Processing and Sectioning Undecalcified Murine Bone Specimens.

Preparation of mineralized tissue specimens for bone-specific staining encompasses a critical sequence of histological techniques that provides visualization of tissue and cellular morphology. Bone specimens are fixed in 10% neutral buffered formalin (NBF), dehydrated in graded ethanol (EtOH) solutions (and optionally cleared in xylene), infiltrated and embedded in polymethyl methacrylate (methyl methacrylate or MMA), classically sliced into 4-10 micrometer (μm) sections, and stained with bone-specific histological stains such as von Kossa (with either nuclear fast red solution counterstain or MacNeal's tetrachrome counterstain), modified Goldner's trichrome, Alizarin Red S, Safranin O, and tartrate-resistant acid phosphatase (TRAP) stain. Here, we describe the tissue processing of mineralized mouse bones from dissection to staining for histological analysis by light microscopy.

Glucocorticoid-Induced Bone Fragility Is Prevented in Female Mice by Blocking Pyk2/Anoikis Signaling.

Excess of glucocorticoids (GCs) is a leading cause of bone fragility, and therapeutic targets are sorely needed. We report that genetic deletion or pharmacological inhibition of proline-rich tyrosine kinase 2 (Pyk2) prevents GC-induced bone loss by overriding GC effects of detachment-induced bone cell apoptosis (anoikis). In wild-type or vehicle-treated mice, GCs either prevented osteoclast apoptosis or promoted osteoblast/osteocyte apoptosis.

Observations on spontaneous tumor formation in mice overexpressing mitotic kinesin Kif14.

The KIF14 locus is gained and overexpressed in various malignancies, with prognostic relevance. Its protein product, a mitotic kinesin, accelerates growth of normal mammary epithelial cells in vitro and retinoblastoma tumours in a mouse model, while KIF14 knockdown blocks growth of brain, liver, ovarian, breast, prostate, and other tumour cells and xenografts. However, the tumour-initiating effects of Kif14 overexpression have not been studied.

Microcrack-associated bone remodeling is rarely observed in biopsies from athletes with medial tibial stress syndrome.

The pathology of medial tibial stress syndrome (MTSS) is unknown. Studies suggest that MTSS is a bony overload injury, but histological evidence is sparse. The presence of microdamage, and its potential association with targeted remodeling, could provide evidence for the pathogenesis of MTSS.

Osteocytic perilacunar/canalicular turnover in hemodialysis patients with high and low serum PTH levels.

Osteocytic perilacunar/canalicular turnover in hemodialysis patients has not yet been reported. Osteocyte lacunae in lamellar bone and woven bone were classified as eroded surface-, osteoid surface-, and quiescent surface-predominant osteocyte lacunae (ES-Lc, OS-Lc, QS-Lc, respectively) in 55 hemodialysis patients with either high- (n = 45) or low- (n = 10) parathyroid hormone levels, and 19 control subjects without chronic kidney disease. We calculated the area and number of ES-Lc, OS-Lc, and QS-Lc.

Rad GTPase is essential for the regulation of bone density and bone marrow adipose tissue in mice.

The small GTP-binding protein Rad (RRAD, Ras associated with diabetes) is the founding member of the RGK (Rad, Rem, Rem2, and Gem/Kir) family that regulates cardiac voltage-gated Ca channel function. However, its cellular and physiological functions outside of the heart remain to be elucidated. Here we report that Rad GTPase function is required for normal bone homeostasis in mice, as Rad deletion results in significantly lower bone mass and higher bone marrow adipose tissue (BMAT) levels.

Improving Combination Osteoporosis Therapy in a Preclinical Model of Heightened Osteoanabolism.

Combining anticatabolic agents with parathyroid hormone (PTH) to enhance bone mass has yielded mixed results in osteoporosis patients. Toward the goal of enhancing the efficacy of these regimens, we tested their utility in combination with loss of the transcription factor Nmp4 because disabling this gene amplifies PTH-induced increases in trabecular bone in mice by boosting osteoblast secretory activity. We addressed whether combining a sustained anabolic response with an anticatabolic results in superior bone acquisition compared with PTH monotherapy.

Protection From Glucocorticoid-Induced Osteoporosis by Anti-Catabolic Signaling in the Absence of Sost/Sclerostin.

Excess of glucocorticoids, either due to disease or iatrogenic, increases bone resorption and decreases bone formation and is a leading cause of osteoporosis and bone fractures worldwide. Improved therapeutic strategies are sorely needed. We investigated whether activating Wnt/β-catenin signaling protects against the skeletal actions of glucocorticoids, using female mice lacking the Wnt/β-catenin antagonist and bone formation inhibitor Sost.

Exploring the Bone Proteome to Help Explain Altered Bone Remodeling and Preservation of Bone Architecture and Strength in Hibernating Marmots.

Periods of physical inactivity increase bone resorption and cause bone loss and increased fracture risk. However, hibernating bears, marmots, and woodchucks maintain bone structure and strength, despite being physically inactive for prolonged periods annually. We tested the hypothesis that bone turnover rates would decrease and bone structural and mechanical properties would be preserved in hibernating marmots (Marmota flaviventris).

Combination Therapy with Zoledronic Acid and Parathyroid Hormone Improves Bone Architecture and Strength following a Clinically-Relevant Dose of Stereotactic Radiation Therapy for the Local Treatment of Canine Osteosarcoma in Athymic Rats.

Clinical studies using definitive-intent stereotactic radiation therapy (SRT) for the local treatment of canine osteosarcoma (OSA) have shown canine patients achieving similar median survival times as the current standard of care (amputation and adjuvant chemotherapy). Despite this, there remains an unacceptable high risk of pathologic fracture following radiation treatment. Zoledronic acid (ZA) and parathyroid hormone (PTH) are therapeutic candidates for decreasing this fracture risk post-irradiation.

Outer surface protein OspC is an antiphagocytic factor that protects Borrelia burgdorferi from phagocytosis by macrophages.

Outer surface protein C (OspC) is one of the major lipoproteins expressed on the surface of Borrelia burgdorferi during tick feeding and the early phase of mammalian infection. OspC is required for B. burgdorferi to establish infection in both immunocompetent and SCID mice and has been proposed to facilitate evasion of innate immune defenses.

Inhibition of osteocyte apoptosis prevents the increase in osteocytic receptor activator of nuclear factor κB ligand (RANKL) but does not stop bone resorption or the loss of bone induced by unloading.

Apoptosis of osteocytes and osteoblasts precedes bone resorption and bone loss with reduced mechanical stimulation, and receptor activator of NF-κB ligand (RANKL) expression is increased with unloading in mice. Because osteocytes are major RANKL producers, we hypothesized that apoptotic osteocytes signal to neighboring osteocytes to increase RANKL expression, which, in turn, increases osteoclastogenesis and bone resorption. The traditional bisphosphonate (BP) alendronate (Aln) or IG9402, a BP analog that does not inhibit resorption, prevented the increase in osteocyte apoptosis and osteocytic RANKL expression.

Osteocytes mediate the anabolic actions of canonical Wnt/β-catenin signaling in bone.

Osteocytes, >90% of the cells in bone, lie embedded within the mineralized matrix and coordinate osteoclast and osteoblast activity on bone surfaces by mechanisms still unclear. Bone anabolic stimuli activate Wnt signaling, and human mutations of components along this pathway underscore its crucial role in bone accrual and maintenance. However, the cell responsible for orchestrating Wnt anabolic actions has remained elusive.

Tips and techniques for processing and sectioning undecalcified murine bone specimens.

Preparation of mineralized tissue specimens for bone-specific staining encompasses a critical sequence of histological techniques that provides visualization of tissue and cellular morphology. Bone specimens are fixed in 10 % neutral-buffered formalin, dehydrated in graded ethanol (EtOH) solutions (and optionally cleared in xylene), infiltrated and embedded in polymethyl methacrylate (methyl methacrylate), classically sliced into 4-10 micrometer (μm) sections, and stained with bone-specific histological stains such as von Kossa (with either nuclear fast red solution counterstain or MacNeal's tetrachrome counterstain), modified Goldner's trichrome, and alizarin red S stain. Here, we describe the tissue processing of mineralized mouse bones from dissection to staining for histological analysis by light microscopy.

Resorption controls bone anabolism driven by parathyroid hormone (PTH) receptor signaling in osteocytes.

The contribution of remodeling-based bone formation coupled to osteoclast activity versus modeling-based bone formation that occurs independently of resorption, to the anabolic effect of PTH remains unclear. We addressed this question using transgenic mice with activated PTH receptor signaling in osteocytes that exhibit increased bone mass and remodeling, recognized skeletal effects of PTH elevation. Direct inhibition of bone formation was accomplished genetically by overexpressing the Wnt antagonist Sost/sclerostin; and resorption-dependent bone formation was inhibited pharmacologically with the bisphosphonate alendronate.

Black bear parathyroid hormone has greater anabolic effects on trabecular bone in dystrophin-deficient mice than in wild type mice.

Duchenne muscular dystrophy (DMD) is an X-linked neuromuscular disease that has deleterious consequences in muscle and bone, leading to decreased mobility, progressive osteoporosis, and premature death. Patients with DMD experience a higher-than-average fracture rate, particularly in the proximal and distal femur and proximal tibia. The dystrophin-deficient mdx mouse is a model of DMD that demonstrates muscle degeneration and fibrosis and osteoporosis.

Resolution of inflammation induces osteoblast function and regulates the Wnt signaling pathway.

Objective: Inflammation in the bone microenvironment stimulates osteoclast differentiation, resulting in uncoupling of resorption and formation. Mechanisms contributing to the inhibition of osteoblast function in inflammatory diseases, however, have not been elucidated. Rheumatoid arthritis (RA) is a prototype of an inflammatory arthritis that results in focal loss of articular bone.

Sost downregulation and local Wnt signaling are required for the osteogenic response to mechanical loading.

Sclerostin, the Wnt signaling antagonist encoded by the Sost gene, is secreted by osteocytes and inhibits bone formation by osteoblasts. Mechanical stimulation reduces sclerostin expression, suggesting that osteocytes might coordinate the osteogenic response to mechanical force by locally unleashing Wnt signaling. To investigate whether sclerostin downregulation is a pre-requisite for load-induced bone formation, we conducted experiments in transgenic mice (TG) engineered to maintain high levels of SOST expression during mechanical loading.

Cell autonomous requirement of connexin 43 for osteocyte survival: consequences for endocortical resorption and periosteal bone formation.

Connexin 43 (Cx43) mediates osteocyte communication with other cells and with the extracellular milieu and regulates osteoblastic cell signaling and gene expression. We now report that mice lacking Cx43 in osteoblasts/osteocytes or only in osteocytes (Cx43(ΔOt) mice) exhibit increased osteocyte apoptosis, endocortical resorption, and periosteal bone formation, resulting in higher marrow cavity and total tissue areas measured at the femoral mid-diaphysis. Blockade of resorption reversed the increased marrow cavity but not total tissue area, demonstrating that endocortical resorption and periosteal apposition are independently regulated.

Increased strontium uptake in trabecular bone of ovariectomized calcium-deficient rats treated with strontium ranelate or strontium chloride.

Based on clinical trials showing the efficacy to reduce vertebral and non-vertebral fractures, strontium ranelate (SrR) has been approved in several countries for the treatment of postmenopausal osteoporosis. Hence, it is of special clinical interest to elucidate how the Sr uptake is influenced by dietary Ca deficiency as well as by the formula of Sr administration, SrR versus strontium chloride (SrCl(2)). Three-month-old ovariectomized rats were treated for 90 days with doses of 25 mg kg(-1) d(-1) and 150 mg kg(-1) d(-1) of SrR or SrCl(2) at low (0.

A scaffold-free multicellular three-dimensional in vitro model of osteogenesis.

In vitro models of osteogenesis are essential for investigating bone biology and the effects of pharmaceutical, chemical, and physical cues on bone formation. Osteogenesis takes place in a complex three-dimensional (3D) environment with cells from both mesenchymal and hematopoietic origins. Existing in vitro models of osteogenesis include two-dimensional (2D) single type cell monolayers and 3D cultures.

PTH receptor signaling in osteocytes governs periosteal bone formation and intracortical remodeling.

The periosteal and endocortical surfaces of cortical bone dictate the geometry and overall mechanical properties of bone. Yet the cellular and molecular mechanisms that regulate activity on these surfaces are far from being understood. Parathyroid hormone (PTH) has profound effects in cortical bone, stimulating periosteal expansion and at the same time accelerating intracortical bone remodeling.

Grizzly bears (Ursus arctos horribilis) and black bears (Ursus americanus) prevent trabecular bone loss during disuse (hibernation).

Disuse typically causes an imbalance in bone formation and bone resorption, leading to losses of cortical and trabecular bone. In contrast, bears maintain balanced intracortical remodeling and prevent cortical bone loss during disuse (hibernation). Trabecular bone, however, is more detrimentally affected than cortical bone in other animal models of disuse.

Osteoblast function is compromised at sites of focal bone erosion in inflammatory arthritis.

In rheumatoid arthritis (RA), synovial inflammation results in focal erosion of articular bone. Despite treatment attenuating inflammation, repair of erosions with adequate formation of new bone is uncommon in RA, suggesting that bone formation may be compromised at these sites. Dynamic bone histomorphometry was used in a murine model of RA to determine the impact of inflammation on osteoblast function within eroded arthritic bone.

Mechanical stimulation of bone in vivo reduces osteocyte expression of Sost/sclerostin.

Sclerostin, the protein product of the Sost gene, is a potent inhibitor of bone formation. Among bone cells, sclerostin is found nearly exclusively in the osteocytes, the cell type that historically has been implicated in sensing and initiating mechanical signaling. The recent discovery of the antagonistic effects of sclerostin on Lrp5 receptor signaling, a crucial mediator of skeletal mechanotransduction, provides a potential mechanism for the osteocytes to control mechanotransduction, by adjusting their sclerostin (Wnt inhibitory) signal output to modulate Wnt signaling in the effector cell population.