The peripheral nervous system modulates bone repair under physiological and pathophysiological conditions. Previously, we reported an essential role for sensory neuropeptide substance P (SP) and sympathetic nerve fibers (SNF) for proper fracture healing and bone structure in a murine tibial fracture model. A similar distortion of bone microarchitecture has been described for mice lacking the sensory neuropeptide α-calcitonin gene-related peptide (α-CGRP). Here, we hypothesize that loss of SP, α-CGRP, and SNF modulates inflammatory and pain-related processes and also affects bone regeneration during fracture healing under postmenopausal conditions. Intramedullary fixed femoral fractures were set to 28 days after bilateral ovariectomy (OVX) in female wild type (WT), SP-, α-CGRP-deficient, and sympathectomized (SYX) mice. Locomotion, paw withdrawal threshold, fracture callus maturation and numbers of TRAP-, CD4-, CD8-, F4/80-, iNos-, and Arg1-positive cells within the callus were analyzed. Nightly locomotion was reduced in unfractured SP-deficient and SYX mice after fracture. Resistance to pressure was increased for the fractured leg in SP-deficient mice during the later stages of fracture healing, but was decreased in α-CGRP-deficient mice. Hypertrophic cartilage area was increased nine days after fracture in SP-deficient mice. Bony callus maturation was delayed in SYX mice during the later healing stages. In addition, the number of CD 4-positive cells was reduced after five days and the number of CD 8-positive cells was additionally reduced after 21 days in SYX mice. The number of Arg1-positive M2 macrophages was higher in α-CGRP-deficient mice five days after fracture. The alkaline phosphatase level was increased in SYX mice 16 days after fracture. Absence of α-CGRP appears to promote M2 macrophage polarization and reduces the pain threshold, but has no effect on callus tissue maturation. Absence of SP reduces locomotion, increases the pain-threshold, and accelerates hypertrophic callus tissue remodeling. Destruction of SNF reduces locomotion after fracture and influences bony callus tissue remodeling during the later stages of fracture repair, whereas pain-related processes are not affected. Full article

Patients with inflammatory bowel disease (IBD) often present poor bone health and are 40% more at risk of bone fracture. Studies have implicated autophagy in IBD pathology and drugs used to treat IBD stimulate autophagy in varying degrees, however, their effect on the skeleton is currently unknown. Here, we have utilised the dextran sulphate sodium (DSS) model of colitis in mice to examine the effects of the thiopurine drug azathioprine on the skeleton. Ten-week-old male mice (n = 6/group) received 3.0% DSS in their drinking water for four days, followed by a 14-day recovery period. Mice were treated with 10 mg/kg/day azathioprine or vehicle control. Histopathological analysis of the colon from DSS mice revealed significant increases in scores for inflammation severity, extent, and crypt damage (p < 0.05). Azathioprine provided partial protection to the colon, as reflected by a lack of significant difference in crypt damage and tissue regeneration with DSS treatment. MicroCT of vehicle-treated DSS mice revealed azathioprine treatment had a significant detrimental effect on the trabecular bone microarchitecture, independent of DSS treatment. Specifically, significant decreases were observed in bone volume/tissue volume (p < 0.01), and trabecular number (p < 0.05), with a concurrent significant increase in trabecular pattern factor (p < 0.01). Immunohistochemical labelling for LC3 revealed azathioprine to induce autophagy in the bone marrow. Together these data suggest that azathioprine treatment may have a deleterious effect on IBD patients who may already be at increased risk of osteoporotic bone fractures and thus will inform on future treatment strategies for patient stratification. Full article

Bone is one of the most important organs in the human body. It provides structure, function, and protection for other vital organs; therefore, bone maintenance and homeostasis are critical processes. As humans age, their bone mineral density decreases, which leads to diseases like osteoporosis. This disease affects one in two women and one in five men aged 50 and over. As the aging population increases, the interest and significance of studying this debilitating bone disease becomes more relevant. Current therapeutic products for osteoporosis have many side effects and can be taken for a limited number of years. Most therapeutic products only focus on decreasing bone resorption, not increasing bone formation. Bone morphogenetic protein 2 is an essential growth factor that drives osteoblast differentiation and activity and is essential for bone formation. However, usage in the clinic is unsuccessful due to several side effects. Recently, a signaling disparity in bone marrow stromal cells within the bone morphogenetic protein pathway that led to decreased bone morphogenetic protein 2 responsiveness was identified in patients diagnosed with osteoporosis. However, it is unclear how other cell populations, especially osteoblasts, which are key players in bone remodeling, are affected and whether the bone morphogenetic protein pathway is affected during osteoporosis. Our research group designed a novel peptide, casein kinase 2.3, that acts downstream of the bone morphogenetic receptor type Ia and increases bone mineralization in murine cells and primary bovine osteoblasts. The aim of the study presented here was to compare the responsiveness of osteoblasts to bone morphogenetic protein 2 and casein kinase 2.3, especially in patients diagnosed with osteoporosis. Mature osteoblasts were extracted from patients diagnosed with osteoporosis or osteoarthritis from Christiana Care Hospital in Newark, Delaware. They were stimulated with either bone morphogenetic protein 2 or casein kinase 2.3, and their effect on osteoblast activity was determined. The osteoporotic patients showed no mineralization response to bone morphogenetic protein 2 stimulation, while the osteoarthritis patients significantly responded to bone morphogenetic protein 2 stimulation. Furthermore, markers for osteoblast activity were increased by casein kinase 2.3, which was in sharp contrast to bone morphogenetic protein 2. This further supports a major bone morphogenetic protein signaling disparity in both the elderly and those suffering with osteoporosis. Both patient types did significantly respond to casein kinase 2.3. Further analysis of the bone morphogenetic protein pathway could lead to new therapeutic products for osteoporosis. Full article

Notch signaling is one of the evolutionarily conserved signaling pathways in multicellular organisms. It plays an important role in embryonic development. During skeletal development of vertebrates, it regulates bone homeostasis by manipulating both osteoblastogenesis and osteoclastogenesis through different mechanisms. However, due to the different nature of Notch signaling in mesenchymal stem cell and osteoblast, regulation of Notch signaling in bone-related diseases remains unsettled. Previous studies by cell culture and mouse models showed contradictory results regarding the role of Notch signaling in bone homeostasis. To clarify the role of Notch signaling in osteogenesis, we established a zebrafish model, in which Notch1a intracellular domain (N1aICD) was specifically expressed in the osteoblasts. We found that overexpression of N1aICD in osteoblasts caused hyperosteogeny in the column region of zebrafish with the morphology of narrowed neural/hemal canals. Moreover, increased metabolic activity of osteoblasts instead of augmenting osteoblast number led to hyperosteogeny in N1aICD-overexpressed zebrafish. In summary, we successfully established a transgenic zebrafish line overexpressing N1aICD to clarify the in-vivo function of Notch signaling during osteoblastogenesis. In the future, this fish line can serve as a valuable tool to test the therapeutic drugs for hyperosteogeny. Full article

As the population of western societies on average ages, the number of people affected by bone remodeling-associated diseases such as osteoporosis continues to increase. The development of new therapeutics is hampered by the high failure rates of drug candidates during clinical testing, which is in part due to the poor predictive character of animal models during preclinical drug testing. Co-culture models of osteoblasts and osteoclasts offer an alternative to animal testing and are considered to have the potential to improve drug development processes in the future. However, a robust, scalable, and reproducible 3D model combining osteoblasts and osteoclasts for preclinical drug testing purposes has not been developed to date. Here we review various types of osteoblast–osteoclast co-culture models and outline the remaining obstacles that must be overcome for their successful translation. Full article

Due to their potent immunomodulatory anti-inflammatory properties, synthetic glucocorticoids (GCs) are widely utilized in the treatment of chronic inflammatory disease. In this review, we examine our current understanding of how chronic inflammation and commonly used therapeutic GCs interact to regulate bone and muscle metabolism. Whilst both inflammation and therapeutic GCs directly promote systemic osteoporosis and muscle wasting, the mechanisms whereby they achieve this are distinct. Importantly, their interactions in vivo are greatly complicated secondary to the directly opposing actions of GCs on a wide array of pro-inflammatory signalling pathways that underpin catabolic and anti-anabolic metabolism. Several clinical studies have attempted to address the net effects of therapeutic glucocorticoids on inflammatory bone loss and muscle wasting using a range of approaches. These have yielded a wide array of results further complicated by the nature of inflammatory disease, underlying the disease management and regimen of GC therapy. Here, we report the latest findings related to these pathway interactions and explore the latest insights from murine models of disease aimed at modelling these processes and delineating the contribution of pre-receptor steroid metabolism. Understanding these processes remains paramount in the effective management of patients with chronic inflammatory disease. Full article

Extracellular signal-regulated kinase (ERK) is a member of the mitogen-activated protein kinase family of signaling molecules. ERK is predominantly found in two forms, ERK1 (p44) and ERK2 (p42), respectively. There are also several atypical forms of ERK, including ERK3, ERK4, ERK5 and ERK7. The ERK1/2 signaling pathway has been implicated in many and diverse cellular events, including proliferation, growth, differentiation, cell migration, cell survival, metabolism and transcription. ERK1/2 is activated (i.e., phosphorylated) in the cytosol and subsequently translocated to the nucleus, where it activates transcription factors including, but not limited to, ETS, c-Jun, and Fos. It is not surprising that the ERK1/2 signaling cascade has been implicated in many pathological conditions, namely, cancer, arthritis, chronic inflammation, and osteoporosis. This narrative review examines many of the cellular events in which the ERK1/2 signaling cascade plays a critical role. It is anticipated that agents designed to inhibit ERK1/2 activation or p-ERK1/2 activity will be developed for the treatment of those diseases characterized by dysregulated gene expression through ERK1/2 activation. Full article