Bone

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.

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.

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.

Low-intensity vibration (LIV) is a non-invasive mechanical stimulus capable of regulating skeletal adaptation and cellular signaling pathways involved in bone remodeling. Despite growing interest in LIV, substantial methodological heterogeneity persists in the selection of experimental vibration parameters such as frequency, expressed in Hertz (Hz) and intensity, defined as earth's gravitational field (g) (9.81 m/s2). Focusing on micro-computed tomography (CT) derived trabecular bone volume fraction (BV/TV) as the main outcome measure, this study sought to synthesize the effects of different LIV frequency and intensity on BV/TV in small rodents (mice and rats) as they remain as the most studied pre-clinical model. To accomplish this, we performed a systematic review searching for publications in English on PubMed, Web of Science, CINAHL, and Embase databases. Two independent investigators followed inclusion criteria to select only peer-reviewed studies with mature mice, using whole-body vibration experiments without other co-variables. We further restricted to include studies that analyzed non-fractured bones and compared pre- and post-intervention or control values. In addition to these core criteria, a detailed hierarchical screening framework was applied during full-text review. The two independent investigators extracted data independently and considered the characteristics of the study, animals' characteristics, intervention characteristics, and results. For this study we considered load-bearing hindlimbs, femur and tibia, separately but did not include vertebrae in the analysis. A Bayesian network meta-analysis and a revised SYRCLE risk of bias (RoB) tool were used to evaluate the risk of bias across included studies. Seven studies met the inclusion criteria. Results showed that an LIV regime applied at 45Hz at 2g presented higher chances to increase trabecular BV/TV of the mouse tibia (estimated effect 3.22 [CrI 1.98, 4.45]), while LIV regimes applied to the femur at 90Hz and 1.4g (estimated effect 3.08 [CrI -1.99, 7.97]) present better chances to increase trabecular BV/TV results compared to other interventions but with no significant differences. Finally, we applied 45Hz at 0.2g LIV to 5 month old male C57BL/6 for 5 weeks (n=10/group) which showed significantly increased Trabecular Thickness (Tb.Th) for both the tibia (10%, p<0.01) and femur (17%, p<0.001), with the femur showing further increases in trabecular BV/TV (32%, p<0.05) compared to non-LIV controls. We conclude that changes in the microarchitectures of the tibia and femur respond differently to the same application of LIV (45Hz, 0.2g) in mice and rats.

Fracture nonunion remains a major cause of morbidity, yet patient-specific factors associated with impaired healing remain incompletely characterized. Anemia has been associated with adverse orthopaedic outcomes, but its relationship with fracture nonunion is poorly understood. We examined whether pre-fracture anemia, anemia burden, and clinically relevant anemia subtypes were associated with nonunion following tibial or femoral fractures. Using commercial and Medicare fee-for-service claims from 2016 through 2023, we identified adults aged 19 years or older with a tibial or femoral fracture, continuous enrollment during the preceding year and for at least six months after fracture, and no baseline cancer. Pre-fracture anemia was evaluated as any anemia, the number of distinct anemia diagnoses, and nutritional, hemolytic, aplastic, and other anemia subgroups. Nonunion occurring six to eighteen months after fracture was assessed using incidence rates and multivariable-adjusted hazard models. Among 326,673 adults, 149,704 had pre-fracture anemia and 176,969 did not. The crude incidence of nonunion was 42% higher among individuals with anemia than among those without anemia (incidence rate ratio, 1.42; 95% confidence interval, 1.32 to 1.53) and increased with greater anemia burden. After adjustment for demographic and clinical characteristics, including prior fractures at other anatomical sites, pre-fracture anemia remained associated with nonunion following tibial and femoral fractures, with hazard ratios of 1.83 (95% confidence interval, 1.54 to 2.18) and 1.38 (95% confidence interval, 1.26 to 1.50), respectively. Associations were also observed for nutritional and other anemias, whereas estimates for hemolytic and aplastic anemias were limited by few nonunion events. Within the femur, the association was strongest for distal fractures. These findings demonstrate that pre-fracture anemia is independently associated with nonunion. The increase in risk with greater anemia burden and findings across evaluable subgroups suggest that pre-fracture anemia may help identify patients at increased risk of impaired fracture healing.

Bone remodeling is a tightly regulated physiological process that maintains bone health through coordinated action of bone-resorbing osteoclasts and bone-forming osteoblasts. Disruption of this balance, such as the one induced by estrogen decline after menopause, results in bone loss and osteoporosis. Genetic factors play an important role in determining bone mineral density (BMD) loss over time. However, translating genetic associations into individualized risk prediction remains challenging due to small effect size of individuals variants and non-linear interactions within the bone remodeling unit. Here, we present a bone cell population dynamics model that includes major regulatory pathways, such as the RANK/RANKL/OPG axis, Wnt signaling, and hormonal regulation by estrogen, parathyroid hormone, and TGF-{beta}. We calibrate the model on clinical data from healthy postmenopausal women, and women with reduced BMD undergoing anti-osteoporotic therapy. The calibrated model captures healthy BMD decline in postmenopausal women and therapeutic response to anti-osteoporotic medications. We mechanistically incorporate the effect of 22 variants across 8 genes involved in bone remodeling and simulate BMD trajectories in 1,000 virtual subjects differing by ancestry and genetic makeup. The median predicted 5-year BMD loss was 3.57% (95% prediction interval: 1.31-5.24), consistent with the values reported in the literature. The virtual individuals with African ancestry were predicted to experience the highest average 5-year BMD loss. The strongest genetic risk factors for bone loss were predicted to be CYP19A1 rs727479 and OPG rs3102735, while LRP5 rs11228240 emerged as a protective factor that could partially counteract the detrimental effects of other variants. Several epistatic effects were observed in the genetic interaction analysis. Mechanistically, our model suggested that estrogen exerts its effect on bone remodeling primarily by modulating osteoclast apoptosis. Overall, this framework demonstrates a proof-of-concept for integration of genetic risk factors into mechanistic models of disease and can be extended to other conditions with polygenic inheritance.

Background: Viable pediatric human growth plate (GP) tissue is rarely available for translational research, limiting direct investigation of human longitudinal bone growth and pediatric growth disorders. In this proof-of-concept study, we aimed to determine whether it is feasible to establish a clinically integrated ex vivo human GP model using tissue obtained during routine percutaneous epiphysiodesis (PE) procedures in adolescents treated for extreme tall stature or leg length difference due to trauma. Methods: GP tissue and cells were collected during PE and processed using protocols adapted from established methods of human osteoarthritic cartilage processing within the RAAK study. Feasibility was assessed by evaluating tissue collection, cell isolation, contamination rate, monolayer expansion, and generation of three-dimensional cartilage pellets. Proliferation of GP-derived chondrocytes was compared with osteoarthritis-derived articular chondrocytes, and histological assessment was performed to evaluate cartilage-like matrix formation. Results: Across consecutive surgical procedures, viable GP tissue could be obtained reproducibly, with only few samples failing to yield cells and no relevant contamination issues. Isolated GP chondrocytes expanded successfully in two-dimensional culture and showed a strong early proliferative response compared with RAAK-derived chondrocytes. In addition, GP-derived cells formed three-dimensional organoids and histology confirmed cartilage-like matrix deposition supporting their capacity to generate neo-cartilage tissue in vitro. Conclusion: This study demonstrates feasibility to obtain, culture, and functionally assess viable human GP tissue from routine PE surgery. As such, the Leiden ex vivo human GP model provides a unique platform to study local mechanisms of endochondral bone growth, link genetic determinants of height to functional GP biology, and support future therapeutic research in pediatric growth disorders.

Teriparatide (PTH 1-34) is an anabolic agent used to treat osteoporosis, yet clinical response varies widely among patients. To investigate genetic and sex-specific determinants of skeletal response, we administered intermittent PTH to male and female mice from eight genetically diverse inbred strains. Mice were treated for four weeks, and bone phenotypes were assessed via DXA, microCT, and mechanical testing. Response to PTH was highly strain- and sex-dependent, with some strains responding at the femur but not the spine, and vice versa. Heritability estimates for PTH-induced changes in bone mineral density (BMD), cortical area, breaking strength, and trabecular bone volume fraction (BV/TV) ranged from moderate to high, with BV/TV showing the strongest genetic influence. Cortical bone response mechanisms differed by sex: males exhibited periosteal expansion, while females showed endosteal remodeling. These findings mirror clinical observations where hip non-response is more prevalent than spine non-response and suggest that genetic background and sex significantly influence therapeutic outcomes. Our data support the use of genetically diverse mouse models to elucidate the genetic architecture of PTH response and highlight the potential for personalized approaches in osteoporosis treatment. Future genome-wide association studies in outbred mice may identify specific loci mediating skeletal responsiveness to PTH, advancing precision medicine strategies for bone anabolic therapies. LAY SUMMARYTeriparatide, a drug used to treat osteoporosis, consists of the active portion of parathyroid hormone (PTH). Information from clinical studies suggests that not all patients will respond to this medication. We used eight strains of inbred mice to study the impact of genetic background and sex on the response to PTH. We learned that response to PTH is driven by both genetics and sex. Some strains responded at the femur, but not the spine and vice versa. These results may explain why a failure to respond at the hip in humans is more common than at the spine.

Background: Acute osteoporotic vertebral fractures (OVFs) may be difficult to detect on conventional radiographs, particularly before substantial vertebral collapse occurs. Comparing supine and sitting lateral radiographs may reveal load-dependent vertebral mobility. This preliminary study evaluated the diagnostic accuracy of supine to sitting dynamic radiography for detecting MRI confirmed acute OVFs. Methods: This retrospective, single center diagnostic accuracy study included consecutive patients who underwent paired supine and sitting lateral radiography and MRI of the same spinal region between April 2024 and July 2026. Dynamic radiographs were interpreted by a board certified orthopedic and spine surgeon who was blinded to the MRI findings. MRI was independently interpreted by a second board certified orthopedic surgeon and served as the reference standard. The primary outcome was patient-level sensitivity and specificity. Vertebra level diagnostic accuracy was evaluated secondarily, with patient cluster bootstrap confidence intervals used to account for within patient correlation. Results: Sixty three patients (mean age, 80.6 years; 51 women [81.0%]) and 490 evaluable vertebrae were analyzed. MRI identified acute OVFs in 34 patients and 36 vertebrae. At the patient level, dynamic radiography yielded 31 true positive, no false-positive, three false negative, and 29 true negative results. Sensitivity was 91.2% (95% confidence interval [CI], 76.3%-98.1%), specificity was 100.0% (95% CI, 88.1%-100.0%), positive predictive value was 100.0%, negative predictive value was 90.6%, and overall accuracy was 95.2%. At the vertebral level, sensitivity was 91.7% (33/36; patient cluster bootstrap 95% CI, 81.3%-100.0%) and specificity was 100.0% (454/454). The three missed fractures involved T9, L2, and L3. No false-positive vertebrae were observed. Conclusions: Supine to sitting dynamic radiography demonstrated high patient level sensitivity and no observed false positive findings for MRI confirmed acute OVFs. It may provide a practical complementary diagnostic option when MRI is not immediately available. However, a negative dynamic radiographic examination does not exclude an acute fracture, and the apparent perfect specificity requires validation in larger, prospective multi-reader studies.

Critical sized defects (CSDs) are a serious challenge in orthopedics that require the development of more robust and effective treatments to improve quality of life for patients. Current CSD research is limited by the applicable and affordable animal models available. Mice would be the preferred species as they are cheaply housed and have many transgenic variations readily available; however, their small size makes CSD surgeries difficult and expensive. We propose the use of cerclage wires to achieve internal plate fixation. PEEK plates were secured to the right femur of 26 C57BL/6 mice using four cerclage wires to achieve modified double-loop fixation implemented through bicortical holes and defects were created. 10 received 3mm defects and 6 received 4mm defects that were left empty and were taken out to 20 weeks (Group E3, E4). Another 10 received 3mm defects that were filled with a morselized bone graft and were taken out to 8 weeks (Group G3). Blinded longitudinal x-ray grading by orthopedic surgeons was conducted on the empty defects for plate stability and wire fixation. All samples received microCT analysis at their endpoints. There were no significant differences in plate or wire stability between the empty groups and wire scores worsened negligibly over time. MicroCT analysis further supported wire integration as bone growth directly upon the wires was observed in all samples. The efficacy of this model in achieving non-union when left untreated was also confirmed via microCT. Further, only three mice in group G3 achieved union and two of these unions were not optimal. Our study is the first to successfully show that cerclage wire can be used in a murine CSD model to achieve affordability and clinical relevancy.

Purpose: The primary objective of the Stanford Knee Osteoarthritis PET/MRI Evaluation (SKOPE) study is to develop and evaluate a multimodal, dynamic [18F]NaF PET-MRI framework for characterizing whole-joint physiology and its relationship to osteoarthritis (OA) risk, pain, and disease progression. Specifically, we aim to integrate dynamic PET with quantitative and anatomical MRI, to characterize structural, compositional, and metabolic features across the knee and surrounding musculoskeletal system, evaluate acute tissue responses to exercise, and identify imaging biomarkers associated with OA risk, pain, and disease progression. Methods: The SKOPE study includes multimodal PET-MRI of the knee and surrounding musculoskeletal tissues, with imaging of the knee, tibia, ankle, thigh, hip, pelvis, and lumbosacral spine. Dynamic [18F]NaF PET is combined with conventional anatomical MRI and quantitative MRI techniques, including quantitative double-echo steady-state (qDESS) T2 mapping of cartilage, Dixon fat-fraction imaging, ultrashort echo time (UTE) T2* mapping of short-T2 tissues, UTE imaging of tibial bone, and zero echo time (ZTE) imaging for bone morphology and pseudo-CT generation. Additional MRI sequences characterize muscle composition, bone and joint anatomy, intervertebral discs, and regional vascular anatomy. Selected scans are acquired before and after a standardized exercise protocol to assess the acute physiological response of the joint. Automated segmentation is used to generate subject-specific masks of muscles, bones, vertebrae, and intervertebral discs. A subset of the MRI protocol is repeated at 1- and 2-year follow-up to assess longitudinal changes. Expected Impact: By combining dynamic bone metabolic imaging with quantitative measures of cartilage, menisci, muscle, bone, fat, vascular structures, and the spine and hip, the SKOPE protocol provides a whole-joint and multijoint framework for studying the structural, metabolic, and physiological processes associated with OA and pain. Exercise and longitudinal imaging further enable assessment of acute tissue responses and changes over time, supporting the development of quantitative imaging biomarkers for OA risk, pain, and disease progression.

Vitamin D receptor (VDR) signaling is essential for osteoblast maturation and skeletal mineralization, yet the intracellular mechanisms linking VDR activity to matrix production remain poorly defined. Here, we show that vdr-/- calvarial osteoblasts initiate differentiation but fail to complete the transition to a mature, mineralizing state, exhibiting suppressed late-stage markers such as Dmp1, Phex, and Col1a1 and defective nodule formation both in vivo and in vitro. Transcriptomic profiling revealed a SMAD network imbalance, with elevated inhibitory SMADs and attenuated phosphorylation of both SMAD1/5/9 and SMAD2/3. Whereas SMAD1/5/9 activation was preserved in vivo and restored by exogenous BMP2 in vitro, consistent with in vivo paracrine BMP availability, reduced SMAD2/3 phosphorylation persisted in both contexts, indicating a cell-autonomous defect. Mechanistically, vdr-/- osteoblasts displayed impaired intracellular Ca{superscript 2} dynamics and diminished CaMKII activation, with VDR/RXR occupancy detected near the Camk2g locus. Pharmacological CaMKII inhibition (KN-93) recapitulated the phenotype, reducing SMAD2/3 phosphorylation and mineralization and establishing CaMKII as an upstream regulator of SMAD2/3. A milk-based diet rescued skeletal defects in vdr-/- mice in a calcium-independent manner and selectively restored CaMKII-SMAD2/3 signaling. Metabolomic profiling identified the omega-6 fatty acid {gamma}-linolenic acid (GLA) as an elevated circulating mediator; exogenous GLA restored Ca{superscript 2} flux, CaMKII activation, SMAD2/3 phosphorylation, matrix production, and mineralization, without reactivating Runx2, Sp7, or BMP-SMAD1/5/9 signaling, and dependent on CaMKII activity. These findings reveal a VDR-independent, GLA-inducible CaMKII-SMAD2/3 mineralization program, positioning metabolic modulation of calcium signaling as a strategy to restore osteoblast function under impaired vitamin D signaling.

Bone adapts to its mechanical and physiological environment through the coordinated actions of osteocytes and marrow-resident cell populations. Because these processes depend on complex interactions within native tissue microenvironments, intravital imaging offers a unique opportunity to reveal cellular behaviors that cannot be fully captured ex vivo. However, the highly scattering nature of mineralized bone limits imaging depth and direct observation of cells within intact tissue. Two-photon (2P) microscopy has enabled important advances in bone biology, while three-photon (3P) microscopy has been proven to extend imaging depth and image quality in skeletal tissues. Here, we directly compared the performance of 2P and 3P microscopy in ex vivo mouse long bones by quantifying laser attenuation, signal-to-noise ratio, signal-to-background ratio, and spatial resolution, as well as assessed the impact of wave-front correction. We found that 2P and 3P microscopy generated comparable image quality through approximately 50 {micro}m of cortical bone. Beyond this depth, however, 3P microscopy provided superior brightness, contrast, and resolution, enabling improved visualization of structures deep within the cortex and at the cortical-marrow interface. To assess compatibility with intravital imaging, we evaluated endogenous markers of cellular stress during 3P imaging. Although prolonged continuous imaging decreased osteocyte spontaneous calcium signaling magnitude and increased autofluorescence, short intermittent imaging bouts produced negligible evidence of cellular damage compared to controls. Leveraging the enhanced penetration depth of 3P microscopy, we further show visualization of immune cell migration within the marrow cavity through intact cortical bone in both the third metatarsal (MT3) and tibia. Together, these findings affirm 3P microscopy as a powerful tool for studying cellular dynamics in living bone. By extending imaging beyond superficial cortical regions and enabling direct visualization of marrow-resident cells through intact bone, 3P microscopy expands opportunities to investigate osteocyte biology, marrow niche function, and skeletal adaptation in vivo.

Purpose Radiation injury to growth plates commonly leads to skeletal late complications including short stature, limb length-discrepancy, and scoliosis/kyphosis in pediatric oncology patients. We aimed to understand the acute responses of direct growth plate irradiation that result in skeletal late complications. Materials and methods We first established an in vivo model of focal growth plate irradiation that recapitulates the clinical development of skeletal late complications and used it to explore the responses of growth plate chondrocytes within the first 72 hours of radiation exposure. To monitor acute effects of radiation exposure on human chondrocytes, rare human growth plate biopsies were exposed to ionizing radiation ex vivo. Using these approaches, we applied clonal genetic tracing and immunofluorescence to monitor changes at the cellular and molecular levels. Functional in vivo perturbations were conducted with clinically-relevant autophagy inhibitor, hydroxychloroquine. Results Growth plate irradiation disrupted the continuous production of chondrocytes required for bone elongation and was associated with DNA damage throughout the growth plate. Indicators of growth plate activity, SOX9 and the phosphorylated form of ribosomal protein S6, decreased during a 6- and 24-hour post-irradiation window but returned to normal levels 72 hours after irradiation. We identified a surge in autophagic flux throughout the growth plate during this window, based on temporal SQSTM1 and LAMP1 protein levels. The earliest stages of these response mechanisms are conserved between species and relevant to humans. Hydroxychloroquine treatment immediately after radiation injury in mice impaired growth plate regeneration, resulting in more severe late complications. Conclusion Our findings demonstrate that autophagy is an important acute response to irradiation in growth plate chondrocytes, revealing a novel potential therapeutic target for preventing radiation-induced skeletal late complications.

Abstract Fractures in older adults cause high morbidity and mortality. This study aims to identify fracture incidence and risk factors, and to develop the Fracture Risk Model-Abu Dhabi (FRM-AD). Method This retrospective cohort study of 1757 males and females aged 40+ from Abu Dhabi, United Arab Emirates, who participated in a screening program from 2016 to 2020, followed until 2024 for 4.7 years, SD =1.8. It utilized Electronic Medical Records (EMRs) data and telephone interviews. The outcome assessed was the occurrence of fractures. Results There were 58 out of the 1149 females (5.0%) and 26 out of the 608 males (4.3%) who had at least one incident fracture. Hip and vertebral fractures accounted for 12.1% and 11.5%, respectively. There was an annual incidence of 10.5 fractures per 1,000 person-years, 10.7 cases among females and 9.9 among males, rising after age 80 to 39.1 among females and 25.3 among males. Fracture Predictors identified using Poisson regression analysis were older age, smoking, history of previous fracture(s), interaction of history of fracture in a parent with a family history of osteoporosis, positive history of hip fracture in a parent after the age of 40, lack of dyslipidemia diagnosis and the interaction of diabetes mellitus diagnosis and family history of osteoporosis. A higher Body Mass Index (BMI) increased the risk of fractures in this cohort, especially in obese males. Logistic regression of variables at the end of follow-up showed that lower latest vitamin D levels were associated with increased fracture risk. FRM-AD, derived using Poisson regression, performed well in predicting fractures, with Receiver Operating Characteristic (ROC) curves of 0.736 (0.677-0.794) and 0.742 (0.684-0.800) without and with BMD, respectively. The FRAX AUC to predict MOF and hip fracture ranged between 0.624 and 0.683. Conclusion In this Emirati cohort, the locally derived FRM-AD showed moderate discrimination for incident fracture and outperformed FRAX-AD, suggesting that a locally derived model may improve fracture risk stratification. Several risk factors and predictors were identified that can be targeted for prevention.

Sutures are the primary sites of cranial bone growth, allowing the skull to accommodate the growing brain. External mechanical stimulus has been shown to delay suture fusion and induce tissue remodelling. Recent studies suggest that in vivo cyclic bone loading increased proliferation of mesenchymal stem cells (MSC) in the coronal suture. The overall aim of this study was to understand how many loading sessions (exposure-response) and how long after loading (time-course) did MSC proliferation increase in the coronal suture. In the exposure-response analysis, mice underwent 1, 3, or 5 loading sessions between Postnatal day 7 (P7) and P11, and in the time-course analysis, treated mice underwent 10 loading sessions between P7 and P21. Loading sessions were 10 minutes at a frequency of 1 Hz and a force of 10 g (0.1 N). The loading tip was positioned on the posterior aspect of the left frontal bone, dorsal to the coronal suture. The EdU marker shows a statistically significant increase in proliferation after one loading session and a decrease after three loading sessions. The PCNA marker shows a statistically significant increase after three and five loading sessions. The exposure-response analysis showed that when the results of both markers are combined, levels of proliferation cannot be interpreted until at least five loading sessions have been completed, after which a clear increase in proliferation was observed. In the time-course analysis, proliferation was highest immediately after the final treatment session and 24 hours after the final loading session the effects of mechanical bone loading gradually returned to baseline.

Abstract Introduction: Proximal humerus fractures account for approximately 5-6% of all adult fractures and are primarily managed nonoperatively. Healing is conventionally monitored with radiographs, with radiopaque callus formation indicating healing. Visible radiographic callus appears weeks after biological union begins. Ultrasound provides a dynamic, radiation-free, and cost-effective method that can detect early callus formation before x-ray visibility. Although ultrasound has demonstrated utility for fracture healing in the clavicle and humeral shaft, its role in proximal humerus fractures remains unclear. Methods: This single-centre prospective study will be conducted in two phases. The pilot phase will measure inter-rater reliability for ultrasound detection of early callus formation at 2 and 4 weeks post-injury. Ten patients with proximal humerus fractures treated nonoperatively will undergo standardized anterior and lateral scans. Each patient will generate four saved images (short- and long-axis views), producing forty anonymized images independently reviewed by two raters. The prospective cohort phase will recruit approximately thirty additional patients. Results: Reliability will be quantified using Cohens kappa. A power calculation will be performed after pilot analysis. Results from the prospective cohort phase will help determine the association and predictive value of early ultrasound-detected bridging callus for radiographic and clinical union at three and six months. Patient reported outcome measures will be assessed using the Quick Disabilities of Arm, Shoulder and Hand (QuickDASH) questionnaire. Discussion: This study will develop and validate a standardized ultrasound protocol for assessing early fracture healing in proximal humerus fractures. By establishing both inter-rater reliability and predictive value, the findings may support ultrasound as a reproducible, radiation-free adjunct to conventional imaging and enable earlier identification of union status.

Pseudoxanthoma elasticum (PXE) is a rare inherited disorder characterized by progressive ectopic calcification of soft connective tissues, including skin, arteries, and eyes, leading to significant morbidity. PXE results from loss of functional ABCC6, a liver specific ATP efflux conduit. Reduced ATP release into the circulation limits its conversion into AMP and the mineralization inhibitor pyrophosphate (PPi). Consequently, low plasma PPi levels drive ectopic calcification in PXE. Although oral PPi supplementation can inhibit ectopic calcification in Abcc6-/- mice, impractically high doses are needed, due to its rapid hydrolysis in the gastrointestinal tract. Here, we evaluated phosphocitrate, an exceedingly more potent mineralization inhibitor, in vitro and in Abcc6-/- mice. In ATDC5 cells, 1 {micro}M phosphocitrate significantly inhibited mineralization following induction, comparable to approximately tenfold higher concentrations of PPi. In vivo, daily intraperitoneal administration of phosphocitrate (4.7 {micro}mol/kg bw) markedly reduced calcification in muzzle skin and kidneys, whereas an at least fivefold higher dose of PPi was needed to achieve a similar effect. Oral administration required substantially higher doses of both compounds ([~]2.4 mmol/kg bw), but PC remained more effective than PPi at inhibiting soft tissue calcification in Abcc6-/- mice. Importantly, unlike PPi, oral phosphocitrate did not adversely affect skeletal strength or stiffness, even at supraphysiological doses. In summary, phosphocitrate is a more potent inhibitor of ectopic calcification than PPi in Abcc6-/- mice. While optimization of oral delivery remains necessary, its increased potency supports the potential of alternative administration routes, including subcutaneous delivery, as a practical therapeutic strategy for PXE.

Background and rationale: Knee osteoarthritis (KOA) is a leading cause of lower limb disability worldwide, characterized by functional limitations, stiffness and pain. The incidence of KOA is especially tied to age and obesity. It is a disabling disease that often makes patients less physically active, thus increasing the risk of other diseases and mortality1. The clinical diagnosis of KOA is based on the symptoms and functional limitations of the joint. The diagnosis is usually supported with a radiograph (X-ray) of the weight-bearing knee. Radiographic features, such as Kellgren-Lawrence grade, are used as eligibility criteria for clinical studies while other features, such as joint space width (JSW), are used as endpoints for structural KOA progression2,3. While the use of these radiographic features is standard in academia, the use of JSW as a structural biomarker has received criticism. Critics point out that JSW is an indirect and projection dependent measure of cartilage deterioration which is sensitive to technical factors such as the angulation of the X-ray beam and the positioning of the knee. Small differences in these factors can alter the measured joint space and may not reflect true disease progression4,5. Despite limitations, minimum joint space width (mJSW) remains as one of the most widely used structural biomarkers in KOA trials and is currently one of the only structural imaging accepted in regulatory guidance as evidence of disease modification in OA drug development3. For JSW to be reliable and consistent in determining the advancement of KOA, the use of fixed-flexion devices is crucial to reduce the risk of unwanted narrowing or widening of the radiographic joint space width6,7. The LOSEIT trial, which the present study is based on, acknowledges the angulation problem and uses a standard clinical fixed-flexion device in weight-bearing PA views to get reliable JSW results8. Historically, a radiologist would draw on and grade radiographs of the knee-joint to extract the features. However, manual reading and annotation is time consuming with notable interobserver variance9. With increasing computational power and the use of deep neural networks, off-the-shelf artificial intelligence (AI) tools have become available for automatic extraction of radiograph features. Automation would free up time from radiologists and provide more consistent measurements due to the reproducible nature of the models10. These tools have received regulatory approval for commercial use, however, regulatory approval does not guarantee uniform or bias free performance when used on real-world data11. Furthermore, in a large multi-hospital chest X-ray study, Zech et al., showed that convolutional neural networks achieved worse results on data from other hospitals than on the original hospitals in which it was tested12. This highlights the risk of overestimating the accuracy of AI tools when only internally validated. It is therefore apparent that external validation is required when testing these AI models. Objectives: The aim of this analysis is to evaluate the agreement of a commercially available AI tool for measuring JSW with the best practice radiologist annotation in the tibiofemoral joint of the knee in radiographs stabilized with a fixed-flexion device and acquired as part of a clinical trial. Methods: This study is a secondary analysis of the data from the LOSEIT trial, a randomized, double-blind, placebo-controlled, single-center trial, where patients were randomized to either liraglutide or identically appearing placebo after an initial weight-loss period to investigate the effects on KOA. Radiographs of the tibiofemoral joint were acquired at enrollment (week -8) and at end-of-trial (week 52) for a total acquisition-to-acquisition time of 60 weeks13. The primary analysis will assess agreement between AI-derived and reference-derived change in JSW from enrolment to follow-up. Change will be calculated as follow-up minus enrolment separately for the AI tool and the reference measurement. The main measure of interest will be the change in medial minimal JSW (mmJSW), with change in lateral minimal JSW (lmJSW), medial fixed JSW (mfJSW) and lateral fixed JSW (lfJSW) as secondary measures. This study will follow an equivalence framework using the two one-sided tests (TOST) approach with a Bland-Altman analysis as the main outcome. The equivalence margin will be set at {delta} = 0.5 mm. Agreement consistent with equivalence will be considered established if the upper limit of the 95% confidence interval (95% CI) for the upper limit of agreement (LoA) and the lower limit of the 95% CI for the lower LoA are within the established margins. The reference JSW will be the average measurement of two independent resident radiologists. If there is a mismatch in the measurements of more than 0.40 mm between the two radiologists, the radiologists will re-annotate the case independently. If the difference remains greater than 0.40 mm, a musculoskeletal radiology consultant will review the radiograph and establish the reference JSW. The index test will be the measurements output by the AI tool. Populations: Patients aged 18 to 74 with symptomatic knee osteoarthritis, radiographically confirmed KL grade 1-3, with a BMI [&ge;]27, motivated for weight loss and in accordance with the LOSEIT trial inclusion criteria Further statistical details Sample size: Not applicable as this is a secondary analysis. Framework: This is an agreement study assessing the equivalence of a commercially available AI tool for radiographic evaluation of knee osteoarthritis with best practice radiologist measurements. Confidence intervals and P values: All 95% confidence intervals and P-values will be two-sided. Statistical software: SAS Studio and/or R version 4.2.2 (or newer).

Motion artifacts present a major challenge for intravital imaging of tissues undergoing physiological movement or mechanical loading. Blurring or artificial changes in image intensity due to shifting on the z-axis reduce data reproducibility and reliability. Existing post-acquisition approaches can partially compensate for motion, but they increase experimental complexity and are not well suited for use in mechanically loaded bone. Therefore, we developed a novel method for real-time correction of axial motion during mechanical loading of bone by synchronizing the movement of the objective to the actuator. Synchronization was achieved by linking the position of the actuator piezo motor to the objective piezo motor with a user refined reduction via potentiometer. Applying axial motion correction effectively removed artificial changes in fluorescent intensity in a static fluorescent marker up to 3000{varepsilon} in bone as measured by similarity and average intensity before and during loading. This improvement was reflected in the improved accuracy in capture of a dynamic fluorescent calcium indicator (GCaMP6f) in osteocytes. Our system provides a user-friendly, robust framework that can be easily adapted to other mechanically loaded tissues, improving data collection and expanding the utility of two-photon imaging across a variety of biological applications.