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CSF‐1 is a key factor in regulating bone remodeling; osteocytes express CSF‐1 and its receptor. Viable osteocytes are essential for bone remodeling through cell–cell contact and secretion of factors that regulate osteoblasts and osteoclasts. Increased oxidative stress contributes to osteocyte death and correlates with bone loss during aging. The NADPH oxidase Nox4 is a major source of ROS in bone. CSF‐1 decreases Nox4, suggesting that CSF‐1 protects against oxidative stress. Here, we show that osteocyte apoptosis previously reported in our global CSF‐1KO mice is associated with increased Nox4, as well as 4‐HNE expression in osteocytes. Osteocytes isolated from CSF‐1KO mice were less viable and showed increased intracellular ROS, elevated NADPH oxidase activity/Nox4 protein, activation of mTOR/S6K, and downstream apoptosis signals compared with WT osteocytes. Nox4 expression was also increased in CSF‐1KO osteocytes and colocalized with MitoTracker Red in mitochondria. Notably, CSF‐1 inhibited Nox4 expression and apoptosis cascade signals. In additional studies, shNox4 decreased these signals in CSF‐1KO osteocytes, whereas overexpression of Nox4 in WT osteocytes activated the apoptosis pathway. To determine the role of CSF‐1 in osteocytes, DMP1Cre‐CSF‐1cKO (CSF‐1cKO) mice that lack CSF‐1 in osteocytes/late osteoblasts were developed. Osteocyte defects in CSF‐1cKO mice overlapped with those in CSF‐1KO mice, including increased apoptosis, Nox4, and 4‐HNE‐expressing osteocytes. CSF‐1cKO mice showed unbalanced cancellous bone remodeling with decreased bone formation and resorption. Continued exposure to high Nox4/ROS levels may further compromise bone formation and predispose to bone loss and skeletal fragility. Taken together, our findings suggest a novel link between CSF‐1, Nox4‐derived ROS, and osteocyte survival/function that is crucial for osteocyte‐mediated bone remodeling. Results reveal new mechanisms by which CSF‐1/oxidative stress regulate osteocyte homeostasis, which may lead to therapeutic strategies to improve skeletal health in aging. © 2018 American Society for Bone and Mineral Research

Hydration status significantly affects the toughness of bone. In addition to the collagen phase, recent evidence shows that glycosaminoglycans (GAGs) of proteoglycans (PGs) in the extracellular matrix also play a pivotal role in regulating the tissue-level hydration status of bone, thereby affecting the tissue-level toughness of bone. In this study, we hypothesized that the amount of GAGs in bone matrix decreased with age and such changes would lead to reduction in bound water and subsequently result in a decrease in the tissue-level toughness of bone. To test the hypothesis, nanoscratch tests were conducted to measure the tissue-level toughness of human cadaveric bone specimens, which were procured only from male donors in three different age groups: young (26 ± 6 years old), mid-aged (52 ± 5 years old) and elderly (73 ± 5 years old), with six donors in each group. Biochemical and histochemical assays were performed to determine the amount and major subtypes of GAGs and proteoglycans in bone matrix. In addition, low-field NMR measurements were implemented to determine bound water content in bone matrix. The results demonstrated that aging resulted in a statistically significant reduction (17%) of GAGs in bone matrix. Concurrently, a significant deterioration (20%) of tissue-level toughness of bone with age was observed. Most importantly, the deteriorated tissue-level toughness of bone was associated significantly with the age-related reduction (40%) of bound water, which was partially induced by the decrease of GAGs in bone matrix. Furthermore, we identified that chondroitin sulfate (CS) was a major subtype of GAGs and the amount of CS decreased with aging in accompany with a decrease of biglycan that is a major subtype of PGs in bone. The findings of this study suggests that reduction of GAGs in bone matrix is likely one of the molecular origins for age-related deterioration of bone quality.

Proteoglycans (PGs) are one type of noncollagenous proteins in the extracellular matrix of bone that primarily contain a core protein and glycosaminoglycans (GAGs). GAGs are highly polar and negatively charged, thus have a strong tendency to attract water molecules into the matrix. We hypothesized in this study that PGs in bone play a pivotal role in sustaining the toughness of the tissue only when water is present. To test the hypothesis, we used a novel nanoscratch test to measure the in situ toughness of human cadaveric bone treated with and without peptide-N-glycosidase F (PNGase F), an enzyme that specifically removes the N-linked oligosaccharides of GAGs from core proteins. Cortical bone specimens were prepared from the posterior aspect of mid-diaphyseal femurs of six (n = 6) male human donors between 51.5 ± 5.17 years old. Biochemical and histochemical assays were used to verify whether N-linked oligosaccharides were removed from bone matrix by PNGase F. By testing wet and dehydrated bone specimens, the coupling effect between water and PGs on the in situ toughness of bone was investigated. The two-way ANOVA analyses showed that removal of GAGs had significant effects on the in situ toughness of wet bone samples. In contrast, the removal of GAGs did not show significant effects on the toughness of dry bone. The results of this study, for the first time, suggest that GAGs play a pivotal role in the in situ toughness of bone only when water is present, and vice versa, water functions as a plasticizer in bone only when GAGs are present. © 2015 American Society for Bone and Mineral Research.

Norrie-Warburg syndrome (NWS) is a rare X-linked disorder characterized by blindness, which is invariable, deafness and mental disturbances, which are present occasionally. We describe here two novel mutations, a missense mutation (C126S) and a 1-base pair insertion (insT466/T467), together with a recurrent mutation (M1V), found in patients presenting with the classical clinical phenotype of NWS. All three mutations are likely to result in prominent structural changes of the norrin protein.