A Review of CT-Based Fracture Risk Assessment with Finite Element Modeling and Machine Learning.

PURPOSE OF REVIEW: We reviewed advances over the past 3 years in assessment of fracture risk based on CT scans, considering methods that use finite element models, machine learning, or a combination of both. RECENT FINDINGS: Several studies have demonstrated that CT-based assessment of fracture risk, using finite element modeling or biomarkers derived from machine learning, is equivalent to currently used clinical tools. Phantomless calibration of CT scans for bone mineral density enables accurate measurements from routinely taken scans. This opportunistic use of CT scans for fracture risk assessment is facilitated by high-quality automated segmentation with deep learning, enabling workflows that do not require user intervention. Modeling of more realistic and diverse loading conditions, as well as improved modeling of fracture mechanisms, has shown promise to enhance our understanding of fracture processes and improve the assessment of fracture risk beyond the performance of current clinical tools. CT-based screening for fracture risk is effective and, by analyzing scans that were taken for other indications, could be used to expand the pool of people screened, therefore improving fracture prevention. Finite element modeling and machine learning both provide valuable tools for fracture risk assessment. Future approaches should focus on including more loading-related aspects of fracture risk.

Trabecular Architecture and Mechanical Heterogeneity Effects on Vertebral Body Strength.

PURPOSE OF REVIEW: We aimed to synthesize the recent work on the intra-vertebral heterogeneity in density, trabecular architecture and mechanical properties, its implications for fracture risk, its association with degeneration of the intervertebral discs, and its implications for implant design. RECENT FINDINGS: As compared to the peripheral regions of the centrum, the central region of the vertebral body exhibits lower density and more sparse microstructure. As compared to the anterior region, the posterior region shows higher density. These variations are more pronounced in vertebrae from older persons and in those adjacent to degenerated discs. Mixed results have been reported in regard to variation along the superior-inferior axis and to relationships between the heterogeneity in density and vertebral strength and fracture risk. These discrepancies highlight that, first, despite the large amount of study of the intra-vertebral heterogeneity in microstructure, direct study of that in mechanical properties has lagged, and second, more measurements of vertebral loading are needed to understand how the heterogeneity affects distributions of stress and strain in the vertebra. These future areas of study are relevant not only to the question of spine fractures but also to the design and selection of implants for spine fusion and disc replacement. The intra-vertebral heterogeneity in microstructure and mechanical properties may be a product of mechanical adaptation as well as a key determinant of the ability of the vertebral body to withstand a given type of loading.

Bone Mechanical Properties in Healthy and Diseased States.

The mechanical properties of bone are fundamental to the ability of our skeletons to support movement and to provide protection to our vital organs. As such, deterioration in mechanical behavior with aging and/or diseases such as osteoporosis and diabetes can have profound consequences for individuals' quality of life. This article reviews current knowledge of the basic mechanical behavior of bone at length scales ranging from hundreds of nanometers to tens of centimeters. We present the basic tenets of bone mechanics and connect them to some of the arcs of research that have brought the field to recent advances. We also discuss cortical bone, trabecular bone, and whole bones, as well as multiple aspects of material behavior, including elasticity, yield, fracture, fatigue, and damage. We describe the roles of bone quantity (e.g., density, porosity) and bone quality (e.g., cross-linking, protein composition), along with several avenues of future research.

Tributyltin induces distinct effects on cortical and trabecular bone in female C57Bl/6J mice.

The retinoid X receptors (RXR), peroxisome proliferator activated receptor gamma (PPARγ), and liver X receptors (LXR) all have been shown to regulate bone homeostasis. Tributyltin (TBT) is an environmental contaminant that is a dual RXRα/ß and PPARγ agonist. TBT induces RXR, PPARγ, and LXR-mediated gene transcription and suppresses osteoblast differentiation in vitro. Bone marrow multipotent mesenchymal stromal cells derived from female C57BL/6J mice were more sensitive to suppression of osteogenesis by TBT than those derived from male mice. In vivo, oral gavage of 12 week old female, C57Bl/6J mice with 10 mg/kg TBT for 10 weeks resulted in femurs with a smaller cross-sectional area and thinner cortex. Surprisingly, TBT induced significant increases in trabecular thickness, number, and bone volume fraction. TBT treatment did not change the Rankl:Opg RNA ratio in whole bone, and histological analyses showed that osteoclasts in the trabecular space were minimally reduced. In contrast, expression of cardiotrophin-1, an osteoblastogenic cytokine secreted by osteoclasts, increased. In primary bone marrow macrophage cultures, TBT marginally inhibited the number of osteoclasts that differentiated, in spite of significantly suppressing expression of osteoclast markers Nfatc1, Acp5, and Ctsk and resorptive activity. TBT induced expression of RXR- and LXR-dependent genes in whole bone and in vitro osteoclast cultures. However, only an RXR antagonist, but not an LXR antagonist, significantly inhibited TBTs ability to suppress osteoclast differentiation. These results suggest that TBT has distinct effects on cortical versus trabecular bone, likely resulting from independent effects on osteoblast and osteoclast differentiation that are mediated through RXR.

Differences in Trabecular Microarchitecture and Simplified Boundary Conditions Limit the Accuracy of Quantitative Computed Tomography-Based Finite Element Models of Vertebral Failure.

Vertebral fractures are common in the elderly, but efforts to reduce their incidence have been hampered by incomplete understanding of the failure processes that are involved. This study's goal was to elucidate failure processes in the lumbar vertebra and to assess the accuracy of quantitative computed tomography (QCT)-based finite element (FE) simulations of these processes. Following QCT scanning, spine segments (n = 27) consisting of L1 with adjacent intervertebral disks and neighboring endplates of T12 and L2 were compressed axially in a stepwise manner. A microcomputed tomography scan was performed at each loading step. The resulting time-lapse series of images was analyzed using digital volume correlation (DVC) to quantify deformations throughout the vertebral body. While some diversity among vertebrae was observed on how these deformations progressed, common features were large strains that developed progressively in the superior third and, concomitantly, in the midtransverse plane, in a manner that was associated with spatial variations in microstructural parameters such as connectivity density. Results of FE simulations corresponded qualitatively to the measured failure patterns when boundary conditions were derived from DVC displacements at the endplate. However, quantitative correspondence was often poor, particularly when boundary conditions were simplified to uniform compressive loading. These findings suggest that variations in trabecular microstructure are one cause of the differences in failure patterns among vertebrae and that both the lack of incorporation of these variations into QCT-based FE models and the oversimplification of boundary conditions limit the accuracy of these models in simulating vertebral failure.

Local Changes to the Distal Femoral Growth Plate Following Injury in Mice.

Injury to the growth plate is associated with growth disturbances, most notably premature cessation of growth. The goal of this study was to identify spatial changes in the structure and composition of the growth plate in response to injury to provide a foundation for developing therapies that minimize the consequences for skeletal development. We used contrast-enhanced microcomputed tomography (CECT) and histological analyses of a murine model of growth plate injury to quantify changes in the cartilaginous and osseous tissue of the growth plate. To distinguish between local and global changes, the growth plate was divided into regions of interest near to and far from the injury site. We noted increased thickness and CECT attenuation (a measure correlated with glycosaminoglycan (GAG) content) near the injury, and increased tissue mineral density (TMD) of bone bridges within the injury site, compared to outside the injury site and contralateral growth plates. Furthermore, we noted disruption of the normal zonal organization of the physis. The height of the hypertrophic zone was increased at the injury site, and the relative height of the proliferative zone was decreased across the entire injured growth plate. These results indicate that growth plate injury leads to localized disruption of cellular activity and of endochondral ossification. These local changes in tissue structure and composition may contribute to the observed retardation in femur growth. In particular, the changes in proliferative and hypertrophic zone heights seen following injury may impact growth and could be targeted when developing therapies for growth plate injury.

Sex-Linked Skeletal Phenotype of Lysyl Oxidase Like-1 Mutant Mice.

Lysyl oxidases are required for collagen and elastin cross-linking and extracellular matrix maturation including in bone. The lysyl oxidase family consists of lysyl oxidase (LOX) and 4 isoforms (LOXL1-4). Here we investigate whether deletion of LOXL1, which has been linked primarily to elastin maturation, leads to skeletal abnormalities. Left femurs (n = 8), L5 vertebrae (n = 8), and tibiae (n = 8) were analyzed by micro-computed tomography in 13-week-old wild-type (WT) and LOXL1-/- male and female mice. Right femurs (n = 8) were subjected to immunohistochemistry for LOXL1, and histochemical/histology analyses of osteoclasts and growth plates. Sera from all mice were analyzed for bone turnover markers. Results indicate strong expression of LOXL1 in wild-type growth plates in femurs. Significant deterioration of trabecular bone structure in long bones and vertebrae from female was observed but not from male, mutant mice compared with WT. Decreases in BV/TV, Conn.D, trabecular thickness, and number in the femoral distal metaphysis were observed in female, but not in male, mutant mice. Trabecular spacing was increased significantly in femurs of female mutant mice. Findings were similar in trabeculae of L5 vertebrae from female mutant mice. The number of TRAP positive osteoclasts at the trabecular bone surface was increased in female mutant mice compared with WT females, consistent with increased serum RANKL and decreased OPG levels. Analysis of bone turnover markers confirmed increased bone resorption as indicated by significantly elevated CTX-1 in the serum of female LOXL1-/- mice compared to their wild-type counterparts, as well as decreased bone formation as measured by decreased serum levels of PINP. Picrosirius red staining revealed a loss of heterogeneity in collagen organization in female LOXL1-/- mice only, with little to no yellow and orange birefringence. Organization was also impaired in chondrocyte columns in both female and male LOXL1-/- mice, but to a greater extent in females. Data indicate that LOXL1-/- mutant mice develop appendicular and axial skeletal phenotypes characterized by decreased bone volume fraction and compromised trabecular microstructure, predominantly in females.

Intrinsic Sex-Linked Variations in Osteogenic and Adipogenic Differentiation Potential of Bone Marrow Multipotent Stromal Cells.

Bone formation and aging are sexually dimorphic. Yet, definition of the intrinsic molecular differences between male and female multipotent mesenchymal stromal cells (MSCs) in bone is lacking. This study assessed sex-linked differences in MSC differentiation in 3-, 6-, and 9-month-old C57BL/6J mice. Analysis of tibiae showed that female mice had lower bone volume fraction and higher adipocyte content in the bone marrow compared to age-matched males. While both males and females lost bone mass in early aging, the rate of loss was higher in males. Similar expression of bone- and adipocyte-related genes was seen in males and females at 3 and 9 months, while at 6 months, females exhibited a twofold greater expression of these genes. Under osteogenic culture conditions, bone marrow MSCs from female 3- and 6-month-old mice expressed similar levels of bone-related genes, but significantly greater levels of adipocyte-related genes, than male MSCs. Female MSCs also responded to rosiglitazone-induced suppression of osteogenesis at a 5-fold lower (10 nM) concentration than male MSCs. Female MSCs grown in estrogen-stripped medium showed similar responses to rosiglitazone as MSCs grown in serum containing estrogen. MSCs from female mice that had undergone ovariectomy before sexual maturity also were sensitive to rosiglitazone-induced effects on osteogenesis. These results suggest that female MSCs are more sensitive to modulation of differentiation by PPARγ and that these differences are intrinsic to the sex of the animal from which the MSCs came. These results also may explain the sensitivity of women to the deleterious effects of rosiglitazone on bone.

Macrophage-specific TLR2 signaling mediates pathogen-induced TNF-dependent inflammatory oral bone loss.

Porphyromonas gingivalis is a primary etiological agent of chronic periodontal disease, an infection-driven chronic inflammatory disease that leads to the resorption of tooth-supporting alveolar bone. We previously reported that TLR2 is required for P. gingivalis-induced alveolar bone loss in vivo, and our in vitro work implicated TNF as a key downstream mediator. In this study, we show that TNF-deficient (Tnf(-/-)) mice are resistant to alveolar bone loss following oral infection with P. gingivalis, and thus establish a central role for TNF in experimental periodontal disease. Using bone marrow-derived macrophages (BMDM) from wild-type and gene-specific knockout mice, we demonstrate that the initial inflammatory response to P. gingivalis in naive macrophages is MyD88 dependent and requires cooperative signaling of TLR2 and TLR4. The ability of P. gingivalis to activate cells via TLR2 or TLR4 was confirmed in TLR2- or TLR4-transformed human embryonic kidney cells. Additional studies using bacterial mutants demonstrated a role for fimbriae in the modulation of TLR-mediated activation of NF-κB. Whereas both TLR2 and TLR4 contributed to TNF production in naive macrophages, P. gingivalis preferentially exploited TLR2 in endotoxin-tolerant BMDM to trigger excessive TNF production. We found that TNF induced surface TLR2 expression and augmented TLR-induced cytokine production in P. gingivalis-stimulated BMDM, establishing a previously unidentified TNF-dependent feedback loop. Adoptive transfer of TLR2-expressing macrophages to TLR2-deficient mice restored the ability of P. gingivalis to induce alveolar bone loss in vivo. Collectively, our results identify a TLR2- and TNF-dependent macrophage-specific mechanism underlying pathogen-induced inflammatory bone loss in vivo.

Elastic Anisotropy of Trabecular Bone in the Elderly Human Vertebra.

Knowledge of the nature of the elastic symmetry of trabecular bone is fundamental to the study of bone adaptation and failure. Previous studies have classified human vertebral trabecular bone as orthotropic or transversely isotropic but have typically obtained samples from only selected regions of the centrum. In this study, the elastic symmetry of human vertebral trabecular bone was characterized using microfinite element (µFE) analyses performed on 1019 cubic regions of side length equal to 5 mm, obtained via thorough sampling of the centrums of 18 human L1 vertebrae (age = 81.17 ± 7.7 yr; eight males and ten females). An optimization procedure was used to find the closest orthotropic representation of the resulting stiffness tensor for each cube. The orthotropic elastic constants and orientation of the principal elastic axes were then recorded for each cube and were compared to the constants predicted from Cowin's fabric-based constitutive model (Cowin, 1985, "The Relationship Between the Elasticity Tensor and the Fabric Tensor," Mech. Mater., 4(2), pp. 137-147.) and the orientation of the principal axes of the fabric tensor, respectively. Deviations from orthotropy were quantified by the "orthotropic error" (van Rietbergen et al., 1996, "Direct Mechanics Assessment of Elastic Symmetries and Properties of Trabecular Bone Architecture," J. Biomech., 29(12), pp. 1653-1657), and deviations from transverse isotropy were determined by statistical comparison of the secondary and tertiary elastic moduli. The orthotropic error was greater than 50% for nearly half of the cubes, and the secondary and tertiary moduli differed from one another (p < 0.0001). Both the orthotropic error and the difference between secondary and tertiary moduli decreased with increasing bone volume fraction (BV/TV; p ≤ 0.007). Considering only the cubes with an orthotropic error less than 50%, only moderate correlations were observed between the fabric-based and the µFE-computed elastic moduli (R2 ≥ 0.337; p < 0.0001). These results indicate that when using a criterion of 5 mm for a representative volume element (RVE), transverse isotropy or orthotropy cannot be assumed for elderly human vertebral trabecular bone. Particularly at low values of BV/TV, this criterion does not ensure applicability of theories of continuous media. In light of the very sparse and inhomogeneous microstructure found in the specimens analyzed in this study, further work is needed to establish guidelines for selecting a RVE within the aged vertebral centrum.

Geometric determinants of the mechanical behavior of image-based finite element models of the intervertebral disc.

The intervertebral disc is an important structure for load transfer through the spine. Its injury and degeneration have been linked to pain and spinal fractures. Disc injury and spine fractures are associated with high stresses; however, these stresses cannot be measured, necessitating the use of finite element (FE) models. These models should include the disc's complex structure, as changes in disc geometry have been linked to altered mechanical behavior. However, image-based models using disc-specific structures have yet to be established. This study describes a multiphasic FE modeling approach for noninvasive estimates of subject-specific intervertebral disc mechanical behavior based on medical imaging. The models (n = 22) were used to study the influence of disc geometry on the predicted global mechanical response (moments and forces), internal local disc stresses, and tractions at the interface between the disc and the bone. Disc geometry was found to have a strong influence on the predicted moments and forces on the disc (R2 = 0.69-0.93), while assumptions regarding the side curvature (bulge) of the disc had only a minor effect. Strong variability in the predicted internal disc stresses and tractions was observed between the models (mean absolute differences of 5.1%-27.7%). Disc height had a systematic influence on the internal disc stresses and tractions at the disc-to-bone interface. The influence of disc geometry on mechanics highlights the importance of disc-specific modeling to estimate disc injury risk, loading on the adjacent vertebral bodies, and the mechanical environment present in disc tissues.

Superlative mechanical energy absorbing efficiency discovered through self-driving lab-human partnership.

Energy absorbing efficiency is a key determinant of a structure's ability to provide mechanical protection and is defined by the amount of energy that can be absorbed prior to stresses increasing to a level that damages the system to be protected. Here, we explore the energy absorbing efficiency of additively manufactured polymer structures by using a self-driving lab (SDL) to perform >25,000 physical experiments on generalized cylindrical shells. We use a human-SDL collaborative approach where experiments are selected from over trillions of candidates in an 11-dimensional parameter space using Bayesian optimization and then automatically performed while the human team monitors progress to periodically modify aspects of the system. The result of this human-SDL campaign is the discovery of a structure with a 75.2% energy absorbing efficiency and a library of experimental data that reveals transferable principles for designing tough structures.

Effect of specimen-specific anisotropic material properties in quantitative computed tomography-based finite element analysis of the vertebra.

Intra- and inter-specimen variations in trabecular anisotropy are often ignored in quantitative computed tomography (QCT)-based finite element (FE) models of the vertebra. The material properties are typically estimated solely from local variations in bone mineral density (BMD), and a fixed representation of elastic anisotropy ("generic anisotropy") is assumed. This study evaluated the effect of incorporating specimen-specific, trabecular anisotropy on QCT-based FE predictions of vertebral stiffness and deformation patterns. Orthotropic material properties estimated from microcomputed tomography data ("specimen-specific anisotropy"), were assigned to a large, columnar region of the L1 centrum (n = 12), and generic-anisotropic material properties were assigned to the remainder of the vertebral body. Results were compared to FE analyses in which generic-anisotropic properties were used throughout. FE analyses were also performed on only the columnar regions. For the columnar regions, the axial stiffnesses obtained from the two categories of material properties were uncorrelated with each other (p = 0.604), and the distributions of minimum principal strain were distinctly different (p ≤ 0.022). In contrast, for the whole vertebral bodies in both axial and flexural loading, the stiffnesses obtained using the two categories of material properties were highly correlated (R2 > 0.82, p < 0.001) with, and were no different (p > 0.359) from, each other. Only moderate variations in strain distributions were observed between the two categories of material properties. The contrasting results for the columns versus vertebrae indicate a large contribution of the peripheral regions of the vertebral body to the mechanical behavior of this bone. In companion analyses on the effect of the degree of anisotropy (DA), the axial stiffnesses of the trabecular column (p < 0.001) and vertebra (p = 0.007) increased with increasing DA. These findings demonstrate the need for accurate modeling of the peripheral regions of the vertebral body in analyses of the mechanical behavior of the vertebra.

Harnessing mechanical cues in the cellular microenvironment for bone regeneration.

At the macroscale, bones experience a variety of compressive and tensile loads, and these loads cause deformations of the cortical and trabecular microstructure. These deformations produce a variety of stimuli in the cellular microenvironment that can influence the differentiation of marrow stromal cells (MSCs) and the activity of cells of the MSC lineage, including osteoblasts, osteocytes, and chondrocytes. Mechanotransduction, or conversion of mechanical stimuli to biochemical and biological signals, is thus part of a multiscale mechanobiological process that drives bone modeling, remodeling, fracture healing, and implant osseointegration. Despite strong evidence of the influence of a variety of mechanical cues, and multiple paradigms proposed to explain the influence of these cues on tissue growth and differentiation, even a working understanding of how skeletal cells respond to the complex combinations of stimuli in their microenvironments remains elusive. This review covers the current understanding of what types of microenvironmental mechanical cues MSCs respond to and what is known about how they respond in the presence of multiple such cues. We argue that in order to realize the vast potential for harnessing the cellular microenvironment for the enhancement of bone regeneration, additional investigations of how combinations of mechanical cues influence bone regeneration are needed.

Multiscale theoretical model shows that aging-related mechanical degradation of cortical bone is driven by microstructural changes in addition to porosity.

This study aims to gain mechanistic understanding of how aging-related changes in the microstructure of cortical bone drive mechanical consequences at the macroscale. To that end, cortical bone was modeled as a bundle of elastic-plastic, parallel fibers, which represented osteons and interstitial tissue, loaded in uniaxial tension. Distinct material properties were assigned to each fiber in either the osteon or interstitial fiber "families." Models representative of mature (20-60 yrs.) bone, and elderly (60+) bone were created by modeling aging via the following changes to the input parameters: (i) increasing porosity from 5% to 15%, (ii) increasing the ratio of the number of osteon fibers relative to interstitial fibers from 40% to 50%, and (iii) changing the fiber material properties from representing mature bone samples to representing elderly bone samples (i.e., increased strength and decreased toughness of interstitial fibers together with decreased toughness of osteon fibers). To understand the respective contributions of these changes, additional models isolating one or two of each of these were also created. From the computed stress-strain curve for the fiber bundle, the yield point (ϵy, σy), ultimate point (ϵu, σu), and toughness (UT) for the bundle as a whole were measured. We found that changes to all three input parameters were required for the model to capture the aging-related decline in cortical bone mechanical properties consistent with those previously reported in the literature. In both mature and elderly bundles, rupture of the interstitial fibers drove the initial loss of strength following the ultimate point. Plasticity and more gradual rupture of the osteons drove the remainder of the response. Both the onset and completion of interstitial fiber rupture occurred at lower strains in the elderly vs. mature case. These findings point to the importance of studying microstructural changes beyond porosity, such as the area fraction of osteons and the material properties of osteon and interstitial tissue, in order to further understanding of aging-related changes in bone.

Biomechanical structure-function relations for human trabecular bone - comparison of calcaneus, femoral neck, greater trochanter, proximal tibia, and vertebra.

MicroCT-based finite element models were used to compute power law relations for uniaxial compressive yield stress versus bone volume fraction for 78 cores of human trabecular bone from five anatomic sites. The leading coefficient of the power law for calcaneus differed from those for most of the other sites (p < 0.05). However, after normalizing by site-specific mean values, neither the leading coefficient (p > 0.5) nor exponent (p > 0.5) differed among sites, suggesting that a given percentage deviation from mean bone volume fraction has the same mechanical consequence for all sites investigated. These findings help explain the success of calcaneal x-ray and ultrasound measurements for predicting hip fracture risk.

An in silico micro-multiphysics agent-based approach for simulating bone regeneration in a mouse femur defect model.

Bone defects represent a challenging clinical problem as they can lead to non-union. In silico models are well suited to study bone regeneration under varying conditions by linking both cellular and systems scales. This paper presents an in silico micro-multiphysics agent-based (micro-MPA) model for bone regeneration following an osteotomy. The model includes vasculature, bone, and immune cells, as well as their interaction with the local environment. The model was calibrated by time-lapsed micro-computed tomography data of femoral osteotomies in C57Bl/6J mice, and the differences between predicted bone volume fractions and the longitudinal in vivo measurements were quantitatively evaluated using root mean square error (RMSE). The model performed well in simulating bone regeneration across the osteotomy gap, with no difference (5.5% RMSE, p = 0.68) between the in silico and in vivo groups for the 5-week healing period - from the inflammatory phase to the remodelling phase - in the volume spanning the osteotomy gap. Overall, the proposed micro-MPA model was able to simulate the influence of the local mechanical environment on bone regeneration, and both this environment and cytokine concentrations were found to be key factors in promoting bone regeneration. Further, the validated model matched clinical observations that larger gap sizes correlate with worse healing outcomes and ultimately simulated non-union. This model could help design and guide future experimental studies in bone repair, by identifying which are the most critical in vivo experiments to perform.

Spatial assessment of femoral neck bone density and microstructure in hip osteoarthritis.

Osteoarthritis (OA) is known to involve profound changes in bone density and microstructure near to, and even distal to, the joint. Critically, however, a full, spatial picture of these abnormalities has not been well documented in a quantitative fashion in hip OA. Here, micro-computed tomography (44.8 µm/voxel) and data-driven computational anatomy were used to generate 3-D maps of the distribution of bone density and microstructure in human femoral neck samples with early (6F/4M, mean age = 51.3 years), moderate (14F/8M, mean age = 60 years), and severe (16F/6M, mean age = 63.3 years) radiographic OA. With increasing severity of radiographic OA, there was decreased cortical bone mineral density (BMD) (p=0.003), increased cortical thickness (p=0.001), increased cortical porosity (p=0.0028), and increased cortical cross-sectional area (p=0.0012, due to an increase in periosteal radius (p=0.018)), with no differences detected in the total femoral neck or trabecular compartment measures. No OA-related region-specific differences were detected through Statistical Parametric Mapping, but there were trends towards decreased tissue mineral density (TMD) in the inferior femoral neck with increasing OA severity (0.050 < p ≤ 0.091), possibly due to osteophytes. Overall, the lack of differences in cortical TMD among radiographic OA groups indicated that the decrease in cortical BMD with increasing OA severity was largely due to the increased cortical porosity rather than decreased tissue mineralization. As porosity is inversely associated with stiffness and strength in cortical bone, increased porosity may offset the effect that increased cortical cross-sectional area would be expected to have on reducing stresses within the femoral neck. The use of high-resolution imaging and quantitative spatial assessment in this study provide insight into the heterogeneous and multi-faceted changes in density and microstructure in hip OA, which have implications for OA progression and fracture risk.

Identification of Known and Novel Long Noncoding RNAs Potentially Responsible for the Effects of Bone Mineral Density (BMD) Genomewide Association Study (GWAS) Loci.

Osteoporosis, characterized by low bone mineral density (BMD), is the most common complex disease affecting bone and constitutes a major societal health problem. Genome-wide association studies (GWASs) have identified over 1100 associations influencing BMD. It has been shown that perturbations to long noncoding RNAs (lncRNAs) influence BMD and the activities of bone cells; however, the extent to which lncRNAs are involved in the genetic regulation of BMD is unknown. Here, we combined the analysis of allelic imbalance (AI) in human acetabular bone fragments with a transcriptome-wide association study (TWAS) and expression quantitative trait loci (eQTL) colocalization analysis using data from the Genotype-Tissue Expression (GTEx) project to identify lncRNAs potentially responsible for GWAS associations. We identified 27 lncRNAs in bone that are located in proximity to a BMD GWAS association and harbor single-nucleotide polymorphisms (SNPs) demonstrating AI. Using GTEx data we identified an additional 31 lncRNAs whose expression was associated (false discovery rate [FDR] correction < 0.05) with BMD through TWAS and had a colocalizing eQTL (regional colocalization probability [RCP] > 0.1). The 58 lncRNAs are located in 43 BMD associations. To further support a causal role for the identified lncRNAs, we show that 23 of the 58 lncRNAs are differentially expressed as a function of osteoblast differentiation. Our approach identifies lncRNAs that are potentially responsible for BMD GWAS associations and suggest that lncRNAs play a role in the genetics of osteoporosis. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Identification and Characterization of a Novel Long Noncoding RNA that Regulates Osteogenesis in Diet-Induced Obesity Mice.

As a precursor to type 2 diabetes mellitus (T2D), obesity adversely alters bone cell functions, causing decreased bone quality. Currently, the mechanisms leading to alterations in bone quality in obesity and subsequently T2D are largely unclear. Emerging evidence suggests that long noncoding RNAs (lncRNAs) participate in a vast repertoire of biological processes and play essential roles in gene expression and posttranscriptional processes. Mechanistically, the expression of lncRNAs is implicated in pathogenesis surrounding the aggregation or alleviation of human diseases. To investigate the functional link between specific lncRNA and obesity-associated poor bone quality and elucidate the molecular mechanisms underlying the interaction between the two, we first assessed the structure of the bones in a diet-induced obese (DIO) mouse model. We found that bone microarchitecture markedly deteriorated in the DIO mice, mainly because of aberrant remodeling in the bone structure. The results of in vitro mechanistic experiments supported these observations. We then screened mRNAs and lncRNAs from DIO bones and functionally identified a specific lncRNA, Gm15222. Further analyses demonstrated that Gm15222 promotes osteogenesis and inhibits the expression of adipogenesis-related genes in DIO via recruitment of lysine demethylases KDM6B and KDM4B, respectively. Through this epigenetic pathway, Gm15222 modulates histone methylation of osteogenic genes. In addition, Gm15222 showed a positive correlation with the expression of a neighboring gene, BMP4. Together, the results of this study identified and provided initial characterization of Gm15222 as a critical epigenetic modifier that regulates osteogenesis and has potential roles in targeting the pathophysiology of bone disease in obesity and potential T2D.