Abstract Low bone mineral density (BMD) and impaired bone strength are established risk factors for fractures in older adults. Decreased muscle size also contributes to fracture risk, however, the relationship between muscle size and bone density, microarchitecture, and strength using state-of-the-art assessment methods is not clear. In The Study of Muscle, Mobility and Aging (SOMMA), muscle size was assessed using whole-body muscle mass (kg, deuterated creatine (D3Cr) dilution method) and thigh muscle volume (L, by magnetic resonance imaging (MRI)). We investigated cross-sectional associations between baseline D3Cr muscle mass and MRI thigh muscle volume with bone volumetric density, microarchitecture, and strength from high-resolution peripheral quantitative computed tomography (HR-pQCT) at the distal tibia (DT) and radius (DR), and hip areal BMD from dual-energy X-ray absorptiometry (DXA) at the first annual follow-up visit (Year 1). Muscle and bone parameters were standardized within sex and analyses were stratified by sex. Linear regression models were adjusted for age, race, weight, ≥1 alcoholic drink/wk, ever cigarette smoker, total activity from wrist-worn accelerometry, multimorbidity count (0-11), arthritis, and tibia or ulna length. In 181 women (age 76.2 ± 4.7 yr, 86% White) and 118 men (age 76.1 ± 4.3 yr, 93% White), higher thigh muscle volume (per SD: 1.1 L women; 1.5 L men) was associated with higher DT and DR failure load (p < .05). Greater thigh muscle volume was associated with higher DXA total hip BMD in men but not in women. Greater D3Cr muscle mass (per SD: 4.4 kg women; 5.5 kg men) was associated with higher DT and DR failure load (p < .05) in women only. Associations of muscle size with microarchitecture were variable and differed by sex. Given that failure load is a strong predictor of fracture risk, future studies should investigate whether interventions that target muscle size may impact fracture risk in older adults.
Extremely premature infants are at high risk of metabolic bone disease of prematurity. Current diagnostic tools require radiation exposure, additional risks to patients, and likely provide late diagnosis. Serum markers of minerals are the current standard of care, but there are no universal diagnostic definitions. QUS is a potential diagnostic method that is non-invasive, provides immediate results, can be performed in the incubator, and has no radiation exposure. QUS has been studied in preterm infants, but few studies have evaluated the intra-rater and inter-rater reliability of QUS in preterm infants. Our study evaluated the inter-rater and intra-rater reliability in infants less than 28 wk or <1000 g at birth at a single level IV academic center. Three operators performed QUS on the tibia in infants with 3 separate measurements per operator at one month post birth (n = 73) and at 36 wk postmenstrual age (PMA) (n = 81). The MiniOmni was the QUS device, and the smallest probe, CS probe (length 24 mm, range 2200-4700 m/s, and max depth 4.5 mm), was used for all subjects. Measurements were taken at the mid-tibia location. Inter-rater reliability was calculated using 2-way random-effects intraclass correlation coefficients (ICC); intra-rater reliability was calculated using 2-way mixed-effects ICC. 95% CI were calculated for each. Value of ICC estimates less than 0.5, between 0.5 and 0.75, between 0.75 and 0.9, and greater than 0.90 are indicative of poor, moderate, good, and excellent reliability, respectively. Intra-rater reliability at 1 mo post-birth and 36 wk PMA was excellent across all operators. Inter-rater reliability was more variable with good reliability at 1-mo measurements (0.84, 0.78-0.90, 95% CI) and excellent reliability at 36 wk PMA (0.94, 0.90-0.96, 95% CI). Our study demonstrated that QUS is a reliable diagnostic tool and a feasible potential alternative to current diagnostic methodologies for MBPD in extremely preterm infants.
Sestrin2 is a stress-inducible protein that plays a significant role in maintaining antioxidant balance and metabolic homeostasis via the AMPK/mTOR pathways. Animal models suggested that Sestrin2 deficiency may be associated with high bone mass; however, how this may translate into humans, especially in aging and stress conditions, is unknown. We performed a cross-sectional analysis of 910 adults recruited from the Qatar Biobank cohort. Participants were stratified into 3 groups: younger than 40 yr, 40-55 yr, and older than 55 yr. We investigated the relationship between plasma Sestrin2 and FN BMD T-score in adults using multiple linear regression, controlling for age, sex, BMI, diabetes, and estimated glomerular filtration rate. We observed a significant interaction between Sestrin2 and age (p < .001). In younger and middle-aged adults, Sestrin2 was not correlated with BMD. However, in adults aged >55 yr, Sestrin2 was strongly and positively correlated with FN T-scores (β = .131, 95% CI: 0.062-0.201, p < .001). This association remained significant after adjustment for all possible confounders, with an effect size reduction of only 7.7%. Further analysis showed that this association is more pronounced in males (β = .155, p < .001) but is not significant in females (β = -.008, p = .935). Our study indicates that Sestrin2 and BMD are significantly correlated but only in aged adults. In contrast to animal models, we showed that Sestrin2 in aged adults is positively correlated with bone density. This suggests that Sestrin2 may serve as a biomarker of resilience against aging-induced stress and osteosenescence. Furthermore, this resilience mechanism appears to be sex-specific, offering protective effects primarily in aging males, likely due to differences in hormonal preservation.
Cognitive impairment is common in patients undergoing hemodialysis; however, evidence linking routinely measured bone turnover markers to cognition remains limited. In this exploratory cross-sectional study, we aimed to evaluate whether bone-specific alkaline phosphatase (BAP), total alkaline phosphatase (ALP), and tartrate-resistant acid phosphatase 5b (TRACP-5b) are related to global cognitive function in older patients undergoing hemodialysis. Adults aged ≥65 yr receiving maintenance hemodialysis were enrolled at seven centers in Tokyo and Chiba, Japan (November 2021-November 2022). Global cognition was assessed using the Montreal Cognitive Assessment (MoCA) and Mini-Mental State Examination (MMSE). Multivariable linear regression with robust SEs and facility fixed effects was used to examine associations with MoCA scores across sequentially adjusted models. Because of a ceiling effect, MMSE was analyzed using logistic regression with MMSE ≤23 as the outcome (unadjusted and age-/sex-adjusted). For statistical analyses, bone turnover markers were natural log-transformed. Among 380 participants (median age 74 yr; 67.6% male), higher BAP was consistently associated with lower MoCA scores (fully adjusted β -1.03; 95% CI, -1.90 to -0.16). Total ALP showed inverse associations in less-adjusted models; however, these associations attenuated after full adjustment (β -.88; 95% CI, -1.89 to 0.14). TRACP-5b was not clearly associated with MoCA scores. Higher BAP and total ALP were associated with higher odds of MMSE-defined cognitive dysfunction, whereas TRACP-5b was not. These findings suggest a potential association between bone formation-related markers and cognitive performance in older patients undergoing hemodialysis. Prospective and mechanistic studies are warranted to clarify causality and clinical utility.
Abstract This retrospective new-user cohort study evaluated the association between denosumab 60 mg and zoledronic acid 5 mg with first hospitalized vertebral fracture in adults aged ≥50 years receiving routine care in a Chinese hospital system. Eligible treatment initiators were identified from hospital injection and administration records at the Affiliated Kunshan Hospital of Jiangsu University between October 2021 and December 2024. Patients with a history of vertebral fracture, cancer, Paget disease, or previous alendronate use were excluded. The primary outcome was the first hospitalized vertebral fracture occurring >30 days after treatment initiation. The complete-case cohort consisted of 3,116 treatment initiators, with a total of 60 events, including 27 among patients receiving zoledronic acid and 33 among those receiving denosumab. In crude analysis, denosumab was associated with a higher fracture hazard than zoledronic acid (HR 3.02, 95% CI 1.46–6.26). In the primary overlap-weighted analysis, which estimated the average treatment effect in the overlap population, the HR was 2.05 (95% CI, 0.88–4.78). The doubly robust overlap-weighted analysis yielded an HR of 1.64 (95% CI, 0.65–4.17). All adjusted confidence intervals included the null; therefore, the adjusted analyses did not demonstrate a statistically significant difference between treatments. These findings should be interpreted with caution because of the limited number of events, treatment channeling, calendar imbalance, incomplete information on treatment persistence, and the potential for residual confounding.
Abstract Hereditary hypophosphatemic rickets with hypercalciuria (HHRH) is a rare FGF23-independent renal phosphate–wasting disorder caused by biallelic variants in SLC34A3, typically diagnosed in childhood or early adulthood. Delayed recognition in elderly individuals is uncommon, and the clinical features leading to diagnosis in this population remain poorly characterized. We report a case of a 77-year-old Japanese man with HHRH who remained undiagnosed until advanced age and was identified following a proximal humeral fracture sustained after a low-energy fall. The severity of the fracture, together with his history of multiple previous fractures, prompted biochemical evaluation for secondary causes of skeletal fragility. Biochemical assessment revealed hypophosphatemia with preserved renal function, elevated 1,25-dihydroxyvitamin D levels, hypercalciuria, and reduced renal phosphate reabsorption. Intact FGF23 was mildly above the assay reference limit and was not appropriately suppressed in the setting of hypophosphatemia. Bone mineral density was in the osteoporotic range at the femoral neck but did not distinguish osteoporosis from an underlying mineralization disorder. Genetic analysis identified a homozygous synonymous variant in SLC34A3 (c.942G>C, p.Ala314=) in the context of a characteristic biochemical phenotype consistent with HHRH. Oral phosphate supplementation alone resulted in rapid normalization of serum phosphate levels, without deterioration in renal function or worsening hypercalciuria. This case suggests that SLC34A3-related phosphate-wasting disorders may remain clinically unrecognized until late adulthood. Importantly, it highlights the value of biochemical evaluation, including measurement of serum phosphate and assessment of renal phosphate handling, in patients with otherwise unexplained skeletal fragility. Phosphate-wasting disorders should be considered in patients with recurrent fractures or skeletal fragility that is not fully explained by bone mineral density or the reported injury mechanism.
Abstract Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare genetic bone disorder associated with considerable clinical, economic, and societal impacts on patients, their caregivers, and the healthcare system. The aim of this study was to update the estimated prevalence of FOP in France, characterize the FOP patient population and impact of the disease, assess associated mortality rates, and quantify healthcare resource utilization (HCRU) and socioeconomic costs for individuals with FOP. An observational, retrospective case-control study was conducted using linked data for individuals with FOP from the French National Rare Diseases Registry (BNDMR) and the French National Healthcare System Database (SNDS). Results were compared with the SNDS for a control group of individuals with any disease other than FOP. Prevalence of FOP was estimated at 1.39 per million. Data were available for 77 individuals with FOP and 769 controls without FOP. Of 70 individuals with FOP and a social security number at the index date, 50.0% (n = 35/70) were female and mean (SD) age was 25.3 (15.7) years. Comorbidities were consistently more prevalent in individuals with FOP than people without FOP. Consultations with general practitioners and non-physician healthcare providers, hospitalizations, and use of medical devices and treatments were significantly greater in individuals with vs without FOP (p < .05 for all). Mean total annual healthcare expenditure was more than 10 times greater in individuals with FOP than those without FOP from a societal perspective (largely driven by outpatient costs) and 14 times greater from a payer perspective. Overall survival was significantly worse in individuals with FOP than individuals without (p < .0001). Findings from this study reinforce the clinical, economic, and societal impacts associated with FOP. Improving disease awareness and developing new interventions to support a reduction in these impacts should be considered key priorities for the FOP community and healthcare providers.
Abstract Chronic kidney disease (CKD) is associated with alterations in cortical bone structure and composition that contribute to increased fracture risk but are incompletely captured by standard clinical imaging, including dual-energy X-ray absorptiometry (DXA). This study evaluated whether radiomics combined with machine learning can enhance detection of CKD-related cortical bone characteristics using high-resolution peripheral quantitative computed tomography (HR-pQCT). HR-pQCT images (60.7 μm isotropic resolution; 168-slice stacks acquired at 7.3% and 30% proximal to the tibial endplate) were analyzed from 72 participants (38 non-CKD controls and 34 individuals with advanced CKD), yielding 24 192 cortical bone image slices. Cortical bone was segmented using a pre-trained neural network optimized through transfer learning. Radiomic features were extracted using Gray Level Co-Occurrence Matrix (GLCM), Local Binary Pattern (LBP), and combined GLCM+LBP feature sets, and paired with seven machine-learning classifiers to systematically evaluate 21 feature-classifier combinations at distal and diaphyseal tibial sites. To address optimism associated with correlated slice-level data, radiomic features were aggregated per patient prior to model training and evaluated using patient-level splits (~7 test patients); this patient-level analysis constitutes the primary evaluation framework of this study. Under patient-level evaluation, classification performance was more variable across feature-classifier combinations and tibial sites, with wide confidence intervals reflecting the limited sample size. At the slice level, hybrid GLCM+LBP features combined with XGBoost demonstrated the strongest classification performance across both tibial regions (distal AUC = 0.999; diaphyseal AUC = 0.999), providing methodological context for the systematic evaluation of all 21 feature-classifier combinations. Radiomics-derived texture features identified cortical heterogeneity that was not consistently reflected by conventional HR-pQCT metrics. These findings support radiomics as a promising methodological framework for characterizing CKD-associated cortical bone alterations while highlighting the importance of larger patient-level validation studies.
FRAXplus complements the fracture risk assessment tool (FRAX) by incorporating additional risk factors to adjust 10-yr probabilities of major osteoporotic and hip fractures. We aimed to identify which adjustments most strongly influence fracture risk estimates and how they affect risk classification and treatment recommendations in Switzerland. We analyzed data from the Swiss Osteoporosis Registry (2015-2023), which captures all standard FRAX variables plus lumbar spine (LS) BMD, trabecular bone score (TBS), fall history in the previous year, oral glucocorticoid dosages, and the location and recency of prior fractures. Data on hip axis length and duration of type 2 diabetes were not available. A total of 28 708 individuals (88% women; mean age 65.8 ± 11.4 yr) were included, with a median 10-yr probabilities for major osteoporotic fracture (MOF) of 16% (interquartile range [IQR]: 9-24) using standard FRAX and 18% [IQR: 11-28] with FRAXplus adjustments. Most individuals (72%) had higher probabilities with FRAXplus (mean absolute increase: +4.8%), while 27% had lower probabilities (mean absolute decrease: -1.0%). TBS and LS minus FN T-score (LS - FN) influenced nearly all risk estimates, but differences between FRAX and FRAXplus were generally modest. At the individual level, ≥1 fall and recent vertebral or hip fractures produced the largest differences. Applying FRAXplus with Swiss intervention thresholds led to an upward reclassification in 16% of MOF estimates and a downward reclassification in 1%. In summary, FRAXplus adjustments significantly alter fracture risk estimates for most individuals. While LS BMD and TBS affect most probabilities, falls and recent vertebral or hip fractures have the largest absolute impact at the individual level, leading to clinically relevant changes in treatment recommendations according to Swiss guidelines.
Abstract Bone regeneration remains a significant clinical challenge, requiring a deeper understanding of the cellular and molecular mechanisms underlying bone repair. Osteocytes, which represent 90-95% of bone cells, form a connective dendritic network and play a key role in skeletal homeostasis. Embedded within the mineralized matrix, they experience unique metabolic constraints, including reduced oxygen availability. Hypoxia may therefore influence their function, but its impact and potential role in regulating angiogenesis during bone regeneration remain poorly understood. This study aimed to investigate i) how hypoxia affects osteocytes and ii) their paracrine communication with endothelial cells. To model this interaction, we cultured the osteocytic cell line MLO-Y4 under normoxia (21% O₂), moderate hypoxia (5% O₂), or severe hypoxia (1% O₂) and collected the conditioned media after 48 hours. Mouse endothelial cells (1×104 cells/cm2) were cultured for 48 hours in a 1:1 ratio of endothelial cell medium and conditioned media from normoxic or hypoxic (5% and 1% O₂) osteocytes. Following optimization of co-culture conditions, RNA sequencing was performed to evaluate osteocyte and endothelial cells' gene expression changes. Our findings indicate that hypoxia contributes to the development of the osteocyte dendritic network and upregulates neurogenesis-associated genes. Moreover, oxygen levels differentially influence osteocyte behavior by modulating their hypoxic response and metabolism. Indeed, severe hypoxia (1% O₂) induces hypoxia-responsive genes and glycolysis more strongly than moderate hypoxia (5% O₂), resulting in distinct metabolic and transcriptional profiles. However, osteocyte-derived factors did not significantly alter gene expression in endothelial cells, suggesting that paracrine signalling, possibly not modulated by hypoxia, is not the principal mode of communication involved. Further studies are required to clarify the mechanisms by which oxygen levels shape osteocyte physiology.
PTH-related peptide (PTHrP) and its receptor, the PTH/PTHrP receptor type 1 (PTH1R), are essential for chondrocyte differentiation and epiphyseal cartilage homeostasis. To evaluate the functional differences between PTHrP and PTH1R derived from mesenchymal progenitor cells (MPCs) in skeletal development, we generated MPC-specific Pthrp conditional KO mice (PTHrP cKO; Prrx1-Cre; Pthrp flox/flox) or Pth1r cKO mice (PTH1R cKO; Prrx1-Cre; Pth1r flox/flox) using the Prrx1-Cre driver and analyzed their skeletal phenotypes. Both PTHrP cKO mice and PTH1R cKO mice exhibited severe shortening of long bones and impaired growth plate formation immediately after birth. However, in the digits, only PTHrP cKO mice showed distinctive digit deviations in the toes, which were similar to those seen in human diastrophic dysplasia (DTD). These results suggest that MPC-derived PTHrP and PTH1R play distinct roles. Both are essential for longitudinal bone growth, but PTHrP plays an important role in regulating toe position and morphology. This phenotypic difference suggests a novel PTH1R-independent function of PTHrP in skeletal development. Thus, our study provides genetic evidence for this mechanism, offering insights into the pathogenesis of congenital digit deformities such as those seen in DTD.
Pain in fibrous dysplasia/McCune-Albright syndrome (FD/MAS) remains inadequately treated. The pilot study was aimed to determine the feasibility (ie, completion, retention, and acceptability) and to explore the effectiveness of an intensive interdisciplinary pain treatment (IIPT) using a biopsychosocial approach in patients with FD/MAS. Five patients (4 females and 1 male; 22-30 yr old) with FD/MAS, with baseline pain severity of 4+ (0-10 scale) were enrolled. A 3-wk intervention (4 d/wk; 3-4 h/d) integrating psychological, physical, and occupational therapies was administered to patients. Study measures included patient-reported outcomes at treatment admission (week 0), discharge (week 3), and follow-up (week 15). Physician Global Assessments (PGA) at weeks 0 and 3 were also collected. All 5 patients completed the 3-wk intervention and follow-up assessment and reported a high degree of acceptability, appropriateness, and feasibility of the intervention. Average pain severity decreased after treatment, with mean changes of -3.0 and -3.2 points at weeks 3 and 15, respectively. Patients demonstrated improvements in pain interference, external pain expression (measured with the pain behavior scale), and pain catastrophizing. Trends of decreased anxiety and depression were observed. Overall health-related quality of life improved at treatment discharge, which was consistent with the PGA (ie, +2 to +3) scores. This pilot study demonstrates that IIPT was feasible and showed initial clinical benefit across multiple dimensions in a small cohort of patients with FD/MAS. Larger-scale, controlled trials are needed for further validation.
Burosumab has shown benefits in treating X-linked hypophosphatemia (XLH), a rare genetic bone disorder that causes rickets among pediatric patients. However, data in Chinese children with XLH are lacking. Therefore, we evaluated the effectiveness, safety, and pharmacokinetics of burosumab among Chinese children (aged 1-12 yr) with XLH in this open-label, multi-center, single-arm, phase 4 study. Of the 28 patients enrolled (12 male, 16 female), 27 completed the study. All patients received s.c. injections of burosumab at a starting dose of 0.8 mg/kg every 2 wk for 64 wk. The mean ± SD patient age was 5.68 ± 3.07 yr. Burosumab treatment increased the serum phosphorus level by 0.96 ± 0.27 mg/dL from baseline during the burosumab dose cycles (average of week 2, 4, 8, 12, 16, 24, 32, 40, 52, and 64). The improvement from baseline (2.33 ± 0.24 mg/dL) to week 64 (3.29 ± 0.30 mg/dL) was significant (p < .05). The Radiograph Global Impression of Change was significantly improved at week 64 (1.90 ± 0.48; p < .0001 vs baseline), and the percentage predicted 6-min walking test among patients aged ≥5 yr with post-baseline values (n = 16) was significantly increased from 67.32% ± 8.27% at baseline to 71.91% ± 8.25% at week 64 (p < .05). All patients experienced treatment-emergent adverse events; most were mild to moderate. In conclusion, burosumab treatment in Chinese children with XLH corrected serum phosphorus levels and improved clinical parameters with tolerable safety profiles.
Human bone marrow-derived stromal cells (hMSCs) are a great resource for studying how genes influence cell fate and differentiation into various cell types like osteoblasts, adipocytes, and chondrocytes, among other cell types. However, genetic manipulation of primary hMSCs has been challenging due to their short lifespan and cellular senescence after limited passaging. Their low and unstable transfection efficiency also complicates gene delivery or inactivation, hindering long-term functional studies. The limited lifespan has been effectively solved by immortalizing hMSCs with telomerase reverse transcriptase (hMSCs-TERT). The use of these cells is ideal for functional studies of osteoblast and adipocyte differentiation through genetic manipulation, providing a stable and reliable model. Here, we have engineered a stable CAS9 expressing hMSC-TERT cell line (hMSC-TERTCAS9) via lentiviral transduction. The constitutive expression of spCas9 enables efficient and reproducible gene editing. We demonstrate the potential of these hMSC-TERTCAS9 cells for generating gene disruptions using plasmid delivery of guide RNAs as a fast and efficient strategy for targeted genome editing. The edited cells can be sorted and expanded as single cells to obtain homogenous clonal cell lines with mono- as well as bi-allelic gene deletions, a crucial step for producing reliable experimental results. We further validate this cell line as a powerful tool for studying gene function during hMSC proliferation and differentiation, providing 3 distinct examples of its utility. Through the generation of indels, single-cell sorting, and clonal selection, we have efficiently inactivated the vitamin D receptor and created both larger (256 nucleotides) gene disruptions in Forkhead box protein O1 and precise removals of a small genomic sequence (73 nucleotides) coding for microRNA MIR675. This novel hMSC-TERTCAS9 cell line represents a significant advancement, offering a stable, efficient, and versatile platform for advanced genetic studies, high-throughput screening, and the creation of reliable cellular disease models.
Sarcopenia is common among older adults with type 2 diabetes mellitus (T2DM) and is associated with increased risks of functional decline and adverse health outcomes. Phase angle (PhA), derived from bioelectrical impedance analysis, has emerged as a potential marker of muscle health. This cross-sectional study aimed to evaluate the predictive value of PhA for detecting sarcopenia in older adults with T2DM and to determine sex-specific cutoff values for sarcopenia screening. A total of 390 patients aged ≥60 yr with T2DM were included. Sarcopenia was defined according to the Asian Working Group for Sarcopenia 2019 criteria. Lower PhA values were significantly associated with sarcopenia, and PhA showed good discriminatory performance for detecting sarcopenia. Sex-specific cutoff values improved the diagnostic accuracy of PhA for sarcopenia screening. These findings suggest that PhA may serve as a practical and noninvasive tool for identifying sarcopenia in older adults with T2DM. Further longitudinal studies are warranted to confirm its prognostic value.
Osteoporosis is a skeletal disease that significantly increases fracture risk and imposes a growing public health and economic burden. Notably, hip fractures are associated with high mortality, long-term disability, and loss of independence. When evaluating osteoporosis and estimating fracture risk, DXA is commonly used to determine the BMD. To capture geometrical morphology from these DXA scans as factors in fracture risk prediction, landmarking around the femur is conducted manually around the ROI. However, this process is labor-intensive and prone to variability. This study presents the development of a fully automated femoral landmarking tool that integrates a U-Net convolutional neural network for femur contour segmentation with a geometric algorithm for consistent landmark placement. A heterogeneous dataset of 555 DXA scans was used to train and evaluate the U-Net model, achieving a pixel-wise accuracy of 97.55%, an Intersection over Union of 91.55%, and a Dice coefficient of 95.56% on the test set. When incorporated into a statistical shape and appearance modeling (SSAM) framework for fracture risk prediction, predictions using the automatically generated landmarks achieved a test AUC of 0.831 (95% CI: 0.698-0.965), compared with 0.780 (95% CI: 0.635-0.925) for manual landmarks; a paired DeLong test showed no significant difference (p = .53), indicating a comparable performance. The proposed pipeline produces anatomically relevant, reproducible landmarks, and supports fracture prediction performance similar to manual methods. It presents a scalable and objective solution for morphometric analysis in DXA imaging.
Early-stage osteochondrosis (OC) lesions are focal failures of endochondral ossification in the physeal and epiphyseal regions. Lesions are initially detected and quantified as ossification variants using non-invasive imaging methods. The etiology of OC lesions is not understood, as early-stage lesions are rarely imaged in children or animals until symptoms advance to stages that require intervention. The distal femur, a common site for OC lesions, provides an easily accessible, anatomical location to study early-stage development. Non-invasive CT and MRI techniques for distinguishing early-stage lesions were compared in images collected from Cross-sectional and Longitudinal Studies. Osteochondrosis lesion traits in the physeal and epiphyseal regions of swine distal femurs were quantified. Cross-sectional subjects were randomly selected from asymptomatic, crossbred swine at 7, 12, and 24 wk of age. A longitudinal cohort was repeatedly imaged at 6, 12, and 18 wk of age. Subsets of femurs from each age group were selected for histological analysis. Ossification variants defined as abnormal physeal widths without defined OC lesions, were observed at 7 wk. At 12 wk, 2.7 ± 1.8 physeal lesions per pig with an average volume = 9.8 ± 10.0 mm3 were quantified but no epiphyseal lesions were detected. At 24 wk, similar numbers (2.1 ± 1.4) of epiphyseal and physeal lesions were observed. Epiphyseal lesions had greater volumes (44.5 ± 53.5 mm3). At 24 wk, atypical ossification variants not previously characterized, were unexpectedly observed in over 40% of the femurs. These large, undefined regions characterized by extreme undulations and irregular mineralization without physeal widening were labeled irregular physeal lesions. Although the quantitative outputs differed, both CT and MRI methods yielded consistent inferences regarding the developmental age and location (primary vs secondary centers of ossification) of OC lesion progression across Cross-sectional and Longitudinal Studies. These outcomes support future research efforts to identify pivotal molecular signals controlling the initiation, progression, and regression of early-stage lesion development.
Bone in adult mammals plays critical structural and endocrine functions, both largely rely on osteocytes—the most abundant bone cells and principal source of bone-derived hormones such as fibroblast growth factor 23 and sclerostin. The widely used 10-kb promoter-driven transgenic Tg(Dmp1-Cre) and Tg(Dmp1-CreERT2) lines, while widely used and helpful, utilize transgenic approaches with transgene expression driven by the Dmp1 promoter. The transgene expression suffers from positional effects with off-target expression in muscle, brain, and other tissues, limiting their applications, causing a major technical gap in the investigation of osteocyte-specific functions. Here we generated two novel mouse lines — Dmp1em1(CreERT2) and Dmp1em2(ZsGreen) —via CRISPR–Cas9 mediated “knock-in” approaches to insert an IRES–CreERT2 or IRES–ZsGreen cassette into the Dmp1 locus, allowing CreERT2 or ZsGreen expression under the genetic control of the endogenous Dmp1 locus while preserving Dmp1 gene expression. We show that the Dmp1em1(CreERT2) enabled highly efficient tamoxifen-inducible recombination (>90% in cortical and trabecular osteocytes) with minimal off-target expression in non-bone tissues. The Dmp1em2(ZsGreen) line showed robust, fixation- and decalcification-resistant green fluorescence in osteocytes from birth through adulthood, faithfully reflecting endogenous Dmp1 expression. Lineage tracing using the Dmp1em1(CreERT2) line further revealed that the cortical osteocytes are long-lived, whereas trabecular osteocytes undergo continuous renewal, uncovering compartment-specific differences in osteocyte lifespan. By eliminating off-target recombination or gene expression in skeletal muscle, brain, gastrointestinal tract, and marrow compartments, the two mouse lines offer powerful tools for rigorous studies in osteocyte biology, mechanotransduction, and bone regulation of systemic physiology that impact health, aging, and diseases, while minimizing complications due to off-target transgene expression.
Micro-CT enables high-resolution, 3D, and non-destructive visualization of bone microarchitecture in small preclinical animal models. It is widely applied to assess mineralized tissues in joints, making it a valuable tool for monitoring disease progression. Murine models are particularly prevalent in joint research due to their cost-effectiveness and disease tunability. Established guidelines for murine bone microstructure assessment provide a standardized framework for morphometric analysis and facilitate cross-study comparisons. However, these recommendations were developed for trabecular and cortical bone in long bones, and may not fully address the unique characteristics of mineralized tissues within joints. This review focuses on murine studies, and aims to: (1) examine reported methodologies for volume of interest selection; (2) outline commonly evaluated parameters; and (3) propose adaptations to expand existing guidelines for quantitative analysis of the osteochondral unit. In the absence of specific guidelines for osteochondral unit analysis, reported volumes of interest vary considerably and are influenced by anatomical differences across strains, sexes, ages, and disease stages. Many studies target the subchondral bone plate (SBP); however, the spatial resolution of desktop microCT is insufficient to distinguish SBP from calcified cartilage; hence, they are measured as 1 entity. To enhance reproducibility and comparability, we recommend standardized volumes of interest that include all mineralized tissues from the articular surface to the growth plate. Given the structural differences between SBP and subchondral trabecular bone, a consistent method for defining their boundary is strongly recommended. Data should be reported using robust, easily implementable metrics such as volume and thickness, with additional parameters (eg, porosity, mineral density) included as appropriate. Commonly used metrics such as SBP thickness and volume should continue to be used. When SBP and calcified cartilage cannot be distinguished, we recommend using the subchondral mineralized plate nomenclature.
