The roles of Orai and Stim in bone health and disease

Orai and Stim proteins are the mediators of calcium release-activated calcium signaling and are important in the regulation of bone homeostasis and disease. This includes separate regulatory systems controlling mesenchymal stem cell differentiation to form osteoblasts, which make bone, and differentiation and regulation of osteoclasts, which resorb bone. These systems will be described separately, and their integration and relation to other systems, including Orai and Stim in teeth, will be briefly discussed at the end of this review.     

Regulatory T cells as a potent target for controlling bone loss

Metabolic bone diseases, such as rheumatoid arthritis (RA) and osteoporosis, affect hundreds and millions of people worldwide leading causes of long-term pain and disability. Effective clinical treatment for bone destruction in bone diseases is lacking because the knowledge about molecular mechanisms leading to bone destruction are incompletely understood. Recently, it has been confirmed that regulatory T cells (Tregs) play a crucial role in suppressing the immune response in the pathogenesis of various autoimmune diseases. In vitro, Tregs directly inhibit osteoclasts and differentiation and function. In mice, the injection of Tregs into the TNF transgenic results in enhanced systemic bone density. In addition, it has been shown that increase of Tregs numbers by overexpressing the FoxP3 is effective in the prevention of local and systemic bone destruction. In vivo treatment with anti-CD28 superagonist antibody leading to a stronger increase in Tregs numbers protect against TNF-a-induced bone loss in TNF-transgenic mice. In agreement, Tregs can control ovariectomy-induced bone loss in FoxP3-transgenic mice. In this paper, we will briefly discuss the biological features of Tregs and summarize recent advances on the role of Tregs in the pathogenesis and treatment of bone loss in metabolic bone diseases.     

Zinc supplements and bone health: The role of the RANKL-RANK axis as a therapeutic target

BackgroundTo this day, empirical data suggests that zinc has important roles in matrix synthesis, bone turnover, and mineralization and its beneficial effects on bone could be mediated through different mechanisms. The influence of zinc on bone turnover could be facilitated via regulating RANKL/RANK/OPG pathway in bone tissue. Therefore, the aim of the study was to conduct a review to investigate the possible effect of the zinc mediated bone remodeling via RANKL/RANK/OPG pathway.MethodsA comprehensive systematic search was performed in MEDLINE/PubMed, Cochrane Library, SCOPUS, and Google Scholar to explore the studies investigating the effect of zinc as a bone remodeling factor via RANKL/RANK/OPG pathway regulation. Subsequently, the details of the pathway and the impact of zinc supplements on RANKL/RANK/OPG pathway regulation were discussed.ResultsThe pathway could play an important role in bone remodeling and any imbalance between RANKL/RANK/OPG components could lead to extreme bone resorption. Although the outcomes of some studies are equivocal, it is evident that zinc possesses protective properties against bone loss by regulating the RANKL/RANK/OPG pathway. There are several experiments where zinc supplementation resulted in upregulation of OPG expression or decreases RANKL level. However, the results of some studies oppose this.ConclusionIt is likely that sufficient zinc intake will elicit positive effects on bone health by RANKL/RANK/OPG regulation. Although the outcomes of a few studies are equivocal, it seems that zinc can exert the protective properties against bone loss by suppressing osteoclastogenesis via downregulation of RANKL/RANK. Additionally, there are several experiments where zinc supplementation resulted in upregulation of OPG expression. However, the results of limited studies oppose this. Therefore, aside from the positive role zinc possesses in preserving bone mass, further effects of zinc in RANKL/RANK/OPG system requires further animal/human studies.     

Female reproductive system and bone

The female reproductive system plays a major role in regulating the acquisition and loss of bone by the skeleton from menarche through senescence. Onset of gonadal sex steroid secretion at puberty is the major factor responsible for skeletal longitudinal and radial growth, as well as significant gain in bone density, until peak bone density is achieved in third decade of life. Gonadal sex steroids then help maintain peak bone density until menopause, including during the transient changes in skeletal mineral content associated with pregnancy and lactation. At menopause, decreased gonadal sex steroid production normally leads to rapid bone loss. The most rapid bone loss associated with decreased estrogen levels occurs in the first 8-10 years after menopause, with slower age-related bone loss occurring during later life. Age-related bone loss in women after the early menopausal phase of bone loss is caused by ongoing gonadal sex steroid deficiency, vitamin D deficiency, and secondary hyperparathyroidism. Other factors also contribute to age-related bone loss, including intrinsic defects in osteoblast function, impairment of the GH/IGF axis, reduced peak bone mass, age-associated sarcopenia, and various sporadic secondary causes. Further understanding of the relative contributions of the female reproductive system and each of the other factors to development and maintenance of the female skeleton, bone loss, and fracture risk will lead to improved approaches for prevention and treatment of osteoporosis.     

Sex hormones and bone health in males

Sex steroids play a key role in maintaining skeletal integrity lifelong, through a complex variety of endocrine, but also paracrine and possibly autocrine actions. The current knowledge that androgens may act as pro-hormones for estrogens has seriously challenged many traditional views, so that, at least for their skeletal actions, these can no longer be considered exclusively “male” or “female” hormones.     

Further evidence for direct pro-resorptive actions of FSH

We confirm that FSH stimulates osteoclast formation, function and survival to enhance bone resorption. It does so via the activation of a pertussis toxin-sensitive G_i-coupled FSH receptor that we and others have identified on murine and human osteoclast precursors and mature osteoclasts. FSH additionally enhances the production of several osteoclastogenic cytokines, importantly TNFα, likely within the bone marrow microenvironment, to augment its pro-resorptive action. FSH levels in humans rise before estrogen falls, and this hormonal change coincides with the most rapid rates of bone loss. On the basis of accumulating evidence, we reaffirm that FSH contributes to the rapid peri-menopausal and early post-menopausal bone loss, which might thus be amenable to FSH blockade.     

Piecewise Whittle estimator for trabecular bone radiograph characterization

Osteoporosis is a disease in which low bone mass and microarchitectural deterioration of bone tissue lead to increased bone fragility and a consequent increase in fracture risk. The objective of this paper is to develop and validate a new method to assess bone microarchitecture on radiographs. Taking into account the piecewise fractal nature of bone radiograph images, an appropriate fractal model (piecewise fractional Brownian motion) is used to characterize the trabecular bone network. Based on the Whittle estimator, a new method for calculating the Hurst exponent H is developed to better consider the piecewise fractal nature of the data. Different estimators are used and compared to the proposed method to discriminate two populations composed of healthy controls and osteoporotic patients. Our findings demonstrate that the new estimator proposed here provides effective results in terms of discrimination of the subjects and is better adapted to bone radiograph image analysis.     

Oral administration of phytocomponent p-hydroxycinnamic acid prevents bone loss in ovariectomized rats

The preventive effect of phytocomponent p-hydroxycinnamic acid (HCA) on ovariectomy (OVX)-induced bone loss was investigated. HCA (250 or 500 μg/100 g body weight) was orally administered once daily for 30 days to OVX rats. The analysis using a peripheral quantitative computed tomography (pQCT) showed that OVX caused bone loss in the femoral-metaphyseal tissues. This change was significantly restored after the administration of HCA (250 or 500 μg/100 g body weight) to OVX rats. Mineral content, mineral density, and polar strength strain index in the femoral-metaphyseal tissues were significantly decreased in OVX rats. These decreases were significantly restored after the administration of HCA (500 μg/100 g) to OVX rats. Moreover, OVX caused a significant decrease in calcium content or alkaline phosphatase activity in the femoral-diaphyseal and -metaphyseal tissues. These decreases were significantly restored after the administration of HCA (250 or 500 μg/100 g) to OVX rats. Deoxyribonucleic acid (DNA) content in the diaphyseal or metaphyseal tissues was significantly increased in OVX rats. These increases were significantly restored after oral administration of HCA (500 μg/100 g). This study demonstrates that HCA has preventive effects on OVX-induced bone loss of rats in vivo.     

Stress to endoplasmic reticulum of mouse osteoblasts induces apoptosis and transcriptional activation for bone remodeling

ATF4 is an essential regulator in osteogenesis as well as in stress responses to the endoplasmic reticulum (ER). We addressed a question: Does ER stress to osteoblasts upregulate ATF4 expression? If so, do they exhibit ATF4-mediated bone remodeling or apoptosis? ER stress, induced by Thapsigargin and tunicamycin, elevated a phosphorylated form of eIF2alpha and ATF4, but the cellular fate depended on treatment duration. The treatment for 1h, for instance, activated Runx2, and type I collagen, while the treatment for 24h induced apoptosis. Our observations suggest that there is a threshold for ER stress and osteoblasts present a bi-phasic pattern of their fate.     

A supra-cellular model for coupling of bone resorption to formation during remodeling: lessons from two bone resorption inhibitors affecting bone formation differently

The bone matrix is maintained functional through the combined action of bone resorbing osteoclasts and bone forming osteoblasts, in so-called bone remodeling units. The coupling of these two activities is critical for securing bone replenishment and involves osteogenic factors released by the osteoclasts. However, the osteoclasts are separated from the mature bone forming osteoblasts in time and space. Therefore the target cell of these osteoclastic factors has remained unknown. Recent explorations of the physical microenvironment of osteoclasts revealed a cell layer lining the bone marrow and forming a canopy over the whole remodeling surface, spanning from the osteoclasts to the bone forming osteoblasts. Several observations show that these canopy cells are a source of osteoblast progenitors, and we hypothesized therefore that they are the likely cells targeted by the osteogenic factors of the osteoclasts. Here we provide evidence supporting this hypothesis, by comparing the osteoclast-canopy interface in response to two types of bone resorption inhibitors in rabbit lumbar vertebrae. The bisphosphonate alendronate, an inhibitor leading to low bone formation levels, reduces the extent of canopy coverage above osteoclasts. This effect is in accordance with its toxic action on periosteoclastic cells. In contrast, odanacatib, an inhibitor preserving bone formation, increases the extent of the osteoclast-canopy interface. Interestingly, these distinct effects correlate with how fast bone formation follows resorption during these respective treatments. Furthermore, canopy cells exhibit uPARAP/Endo180, a receptor able to bind the collagen made available by osteoclasts, and reported to mediate osteoblast recruitment. Overall these observations support a mechanism where the recruitment of bone forming osteoblasts from the canopy is induced by osteoclastic factors, thereby favoring initiation of bone formation. They lead to a model where the osteoclast-canopy interface is the physical site where coupling of bone resorption to bone formation occurs.     

T cell-mediated increased osteoclast formation from peripheral blood as a mechanism for Crohn's disease-associated bone loss

The pathophysiology of osteoporosis in patients with Crohn's disease (CD) is still not completely elucidated. In this study, we evaluated osteoclastogenesis from peripheral blood cells of CD patients and studied the role of lymphocytes and inflammatory cytokines in this process. Peripheral blood mononuclear cells from seven patients with quiescent CD and matched healthy controls were isolated, and separated into T cells, B cells, and a T- and B-cell depleted fraction. In various culture combinations, osteoclast formation in the absence of the osteoclastogenic factors RANKL and M-CSF was assessed by scoring the number of tartrate-resistant acid phosphatase (TRACP) positive multinucleated cells (MNCs). Cytokine levels in culture supernatants were measured. Formation of heterogeneous cell clusters in culture was noticed; a process that was inhibited by anti-LFA-1. In CD cultures, mean cluster area was up to threefold higher than in control cultures, and shown to be induced by T cells. Over tenfold higher numbers of TRACP(+) MNCs were found in CD cultures, but exclusively in cultures containing T cells. Formation of cell clusters correlated strongly with formation of TRACP(+) MNCs. Both cell cluster formation and osteoclast formation were related to IL-17 levels in vitro. In conclusion, osteoclastogenesis, preceded by cell cluster formation, is T cell-mediated and increased in patients with quiescent CD. Our findings suggest heterotypic interactions between osteoclast precursors and T cells to be a triggering step in osteoclast formation in CD. Furthermore, our results propose a possible role for IL-17 in osteoclastogenesis in CD patients, and as such in CD-associated bone loss.     

A sensitive stain for aluminum in undecalcified cancellous bone

Aluminum is known to accumulate with age in bone and other tissues of humans, even in the absence of renal disease. Our study aimed to develop a histological staining method sufficiently sensitive to detect aluminum in plastic sections of undecalcified bone biopsies from healthy volunteers as well as from patients with renal and non-renal bone diseases. We used quantitative histomorphometry to measure the percentage of trabecular surface stained by aluminum and found that our new method was approximately 50% more sensitive for detecting aluminum than the Acid Solochrome Azurine (ASA) method which in turn was significantly more sensitive than the Aluminon method. Aluminon is widely used in pathology laboratories for diagnostic purposes despite concerns in the literature about Aluminon's limited sensitivity for aluminum. Our histomorphometric results showed that the newly developed method stained approximately 10% of the trabecular surface in bone sections from healthy controls, 38% from renal patients, 26% from patients with vitamin D deficiency, and 29% from patients with osteoporosis. Histomorphometric measurements of aluminum-stained trabecular surfaces in sections stained with ASA were consistent with those obtained in Walton-stained sections but proportionately lower. Moreover, the Walton and ASA methods stained aluminum at similar locations in adjacent bone sections. As the ASA and Walton methods are considerably more sensitive for bone aluminum than the Aluminon method, we recommend that either of them should be used in place of the Aluminon method for routine diagnostic purposes.     

Human demineralised bone matrix as a bone substitute for reconstruction of cystic defects of the lower jaw

Ina retrospective study validated by a standardized clinical and radiologicalexamination, the bone regeneration in 90 patients with cystic mandibulardefectswas examined. In 50 patients bony defect reconstructions with humandemineralised bone matrix (HDBM) were carried out, while in a comparable groupof 40 patients the hollow pockets were left to regenerate bone spontaneously.The bone regeneration after the implantation of human demineralised bone matrix(HDBM) was subjected to a comparative validation. Osteoinductive proteinspresent in HDBM (bone morphogenetic proteins) can diffuse into the implant seatand induce new bone formation (osteoinduction). A markedly faster and morethorough bone regeneration was demonstrated after the surgical therapy ofcysticmandibular lesions with HDBM than without. HDBM also proved to be exceptionallybiocompatible.     

Encapsulated boron as an osteoinductive agent for bone scaffolds

The aim of this study was to develop boron (B)-releasing polymeric scaffold to promote regeneration of bone tissue. Boric acid-doped chitosan nanoparticles with a diameter of approx. 175 nm were produced by tripolyphosphate (TPP)-initiated ionic gelation process. The nanoparticles strongly attached via electrostatic interactions into chitosan scaffolds produced by freeze-drying with approx. 100 μm pore diameter. According to the ICP-OES results, following first 5 h initial burst release, fast release of B from scaffolds was observed for 24 h incubation period in conditioned medium. Then, slow release of B was performed over 120 h. The results of the cell culture studies proved that the encapsulated boron within the scaffolds can be used as an osteoinductive agent by showing its positive effects on the proliferation and differentiation of MC3T3-E1 preosteoblastic cells.     

Opportunities for biomineralization research using multiscale computed X-ray tomography as exemplified by bone imaging

Biominerals typically have complex hierarchical structures traversing many length scales. This makes their structural characterization complicated, since it requires 3D techniques that can probe full specimens at down to nanometer-resolution, a combination that is difficult – if not impossible – to achieve simultaneously. One challenging example is bone, a mineralized tissue with a highly complex architecture that is replete with a network of cells. X-ray computed tomography techniques enable multiscale structural characterization through the combination of various equipment and emerge as promising tools for characterizing biominerals. Using bone as an example, we discuss how combining different X-ray imaging instruments allow characterizing bone structures from the nano- to the organ-scale. In particular, we compare and contrast human and rodent bone, emphasize the importance of the osteocyte lacuno-canalicular network in bone, and finally illustrate how combining synchrotron X-ray imaging with laboratory instrumentation for computed tomography is especially helpful for multiscale characterization of biominerals.     

Dynamic culture improves MSC adhesion on freeze-dried bone as a scaffold for bone engineering

AIM:To investigate the interaction between mesenchymal stem cells(MSCs) and bone grafts using two different cultivation methods:static and dynamic.METHODS:MSCs were isolated from rat bone marrow.MSC culture was analyzed according to the morphology,cell differentiation potential,and surface molecular markers.Before cell culture,freeze-dried bone(FDB) was maintained in culture for 3 d in order to verify culture medium pH.MSCs were co-cultured with FDB using two different cultivation methods:static co-culture(two-dimensional) and dynamic co-culture(threedimensional).After 24 h of cultivation by dynamic or static methods,histological analysis of Cell adhesion on FDB was performed.Cell viability was assessed by the Trypan Blue exclusion method on days 0,3 and 6 after dynamic or static culture.Adherent cells were detached from FDB surface,stained with Trypan Blue,and quantified to determine whether the cells remained on the graft surface in prolonged non-dynamic culture.Statistical analyses were performed with SPSS and a P < 0.05 was considered significant.RESULTS:The results showed a clear potential for adipogenic and osteogenic differentiation of MSC cultures.Rat MSCs were positive for CD44,CD90 and CD29 and negative for CD34,CD45 and CD11bc.FDBs were maintained in culture for 3 d and the results showed there was no significant variation in the culture medium pH with FDB compared to pure medium pH(P > 0.05).In histological analysis,there was a significant difference in the amount of adhered cells on FDB between the two cultivation methods(P < 0.05).The MSCs in the dynamic co-culture method demonstrated greater adhesion on the bone surface than in static co-culture method.On day 0,the cell viability in the dynamic system was significantly higher than in the static system(P < 0.05).There was a statistical difference in cell viability between days 0,3 and 6 after dynamic culture(P < 0.05).In static culture,cell viability on day 6 was significantly lower than on day 3 and 0(P < 0.05).CONCLUSION:An alternative cultivation method was developed to improve the MSCs adhesion on FDB,demonstrating that dynamic co-culture provides a superior environment over static conditions.     

A novel carborane analog, BE360, with a carbon-containing polyhedral boron-cluster is a new selective estrogen receptor modulator for bone

Carboranes are a class of carbon-containing polyhedral boron-cluster compounds with globular geometry and hydrophobic surface that interact with hormone receptors. Estrogen deficiency results in marked bone loss due to increased osteoclastic bone resorption in females, but estrogen replacement therapy is not generally used for postmenopausal osteoporosis due to the risk of uterine cancer. We synthesized a novel carborane compound BE360 to clarify its anti-osteoporosis activity. BE360 showed a high binding affinity to estrogen receptors (ER), ERα and ERβ. In ovariectomized (OVX) mice, femoral bone volume was markedly reduced and BE360 dose-dependently restored bone loss in OVX mice. However, BE360 did not exhibit any estrogenic activity in the uterus. BE360 also restored bone loss in orchidectomized mice without androgenic action in the sex organs. Therefore, BE360 is a novel selective estrogen receptor modulator (SERM) that may offer a new therapy option for osteoporosis.     

The earliest evidence for the use of human bone as a tool

We report on the analysis of three human cranial fragments from a Mousterian context at the site of La Quina (France), which show anthropogenic surface modifications. Macroscopic and microscopic analyses, including SEM observation, demonstrate that the modifications visible on one of these fragments are similar to those produced on bone fragments used experimentally to retouch flakes. The microscopic analysis also identified ancient scraping marks, possibly resulting from the cleaning of the skull prior to its breakage and utilisation of a resulting fragment as a tool. The traces of utilisation and the dimensions of this object are compared to those on a sample of 67 bone retouchers found in the same excavation area and layer. Results show that the tool size, as well as the dimensions and location of the utilised area, fall well within the range of variation observed on faunal shaft fragments from La Quina that were used as retouchers. This skull fragment represents the earliest known use of human bone as a raw material and the first reported use of human bone for this purpose by hominins other than modern humans. The two other skull fragments, which probably come from the same individual, also bear anthropogenic surface modifications in the form of percussion, cut, and scraping marks. The deliberate versus unintentional hypotheses for the unusual choice of the bone are presented in light of contextual information, modifications identified on the two skull fragments not used as tools, and data on bone retouchers from the same layer, the same site, and other Mousterian sites.     

Noggin is novel inducer of mesenchymal stem cell adipogenesis: implications for bone health and obesity

Noggin is a glycosylated-secreted protein known so far for its inhibitory effects on bone morphogenetic protein (BMP) signaling by sequestering the BMP ligand. We report here for the first time a novel mechanism by which noggin directly induces adipogenesis of mesenchymal stem cells independently of major human adipogenic signals through C/EBPδ, C/EBPα and peroxisome proliferator-activated receptor-γ. Evaluation of a possible mechanism for noggin-induced adipogenesis of mesenchymal stem cells identified the role of Pax-1 in mediating such differentiation. The relevance of elevated noggin levels in obesity was confirmed in a preclinical, immunocompetent mouse model of spontaneous obesity and in human patients with higher body mass index. These data clearly provide a novel role for noggin in inducing adipogenesis and possibly obesity and further indicates the potential of noggin as a therapeutic target to control obesity.