Bone

Bone is a dynamic tissue that continuously adapts its structure in response to mechanical loading, an essential process for maintaining skeletal health. However, this adaptive capacity declines with aging, contributing to increased fragility and fracture risk. Developing therapeutic strategies that preserve or restore bone mechanoadaptation in patients with increased bone fragility requires identifying key molecular regulators of this process. We applied spatial transcriptomics (GeoMx, NanoString) to characterize gene expression changes induced by mechanical loading in the murine tibia, focusing on periosteal and bone compartments in regions under tension and compression. Spatial data were validated and cross-compared with previously published bulk RNA-seq and laser-capture microdissection datasets, identifying a set of 12 genes consistently regulated by loading across independent platforms and laboratories. As part of a functional analysis, we selected Slc13a5, a citrate transporter implicated in bone mineralization and metabolism. Conditional deletion of Slc13a5 in osteolineage cells using Osteocalcin-Cre significantly increased the loading-induced mineralizing surface in tensile regions compared with Cre- Slc13a5fl/fl littermates. In addition, Slc13a5 cKO mice exhibited lower resorption around the neutral axis after loading compared to controls. Together, these findings identify Slc13a5 as a regulator of bone adaptation in regions experiencing low mechanical stimulation and suggest it as a potential therapeutic target for conditions characterized by impaired mechanoadaptive responses. This study highlights spatial transcriptomics as a powerful gene discovery framework for bone, enabling identification of novel targets to understand mechanisms and develop therapies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=181 SRC="FIGDIR/small/711126v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@5bf180org.highwire.dtl.DTLVardef@4c33b7org.highwire.dtl.DTLVardef@d75668org.highwire.dtl.DTLVardef@169fa97_HPS_FORMAT_FIGEXP M_FIG C_FIG

The porous bone cortex comprises an interconnected network of intracortical vascular canals and osteocyte lacunae, embedded within the bone mineral. Increases in cortical porosity reduce bone strength and increase fracture risk. To date, our understanding of mechanisms coupling the arrangements of the vascular: lacunar network in the bone cortex is poorly understood yet it could be key in establishing regulation of cortical porosity evident with age. Using synchrotron radiation-based computed tomography we develop automated tools to characterise the 3D spatial organisation and morphology of osteocyte lacunae, and the bone vasculature at the tibiofibular junction (TFJ), defining posterior, medial, lateral, and anterior regions in male C57BL/6 mice (n = 3). We also investigate the role of osteoblast-derived VEGF in regulating the 3D spatial arrangement by conditional disruption of VEGF in osteocalcin-expressing cells (OcnVEGFKO versus WT, n = 3). Regional lacunar phenotypes were assessed by 3D distance mapping of lacunar organisation surrounding the vascular compartments, including endosteal and periosteal surfaces, or intracortical canals. Surface-associated lacunae were indistinct in size across posterior, medial, lateral and anterior regions. However, lacunae associated with intracortical canals were significantly larger exclusively within the posterior region. In the absence of VEGF, the increased lacunar volume associated with posterior intracortical canals was lost. Our results suggest that the influence of intracortical canals on lacunar volumes is spatially regulated and sensitive to locally produced growth factors such as osteoblast-derived VEGF.

In adult mice, new bone accrual following mechanical load is mediated by the neurotrophin nerve growth factor (NGF) that is expressed by osteoblasts on the bone surface. NGF can bind to its high affinity receptor, neurotrophic tyrosine kinase receptor type 1 (TrkA), on peripheral sensory nerves resident in bone and support new bone formation. However, the osteoanabolic therapeutic potential of NGF-TrkA signaling to repair bone is limited due to the long-lasting thermal and mechanical hyperalgesia induced by administration of NGF in mice and humans. Here, we investigated whether 1) mature osteoblasts are the primary source of NGF required for bone accrual following loading, and 2) a small molecule TrkA receptor agonist - gambogic amide - can harness the downstream osteoanabolic potential of NGF-TrkA signaling in the absence of endogenous NGF. Loss of Ngf transcription in mature osteoblasts did not appear to affect bone structure or bone mass in adulthood. However, Ngf knockout mice significantly reduced periosteal bone accrual and osteogenic Wnt transcription in response to loading compared to wildtype mice. Intraperitoneal injection of gambogic amide prior to loading was unable to produce its osteoanabolic effects in Ngf knockout mice, suggesting that gambogic amide primarily functions in collaboration with endogenous NGF in bone. In total, our study reveals an important role for osteoblastic NGF in the skeletal adaptation of bone to mechanical forces.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a crucial connective component between the nuclear envelope and the cytoskeleton involving various cellular processes including nuclear positioning, nuclear architecture, and mechanotransduction. How LINC complexes regulate bone formation in vivo, however, is not well understood. To start bridging this gap, here we created a LINC disruption murine model using transgenic mice expressing Cre recombinase enzyme under the control of the Osterix (Osx-Cre) which is primarily active in pre-osteoblasts and floxed Tg(CAG-LacZ/EGFP-KASH2) mice. Tg(CAG-LacZ/EGFP-KASH2) mice contain a lox-STOP-lox flanked LacZ gene which is deleted upon cre recombination allowing for the overexpression of an EGFP-KASH2 fusion protein. This overexpressed protein disrupts endogenous Nesprin-Sun binding leading to disruption of LINC complexes. Thus, crossing these two lines results in a Osx-driven LINC disruption (ODLD) specific to pre-osteoblasts. In this study, we investigated how this LINC disruption affects exercise induced bone accrual. ODLD cells had decreased osteogenic and adipogenic potential in vitro compared to non-disrupted controls and sedentary ODLD mice showed decreased bone quality at 8-weeks. Upon access to a voluntary running wheel ODLD animals showed increased running time and distance; however, our 6-week exercise intervention did not significantly affect bone microarchitecture and bone mechanical properties.

INTRODUCTION: Osteogenesis imperfecta (OI) is a genetic disorder primarily due to mutations in collagen type I-encoding genes, resulting in fragile bones, frequent fractures, pain, and mobility issues. Disease severity and phenotype vary widely, even with the same mutation, suggesting the importance of other factors within the bone microenvironment that influence disease severity. To study the role of such factors, we analyzed bone samples from OI patients and healthy controls using single-cell RNA sequencing to reveal if RNA expression profiles may uncover mechanisms behind OI phenotype. METHODS: Bone samples from surgeries of OI patients and healthy individuals isolated and RNA single-cell sequencing was performed, followed by quality control and bioinformatics analysis. Two healthy and three OI patients were included: two with type-I OI, characterized by a mutation in COL1A1 (collagen type I), and another with type-VIII OI, associated with LEPRE1 mutations, which disrupt the 3-hydroxylation of type I collagen. RESULTS: Clustering and differential expression analysis showed distinct subpopulations in mesenchymal and immune cells. In all OI samples, mesenchymal stromal cell (MSC) proportions were reduced compared to healthy controls. OI type-I patients showed decreased osteoblast numbers alongside an increase in osteoclast precursor cells. Whereas in OI type-VIII, all bone turnover-related cells (osteoblast, osteoclast precursor, and osteoclast) were elevated. Notably, BMP5 and RUNX1 were downregulated in MSCs from both OI types. DISCUSSION: This study demonstrates that the bone marrow microenvironment in OI is significantly altered beyond the known collagen defects. Single-cell RNA sequencing revealed reduced MSC numbers and downregulated osteogenic gene expression. Furthermore, alterations are patient-specific: OI type-I is characterized by reduced osteoblast counts, whereas OI type-VIII exhibits increased osteoblasts and osteoclasts. These findings highlight the critical role of impaired osteogenic differentiation and an abnormal bone remodeling environment in the pathology of OI.

Osteoclasts derived from hematopoietic stem cells control bone resorption. Identifying novel molecules that can epigenetically regulate osteoclastogenesis has been an important basic and clinical issue. The polycomb group (PcG) protein enhancer of zeste homologue 2 (Ezh2), a histone lysine methyltransferase is associated with epigenetic regulation of numerous cellular processes, it is not yet clear on its involvement in bone cell development and homeostasis. Here, we crossed LysM-Cre mice with Ezh2flox/flox mice to delete Ezh2 in myeloid cell lineage mature macrophages. Conditional deletion of Ezh2 in macrophages resulted in significant increases in postnatal bone growth in the first 6 months of life, but tibia length and body weight gains were not different in knockout mice compared with their wild type controls. Significantly decreased osteoclastogenesis but increased bone mass without osteopetrosis were found in Ezh2 conditional knockout (CKO) mice. In contrast to female mice, one floxed Ezh2 gene copy recombinant with LysM-Cre+ (Ezh2flox/+LysM-Cre+) produced increased bone mass in young adult male mice compared with control mice (Ezh2flox/flox, LysM-Cre+ and wild type). Inflammatory milieu in bone was significantly lower in both male and female CKO mice compared with their respective controls. Deletion of Ezh2 in macrophages triggered increased gene expression of osteoclast suppressors, IRF8, MafB and Arg1 due to decreased Ezh2-induced trimethylation of H3K27me3. Conversely, NFATc1 and Cathepsin k expression were decreased. These findings suggest that pre-osteoclastic cell differentiation is under epigenetic control of osteoclast suppressive gene expression via an Ezh2-dependent mechanisms.

Micro-computed tomography ({micro}CT) is widely used to assess trabecular bone microarchitecture, with trabecular separation (Tb.Sp) among the core parameters recommended for reporting. Tb.Sp is typically expressed as a single volume-weighted mean derived from maximal sphere fitting, although the underlying distribution of local separation values is rarely examined. Here, we show that Tb.Sp distributions in metaphyseal trabecular bone are frequently non-Gaussian and bimodal or multimodal. Using {micro}CT datasets from three established models of osteoporosis, spinal cord injury (SCI), ovariectomy (OVX), and ageing, we demonstrate that this behaviour is most evident in metaphyseal trabecular bone and is less apparent in epiphyseal trabecular bone or trabecular thickness distributions. We further show that multimodal metaphyseal Tb.Sp distributions correspond to two spatially distinct contributions within the marrow space: lower-diameter local separation within the residual trabecular network, and higher-diameter regions associated with larger contiguous marrow cavities. Based on this observation, we introduce a simple extension to standard morphometric analysis in which Tb.Sp is decomposed into local trabecular separation (Tb.SpL) and marrow cavity separation (Tb.SpM). Tb.Sp decomposition revealed model-specific patterns of trabecular deterioration. SCI was characterised predominantly by increased Tb.SpM, consistent with expansion of larger marrow cavities, whereas OVX showed a more subtle or distributed alteration. Ageing showed changes in both Tb.SpL and Tb.SpM, with the higher-diameter component becoming most prominent in older animals. Together, these findings demonstrate that mean Tb.Sp can mask structurally distinct forms of metaphyseal marrow-space organisation and support reporting distributional descriptors, and where appropriate Tb.SpL and Tb.SpM, alongside conventional Tb.Sp.

Schwann cells (SCs) provide support for nerves throughout the body. Despite importance for nerve function and repair, the morphology and distribution of SCs in bone remains largely undefined. In this study we used a "Schwann Cell Mapper" mouse (Mpz-Cre+/-;TdT+/+;Ngfr-eGFP+/+) and a "p75 Lineage Tracer" mouse (Ngfr-CreERT2+/-;ZsGreen1+/+) to study SC localization and morphology within the adult mouse calvaria, limb, and vertebrae. We found that all nerves in bone were covered by mature myelinating or non-myelinating SCs, labeled by MPZ and p75-NGFR, respectively. Mature SCs populated the periosteum and entered the bone marrow through transcortical canals. Non-myelinating SCs outnumbered myelinating SCs in bone, with a ratio of [~]2:1 by length density. Non-myelinating SCs in periosteum had more branching and increased size relative to myelinating SCs. In addition, we identified two candidate populations of MPZ lineage+ and p75-NGFR+ immature SCs (iSCs) that were distributed throughout the calvarial periosteum. Similar to mature SCs, p75-NGFR+ candidate iSCs were more prevalent at a ratio of [~]3:1. Overall, neural crest-derived calvarial bone had evidence of increased SC maturity relative to mesoderm-derived sites, identifying niche-level differences in SC maturation. Lastly, lineage tracing revealed that mesenchymal lineages in bone were largely negative for both MPZ and p75-NGFR (<0.1% labeling). These findings provide a framework of SC organization in bone and highlight previously unrecognized diversity across skeletal compartments. By defining distribution and morphology, this work lays the foundation for future studies investigating how SCs contribute to bone biology, including roles in repair, pain, and homeostasis.

Neutralizing sclerostin antibodies (Scl-Ab) mitigate bone loss and promote bone formation to address fracture risk in postmenopausal osteoporosis. Clinically, this treatment is administered monthly for women at high risk of fragility fractures, who are often years into menopause. Preclinical studies have demonstrated that dampening of bone formation occurs with continuous dosing at supraphysiological doses. Osteoporotic bone loss occurs rapidly during early menopause, followed by longer-term changes in bone mineralization and osteocyte activity. Whether earlier administration of lower-exposure Scl-Ab can mitigate bone loss and osteocyte-driven secondary mineralisation is unknown. The objective of this study was to evaluate the effects of early intermittent low-dose Scl-Abon: (1) osteoclastogenesis and bone resorption, (2) perilacunar remodelling, (3) secondary mineralization, and (4) osteocyte mechanosensitivity. Female retired breeder Wistar rats underwent bilateral ovariectomy and received monthly low-dose Scl-Ab injections (2 mg/kg/month) from 3 to 14 weeks post-OVX, while a control group remained untreated. Early intermittent low-dose Scl-Ab treatment increased bone formation and reduced osteoclastogenesis and catabolic gene expression ((Sost, Ctsk, Mmp9) compared to untreated rats. Treatment also decreased the percentage of empty lacunae and the number of MMP14+ osteocytes, accompanied by lower perilacunar mineral density and smaller lacunar size, indicating improved osteocyte survival and reduced perilacunar remodelling. Conversely, expression of osteocyte-mediated mineralization genes (DMP1, PHEX, OPN, ALP) and mechanotransduction-related genes (Vcl, integrins 5, V, {beta}1, CX43, Axin2, IFT88, Adcy6, Pkd1, Cav1) were reduced. Together, these findings suggest that early intermittent low-dose Scl-Ab therapy promotes surface bone formation while attenuating osteocyte-mediated secondary mineralization after initial bone loss. Mini AbstractEarly intermittent low-dose sclerostin antibody treatment reduced osteoclastogenesis, bone resorption, and perilacunar remodelling, while promoting bone formation, decreasing osteocyte apoptosis, and downregulating genes associated with secondary mineralization and mechanosensitivity in ovariectomized rats. These findings suggest early intervention with Scl-Ab enhances bone formation and limits osteocyte apoptosis and subsequent secondary mineralization.

BackgroundPalliative radiation therapy (RT) for metastatic spine disease significantly increases the risk of vertebral fractures. However, the temporal mechanisms underlying radiation-induced vertebral bone fragility remain poorly understood. ObjectiveTo evaluate the longitudinal effects of a single high-dose irradiation, simulating palliative RT, on vertebral bone mechanical, architectural, and compositional properties in a healthy, skeletally mature rat model. MethodsThirty-one male Sprague Dawley rats received a single 15 Gy lumbar spine irradiation (IR). L4 vertebrae were assessed across all groups (irradiation: 7, 14, and 28 days post-IR, controls: at 0 and 28 days post-IR) for compressive strength and stiffness, micro-CT-derived bone composition and trabecular indices, serum bone turnover markers (NTX and BAP) and advanced glycation endproducts (AGEs). ResultsIrradiation induced progressive deterioration of vertebral bone mechanical properties, with strength decreasing up to 44% and stiffness up to 38% by 28 days post-IR, compared to 0- day controls. Trabecular bone exhibited reduced BMD, BV/TV, and Tb.N with increased Tb.Sp, a shift toward a more rod-like structure. Early post-IR changes suggested disrupted bone remodeling, characterized by elevated NTX and AGEs, but decreased BAP. Multivariable regression demonstrated that Tb.Th and AGEs were independent predictors of stiffness, collectively explaining 61% of its variance. DiscussionHigh-dose irradiation induces sustained temporal degradation of vertebral mechanical properties driven by both trabecular architectural deterioration and alterations in bone matrix quality. Measures of bone composition and non-enzymatic bone turnover suggest this early damage was driven by disruption of bone cellular homeostasis, favoring increased resorption over formation. These findings support that radiation impairs both structural integrity and pre-yield mechanical behavior, providing mechanistic insight into the elevated fracture risk observed clinically after irradiation for metastatic spine disease. Lay summaryThis study used a rat model to mimic palliative radiation therapy for cancer that has spread to the spine and evaluated the changes in bone quality up to 28 days post-therapy. We found that irradiation progressively weakened the structural integrity and composition of the bones in the spine and disrupted the normal balance of bone breakdown and repair, leading to greater bone loss and fragility. Our findings provide insight into the increased risk of fractures observed in patients receiving radiation therapy to the spine and may support efforts to better protect bone health during treatment.

Osteoarthritis (OA) is the most common joint disease globally. In OA, articular cartilage degradation is often accompanied with sclerosis of the subchondral bone. However, the association between OA and tissue mineralization at the nanostructural level is currently not understood. Especially, it is technically challenging to identify calcified cartilage, where relevant but poorly understood pathological processes like tidemark multiplication and advancement occur. Here, we used state-of-the-art micro-focus small-angle X-ray scattering with high 5{micro}m spatial resolution to determine mineral crystal thickness in human subchondral bone and calcified cartilage. Specimens with a wide spectrum of OA severities were acquired from the medial and lateral compartments of medial compartment knee OA patients (n=15) and cadaver knees (n=10). For the first time, we identified a well-defined layer of calcified cartilage associated with pathological tidemark multiplication, containing 0.32nm thicker crystals compared to the rest of calcified cartilage. In addition, we found 0.2nm thicker mineral crystals in both tissues of the lateral compartment in OA compared with healthy knees, indicating a loading-related disease process since the lateral compartment is typically less loaded in medial compartment knee OA. Furthermore, the crystal thickness of the subchondral bone was lower with increasing histopathological OA severity. In summary, we report novel changes in mineral crystal thickness during OA. Our data suggest that unloading in the knee is associated with the growth of mineral crystals, which is especially evident in the calcified cartilage. In the subchondral bone, mineral crystals become thinner with increasing OA severity, which indicates new bone formation with sclerosis. One Sentence SummaryMineral crystal thickness increases with osteoarthritis in the lateral condyle that is typically unloaded.

Approximately 85-90% of individuals with Osteogenesis Imperfecta (OI) have dominant pathogenic variants in the COL1A1 or COL1A2 genes. This leads to decreased or abnormal Collagen type I production. Subsequently, bone formation is strongly reduced, causing bone fragility and liability to fractures throughout life. OI is clinically classified in 5 types with the severity ranging from mild to lethal depending on the gene and the type and location of the OI-causative variant and the subsequent effect on (pro) collagen type I synthesis. However, the specific effects on the phenotype and function of osteoblasts are not fully understood. To investigate this, the OI murine model was used, with the oim/oim (OIM) mice closest resembling severely deforming OI type 3 in humans. We showed that in OIM, COL1 mutation results in a multifactorial inhibition of the osteogenic differentiation and maturation as well as inhibition of osteoclastogenesis. The phenotype of differentiated OIM osteoblasts also differs from that of wild type mature osteoblasts, with upregulated oxidative cell stress and autophagy pathways, possibly in response to the intracellular accumulation of type I collagen mRNA. The extracellular accumulation of defective type I collagen fibres contributes to activation of the TGF-{beta} signalling pathway and activates the inflammatory pathway. These effects combine to destabilise the balance of bone turnover, increasing bone fragility. Together, these findings identify the complex mechanisms underlying OI bone fragility in the OIM model of severe OI and can potentially enable identification of clinically relevant endpoints to assess the efficacy of innovative pro-osteogenic treatment for patients with OI.

PurposeLamellar bone that forms in moderate and severe osteogenesis imperfecta (OI) is often composed of structurally irregular lamellae compared to those in normal bone. Polarization light microscopy (PLM) demonstrates lamellar bone well but has rarely been used for quantitative studies; information available on normal bone lamellae tends to be variable and studies specifically assessing OI bone lamellae have not been done. We report on PLM histomorphometry quantifying bright and dark lamellar thicknesses in normal and OI bone. Manual measurements of individual lamellar thicknesses have been made on histologic sections using the cellSens image analysis system; in an effort to augment the number of measurements we also developed a method of automated thickness averaging in quantifying regions of lamellae. MethodsFemoral and tibial cortical bone fragments from 5 individuals 5 - 26 years old (without molecular bone disorders) and 8 individuals 5 - 16 years old with progressively deforming (Sillence III) OI were obtained. The fragments were decalcified, infiltrated in JB4 solution, embedded in JB4 plastic, sectioned at 5 thickness and stained with 1% toluidine blue for light and polarizing microscopy. Manual measurements: Strict criteria for measurement, primarily to eliminate oblique lamellae, included accumulations of 16-20 bright and dark lamellae under PLM with a relatively narrow range of thicknesses, flattened elliptical osteocytes along the longitudinal axis of the lamellae and canaliculi passing from the walls of the osteocyte lacunae at right angles to the lamellae. Histomorphometric measurements of bright and dark lamellae by PLM were made at 20X magnification. Automated measurements: A script for automated measurement of average lamellar thicknesses from PLM images was developed in MATLAB (Mathworks, Natick, MA) to make measurement faster and less subjective. The script isolates a region from an image for measurement and marks each pixel as either bright or dark based on a local average intensity threshold. It then takes multiple pixel measurements along the length of the lamellae in the image and returns the average thickness of each in m. Results1. OI bone mean lamellar thickness values are always less than those in normal bone. The mean value for all OI bright and dark lamellae combined is 1.80 {+/-} 0.72 m and the value in normal bone is 2.54 {+/-} 0.92 m. 2. Mean value for the bright lamellae is less than that for the dark lamellae in both normal and OI bone. The mean value for bright lamellae in OI is 1.47 {+/-} 0.53 m and for dark lamellae 2.18 {+/-} 0.72 m; in normal bone the mean value for bright lamellae is 2.06 {+/-} 0.54 m and for dark lamellae 3.07 {+/-} 0.96 m. The differences are statistically significant: between groups of normal and OI lamellae (p<0.001), normal and OI light bands (p<0.001), and normal and OI dark bands (p<0.001). 3. Ratio of mean values for bright/dark lamellar thicknesses is the same in OI and normal bone. The ratio in OI bone is 0.67 (range: 0.54 - 0.83) and in normal bone 0.67 (range: 0.60 - 0.88). 4. Validation of automated vs. manual datasets: For each lamella in the validation dataset, the percent difference between the automated and manual measurements was calculated. The mean of the absolute values of these percent differences was 18.9%, a statistically non-significant difference (p = 0.0518). Discussion and conclusionsLamellar bone that forms in moderate and severe OI is composed of thinner and less regular lamellae than those in normal bone. i) PLM histomorphometry shows mean lamellar thicknesses (bright and dark merged) are statistically significantly decreased in OI compared to normal bone as are bright and dark lamellar thicknesses measured independently. ii) The automated method can be adapted readily to the assessment process for lamellar thicknesses and is, most likely, more accurate since it averages a greatly increased number of measurements per individual lamella. iii) Lamellar thickness measurements can be helpful in assessing the effect of specific collagen mutations on OI bone synthesis and warrant inclusion in both research and clinical histomorphometric assessments.

Bones adapt to external mechanical loads through a process known as mechanoadaptation. Osteocytes are the bone cells that sense the mechanical environment and initiate a biological response. Investigating the changes in osteocyte molecular expression following mechanical loading has been instrumental in characterizing the regulatory pathways involved in bone adaptation. However, current methods for examining osteocyte molecular expression do not preserve the three-dimensional structure of the bone, which plays a critical role in the mechanical stimuli sensed by the osteocytes and their spatially controlled biological responses. In this study, we used WISH-BONE to investigate the spatial distribution of Sost-mRNA transcripts and its encoded protein, sclerostin, in 3D mouse tibia midshaft following in vivo tibia loading. Our findings showed a decrease in the percentage of Sost-positive osteocytes predominantly at 25% and 37% of the bone length, and in the posterior-lateral side of the tibia after loading. Sclerostin-positive osteocytes in the loaded legs were found to be similar to the contralateral legs after 2 weeks of loading. This work is the first to provide a 3D analysis of Sost and sclerostin distribution in loaded versus contralateral mouse tibia midshafts. It also highlights the importance of the bone region analyzed and the method utilized when interpreting mechanoadaptation results. WISH-BONE represents a powerful tool for further characterization of mechanosensitive genes regulation in bone and holds potential for advancing the development of new treatments targeting mechanosensitivity-related bone disorders.

RNF43 and ZNRF3 are transmembrane E3 ubiquitin ligases that negatively regulate Wnt signaling by promoting ubiquitination and degradation of Frizzled receptors. Loss of either gene enhances Wnt/{beta}-catenin signaling and has been linked to tumorigenesis. Wnt signaling is a key regulator of skeletal development and bone homeostasis, and pharmacologic activation of this pathway is an established therapy for osteoporosis. In Xenopus laevis, simultaneous disruption of rnf43 and znrf3 results in supernumerary limb formation; however, their roles in mammalian limb development and skeletal maintenance remain unclear. We demonstrate that mice homozygous for null alleles of both Rnf43 and Znrf3 do not develop supernumerary limbs. Because activation of Wnt/{beta}-catenin signaling in osteoblasts increases bone mass, we hypothesized that osteoblast-specific deletion of Rnf43 and/or Znrf3 would produce a high-bone-mass phenotype. Instead, osteoblast-specific loss of Znrf3 resulted in age-and sex-dependent reductions in trabecular bone mass, characterized by decreased bone mineral density and bone volume fraction, reduced trabecular number, and increased trabecular separation. Cortical bone exhibited increased cross-sectional size with reduced cortical area fraction and altered structural properties, while tissue mineral density was unchanged. In contrast, deletion of Rnf43 had minimal skeletal effects, and combined deletion of both genes did not exacerbate the phenotype observed with loss of Znrf3 alone. These findings identify Znrf3 as the dominant functional paralog regulating bone architecture in mature osteoblasts and underscore the importance of evaluating skeletal geometry when modulating upstream Wnt regulators.

Tartrate-resistant acid phosphatase (TRAP/ACP5), primarily known as an osteoclast marker, has emerged as a critical regulator of skeletal integrity, regulating sex-specific bone growth, and bones response to mechanical load in young adult male mice. In this study, we investigated the sex-specific roles of TRAP in bone structure and response to mechanical stimuli in old (19-month-old) wild-type (WT) and TRAP-deficient (TRAP-/-) mice using micro-computed tomography, serum bone turnover markers, in vivo axial mechanical loading, and in vitro mechanotransduction assays. Our findings revealed that TRAP-/- mice of both sexes maintained shorter tibiae than WT mice independent of sex. Notably, male, but not female, TRAP-/- mice have increased trabecular bone volume fraction and cortical bone area compared to WT, indicative of disrupted bone remodelling processes in male mice. Interestingly, TRAP-deficiency substantially impaired the anabolic bone response to mechanical loading, affecting both trabecular and cortical compartments in both sexes, indicating that when challenged, TRAP is important for bone formation also in female mice. Mechanical stimulation in vitro of hematopoietic progenitor cells from WT and TRAP-/- mice revealed that the increased ATP-release in response to mechanical stimulation was only disrupted in male mice, while mechanically induced increase in osteoclast formation was inhibited in TRAP-/- mice of both sexes. These results highlight the importance of TRAP in maintaining trabecular architecture and cortical bone in male mice and underscore its critical function in mediating adaptive responses to mechanical loading of both sexes, during aging. Future investigations should focus on elucidation of TRAP-dependent pathways as potential therapeutic targets to counteract age-related deficits in bone adaptation and remodelling.

Radiation therapy is an essential cancer treatment, yet collateral damage to normal tissues remains a major clinical challenge. In bone, radiation-induced toxicity is characterized by loss of hematopoietic function, reduced bone volume, and increased marrow adipose tissue (MAT). Importantly, cancer patients who undergo radiotherapy exhibit significantly higher fracture risk compared to those receiving similar treatments without radiation exposure, underscoring the clinical consequences of bone microenvironment (BME) injury. The BME is inherently hypoxic resulting in the activation of by hypoxia-inducible factor (HIF) signaling. Here, we demonstrate that radiation induces a rapid and persistent accumulation of MAT, with adipocytes localizing preferentially to hypoxic regions of the marrow. To investigate the role of hypoxia/HIF signaling in this process, we generated aP2Cre;Hif-1 fl/fl;Hif-2fl/fl conditional knockout mice. Surprisingly, these mice exhibited increased MAT expansion following radiation compared to controls, suggesting that HIF deletion in aP2-expressing cells exacerbates radiation-induced adipogenesis. Analysis aP2CreRosa26tdTomato/+ mice revealed that most aP2-expressing cells did not give rise to mature adipocytes, macrophages, or endothelial cells, pointing instead to an uncharacterized stromal population that influences MAT formation. In contrast, conditional ablation of HIF transcription factors in LepRCre-expressing skeletal stem cells, which contain a subpopulation of skeletal progenitors which directly contribute to marrow adipocytes, had no effect on radiation-induced MAT expansion. Collectively, these findings identify a previously unrecognized population of adipocyte-regulatory cells whose HIF-dependent activity constrains stress-induced marrow adiposity. This work provides new mechanistic insight into how radiation disrupts the marrow microenvironment and expands MAT, advancing our understanding of the cellular and molecular drivers of radiation-induced bone fragility. One Sentence SummaryLoss of HIF signaling in aP2Cre expressing cells enhances radiation induced marrow adiposity.

Obesity is a major public health issue worldwide. Despite various approaches to weight loss, the most effective technique for reducing obesity, as well as diabetes and associated diseases, is bariatric surgery. Increasingly, young women without children are undergoing bariatric surgery, vertical sleeve gastrectomy (VSG) being the most common procedure nowadays. However, despite several reports suggesting bone loss after VSG, little is known about the potential additive effects of gestation and lactation after VSG to bone health. This study investigated the combined effects of pre-gestational VSG and subsequent gestation/lactation on bone metabolism in a rat model fed a high fat high sugar (HFHS) diet, with a focus on bone biomechanics, mass, microarchitecture and material properties. Furthermore, bone mass and remodelling was followed longitudinally by microCT prior to surgery, 4 weeks post-surgery, after weaning and at sacrifice. Significant alterations in bone mass and microarchitecture, characterized by changes in trabecular thickness and number, as well as changes in bone formation and resorption were influenced by both surgery and reproductive demands. Mechanical testing at sacrifice demonstrated compromised long bone fragility, in rat with HFHS regardless of the surgical procedure (Sham or VSG). Furthermore, analysis of bone material properties highlighted potential disruptions in the pattern of bone mineralization in sham and VSG animals fed a HFHS diet. These findings underscore the complex interplay between pre-gestational VSG and subsequent gestation/lactation in modulating bone metabolism. Understanding these combined effects is essential for optimizing surgical strategies and developing targeted interventions to mitigate potential bone-related complications associated with VSG in reproductive-aged individuals.

Type 2 diabetes mellitus (T2DM) is associated with an increased fracture risk independent of bone mass. The exact origin of this increased fracture risk is still not well understood. Using a polygenic diabetic rat model, synchrotron radiation micro-computed tomography (SRCT), and in situ scanning electron microscope (SEM) fracture toughness, we related the changes at the microscale to toughness and material properties of diabetic rat femurs. The diabetic rat model (ZDSD) displayed overnight fasting hyperglycemia and an increased AGEs content. Additionally, we measured the impairment of post-yield properties and toughness in diabetic rats. The cortical geometry and porosity were also affected in this ZDSD model. We measured a decrease in osteocyte lacunar density associated with a decreased lacunar volume. Moreover, we found decreased canal density while maintaining a similar canal diameter. These results indicate that diabetes impairs bone remodeling, affecting bone microstructure. Because canals and lacunae are also linked with extrinsic toughening mechanisms, we attribute the decreased toughness largely to these microstructural changes. In conclusion, we showed that changes in lacunae and canal density, combined with AGEs accumulation, decreased toughness in T2DM rat bone.

Three-dimensional (3D) imaging of osteocyte lacunae has recently substantiated the connection between lacunar shape and size, and osteocyte age, viability, and mechanotransduction. Yet it remains unclear why individual osteocytes reshape their lacunae and how networks of osteocytes change in response to local alterations in mechanical loads. We evaluated the effects of local mechanical stimuli on osteocyte lacunar morphometrics in tibial cortical bone from young female mice flown on the Space Shuttle for [~]13 days. We optimized scan parameters, using a laboratory-based submicrometer-resolution X-Ray Microscope, to achieve large [~] 0.3 mm3 fields of view with sufficient resolution ([&ge;] 0.3 m) to visualize and measure thousands of lacunae per scan. Our novel approach avoids large measurement errors that are inherent in 2D and enables a facile 3D solution as compared to the lower resolution from benchtop micro-computed tomography (CT) systems or the cost and inaccessibility of synchrotron-based CT. Osteocyte lacunae were altered following microgravity exposure in a region-specific manner: more elongated (+7.0% Stretch) in predominately tensile-loaded bone as compared to those in compressively-loaded regions. In compressively-loaded bone, lacunae formed in microgravity were significantly larger (+6.9% Volume) than in the same region formed on Earth. We also evaluated lacunar heterogeneity (i.e., spatial autocorrelation of lacunar morphometric parameters) via kriging models. These statistical models demonstrated that heterogeneity varied with underlying spatial contributors, i.e. the local mechanical and biological environment. Yet in the absence of gravitational loading, osteocyte lacunae in newly formed bone were larger and were collectively more homogenous than in bone formed on Earth. Overall, this study shows that osteocyte reshape their lacunae in response to changes, or absence, in local mechanical stimuli and different biological environments. Additionally, spatial relationships among osteocytes are complex and necessitate evaluation in carefully selected regions of interest and of large cell populations. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/474962v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@f3cc2aorg.highwire.dtl.DTLVardef@1e0fbbdorg.highwire.dtl.DTLVardef@1af3f7dorg.highwire.dtl.DTLVardef@7133b0_HPS_FORMAT_FIGEXP M_FIG C_FIG