Cancellous Bone Volume Fraction Research Articles

Diet composition influences the effect of high fat diets on bone in growing male mice.

The effect of diet-induced obesity on bone in rodents is variable, with bone mass increases, decreases, and no impact reported. The goal of this study was to evaluate whether the composition of obesogenic diet may influence bone independent of its effect on body weight. As proof-of-principle, we used a mouse model to compare the skeletal effects of a commonly used high fat 'Western' diet and a modified high fat diet. The modified high fat diet included ground English walnut and was isocaloric for macronutrients, but differed in fatty acid composition and contained nutrients (e.g. polyphenols) not present in the standard 'Western' diet. Eight-week-old mice were randomized into 1 of 3 dietary treatments (n=8/group): (1) low fat control diet (LF; 10%kcal fat); (2) high fat 'Western' diet (HF; 46%kcal fat as soybean oil and lard); or (3) modified high fat diet supplemented with ground walnuts (HF+walnut; 46%kcal fat as soybean oil, lard, and walnut) and maintained on their respective diets for 9weeks. Bone response in femur was then evaluated using dual energy x-ray absorptiometry, microcomputed tomography, and histomorphometry. Consumption of both obesogenic diets resulted in increased weight gain but differed in impact on bone and bone marrow adiposity in distal femur metaphysis. Mice consuming the high fat 'Western' diet exhibited a tendency for lower cancellous bone volume fraction and connectivity density, and had lower osteoblast-lined bone perimeter (an index of bone formation) and higher bone marrow adiposity than low fat controls. Mice fed the modified high fat diet did not differ from mice fed control (low fat) diet in cancellous bone microarchitecture, or osteoblast-lined bone perimeter, and exhibited lower bone marrow adiposity compared to mice fed the 'Western' diet. This proof-of-principal study demonstrates that two obesogenic diets, similar in macronutrient distribution and induction of weight gain, can have different effects on cancellous bone in distal femur metaphysis. Because the composition of the diets used to induce obesity in rodents does not recapitulate a common human diet, our finding challenges the translatability of rodent studies evaluating the impact of diet-induced obesity on bone.

Small changes in thermoregulation influence cancellous bone turnover balance in distal femur metaphysis in growing female mice

Mice are typically housed at temperatures well below their thermoneutral zone. When individually housed at room temperature (~22 °C) mice experience cold stress which results in cancellous bone loss and has the potential to alter the skeletal response to treatment. It is not clear if there is a threshold temperature for cold stress-induced bone loss. It is also not clear if alternative strategies for attenuating cold stress, such as group housing, influence bone accrual and turnover. This study aimed to determine how small differences in temperature (4 °C) or heat loss (individual versus group housing with nestlets) influence bone in growing female C57BL/6 J mice. Five-week-old mice were randomized by weight to 1 of 4 treatment groups (N = 10/group): 1) baseline, 2) single housed at 22 °C, 3) single housed at 26 °C, or 4) group housed (n = 5/cage) with nestlets at 22 °C. Mice in the baseline group were sacrificed 1 week later, at 6 weeks of age. The other 3 groups of mice were maintained at their respective temperatures and housing conditions for 13 weeks until 18 weeks of age. Compared to baseline, mice single housed at room temperature had increased body weight and femur size, but dramatically decreased cancellous bone volume fraction in distal femur metaphysis. The cancellous bone loss was attenuated but not prevented in mice individually housed at 26 °C or group housed at 22 °C. In conclusion, by impacting thermogenesis or heat loss, modest differences in housing conditions could influence experimental results.

Effects of Subthermoneutral Housing on Bone in Female C57BL/6J Mice

ObjectiveMice are facultative daily heterotherms with a thermoneutral point of ~32°C and when housed at standard room temperature (~22°C) experience cold stress and premature cancellous bone loss. It is unclear whether housing mice at the maximum recommended temperature of 26°C or group housing affects bone. The objectives of this study were to determine (1) how a 4°C difference in housing temperature (22°C vs. 26°C) and (2) how single versus group housing influence bone metabolism in female mice.HypothesisHousing mice at room temperature (22°C) will result in premature cancellous bone loss in long bones of growing female mice, and housing mice at 26°C or group housing mice at 22°C will attenuate the bone loss.MethodsFive‐week‐old female C57BL/6J mice were randomized by weight in one of 4 treatment groups (N=10/group): 1) baseline, 2) single housed at 22°C, 3) single housed at 26°C, or 4) group housed (2 cages; n=5/cage) at 22°C. Baseline mice were sacrificed and the remaining mice were maintained at their respective temperatures and housing conditions for 13 weeks until 18 weeks of age. Total femur mass and cortical (femur midshaft) and cancellous (distal femur metaphysis) bone microarchitecture were assessed using dual energy absorptiometry and microcomputed tomography, respectively. Osteocalcin, a marker of global bone turnover, was measured in serum using an enzyme‐linked immunoassay.ResultsFemur length, area, bone mineral content, and bone mineral density increased in all groups compared to baseline. Cortical volume, marrow volume, and cortical thickness in midshaft femur increased in all groups compared to baseline. No or minimal differences were observed in total femur mass or midshaft femur microarchitecture among mice single housed at 22°C, single housed at 26°C, or group housed at 22°C. In contrast, there were large reductions in cancellous bone volume fraction, connectivity density, trabecular number and an increase in trabecular separation in the distal femur metaphysis in all groups compared to baseline. Single housing mice at 26°C and group housing mice at 22°C attenuated, but did not prevent, cold stress‐induced bone loss. Cancellous bone volume fraction and trabecular number were higher and trabecular separation was lower in mice single housed at 26°C compared to mice single housed at 22°C, while cancellous bone volume fraction and trabecular thickness were higher in mice group housed at 22°C compared to mice single housed at 22°C. Differences between mice single housed at 26°C and group housed at 22°C were not detected for any of the cancellous endpoints evaluated. Levels of serum osteocalcin were decreased in all groups compared to baseline, but tended to be higher in mice single housed at 26°C (p=0.092) and in mice group housed at 22°C (p=0.076) compared to mice single housed at 22°C.ConclusionsHousing mice at 26°C and group housing mice at 22°C slows, but does not prevent, cold stress‐induced premature cancellous bone loss in distal femur metaphysis, a commonly evaluated anatomical site. These findings demonstrate that small differences in housing conditions can significantly influence cancellous bone microarchitecture.

Bone quality affects stability of orthodontic miniscrews

The objective of this study was to evaluate the effect of bone–miniscrew contact percentage (BMC%) and bone quality and quantity on orthodontic miniscrew stability and the maximum insertion torque value (ITV). Orthodontic miniscrews of five different dimensions and several bovine iliac bone specimens were used in the evaluation. Miniscrews of each dimension group were inserted into 20 positions in bovine iliac bone specimens. The experiment was divided into three parts: (1) Bone quality and quantity were evaluated using cone-beam computed tomography (CBCT) and microcomputed tomography. (2) The 3D BMC% was calculated. (3) The ITVs during miniscrew insertion were recorded to evaluate the stability of the orthodontic miniscrews. The results indicated that longer and thicker miniscrews enabled higher ITVs. CBCT was used to accurately measure cortical bone thickness (r = 0.939, P < 0.05) and to predict the bone volume fraction of cancellous bone (r = 0.752, P < 0.05). BMC% was significantly influenced by miniscrew length. The contribution of cortical bone thickness to the ITV is greater than that of cancellous bone structure, and the contribution of cortical bone thickness to BMC% is greater than that of cancellous bone structure. Finally, the higher is BMC%, the greater is the ITV. This study concludes that use of CBCT may predict the mechanical stability of orthodontic miniscrews.

Neuroskeletal Effects of Chronic Bioelectric Nerve Stimulation in Health and Diabetes.

Background/AimsBioelectric nerve stimulation (eStim) is an emerging clinical paradigm that can promote nerve regeneration after trauma, including within the context of diabetes. However, its ability to prevent the onset of diabetic peripheral neuropathy (DPN) has not yet been evaluated. Beyond the nerve itself, DPN has emerged as a potential contributor to sarcopenia and bone disease; thus, we hypothesized that eStim could serve as a strategy to simultaneously promote neural and musculoskeletal health in diabetes.MethodsTo address this question, an eStim paradigm pre-optimized to promote nerve regeneration was applied to the sciatic nerve, which directly innervates the tibia and lower limb, for 8 weeks in control and streptozotocin-induced type 1 diabetic (T1D) rats. Metabolic, gait, nerve and bone assessments were used to evaluate the progression of diabetes and the effect of sciatic nerve eStim on neuropathy and musculoskeletal disease, while also considering the effects of cuff placement and chronic eStim in otherwise healthy animals.ResultsRats with T1D exhibited increased mechanical allodynia in the hindpaw, reduced muscle mass, decreased cortical and cancellous bone volume fraction (BVF), reduced cortical bone tissue mineral density (TMD), and decreased bone marrow adiposity. Type 1 diabetes also had an independent effect on gait. Placement of the cuff electrode alone resulted in altered gait patterns and unilateral reductions in tibia length, cortical BVF, and bone marrow adiposity. Alterations in gait patterns were restored by eStim and tibial lengthening was favored unilaterally; however, eStim did not prevent T1D-induced changes in muscle, bone, marrow adiposity or mechanical sensitivity. Beyond this, chronic eStim resulted in an independent, bilateral reduction in cortical TMD.ConclusionOverall, these results provide new insight into the pathogenesis of diabetic neuroskeletal disease and its regulation by eStim. Though eStim did not prevent neural or musculoskeletal complications in T1D, our results demonstrate that clinical applications of peripheral neuromodulation ought to consider the impact of device placement and eStim on long-term skeletal health in both healthy individuals and those with metabolic disease. This includes monitoring for compounded bone loss to prevent unintended consequences including decreased bone mineral density and increased fracture risk.

Effects of spaceflight on cancellous and cortical bone in proximal femur in growing rats

Mechanical loading of the skeleton during normal weight bearing plays an important role in bone accrual and turnover balance. We recently evaluated bone microarchitecture in the femoral head in 5.6-week-old male Sprague Dawley rats subjected to a 4-day spaceflight aboard STS-41. Compared to weight bearing ground controls, cancellous bone volume fraction was dramatically lower in animals subjected to microgravity. The effects of spaceflight on the rat skeleton are potentially influenced by factors such as age, duration of flight, strain and sex. To test the generalizability of our initial observation, we evaluated archived proximal femora from two additional spaceflight missions: a 10-day mission (STS-57) with 7.5-week-old male Fisher 344 rats, and a 14-day mission (STS-62) with 12-week-old ovariectomized (ovx) female Fisher 344 rats. Cancellous microarchitecture and cortical thickness were assessed using x-ray microtomography/microcomputed tomography. In male rats, cancellous bone volume fraction (bone volume/tissue volume) was lower in flight animals compared to flight controls, but differences were not significant compared to baseline. In ovx female rats, cancellous bone volume fraction was lower in flight animals compared to flight controls and baseline, indicating net bone loss. Cortical thickness did not differ among groups in either experiment. In summary, findings from three separate studies support the conclusion that spaceflight results in cancellous osteopenia in femoral head of growing rats.

Effects of Raloxifene and tibial loading on bone mass and mechanics in male and female mice

ABSTRACT Purpose: Raloxifene (RAL) is a selective estrogen receptor modulator (SERM) that has previously been shown to cause acellular benefits to bone tissue. Due to these improvements, RAL was combined with targeted tibial loading to assess if RAL treatment during periods of active bone formation would allow for further mechanical enhancements. Methods: Structural, mechanical, and microstructural effects were assessed in bone from C57BL/6 mice that were treated with RAL (0.5 mg/kg), tibial loading, or both for 6 weeks, beginning at 10 weeks of age. Results: Ex vivo microcomputed tomography (CT) images indicated RAL and loading work together to improve bone mass and architecture, especially within the cancellous region of males. Increases in cancellous bone volume fraction were heavily driven by increases in trabecular thickness, though there were some effects on trabecular spacing and number. In the cortical regions, RAL and loading both increased cross-sectional area, cortical area, and cortical thickness. Whole-bone mechanical testing primarily indicated the effects of loading. Further characterization through Raman spectroscopy and nanoindentation showed load-based changes in mineralization and micromechanics, while both loading and RAL caused changes in the secondary collagen structure. In contrast to males, in females, there were large load-based effects in the cancellous and cortical regions, resulting in increased whole-bone mechanical properties. RAL had less of an effect on cancellous and cortical architecture, though some effects were still present. Conclusion: RAL and loading work together to impact bone architecture and mechanical integrity, leading to greater improvements than either treatment individually.

Effects of Propranolol on Bone, White Adipose Tissue, and Bone Marrow Adipose Tissue in Mice Housed at Room Temperature or Thermoneutral Temperature.

Growing female mice housed at room temperature (22°C) weigh the same but differ in body composition compared to mice housed at thermoneutrality (32°C). Specifically, mice housed at room temperature have lower levels of white adipose tissue (WAT). Additionally, bone marrow adipose tissue (bMAT) and cancellous bone volume fraction in distal femur metaphysis are lower in room temperature-housed mice. The metabolic changes induced by sub-thermoneutral housing are associated with lower leptin levels in serum and higher levels of Ucp1 gene expression in brown adipose tissue. Although the precise mechanisms mediating adaptation to sub-thermoneutral temperature stress remain to be elucidated, there is evidence that increased sympathetic nervous system activity acting via β-adrenergic receptors plays an important role. We therefore evaluated the effect of the non-specific β-blocker propranolol (primarily β1 and β2 antagonist) on body composition, femur microarchitecture, and bMAT in growing female C57BL/6 mice housed at either room temperature or thermoneutral temperature. As anticipated, cancellous bone volume fraction, WAT and bMAT were lower in mice housed at room temperature. Propranolol had small but significant effects on bone microarchitecture (increased trabecular number and decreased trabecular spacing), but did not attenuate premature bone loss induced by room temperature housing. In contrast, propranolol treatment prevented housing temperature-associated differences in WAT and bMAT. To gain additional insight, we evaluated a panel of genes in tibia, using an adipogenesis PCR array. Housing temperature and treatment with propranolol had exclusive as well as shared effects on gene expression. Of particular interest was the finding that room temperature housing reduced, whereas propranolol increased, expression of the gene for acetyl-CoA carboxylase (Acacb), the rate-limiting step for fatty acid synthesis and a key regulator of β-oxidation. Taken together, these findings provide evidence that increased activation of β1 and/or β2 receptors contributes to reduced bMAT by regulating adipocyte metabolism, but that this pathway is unlikely to be responsible for premature cancellous bone loss in room temperature-housed mice.

Voluntary Chronic Heavy Alcohol Consumption in Male Rhesus Macaques Suppresses Cancellous Bone Formation and Increases Bone Marrow Adiposity.

Chronic heavy alcohol consumption is an established risk factor for bone fracture, but comorbidities associated with alcohol intake may contribute to increased fracture rates in alcohol abusers. To address the specific effects of alcohol on bone, we used a nonhuman primate model and evaluated voluntary alcohol consumption on: (i) global markers of bone turnover in blood and (ii) cancellous bone mass, density, microarchitecture, turnover, and microdamage in lumbar vertebra. Following a 4-month induction period, 6-year-old male rhesus macaques (Macaca mulatta, n=13) voluntarily self-administered water or ethanol (EtOH; 4% w/v) for 22h/d, 7 d/wk, for a total of 12months. Control animals (n=9) consumed an isocaloric maltose-dextrin solution. Tetracycline hydrochloride was administered orally 17 and 3days prior to sacrifice to label mineralizing bone surfaces. Global skeletal response to EtOH was evaluated by measuring plasma osteocalcin and carboxyterminal collagen cross-links (CTX). Local response was evaluated in lumbar vertebra using dual-energy X-ray absorptiometry, microcomputed tomography, static and dynamic histomorphometry, and histological assessment of microdamage. Monkeys in the EtOH group consumed an average of 2.8±0.2 (mean±SE) g/kg/d of EtOH (30±2% of total calories), resulting in an average blood EtOH concentration of 88.3±8.8mg/dl 7hours after the session onset. Plasma CTX and osteocalcin tended to be lower in EtOH-consuming monkeys compared to controls. Significant differences in bone mineral density in lumbar vertebrae 1 to 4 were not detected with treatment. However, cancellous bone volume fraction (in cores biopsied from the central region of the third vertebral body) was lower in EtOH-consuming monkeys compared to controls. Furthermore, EtOH-consuming monkeys had lower osteoblast perimeter and mineralizing perimeter, no significant difference in osteoclast perimeter, and higher bone marrow adiposity than controls. No significant differences between groups were detected in microcrack density (2nd lumbar vertebra). Voluntary chronic heavy EtOH consumption reduces cancellous bone formation in lumbar vertebra by decreasing osteoblast-lined bone perimeter, a response associated with an increase in bone marrow adiposity.

Thermoneutral housing attenuates premature cancellous bone loss in male C57BL/6J mice.

Mice are a commonly used model to investigate aging-related bone loss but, in contrast to humans, mice exhibit cancellous bone loss prior to skeletal maturity. The mechanisms mediating premature bone loss are not well established. However, our previous work in female mice suggests housing temperature is a critical factor. Premature cancellous bone loss was prevented in female C57BL/6J mice by housing the animals at thermoneutral temperature (where basal rate of energy production is at equilibrium with heat loss). In the present study, we determined if the protective effects of thermoneutral housing extend to males. Male C57BL/6J mice were housed at standard room temperature (22°C) or thermoneutral (32°C) conditions from 5 (rapidly growing) to 16 (slowly growing) weeks of age. Mice housed at room temperature exhibited reductions in cancellous bone volume fraction in distal femur metaphysis and fifth lumbar vertebra; these effects were abolished at thermoneutral conditions. Mice housed at thermoneutral temperature had higher levels of bone formation in distal femur (based on histomorphometry) and globally (serum osteocalcin), and lower global levels of bone resorption (serum C-terminal telopeptide of type I collagen) compared to mice housed at room temperature. Thermoneutral housing had no impact on bone marrow adiposity but resulted in higher abdominal white adipose tissue and serum leptin. The overall magnitude of room temperature housing-induced cancellous bone loss did not differ between male (current study) and female (published data) mice. These findings highlight housing temperature as a critical experimental variable in studies using mice of either sex to investigate aging-related changes in bone metabolism.

Effect of Leptin Deficiency on the Skeletal Response to Hindlimb Unloading in Adult Male Mice

Based on body weight, morbidly obese leptin-deficient ob/ob mice have less bone than expected, suggesting that leptin plays a role in the skeletal response to weight bearing. To evaluate this possibility, we compared the skeletal response of wild type (WT) and ob/ob mice to hindlimb unloading (HU). Mice were individually housed at 32 °C (thermoneutral) from 4 weeks of age (rapidly growing) to 16 weeks of age (approaching skeletal maturity). Mice were then randomized into one of 4 groups (n = 10/group): (1) WT control, (2) WT HU, (3) ob/ob control, and (4) ob/ob HU and the results analyzed by 2-way ANOVA. ob/ob mice pair-fed to WT mice had normal cancellous bone volume fraction (BV/TV) in distal femur, lower femur length and total bone area, mineral content (BMC) and density (BMD), and higher cancellous bone volume fraction in lumbar vertebra (LV). HU resulted in lower BMC and BMD in total femur, and lower BV/TV in distal femur and LV in both genotypes. Cancellous bone loss in femur in both genotypes was associated with increases in osteoclast-lined bone perimeter. In summary, leptin deficiency did not attenuate HU-induced osteopenia in male mice, suggesting that leptin is not required for bone loss induced by unweighting.

Botox-induced muscle paralysis alters intracortical porosity and osteocyte lacunar density in skeletally mature rats.

Reduced mechanical loading can lead to disuse osteoporosis, resulting in bone fragility. Disuse models report macroscopic bone loss due to muscle inactivity and immobilization, yet only recently has there been quantification of the effects of disuse on the vascular pores and osteocyte network, which are believed to play an important role in mechanotransduction via interstitial fluid flow. The goal of this study was to perform a high-resolution analysis of the effects of muscle inactivity on intracortical porosity and osteocyte lacunar density in skeletally mature rats. Muscle paralysis was induced in 20-week-old female Sprague Dawley rats by injection of botulinum neurotoxin. Rats were injected in the right hindlimb muscles with either Botox (BTX, n = 8) or saline solution (CTRL, n = 8), with a third group used as baseline controls (n = 8). Four weeks after injection, Botox caused a ∼60% reduction in hindlimb muscle mass. High-resolution micro-CT analysis showed that Botox-induced muscle paralysis increased vascular canal porosity and reduced osteocyte lacunar density within the tibial metaphysis cortex. Cortical thickness and other areal properties were diminished in the proximal tibial metaphysis, whereas no differences were found in the mid-diaphysis. Within the BTX group, the injected limbs showed a lower cancellous bone volume fraction relative to the contralateral limb. These results indicate that diminished muscle activity alters the vascular canal porosity and osteocyte lacunar density in cortical bone, which could alter interstitial fluid flow, affecting molecular transport and the transmission of mechanical signals to osteocytes. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

Effects of a Four-day Spaceflight and Recombinant Human Growth Hormone on Cancellous Bone Microarchitecture in Femoral Head of Rapidly Growing Male Rats

Spaceflight results in reduced bone accrual and muscle atrophy in growing rodents. Some studies suggest that the detrimental effects of spaceflight are due, in part, to impaired growth hormone (GH) signaling. An experiment flown aboard STS-41 (October 6–10, 1990) evaluated the efficacy of recombinant human growth hormone (rhGH) in ameliorating the detrimental effects of spaceflight on the musculoskeletal system in male Sprague Dawley rats. The rats were 39 days old at launch and sacrificed following the 4–day flight. Ground controls (n=11/treatment group) and flight animals (n=8/treatment group) were treated with rhGH or excipient delivered using osmotic pumps implanted subcutaneously one day prior to launch. For the present examination, cancellous bone in the femoral head was evaluated using X-ray microtomography (microcomputed tomography), a technology not available when the study was performed. Spaceflight resulted in lower cancellous bone volume fraction, connectivity density, trabecular thickness and trabecular number, and higher trabecular separation. rhGH had no independent effect on cancellous bone architecture and there were no spaceflight by rhGH interactions. These findings suggest that a short interval of microgravity during rapid growth was sufficient to reduce accrual of cancellous bone and alter bone microarchitecture at an important weight bearing skeletal site. Additionally, increasing growth hormone levels was ineffective in preventing cancellous osteopenia in flight animals and did not increase cancellous bone volume fraction in ground controls. (Less)

Androgen Receptor in Neurons Slows Age-Related Cortical Thinning in Male Mice.

Androgens via the androgen receptor (AR) are required for optimal male bone health. The target cell(s) for the effects of androgens on cortical bone remain(s) incompletely understood. In females, estrogen receptor alpha in neurons is a negative regulator of cortical and trabecular bone. Whether neuronal AR regulates bone mass in males remains unexplored. Here, we inactivated AR in neurons using a tamoxifen-inducible CreERT2 under the control of the neuronal promoter Thy1. Tamoxifen induced a 70% to 80% reduction of AR mRNA levels in Thy1-CreERT2-positive brain regions cerebral cortex and brainstem as well as in the peripheral nervous tissue of male neuronal AR knockout (N-ARKO) mice. Hypothalamic AR mRNA levels were only marginally reduced and the hypothalamic-pituitary-gonadal axis remained unaffected, as determined by normal levels of serum testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). In contrast to orchidectomy, deletion of neuronal AR did not alter body weight, body composition, hindlimb muscle mass, grip strength, or wheel running. MicroCT analysis of the femur revealed no changes in bone accrual during growth in N-ARKO mice. However, 36- and 46-week-old N-ARKO mice displayed an accelerated age-related cortical involution, namely a more pronounced loss of cortical thickness and strength, which occurred in the setting of androgen sufficiency. Neuronal AR inactivation decreased the cancellous bone volume fraction in L5 vertebra but not in the appendicular skeleton of aging mice. MicroCT findings were corroborated in the tibia and after normalization of hormonal levels. Serum markers of bone turnover and histomorphometry parameters were comparable between genotypes, except for a 30% increase in osteoclast surface in the trabecular compartment of 36-week-old N-ARKO mice. Cortical bone loss in N-ARKO mice was associated with an upregulation of Ucp1 expression in brown adipose tissue, a widely used readout for sympathetic tone. We conclude that androgens preserve cortical integrity in aging male mice via AR in neurons. © 2018 American Society for Bone and Mineral Research.

Metformin Alleviates the Bone Loss Induced by Ketogenic Diet: An In Vivo Study in Mice.

Metformin (Met), an anti-diabetes drug, has also shown therapeutic effects for ovariectomy-induced (OVX) osteoporosis. However, similar effects against bone loss induced by a ketogenic diet (KD) have not been tested. This study was aimed to evaluate the microarchitectures and biomechanics of KD-induced osteoporosis with and without administration of Met, and compare the effect of Met on bone loss induced by KD with OVX. Forty female C57BL/6J mice were randomly divided into Sham, OVX, OVX + Met (100mg/kg/day), KD (3:1 ratio of fat to carbohydrate and protein), and KD + Met (100mg/kg/day) groups. After 12 weeks, the bone mass and biomechanics were measured in distal cancellous bone and in mid-shaft cortical bone of the femur. The activities of serum alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), together with immunohistochemistry staining of osteocalcin (OCN) and TRAP, were evaluated. Both OVX and KDinduced significant bone loss and compromised biomechanical properties in the cancellous bone, but no effect was found in cortical bone. The administration of Met increased the cancellous bone volume fraction (BV/TV) from 3.78 to 5.23% following the OVX and from 4.04 to 6.33% following the KD, it also enhanced the compressive stiffness from 47 to 160N/mm following the OVX and from 35 to 340N/mm with the KD. Met effectively increased serum ALP in the KD group while decreased serum TRAP in the OVX group, but up-regulated expression of OCN and down-regulated expression of TRAP in both OVX and KD groups. The present study demonstrated that Met effectively attenuated the cancellous bone loss induced by KD and maintained the biomechanical properties of long bones, providing evidence for Met as a treatment of by KD-induced osteoporosis in teenage skeleton.

CBCT-based bone quality assessment in decompression of large odontogenic cystic lesions.

Bone quality comprises bone mineral density and trabecular microstructure. The aim of this study was to explore the effectiveness of cone-beam computed tomography (CBCT) in evaluating bone quality of large odontogenic cystic lesions after decompression using CBCT and BoneJ software, and to determine whether secondary definitive surgery can be guided using CBCT data. Twenty-seven patients with large odontogenic cystic lesions treated by decompression were evaluated by CBCT. Medical history and perioperative details were analyzed. The [Formula: see text]CT values for all patients with cystic lesions decreased after decompression, with no differences for age, sex, and histology (p > 0.05). Bone volume fraction and trabecular number of new cancellous bone (0.012%, 0.17/mm3) were lower than those of normal cancellous bone (0.189%, 0.47/mm3) (p < 0.05), while new cancellous bone trabecular separation (11.344 ± 2.556mm) was stronger than normal cancellous bone trabecular separation (4.833 ± 2.232mm) (p < 0.05). There were no differences in trabecular thickness between new cancellous bone (3.812 ± 1.593mm) and normal cancellous bone (4.598 ± 3.573mm) (p = 0.746). The [Formula: see text]CT values of five patients with favorable osteogenesis were - 72, -86, - 86, -47, and - 55, those of three patients with moderate osteogenesis were - 107, -120, and - 71, and those of two patients with poor osteogenesis were - 165 and - 127 during secondary definitive surgery. CBCT is considered beneficial for evaluating bone quality of large odontogenic cystic lesions after decompression, while providing potentially useful information for referral to secondary definitive surgery.

Role of estrogen receptor signaling in skeletal response to leptin in female ob/ob mice.

Leptin, critical in regulation of energy metabolism, is also important for normal bone growth, maturation and turnover. Compared to wild type (WT) mice, bone mass is lower in leptin-deficient ob/ob mice. Osteopenia in growing ob/ob mice is due to decreased bone accrual, and is associated with reduced longitudinal bone growth, impaired cancellous bone maturation and increased marrow adipose tissue (MAT). However, leptin deficiency also results in gonadal dysfunction, disrupting production of gonadal hormones which regulate bone growth and turnover. The present study evaluated the role of increased estrogen in mediating the effects of leptin on bone in ob/ob mice. Three-month-old female ob/ob mice were randomized into one of the 3 groups: (1) ob/ob + vehicle (veh), (2) ob/ob + leptin (leptin) or (3) ob/ob + leptin and the potent estrogen receptor antagonist ICI 182,780 (leptin + ICI). Age-matched WT mice received vehicle. Leptin (40 µg/mouse, daily) and ICI (10 µg/mouse, 2×/week) were administered by subcutaneous injection for 1 month and bone analyzed by X-ray absorptiometry, microcomputed tomography and static and dynamic histomorphometry. Uterine weight did not differ between ob/ob mice and ob/ob mice receiving leptin + ICI, indicating that ICI successfully blocked the uterine response to leptin-induced increases in estrogen levels. Compared to leptin-treated ob/ob mice, ob/ob mice receiving leptin + ICI had lower uterine weight; did not differ in weight loss, MAT or bone formation rate; and had higher longitudinal bone growth rate and cancellous bone volume fraction. We conclude that increased estrogen signaling following leptin treatment is dispensable for the positive actions of leptin on bone and may attenuate leptin-induced bone growth.

High-Dietary Alpha-Tocopherol or Mixed Tocotrienols Have No Effect on Bone Mass, Density, or Turnover in Male Rats During Skeletal Maturation.

High levels of alpha-tocopherol, the usual vitamin E supplement, are reported to decrease bone mass in rodents; however, the effects of other vitamin E forms on the skeleton are unknown. To test the hypothesis that high intakes of various vitamin E forms or the vitamin E metabolite, carboxyethyl hydroxy chromanol, were detrimental to bone status, Sprague-Dawley rats (n = 6 per group, 11-week males) for 18 weeks consumed semipurified diets that contained adequate alpha-tocopherol, high alpha-tocopherol (500 mg/kg diet), or 50% Tocomin (250 mg mixed tocopherols and tocotrienols/kg diet). Vitamin E status was evaluated by measuring plasma, liver, and bone marrow vitamin E concentrations. Bone density, microarchitecture (cross-sectional volume, cortical volume, marrow volume, cortical thickness, and cancellous bone volume fraction, trabecular number, thickness, and spacing), and cancellous bone formation were assessed in the tibia using dual-energy X-ray absorptiometry, microcomputed tomography, and histomorphometry, respectively. In addition, serum osteocalcin was assessed as a global marker of bone turnover; gene expression in response to treatment was evaluated in the femur using targeted (osteogenesis related) gene profiling. No significant differences were detected between treatment groups for any of the bone endpoints measured. Vitamin E supplementation, either as alpha-tocopherol or mixed tocotrienols, while increasing vitamin E concentrations both in plasma and tissues, had no effect on the skeleton in rats.

Effects of High-LET Radiation Exposure and Hindlimb Unloading on Skeletal Muscle Resistance Artery Vasomotor Properties and Cancellous Bone Microarchitecture in Mice.

Weightlessness during spaceflight leads to functional changes in resistance arteries and loss of cancellous bone, which may be potentiated by radiation exposure. The purpose of this study was to assess the effects of hindlimb unloading (HU) and total-body irradiation (TBI) on the vasomotor responses of skeletal muscle arteries. Male C57BL/6 mice were assigned to control, HU (13-16 days), TBI (1 Gy (56)Fe, 600 MeV, 10 cGy/min) and HU-TBI groups. Gastrocnemius muscle feed arteries were isolated for in vitro study. Endothelium-dependent (acetylcholine) and -independent (Dea-NONOate) vasodilator and vasoconstrictor (KCl, phenylephrine and myogenic) responses were evaluated. Arterial endothelial nitric oxide synthase (eNOS), superoxide dismutase-1 (SOD-1) and xanthine oxidase (XO) protein content and tibial cancellous bone microarchitecture were quantified. Endothelium-dependent and -independent vasodilator responses were impaired in all groups relative to control, and acetylcholine-induced vasodilation was lower in the HU-TBI group relative to that in the HU and TBI groups. Reductions in endothelium-dependent vasodilation correlated with a lower cancellous bone volume fraction. Nitric oxide synthase inhibition abolished all group differences in endothelium-dependent vasodilation. HU and HU-TBI resulted in decreases in eNOS protein levels, while TBI and HU-TBI produced lower SOD-1 and higher XO protein content. Vasoconstrictor responses were not altered. Reductions in NO bioavailability (eNOS), lower anti-oxidant capacity (SOD-1) and higher pro-oxidant capacity (XO) may contribute to the deficits in NOS signaling in skeletal muscle resistance arteries. These findings suggest that the combination of insults experienced in spaceflight leads to impairment of vasodilator function in resistance arteries that is mediated through deficits in NOS signaling.

Effects of Spaceflight on Bone Microarchitecture in the Axial and Appendicular Skeleton in Growing Ovariectomized Rats.

This study investigated the effects of a 14-day spaceflight on bone mass, density and microarchitecture in weight bearing (femur and humerus) and non-weight bearing (2nd lumbar vertebra and calvarium) bones in the context of ovarian hormone insufficiency. 12-week-old Fisher 344 rats were ovariectomized 2 weeks before flight and randomized into one of three groups: 1) baseline (n = 6), 2) ground control (n = 12) or 3) spaceflight (n = 12). Additional ground-based ovary-intact rats provided age-matched reference values at baseline (n = 8) and landing (n = 10). Ovariectomy resulted in bone- and bone compartment-specific deficits in cancellous bone volume fraction. Spaceflight resulted in lower cortical bone accrual in the femur but had no effect on cortical bone in the humerus or calvarium. Cancellous bone volume fraction was lower in flight animals compared to ground control animals in lumbar vertebra and distal femur metaphysis and epiphysis; significant differences were not detected in the distal humerus. Bone loss (compared to baseline controls) in the femur metaphysis was associated with lower trabecular number, whereas trabecular thickness and number were lower in the epiphysis. In summary, the effect of spaceflight on bone microarchitecture in ovariectomized rats was bone-and bone compartment-specific but not strictly related to weight bearing.