Cortical Bone Area Research Articles

Use of computer tomography imaging for analyzing bone remodeling around a percutaneous osseointegrated implant.

Osseointegration (OI) is being used for the direct skeletal attachment of prosthetic limbs using an intramedullary stem that extends percutaneously from the subject's residual limb. For this technology to be successful, bone ingrowth and remodeling around the implant must occur. Physicians need an effective way to assess bone remodeling to make informed treatment and rehabilitation decisions. Previous studies utilizing two-dimensional imaging X-ray as a tool to monitor bone-remodeling around OI devices have limitations. This study describes methodology that was developed utilizing computed tomography (CT) imaging as a tool for analyzing bone remodeling around a percutaneous OI implant. Six transfemoral amputees implanted with a percutaneous osseointegrated prosthesis (POP) had CT scans taken of their residual femur at 6 and 52 weeks postoperatively. Three-dimensional femoral models were processed using custom MATLAB script to collect cortical and medullary morphology measurements. Morphology data from 6-and 52-week scans were compared to quantify bone remodeling around the POP implant. Fifty-two weeks after implantation of the POP device, increases in cortical bone area and thickness were observed around the porous-coated stem. Minimal changes were observed in the medullary canal parameters within the periprosthetic regions. This study successfully utilized CT imaging and three-dimensionalmodeling techniques to analyze longitudinal data of bone remodeling around a transfemoral percutaneous implant. These methods have the potential to be used as a clinical tool for evaluating orthopedic implants in vivo. Data collected suggests that the POP device achieved the desired bone remodeling around the porous-coated region of the implanted stem.

Untargeted metabolomics study on the effects of rearing ducks in cages on bone quality

The cage rearing model of the modern poultry industry makes the bones of birds more vulnerable to deterioration. In this study, at 8 wk of age, a total of 60 birds were randomly allocated to 2 groups, including the floor rearing group (FRD) and cage rearing group (CRD), and their body weight was measured every 2 wk. At the age of 20 wk, the tibia, femur, and humerus were collected from each group (n = 12) to determine the bone quality parameters such as weight, size, bone mineral density (BMD), breaking strength, cortical thickness, and area, ash content, calcium (Ca) content, and phosphorus (P) content. Meanwhile, the serum metabolome composition of both groups was detected by untargeted metabolome technology. The results showed that there were no significant differences in body weight, bone weight, and size between the 2 groups (P > 0.05), but the humerus mineral density and the breaking strength, cortical bone thickness, cortical bone area percentage of tibia, femur, and humerus of CRD was significantly lower than those of FRD (P < 0.05), indicating that the cage rearing system caused the deterioration of bone quality. Based on nontarget metabolomics, 49 metabolites were correlated with bone quality parameters, and 10 key metabolites were strongly correlated, including erucic acid, citric acid, and ketoleucine. In addition, the KEGG analysis showed that the caged system mainly perturbed amino acid metabolism, lipid metabolism, and energy metabolism, which led to changes in related metabolite levels, produced ROS, and altering energy supply, thus leading to a deterioration of bone quality of cage rearing ducks. Therefore, our findings were helpful to further understand the potential mechanism of the deterioration of duck bone quality in cage rearing system, provided a theoretical basis for reducing the occurrence of poultry osteoporosis, and ensuring the healthy development of poultry breeding.

The Novel Role of Checkpoint Inhibitor PD-1H/VISTA in Osteoclast Activation and Multiple Myeloma Bone Disease

IntroductionMultiple myeloma (MM)-induced bone disease remains one of its most devastating complications, caused by increased bone resorption by overactivated osteoclasts coupled with impaired bone formation. MM cells produce osteoclast-activating factors that induce osteoclast activation and extensive bone resorption. Our previous work demonstrated that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor that is highly secreted by MM cells (Fu J etc. JCI. 2016). We also identified that the checkpoint inhibitor, programmed death-1 homolog (PD-1H/VISTA), serves as the MMP-13 receptor in osteoclasts and mediates MMP-13-dependent osteoclastogenic function which is largely blocked in Pd-1h -/-osteoclasts (Fu J etc. ASH 2019, 2020). While the inhibitory role of PD-1H/VISTA in T-cells has recently been described (ElTanbouly MA etc. Science 2020), its cellular binding proteins remain unclear, and its role in osteoclast activation andMM bone disease have not been addressed.Methods and ResultsTo identify its interacting proteins, PD-1H-His 6 recombinant protein was expressed in mouse bone marrow mononuclear cells, the associated proteins pulled down by Ni-NTA agarose beads from cell lysates and identified by mass spectrometry. Functional annotation charting of the 75 proteins enriched in PD-1H pull-down samples (with signal ratio of PD-1H-His 6 pull-down vs control >2) indicated that almost 30% of the interacting targets were either cytoskeletal or cytoskeleton-associated proteins.Given that the F-actin cytoskeleton undergoes dynamic reorganization during osteoclast differentiation and plays critical roles in bone resorption, we further addressed the role of PD-1H in F-actin cytoskeleton regulation. Initially, osteoclasts form F-actin-rich adhesive structures, termed podosomes. At later stages, podosomes collectively rearrange into clusters and rings and finally into sealing belts to mediate osteoclast spreading, migration and bone resorption (Teitelbaum SL. Ann N Y Acad Sci. 2011). By confocal immunofluorescence microscopy, we found that PD-1H co-localized with F-actin podosome clusters, rings and sealing belts during osteoclast differentiation (Figure 1A). The functional role of PD-1H in F-actin cytoskeleton reorganization was addressed using Pd-1h -/- osteoclast wherein Pd-1h knockout lead to the disruption of podosome clusters at early stages relative to WT controls, while at later stages, Pd-1h -/-osteoclasts exhibited significantly fewer F-actin rings and belts (Figure 1B). Further, binding of MMP-13 to PD-1H increased the number of osteoclasts forming F-actin rings and belts, as well as the size of F-actin belts, which was blocked in Pd-1h -/- osteoclasts.To determine the role of PD-1H in the development of myeloma-induced lytic bone lesions, 5TGM1 myeloma cells were bilaterally intratibially injected into Pd-1h wtRag2 -/- or Pd-1h -/-Rag2 -/- mice (n=10) to induce lytic bone lesions. Three weeks following intratibial 5TGM1 injection, tibiae were harvested for micro-computed tomography. Subsequent quantitative histomorphology analyses of the trabecular and cortical bones confirmed that the knockout of Pd-1h reduced MM-induced bone destruction with significantly less decrease in trabecular bone volume (Tb. [BV/TV]), trabecular bone number (Tb. N.), trabecular bone thickness (Tb. Th.), as well as less increase in trabecular bone spacing (Tb. Sp.) and bone specific surface (Tb. [BS/BV]) compared to Pd-1h wtRag2 -/- mice. Similar effects were observed in cortical bone with less decrease in cortical bone thickness (CT. Th.), cortical bone area fraction (CT. [BA/TA]), and cortical tissue mineral density (CT. TMD) in 5TGM1 bearing Pd-1h -/-Rag2 -/- mice vs Pd-1h wtRag2 -/- mice (Table 1).ConclusionsTaken together, our study, for the first time, reveals the novel role of checkpoint inhibitor, PD-1H/VISTA, in osteoclasts and myeloma bone disease. PD-1H associates with cytoskeleton proteins and regulates the F-actin cytoskeleton reorganization which is critical for osteoclast bone resorption activity. Further, PD-1H mediates MMP-13-induced osteoclast fusion, F-actin belts formation, and osteoclast activation. Pd-1h -/-in recipient mice significantly impairs MM-induced bone loss, demonstrating that PD-1H/VISTA plays a critical role in MM bone disease. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

An antibody against Siglec-15 promotes bone formation and fracture healing by increasing TRAP+ mononuclear cells and PDGF-BB secretion

Osteoporosis (OP) is a common age-related disease characterized by a deterioration of bone mass and structure that predisposes patients to fragility fractures. Pharmaceutical therapies that promote anabolic bone formation in OP patients and OP-induced fracture are needed. We investigated whether a neutralizing antibody against Siglec-15 can simultaneously inhibit bone resorption and stimulate bone formation. We found that the multinucleation of osteoclasts was inhibited in SIGLEC-15 conditional knockout mice and mice undergoing Siglec-15 neutralizing antibody treatment. The secretion of platelet-derived growth factor-BB (PDGF-BB), the number of tartrate-resistant acid phosphatase-positive (TRAP+) mononuclear cells, and bone formation were significantly increased in the SIGLEC-15 conditional knockout mice and antibody-treated mice. The anabolic effect of the Siglec-15 neutralizing antibody on bone formation was blunted in mice with Pdgfb deleted in TRAP+ cells. These findings showed that the anabolic effect of the Siglec-15 neutralizing antibody was mediated by elevating PDGF-BB production of TRAP+ mononuclear cells. To test the therapeutic potential of the Siglec-15 neutralizing antibody, we injected the antibody in an ovariectomy-induced osteoporotic mouse model, which mimics postmenopausal osteoporosis in women, and in two fracture healing models because fracture is the most serious health consequence of osteoporosis. The Siglec-15 neutralizing antibody effectively reduced bone resorption and stimulated bone formation in estrogen deficiency-induced osteoporosis. Of note, the Siglec-15 neutralizing antibody promoted intramembranous and endochondral ossification at the damaged area of cortical bone in fracture healing mouse models. Thus, the Siglec-15 neutralizing antibody shows significant translational potential as a novel therapy for OP and bone fracture.

Tributyltin protects against ovariectomy-induced trabecular bone loss in C57BL/6J mice with an attenuated effect in high fat fed mice

Risk factors for poor bone quality include estrogen loss at menopause, a high fat diet and exposures to drugs/chemicals that activate peroxisome proliferator activated receptor gamma (PPARγ). We previously reported that the PPARγ and retinoid X receptor dual ligand, tributyltin (TBT), repressed periosteal bone formation but enhanced trabecular bone formation in vivo. Here, we examined the interaction of diet, ovariectomy (OVX) and TBT exposure on bone structure. C57BL/6J mice underwent either sham surgery or OVX at 10 weeks of age. At 12 weeks of age, they were placed on a low (10% kcal) or high (45% kcal) fat, sucrose-matched diet and treated with vehicle or TBT (1 or 5 mg/kg) for 14 weeks. OVX increased body weight gain in mice on either diet. TBT enhanced body weight gain in intact mice fed a high fat diet, but decreased weight gain in OVX mice. Elemental tin concentrations increased dose-dependently in bone. TBT had marginal effects on cortical and trabecular bone in intact mice fed either diet. OVX caused a reduction in cortical and trabecular bone, regardless of diet. In high fat fed OVX mice, TBT further reduced cortical thickness, bone area and total area. Interestingly, TBT protected against OVX-induced trabecular bone loss in low fat fed mice. The protective effect of TBT was nullified by the high fat. These results show that TBT protects against trabecular bone loss, even in the presence of a strongly resorptive environment, at an even lower level of exposure than we showed repressed homeostatic resorption.

Trends in Ancient Populations’ Osteobiography during the Holocene: the Levantine Perspective

Abstract. The rapid changes in our surroundings during the Holocene challenge the human body. The current study, carried out on the terminal Pleistocene-Holocene populations of the Levant, presents several examples of how changes in the habitation and diet have affected the disease patterns and the gross morphology of long bones and mandibles. Our major findings were that the prehistoric Natufian (67%) and the protohistoric Chalcolithic (80%) populations exhibited a much higher rate of ear inflammation (otitis media) than all other populations: Neolithic, Roman/Byzantine, and Ottoman (50% ± 5%). A general continuous significant decrease in the relative femoral bone cortical area from the Natufian (77.8%) to present times (64.7%) was noticed (the Chalcolithic population was the exception). Mandibular shape has changed considerably, mainly between the pre-agricultural population (the Natufian) and the succeeding ones (i.e., the ramus became taller and narrower, the coronoid higher and pointed, the mandibular notch narrower and deeper, and the body more triangular). Possible behavioral, economic, and environmental factors associated with these changes are discussed.

OAB-020: The role of checkpoint inhibitor PD-1H/VISTA in Multiple Myeloma bone disease

<h3>Background</h3> Multiple myeloma (MM) bone disease remains one of the most devastating complications of this incurable cancer, causing bone fractures, pain, mobility issues, and neurological deficits. MM cells secret pro-osteoclastogenic factors which lead to osteoclast (OCL) activation. Our previous work showed that matrix metalloproteinase 13 (MMP-13) is a critical osteoclastogenic factor highly secreted by MM cells and induces OCL fusion and bone resorption independently of its proteolytic activity (JCI 2016). We recently reported that MMP-13 binds to checkpoint inhibitor programmed death-1 homolog (PD-1H/VISTA), a surface receptor that is expressed on OCLs and mediates MMP-13 induced OCL fusion and bone resorption activity (ASH 2019). Bone resorption activity is significantly impaired in Pd-1h-/- OCLs in vitro. However, the function of PD-1H in MM bone disease has not been defined. <h3>Methods</h3> The role of PD-1H in MM bone disease was investigated using the intratibial 5TGM1 Rag2-/- MM bone disease mice model. Pd-1h-/-Rag2-/- mice were generated by crossbreeding Pd-1h-/- with Rag2-/- mice. Firefly luciferase-expressing 5TGM1 cells were intratibially injected into age and sex-paired Rag2-/- or Pd-1h-/-Rag2-/- mice (N=10). 3 weeks later, tibiae were harvested for quantitative micro-CT followed by histological analysis. <h3>Results</h3> Morphological analyses of trabecular and cortical bones confirmed that Pd-1h-/- recipient mice exhibited significantly less 5TGM1-induced bone loss (P<0.05). In trabecular bone, 5TGM1 induced 40.0% decrease of bone volume fraction in Rag2-/- mice vs 12.5% in Pd-1h-/-Rag2-/- mice, 7.3% decrease of trabecular bone numbers in Rag2-/- mice vs 0.6% in Pd-1h-/-Rag2-/- mice, 12.1% decrease of trabecular bone thickness in Rag2-/- mice vs 4.2% in Pd-1h-/-Rag2-/- mice, 8.1% increase of trabecular bone spacing in Rag2-/- mice vs 0.6% in Pd-1h-/-Rag2-/- mice, and 22.2% increase of specific bone surface in Rag2-/- mice vs 5.5% in Pd-1h-/-Rag2-/-mice. Similar effects were observed in cortical bone. 5TGM1 induced 18.9% decrease of cortical bone thickness in Rag2-/- mice vs 1.6% in Pd-1h-/-Rag2-/- mice, 7.28% decrease of cortical bone area fraction in Rag2-/- mice vs 2.7% in Pd-1h-/-Rag2-/- mice, and 8.19% decrease of cortical tissue mineral density in Rag2-/- mice vs 1.7% in Pd-1h-/-Rag2-/- mice. <h3>Conclusions</h3> Taken together, our study, for the first time, reveals that checkpoint inhibitor PD-1H/VISTA is the critical receptor for MMP-13 in osteoclasts, thereby mediating MMP-13-induced osteoclast fusion, activation, and bone resorption. MM-induced trabecular bone loss was significantly lower in Pd-1h-/- mice, demonstrating that PD-1H/VISTA plays a critical role in MMP-13-induced MM bone disease. Given the checkpoint role of PD-1H/VISTA in cancer immunosuppression, we further posit that targeting the interaction of MMP-13 and PD-1H may represent a novel therapeutic strategy to treat MM bone disease and modulate the MM immune environment.

Effects of zinc-containing diamond-like carbon coated splints on the healing of fractures in mice: A pilot study

Diamond-like carbon (DLC) coatings are highly biocompatible. They have been attracting attention because they can be used in the surface treatment of body-implanted devices. Zinc (Zn) promotes the metabolism of skin and bones. For these reasons, we developed Zn-doped DLC (Zn-DLC), a highly biocompatible DLC, as a coating material to promote bone formation. In our previous study, in vitro tests using osteoblast cells revealed that Zn-DLC promoted calcification associated with osteogenesis. Therefore, in the present study, the ability of Zn-DLC to promote osteogenesis was assessed in vivo using a Zn-deficient mouse model.We compared the healing stages of the bone fractures between Zn-DLC splints group and DLC splints group (N = 7 mice in each group), using computed tomography (CT)-created 3D models of the fractured parts. The number of mice that were fully healed by 4 weeks when fixed with DLC and Zn-DLC splints was two and four, respectively. Then, we investigated the bone features (the bone volume, bending strength, twisting strength, cortical bone thickness, cortical bone area ratio, and spongiosa bone density) after the in vivo test. These results suggested that Zn-DLC had tendencies to improve these parameters compared with DLC, though these differences were not significant statistically. Moreover, we confirmed Zn elution from Zn-DLC, using XPS analysis.These results suggested that Zn-DLC can supply Zn in vivo condition and may improve the therapeutic effect of conventional implants used in the treatment of fractures.

Targeted Remodeling of the Tumor Microenvironment Via Mechanical Stimulation Defines a Novel Therapeutic Strategy in Myeloma Bone Disease

Multiple myeloma (MM) patients develop devastating osteolytic bone disease causing non-healing bone fractures and pain. MM cells in the bone marrow microenvironment stimulate osteoclast activity and inhibit osteoblast and osteocyte function leading to massive bone resorption and therefore destruction. Bones have the capacity to locally adapt to changing loads and for self-repair and remodeling as dramatically seen in athletes during training. In this study we determined if mechanical loading can counterbalance osteolytic bone disease in the presence of established MM. We hypothesized that mechanical stimulation increases bone formation and decreases bone resorption by activating known bone (re)modeling pathways, which are deleteriously altered by MM cells in bone. We performed in vivo cyclic axial compressive tibial loading (Fig. 1) to MOPC315.BM BALB/c injected mice (n=19) with established MM bone disease (Fig. 2, bioluminescence imaging). The bone (re)modeling response to mechanical loading (5d/wk, over 3 wks) was investigated by longitudinal in vivo microCT imaging at days 14, 18, 23, 28 and 33 (Fig. 3, 4). Loading was administered to the left tibia starting 14 days after i.t. injection of left limbs of MOPC315.BM (n=10) and PBS (n=7) mice (left limb: εmax=2000µε determined by strain gauging, right limb was nonloaded). 15 mice served as nonloaded control mice (Fig. 3). We also analyzed a group of loaded, but not injected mice (n=10). MicroCT data (Fig. 3), such as cortical thickness (Ct.Th), cortical bone area (Ct.Ar) of loaded left limbs demonstrate that metaphyseal cortical bone formation was significantly greater and cortical porosity (Ct.Po) was lower compared to nonloaded control limbs. Our data provide for the first time an indication that mechanical loading counteracts MM induced bone loss over time. To further study mechanisms of mechanotransduction we used a small scale cell culture bioreactor system to analyze mechanical stimulation of human mesenchymal stem cells (hMSCs) and the translation of mechanical forces into biochemical signals. The bioreactor system allows for analysis of molecular mechanisms of mechanotransduction using a transcription factor activator protein (AP1) luciferase reporter gene construct. Cyclic stretching of telomerase immortalized hMSCs (hMSC-TERT cells) which were stably transfected by an AP1 construct resulted in stimulation of AP1 activity as measured by luciferase gene expression. We analyzed how MM cells interfere with mechanotransduction when co-cultured with hMSC-TERT cells. Our data show that in presence of MOPC315.BM cells and 2 (MM1S, INA-6) out of 4 human MM cell lines hMSCs can be mechanically stimulated and have osteogenic activity. In contrast the human MM cell lines Amo-1 and U266 inhibit the hMSC-TERT response to mechanical stimulation under these conditions. Biochemical and molecular genetic studies are underway to determine how MM cells in bone alter known bone (re)modeling pathways and which yet unanticipated factors regulate the anabolic response to mechanical loading in the MOPC315.BM model. Our study provides a first fundamental understanding of the mechano-biological mechanisms of anabolic bone adaption during MM bone disease in response to mechanical stimuli. We conclude that mechanical loading in the form of exercise has the potential to be a potent novel anabolic strategy for primary and secondary prevention of MM bone disease. Moreover dissection of the underlying molecular pathways will identify new targets for innovative antimyeloma strategies. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Efficacy of Creatine Supplementation and Resistance Training on Area and Density of Bone and Muscle in Older Adults.

To examine the efficacy of creatine (Cr) supplementation and any sex differences during supervised whole-body resistance training (RT) on properties of bone and muscle in older adults. Seventy participants (39 men, 31 women; mean age ± standard deviation: 58 ± 6 yr) were randomized to supplement with Cr (0.1 g·kg-1·d-1) or placebo (Pl) during RT (3 d·wk-1 for 1 yr). Bone geometry (radius and tibia) and muscle area and density (forearm and lower leg) were assessed using peripheral quantitative computed tomography. Compared with Pl, Cr increased or maintained total bone area in the distal tibia (Cr, Δ +17 ± 27 mm2; Pl, Δ -1 ± 22 mm2; P = 0.031) and tibial shaft (Cr, Δ 0 ± 9 mm2; Pl, Δ -5 ± 7 mm2; P = 0.032). Men on Cr increased trabecular (Δ +28 ± 31 mm2; P < 0.001) and cortical bone areas in the tibia (Δ +4 ± 4 mm2; P < 0.05), whereas men on Pl increased trabecular bone density (Δ +2 ± 2 mg·cm-3; P < 0.01). There were no bone changes in the radius (P > 0.05). Cr increased lower leg muscle density (Δ +0.83 ± 1.15 mg·cm-3; P = 0.016) compared with Pl (Δ -0.16 ± 1.56 mg·cm-3), with no changes in the forearm muscle. One year of Cr supplementation and RT had some favorable effects on measures of bone area and muscle density in older adults.

Interleukin (IL)-10 Is Important in the Maintenance of Trabecular and Cortical Bone and Protects Against Western Diet-Induced Disruption in Bone Remodeling in Mice

ObjectivesThe purpose of this study was to investigate if consumption of a western diet (WD) exacerbates the effects of loss of function of IL-10, an anti-inflammatory cytokine, on biomarkers of bone metabolism and microarchitecture. MethodsSix-week-old male B6.129P2-Il10tm1Cgn/J (IL-10 KO) and C57BL/6 mice (WT) were randomized to treatment in a 2 × 2 factorial with diet (AIN-93 control diet CD vs WD) and strain (IL-10 KO vs WT) as factors. Due to potential influence of high fat on intestinal Ca absorption, a WD diet with added Ca (1.2 g/kg) was used. After 12 wks, whole body dual-energy x-ray absorptiometry scans were performed to assess bone density and body composition, and micro-computed x-ray tomography was used to evaluate trabecular and cortical bone microarchitecture in the femur and lumbar vertebra. Serum biomarkers of bone formation, procollagen 1 intact N-terminal propeptide (P1NP), and resorption, c-terminal telopeptide of type I collagen (CTX-1) were assessed. ResultsBody weight, but not % body fat, was lower (P < 0.05) in IL-10 KO mice relative to WT controls. 12 weeks of WD increased (P < 0.05) body weight and % fat, but the response was not as great in the IL-10 KO mice. Bone mineral density and content were lower in IL-10 KO mice compared to WT, and the WD had no effect on these parameters. The IL-10 KO mice exhibited a decrease in trabecular bone volume, thickness, and number, and an increase in trabecular separation and structure model index compared to WT mice within the femur and vertebrae. The WD had no effect on these trabecular bone parameters. Cortical bone thickness and area were reduced (P < 0.05) and porosity increased in both the femur and vertebra of IL-10 KO mice relative to their WT counterparts. This strain effect was not altered by the WD. IL-10 KO mice exhibited a significantly lower serum PINP and higher CTX-1 compared to the WT mice. Despite the lack of structural changes in bone after 12 wks, the WD increased (P < 0.05) CTX-1 and tended to suppress P1NP (P = 0.051) in the IL-10 KO mice compared to WT. ConclusionsWe conclude that IL-10 plays an important role in bone metabolism and maintaining structural properties and in the absence of IL-10, WD negatively affects both osteoclast and osteoblast activity. Further studies are warranted to determine if structural changes occur with longer exposure to WD. Funding SourcesOklahoma Agricultural Experiment Station.

Endocrine, metabolic and apical effects of in utero and lactational exposure to non-dioxin-like 2,2′,3,4,4′,5,5′-heptachlorobiphenyl (PCB 180): A postnatal follow-up study in rats

PCB 180 is a persistent and abundant non-dioxin-like PCB (NDL-PCB). We determined the developmental toxicity profile of ultrapure PCB 180 in developing offspring following in utero and lactational exposure with the focus on endocrine, metabolic and retinoid system alterations. Pregnant rats were given total doses of 0, 10, 30, 100, 300 or 1000 mg PCB 180/kg bw on gestational days 7−10 by oral gavage, and the offspring were sampled on postnatal days (PND) 7, 35 and 84. Decreased serum testosterone and triiodothyronine concentrations on PND 84, altered liver retinoid levels, increased liver weights and induced 7-pentoxyresorufin O-dealkylase (PROD) activity were the sensitive effects used for margin of exposure (MoE) calculations. Liver weights were increased together with induction of the metabolizing enzymes cytochrome P450 (CYP) 2B1, CYP3A1, and CYP1A1. Less sensitive effects included decreased serum estradiol and increased luteinizing hormone levels in females, decreased prostate and seminal vesicle weight and increased pituitary weight in males, increased cortical bone area and thickness of tibial diaphysis in females and decreased cortical bone mineral density in males. Developmental toxicity profiles were partly different in male and female offspring, males being more sensitive to increased liver weight, PROD induction and decreased thyroxine concentrations. MoE assessment indicated that the 95th percentile of current maternal PCB 180 concentrations do not exceed the estimated tolerable human lipid-based PCB 180 concentration. Although PCB 180 is much less potent than dioxin-like compounds, it shares several toxicological targets suggesting a potential for interactions.

Adenine-induced chronic kidney disease induces a similar skeletal phenotype in male and female C57BL/6 mice with more severe deficits in cortical bone properties of male mice.

Chronic kidney disease (CKD) causes bone loss, particularly in cortical bone, through formation of cortical pores which lead to skeletal fragility. Animal models of CKD have shown variability in the skeletal response to CKD between males and females suggesting sex may play a role in this variation. Our aim was to compare the impact of adenine-induced CKD on cortical parameters in skeletally mature male and female C57Bl/6 mice. After 10-weeks of adenine-induced CKD, both male and female adenine mice had high serum parathyroid hormone (PTH), high bone turnover, and cortical porosity compared to non-CKD controls. Both sexes had lower cortical thickness, but only male mice had lower cortical bone area. CKD imparted greater deficits in mechanical properties of male mice compared to female mice. These data demonstrate that both male and female mice develop high PTH/high bone turnover in response to adenine-induced CKD and that cortical bone phenotypes are slightly more severe in males, particularly in mechanical properties deficits.

Teriparatide and Abaloparatide Have a Similar Effect on Bone in Mice.

Three bone anabolic pharmaceuticals are currently approved for treatment of osteoporosis, teriparatide (PTH (1–34)), the parathyroid hormone-related protein analog abaloparatide (ABL), and romosozumab. The present study compared the effect of intermittent PTH (1–34) and ABL on bone tissue directly mole-to-mole in female mice. Forty-seven C57BL/6 mice were randomly allocated to the following groups: Baseline (n = 11), Control (Ctrl) (n = 12), PTH (n = 12), and ABL (n = 12). The mice were injected s.c. with PTH (100 µg/kg), ABL (96 µg/kg), or saline (Ctrl) five days a week for three weeks. To assess the effect of PTH and ABL, the hindlimb bones were analyzed with DXA, µCT, mechanical testing, dynamic bone histomorphometry, and histological quantification of bone cells. In addition, serum calcium concentration was determined. PTH and ABL significantly increased femoral areal bone mineral density (aBMD) (borderline significant p = 0.06 for PTH), femoral mid-diaphyseal bone strength, femoral metaphyseal and epiphyseal and vertebral bone volume fraction (BV/TV), connectivity density, volumetric bone mineral density (vBMD), and bone formation rate (BFR/BS) compared to Ctrl. In addition, ABL also significantly increased mid-diaphyseal cortical thickness and bone area compared to Ctrl. Neither PTH nor ABL significantly increased bone strength at the femoral neck. In conclusion, abaloparatide and PTH have similar bone anabolic properties when compared directly mole-to-mole in mice.

Compromised Volumetric Bone Density and Microarchitecture in Men With Congenital Hypogonadotropic Hypogonadism.

Men with congenital hypogonadotropic hypogonadism (CHH) and Kallmann syndrome (KS) have both low circulating testosterone and estradiol levels. Whether bone structure is affected remains unknown. To characterize bone geometry, volumetric density and microarchitecture in CHH/KS. This cross-sectional study, conducted at a single French tertiary academic medical center, included 51 genotyped CHH/KS patients and 40 healthy volunteers. Among CHH/KS men, 98% had received testosterone and/or combined gonadotropins. High-resolution peripheral quantitative computed tomography (HR-pQCT), dual-energy x-ray absorptiometry (DXA), and measurement of serum bone markers were used to determine volumetric bone mineral density (vBMD) and cortical and trabecular microarchitecture. CHH and controls did not differ for age, body mass index, and levels of vitamin D and PTH. Despite long-term hormonal treatment (10.8 ± 6.8 years), DXA showed lower areal bone mineral density (aBMD) in CHH/KS at lumbar spine, total hip, femoral neck, and distal radius. Consistent with persistently higher serum bone markers, HR-pQCT revealed lower cortical and trabecular vBMD as well as cortical thickness at the tibia and the radius. CHH/KS men had altered trabecular microarchitecture with a predominant decrease of trabecular thickness. Moreover, CHH/KS men exhibited lower cortical bone area, whereas total and trabecular areas were higher only at the tibia. Earlier treatment onset (before age 19 years) conferred a significant advantage for trabecular bone volume/tissue volume and trabecular vBMD at the tibia. Both vBMD and bone microarchitecture remain impaired in CHH/KS men despite long-term hormonal treatment. Treatment initiation during adolescence is associated with enhanced trabecular outcomes, highlighting the importance of early diagnosis.

Type 1 diabetic Akita mice have low bone mass and impaired fracture healing.

Type 1 diabetes (T1DM) impairs bone formation and fracture healing in humans. Akita mice carry a mutation in one allele of the insulin-2 (Ins2) gene, which leads to pancreatic beta cell dysfunction and hyperglycemia by 5-6weeks age. We hypothesized that T1DM in Akita mice is associated with decreased bone mass, weaker bones, and impaired fracture healing. Ins2±(Akita) and wildtype (WT) males were subjected to femur fracture at 18-weeks age and healing assessed 3-21days post-fracture. Non-fractured left femurs were assessed for morphology (microCT) and strength (bending or torsion) at 19-21weeks age. Fractured right femurs were assessed for callus mechanics (torsion), morphology and composition (microCT and histology) and gene expression (qPCR). Both Akita and WT mice gained weight from 3 to 18weeks age, but Akita mice weighed less starting at 5weeks (-5.2%, p<0.05). At 18-20weeks age Akita mice had reduced serum osteocalcin (-30%), cortical bone area (-16%), and thickness (-17%) compared to WT, as well as reduced cancellous BV/TV (-39%), trabecular thickness (-23%) and vBMD (-31%). Mechanical testing of non-fractured femurs showed decreased structural (stiffness, ultimate load) and material (ultimate stress) properties of Akita bones. At 14 and 21days post fracture Akita mice had a significantly smaller callus than WT mice (~30%), with less cartilage and bone area. Assessment of torsional strength showed a weaker callus in Akita mice with lower stiffness (-42%), maximum torque (-44%) and work to fracture (-44%). In summary, cortical and cancellous bone mass were reduced in Akita mice, with lower bone mechanical properties. Fracture healing in Akita mice was impaired by T1DM, with a smaller, weaker fracture callus due to decreased cartilage and bone formation. In conclusion, the Akita mouse mimics some of the skeletal features of T1DM in humans, including osteopenia and impaired fracture healing, and may be useful to test interventions.

Abstract PS9-23: Second-generation HR-pQCT analysis of bone mineral density and microstructure in women with breast cancer who underwent adjuvant endocrine therapy

Abstract Background: Adjuvant endocrine therapy for 5-10 years reduces breast cancer (BC) mortality in women with hormone receptor-positive BC. However, aromatase inhibitors (AIs) decrease bone mineral density (BMD), and increase fractures, joint stiffness, and joint pain. Although dual energy X-ray absorptiometry (DXA) is widely used to assess BMD, it is a two-dimensional method and thus cannot analyze trabecular and cortical bone microstructure. High-resolution peripheral quantitative computed tomography (HR-pQCT) allows in vivo analysis of peripheral bone microarchitecture with high spatial resolution and low radiation exposure. We used HR-pQCT to evaluate changes in volumetric bone density and microstructure consisting of trabecular and cortical bone associated with AI treatment in patients with early BC. Methods: This was a prospective single-center observational study of nonosteoporotic, postmenopausal women with hormone receptor-positive BC, whose baseline DXA lumber spine and femoral neck T-scores were &amp;gt; −2.5. All patients were scheduled to receive DXA and HR-pQCT at baseline and at 6 and 12 months after starting AI therapy. The primary endpoint was to examine changes in total volumetric BMD (Tt. vBMD), trabecular volumetric BMD (Tb. vBMD), cortical volumetric BMD (Ct. vBMD) change at the distal radius and the distal radius between baseline and 6 and 12 months after starting endocrine therapy. Results: Of the 20 women in the study (median age: 57.5 years; range: 55-72 years), 8 had undergone chemotherapy before registration. Their mean change in Tt. vBMD between baseline and 1 year were distal radius: −5.7% (SD: 2.5%, P&amp;lt;0.01); distal tibia: −3.8% (SD: 1.8%, P&amp;lt;0.01), Tb. vBMD distal radius: −4.0% (SD: 3.9%, P&amp;lt;0.01); distal tibia: −0.9% (SD: 2.4%, P&amp;lt;0.05), Ct. vBMD distal radius: −3.1% (SD: 1.6%, P&amp;lt;0.01); distal tibia: −2.9% (SD: 1.7%, P&amp;lt;0.01). Furthermore, at 1 year in trabecular bone, mean trabecular bone volume fraction (p&amp;lt;0.01), trabecular thickness (p&amp;lt;0.05), and trabecular number (p&amp;lt;0.01) were significantly decreased, and trabecular separation (p&amp;lt;0.01) increased, in both the distal radius and tibia. At 1 year in cortical bone, mean changes in cortical thickness (p&amp;lt;0.01) and cortical bone area (p&amp;lt;0.01) were significantly decreased in both the distal radius and tibia; however, the endocortical perimeter of cortical bone did not significantly change and cortical porosity (p&amp;lt;0.05) was significantly greater than that at baseline. At 1 year, DXA decreased at the total hip (−4.4%, P&amp;lt;0.01) and femoral neck (−0.6%, P=0.03) compared with baseline. However, no significant difference was seen in median values for lumbar spine BMD at 1 year, or for tartrate-resistant acid phosphatase-5b (TRACP-5b) or procollagen type-I N-terminal propeptide (PINP). Conclusion: Postmenopausal women who took AIs for early BC experience volumetric bone density decline and both trabecular and cortical bone deterioration. A decade of AI treatment is a standard treatment for early BC; however, healthcare professionals should monitor bone health during and after these treatments. Citation Format: Sayaka Kuba, Konosuke Watanabe, Megumi Matsumoto, Ko Chiba, Kosho Yamanouchi, Ayako Fukushima, Michi Morita, Ryota Otsubo, Hiroshi Yano, Kengo Kanetaka, Makoto Osaki, Takeshi Nagayasu, Susumu Eguchi. Second-generation HR-pQCT analysis of bone mineral density and microstructure in women with breast cancer who underwent adjuvant endocrine therapy [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS9-23.

Quantifying cortical bone in fragmentary archeological second metacarpals.

Skeletal variation in cortical bone thickness is an indicator of bone quality and health in archeological populations. Second metacarpal radiogrammetry, which measures cortical thickness at the shaft midpoint, is traditionally used to evaluate bone loss in bioarcheological and some clinical contexts. However fragmentary elements are regularly omitted because the midpoint cannot be determined. This methodological limitation reduces sample sizes and biases them against individuals prone to fracture, such as older individuals with low bone mass. This study introduces a new technique for measuring cortical bone in second metacarpals, the "Region of Interest" (ROI) method, which quantifies bone in archeological remains with less-than-ideal preservation while accounting for cortical heterogeneity. The ROI method was adapted from digital X-ray radiogrammetry (DXR), a clinical method used to estimate bone mineral density, and tested using second metacarpals from Middenbeemster, Netherlands, a 19th century known age and sex skeletal collection. The ROI method quantifies cortical bone area within a 1.9 cm-long, mid-diaphyseal region, standardized for body size differences using total area (CAIROI ). CAIROI values were compared to traditional radiogrammetric cortical indices (CI) to assess the method's ability to identify age-related bone loss. CAIROI values have high intra- and interobserver replicability and are strongly and significantly correlated with CI values for both males (r[n = 39] = 0.906, p = 0.000) and females (r[n = 58] = 0.925, p = 0.000). The ROI method complements traditional radiogrammetry analyses and provides a reliable way to quantify cortical bone in incomplete second metacarpals, thereby maximizing sample sizes, allowing patterns in bone acquisition and loss to be more comprehensively depicted in archeological assemblages.

Low Energy Availability Interferes With Exercise-Associated Bone Effects in Female Long-Distance Triathletes as Detected by HR-pQCT

Female Athlete Triad, initially described as the association of disordered eating, amenorrhea and osteoporosis, was further redefined to focus on low energy availability (EA), which has a central role in development of hypoestrogenism and low bone mineral density (BMD). However, the contribution of each variable, that is, low EA and hypoestrogenism, for bone derangements is still an open question. To evaluate body composition and bone status in long-distance triathletes without hypoestrogenism, as compared to non-athletes, using DXA and HR-pQCT, and the influence of EA. Population comprised 23 triathletes who had completed at least one long-distance race in the previous year, and 17 non-athletic healthy controls. The athletes denied previous oligo-amenorrhea and had spontaneous regular menses or were on hormonal contraceptives. Control patients also had regular menses. Energy deficiency (low EA) was defined as energy intake below the recommended level for athletes, that is, 45 kcal/kg free fat mass/day. Only femoral neck BMD Z-score measured by DXA trended higher in athletes (p = 0.05), whereas high-resolution peripheral quantitative computed tomography detected significantly higher values of entire bone and trabecular bone area, cortical perimeter, trabecular vBMD and trabecular bone volume/tissue volume, and lower trabecular separation and trabecular inhomogeneity in athletes. No difference was found between athletes with spontaneous menses and those on hormone contraceptives in respect to all parameters. The effects of exercise on bone were not so pronounced in athletes with low EA, although they still had better bone parameters than controls. Stress fractures were reported by 4:12 athletes with low EA and by 2:11 athletes with adequate EA. Long-distance female triathletes without hypoestrogenism show higher values of cortical perimeter, bone area, volumetric density and trabecular microstructure, but low EA interferes with exercise-associated bone effects. These innovative findings reinforce the importance of adequate EA in female athletes to guarantee skeletal health.

Chlorthalidone with potassium citrate decreases calcium oxalate stones and increases bone quality in genetic hypercalciuric stone-forming rats

To study human idiopathic hypercalciuria we developed an animal model, genetic hypercalciuric stone-forming rats, whose pathophysiology parallels that of human idiopathic hypercalciuria. Fed the oxalate precursor, hydroxyproline, every rat in this model develops calcium oxalate stones. Using this rat model, we tested whether chlorthalidone and potassium citrate combined would reduce calcium oxalate stone formation and improve bone quality more than either agent alone. These rats (113 generation) were fed a normal calcium and phosphorus diet with hydroxyproline and divided into four groups: diets plus potassium chloride as control, potassium citrate, chlorthalidone plus potassium chloride, or potassium citrate plus chlorthalidone. Urine was collected at six, 12, and 18 weeks and kidney stone formation and bone parameters were determined. Compared to potassium chloride, potassium citrate reduced urinary calcium, chlorthalidone reduced it further and potassium citrate plus chlorthalidone even further. Potassium citrate plus chlorthalidone decreased urine oxalate compared to all other groups. There were no significant differences in calcium oxalate supersaturation in any group. Neither potassium citrate nor chlorthalidone altered stone formation. However, potassium citrate plus chlorthalidone significantly reduced stone formation. Vertebral trabecular bone increased with chlorthalidone and potassium citrate plus chlorthalidone. Cortical bone area increased with chlorthalidone but not potassium citrate or potassium citrate plus chlorthalidone. Mechanical properties of trabecular bone improved with chlorthalidone, but not with potassium citrate plus chlorthalidone. Thus in genetic hypercalciuric stone-forming rats fed a diet resulting in calcium oxalate stone formation, potassium citrate plus chlorthalidone prevented stone formation better than either agent alone. Chlorthalidone alone improved bone quality, but adding potassium citrate provided no additional benefit.