Is insulin an anabolic agent in bone? Dissecting the diabetic bone for clues

Diabetic osteoporosis is increasingly recognized as a significant comorbidity of type 1 diabetes mellitus. In contrast, type 2 diabetes mellitus is more commonly associated with modest increases in bone mineral density for age. Despite this dichotomy, clinical, in vivo, and in vitro data uniformly support the concept that new bone formation as well as bone microarchitectural integrity are altered in the diabetic state, leading to an increased risk for fragility fracture and inadequate bone regeneration following injury. In this review, we examine the contribution that insulin, as a potential anabolic agent in bone, may make to the pathophysiology of diabetic bone disease. Specifically, we have assimilated human and animal data examining the effects of endogenous insulin production, exogenous insulin administration, insulin sensitivity, and insulin signaling on bone. In so doing, we present evidence that insulin, acting as an anabolic agent in bone, can preserve and increase bone density and bone strength, presumably through direct and/or indirect effects on bone formation.     

Role of carotenoid β-cryptoxanthin in bone homeostasis

Bone homeostasis is maintained through a balance between osteoblastic bone formation and osteoclastic bone resorption. Aging induces bone loss due to decreased osteoblastic bone formation and increased osteoclastic bone resorption. Osteoporosis with its accompanying decrease in bone mass is widely recognized as a major public health problem. Nutritional factors may play a role in the prevention of bone loss with aging. Among various carotenoids (carotene and xanthophylls including beta (β)-cryptoxanthin, lutein, lycopene, β-carotene, astaxanthin, and rutin), β-cryptoxanthin, which is abundant in Satsuma mandarin orange (Citrus unshiu MARC.), has been found to have a stimulatory effect on bone calcification in vitro. β-cryptoxanthin has stimulatory effects on osteoblastic bone formation and inhibitory effects on osteoclastic bone resorption in vitro, thereby increasing bone mass. β-cryptoxanthin has an effect on the gene expression of various proteins that are related osteoblastic bone formation and osteoclastic bone resororption in vitro. The intake of β-cryptoxanthin may have a preventive effect on bone loss in animal models for osteoporosis and in healthy human or postmenopausal women. Epidemiological studies suggest a potential role of β-cryptoxanthin as a sustainable nutritional approach to improving bone health of human subjects. β-Cryptoxanthin may be an osteogenic factor in preventing osteoporosis in human subjects.     

NF-κB modulators in osteolytic bone diseases

Osteoclasts are responsible for bone resorption and play a pivotal role in the pathogenesis of osteolytic disorders. NF-κB is a set of nuclear factors that bind to consensus DNA sequences called κB sites, and is essential for osteoclast formation and survival. NF-κB signalling pathways are strictly regulated to maintain bone homeostasis by cytokines such as RANKL, TNF-α and IL-1, which differentially regulate classical and/or alternative NF-κB pathways in osteoclastic cells. These pathways are also modulated by NF-κB mediators, including TRAF6, aPKC, p62/SQSTM1 and deubiquitinating enzyme CYLD that are involved in the ubiquitin–proteasome system during RANK-mediated osteoclastogenesis. Abnormal activation of NF-κB signalling in osteoclasts has been associated with excessive osteoclastic activity, and frequently observed in osteolytic conditions, including periprosthetic osteolysis, arthritis, Paget's disease of bone, and periodontitis. NF-κB modulators such as parthenolide and NEMO-binding domain peptide demonstrate therapeutic effects on inflammation-induced bone destruction in mouse models. Unravelling the structure and function of NF-κB pathways in osteoclasts and other cell types will be important in developing new strategies for treatments of bone diseases.     

TGF-β signalling-related markers in cancer patients with bone metastasis

Abstract

We measured transforming growth factor (TGF)-β-dependent biomarkers in plasma and in peripheral blood mononuclear cells (PBMCs) to identify suitable pharmacodynamic markers for future clinical trials with TGF-β inhibitors. Forty-nine patients with bone metastasis were enrolled in the study, including patients with breast (n=23) and prostate cancer (n=15). Plasma TGF-β1 levels were elevated in more than half of the cancer patients (geometric mean 2.63 ng ml^?1) and positively correlated with increased platelet factor 4 (PF4) levels, parathyroid-related protein (PTHrP), von Willebrand Factor (vWF) and interleukin (IL)-10. PBMC were stimulated ex vivo to determine the individual biological variability of an ex vivo assay measuring pSMAD expression. This assay performed sufficiently well to allow its future use in a clinical trial of a TGF-β inhibitor.     

What do we currently know from in vivo bone strain measurements in humans?

Bone strains are the most important factors for osteogenic adaptive responses. During the past decades, scientists have been trying to describe the relationship between bone strain and bone osteogenic responses quantitatively. However, only a few studies have examined bone strains under physiological condition in humans, owing to technical difficulty and ethical restrictions. The present paper reviews previous work on in vivo bone strain measurements in humans, and the various methodologies adopted in these measurements are discussed. Several proposals are made for future work to improve our understanding of the human musculoskeletal system. Literature suggests that strains and strain patterns vary systematically in response to different locomotive activities, foot wear, and even different venues. The principal compressive, tension and engineering shear strain, compressive strain rate and shear strain rate in the tibia during running seem to be higher than those during walking. The high impact exercises, such as zig-zag hopping and basketball rebounding induced greater principal strains and strain rates in the tibia than normal activities. Also, evidence suggests an increase of tibia strain and strain rate after muscle fatigue, which strongly supports the opinion that muscle contractions play a role on the alteration of bone strain patterns.     

Long-term potentiation in bone – a role for glutamate in strain-induced cellular memory?

Background

The adaptive response of bone cells to mechanical strain is a primary determinant of skeletal architecture and bone mass. In vivo mechanical loading induces new bone formation and increases bone mineral density whereas disuse, immobilisation and weightlessness induce bone loss. The potency of mechanical strain is such that a single brief period of loading at physiological strain magnitude is able to induce a long-lasting osteogenic response that lasts for days. Although the process of mechanotransduction in bone is incompletely understood, observations that responses to mechanical strain outlast the duration of stimulation necessitate the existence of a form of cellular memory through which transient strain episodes are recorded, interpreted and remembered by bone cells. Recent evidence supports the existence of a complex multicellular glutamate-signalling network in bone that shares functional similarities to glutamatergic neurotransmission in the central nervous system. In neurones, these signalling molecules coordinate synaptic communication required to support learning and memory formation, through a complex process of long-term potentiation.

Presentation of the hypothesis

We hypothesise that osteoblasts use a cellular mechanism similar or identical to neuronal long-term potentiation in the central nervous system to mediate long-lasting changes in osteogenesis following brief periods of mechanical strain.

Testing the hypothesis

N-methyl-D-aspartate (NMDA) receptor antagonism should inhibit the saturating response of mechanical strain and reduce the enhanced osteogenicity of segregated loading to that of an equivalent period of uninterrupted loading. Changes in α-amino-3-hydroxy-5-methyl-isoxazole propionate (AMPA) receptor expression, localisation and electrophysiological responses should be induced by mechanical strain and inhibited by modulators of neuronal long-term potentiation.

Implications of the hypothesis

If true, this hypothesis would provide a mechanism through which the skeleton could be pharmacologically primed to enhance or retrieve the normal osteogenic response to exercise. This would form a basis through which novel therapies could be developed to target osteoporosis and other prevalent bone disorders associated with low bone mass.

     

Methotrexate chemotherapy–induced damages in bone marrow sinusoids: An in vivo and in vitro study

Chemotherapeutic agents are very well evident extrinsic stimuli for causing damage to endothelial cells. Methotrexate is an antimetabolite commonly used to treat solid tumours and paediatric cancers. However, studies on the effect(s) of methotrexate on bone marrow microvascular system are inadequate. In the current study, we observed a significant bone marrow microvascular dilation following methotrexate therapy in rats, accompanied by apoptosis induction in bone marrow sinusoidal endothelial cells, and followed by recovery of bone marrow sinusoids associated with increased proliferation of remaining bone marrow sinusoidal endothelial cells. Our in vitro studies revealed that methotrexate is cytotoxic for cultured sinusoidal endothelial cells and can also induce apoptosis which is associated with upregulation of expression ratio of Bax and Bcl-2 genes and Bax/Bcl-2 expression ratio. Furthermore, it was shown that methotrexate can negatively affect proliferation of cultured sinusoidal endothelial cells and also inhibit their abilities of migration and formation of microvessel like tubes. The data from this study indicates that methotrexate can cause significant bone marrow sinusoidal endothelium damage in vivo and induce apoptosis and inhibit proliferation, migration and tube-forming abilities of sinusoidal endothelial cells in vitro.     

TGF-β and BMP signaling in osteoblast differentiation and bone formation

Transforming growth factor-beta (TGF-β)/bone morphogenic protein (BMP) signaling is involved in a vast majority of cellular processes and is fundamentally important throughout life. TGF-β/BMPs have widely recognized roles in bone formation during mammalian development and exhibit versatile regulatory functions in the body. Signaling transduction by TGF-β/BMPs is specifically through both canonical Smad-dependent pathways (TGF-β/BMP ligands, receptors and Smads) and non-canonical Smad-independent signaling pathway (e.g. p38 mitogen-activated protein kinase pathway, MAPK). Following TGF-β/BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation. The coordinated activity of Runx2 and TGF-β/BMP-activated Smads is critical for formation of the skeleton. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of TGF-β/BMP signaling in bone and in the signaling networks underlying osteoblast differentiation and bone formation. This review summarizes the recent advances in our understanding of TGF-β/BMP signaling in bone from studies of genetic mouse models and human diseases caused by the disruption of TGF-β/BMP signaling. This review also highlights the different modes of cross-talk between TGF-β/BMP signaling and the signaling pathways of MAPK, Wnt, Hedgehog, Notch, and FGF in osteoblast differentiation and bone formation.     

Implant–bone interface healing and adaptation in resurfacing hip replacement

Hip resurfacing demonstrates good survivorship as a treatment for young patients with osteoarthritis, but occasional implant loosening failures occur. On the femoral side there is radiographic evidence suggesting that the implant stem bears load, which is thought to lead to proximal stress shielding and adaptive bone remodelling. Previous attempts aimed at reproducing clinically observed bone adaptations in response to the implant have not recreated the full set of common radiographic changes, so a modified bone adaptation algorithm was developed in an attempt to replicate more closely the effects of the prosthesis on the host bone. The algorithm features combined implant–bone interface healing and continuum bone remodelling. It was observed that remodelling simulations that accounted for progressive gap filling at the implant–bone interface predicted the closest periprosthetic bone density changes to clinical X-rays and DEXA data. This model may contribute to improved understanding of clinical failure mechanisms with traditional hip resurfacing designs and enable more detailed pre-clinical analysis of new designs.     

"Whither flows the fluid in bone?" An osteocyte's perspective

Bone represents a porous tissue containing a fluid phase, a solid matrix, and cells. Movement of the fluid phase within the pores or spaces of the solid matrix translates endogenous and exogenous mechanobiological, biochemical and electromechanical signals from the system that is exposed to the dynamic external environment to the cells that have the machinery to remodel the tissue from within. Hence, bone fluid serves as a coupling medium, providing an elegant feedback mechanism for functional adaptation. Until recently relatively little has been known about bone fluid per se or the influences governing the characteristics of its flow. This work is designed to review the current state of this emerging field. The structure of bone, as an environment for fluid flow, is discussed in terms of the properties of the spaces and channel walls through which the fluid flows and the influences on flow under physiological conditions. In particular, the development of the bone cell syncytium and lacunocanalicular system are presented, and pathways for fluid flow are described from the systemic to the organ, tissue, cellular and subcellular levels. Finally, exogenous and endogenous mechanisms for pressure-induced fluid movement through bone, including mechanical loading, vascular derived pressure gradients, and osmotic pressure gradients are discussed. The objective of this review is to survey the current understanding of the means by which fluid flow in bone is regulated, from the level of the skeletal system down to the level of osteocyte, and to provide impetus for future research in this area of signal transduction and coupling. An understanding of this important aspect of bone physiology has profound implications for restoration of function through innovative treatment modalities on Earth and in space, as well as for engineering of biomimetic replacement tissue.     

Do microcracks decrease or increase fatigue resistance in cortical bone?

Fatigue of cortical bone produces microcracks; it has been hypothesized that these cracks are analogous to those occurring in engineered composite materials and constitute a similar mechanism for fatigue resistance. However, the numbers of these linear microcracks increase substantially with age, suggesting that they contribute to increased fracture incidence among the elderly. To test these opposing hypotheses, we fatigued 20 beams of femoral cortical bone from elderly men and women in load-controlled four point bending having initial strain ranges of 3000 or 5000 microstrain. Loading was stopped at fracture or 10(6) cycles, whichever occurred first, and microcrack density and length were measured in the loaded region and in a control region that was not loaded. We studied the dependence of fatigue life and induced microdamage on initial microdamage, cortical region, subject gender and age, and several other variables. When the effect of modulus variability was controlled, longer fatigue life was associated with higher rather than lower initial crack density, particularly in the medial cortex. The increase in crack density following fatigue loading was greater in specimens from older individuals and those initially having longer microcracks. Crack density increased as much in specimens fatigued short of the failure point as in those that fractured, and microcracks were, on average, shorter in specimens with greater numbers of resorption spaces, a measure of remodeling rate.     

Ecto-5′-nucleotidase (CD73) regulates bone formation and remodeling during intramembranous bone repair in aging mice

Ecto-5′-nucleotidase (CD73) generates adenosine, an osteoblast activator and key regulator of skeletal growth. It is unknown, however, if CD73 regulates osteogenic differentiation during fracture healing in adulthood, and in particular how CD73 activity regulates intramembranous bone repair in the elderly. Monocortical tibial defects were created in 46–52-week-old wild type (WT) and CD73 knock-out mice (CD73^?/?) mice. Injury repair was analyzed at post-operative days 5, 7, 14 and 21 by micro-computed tomography (micro-CT), histomorphometry, proliferating cell nuclear antigen (PCNA) immunostaining, alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) histochemistry. Middle-aged CD73 knock-out mice exhibited delayed bone regeneration and significantly reduced bone matrix deposition detected by histomorphometry and micro-CT. Cell proliferation, ALP activity and osteoclast number were reduced in the CD73^?/? mice, suggesting a combined defect in bone formation and resorption due the absence of CD73 activity in this model of intramembranous bone repair. Results from this study demonstrate that osteoblast activation through CD73 activity is essential during bone repair in aging mice, and it may present a drugable target for future biomimetic therapeutic approaches that aim at enhancing bone formation in the elderly patients.     

Conditional expression of constitutively active estrogen receptor α in chondrocytes impairs longitudinal bone growth in mice

Estrogen plays important roles in the regulation of chondrocyte proliferation and differentiation, which are essential steps for longitudinal bone growth; however, the mechanisms of estrogen action on chondrocytes have not been fully elucidated. In the present study, we generated conditional transgenic mice, designated as caERα(ColII), expressing constitutively active mutant estrogen receptor (ER) α in chondrocytes, using the chondrocyte-specific type II collagen promoter-driven Cre transgenic mice. caERα(ColII) mice showed retardation in longitudinal growth, with short bone lengths. BrdU labeling showed reduced proliferation of hypertrophic chondrocytes in the proliferating layer of the growth plate of tibia in caERα(ColII) mice. In situ hybridization analysis of type X collagen revealed that the maturation of hypertrophic chondrocytes was impaired in caERα(ColII) mice. These results suggest that ERα is a critical regulator of chondrocyte proliferation and maturation during skeletal development, mediating longitudinal bone growth in vivo.     

Conditional expression of constitutively active estrogen receptor α in osteoblasts increases bone mineral density in mice

Estrogen plays an important role in maintaining bone density in women. Estrogen receptor (ER) is expressed in osteoblasts and osteoclasts; however, the precise mechanism of ER in bone is not fully understood. In the present study, we generated a conditional transgenic mouse caERα(ColI) that expresses the constitutively active ERα in osteoblasts using collagen type I promoter-driven Cre transgenic mice. The caERα(ColI) mice showed increased bone mineral density (BMD). Osteoblasts prepared from caERα(ColI) mice expressed high levels of osteoprotegerin and decreased levels of IL-6, both of which are known to regulate osteoclast differentiation. These results suggest that ERα regulates osteoprotegerin and IL-6 production in osteoblasts and modulates BMD. The conditional transgenic mouse model is useful for understanding the in vivo function of ERα.     

Are bone losers distinguishable from bone formers in a skeletal series? Implications for adult age at death assessment methods.

Clinical studies indicate that genetic factors play a crucial role in primary osteoarthritis and osteoporosis. In addition, it has been suggested that these two diseases are inversely related. Within a population, one can find two sub-groups: the "bone formers" and the "bone losers". The changes to the joint surfaces used to assess adult age at death are related to the loss of bone substance and to bone formation (osteophytes). The modification of these indicators with age differs between bone formers and bone losers. Therefore, age-at-death assessment methods should make use of two standards, one for each sub-group. A preliminary study examining the possibility of distinguishing those who lose cortical bone from those who show signs of bony formation was conducted on a series of skeletons from Portugal, dating to the end of 19th century and the beginning of the 20th. Bone loss was evaluated using the cortical index (CI) of the second metacarpal on X-rays. The presence of osteophytes on dry bones was assessed macroscopically. Our study indicates that females' CI decreases with age, whereas the presence of osteophytes is strongly related to age in both sexes. But we have failed to find the inverse relationship between osteophytes and bone loss. Our study, however, shows that within a population, some individuals are not likely to develop osteophytes.     

Mollugin from Rubea cordifolia suppresses receptor activator of nuclear factor-κB ligand-induced osteoclastogenesis and bone resorbing activity in vitro and prevents lipopolysaccharide-induced bone loss in vivo

Osteopenic diseases, such as osteoporosis, are characterized by progressive and excessive bone resorption mediated by enhanced receptor activator of nuclear factor-κB ligand (RANKL) signaling. Therefore, downregulation of RANKL downstream signals may be a valuable approach for the treatment of bone loss-associated disorders. In this study, we investigated the effects of the naphthohydroquinone mollugin on osteoclastogenesis and its function in vitro and in vivo. Mollugin efficiently suppressed RANKL-induced osteoclast differentiation of bone marrow macrophages (BMMs) and bone resorbing activity of mature osteoclasts by inhibiting RANKL-induced c-Fos and NFATc1 expression. Mollugin reduced the phosphorylation of signaling pathways activated in the early stages of osteoclast differentiation, including the MAP kinase, Akt, and GSK3β and inhibited the expression of different genes associated with osteoclastogenesis, such as OSCAR, TRAP, DC-STAMP, OC-STAMP, integrin αν, integrin β3, cathepsin K, and ICAM-1. Furthermore, mice treated with mollugin showed significant restoration of lipopolysaccharide (LPS)-induced bone loss as indicated by micro-CT and histological analysis of femurs. Consequently, these results suggested that mollugin could be a novel therapeutic candidate for bone loss-associated disorders including osteoporosis, rheumatoid arthritis, and periodontitis.     

PDGFBB promotes PDGFRα-positive cell migration into artificial bone in vivo

Bone defects caused by traumatic bone loss or tumor dissection are now treated with auto- or allo-bone graft, and also occasionally by artificial bone transplantation, particularly in the case of large bone defects. However, artificial bones often exhibit poor affinity to host bones followed by bony union failure. Thus therapies combining artificial bones with growth factors have been sought. Here we report that platelet derived growth factor bb (PDGFBB) promotes a significant increase in migration of PDGF receptor α (PDGFRα)-positive mesenchymal stem cells/pre-osteoblastic cells into artificial bone in vivo. Growth factors such as transforming growth factor beta (TGFβ) and hepatocyte growth factor (HGF) reportedly inhibit osteoblast differentiation; however, PDGFBB did not exhibit such inhibitory effects and in fact stimulated osteoblast differentiation in vitro, suggesting that combining artificial bones with PDGFBB treatment could promote host cell migration into artificial bones without inhibiting osteoblastogenesis.