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Radiation-induced bystander effects have been implicated in contributing to the growth delay of disseminated tumor cells (DTC) caused by 223 RaCl 2 , an alpha particle-emitting radiopharmaceutical. To understand how 223 RaCl 2 affects the growth, we have quantified biological changes caused by direct effects of radiation and bystander effects caused by the emitted radiations on DTC and osteocytes. Characterizing these effects contribute to understanding the efficacy of alpha particle-emitting radiopharmaceuticals and guide expansion of their use clinically. MDA-MB-231 or MCF-7 human breast cancer cells were inoculated intratibially into nude mice that were previously injected intravenously with 50 or 600 kBq/kg 223 RaCl 2 . At 1-day and 3-days postinoculation, tibiae were harvested and examined for DNA damage (γ-H2AX foci) and apoptosis in osteocytes and cancer cells located within and beyond the range (70 μm) of alpha particles emitted from the bone surface. Irradiated and bystander MDA-MB-231 and MCF-7 cells harbored DNA damage. Bystander MDA-MB-231 cells expressed DNA damage at both treatment levels while bystander MCF-7 cells required the higher administered activity. Osteocytes also had DNA damage regardless of inoculated cancer cell line. The extent of DNA damage was quantified by increases in low (1-2 foci), medium (3-5 foci), and high (5+ foci) damage. MDA-MB-231 but not MCF-7 bystander cells showed increases in apoptosis in 223 RaCl 2 -treated animals, as did irradiated osteocytes. In summary, radiation-induced bystander effects contribute to DTC cytotoxicity caused by 223 RaCl 2 . IMPLICATIONS: This observation supports clinical investigation of the efficacy of 223 RaCl 2 to prevent breast cancer DTC from progressing to oligometastases., (©2021 American Association for Cancer Research.)

Rationale: The role of radiation-induced bystander effects in cancer therapy with alpha-particle emitting radiopharmaceuticals remains unclear. With renewed interest in using alpha-particle emitters to sterilize disseminated tumor cells, micrometastases, and tumors, a better understanding of the direct effects of alpha particles and the contribution of the bystander responses they induce is needed to refine dosimetric models that help predict clinical benefit. Accordingly, this work models and quantifies the relative importance of direct effects (DE) and bystander effects (BE) in the growth delay of human breast cancer xenografts observed previously in the tibiae of mice treated with 223 RaCl 2 ., Methods: A computational model of MDA-MB-231 and MCF-7 human breast cancer xenografts in the tibial bone marrow of mice administered 223 RaCl 2 was created. A Monte Carlo radiation transport simulation was performed to assess individual cell absorbed doses. The responses of the breast cancer cells to direct alpha particle irradiation and gamma irradiation were needed as input data for the model and were determined experimentally using a colony-forming assay and compared to the responses of preosteoblast MC3T3-E1 and osteocyte-like MLO-Y4 bone cells. Using these data, a scheme was devised to simulate the dynamic proliferation of the tumors in vivo , including DE and BE propagated from the irradiated cells. The parameters of the scheme were estimated semi-empirically to fit experimental tumor growth., Results: A robust BE component, in addition to a much smaller DE component, was required to simulate the in vivo tumor proliferation. We also found that the relative biological effectiveness (RBE) for cell killing by alpha particle radiation was greater for the bone cells than the tumor cells., Conclusion: This modeling study demonstrates that DE of radiation alone cannot explain experimental observations of 223 RaCl 2 -induced growth delay of human breast cancer xenografts. Furthermore, while the mechanisms underlying BE remain unclear, the addition of a BE component to the model is necessary to provide an accurate prediction of the growth delay. More complex models are needed to further comprehend the extent and complexity of 223 RaCl 2 -induced BE.

Fructose is metabolized in the cytoplasm by the enzyme ketohexokinase (KHK), and excessive consumption may affect bone health. Previous work in calcium-restricted, growing mice demonstrated that fructose disrupted intestinal calcium transport. Thus, we hypothesized that the observed effects on bone were dependent on fructose metabolism and took advantage of a KHK knockout (KO) model to assess direct effects of high plasma fructose on the long bones of growing mice. Four groups (n = 12) of 4-week-old, male, C57Bl/6 background, congenic mice with intact KHK (wild-type, WT) or global knockout of both isoforms of KHK-A/C (KHK-KO), were fed 20% glucose (control diet) or fructose for 8 weeks. Dietary fructose increased by 40-fold plasma fructose in KHK-KO compared to the other three groups (p < 0.05). Obesity (no differences in epididymal fat or body weight) or altered insulin was not observed in either genotype. The femurs of KHK-KO mice with the highest levels of plasma fructose were shorter (2%). Surprisingly, despite the long-term blockade of KHK, fructose feeding resulted in greater bone mineral density, percent volume, and number of trabeculae as measured by µCT in the distal femur of KHK-KO. Moreover, higher plasma fructose concentrations correlated with greater trabecular bone volume, greater work-to-fracture in three-point bending of the femur mid-shaft, and greater plasma sclerostin. Since the metabolism of fructose is severely inhibited in the KHK-KO condition, our data suggest mechanism(s) that alter bone growth may be related to the plasma concentration of fructose.

The role of radiation-induced bystander effects in radiation therapy remains unclear. With renewed interest in therapy with α-particle emitters, and their potential for sterilizing disseminated tumor cells (DTCs), it is critical to determine the contribution of bystander effects to the overall response so they can be leveraged for maximum clinical benefit. Methods: Female Foxn1 nu athymic nude mice were administered 0, 50, or 600 kBq/kg 223 RaCl 2 to create bystander conditions. At 24 hours after administration, MDA-MB-231 or MCF-7 human breast cancer cells expressing luciferase were injected into the tibial marrow compartment. Tumor burden was tracked weekly via bioluminescence. Results: The MDA-MB-231 xenografts were observed to have a 10-day growth delay in the 600 kBq/kg treatment group only. In contrast, MCF-7 cells had 7- and 65-day growth delays in the 50 and 600 kBq/kg groups, respectively. Histologic imaging of the tibial marrow compartment, α-camera imaging, and Monte Carlo dosimetry modeling revealed DTCs both within and beyond the range of the α-particles emitted from 223 Ra in bone for both MCF-7 and MDA-MB-231 cells. Conclusion: Taken together, these results support the participation of 223 Ra-induced antiproliferative/cytotoxic bystander effects in delayed growth of DTC xenografts. They indicate that the delay depends on the injected activity and therefore is dose-dependent. They suggest using 223 RaCl 2 as an adjuvant treatment for select patients at early stages of breast cancer., (© 2020 by the Society of Nuclear Medicine and Molecular Imaging.)

Current fructose consumption levels often overwhelm the intestinal capacity to absorb fructose. We investigated the impact of fructose malabsorption on intestinal endocrine function and addressed the role of the microbiota in this process. To answer this question, a mouse model of moderate fructose malabsorption [ketohexokinase mutant (KHK) -/- ] and wild-type (WT) littermate mice were used and received a 20%-fructose (KHK-F and WT-F) or 20%-glucose diet. Cholecystokinin ( Cck ) mRNA and protein expression in the ileum and cecum, as well as preproglucagon ( Gcg ) and neurotensin ( Nts ) mRNA expression in the cecum, increased in KHK-F mice. In KHK-F mice, triple-label immunohistochemistry showed major up-regulation of CCK in enteroendocrine cells (EECs) that were glucagon-like peptide-1 (GLP-1) + /Peptide YY (PYY - ) in the ileum and colon and GLP-1 - /PYY - in the cecum. The cecal microbiota composition was drastically modified in the KHK-F in association with an increase in glucose, propionate, succinate, and lactate concentrations. Antibiotic treatment abolished fructose malabsorption-dependent induction of cecal Cck mRNA expression and, in mouse GLUTag and human NCI-H716 cells, Cck mRNA expression levels increased in response to propionate, both suggesting a microbiota-dependent process. Fructose reaching the lower intestine can modify the composition and metabolism of the microbiota, thereby stimulating the production of CCK from the EECs possibly in response to propionate.-Zhang, X., Grosfeld, A., Williams, E., Vasiliauskas, D., Barretto, S., Smith, L., Mariadassou, M., Philippe, C., Devime, F., Melchior, C., Gourcerol, G., Dourmap, N., Lapaque, N., Larraufie, P., Blottière, H. M., Herberden, C., Gerard, P., Rehfeld, J. F., Ferraris, R. P., Fritton, J. C., Ellero-Simatos, S., Douard, V. Fructose malabsorption induces cholecystokinin expression in the ileum and cecum by changing microbiota composition and metabolism.

Recent studies have demonstrated matrix-mineral alterations in bone tissue surrounding osteocytes in estrogen-deficient animals. While cortical bone porosity has been shown to be a contributor to the mechanical properties of bone tissue, little analysis has been done to investigate the effects of estrogen deficiency on bone's microporosities, including the vascular and osteocyte lacunar porosities. In this study we examined alterations in cortical bone microporosity, mineralization, and cancellous bone architecture due to estrogen deficiency in the ovariectomized rat model of postmenopausal osteoporosis. Twenty-week-old female Sprague-Dawley rats were subjected to either ovariectomy or sham surgery. Six weeks post-surgery tibiae were analyzed using high-resolution micro-CT, backscattered electron imaging, nanoindentation, and dynamic histomorphometry. Estrogen deficiency caused an increase in cortical bone vascular porosity, with enlarged vascular pores and little change in tissue mineral density in the proximal tibial metaphysis. Measurements of cancellous architecture corresponded to previous studies reporting a decrease in bone volume fraction, an increase in trabecular separation, and a decrease in trabecular number in the proximal tibia due to estrogen deficiency. Nanoindentation results showed no differences in matrix stiffness in osteocyte-rich areas of the proximal tibia of estrogen-deficient rats, and bone labeling and backscattered electron imaging showed no significant changes in mineralization around the vascular pores. The findings demonstrate local surface alterations of vascular pores due to estrogen deficiency. An increase in cortical vascular porosity may diminish bone strength as well as alter bone mechanotransduction via interstitial fluid flow, both of which could contribute to bone fragility during postmenopausal osteoporosis., (Copyright © 2018 Elsevier Inc. All rights reserved.)

A remodeling cycle sets the size of the osteon and associated lamellae in the basic multicellular unit. Treatments and aging affect these micro-structural features. We previously demonstrated decreased fatigue life with an unexplained mechanism and decreased osteon size in cortical bone treated with high-dose bisphosphonate. Here, three finite element models were examined: type-1: a single osteon, as a homogeneous unit and with heterogeneous lamellae and interlamellae, type-2: a control, interstitial-only tissue and type-3: the osteon with cement line, set within the interstitial tissue. Models were loaded in simulated, sinusoidal bending fatigue. As osteon size was decreased, lamellar number and lamellar thickness were incrementally adjusted for each model. As hypothesized, lamellae within the larger type-1 models attained greater cycles to failure and the addition of an osteon to type-2 models (generating a type-3 model set) yielded increased fatigue life. However, as the osteon size was decreased, the potential for compressive damage nucleation was increased within the lamellae of the osteons versus the interstitium. Also, osteons with fewer, thicker lamellae displayed increased fatigue life. Osteonal microstructure plays a role in damage initiation location, especially when BMU size is smaller. Previous findings by us and others could partially be explained by this further understanding of increased probability for damage nucleation in smaller osteons., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Background Context: Previous studies have found that insulin or insulin-like growth factor treatment can stimulate fracture healing in diabetic and normal animal models, and increase fusion rates in a rat spinal fusion model. Insulin-mimetic agents, such as zinc, have demonstrated antidiabetic effects in animal and human studies, and these agents that mimic the effects of insulin could produce the same beneficial effects on bone regeneration and spinal fusion., Purpose: The purpose of this study was to analyze the effects of locally applied zinc on spinal fusion in a rat model., Study Design/setting: Institutional Animal Care and Use Committee-approved animal study using Sprague-Dawley rats was used as the study design., Methods: Thirty Sprague-Dawley rats (450-500 g) underwent L4-L5 posterolateral lumbar fusion (PLF). After decortication and application of approximately 0.3 g of autograft per side, one of three pellets were added to each site: high-dose zinc calcium sulfate (ZnCaSO4), low-dose ZnCaSO4 (half of the high dose), or a control palmitic acid pellet (no Zn dose). Systemic blood glucose levels were measured 24 hours postoperatively. Rats were sacrificed after 8weeks and the PLFs analyzed qualitatively by manual palpation and radiograph review, and quantitatively by micro-computed tomography (CT) analysis of bone volume and trabecular thickness. Statistical analyses with p-values set at .05 were accomplished with analysis of variance, followed by posthoc tests for quantitative data, or Mann-Whitney rank tests for qualitative assessments., Results: Compared with controls, the low-dose zinc group demonstrated a significantly higher manual palpation grade (p=.011), radiographic score (p=.045), and bone formation on micro-CT (172.9 mm(3) vs. 126.7 mm(3) for controls) (p<.01). The high-dose zinc also demonstrated a significantly higher radiographic score (p=.017) and bone formation on micro-CT (172.7 mm(3) vs. 126.7 mm(3)) (p<.01) versus controls, and was trending toward higher manual palpation scores (p=.058)., Conclusions: This study demonstrates the potential benefit of a locally applied insulin-mimetic agent, such as zinc, in a rat lumbar fusion model. Previous studies have demonstrated the benefits of local insulin application in the same model, and it appears that zinc has similar effects., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Osteocyte apoptosis is required to induce intracortical bone remodeling after microdamage in animal models, but how apoptotic osteocytes signal neighboring "bystander" cells to initiate the remodeling process is unknown. Apoptosis has been shown to open pannexin-1 (Panx1) channels to release adenosine diphosphate (ATP) as a "find-me" signal for phagocytic cells. To address whether apoptotic osteocytes use this signaling mechanism, we adapted the rat ulnar fatigue-loading model to reproducibly introduce microdamage into mouse cortical bone and measured subsequent changes in osteocyte apoptosis, receptor activator of NF-κB ligand (RANKL) expression and osteoclastic bone resorption in wild-type (WT; C57Bl/6) mice and in mice genetically deficient in Panx1 (Panx1KO). Mouse ulnar loading produced linear microcracks comparable in number and location to the rat model. WT mice showed increased osteocyte apoptosis and RANKL expression at microdamage sites at 3 days after loading and increased intracortical remodeling and endocortical tunneling at day 14. With fatigue, Panx1KO mice exhibited levels of microdamage and osteocyte apoptosis identical to WT mice. However, they did not upregulate RANKL in bystander osteocytes or initiate resorption. Panx1 interacts with P2X7 R in ATP release; thus, we examined P2X7 R-deficient mice and WT mice treated with P2X7 R antagonist Brilliant Blue G (BBG) to test the possible role of ATP as a find-me signal. P2X7 RKO mice failed to upregulate RANKL in osteocytes or induce resorption despite normally elevated osteocyte apoptosis after fatigue loading. Similarly, treatment of fatigued C57Bl/6 mice with BBG mimicked behavior of both Panx1KO and P2X7 RKO mice; BBG had no effect on osteocyte apoptosis in fatigued bone but completely prevented increases in bystander osteocyte RANKL expression and attenuated activation of resorption by more than 50%. These results indicate that activation of Panx1 and P2X7 R are required for apoptotic osteocytes in fatigued bone to trigger RANKL production in neighboring bystander osteocytes and implicate ATP as an essential signal mediating this process., (© 2016 American Society for Bone and Mineral Research.)

Redirecting the adipogenic potential of bone marrow-derived mesenchymal stem cells to other lineages, particularly osteoblasts, is a key goal in regenerative medicine. Controlling lineage selection through chromatin remodeling complexes such as SWI/SNF, which act coordinately to establish new patterns of gene expression, would be a desirable intervention point, but the requirement for the complex in essentially every lineage pathway has generally precluded selectivity. However, a novel approach now appears possible by targeting the subset of SWI/SNF powered by the alternative ATPase, mammalian brahma (BRM). BRM is not required for development, which has hindered understanding of its contributions, but knockdown genetics here, designed to explore the hypothesis that BRM-SWI/SNF has different regulatory roles in different mesenchymal stem cell lineages, shows that depleting BRM from mesenchymal stem cells has a dramatic effect on the balance of lineage selection between osteoblasts and adipocytes. BRM depletion enhances the proportion of cells expressing markers of osteoblast precursors at the expense of cells able to differentiate along the adipocyte lineage. This effect is evident in primary bone marrow stromal cells as well as in established cell culture models. The altered precursor balance has major physiological significance, which becomes apparent as protection against age-related osteoporosis and as reduced bone marrow adiposity in adult BRM-null mice., (© 2015 The Authors STEM CELLS published by Wiley Periodicals, Inc. on behalf of AlphaMed Press.)

The biomechanics literature contains many well-understood mechanisms behind typical fracture types that have important roles in treatment planning. The recent association of "atypical" fractures with long-term use of drugs designed to prevent osteoporosis has renewed interest in the effects of agents on bone tissue-level quality. While this class of fracture was recognized prior to the introduction of the anti-resorptive bisphosphonate drugs and recently likened to stress fractures, the mechanism(s) that lead to atypical fractures have not been definitively identified. Thus, a causal relationship between these drugs and atypical fracture has not been established. Physicians, bioengineers and others interested in the biomechanics of bone are working to improve fracture-prevention diagnostics, and the design of treatments to avoid this serious side-effect in the future. This review examines the mechanisms behind the bone tissue damage that may produce the atypical fracture pattern observed increasingly with long-term bisphosphonate use. Our recent findings and those of others reviewed support that the mechanisms behind normal, healthy excavation and tunnel filling by bone remodeling units within cortical tissue strengthen mechanical integrity. The ability of cortical bone to resist the damage induced during cyclic loading may be altered by the reduced remodeling and increased tissue age resulting from long-term bisphosphonate treatment. Development of assessments for such potential fractures would restore confidence in pharmaceutical treatments that have the potential to spare millions in our aging population from the morbidity and death that often follow bone fracture., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Bisphosphonates are the most prescribed preventative treatment for osteoporosis. However, their long-term use has recently been associated with atypical fractures of cortical bone in patients who present with low-energy induced breaks of unclear pathophysiology. The effects of bisphosphonates on the mechanical properties of cortical bone have been exclusively studied under simple, monotonic, quasi-static loading. This study examined the cyclic fatigue properties of bisphosphonate-treated cortical bone at a level in which tissue damage initiates and is accumulated prior to frank fracture in low-energy situations. Physiologically relevant, dynamic, 4-point bending applied to beams (1.5 mm × 0.5 mm × 10 mm) machined from dog rib (n=12/group) demonstrated mechanical failure and micro-architectural features that were dependent on drug dose (3 groups: 0, 0.2, 1.0mg/kg/day; alendronate [ALN] for 3 years) with cortical bone tissue elastic modulus (initial cycles of loading) reduced by 21% (p<0.001) and fatigue life (number of cycles to failure) reduced in a stress-life approach by greater than 3-fold with ALN1.0 (p<0.05). While not affecting the number of osteons, ALN treatment reduced other features associated with bone remodeling, such as the size of osteons (-14%; ALN1.0: 10.5±1.8, VEH: 12.2±1.6, ×10(3) μm2; p<0.01) and the density of osteocyte lacunae (-20%; ALN1.0: 11.4±3.3, VEH: 14.3±3.6, ×10(2) #/mm2; p<0.05). Furthermore, the osteocyte lacunar density was directly proportional to initial elastic modulus when the groups were pooled (R=0.54, p<0.01). These findings suggest that the structural components normally contributing to healthy cortical bone tissue are altered by high-dose ALN treatment and contribute to reduced mechanical properties under cyclic loading conditions., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Excessive fructose consumption inhibits adaptive increases in intestinal Ca2+ transport in lactating and weanling rats with increased Ca2+ requirements by preventing the increase in serum levels of 1,25(OH)2D3. Here we tested the hypothesis that chronic fructose intake decreases 1,25(OH)2D3 levels independent of increases in Ca2+ requirements. Adult mice fed for five wk a high glucose-low Ca2+ diet displayed expected compensatory increases in intestinal and renal Ca2+ transporter expression and activity, in renal CYP27B1 (coding for 1α-hydroxylase) expression as well as in serum 1,25(OH)2D3 levels, compared with mice fed isocaloric glucose- or fructose-normal Ca2+ diets. Replacing glucose with fructose prevented these increases in Ca2+ transporter, CYP27B1, and 1,25(OH)2D3 levels induced by a low Ca2+ diet. In adult mice fed for three mo a normal Ca2+ diet, renal expression of CYP27B1 and of CYP24A1 (24-hydroxylase) decreased and increased, respectively, when the carbohydrate source was fructose instead of glucose or starch. Intestinal and renal Ca2+ transporter activity and expression did not vary with dietary carbohydrate. To determine the time course of fructose effects, a high fructose or glucose diet with normal Ca2+ levels was fed to adult rats for three mo. Serum levels of 1,25(OH)2D3 decreased and of FGF23 increased significantly over time. Renal expression of CYP27B1 and serum levels of 1,25(OH)2D3 still decreased in fructose- compared to those in glucose-fed rats after three mo. Serum parathyroid hormone, Ca2+ and phosphate levels were normal and independent of dietary sugar as well as time of feeding. Thus, chronically high fructose intakes can decrease serum levels of 1,25(OH)2D3 in adult rodents experiencing no Ca2+ stress and fed sufficient levels of dietary Ca2+. This finding is highly significant because fructose constitutes a substantial portion of the average diet of Americans already deficient in vitamin D.

Concern that long-term bisphosphonate therapy may significantly undermine bone quality in osteoporotic patients has been heightened by rare instances of low-impact atypical femoral fractures that are often bilateral. Reduced fracture toughness is believed to result from reduced bone remodeling, leading to increased homogeneity in bone microarchitecture, narrowed bone mineral density distribution, and increased bone tissue microdamage burden. We postulate that these long-term alterations in bone quality may undermine the ongoing remodeling surrounding osteointegrated endosseous dental implants as well. To illustrate our hypothesis, we report the catastrophic failure of multiple, successfully osteointegrated dental implants in an osteopenic patient following long-term bisphosphonate treatment. This clinical presentation may reflect underlying adverse changes in bone quality, in a manner analogous to atypical fractures in a small percentage of patients on bisphosphonates. This report highlights the need for multidisciplinary care of patients who have dental implants and begin or are receiving long-term bisphosphonate therapy., (Copyright © 2012 Elsevier Inc. All rights reserved.)

There is growing evidence that insulin-like growth factor 1 (IGF-1) and parathyroid hormone (PTH) have synergistic actions on bone and that part of the anabolic effects of PTH is mediated by local production of IGF-1. In this study we analyzed the skeletal response to PTH in mouse models with manipulated endocrine or autocrine/paracrine IGF-1. We used mice carrying a hepatic IGF-1 transgene (HIT), which results in a threefold increase in serum IGF-1 levels and normal tissue IGF-1 expression, and Igf1 null mice with blunted IGF-1 expression in tissues but threefold increases in serum IGF-1 levels (KO-HIT). Evaluation of skeletal growth showed that elevations in serum IGF-1 in mice with Igf1 gene ablation in all tissues except the liver (KO-HIT) resulted in a restoration of skeletal morphology and mechanical properties by adulthood. Intermittent PTH treatment of adult HIT mice resulted in increases in serum osteocalcin levels, femoral total cross-sectional area, cortical bone area and cortical bone thickness, as well as bone mechanical properties. We found that the skeletal response of HIT mice to PTH was significantly higher than that of control mice, suggesting synergy between IGF-1 and PTH on bone. In sharp contrast, although PTH-treated KO-HIT mice demonstrated an anabolic response in cortical and trabecular bone compartments compared with vehicle-treated KO-HIT mice, their response was identical to that of PTH-treated control mice. We conclude that (1) in the presence of elevated serum IGF-1 levels, PTH can exert an anabolic response in bone even in the total absence of tissue IGF-1, and (2) elevations in serum IGF-1 levels synergize PTH action on bone only if the tissue IGF-1 axis is intact. Thus enhancement of PTH anabolic actions depends on tissue IGF-1., (© 2010 American Society for Bone and Mineral Research.)

Age-related osteoporosis is accompanied by an increase in marrow adiposity and a reduction in serum insulin-like growth factor-1 (IGF-1) and the binding proteins that stabilize IGF-1. To determine the relationship between these proteins and bone marrow adiposity, we evaluated the adipogenic potential of marrow-derived mesenchymal stromal cells (MSCs) from mice with decreased serum IGF-1 due to knockdown of IGF-1 production by the liver or knock-out of the binding proteins. We employed 10-16-week-old, liver-specific IGF-1-deficient, IGFBP-3 knock-out (BP3KO) and acid-labile subunit knock-out (ALSKO) mice. We found that expression of the late adipocyte differentiation marker peroxisome proliferator-activated receptor gamma was increased in marrow isolated from ALSKO mice. When induced with adipogenic media, MSC cultures from ALSKO mice revealed a significantly greater number of differentiated adipocytes compared with controls. MSCs from ALSKO mice also exhibited decreased alkaline-phosphatase positive colony size in cultures that were stimulated with osteoblast differentiation media. These osteoblast-like cells from ALSKO mice failed to induce osteoclastogenesis of control cells in co-culture assays, indicating that impairment of IGF-1 complex formation with ALS in bone marrow alters cell fate, leading to increased adipogenesis.

Early after estrogen loss in postmenopausal women and ovariectomy (OVX) of animals, accelerated endosteal bone resorption leads to marrow expansion of long bone shafts that reduce mechanical integrity. Both growth hormone (GH) and insulin-like growth factor (IGF-1) are potent regulators of bone remodeling processes. To investigate the role of the GH/IGF-1 axis with estrogen deficiency, we used the liver IGF-1-deficient (LID) mouse. Contrary to deficits in controls, OVX of LID mice resulted in maintenance of cortical bone mechanical integrity primarily owing to an enhanced periosteal expansion affect on cross-sectional structure (total area and cortical width). The serum balance in LID that favors GH over IGF-1 diminished the effects of ablated ovarian function on numbers of osteoclast precursors in the marrow and viability of osteocytes within the cortical matrix and led to less endosteal resorption in addition to greater periosteal bone formation. Interactions between estrogen and the GH/IGF-1 system as related to bone remodeling provide a pathway to minimize degeneration of bone tissue structure and osteoporotic fracture., (Copyright 2010 American Society for Bone and Mineral Research.)

Purpose: The aim was to investigate that a bio-degradable alginate and poly lactide-co-glycolide (PLG) system capable of delivering growth factors sequentially would be superior to single growth factor delivery in promoting neovascularization and improving perfusion., Methods: Three groups of apoE null mice underwent unilateral hindlimb ischemia surgery and received ischemic limb intramuscular injections of alginate (Blank), alginate containing VEGF(165) (VEGF), or alginate containing VEGF(165) combined with PLG microspheres containing PDGF-BB (VEGF/PDGF). Vascularity in the ischemic hindlimb was assessed by morphologic and immunohistochemical end-points, while changes in blood flow were assessed by Laser Doppler Perfusion Index. Muscle VEGF and PDGF content was assessed at multiple time points., Results: In the VEGF/PDGF group, local tissue VEGF and PDGF levels peaked at week 2 and 4, respectively, with detectable PDGF levels at week 6. At week 6, mean vessel mean diameter was significantly greater in the VEGF/PDGF group compared to the VEGF or Blank groups with evidence of well-formed smooth muscle-lined arterioles., Conclusions: Sequential delivery of VEGF and PDGF using an injectable, biodegradable platform resulted in stable and sustained improvements in perfusion. This sustained, control-released, injectable alginate polymer system is a promising approach for multiple growth factor delivery in clinical application.

Type 2 diabetics often demonstrate normal or increased bone mineral density, yet are at increased risk for bone fracture. Furthermore, the anti-diabetic oral thiazolidinediones (PPARgamma agonists) have recently been shown to increase bone fractures. To investigate the etiology of possible structural and/or material quality defects, we have utilized a well-described mouse model of Type 2 diabetes (MKR). MKR mice exhibit muscle hypoplasia from birth with reduced mass by the pre-diabetic age of 3 weeks. A compensatory hyperplasia ensues during early (5 weeks) development; by 6-8 weeks muscle is normal in structure and function. Adult whole-bone mechanical properties were determined by 4-point bending to test susceptibility to fracture. Micro-computed tomography and cortical bone histomorphometry were utilized to assess static and dynamic indices of structure, bone formation and resorption. Osteoclastogenesis assays were performed from bone marrow-derived non-adherent cells. The 8-week and 16-week, but not 3-week, male MKR had slender (i.e., narrow relative to length) femurs that were 20% weaker (p<0.05) relative to WT control femurs. Tissue-level mineral density was not affected. Impaired periosteal expansion during early diabetes resulted from 250% more, and 40% less of the cortical bone surface undergoing resorption and formation, respectively (p<0.05). Greater resorption persisted in adult MKR on both periosteal and endosteal surfaces. Differences were not limited to cortical bone as the distal femur metaphysis of 16 week MKR contained less trabecular bone and trabecular separation was greater than in WT by 60% (p<0.05). At all ages, MKR marrow-derived cultures demonstrated the ability for enhanced osteoclast differentiation in response to M-CSF and RANK-L. Taken together, the MKR mouse model suggests that skeletal fragility in Type 2 diabetes may arise from reduced transverse bone accrual and increased osteoclastogenesis during growth that is accelerated by the diabetic/hyperinsulinemic milieu. Further, these results emphasize the importance of evaluating diabetic bone based on morphology in addition to bone mass.

Introduction: The study of adaptation to mechanical loading under osteopenic conditions is relevant to the development of osteoporotic fracture prevention strategies. We previously showed that loading increased cancellous bone volume fraction and trabecular thickness in normal male mice. In this study, we tested the hypothesis that cyclic mechanical loading of the mouse tibia inhibits orchidectomy (ORX)-associated cancellous bone loss., Materials and Methods: Ten-week-old male C57BL/6 mice had in vivo cyclic axial compressive loads applied to one tibia every day, 5 d/wk, for 6 wk after ORX or sham operation. Adaptation of proximal cancellous and diaphyseal cortical bone was characterized by muCT and dynamic histomorphometry. Comparisons were made between loaded and nonloaded contralateral limbs and between the limbs of ORX (n = 10), sham (n = 11), and basal (n = 12) groups and tested by two-factor ANOVA with interaction., Results: Cyclic loading inhibited bone loss after ORX, maintaining absolute bone mass at age-matched sham levels. Relative to sham, ORX resulted in significant loss of cancellous bone volume fraction (-78%) and trabecular number (-35%), increased trabecular separation (67%), no change in trabecular thickness, and smaller loss of diaphyseal cortical properties, consistent with other studies. Proximal cancellous bone volume fraction was greater with loading (ORX: 290%, sham: 68%) than in contralateral nonloaded tibias. Furthermore, trabeculae thickened with loading (ORX: 108%, sham: 48%). Dynamic cancellous bone histomorphometry indicated that loading was associated with greater mineral apposition rates (ORX: 32%, sham: 12%) and smaller percent mineralizing surfaces (ORX: -47%, sham: -39%) in the final week. Loading resulted in greater BMC (ORX: 21%, sham: 15%) and maximum moment of inertia (ORX: 39%, sham: 24%) at the cortical midshaft., Conclusions: This study shows that cancellous bone mass loss can be prevented by mechanical loading after hormonal compromise and supports further exploration of nonpharmacologic measures to prevent rapid-onset osteopenia and associated fractures.

Background: Observational studies suggest a strong relationship between menopause and vascular calcification. Receptor activator of nuclear factor-kappaBeta ligand (RANKL) and osteoprotegerin (OPG) are critical regulators of bone remodelling and modulate vascular calcification. We assessed the hypothesis that ovariectomy increases vascular calcification via the OPG/RANKL axis., Materials and Methods: Age-matched sexually mature rabbits were randomized to ovariectomy (OVX, n = 12) or sham procedure (SHAM, n = 12). One month post-procedure, atherosclerosis was induced by 15 months 0.2%-cholesterol diet and endothelial balloon denudations (at months 1 and 3). Aortic atherosclerosis was assessed in vivo by magnetic resonance imaging (MRI) at months 9 and 15. At sacrifice, aortas were harvested for ex vivo microcomputed tomography (microCT) and molecular analysis of the vascular tissue., Results: Vascular calcification density and calcific particle number were significantly greater in OVX than SHAM (8.4 +/- 2.8 vs. 1.9 +/- 0.6 mg cm(-3), P = 0.042, and 94 +/- 26 vs. 33 +/- 7 particles cm(-3), P = 0.046, respectively). Calcification morphology, as assessed by the arc angle subtended by the largest calcific particle, showed no difference between groups (OVX 33 +/- 7 degrees vs. SHAM 33 +/- 5 degrees , P = 0.99). By Western blot analysis, OVX increased the vascular OPG:RANKL ratio by 66%, P = 0.029, primarily by decreasing RANKL (P = 0.019). At month 9, MRI demonstrated no difference in atheroma volume between OVX and SHAM, and no significant change was seen by the end of the study., Conclusions: In contrast to bone, vascular OPG:RANKL ratio increased in response to ovariectomy with a corresponding fourfold increase in arterial calcification. This diametrical organ-specific response may explain the comorbid association of osteoporosis with calcifying atherosclerosis in post-menopausal women.

Adaptation to mechanical loading has been studied extensively in cortical, but not cancellous bone. However, corticocancellous sites are more relevant to osteoporosis and related fracture risk of the hip and spine. We tested the hypotheses that adaptation in a long bone would be greater at cancellous than cortical sites and would depend on the term of daily in vivo cyclic axial loading. We applied compressive loads to the adolescent, 10-week old, male C57BL/6 mouse tibia to examine the skeletal response immediately prior to attainment of peak bone mass. Adaptation was quantified at the completion of either 2-week (n = 8) or 6-week (n = 12) loading terms by directly comparing volumetric bone mineral content between loaded and contralateral limbs by microcomputed tomography. The increase in mineral content was site specific with a greater response found in the corticocancellous proximal metaphysis (14%) than the cortical mid-shaft (2%) after 6 weeks of loading. Furthermore, bone volume fraction and average trabecular thickness of cancellous bone in the proximal tibia increased after 6 weeks by 15% and 12% respectively. Diaphyseal response was only evident proximal to the mid-shaft as indicated by an 8% increase in maximum principal moment of inertia. Both loading terms produced similar results for mineral content, volume fraction, and moments of inertia. Our finding that non-invasive loading increases the bone volume and fraction at a corticocancellous site by as much as 15% motivates exploring the use of mechanical loading to attain greater peak bone mass and inhibit osteoporosis.

Study Design: Experiments were undertaken to determine the degree to which high-frequency (15-35 Hz) ground-based, whole-body vibration are transmitted to the proximal femur and lumbar vertebrae of the standing human., Objectives: To establish if extremely low-level (<1 g, where 1 g = earth's gravitational field, or 9.8 ms-2) mechanical stimuli can be efficiently delivered to the axial skeleton of a human., Summary of Background Data: Vibration is most often considered an etiologic factor in low back pain as well as several other musculoskeletal and neurovestibular complications, but recent in vivo experiments in animals indicates that extremely low-level mechanical signals delivered to bone in the frequency range of 15 to 60 Hz can be strongly anabolic. If these mechanical signals can be effectively and noninvasively transmitted in the standing human to reach those sites of the skeleton at greatest risk of osteoporosis, such as the hip and lumbar spine, then vibration could be used as a unique, nonpharmacologic intervention to prevent or reverse bone loss., Materials and Methods: Under sterile conditions and local anesthesia, transcutaneous pins were placed in the spinous process of L4 and the greater trochanter of the femur of six volunteers. Each subject stood on an oscillating platform and data were collected from accelerometers fixed to the pins while a vibration platform provided sinusoidal loading at discrete frequencies from 15 to 35 Hz, with accelerations ranging up to 1 g(peak-peak)., Results: With the subjects standing erect, transmissibility at the hip exceeded 100% for loading frequencies less than 20 Hz, indicating a resonance. However, at frequencies more than 25 Hz, transmissibility decreased to approximately 80% at the hip and spine. In relaxed stance, transmissibility decreased to 60%. With 20-degree knee flexion, transmissibility was reduced even further to approximately 30%. A phase-lag reached as high as 70 degrees in the hip and spine signals., Conclusions: These data indicate that extremely low-level, high-frequency mechanical accelerations are readily transmitted into the lower appendicular and axial skeleton of the standing individual. Considering the anabolic potential of exceedingly low-level mechanical signals in this frequency range, this study represents a key step in the development of a biomechanically based treatment for osteoporosis.

Whole-body vibration (WBV) has been demonstrated to have a strong influence on physiological systems, ranging from severely destructive to potentially beneficial. Unfortunately, the study of WBV in a controlled manner is commonly constrained by space and budgetary factors, particularly where vibration in the low frequency range is considered. In the work presented here, a small, low-cost device for performing WBV of the human skeleton is developed to assist in studies of vertical acceleration in a clinical setting. The device design consists of a spring-supported plate driven by an 18 N peak-force electromagnetic actuator, and the associated driving and monitoring electronics. Animal and human lumped-mass models have been coupled with a model of the loading device to seek a resonance response in the vicinity of 30 Hz. This approach minimizes the loading requirements of such a device, and thus a major component of the cost, yet can provide peak accelerations of 0.15 g at a frequency of 30 Hz in a small, lightweight package capable of use in a clinical or laboratory setting.