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 affect bone structure or bone mass in adulthood. However, Ngf knockout mice displayed significantly reduced periosteal bone accrual and osteogenic Wnt transcription in response to axial forelimb compression as compared to wildtype mice. Administration of a single dose of gambogic amide prior to the first of three bouts of axial forelimb compression significantly increased load-induced bone formation in wildtype mice, but failed to do so in either male or female Ngf knockout mice. Furthermore, increasing the frequency of gambogic amide administration to three daily doses resulted in a similar osteoanabolic effect without any adverse events. In total, our study reveals an important role for osteoblastic NGF in the skeletal adaptation of bone to mechanical forces, suggests that gambogic amide primarily functions in collaboration with endogenous NGF in bone, and provides insights for dose optimization of gambogic amide in future therapeutic applications.

Prolonged unloading of the shoulder, most seen after stroke related hemiparesis, accelerates degeneration of the supraspinatus enthesis in part through localized bone loss at the tendon to bone interface. Because bisphosphonates are often used in populations experiencing disuse, it remains unclear whether they can mitigate bone deterioration at the supraspinatus enthesis. In this study, we examined how unloading and alendronate treatment influence bone architecture, mineral composition, cellular activity, and mechanical behavior at the supraspinatus enthesis using a 24-day murine model of botulinum toxin induced supraspinatus paralysis. Unloading produced significant trabecular deterioration, including reduced bone volume fraction and trabecular thickness, while fibrocartilage morphology and tendon area remained unchanged. Alendronate did not prevent structural bone loss, though it produced clear compositional changes such as increased carbonate substitution. Mineral to matrix ratio, crystallinity, and phosphate peak position did not differ significantly across any of the four treatment groups: under disuse- conditions, bisphosphonate treatment, or combined condition with bisphosphonate. Histomorphometry showed no differences in osteoblast or osteoclast number or surface, suggesting that bone loss occurs primarily through suppressed formation rather than elevated resorption. Because osteoclast number and surface were unchanged, elevated resorption is unlikely to account for observed bone loss. Osteoblast number and surface were similarly unaffected, indicating that any deficit in formation is more likely attributable to reduced activity per cell rather than to a reduction in osteoblast recruitment or abundance. Mechanical testing demonstrated that unloading reduced supraspinatus humerus complex strength and modulus. Although alendronate did not preserve trabecular structure, it produced a non-significant trend toward higher complex level strength, raising the possibility that material-level changes can influence failure independently of architecture. However, combined unloading and turnover suppression reduced post yield properties, highlighting tradeoffs for enthesis quality under low load conditions. These findings show that disuse induced bone loss at the supraspinatus enthesis is driven by formation and that bisphosphonates, while modifying mineral composition, do not prevent structural degeneration.

Matrix vesicles (MVs), a specialized class of extracellular vesicles, play a central role in skeletal mineralization. Phosphoethanolamine/phosphocholine phosphatase 1 (PHOSPHO1) generates inorganic phosphate (Pi) within the MV lumen and is considered essential for the initiation of mineralization. This study investigated the role of PHOSPHO1 in osteoblast differentiation and MV biology using primary calvarial osteoblasts from 6-day-old wild-type (WT) and Phospho1[-/-] mice. Cells were cultured under osteogenic conditions for 14 and 21 days, followed by analyses of cell viability, mineralization, gene expression, MV biophysical properties, and proteomic analyses. Phospho1[-/-] osteoblasts exhibited threefold higher mitochondrial activity than WT cells. Mineral deposition was detected only in WT cultures at day 21, indicating impaired mineralization in the absence of PHOSPHO1. Gene expression analysis revealed increased Alpl and Smpd3, and decreased Enpp1 expression in Phospho1[-/-] osteoblasts. Consistent with these findings, alkaline phosphatase (ALP) activity in MVs increased 7.5-fold between days 14 and 21 in both genotypes. Atomic force microscopy of MVs derived from Phospho1[-/-] osteoblasts exhibited a 6.5- and 2.1-fold reduction in vesicle volume and surface roughness, respectively, compared with WT MVs. Proteomic analysis revealed reduced tissue non-specific alkaline phosphatase (TNAP) at day 14, followed by increased TNAP levels at day 21, accompanied by decreased collagen XII and collagen VI content in Phospho1[-/-] MV preparations. Collectively, these findings indicate that cellular adaptation is insufficient to restore mineralization in the absence of PHOSPHO1. Instead, PHOSPHO1 appears to play a critical role in establishing the molecular and biophysical properties of MVs necessary for mineral nucleation and propagation.

Pseudoxanthoma elasticum (PXE) is a rare genetic disease characterized by ectopic calcification in the eyes, skin, and arteries. It is caused by pathogenic variants in the ABCC6 gene and leads to reduced plasma levels of inorganic pyrophosphate. Etidronate, a stable analogue of pyrophosphate, has been shown to reduce arterial calcification. However, the long-term skeletal safety of high-dose cyclical etidronate treatment remains unclear. This study aimed to evaluate the skeletal effects of cyclical etidronate administration in adults aged ≥50 years with PXE. This prospective study included patients treated with etidronate (20 mg/kg/day for 14 days every 12 weeks) for ≥1 year and untreated control patients from the Dutch Expertise Center for PXE. Bone density (BD) was measured on whole-body low-dose CT scans in Hounsfield units at the spine and femoral necks. Safety was assessed using bone turnover markers and fracture data. BD changes were compared within treated patients and between the treated and control groups using scanner-adjusted linear mixed models. In total, 71 treated patients (mean age 57.0 ± 7.6 years; 56% female) and 72 untreated patients (mean age 54.2 ± 11.0 years; 69% female) were included. Median etidronate follow-up duration was 5.2 years (IQR 5.1-7.5). Of the 71 treated patients, 66 (93%) completed 5 years of follow-up, and all treated patients completed at least 2.5 years of treatment. In the treated group, BD remained stable during treatment, whereas in the untreated group it declined substantially, resulting in a greater overall loss in the untreated group compared to the treated group. Non-vertebral fracture incidence was low and comparable to controls, and no atypical femur fractures, excessive suppression of bone turnover markers, or medication-related osteonecrosis of the jaw were observed. This study demonstrates that cyclical administration of high-dose etidronate to patients aged ≥50 years with PXE for 5 years did not negatively affect skeletal integrity and metabolism and prevented bone loss at both the spine and the hip.

Autosomal recessive hypophosphatemic rickets type 2 (ARHR2) is a rare metabolic bone disorder caused by biallelic loss-of-function variants in ENPP1, the gene encoding for Ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1), an enzyme essential for generating extracellular pyrophosphate, a key endogenous inhibitor of ectopic calcification. The clinical spectrum of ENPP1 deficiency varies widely, and recognition in adulthood is challenging due to phenotypic heterogeneity and overlap with more common metabolic bone and parathyroid disorders. We describe two brothers in their late 40s with a novel homozygous ENPP1 splice-site pathogenic variant (c.1437 + 1G > C) presenting with chronic hypophosphatemia, elevated serum PTH, extra-skeletal calcifications, and markedly abnormal bone microarchitecture on high-resolution peripheral quantitative computed tomography (HR-pQCT). Brother 1 exhibited long-standing hypophosphatemia, ossification of the posterior longitudinal ligament, conductive hearing loss, congenital hip dysplasia, and aortic valve calcification. Brother 2 presented with progressive myelopathy and spinal stenosis, and underwent parathyroidectomy for presumed normocalcemic primary hyperparathyroidism. Postoperative hypoparathyroidism led to initiation of calcitriol and calcium supplementation, and subsequent nephrolithiasis. Both patients demonstrated FGF23-mediated renal phosphate wasting, low-normal 1,25-dihydroxyvitamin D, elevated parathyroid hormone and normocalcemia, favored to represent secondary rather than primary hyperparathyroidism, and significant abnormalities in cortical and trabecular bone parameters by HR-pQCT. These cases highlight diagnostic pitfalls in adult-onset ARHR2, including delayed recognition of FGF23-mediated phosphate wasting and misdiagnosis as primary hyperparathyroidism. Accurate genetic diagnosis is essential because ARHR2 clinically mimics X-linked hypophosphatemia, underscoring the need for early recognition to guide safe and effective treatment.

Opportunistic osteoporosis screening using artificial intelligence (AI) applied to routine chest radiographs offers a novel, low-burden strategy to improve early detection and treatment initiation. This study evaluated the cost-effectiveness of AI-enhanced opportunistic osteoporosis screening compared with no screening among Australian women aged ≥50 years undergoing chest radiography. A microsimulation-based Markov model was developed to estimate quality-adjusted life years (QALYs) and lifetime costs in 2025 Australian dollars. The model incorporated the diagnostic performance of AI applied to chest radiographs, subsequent DXA confirmation, treatment initiation, and real-world medication persistence. It also explored the effect of enhanced follow-up and adherence on clinical and economic outcomes. AI-enhanced opportunistic screening was associated with improved health outcomes (+0.0025 QALYs and - 0.0043 fractures per patient) and lower lifetime costs (-AUD59) compared with no screening, representing a dominant strategy. When scaled to 10,000 screened women aged ≥50 years, the model projected prevention of 43 fractures, 25 additional life-years, 25 QALYs gained, and AUD590,000 in healthcare savings. Optimizing follow-up and adherence could amplify clinical benefits by four- to five-fold. AI-enhanced opportunistic osteoporosis screening from chest X-rays appears to be a cost-effective strategy within the Australian healthcare system, delivering better outcomes at lower cost. The results also suggest that linkage to DXA confirmation and subsequent treatment initiation may influence the magnitude of health and economic outcomes. These findings highlight that follow-up and persistence are not merely complementary but essential for realizing the full potential of cost-effective AI-driven osteoporosis screening.

Low-intensity vibration (LIV) is a non-invasive mechanical stimulus capable of regulating skeletal adaptation and bone remodeling cellular signaling pathways. Despite growing interest in LIV, substantial methodological heterogeneity persists in the experimental vibration parameters. This study sought to synthesize the effects of different LIV frequencies and intensities on bone volume fraction (BV/TV) in small rodent models. 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 peer-reviewed studies with mature mice and rats, using whole-body vibration experiments without other co-variables. In addition to these core criteria, a detailed hierarchical screening framework was applied for full-text review. We used Bayesian network meta-analysis (NMA) and SYRCLE risk of bias (RoB) to assess the selected studies. For tibia, NMA found 45 Hz at 2 g0 (estimated effect 3.22 [CrI 1.98, 4.45]), 45 Hz at 0.2 g0 (estimated effect 2.32 [CrI 1.01, 3.62]) and 90 Hz at 1 g0 (estimated effect 0.25 [CrI -2.70, 3.26]) as the top three candidates. For femur, NMA identified 90 Hz at 1.4 g0 (estimated effect 3.08 [CrI -1.99, 7.97]), 45 Hz at 2 g0 (estimated effect 1.25 [CrI -1.48, 3.86]), and 45 Hz at 0.2 g0 (estimated effect 1.18 [CrI -1.54, 3.80]) as the regimes with the highest chances to increase trabecular BV/TV. To evaluate a common top performing regimen for both tibia and femur we exposed 5-month-old male C57BL/6 for 4 weeks (n = 10/group) to a 45 Hz at 0.2 g0 LIV regimen. When comparing pre- and post-LIV measurements within each bone, 9 out 18 μCT variables evaluated showed significant changes in the femur, and 5 variables within tibia with two-way ANOVA identifying a greater response in the femur. These findings support NMA as a useful tool for prioritizing LIV regimens in small rodents. Although several μCT measures showed greater femoral responses experimentally, which was not predicted by NMA, LIV effects in both bones fell within the NMA's 95% credible intervals, supporting 45 Hz/0.2 g0 as a promising regimen for both the tibia and femur.

Pycnodysostosis (PYCD) is a rare genetic bone disease characterized by impaired osteoclastic bone resorption. The inability to resorb bone leads to sclerosis, with increased bone mineral density (BMD) but paradoxically increased bone fragility. We report a case of a young female with PYCD with multiple fragility fractures including bilateral atypical femur fractures (AFF). Areal and volumetric bone density were supranormal, measured by dual-energy x-ray absorptiometry (DXA) and high-resolution peripheral quantitative computed tomography (HR-pQCT), respectively. However, bone disorganization was prominent, observed on visual assessment of HR-pQCT images, with microfractures and sclerotic intramedullary lesions. ALIGNOGRAM1.0 is an artificial intelligence (AI) powered disorganization quantifier. It detected femoral lesions (including microfractures) on femoral radiographs up to two years prior to atypical femoral fractures at those sites. These microcracks were not visible on routine hip radiographs without the ALIGNOGRAM1.0 software. We postulate that disorganized bone, including microfractures, may be an underrecognized mechanism of bone fragility in patients with PYCD that could contribute to complications such as delayed fracture healing. Visual assessment via HR-pQCT may detect gross abnormalities but may miss subtle, subclinical changes - e.g., early sclerotic changes - that predate severe lesions and are not visible on routine HR-pQCT analysis. Quantitative tools such as ALIGNOGRAM1.0 may be particularly valuable in disorders characterized by paradoxical skeletal fragility despite normal or elevated bone density and apparently preserved microarchitecture. By quantifying bone disorganization on standard radiographs, these approaches may provide complementary information beyond conventional assessments of bone mass and structure. The paradox of severe fragility despite supranormal bone density has long remained unexplained. This diagnostic paradox is not unique to pycnodysostosis and is encountered across a range of genetic, metabolic, and treatment-related skeletal disorders in which fracture risk appears disproportionate to conventional measures of bone density or structure. Consequently, there is a need for complementary approaches capable of identifying abnormalities that are not captured by standard assessments. In genetic and metabolic bone disorders (e.g., pycnodysostosis or hypophosphatasia) that evade conventional assessments of bone health (e.g., DXA and HR-pQCT), we demonstrate how disorganization may act as a causative mechanism as an independent biomarker of fragility.