The TrkA agonist gambogic amide augments skeletal adaptation to mechanical loading.

The periosteal and endosteal surfaces of mature bone are densely innervated by sensory nerves expressing TrkA, the high-affinity receptor for nerve growth factor (NGF). In previous work, we demonstrated that administration of exogenous NGF significantly increased load-induced bone formation through the activation of Wnt signaling. However, the translational potential of NGF is limited by the induction of substantial mechanical and thermal hyperalgesia in mice and humans. Here, we tested the effect of gambogic amide (GA), a recently identified robust small molecule agonist for TrkA, on hyperalgesia and load-induced bone formation. Behavioral analysis was used to assess pain up to one week after axial forelimb compression. Contrary to our expectations, GA treatment was not associated with diminished use of the loaded forelimb or sensitivity to thermal stimulus. Furthermore, dynamic histomorphometry revealed a significant increase in relative periosteal bone formation rate as compared to vehicle treatment. Additionally, we found that GA treatment was associated with an increase in the number of osteoblasts per bone surface in loaded limbs as well as a significant increase in the fold change of Ngf, Wnt7b, and Axin2 mRNA expression as compared to vehicle (control). To test the effect of GA on osteoblasts directly, we cultured MC3T3-E1 cells for up to 21 days in osteogenic differentiation media containing NGF, GA, or vehicle (control). Media containing GA induced the significant upregulation of the osteoblastic differentiation markers Runx2, Bglap2, and Sp7 in a dose-dependent manner, whereas treatment with NGF was not associated with any significant increases in these markers. Furthermore, consistent with our in vivo findings, we observed that administration of 50 nM of GA upregulated expression of Ngf at both Day 3 and Day 7. However, cells treated with the highest dose of GA (500 nM) had significantly increased apoptosis and impaired cell proliferation. In conclusion, our study indicates GA may be useful for augmenting skeletal adaptation to mechanical forces without inducing hyperalgesia

Toll-like Receptor 4 Signaling in Osteoblasts Is Required for Load-Induced Bone Formation in Mice

In mature bone, NGF is produced by osteoblasts following mechanical loading and signals through resident sensory nerves expressing its high affinity receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), to support bone formation. Here, we investigated whether osteoblastic expression of Toll-like receptor 4 (TLR4), a key receptor in the NF-κB signaling pathway, is required to initiate NGF-TrkA signaling required for load-induced bone formation. Although Tlr4 conditional knockout mice have normal skeletal mass and strength in adulthood, the loss of TLR4 signaling significantly reduced lamellar bone formation following loading. Inhibition of TLR4 signaling reduced Ngf expression in primary osteoblasts and RNA sequencing of bones from Tlr4 conditional knockout mice and wild-type littermates revealed dysregulated inflammatory signaling three days after osteogenic mechanical loading. In total, our study reveals an important role for osteoblastic TLR4 in the skeletal adaptation of bone to mechanical forces

Preclinical single photon emission computed tomography of alpha particle-emitting radium-223

Objective: Dose optimization and pharmacokinetic evaluation of α-particle emitting radium-223 dichloride (223RaCl2) by planar γ-camera or single photon emission computed tomography (SPECT) imaging are hampered by the low photon abundance and injected activities. In this study, we demonstrate SPECT of 223Ra using phantoms and small animal in vivo models. Methods: Line phantoms and mice bearing 223Ra were imaged using a dedicated small animal SPECT by detecting the low-energy photon emissions from 223Ra. Localization of the therapeutic agent was verified by whole-body and whole-limb autoradiography and its radiobiological effect confirmed by immunofluorescence. Results: A state-of-the-art commercial small animal SPECT system equipped with a highly sensitive collimator enables collection of sufficient counts for three-dimensional reconstruction at reasonable administered activities and acquisition times. Line sources of 223Ra in both air and in a water scattering phantom gave a line spread function with a full-width-at-half-maximum of 1.45 mm. Early and late-phase imaging of the pharmacokinetics of the radiopharmaceutical were captured. Uptake at sites of active bone remodeling was correlated with DNA damage from the α particle emissions. Conclusions: This work demonstrates the capability to noninvasively define the distribution of 223RaCl2, a recently approved α-particle-emitting radionuclide. This approach allows quantitative assessment of 223Ra distribution and may assist radiation-dose optimization strategies to improve therapeutic response and ultimately to enable personalized treatment planning

The Role of Nerves in Skeletal Development, Adaptation, and Aging.

The skeleton is well-innervated, but only recently have the functions of this complex network in bone started to become known. Although our knowledge of skeletal sensory and sympathetic innervation is incomplete, including the specific locations and subtypes of nerves in bone, we are now able to reconcile early studies utilizing denervation models with recent work dissecting the molecular signaling between bone and nerve. In total, sensory innervation functions in bone much as it does elsewhere in the body-to sense and respond to stimuli, including mechanical loading. Similarly, sympathetic nerves regulate autonomic functions related to bone, including homeostatic remodeling and vascular tone. However, more study is required to translate our current knowledge of bone-nerve crosstalk to novel therapeutic strategies that can be effectively utilized to combat skeletal diseases, disorders of low bone mass, and age-related decreases in bone quality

Physiological Factors of Female Runners With and Without Stress Fracture Histories: A Pilot Study.

BACKGROUND: Female runners are at increased risk of stress fractures (SFs) compared with men. Literature is lacking with regard to best practice for preventing and treating SFs in women. The purpose of the study was to compare physiological measures and running-related factors between women of various ages and running abilities with and without a history of running-related SFs. HYPOTHESIS: Women with and without SF histories will differ with regard to medical and menstrual history, bone health, body composition, nutrition, and running history. STUDY DESIGN: Prospective cohort study. LEVEL OF EVIDENCE: Level 2. METHODS: A total of 20 female runners with SF histories were matched based on age and running distance with 20 women without SF histories. Data included medical, menstrual, running, injury, and nutritional histories; blood histology related to nutritional, hormonal, and bone-related risk factors; and bone density, fat, and lean tissue using dual energy x-ray absorptiometry. Paired RESULTS: Women with SF histories had lower hip bone mineral density compared with women without SF histories ( CONCLUSION: Female runners with low hip bone mineral density, menstrual changes during peak training, and elevated bone turnover markers may be at increased risk of SF. CLINICAL RELEVANCE: Female runners need routine screening for risks associated with SF occurrence. As bone mineral density and bone turnover markers are not routinely assessed in this population, important risk factors may be missed

Local injections of β-NGF accelerates endochondral fracture repair by promoting cartilage to bone conversion.

There are currently no pharmacological approaches in fracture healing designed to therapeutically stimulate endochondral ossification. In this study, we test nerve growth factor (NGF) as an understudied therapeutic for fracture repair. We first characterized endogenous expression of Ngf and its receptor tropomyosin receptor kinase A (TrkA) during tibial fracture repair, finding that they peak during the cartilaginous phase. We then tested two injection regimens and found that local β-NGF injections during the endochondral/cartilaginous phase promoted osteogenic marker expression. Gene expression data from β-NGF stimulated cartilage callus explants show a promotion in markers associated with endochondral ossification such as Ihh, Alpl, and Sdf-1. Gene ontology enrichment analysis revealed the promotion of genes associated with Wnt activation, PDGF- and integrin-binding. Subsequent histological analysis confirmed Wnt activation following local β-NGF injections. Finally, we demonstrate functional improvements to bone healing following local β-NGF injections which resulted in a decrease in cartilage and increase of bone volume. Moreover, the newly formed bone contained higher trabecular number, connective density, and bone mineral density. Collectively, we demonstrate β-NGF\u27s ability to promote endochondral repair in a murine model and uncover mechanisms that will serve to further understand the molecular switches that occur during cartilage to bone transformation

Activin Receptor Type 2A (ACVR2A) Functions Directly in Osteoblasts as a Negative Regulator of Bone Mass

Bone and skeletal muscle mass are highly correlated in mammals, suggesting the existence of common anabolic signaling networks that coordinate the development of these two anatomically adjacent tissues. The activin signaling pathway is an attractive candidate to fulfill such a role. Here, we generated mice with conditional deletion of activin receptor (ACVR) type 2A, ACVR2B, or both, in osteoblasts, to determine the contribution of activin receptor signaling in regulating bone mass. Immunohistochemistry localized ACVR2A and ACVR2B to osteoblasts and osteocytes. Primary osteoblasts expressed activin signaling components, including ACVR2A, ACVR2B, and ACVR1B (ALK4) and demonstrated increased levels of phosphorylated Smad2/3 upon exposure to activin ligands. Osteoblasts lacking ACVR2B did not show significant changes in vitro. However, osteoblasts deficient in ACVR2A exhibited enhanced differentiation indicated by alkaline phosphatase activity, mineral deposition, and transcriptional expression of osterix, osteocalcin, and dentin matrix acidic phosphoprotein 1. To investigate activin signaling in osteoblasts in vivo, we analyzed the skeletal phenotypes of mice lacking these receptors in osteoblasts and osteocytes (osteocalcin-Cre). Similar to the lack of effect in vitro, ACVR2B-deficient mice demonstrated no significant change in any bone parameter. By contrast, mice lacking ACVR2A had significantly increased femoral trabecular bone volume at 6 weeks of age. Moreover, mutant mice lacking both ACVR2A and ACVR2B demonstrated sustained increases in trabecular bone volume, similar to those in ACVR2A single mutants, at 6 and 12 weeks of age. Taken together, these results indicate that activin receptor signaling, predominantly through ACVR2A, directly and negatively regulates bone mass in osteoblasts

Using CRISPR/Cas9 genome editing in tomato to create a gibberellin-responsive dominant dwarf DELLA allele

The tomato PROCERA gene encodes a DELLA protein, and loss-of-function mutations derepress growth. We used CRISPR/Cas9 and a single guide RNAs (sgRNA) to target mutations to the PROCERA DELLA domain, and recovered several loss-of-function mutations and a dominant dwarf mutation that carries a deletion of one amino acid in the DELLA domain. This is the first report of a dominant dwarf PROCERA allele. This allele retains partial responsiveness to exogenously applied gibberellin. Heterozygotes show an intermediate phenotype at the seedling stage, but adult heterozygotes are as dwarfed as homozygotes

Representations of sport in the revolutionary socialist press in Britain, 1988–2012

This paper considers how sport presents a dualism to those on the far left of the political spectrum. A long-standing, passionate debate has existed on the contradictory role played by sport, polarised between those who reject it as a bourgeois capitalist plague and those who argue for its reclamation and reformation. A case study is offered of a political party that has consistently used revolutionary Marxism as the basis for its activity and how this party, the largest in Britain, addresses sport in its publications. The study draws on empirical data to illustrate this debate by reporting findings from three socialist publications. When sport did feature it was often in relation to high profile sporting events with a critical tone adopted and typically focused on issues of commodification, exploitation and alienation of athletes and supporters. However, readers’ letters, printed in the same publications, revealed how this interpretation was not universally accepted, thus illustrating the contradictory nature of sport for those on the far left