is not performed widely. The foundation of regional bone banks is expected to resolve this problem. Since excision of bone preparations from cadaver donors is not common, bone allografts are not supplied sufficiently at present. It is needed to develop a network connecting bone banks in Japan. The enlightenment activities to the ordinary people and medical institutions will also be required.     

The Additive Effect of Mesenchymal Stem Cells and Bone Morphogenetic Protein 2 on γ-Irradiated Bone Marrow in Mice

Irradiation from γ-rays can cause severe damage to bone marrow and hematopoietic tissues. Presently, the most effective method available to treat severe hematopoietic injury is a bone marrow transplant (BMT). Allogeneic BMT is a difficult technique to perform due to the differences in human leukocyte antigen proteins between the donor and recipient, with acute graft-versus-host disease being a major complication of the technique. This limits the widespread applicability of allogeneic BMT. To develop a novel treatment for acute hematopoietic damage, we transplanted bone marrow derived mesenchymal stem cells (MSCs) into recipient mice and treated them with recombinant human bone morphogenetic protein 2 (rhBMP2) to investigate whether MSCs and rhBMP2 could additively promote the restoration of hematopoietic function. MSCs are vital components of the hematopoietic microenvironment that supports hematopoiesis, and bone morphogenic protein is a key factor in hematopoiesis. The 30-day survival rate as well as the numbers of nucleated cells, bone marrow colony-forming unit-granulocyte macrophages, spleen colony-forming units and peripheral blood cells were enumerated. The results showed that, after γ-irradiation and transplantation, MSCs and rhBMP2 additively promoted and improved hematopoietic restoration and function in vivo and in vitro. This additive effect of MSCs and rhBMP2 may one day provide a novel means of treating acute hematopoietic damage.     

Does Baseline PTH Influence Recovery of Bone Mineral Density,Trabecular Bone Score and Bone Turnover Markers? A Prospective Study Following Curative PArathyroidectomy in Primary Hyperparathyroidism

Objective: This prospective study was carried out to assess trabecular bone score, bone mineral density (BMD), and bone biochemistry in Indian subjects with symptomatic primary hyperparathyroidism (PHPT), and to study the influence of baseline parathyroid hormone (PTH) on recovery of these parameters following curative surgery.Methods: This was a 2-year prospective study conducted at a tertiary care centre in southern India. Baseline assessment included demographic details, mode of presentation, bone mineral biochemistry, BMD, trabecular bone score (TBS), and bone turnover markers (BTMs). These parameters were reassessed at the end of the first and second years following curative parathyroid surgery.Results: Fifty-one subjects (32 men and 19 women) with PHPT who had undergone curative parathyroidectomy were included in this study. The mean (SD) age was 44.6 (13.7) years. The TBS, BTMs, and BMD at lumbar spine and forearm were significantly worse at baseline in subjects with higher baseline PTH (≥250 pg/mL) when compared to the group with lower baseline PTH (<250 pg/mL). At the end of 2 years, the difference between high versus low PTH groups (mean ± SD) persisted only for forearm BMD (0.638 ± 0.093 versus 0.698 ± 0.041 g/cm²; P =.01). However, on follow-up visits in the first and second year after curative parathyroidectomy, there was no significant difference in BTMs, BMD at the femoral neck, lumbar spine, and TBS between the 2 groups stratified by baseline PTH.Conclusion: The BMD at the forearm remained significantly worse in individuals with high baseline PTH even at 2 years after surgery, while other parameters including TBS improved significantly from baseline.Abbreviations: 25(OH)D = 25-hydroxyvitamin D; BMD = bone mineral density; BMI = body mass index; BTMs = Bone turnover markers; CTX = C-terminal telopeptide of type 1 collagen; DXA = dual energy X-ray absorptiometry; P1NP = N-terminal propeptide of type 1 procollagen; PHPT = primary hyperparathyroidism; PTH = parathyroid hormone; TBS = trabecular bone score     

Bone mass increase in puberty: what makes it happen?

It is now thought that the critical property of bone is strength rather than weight, and that control of bone strength is mainly exercised through the effect of the mechanical loads brought to bear on bone. Muscle contraction places the greatest physiological load on bone, and so the strength of bone must be adapted to muscle strength (the functional muscle-bone unit). The Utah paradigm of skeletal physiology [J Hum Biol 1998;10:599-605] provides a model of bone development that describes how bone structure is regulated by local mechanical effects that can be adjusted by the effects of hormones. The DONALD (Dortmund Nutritional and Anthropometric Longitudinally Designed) study analysed the interaction between the muscle and bone systems in males and females before and during puberty. This study found that differences between the genders in bone adaptation during puberty are at least partly driven by the influence of oestrogen in females. Testosterone seems to have no direct relevant effect on bone during puberty, but may be implicated in the greater amount of muscle mass achieved in boys compared with girls.     

Hypoxia and TGF-β Drive Breast Cancer Bone Metastases through Parallel Signaling Pathways in Tumor Cells and the Bone Microenvironment

Background

Most patients with advanced breast cancer develop bone metastases, which cause pain, hypercalcemia, fractures, nerve compression and paralysis. Chemotherapy causes further bone loss, and bone-specific treatments are only palliative. Multiple tumor-secreted factors act on the bone microenvironment to drive a feed-forward cycle of tumor growth. Effective treatment requires inhibiting upstream regulators of groups of prometastatic factors. Two central regulators are hypoxia and transforming growth factor (TGF)- β. We asked whether hypoxia (via HIF-1α) and TGF-β signaling promote bone metastases independently or synergistically, and we tested molecular versus pharmacological inhibition strategies in an animal model.

Methodology/Principal Findings

We analyzed interactions between HIF-1α and TGF-β pathways in MDA-MB-231 breast cancer cells. Only vascular endothelial growth factor (VEGF) and the CXC chemokine receptor 4 (CXCR4), of 16 genes tested, were additively increased by both TGF-β and hypoxia, with effects on the proximal promoters. We inhibited HIF-1α and TGF-β pathways in tumor cells by shRNA and dominant negative receptor approaches. Inhibition of either pathway decreased bone metastasis, with no further effect of double blockade. We tested pharmacologic inhibitors of the pathways, which target both the tumor and the bone microenvironment. Unlike molecular blockade, combined drug treatment decreased bone metastases more than either alone, with effects on bone to decrease osteoclastic bone resorption and increase osteoblast activity, in addition to actions on tumor cells.

Conclusions/Significance

Hypoxia and TGF-β signaling in parallel drive tumor bone metastases and regulate a common set of tumor genes. In contrast, small molecule inhibitors, by acting on both tumor cells and the bone microenvironment, additively decrease tumor burden, while improving skeletal quality. Our studies suggest that inhibitors of HIF-1α and TGF-β may improve treatment of bone metastases and increase survival.     

Doxorubicin-Mediated Bone Loss in Breast Cancer Bone Metastases Is Driven by an Interplay between Oxidative Stress and Induction of TGFβ

Breast cancer patients, who are already at increased risk of developing bone metastases and osteolytic bone damage, are often treated with doxorubicin. Unfortunately, doxorubicin has been reported to induce damage to bone. Moreover, we have previously reported that doxorubicin treatment increases circulating levels of TGFβ in murine pre-clinical models. TGFβ has been implicated in promoting osteolytic bone damage, a consequence of increased osteoclast-mediated resorption and suppression of osteoblast differentiation. Therefore, we hypothesized that in a preclinical breast cancer bone metastasis model, administration of doxorubicin would accelerate bone loss in a TGFβ-mediated manner. Administration of doxorubicin to 4T1 tumor-bearing mice produced an eightfold increase in osteolytic lesion areas compared untreated tumor-bearing mice (P = 0.002) and an almost 50% decrease in trabecular bone volume expressed in BV/TV (P = 0.0005), both of which were rescued by anti-TGFβ antibody (1D11). Doxorubicin, which is a known inducer of oxidative stress, decreased osteoblast survival and differentiation, which was rescued by N-acetyl cysteine (NAC). Furthermore, doxorubicin treatment decreased Cu-ZnSOD (SOD1) expression and enzyme activity in vitro, and treatment with anti-TGFβ antibody was able to rescue both. In conclusion, a combination therapy using doxorubicin and anti-TGFβ antibody might be beneficial for preventing therapy-related bone loss in cancer patients.     

Bone mineral density and physical activity in 50–60-year-old women

The bone mineral density (BMD) of the calcaneus was measured utilizing a single energy photon absorption method in 108 women, aged 50–60 years. The women who participated in vigorous exercise two or more times a week or whose total physical activity amounted to 4 h a week had significantly higher BMD values than those who exercised less than two times a week or did less than 4 h physical activity a week. The physically active women also showed higher values for leg extension force and maximal oxygen uptake. BMD and leg extension force were positively correlated, whereas correlations between BMD and body mass, and the width of the calcaneus were negative. When other life-style variables were taken into account, such as smoking and drinking, a significant difference in BMD was found between physically active and sedentary women, but not between the smokers and non-smokers, or the drinkers and non-drinkers.     

Bone marrow–derived mesenchymal stem cells ameliorate parotid injury in ovariectomized rats

Background aims

Parotid hypofunction causes life-disrupting effects, and there are no effective medications for xerostomia. We hypothesized that mesenchymal stem cells (MSCs) have repairing effects on parotid glands of ovariectomized (OVX) rats.

A Highly Temperature-sensitive Proton Current in Mouse Bone Marrow–derived Mast Cells

Proton (H⁺) conductive pathways are suggested to play roles in the regulation of intracellular pH. We characterized temperature-sensitive whole cell currents in mouse bone marrow–derived mast cells (BMMC), immature proliferating mast cells generated by in vitro culture. Heating from 24 to 36°C reversibly and repeatedly activated a voltage-dependent outward conductance with Q₁₀ of 9.9 ± 3.1 (mean ± SD) (n = 6). Either a decrease in intracellular pH or an increase in extracellular pH enhanced the amplitude and shifted the activation voltage to more negative potentials. With acidic intracellular solutions (pH 5.5), the outward current was detected in some cells at 24°C and Q₁₀ was 6.0 ± 2.6 (n = 9). The reversal potential was unaffected by changes in concentrations of major ionic constituents (K⁺, Cl⁻, and Na⁺), but depended on the pH gradient, suggesting that H⁺ (equivalents) is a major ion species carrying the current. The H⁺ current was featured by slow activation kinetics upon membrane depolarization, and the activation time course was accelerated by increases in depolarization, elevating temperature and extracellular alkalization. The current was recorded even when ATP was removed from the intracellular solution, but the mean amplitude was smaller than that in the presence of ATP. The H⁺ current was reversibly inhibited by Zn²⁺ but not by bafilomycin A₁, an inhibitor for a vacuolar type H⁺-ATPase. Macroscopic measurements of pH using a fluorescent dye (BCECF) revealed that a rapid recovery of intracellular pH from acid-load was attenuated by lowering temperature, addition of Zn²⁺, and depletion of extracellular K⁺, but not by bafilomycin A₁. These results suggest that the H⁺ conductive pathway contributes to intracellular pH homeostasis of BMMC and that the high activation energy may be involved in enhancement of the H⁺ conductance.     

Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans–Induced Bone Resorption in a Rat Model for Periodontal Disease

Aggregatibacter actinomycetemcomitans a causative agent of periodontal disease in humans, forms biofilm on biotic and abiotic surfaces. A. actinomycetemcomitans biofilm is heterogeneous in nature and is composed of proteins, extracellular DNA and exopolysaccharide. To explore the role played by the exopolysaccharide in the colonization and disease progression, we employed genetic reduction approach using our rat model of A. actinomycetemcomitans-induced periodontitis. To this end, a genetically modified strain of A. actinomycetemcomitans lacking the pga operon was compared with the wild-type strain in the rat infection model. The parent and mutant strains were primarily evaluated for bone resorption and disease. Our study showed that colonization, bone resorption/disease and antibody response were all elevated in the wild-type fed rats. The bone resorption/disease caused by the pga mutant strain, lacking the exopolysaccharide, was significantly less (P < 0.05) than the bone resorption/disease caused by the wild-type strain. Further analysis of the expression levels of selected virulence genes through RT-PCR showed that the decrease in colonization, bone resorption and antibody titer in the absence of the exopolysaccharide might be due to attenuated levels of colonization genes, flp-1, apiA and aae in the mutant strain. This study demonstrates that the effect exerted by the exopolysaccharide in A. actinomycetemcomitans-induced bone resorption has hitherto not been recognized and underscores the role played by the exopolysaccharide in A. actinomycetemcomitans-induced disease.     

TNF-α Regulates the Effects of Irradiation in the Mouse Bone Marrow Microenvironment

Background

Secondary bone marrow (BM) myelodysplastic syndromes (MDS) are increasingly common, as a result of radio or chemotherapy administered to a majority of cancer patients. Patients with secondary MDS have increased BM cell apoptosis, which results in BM dysfunction (cytopenias), and an increased risk of developing fatal acute leukemias. In the present study we asked whether TNF-α, known to regulate cell apoptosis, could modulate the onset of secondary MDS.

Principal Findings

We show that TNF-α is induced by irradiation and regulates BM cells apoptosis in vitro and in vivo. In contrast to irradiated wild type (WT) mice, TNF-α deficient (TNF-α KO) mice or WT mice treated with a TNF-α-neutralizing antibody were partially protected from the apoptotic effects of irradiation. Next we established a 3-cycle irradiation protocol, in which mice were sub-lethally irradiated once monthly over a 3 month period. In this model, irradiated WT mice presented loss of microsatellite markers on BM cells, low white blood cell (WBC) counts, reduced megakaryocyte (MK) and platelet levels (thrombocytopenia) and macrocytic anemia, phenoypes that suggest the irradiation protocol resulted in BM dysfunction with clinical features of MDS. In contrast, TNF-α KO mice were protected from the irradiation effects: BM cell apoptosis following irradiation was significantly reduced, concomitant with sustained BM MK numbers and absence of other cytopenias. Moreover, irradiated WT mice with long term (≥5 months) BM dysfunction had increased BM angiogenesis, MMPs and VEGF and NFkB p65, suggestive of disease progression.

Conclusion

Taken together, our data shows that TNF-α induction following irradiation modulates BM cell apoptosis and is a crucial event in BM dysfunction, secondary MDS onset and progression.     

TGF-β-Neutralizing Antibody 1D11 Enhances Cytarabine-Induced Apoptosis in AML Cells in the Bone Marrow Microenvironment

Hypoxia and interactions with bone marrow (BM) stromal cells have emerged as essential components of the leukemic BM microenvironment in promoting leukemia cell survival and chemoresistance. High levels of transforming growth factor beta 1 (TGFβ1) produced by BM stromal cells in the BM niche regulate cell proliferation, survival, and apoptosis, depending on the cellular context. Exogenous TGFβ1 induced accumulation of acute myeloid leukemia (AML) cells in a quiescent G₀ state, which was further facilitated by the co-culture with BM-derived mesenchymal stem cells (MSCs). In turn, TGFβ-neutralizing antibody 1D11 abrogated rhTGFβ1 induced cell cycle arrest. Blocking TGFβ with 1D11 further enhanced cytarabine (Ara-C)–induced apoptosis of AML cells in hypoxic and in normoxic conditions. Additional constituents of BM niche, the stroma-secreted chemokine CXCL12 and its receptor CXCR4 play crucial roles in cell migration and stroma/leukemia cell interactions. Treatment with 1D11 combined with CXCR4 antagonist plerixafor and Ara-C decreased leukemia burden and prolonged survival in an in vivo leukemia model. These results indicate that blockade of TGFβ by 1D11 and abrogation of CXCL12/CXCR4 signaling may enhance the efficacy of chemotherapy against AML cells in the hypoxic BM microenvironment.     

Triple Point-Mutants of Hypoxia-Inducible Factor-1α Accelerate In Vivo Angiogenesis in Bone Defect Regions

To investigate the functions of triple point-mutants of hypoxia-inducible factor 1α (HIF1α) in angiogenesis in bone defect regions under normoxic conditions. 1. Triple point-mutations (in amino acids 402, 564, and 803) in the HIF1α coding sequence (CDS) were induced by polymerase chain reaction. The triple mutant HIF1α (402/564/803) was inserted into the adenovirus pAdEasy-1 system for complete viral packaging and titer measurements. 2. For the in vitro experiment, rabbit bone marrow mesenchymal stem cells (MSCs) were divided into four experimental groups. The efficiency of infection was observed by the expression of human renilla reniformis green fluorescent protein (hrGFP). The HIF1α mRNA, protein and VEGF protein expression levels in infected cells in each experimental group were measured. 3. As in the in vivo experiment, the MSCs were divided into four groups and infected with the viral solutions from each complementary in vitro group and cultured under normoxic conditions. The MSCs were used as seed cells and transplanted into an apatite–wollastonite magnetic bioactive glass–ceramic (AW MGC) vector to construct artificial tissue-engineering scaffolds that were then implanted into the in vivo rabbit radial bone defect model. The animals from each group were killed 8 weeks after the surgery, and the tissues from the implantation region were harvested for the evaluation of the angiogenesis. 1. The 402,564, and 803 amino acids in CDS area were point mutated into alanine; three types of recombinant adenovirus were successfully constructed, packaged, and characterized. 2. The expression levels of HIF1α mRNA in A and B groups were significantly higher than those in the C and D groups (P < 0.05). The HIF1α and VEGF protein expression levels in A group were significantly higher than those in the other three groups (P < 0.05). 3. There was prominent angiogenesis in bone defect regions in group A animals. 1. Triple point-mutants of HIF1α efficiently expressed functional proteins under normoxic conditions. 2. Triple point-mutants HIF1α effectively promoted in vivo angiogenesis in bone defect regions.