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Social isolation stress has numerous known negative health effects, including increased risk for cardiovascular disease, dementia, as well as overall mortality. The impacts of social isolation on skeletal health, however, have not been thoroughly investigated. We previously found that 4 wk of social isolation through single housing led to a significant reduction in trabecular and cortical bone in male, but not female, mice. One possible explanation for these changes in male mice is thermal stress due to sub-thermoneutral housing and sex differences in thermal physiology. Single housing at room temperature (~20 to 25 °C)-below the thermoneutral range of mice (~26 to 34 °C)-may lead to cold stress, which has known negative effects on bone. Therefore, the aim of this study was to test the hypothesis that housing mice near thermoneutrality, thereby ameliorating cold-stress, will prevent social isolation-induced bone loss in male C57BL/6J mice. 16-wk-old mice were randomized into social isolation (1 mouse/cage) or grouped housing (4 mice/cage) at either room temperature (~23 °C) or in a warm temperature incubator (~28 °C) for 4 wk (N = 8/group). As seen in our previous studies, isolated mice at room temperature had significantly reduced bone parameters, including femoral bone volume fraction (-35% BV/TV), bone mineral density (-27% BMD), and cortical thickness (-12%). Contrary to our hypothesis, these negative effects on bone were not fully ameliorated by thermoneutral housing. There was no significant effect of housing or temperature on serum turnover markers. Social isolation increased glucocorticoid-related gene expression in bone and Ucp1 and Pdk4 expression in BAT across temperatures, while thermoneutral housing increased percent lipid area and decreased Ucp1 and Pdk4 expression in BAT across housing conditions. Overall, our data suggest thermal stress from single housing cannot fully explain social isolation-induced bone loss and provide a key insight into the mechanism mediating the effects of isolation on skeletal health.

A 16-yr-old male with a genetically undiagnosed neurodevelopmental disorder (NDD) was admitted to our outpatient clinic for skeletal assessment. DXA and HR-pQCT showed a severely reduced BMD and a pronounced reduction of trabecular and cortical bone mass. Lateral vertebral assessment identified multiple previously unrecognized vertebral fractures of the thoracic and lumbar spine. Laboratory tests indicated an activated bone turnover, which was confirmed by an increased number of osteoclasts and osteoblasts in an undecalcified tibia biopsy of the patient. Treatment of the severe osteoporosis was initiated with neridronate. Trio exome sequencing in the patient and healthy parents did not uncover a genetic cause of the disease. Importantly, however, targeted sequencing of the RNU4-2 gene, which encodes the U4 small nuclear RNA (a major component of the splicing machinery), identified a heterozygous causative variant in the patient. This led to the molecular diagnosis of ReNU syndrome. RNU4-2 pathogenic variants underlie a NDD with multisystemic involvement, including skeletal abnormalities. Therefore, this case not only underlines the relevance of osteologic assessment and therapy in individuals with NDDs, but also highlights the necessity of future research efforts to elucidate the bone pathologies in ReNU syndrome.

Breast cancer cells frequently spread to the bone, causing osteoclast-mediated bone destruction and pathological fractures. Bone anabolic agents, such as abaloparatide, are used clinically to increase bone formation in osteoporotic patients, but their effectiveness against tumor-induced bone destruction is poorly understood. In this study, we present the first evaluation of abaloparatide in preclinical models of breast cancer cells disseminated to the bone marrow and demonstrate that intermittent abaloparatide dramatically increases trabecular bone volume in mice inoculated with triple negative breast cancer cells. Abaloparatide also increases BMD in a model of estrogen receptor positive breast cancer, despite elevated baseline bone volume due to estradiol supplementation. Importantly, abaloparatide does not increase tumor burden or incidence in bone or soft tissue sites in either model. These results suggest that abaloparatide may be effectively used to increase bone mass without stimulating growth of cancer cells in the bone marrow and may be beneficial for cancer patients with low bone mass.

To understand differences that exist between the cell populations in different skeletal sites, we performed an unbiased genetic survey via single-cell RNA sequencing of CD11b+ myeloid cells from mandibular- and femur-derived bone marrow of 2-mo-old C57BL/6 mice. Our results reveal transcriptomic evidence that suggests a uniquely inflammatory genetic profile of the mandibular-derived CD11b+ cells. The monocyte cell population found within the CD11b+ cells analyzed by scRNA-seq expressed Csf1r and Tnfrsf11a suggesting that this population contained osteoclast precursors. Osteoclasts of the craniofacial region facilitate processes such as tooth eruption and jawbone development. Evidence from multiple researchers suggests that craniofacial osteoclasts exhibit differences in size, gene expression, and activity compared to their counterparts within the appendicular skeleton. A biological mechanism to explain the observable difference between craniofacial osteoclasts and osteoclasts in the long bones has not been previously explored. This monocyte population had enhanced inflammatory gene expression by qRT-PCR which correlated with an increase in select areas of open chromatin by assay for transposase-accessible chromatin using sequencing. Further exploration into a specific upregulated gene determined KLF4 was both necessary and important for proper differentiation in mandibular- but not femur-derived cells.

Stromal cells are critical regulators of hematopoietic stem/progenitor cells and skeletal homeostasis. Although precise systems for functional analysis are critical to investigate mechanistically bone and bone marrow (BM)-derived stromal cells, the establishment of reproducible, highly enriched ex vivo methods for stromal cell isolation, culture and evaluation have been challenging, leading to inconsistent data on stromal cell function. In this work, we carefully tested ex vivo culture of murine stromal cells from BM and bone and discovered abundant and persistent contamination of monocytes and macrophages. We succeeded in establishing highly enriched ex vivo culture system for stromal cells by eliminating persistent monocytes and macrophages using selection against the immunological markers F4/80, Ly6C, and CD45. Transcriptional and functional assays of enriched stromal cell culture revealed differential characteristics of stromal cells from different origins, a dormant signature for bone-derived cells and a highly proliferative progenitor-like signature for BM-derived cells. Monocyte and macrophage contamination reduced signatures of immature stromal cells such as expression levels of SOX9 and CD140a as well as the cells' ability to support hematopoietic stem and progenitor cells based on our growth factor-free co-culture system of hematopoietic cells and stromal cells followed by in vivo functional assays. The inhibitory effects of macrophages on stromal cells may be explained by their potent production of inflammatory cytokines such as CXCL2, CCL3, and complement factor (C1q) confirmed by protein immunoassay of culture supernatant, as well as the differential contribution of pre-osteoblasts to the stromal cell population. This study highlights the functional diversity of stromal cells depending on the microenvironment of origin while addressing a critical limitation of murine ex vivo systems. Our robust culture system enables the study of isolated stromal cells function as well as the impact of stromal cells-macrophage crosstalk.

Hypercementosis has been previously reported in mice lacking progressive ankylosis protein (Ank KO mice, or Ank, KO - knockout, WT - wildtype) due to decreased levels of the mineralization inhibitor inorganic pyrophosphate. However, the impact of hypercementosis on alveolar bone remodeling and periodontal ligament (PDL) maintenance from orthodontic forces during orthodontic tooth movement (OTM) remains unclear. To investigate the roles of ANK protein on tooth movement, PDL maintenance, alveolar bone remodeling, and tooth root resorption, we performed a split-mouth model of OTM induced by a closed-coil spring stretched between the maxillary first molar and maxillary incisors in Ank KO and WT mice (including both males and females). Micro-computed tomographic analysis revealed a 36.6% reduction in OTM in Ank KO mice compared with WT mice, although OTM-induced thickening of PDL was found to be similar in both groups. While reduced tissue mineral density (TMD) of the alveolar bone was observed in WT mice, TMD in Ank KO mice was maintained. Loss of Ank leads to wider roots with thicker cementum on the untreated, contralateral side, whereas a significant increase in OTM-induced root resorption was observed on the lateral tension side. Histologic analysis of root resorption confirmed these data and showed increased resorption lacunae located prevalently in the OTM tooth root cementum of Ank KO mice. Using a quantitative PCR array of bone-associated markers to interrogate total RNA harvested from PDL tissues along the root surface, we found alterations in gene expression from OTM in both WT and Ank KO mice, which included genes involved in bone remodeling, calciotropic hormones and receptors, cytokines, growth factors, and receptors. Our findings advance the understanding of the role of Ank in regulating mineralization in the periodontium as well as factors involved in root resorption.

Kynurenine (KYN), a tryptophan metabolite that increases with age, impairs osteoblast function. The aryl hydrocarbon receptor (AhR) has been proposed to mediate KYN's actions in bone. To test whether deletion of AhR in osteoblasts is beneficial for bone, we established an adult-onset AhR conditional knockout (CKO) model using Osx-Cre and examined the effects of AhR CKO at 4.5 and 6 mo of age (representing ~6 and 12 wk of CKO). While BMSC-derived osteoblasts from WT mice demonstrated reduced matrix formation from KYN treatment, AhR CKO osteoblasts were unaffected by KYN. Kynurenine's harmful effects were most pronounced in the middle of an osteoblastic differentiation time course, and these effects could be rescued via the AhR antagonist BAY2416964. In vivo, AhR deletion in Osx-expressing cells promoted sex- and compartment-specific skeletal phenotypes. Trabecular bone was increased in the distal femur of male and female AhR CKO mice at both 4.5 and 6 mo of age, potentially driven by a net decrease in the ratio of trabecular osteoclasts to osteoblasts despite a reduction in mineral apposition rate at 6 mo of age. In contrast, cortical bone phenotypes induced by AhR deletion depended on age and sex. In males, cortical bone volume fraction (Ct.BV/TV) was elevated in AhR CKO mice vs WT littermates at 4.5 mo of age, but differences resolved by 6 mo of age. In contrast, cortical bone was reduced in female AhR CKO as compared to WT littermates at 6 mo of age. These results underscore the complexity of AhR signaling in skeletal biology that must be considered while exploring AhR as a therapeutic target for conditions like osteoporosis and musculoskeletal frailty. Future studies will be needed to test the effects of osteoblastic AhR deletion at advanced ages, when the endogenous AhR ligand KYN is elevated in the circulation and skeletal niche.

Tumor-induced osteomalacia (TIO) is a rare disorder characterized by impaired bone mineralization due to phosphate wasting. Long-term changes in BMD and microarchitecture after surgical cure or medical therapy in TIO are not well understood. This study describes changes in BMD, microarchitecture, and bone strength in patients with TIO following surgical cure or medical therapy. A prospective cohort study included adults diagnosed with TIO from May 2018 to 2024, categorized into those with surgical cure and those on medical therapy. Follow-up assessments were classified as early (median 8 mo), intermediate (median 17 mo), and long-term (median 26 mo). Fifteen patients were included: seven achieved surgical cure, and eight remained on medical therapy. Lumbar spine BMD increased by +19% at early, +27% at intermediate, and +15% at long-term follow-up. Total hip BMD increased by +31%, +36%, and +31% at early, intermediate, and long-term assessments, respectively. All patients achieved a normal lumbar spine BMD, while 91% attained a normal total hip BMD. At the distal tibia, substantial increases in bone microarchitecture parameters-cortical area (Ct.Ar), cortical volumetric density (Ct.vBMD), and cortical thickness (Ct.Th)-were observed. Notably, Ct.Th improved to levels comparable to healthy controls. Bone strength improved by 13% but was not statistically significant, probably due to the small sample size. At the distal radius, most parameters remained stable. Patients with surgical cure showed more rapid and substantial improvements in BMD and cortical microarchitecture than non-cured patients, but these differences did not reach statistical significance. Overall, bone recovery in TIO is gradual, with gains in spine and hip BMD and significant improvements in tibial cortical parameters. However, some aspects of bone microarchitecture remained below control levels, underscoring the need for ongoing monitoring and individualized management strategies.

Denosumab is an injectable osteoporosis medication administered twice per year. Discontinuation of denosumab can result in rapid rebound fractures, but the evidence is limited on real-world persistence with denosumab. We conducted 2 parallel, population-based cohort studies leveraging (1) healthcare administrative data from Ontario, Canada (ON; 100% population) and (2) a 20% random sample of US Medicare beneficiaries (US). The first denosumab claim (US: 1/2010-12/2019; ON: 1/2012-12/2021) was identified using pharmacy claims (ON) and Medicare Parts D and B claims (US). Patients aged <66 yr, residing in long-term care (LTC), or with implausible data (eg, death before first claim) were excluded. We developed and applied an algorithm that used dosing and days between dispensations to clean denosumab claims. We assumed a days supply of 183 d for each dispensation and defined discontinuation as a 60-d gap in coverage. We estimated initial persistence, reinitiation, and switching to other osteoporosis medications using Kaplan-Meier estimators, censoring on death, disenrollment (US only), LTC admission, or study end (12/31/2022 [ON], 12/31/2020 [US]). We also estimated the monthly proportion of patients with an on-time denosumab dose to explore time trends. We identified 168 339 eligible individuals in ON (mean age = 78 yr; 90% female) and 97 595 in the US (mean age = 77 yr; 90% female). In ON, the median time to denosumab discontinuation was longer (median 2.3 yr [ON] vs 1.7 yr [US]; 3-yr persistence: 44% [ON] vs 31% [US]), and time to reinitiation was shorter (median = 0.5 yr [ON] vs 1.9 yr [US]). In both populations, around 10% switched to another osteoporosis medication. Women and those with prior oral bisphosphonate use had longer durations of denosumab treatment in ON but not in the US. The proportion persisting with on-time doses did not increase over time in the US or ON. Research to improve persistence with denosumab and optimize post-denosumab treatment is critical.

Charcot-Marie-Tooth (CMT) disease is a genetic, progressive peripheral nerve disease that commonly manifests in a cavovarus foot deformity. Previously, this foot deformity has been believed to be an alignment change in the foot, but recent research has shown that there are bone morphology differences in individuals with CMT. Differences in bone morphology have been identified in the calcaneus, talus, and medial cuneiform, but have not been consistently analyzed throughout the foot or studied in relation to different genetic subtypes of CMT. This study is a retrospective, cross-sectional analysis of bone morphology in CMT using weight-bearing computed tomography and statistical shape modeling. This analysis identified bone morphology differences between CMT and control groups throughout the hindfoot, midfoot, and forefoot. Bone morphology differences were also present between the 2 primary disease subtypes throughout the foot. Key morphologic findings include the altered shape of the subtalar articular surfaces on the talus, bending of the metatarsals, variation in navicular process morphology, and differences between subtypes in the talus, medial cuneiform, and medial metatarsals. There are several possible theoretical mechanisms for this osseous deformation, including bone remodeling in response to altered loading from alignment change or from decreased musculotendinous forces, but the patterns of morphological variation seen in these data cannot be fully explained by these mechanisms, suggesting that there may be an interaction between the neuronal disease and bone remodeling. Future work is necessary to characterize the progression of bony deformity throughout development and to correlate bone shape with function, gait, muscle morphology and strength to elucidate the mechanism of osseous morphology change in varying subtypes of CMT.