Temporomandibular joint osteoarthritis (TMJ OA) is a prevalent degenerative disease characterized by chronic pain and impaired jaw function. Similarly to other OA affected limb joints, TMJ OA has shown increased prevalence across several demographic and biological factors. These risk factors include sex, with female patients reporting twice the incidence rate and suffering from greater symptom severity compared to male patients. However, due to a lack of relevant preclinical models, there is limited knowledge related to how sex differences impact TMJ OA pathophysiology. With increasing attention in women's health, we aim to understand the molecular mechanisms underlying sexually-dimorphic TMJ OA pathogenesis. Using the burn-synovectomy mouse model to induce TMJ OA, which mimics the elevated inflammatory cytokines associated with chronic stressors and local TMJ derangement, we observed more severe cartilage and subchondral bone abnormalities in females consistent with findings in patients. Single-cell RNA sequencing (scRNA-seq) and histology revealed that females had an increased inflammatory response, both in terms of immune cell abundance as well as in their inflammatory gene signature, compared to male animals. Additional analyses also showed that female mice downregulated major morphogenetic pathways, such as WNT, BMP TGF-β, and FGF, within osteochondral cell populations, while simultaneously increasing expression of extracellular matrix remodeling enzymes. These results demonstrate the sexual-dimorphisms in disease initiation and progression leading to the differentially observed TMJ OA phenotypes. The data herein may explain the incident tendency and symptom severity in female patients and suggest sex-dependent treatment strategies for future patient care.

The choice of segmentation method for HR-pQCT scans influences accuracy of bone microarchitecture measurements. Smaller or under-mineralized bone can present challenges in accurate extraction of bone structure using global thresholding methods, as local variations in intensity are not considered and finer structural details are not detected. This is especially important in small hand bones, where bone structure is finer, or younger populations, where bone tissue may be under mineralized. This study compared accuracy of global thresholding methods using Gaussian and Laplace-Hamming filters, and an adaptive local thresholding (AT) method in HR-pQCT scans of carpal bones. Eight ex vivo human cadaveric forearms (n = 64 carpal bones) were analyzed. Three specimens (n = 24 carpal bones, 2 female, mean age: 82.7 ± 4.6yr) were used for AT parameter optimization, and 5 specimens (n = 40 carpal bones, 3 female, mean age: 82.0 ± 6.4yr) were used to compare trabecular microarchitecture accuracy and spatial agreement relative to micro-CT (μCT, 20μm isotropic resolution). Micro-CT images were segmented using a Gaussian filter and Otsu's method, and HR-pQCT images were segmented using Gaussian filtering and global thresholding, Laplace-Hamming filtering and global thresholding, and the AT method. Trabecular thickness (Tb.Th), separation (Tb.Sp), and bone volume fraction (Tb.BV/TV) accuracy were evaluated, and spatial agreement was assessed using Dice similarity coefficients (DSC), 95th percentile Hausdorff distances (HD95), and average symmetric surface distances (ASSD). The AT method yielded the smallest absolute and relative errors, and lowest bias across all trabecular parameters. Compared to the Gaussian and fixed threshold method, AT reduced mean absolute error by 36% for Tb.Th, 14% for Tb.Sp, and 15% for Tb.BV/TV, and achieved the highest spatial agreement with μCT (DSC = 0.84, HD95 = 0.061mm, and ASSD = 0.018mm). These findings extend prior HR-pQCT segmentation validation studies to carpal bones and demonstrate that AT outperforms the standard and Laplace-Hamming methods.

High-throughput transcriptomic technologies have advanced rapidly, enabling genome-wide gene expression profiling. Microarrays, introduced in 1995, laid the foundation for large-scale analysis but were later surpassed in 2008 by RNA sequencing (RNA-seq), which offers single-nucleotide resolution, detects low-abundance transcripts, and does not require prior sequence knowledge. Bulk RNA-seq provides robust insights into global transcriptomic changes but lacks single-cell resolution. Single-cell RNA-seq (scRNA-seq), introduced in 2009, addressed this limitation by revealing cellular heterogeneity and dynamic gene expression. However, its application in bone research is constrained due to difficulties in releasing bone cells called osteocytes from the mineralized matrix, often resulting in low yield and dissociation-induced artifacts. In order to address these challenges, single-nucleus RNA-seq (snRNA-seq), first introduced in 2016 to enable transcriptomic profiling from isolated nuclei, was used in this study. We developed a protocol for snRNA-seq on bone tissue, achieving high-yield recovery of osteocyte nuclei from snap-frozen, marrow-flushed long bones. This approach minimized dissociation bias and enhanced osteocyte representation. We applied this robust method to long bones from young adult male and female mice, generating a high-resolution map of osteocyte gene expression under physiological conditions. Compared to scRNA-seq datasets, where osteocytes represent only 0.18%-6.64% of cells, our snRNA-seq approach increased osteocyte capture and transcriptomic fidelity to 18.5%. We identified an osteocyte transcriptomic signature highlighting the top 30 genes, including Sost, which is typically undetected or lowly-expressed in scRNA-seq. Notably, 23 of these genes have not been well-characterized in osteocytes, including Tg, Kcnq5, Rapgef4os1, Cacna1a, Egr3, Dok5, and Lgr6, which may represent novel regulators of osteocyte biology. This study represents the first application of snRNA-seq specifically for osteocyte analysis in bone tissue, providing a valuable resource for investigating osteocyte biology and skeletal disorders.

Fibrodysplasia ossificans progressiva (FOP) is an ultra-rare and disabling disease. Preventing unnecessary and/or harmful medical procedures to patients requires early and accurate diagnosis, multidisciplinary care, and access to specialists with FOP expertise. This study aims to assess the needs of healthcare providers (HCPs) when providing care to patients with FOP. Knowledge, confidence, and management practices were measured through a short awareness survey, completed by 400 HCPs from different specialties who may encounter patients with FOP. Complementary qualitative data were obtained from a dialogue session about FOP. Quantitative data was analyzed using descriptive and inferential analysis, while qualitative data underwent thematic coding. Results indicate a need for enhanced knowledge and confidence regarding identifying clinical manifestations of FOP, referral to FOP specialists and subsequent patient management. Only 8.8% of HCPs correctly identified FOP from an open-ended question showing malformed great toes, a classical clinical manifestation of FOP, and only 31.8% correctly identified FOP from a multiple-choice question showing heterotopic ossification on a child’s back, another classical clinical manifestation of FOP. Structural and system-level issues were also identified as barriers to optimal patient care pathways, with limited access to FOP specialists reported as a prominent concern. These results document a need for improvements in HCP knowledge in addition to local care collaboration and healthcare system optimization to meet the needs of patients with rare diseases. Given that FOP is an ultra-rare disease, educational interventions are recommended to focus on rare disease recognition, with FOP included as one topic. In addition, greater team collaboration and system-level approaches to assist HCPs in overcoming barriers that impede patient access to care and ongoing management, as well as interventions to enhance patients’ capacity to have informed communication with their HCPs, are needed.

We aimed to develop a tool to predict the probability of hip fracture given a fall, using biofidelic finite element models (FEMs) built from DXA scans. Additionally, we evaluated the effectiveness of hip protectors, assuming full compliance, by quantifying the reduction in relative risk in-silico. This study included 700 subjects (325 males and 375 females) from the Population Health and Eye Disease Profile in Elderly Singaporeans (PIONEER) cohort, consisting of Chinese, Indian, and Malay ethnicities. The probability of hip fracture was calculated using DXA-based biofidelic FEMs by simulating various falls. The relative risk was determined by comparing fracture probabilities between falls with and without hip protectors. The overall probability of hip fracture in the cohort (mean age: 71.5yr) was 4.49% (SD: 4.71). Chinese males had a significantly higher fracture risk, with odds ratios of 3.15 (95% CI: 1.81-5.49) compared to Malay males, and 4.57 (95% CI, 2.21-9.44) compared to Indian males. Similarly, Chinese females had a higher fracture risk, with odds ratios of 2.22 (95% CI, 1.16-4.24) compared to Malay females, and 7.70 (95% CI, 2.30-25.76) compared to Indian females. The relative risk with hip protectors was 0.38 (SD: 0.28) for males overall, with subgroup values of 0.42 (SD: 0.29) for Chinese, 0.33 (SD: 0.29) for Indians, and 0.38 (SD: 0.26) for Malays. For females, the overall relative risk was 0.43 (SD: 0.28), with subgroup values of 0.41 (SD: 0.21) for Chinese, 0.37 (SD: 0.32) for Indian, and 0.51 (SD: 0.29) for Malay, with no significant differences between ethnicities. In conclusion, we have developed an in-silico tool to assess hip fracture risk and evaluate the effectiveness of hip protectors using clinical DXA scans. These findings support the adoption of hip protectors as a preventive measure to reduce fracture risk.

Children and adolescents with end-stage kidney disease (ESKD) have a high prevalence of bone deformities and fractures, even when bone histomorphometric measures of bone formation, mineralization, and volume are within normal range. We postulated that bone matrix quality and osteocyte differentiation are impaired in ESKD in ways which might be not reflected by alterations in surface-based cell activity. We assessed bone turnover, mineralization, and volume by traditional histomorphometry in bone biopsy cores from 24 adolescent patients with ESKD. We stained sequential sections of the same cores for apoptosis (TUNEL staining) and for osteocyte-specific maturation markers (sclerostin, DMP1, and FGF23). We then performed quantitative backscattered electron imaging (qBEI) to assess bone mineralization density distribution on the whole bone surface and additionally in bone packets containing FGF23-expressing osteocytes. Increased amounts of osteoid (OV/BV > 4.7%) and a true defect in bone surface mineralization (O.Th > 10 μm combined with MLT > 27.1 d) were detectable by histomorphometry in 29% and 17% of bone samples, respectively. By contrast, qBEI detected an excess in primary mineralizing bone (TbCaLow > 6.8%) in 88% of samples and abnormally low bone matrix mineralization (TbCaMean < 22.1 wt% calcium) in 75% of samples. Osteocyte lacunar density and size were within normal range. Unexpectedly, higher numbers of FGF23-expressing osteocytes and a more elongated osteocyte lacunar shape were found in bone samples with greater matrix mineralization. In conclusion, matrix mineralization defects, which are not detected by traditional histomorphometry, are highly prevalent in ESKD bone and reflect poor bone matrix maturation. High numbers of FGF23-expressing osteocytes are found in bone with the most appropriate matrix maturation characteristics. This differs from the bone characteristics of individuals with normal kidney function, in whom large amounts of FGF23 result in low circulating phosphate levels, which in turn triggers severe osteomalacia and hypomineralized periosteocytic lesions.

At present, there are no FDA-approved orally-available bone anabolic agents to treat osteoporosis. PTH stimulates bone formation through an intracellular signaling cascade that involves the inhibition of salt-inducible kinase (SIK) isoforms 2 and 3. Therefore, direct small molecule SIK2/SIK3 inhibitors may represent a strategy to mimic PTH actions to treat different forms of osteoporosis. We previously described the synthesis and characterization of SK-124, a pharmacologic SIK2/SIK3 inhibitor that increases trabecular bone formation in eugonadal mice. However, the efficacy of this agent in osteoporosis mouse models remains unknown. Hypogonadism is an important cause of age-related bone loss. In this study, we investigated the therapeutic potential of SK-124 in a male hypogonadal bone loss model (orchiectomy, ORX) in BALB/c mice. Radiographic and histological analyses revealed that SK-124-treated ORX mice showed reduced bone loss compared to the vehicle-treated ORX mice. Serum bone turnover markers demonstrated that SK-124 treatment increased bone turnover, suggesting that SK-124 acts in a PTH-like manner in ORX mice. Bone RNA-sequencing analysis demonstrated novel pathways associated with increased bone formation in response to SK-124 treatment. These findings indicate that SK-124 prevents bone loss in a hypogonadal bone loss model and holds potential as an orally available therapeutic for treating osteoporosis due to testosterone deficiency.

Secondary osteoporosis is a major global health problem and can be caused by endocrine diseases, including hyperthyroidism. Of note, hyperthyroid mice exhibit both trabecular and cortical bone loss, accompanied by reduced bone strength, as a consequence of a high bone turnover in which bone resorption exceeds bone formation. Recently, we discovered that osteocytes in bones obtained from hyperthyroid mice develop osteoclast-like features. However, the role of thyroid hormone receptor α (TRα) in hyperthyroidism-driven bone resorption remains ill-defined. Here, we rendered male Thra0/0 mice (which lack all TRα isoforms) hyperthyroid, and comprehensively characterized their bone phenotype. Thra0/0 mice with L-thyroxine (T4) treatment displayed trabecular, but not cortical bone loss, and maintained bone strength, while T4-treated WT littermates showed overall reduced bone mass and strength. Serum levels of bone resorption marker tartrate-resistant acid phosphatase (TRAP) and numbers of TRAP-positive osteoclasts were elevated in hyperthyroid WT, but not Thra0/0 mice. We also detected increased numbers of TRAP-positive osteocytes in long bones of T4-treated WT mice. In line, the expression of osteoclast marker genes was upregulated with T4 in bone tissue from WT mice only. While thyroid hormones (THs) have been reported not to directly affect osteoclast activity, our in vitro studies using late osteoblasts/osteocytes derived from Thra0/0 and WT mice revealed that the TH-induced expression of osteoclast markers in these cells was Thra-dependent. Further, the Rankl/Opg ratio linking osteocytes to enhanced osteoclastogenesis was upregulated in hyperthyroid WT, but not Thra0/0 cells. Taken together, we demonstrate that Thra KO can preserve cortical bone mass and bone strength by obstructing an increase of bone-resorbing osteoclasts and osteocytes with osteoclast-like features in hyperthyroid male mice.

The rate of orthodontic tooth movement slows with age, likely due to changes in alveolar bone remodeling. However, the underlying mechanisms remain poorly understood. Osteocytes (OCys), which form the lacunar-canalicular network (LCN), play a key role in bone metabolism; however, the effects of aging on the alveolar LCN are still unclear. This study aimed to clarify age-related alterations in the LCN of alveolar bone and their underlying mechanisms. The LCN was visualized using Ploton silver staining, revealing a significant reduction in 16-mo-old C57BL/6J mice compared to 2-mo-old mice. The number of OCys, as well as the number and length of canaliculi per OCy, decreased significantly with age. These reductions were consistent in both sexes. Periodontal ligament cells (PDLCs) from aged mice showed increased expression of the senescence marker p16INK4A and upregulation of senescence-associated secretory phenotype (SASP) factor mRNAs. To evaluate the effect of SASP factors on LCN formation, a 3D-block culture system was utilized. Conditioned medium (CM) from senescent human PDLCs (over 20 passages) suppressed dendrite formation in the 3D culture as well as the expression of E11/podoplanin, while CM from young PDLCs had no significant effect. To test whether SASP inhibition could preserve LCN, the senolytic agents Dasatinib and Quercetin (D + Q) were administered to 12-mo-old mice for 4 mo. D + Q treatment significantly increased both canalicular number and length per OCy in alveolar bone compared to vehicle controls. In addition, D + Q treatment increased E11 expression and suppressed sclerostin expression on OCys in aged alveolar bones. These findings suggest that SASP factors from senescent PDLCs contribute to the age-related LCN deterioration in alveolar bone, potentially impairing OCy function and bone remodeling. Targeting PDL cellular senescence may therefore offer a novel strategy for preserving alveolar bone integrity with aging.