Archaeometabolomics characterizes phenotypic differences in human cortical bone at a molecular level relating to tobacco use.

Tobacco consumption affects human health, but no studies have investigated its effect on the bone metabolome, or if any changes are traceable after long postmortem intervals. Human osteoarchaeological remains preserve small molecules, making them valuable for studies that aim to examine past conditions. We test if there are molecular differences in the metabolome of cortical bone between archaeological individuals who used tobacco and those who did not, and if these differences are distinct enough to assign tobacco use status to individuals with unknown tobacco use. Cortical bone of 323 known and unknown tobacco users was studied by an untargeted metabolomics assay using a liquid chromatography-high-resolution mass spectrometry platform. We identified 45 discriminating molecular features that differed between tobacco consumers (15 up-regulated features) and nonconsumers (17 up-regulated features). Tobacco consumption leaves a metabolic record in human bone distinctive enough to identify its use in individuals of unknown tobacco consumption. Future work will validate molecular features relating to tobacco consumption.

HIF1 activation safeguards cortical bone formation against impaired oxidative phosphorylation.

Energy metabolism, through pathways such as oxidative phosphorylation (OxPhos) and glycolysis, plays a pivotal role in cellular differentiation and function. Our study investigates the impact of OxPhos disruption in cortical bone development by deleting mitochondrial transcription factor A (TFAM). TFAM controls OxPhos by regulating the transcription of mitochondrial genes. The cortical bone, constituting the long bones' rigid shell, is sheathed by the periosteum, a connective tissue layer populated with skeletal progenitors that spawn osteoblasts, the bone-forming cells. TFAM-deficient mice presented with thinner cortical bone, spontaneous midshaft fractures, and compromised periosteal cell bioenergetics, characterized by reduced ATP levels. Additionally, they exhibited an enlarged periosteal progenitor cell pool with impaired osteoblast differentiation. Increasing hypoxia-inducible factor 1a (HIF1) activity within periosteal cells substantially mitigated the detrimental effects induced by TFAM deletion. HIF1 is known to promote glycolysis in all cell types. Our findings underscore the indispensability of OxPhos for the proper accrual of cortical bone mass and indicate a compensatory mechanism between OxPhos and glycolysis in periosteal cells. The study opens new avenues for understanding the relationship between energy metabolism and skeletal health and suggests that modulating bioenergetic pathways may provide a therapeutic avenue for conditions characterized by bone fragility.

Extraction and untargeted analysis of metabolome from undemineralised cortical bone matrix.

Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) untargeted metabolomics has become the gold standard for the profiling of low-molecular-weight compounds. Recently, this discipline has raised great interest in forensic sciences, especially in the field of toxicology and for post-mortem interval estimation. The current study aims at evaluating three extraction protocols and two LC-MS/MS assays run in both positive and negative modes, to identify the most suitable method to conduct post-mortem metabolomic profiling of bone tissue. A fragment of the anterior tibia of a 82 years-old male sampled from a human taphonomy facility was powdered via freeze-milling. The powdered sub-samples were extracted in five replicates per protocol. Methods tested were (I) a biphasic chloroform-methanol-water protocol, (II) a single phase methanol-water protocol, and (III) a single phase methanol-acetonitrile-water protocol. LC-MS/MS analyses were carried out via high performance liquid chromatography, either on hydrophilic interaction (HILIC) or on reversed-phase (C18) columns in both positive and negative ionisation modes, coupled with a Q-TOF mass spectrometer. Results suggest that the highest consistency between replicates and quality control samples was obtained with the single phase extractions (i.e., methanol-acetonitrile-water), whilst the ideal combination of instrumental set up HILIC chromatography in positive ionisation mode and of C18 chromatography in negative ionisation mode. For the purpose of forensic investigations, a combination of a single phase extraction and the two aforementioned chromatographic and mass spectrometry modes could represent an ideal set up for obtaining bone metabolomic profiles from taphonomically altered bones.

A map of glycation and glycoxidation sites in collagen I of human cortical bone: Effects of sex and type 2 diabetes.

Complications of diabetes is a major health problem affecting multiple organs including bone, where the chronic disease increases the risk of fragility fractures. One hypothesis suggests a pathogenic role for hyperglycemia-induced modification of proteins, a.k.a. advanced glycation end products (AGEs), resulting in structural and functional damage to bone extracellular matrix (ECM). Evidence supporting this hypothesis has been limited by the lack of comprehensive information about the location of AGEs that accumulate in vivo at specific sites within the proteins of bone ECM. Analyzing extracts from cortical bone of cadaveric femurs by liquid chromatography tandem mass spectrometry, we generated a quantitative AGE map of human collagen I for male and female adult donors with and without diabetes. The map describes the chemical nature, sequence position, and levels of four major physiological AGEs, e.g. carboxymethyllysine, and an AGE precursor fructosyllysine within the collagen I triple-helical region. The important features of the map are: 1) high map reproducibility in the individual bone extracts, i.e. 20 male and 20 female donors; 2) localization of modifications to distinct clusters: 10 clusters containing 34 AGE sites in male donors and 9 clusters containing 28 sites in female donors; 3) significant increases in modification levels in diabetes at multiple sites: 26 out of 34 sites in males and in 17 out of 28 sites in females; and 4) generally higher modification levels in male vs. female donors. Moreover, the AGE levels at multiple individual sites correlated with total bone pentosidine levels in male but not in female donors. Molecular dynamics simulations and molecular modeling predicted significant impact of modifications on solvent exposure, charge distribution, and hydrophobicity of the triple helix as well as disruptions to the structure of collagen I fibril. In summary, the AGE map of collagen I revealed diabetes-induced, sex-specific non-enzymatic modifications at distinct triple helical sites that can disrupt collagen structure, thus proposing a specific mechanism of AGE contribution to diabetic complications in human bone.

Direct activation of PI3K in osteoblasts and osteocytes strengthens murine bone through sex-specific actions on cortical surfaces.

Intracellular phosphoinositide 3-kinase (PI3K) signaling is activated by multiple bone-active receptors. Genetic mutations activating PI3K signaling are associated with clinical syndromes of tissue overgrowth in multiple organs, often including the skeleton. While one formation is increased by removing the PI3K inhibitor (phosphatase and TENsin homolog deleted on chromosome 10 (PTEN)), the effect of direct PI3K activation in the osteoblast lineage has not been reported. We introduced a known gain-of-function mutation in Pik3ca, the gene encoding the p110α catalytic subunit of PI3K, in osteocytes and late osteoblasts using the dentin matrix protein-1 Cre (Dmp1Cre) mouse and assessed the skeletal phenotype. Femur shape was grossly normal, but cortical thickness was significantly greater in both male and female Dmp1Cre.Pik3caH1047R mice, leading to almost doubled bone strength at 12 wk of age. Both sexes had smaller marrow areas from 6 wk of age. Female mice also exhibited greater cross-sectional area, which continued to increase until 24 wk of age, resulting in a further increase in bone strength. Although both male and female mice had increased endocortical mineralizing surface, only female mice had increased periosteal mineralizing surface. The bone formed in the Dmp1Cre.Pik3caH1047R mice showed no increase in intracortical remodeling nor any defect in cortical bone consolidation. In contrast, on both endocortical and periosteal surfaces, there was more lamellar bone formation, including highly organized osteocyte networks extending along the entire surface at a greater thickness than in control mice. In conclusion, direct activation of PI3Kα in cells targeted by Dmp1Cre leads to high cortical bone mass and strength with abundant lamellar cortical bone in female and male mice with no increase in intracortical remodeling. This differs from the effect of PTEN deletion in the same cells, suggesting that activating PI3Kα in osteoblasts and osteocytes may be a more suitable target to promote formation of lamellar bone.

Patients with genetic activation of enzymes called phosphoinositide-3 kinase (PI3K) have tissue overgrowth syndromes, where parts of the body become enlarged, sometimes including the skeleton. There are 2 types of mutations that cause this: one that directly activates the PI3K enzyme, and one that removes the normal brake on PI3K signaling (called PTEN). We tested the effect of directly activating a PI3K enzyme specifically in osteoblasts (the cells that form bone) and osteocytes (osteoblasts that make a network inside the bone tissue itself). We found that mice with these mutations had very strong bones with an outer shell that was thicker than usual. In both male and female mice, it became thicker on the inside of the shell, but in female mice it also became thicker on the outside, making the bones even stronger over time. The new bone was well-organized, which likely helped make the increase in bone strength so profound. This is very different to previous studies of mice with the other type of mutation in their bone-forming cells; they had a shell with many large holes (pores). This indicates that directly stimulating PI3K enzyme is more beneficial for bone than removing the PTEN brake.

Calorie restriction in mice impairs cortical but not trabecular peak bone mass by suppressing bone remodeling.

Calorie restriction (CR) can lead to weight loss and decreased substrate availability for bone cells. Ultimately, this can lead to impaired peak bone acquisition in children and adolescence and bone loss in adults. But the mechanisms that drive diet-induced bone loss in humans are not well characterized. To explore those in greater detail, we examined the impact of 30% CR for 4 and 8 wk in both male and female 8-wk-old C57BL/6 J mice. Body composition, areal bone mineral density (aBMD), skeletal microarchitecture by micro-CT, histomorphometric parameters, and in vitro trajectories of osteoblast and adipocyte differentiation were examined. After 8 wk, CR mice lost weight and exhibited lower femoral and whole-body aBMD vs ad libitum (AL) mice. By micro-CT, CR mice had lower cortical bone area fraction vs AL mice, but males had preserved trabecular bone parameters and females showed increased bone volume fraction compared to AL mice. Histomorphometric analysis revealed that CR mice had a profound suppression in trabecular as well as endocortical and periosteal bone formation in addition to reduced bone resorption compared to AL mice. Bone marrow adipose tissue was significantly increased in CR mice. In vitro, the pace of adipogenesis in bone marrow stem cells was greatly accelerated with higher markers of adipocyte differentiation and more oil red O staining, whereas osteogenic differentiation was reduced. qRT-PCR and western blotting suggested that the expression of Wnt16 and the canonical ß-catenin pathway was compromised during CR. In sum, CR causes impaired peak cortical bone mass due to a profound suppression in bone remodeling. The increase in marrow adipocytes in vitro and in vivo is related to both progenitor recruitment and adipogenesis in the face of nutrient insufficiency. Long-term CR may lead to lower bone mass principally in the cortical envelope, possibly due to impaired Wnt signaling.

Calorie restriction led to impaired bone mass and increased accumulation of bone marrow adipose tissue. During the development of bone-fat imbalance due to calorie restriction, bone remodeling was notably inhibited. Calorie restriction may shift the differentiation of bone marrow stem cells toward adipocytes instead of osteoblasts. This process involves a disruption in the canonical Wnt signaling pathway.

Osteocyte ferroptosis induced by ATF3/TFR1 contributes to cortical bone loss during ageing.

Cortical bone loss is intricately associated with ageing and coincides with iron accumulation. The precise role of ferroptosis, characterized by iron overload and lipid peroxidation, in senescent osteocytes remains elusive. We found that ferroptosis was a crucial mode of osteocyte death in cortical bone during ageing. Using a single-cell transcriptome analysis, we identified activating transcription factor 3 (ATF3) as a critical driver of osteocyte ferroptosis. Elevated ATF3 expression in senescent osteocytes promotes iron uptake by upregulating transferrin receptor 1 while simultaneously inhibiting solute carrier family 7-member 11-mediated cystine import. This process leads to an iron overload and lipid peroxidation, culminating in ferroptosis. Importantly, ATF3 inhibition in aged mice effectively alleviated ferroptosis in the cortical bone and mitigated cortical bone mass loss. Taken together, our findings establish a pivotal role of ferroptosis in cortical bone loss in older adults, providing promising prevention and treatment strategies for osteoporosis and fractures.

Immunohistochemical and Morphometric Assessment on the Biological Function and Vascular Endothelial Cells in the Initial Process of Cortical Porosity in Mice With PTH Administration.

To clarify the cellular mechanism of cortical porosity induced by intermittent parathyroid hormone (PTH) administration, we examined the femoral cortical bone of mice that received 40 µg/kg/day (four times a day) human PTH (hPTH) (1-34). The PTH-driven cortical porosity initiated from the metaphyseal region and chronologically expanded toward the diaphysis. Alkaline phosphatase (ALP)-positive osteoblasts in the control mice covered the cortical surface, and endomucin-positive blood vessels were distant from these osteoblasts. In PTH-administered mice, endomucin-reactive blood vessels with TRAP-positive penetrated the ALP-positive osteoblast layer, invading the cortical bone. Statistically, the distance between endomucin-positive blood vessels and the cortical bone surface abated after PTH administration. Transmission electron microscopic observation demonstrated that vascular endothelial cells often pass through the flattened osteoblast layer and accompanied osteoclasts in the deep region of the cortical bone. The cell layers covering mature osteoblasts thickened with PTH administration and exhibited ALP, α-smooth muscle actin (αSMA), vascular cell adhesion molecule-1 (VCAM1), and receptor activator of NF-κB ligand (RANKL). Within these cell layers, osteoclasts were found near endomucin-reactive blood vessels. In PTH-administered femora, osteocytes secreted Dkk1, a Wnt inhibitor that affects angiogenesis, and blood vessels exhibited plasmalemma vesicle-associated protein, an angiogenic molecule. In summary, endomucin-positive blood vessels, when accompanied by osteoclasts in the ALP/αSMA/VCAM1/RANKL-reactive osteoblastic cell layers, invade the cortical bone, potentially due to the action of osteocyte-derived molecules such as DKK1.

Osteomodulin deficiency in mice causes a specific reduction of transversal cortical bone size.

Skeletal growth, modeling, and remodeling are regulated by various molecules, one of them being the recently identified osteoanabolic factor WNT1. We have previously reported that WNT1 transcriptionally activates the expression of Omd, encoding Osteomodulin (OMD), in a murine mesenchymal cell line, which potentially explained the skeletal fragility of mice with mutational WNT1 inactivation, since OMD has been shown to regulate type I collagen fibril formation in vitro. In this study we confirmed the strong induction of Omd expression in a genome-wide expression analysis of transfected cells, and we obtained further evidence for Omd being a direct target gene of WNT1. To assess the in vivo relevance of this regulation, we crossed Omd-deficient mice with a mouse line harboring an inducible, osteoblast-specific Wnt1 transgene. After induction of Wnt1 expression for 1 or 3 weeks, the osteoanabolic potency of WNT1 was not impaired despite the Omd deficiency. Since current knowledge regarding the in vivo physiological function of OMD is limited, we next focused on skeletal phenotyping of wild-type and Omd-deficient littermates, in the absence of a Wnt1 transgene. Here we did not observe an impact of Omd deficiency on trabecular bone parameters by histomorphometry and µCT either. Importantly, however, male and female Omd-deficient mice at the ages of 12 and 24 weeks displayed a slender bone phenotype with significantly smaller long bones in the transversal dimension, while the longitudinal bone growth remained unaffected. Although mechanical testing revealed no significant changes explained by impaired bone material properties, atomic force microscopy of the femoral bone surface of Omd-deficient mice revealed moderate changes at the nanostructural level, indicating altered regulation of collagen fibril formation and aggregation. Taken together, our data demonstrate that, although OMD is dispensable for the osteoanabolic effect of WNT1, its deficiency in mice specifically modulates transversal cortical bone morphology.

We explored the physiological relevance of the protein Osteomodulin (OMD) that we previously found to be induced by the osteoanabolic molecule WNT1. While other studies have shown that OMD is involved in the regulation of collagen fibril formation in vitro, its function in vivo has not been investigated. We confirmed that OMD is directly regulated by WNT1 but surprisingly, when we bred mice lacking OMD with mice engineered to highly express WNT1, we found that the osteoanabolic effect of WNT1 was unaffected by the absence of OMD. Interestingly, mice lacking OMD did show differences in the shape of their bones, particularly in their width, despite no significant changes in bone density or length. Investigation of the bone matrix of mice lacking OMD at the nanostructural level indicated moderate differences in the organization of collagen fibrils. This study provided further insights into the effect of WNT1 on bone metabolism and highlighted a specific function of OMD in skeletal morphology.

Fracture behavior of human cortical bone with high glycation content under dynamic loading.

The present study simulates the fracture behavior of diabetic cortical bone with high levels of advanced glycation end-products (AGEs) under dynamic loading. We consider that the increased AGEs in diabetic cortical bone degrade the materials heterogeneity of cortical bone through a reduction in critical energy release rates of the microstructural features. To simulate the initiation and propagation of cracks, we implement a phase field fracture framework on 2D models of human tibia cortical microstructure. The simulations show that the mismatch between the fracture properties (e.g., critical energy release rate) of osteons and interstitial tissue due to high AGEs contents can change crack growth trajectories. The results show crack branching in the cortical microstructure under dynamic loading is affected by the mismatches related to AGEs. In addition, we observe cortical features such as osteons and cement lines can prevent multiple cracking under dynamic loading even with changing the mismatches due to high AGEs. Furthermore, under dynamic loading, some toughening mechanisms can be activated and deactivated with different AGEs contents. In conclusion, the current findings present that the combination of the loading type and materials heterogeneity of microstructural features can change the fracture response of diabetic cortical bone and its fragility.

Ultrashort echo time MRI detects significantly lower collagen but higher pore water in the tibial cortex of female patients with osteopenia and osteoporosis.

Ultrashort echo time (UTE) MRI can quantify the major proton pool densities in cortical bone, including total (TWPD), bound (BWPD), and pore water (PWPD) proton densities, as well as the macromolecular proton density (MMPD), associated with the collagen content, which is calculated using macromolecular fraction (MMF) from UTE magnetization transfer (UTE-MT) modeling. This study aimed to investigate the differences in water and collagen contents in tibial cortical bone, between female osteopenia (OPe) patients, osteoporosis (OPo) patients, and young participants (Young). Being postmenopausal and above 55 yr old were the inclusion criteria for OPe and OPo groups. The tibial shaft of 14 OPe (72.5 ± 6.8 yr old), 31 OPo (72.0 ± 6.4 yr old), and 31 young subjects (28.0 ± 6.1 yr old) were scanned using a knee coil on a clinical 3T scanner. Basic UTE, inversion recovery UTE, and UTE-MT sequences were performed. Investigated biomarkers were compared between groups using Kruskal-Wallis test. Spearman's correlation coefficients were calculated between the TH DXA T-score and UTE-MRI results. MMF, BWPD, and MMPD were significantly lower in OPo patients than in the young group, whereas T1, TWPD, and PWPD were significantly higher in OPo patients. The largest OPo/Young average percentage differences were found in MMF (41.9%), PWPD (103.5%), and MMPD (64.0%). PWPD was significantly higher (50.7%), while BWPD was significantly lower (16.4%) in OPe than the Young group on average. MMF was found to be significantly lower (27%) in OPo patients compared with OPe group. T1, MMF, TWPD, PWPD, and MMPD values significantly correlated with the TH DXA T-scores (provided by the patients and only available for OPe and OPo patients). DXA T-score showed the highest correlations with PWPD (R = 0.55) and MMF (R = 0.56) values. TWPD, PWPD, and MMF estimated using the UTE-MRI sequences were recommended to evaluate individuals with OPe and OPo.

Ultrashort echo time (UTE) is an MRI technique that can quantify the water and collagen content of cortical bone. Water in the bone can be found residing in pores (pore water) or bound to the bone matrix (bound water). We investigated the differences in water and collagen contents of tibial cortical bone between female osteopenia patients, osteoporosis patients, and young participants. Bound water and collagen contents were significantly lower in osteoporosis patients than in the young group, whereas total water and pore water contents were significantly higher in osteoporosis patients. Pore water was significantly higher, while bound water was significantly lower in osteopenia than in the Young group. Collagen content was found to be significantly lower in osteoporosis patients compared with the osteopenia group. The estimated water and collagen contents were significantly correlated with the TH bone densitometry measures in the patients.

In-silico simulation of nanoindentation on bone using a 2D cohesive finite element model.

This study proposed and validated a 2D finite element (FE) model for conducting in-silico simulations of in-situ nanoindentation tests on mineralized collagen fibrils (MCF) and the extrafibrillar matrix (EFM) within human cortical bone. Initially, a multiscale cohesive FE model was developed by adapting a previous model of bone lamellae, encompassing both MCF and EFM. Subsequently, nanoindentation tests were simulated in-silico using this model, and the resulting predictions were compared to AFM nanoindentation test data to verify the model's accuracy. The FE model accurately predicted nanoindentation results under wet conditions, closely aligning with outcomes obtained from AFM nanoindentation tests. Specifically, it successfully mirrored the traction/separation curve, nanoindentation modulus, plastic energy dissipation, and plastic energy ratio obtained from AFM nanoindentation tests. Additionally, this in-silico model demonstrated its ability to capture alterations in nanoindentation properties caused by the removal of bound water, by considering corresponding changes in mechanical properties of the collagen phase and the interfaces among bone constituents. Notably, significant changes in the elastic modulus and plastic energy dissipation were observed in both MCF and EFM compartments of bone, consistent with observations in AFM nanoindentation tests. These findings indicate that the proposed in-silico model effectively captures the influence of ultrastructural changes on bone's mechanical properties at sub-lamellar levels. Presently, no experimental methods exist to conduct parametric studies elucidating the ultrastructural origins of bone tissue fragility. The introduction of this in-silico model presents an invaluable tool to bridge this knowledge gap in the future.

miR181a/b-1 controls osteocyte metabolism and mechanical properties independently of bone morphology.

Bone derives its ability to resist fracture from bone mass and quality concurrently; however, many questions about the molecular mechanisms controlling bone quality remain unanswered, limiting the development of diagnostics and therapeutics. Despite the increasing evidence on the importance of miR181a/b-1 in bone homeostasis and disease, whether and how osteocyte-intrinsic miR181a/b-1 controls bone quality remains elusive. Osteocyte-intrinsic deletion of miR181a/b-1 in osteocytes in vivo resulted in compromised overall bone mechanical behavior in both sexes, although the parameters affected by miR181a/b-1 varied distinctly based on sex. Furthermore, impaired fracture resistance in both sexes was unexplained by cortical bone morphology, which was altered in female mice and intact in male mice with miR181a/b-1-deficient osteocytes. The role of miR181a/b-1 in the regulation of osteocyte metabolism was apparent in bioenergetic testing of miR181a/b-1-deficient OCY454 osteocyte-like cells and transcriptomic analysis of cortical bone from mice with osteocyte-intrinsic ablation of miR181a/b-1. Altogether, this study demonstrates the control of osteocyte bioenergetics and the sexually dimorphic regulation of cortical bone morphology and mechanical properties by miR181a/b-1, hinting at the role of osteocyte metabolism in the regulation of mechanical behavior.

Delayed cortical bone healing due to impaired nuclear Nrf2 translocation in COPD mice.

The effect of the pathogenesis of chronic obstructive pulmonary disease (COPD) on bone fracture healing is unknown. Oxidative stress has been implicated in the systemic complications of COPD, and decreased activity of Nrf2 signaling, a central component of the in vivo antioxidant mechanism, has been reported. We investigated the process of cortical bone repair in a mouse model of elastase-induced emphysema by creating a drill hole and focusing on Nrf2 and found that the amount of new bone in the drill hole was reduced and bone formation capacity was decreased in the model mice. Furthermore, nuclear Nrf2 expression in osteoblasts was reduced in model mice. Sulforaphane, an Nrf2 activator, improved delayed cortical bone healing in model mice. This study indicates that bone healing is delayed in COPD mice and that impaired nuclear translocation of Nrf2 is involved in delayed cortical bone healing, suggesting that Nrf2 may be a novel target for bone fracture treatment in COPD patients.

Orchiectomy sensitizes cortical bone in male mice to the harmful effects of kynurenine.

Kynurenine (Kyn) is a tryptophan metabolite that increases with age and promotes musculoskeletal dysfunction. We previously found a sexually dimorphic pattern in how Kyn affects bone, with harmful effects more prevalent in females than males. This raises the possibility that male sex steroids might exert a protective effect that blunts the effects of Kyn in males. To test this, orchiectomy (ORX) or sham surgeries were performed on 6-month-old C57BL/6 mice, after which mice received Kyn (10 mg/kg) or vehicle via intraperitoneal injection, once daily, 5×/week, for four weeks. Bone histomorphometry, DXA, microCT, and serum marker analyses were performed after sacrifice. In vitro studies were performed to specifically test the effect of testosterone on activation of aryl hydrocarbon receptor (AhR)-mediated signaling by Kyn in mesenchymal-lineage cells. Kyn treatment reduced cortical bone mass in ORX- but not sham-operated mice. Trabecular bone was unaffected. Kyn's effects on cortical bone in ORX mice were attributed primarily to enhanced endosteal bone resorption activity. Bone marrow adipose tissue was increased in Kyn-treated ORX animals but was unchanged by Kyn in sham-operated mice. ORX surgery increased mRNA expression of the aryl hydrocarbon receptor (AhR) and its target gene Cyp1a1 in the bone, suggesting a priming and/or amplification of AhR signaling pathways. Mechanistic in vitro studies revealed that testosterone blunted Kyn-stimulated AhR transcriptional activity and Cyp1a1 expression in mesenchymal-linage cells. These data suggest a protective role for male sex steroids in blunting the harmful effects of Kyn in cortical bone. Therefore, testosterone may play an important role in regulating Kyn/AhR signaling in musculoskeletal tissues, suggesting crosstalk between male sex steroids and Kyn signaling may influence age-associated musculoskeletal frailty.

Piezo1 opposes age-associated cortical bone loss.

As we age, our bones undergo a process of loss, often accompanied by muscle weakness and reduced physical activity. This is exacerbated by decreased responsiveness to mechanical stimulation in aged skeleton, leading to the hypothesis that decreased mechanical stimulation plays an important role in age-related bone loss. Piezo1, a mechanosensitive ion channel, is critical for bone homeostasis and mechanotransduction. Here, we observed a decrease in Piezo1 expression with age in both murine and human cortical bone. Furthermore, loss of Piezo1 in osteoblasts and osteocytes resulted in an increase in age-associated cortical bone loss compared to control mice. The loss of cortical bone was due to an expansion of the endosteal perimeter resulting from increased endocortical resorption. In addition, expression of Tnfrsf11b, encoding anti-osteoclastogenic protein OPG, decreases with Piezo1 in vitro and in vivo in bone cells, suggesting that Piezo1 suppresses osteoclast formation by promoting Tnfrsf11b expression. Our results highlight the importance of Piezo1-mediated mechanical signaling in protecting against age-associated cortical bone loss by inhibiting bone resorption in mice.

Sostdc1 Suppression in the Absence of Sclerostin Potentiates Anabolic Action of Cortical Bone in Mice.

The development of Wnt-based osteoanabolic agents has progressed rapidly in recent years, given the potent effects of Wnt modulation on bone homeostasis. Simultaneous pharmacologic inhibition of the Wnt antagonists sclerostin and Dkk1 can be optimized to create potentiated effects in the cancellous bone compartment. We looked for other candidates that might be co-inhibited along with sclerostin to potentiate the effects in the cortical compartment. Sostdc1 (Wise), like sclerostin and Dkk1, also binds and inhibits Lrp5/6 coreceptors to impair canonical Wnt signaling, but Sostdc1 has greater effects in the cortical bone. To test this concept, we deleted Sostdc1 and Sost from mice and measured the skeletal effects in cortical and cancellous compartments individually. Sost deletion alone produced high bone mass in all compartments, whereas Sostdc1 deletion alone had no measurable effects on either envelope. Mice with codeletion of Sostdc1 and Sost had high bone mass and increased cortical properties (bone mass, formation rates, mechanical properties), but only among males. Combined administration of sclerostin antibody and Sostdc1 antibody in wild-type female mice produced potentiation of cortical bone gain despite no effect of Sostdc1 antibody alone. In conclusion, Sostdc1 inhibition/deletion can work in concert with sclerostin deficiency to improve cortical bone properties. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Single cell cortical bone transcriptomics define novel osteolineage gene sets altered in chronic kidney disease.

Introduction: Due to a lack of spatial-temporal resolution at the single cell level, the etiologies of the bone dysfunction caused by diseases such as normal aging, osteoporosis, and the metabolic bone disease associated with chronic kidney disease (CKD) remain largely unknown. Methods: To this end, flow cytometry and scRNAseq were performed on long bone cells from Sost-cre/Ai9+ mice, and pure osteolineage transcriptomes were identified, including novel osteocyte-specific gene sets. Results: Clustering analysis isolated osteoblast precursors that expressed Tnc, Mmp13, and Spp1, and a mature osteoblast population defined by Smpd3, Col1a1, and Col11a1. Osteocytes were demarcated by Cd109, Ptprz1, Ramp1, Bambi, Adamts14, Spns2, Bmp2, WasI, and Phex. We validated our in vivo scRNAseq using integrative in vitro promoter occupancy via ATACseq coupled with transcriptomic analyses of a conditional, temporally differentiated MSC cell line. Further, trajectory analyses predicted osteoblast-to-osteocyte transitions via defined pathways associated with a distinct metabolic shift as determined by single-cell flux estimation analysis (scFEA). Using the adenine mouse model of CKD, at a time point prior to major skeletal alterations, we found that gene expression within all stages of the osteolineage was disturbed. Conclusion: In sum, distinct populations of osteoblasts/osteocytes were defined at the single cell level. Using this roadmap of gene assembly, we demonstrated unrealized molecular defects across multiple bone cell populations in a mouse model of CKD, and our collective results suggest a potentially earlier and more broad bone pathology in this disease than previously recognized.

SIRT1 haplo-insufficiency results in reduced cortical bone thickness, increased porosity and decreased estrogen receptor alpha in bone in adult 129/Sv female mice.

Introduction: Sirtuin 1 (SIRT1) is a key player in aging and metabolism and regulates bone mass and architecture. Sexual dimorphism in skeletal effects of SIRT1 has been reported, with an unfavorable phenotype primarily in female mice. Methods: To investigate the mechanisms of gender differences in SIRT1 skeletal effect, we investigated femoral and vertebral cortical and cancellous bone in global Sirt1 haplo-insufficient 129/Sv mice aged 2,7,12 months lacking Sirt1 exons 5,6,7 (Sirt1+/Δ ) and their wild type (WT) counterparts. Results: In females, femoral bone mineral content, peak cortical thickness, and trabecular bone volume (BV/TV%), number and thickness were significantly lower in Sirt1+/Δ compared to WT mice. Increased femoral cortical porosity was observed in 7-month-old Sirt1+/Δ compared to WT female mice, accompanied by reduced biomechanical strength. No difference in vertebral indices was detected between Sirt1+/Δ and WT female mice. SIRT1 decreased with aging in WT female mice and was lower in vertebrae and femur in 18- and 30- versus 3-month-old 129/Sv and C57BL/6J female mice, respectively. Decreased bone estrogen receptor alpha (ERα) was observed in Sirt1+/Δ compared to WT female mice and was significantly higher in Sirt1 over-expressing C3HT101/2 murine mesenchymal stem cells. In males no difference in femoral indices was detected in Sirt1+/Δ versus WT mice, however vertebral BV/TV%, trabecular number and thickness were higher in Sirt1+/Δ vs. WT mice. No difference in androgen receptor (AR) was detected in bone in Sirt1+/Δ vs. WT male mice. Bone SIRT1 was significantly lower in male compared to female WT mice, suggesting that SIRT1 maybe more significant in female than male skeleton. Discussion: These findings demonstrate that 50% reduction in SIRT1 is sufficient to induce the hallmarks of skeletal aging namely, decreased cortical thickness and increased porosity in female mice, highlighting the role of SIRT1 as a regulator of cortical bone quantity and quality. The effects of SIRT1 in cortical bone are likely mediated in part by its regulation of ERα. The age-associated decline in bone SIRT1 positions SIRT1 as a potential therapeutic target to ameliorate age-related cortical bone deterioration in females. The crosstalk between ERα, AR and SIRT1 in the bone microenvironment remains to be further investigated.