Loss of Sex-Specific Difference in Femoral Bone Parameters in Male Leptin Knockout Mice

Sex-dependent differences were identified in the femoral bone parameters of male and female ob/ob (leptin knockout) mice compared with their C57BL/6 wild-type background strain. Total fat, lean weight and body weight were not different between adult male and female leptin knockout mice. However, leptin knockout males exhibited lower lean weights than C57BL/6 males. Peripheral quantitative computerized tomographic measurements at the femoral midshaft revealed that the normal differences in the periosteal circumference, endosteal circumference, total bone mineral content, and polar moment of inertia normally observed between adult male and female wild-type mice were lost between adult male and female ob/ob mice. Significant reductions in these bone parameters were seen in male ob/ob mice compared to male wild-type mice but not in female ob/ob mice compared to female wild-type mice. In prepubertal mice, there were no differences in phenotype and femoral bone parameters between males and females within any strain, suggesting sex hormone functions. Serum free testosterone levels were 5.6-fold higher in adult male ob/ob mice than in adult male C57BL/6 wild-type mice, and serum estradiol levels were 1.8- and 1.3-fold greater in adult male and female ob/ob mice, respectively, than in their wild-type counterparts. Androgen receptor gene expression was not different in femur-derived bone cells of male ob/ob mice compared with wild-type mice. The loss of sex-related differences in these bone parameters in adult male ob/ob mice might result from deficient signaling in the androgen signaling pathway and the fact that leptin functions are permissive for androgen effects on bone development. Xiaoguang Wang, Charles H. Rundle contributed equally to this work.     

Genetic Variations in Bone Density, Histomorphometry, and Strength in Mice

The purpose of this study was to assess breed-related differences in bone histomorphometry, bone biomechanics, and serum biochemistry in three mouse breeds shown to differ in bone mineral density (BMD) (as measured by DXA) and bone mineral content (BMC). Femurs, tibiae, and sera were collected from 16-week-old C3H/HeJ {C3H}, C57BL/6J {BL6}, and DBA/2J {DBA}mice (n = 12/breed). Data collected included BMC and BMD (femora), histomorphometry of cancellous (distal femur) and cortical bone (diaphyseal tibiae and femora), bone strength (femora), and serum alkaline phosphatase (ALP). Consistent with previous reports, BMC and BMD were higher in C3H than in BL6 or DBA mice. The higher BMD in the C3H breed was associated with greater cancellous bone volume, cortical bone area, periosteal bone formation rate, biomechanical strength, and serum ALP. However, mid-diaphyseal total femoral and tibial cross-sectional area and moment of inertia were greatest in BL6, intermediate in C3H, and lowest in DBA mice. The specific distribution of cortical bone in C3H, BL6, DBA mice represents a difference in adaptive response to similar mechanical loads in these breeds. This difference in adaptive response may be intrinsic to the adaptive mechanism, or may be intrinsic to the bone tissue material properties. In either case, the bone-adaptive response to ordinary mechanical loads in the BL6 mice yields bones of lower mechanical efficiency (less stiffness per unit mass of bone tissue) and does not adapt as well as that of the C3H mice where the final product is a bone with greater resistance to bending under load. We suggest that the size, shape, and BMD of the bone are a result of breed-specific genetically regulated cellular mechanisms. Compared with the C3H mice, the lower BMD in BL6 mice is associated with long bones that are weaker because the larger cross-sectional area fails to compensate completely for their lower BMD and BMC. Received: 16 November 1999 / Accepted: 19 April 2000 / Online publication: 27 July 2000     

Effect of Estrogen Deficiency on Cancellous and Cortical Bone Structure and Strength of the Femoral Neck in Rats

Eighty mature Sprague-Dawley rats were weight matched before ovariectomy (Ovx) or Sham surgery (Sham). Sham rats had free access to food and water throughout the experiment, whereas Ovx rats were kept on the pair-fed diet. Rats were euthanized at 4, 8, and 12 weeks after surgery, and had received fluorochrome bone markers at 9 and 2 days prior to euthanasia. In addition 10 rats were euthanized at the time of surgery serving as baseline controls. All rats were also scanned for body composition and bone mineral parameters by DEXA before surgery and euthanasia. Left proximal femurs (femoral necks) were used for bone histomorphometry, whereas right femurs were used for in vitro DEXA measurements and mechanical testing. Despite pair-feeding, ovariectomized rats had increased body weights and fat body mass, whereas the percent lean body mass steadily declined throughout the experiment. Mineral density of the whole femur and femoral neck was significantly higher in the Sham rats relative to Ovx animals. Ovariectomy reduced trabecular number and thickness, and increased trabecular separation and bone marrow space at the femoral midneck location. The structure of the remaining trabeculae was dramatically changed toward simpler struts as revealed by nodal analyses. Cortical thickness in Ovx rats was reduced because of the high endocortical resorption, which, in addition to cancellous bone resorption, resulted in fewer endocortico-trabecular connections. Femoral necks obtained from ovariectomized rats had reduced strength and were less stiff relative to controls. Because of the enormous clinical significance of the proximal femur for osteoporosis in humans, and the opportunity for studying bone BMD, mass, structure, and strength at the same skeletal location, the femoral neck appears superior to other skeletal sites routinely used for bone histomorphometry or mechanical testing in the Ovx rat model. Received: 25 September 1996 / Accepted: 24 March 1997     

Age-Related Changes in Bone Structure and Strength in Female and Male BALB/c Mice

Mice may be useful for studies of skeletal aging, but there are limited data on changes in bone structure and strength over their life span. We obtained bones from female and male BALB/c mice at ages 2, 4, 7, 12, and 20 months and evaluated their structural, densitometric, and mechanical properties. MicroCT of the mid-diaphysis of the femur and radius indicated that during skeletal growth (2–7 months) bone cross-sectional size (area, moment of inertia) increased rapidly; during aging (7–20 months) cortical area was maintained, while moment of inertia continued to increase. Bones from females were smaller than those from males at young ages but not at later ages. Changes in whole-bone stiffness and strength reflected the changes in bone size, with a rapid increase from 2 to 7 months, followed by little or no change. In contrast, energy-to-fracture declined with aging. Cortical tissue mineral density increased during growth and was maintained with aging. MicroCT of trabecular bone revealed age-related changes that were site-dependent. The proximal tibia showed a clear pattern of age-related decline in trabecular BV/TV, with progressive decreases after 4 months in both sexes; lumbar vertebra L5 had more modest age-related declines; in contrast, caudal vertebra Ca7 had increasing BV/TV with aging. Overall, we found no evidence that females had more pronounced age-related deterioration than males. We conclude that bones from aging female and male BALB/c mice exhibit many of the changes seen in humans and are therefore a clinically relevant model for studies of skeletal aging.     

New Autophagy Reporter Mice Reveal Dynamics of Proximal Tubular Autophagy

The accumulation of autophagosomes in postischemic kidneys may be renoprotective, but whether this accumulation results from the induction of autophagy or from obstruction within the autophagic process is unknown. Utilizing the differential pH sensitivities of red fluorescent protein (RFP; pK_a 4.5) and enhanced green fluorescent protein (EGFP; pK_a 5.9), we generated CAG-RFP-EGFP-LC3 mice to distinguish early autophagic vacuoles from autolysosomes. In vitro and in vivo studies confirmed that in response to nutrient deprivation, renal epithelial cells in CAG-RFP-EGFP-LC3 mice produce autophagic vacuoles expressing RFP and EGFP puncta. EGFP fluorescence diminished substantially in the acidic environment of the autolysosomes, whereas bright RFP signals remained. Under normal conditions, nephrons expressed few EGFP and RFP puncta, but ischemia-reperfusion injury (IRI) led to dynamic changes in the proximal tubules, with increased numbers of RFP and EGFP puncta that peaked at 1 day after IRI. The number of EGFP puncta returned to control levels at 3 days after IRI, whereas the high levels of RFP puncta persisted, indicating autophagy initiation at day 1 and autophagosome clearance during renal recovery at day 3. Notably, proliferation decreased in cells containing RFP puncta, suggesting that autophagic cells are less likely to divide for tubular repair. Furthermore, 87% of proximal tubular cells with activated mechanistic target of rapamycin (mTOR), which prevents autophagy, contained no RFP puncta. Conversely, inhibition of mTOR complex 1 induced RFP and EGFP expression and decreased cell proliferation. In summary, our results highlight the dynamic regulation of autophagy in postischemic kidneys and suggest a role of mTOR in autophagy resolution during renal repair.Autophagy is a lysosomal degradation pathway that is essential for cellular stress adaptation and normal homeostasis.^1–3 It involves a series of membrane rearrangements to form autophagosomes, which are double-membraned vacuoles that contain cytoplasmic contents and organelles. Fusion of autophagosomes with lysosomes results in the formation of autolysosomes in which captured materials are degraded for removal of damaged organelles and recycling of nutrients within the cells. Autophagy has been recognized as a protective mechanism after renal ischemia-reperfusion injury (IRI).^4–8 Increased levels of autophagy have been reported in the postischemic kidneys by accumulation of autophagosomes under electron microscopy or increased Atg proteins by immunoblot analysis.^4,7,8 However, electron microscopy can only provide static information and does not distinguish whether the accumulation of autophagosomes is due to the induction of autophagy or a blockage in downstream processes of autophagy. Immunoblot analysis of Atg proteins detects autophagy in a heterogeneous and asynchronous cell population and does not reflect autophagy in individual compartments of the kidney. New tools are needed to study autophagic flux, which will provide a more accurate assessment of autophagic activity in individual cells of the organ.LC3 protein is the mammalian homology of Atg8 in yeasts and is essential for autophagy to occur. LC3 is cleaved to LC3-I immediately after its synthesis. LC3-I is an ubiquitin-like protein that can be conjugated to phosphatidylethanolamine and possibly phosphatidylserine. The lipidated forms are referred to LC3-II, which is present in all autophagic vacuoles. LC3-II is the most widely used Atg protein to quantify autophagic levels by immunoblot analysis.³ Methods for immunostaining of LC3-II have been recently developed.⁹ However, it is not always possible to detect low levels of endogenous LC3-II. Transfection of cells with plasmids expressing enhanced green fluorescent protein (EGFP) fused with LC3 enables the visualization of EGFP puncta in autophagic vacuoles. Transgenic mice expressing EGFP-LC3 fusion protein under the cytomegalovirus immediate-early enhancer and chicken β-actin (CAG) promoter have been generated.¹⁰ The mice are useful to study autophagy induction in many organs, including the kidney, based on the appearance of EGFP puncta identified with fluorescence microscopy.⁵ However, EGFP fluorescence is quenched in the acidic environment of autolysosomes, and thus loses its ability to track the autophagic process.We generated a new strain of autophagy reporter mice that express a tandem red fluorescent protein (RFP)-EGFP-LC3 fusion protein ubiquitously under the CAG promoter to advance our understanding of the dynamics of autophagy. Specifically, the inclusion of a relatively acid-insensitive RFP (pK_a 4.5) and acid-sensitive EGFP (pK_a 5.9) is expected to result in the quenching of EGFP but persistence of RFP signals in the low pH environment of autolysosomes (pH=4–5), whereas both EGFP and RFP fluorescence will be maintained before autophagosomes fuse with lysosomes.^11,12 The mice were used as novel tools to study the dynamics of renal epithelial autophagy under stress conditions such as starvation and ischemic injury.     

Bone Mineral Content and Mechanical Strength of the Femoral Neck

The bone mineral content of the femoral neck of 61 autopsy specimens was assayed by x-ray spectrophotometry. The mechanical strength of the specimens was also determined experimentally by applying a compressive force perpendicularly to the shaft. The ultimate force at fracture was obtained from force/displacement plots. a coefficient of correlation of 0.89 between bone mineral content of the femoral neck and the ultimate force at fracture was found. Even when limited to a group of women aged 67-80 a fairly close correlation was found. This indicates that the bone mineral level, measured in vivo, can be used as a criterion of the risk of fracture in elderly women.     

Sex‐Specific Genetic Loci for Femoral Neck Bone Mass and Strength Identified in Inbred COP and DA Rats

Introduction: Hip fracture is the most devastating osteoporotic fracture type with significant morbidity and mortality. Several studies in humans identified chromosomal regions linked to hip size and bone mass. Animal models, particularly the inbred rat, serve as complementary approaches for studying the genetic influence on hip fragility. The purpose of this study is to identify sex‐independent and sex‐specific quantitative trait loci (QTLs) for femoral neck density, structure, and strength in inbred Copenhagen 2331 (COP) and Dark Agouti (DA) rats. Materials and Methods: A total of 828 (405 males and 423 females) F₂ progeny derived from the inbred COP and DA strains of rats were phenotyped for femoral neck volumetric BMD (vBMD), cross‐sectional area, polar moment of inertia (Ip), neck width, ultimate force, and energy to break. A whole genome screen was performed using 93 microsatellite markers with an average intermarker distance of 20 cM. Recombination‐based marker maps were generated using MAPMAKER/EXP from the COP × DA F₂ data and compared with published Rat Genome Database (RGD) maps. These maps were used for genome‐wide linkage analyses to detect sex‐independent and sex‐specific QTLs. Results: Significant evidence of linkage (p < 0.01) for sex‐independent QTLs were detected for (1) femoral neck vBMD on chromosomes (Chrs) 1, 6, 10, and 12, (2) femoral neck structure on Chrs 5, 7, 10, and 18, and (3) biomechanical properties on Chrs 1 and 4. Male‐specific QTLs were discovered on Chrs 2, 9, and 18 for total vBMD, on Chr 17 for trabecular vBMD, on Chr 9 for total bone area, and on Chr 15 for ultimate force. A female‐specific QTL was discovered on Chr 2 for ultimate force. The effect size of the individual QTL varied between 1% and 4%. Conclusions: We detected evidence that sex‐independent and sex‐specific QTLs contribute to hip fragility in the inbred rat. Several QTLs regions identified in this study are homologous to human chromosomal regions previously linked to QTLs contributing to femoral neck and related phenotypes.     

Developmental Timing of High-Fat Diet Exposure Impacts Glucose Homeostasis in Mice in a Sex-Specific Manner

We previously demonstrated that male, but not female, Swiss Webster mice are susceptible to diabetes, with incidence increased by early overnutrition and high-fat diet (HFD). In this study, we investigated how HFD in Swiss Webster males and females during preweaning, peripubertal, and postpubertal periods alters glucose homeostasis and diabetes susceptibility. In males, HFD throughout life resulted in the highest diabetes incidence. Notably, switching to chow postpuberty was protective against diabetes relative to switching to chow at weaning, despite the longer period of HFD exposure. Similarly, HFD throughout life in males resulted in less liver steatosis relative to mice with shorter duration of postpubertal HFD. Thus, HFD timing relative to weaning and puberty, not simply exposure length, contributes to metabolic outcomes. Females were protected from hyperglycemia regardless of length or timing of HFD. However, postpubertal HFD resulted in a high degree of hepatic steatosis and adipose fibrosis, but glucose regulation and insulin sensitivity remained unchanged. Interestingly, peri-insulitis was observed in the majority of females but was not correlated with impaired glucose regulation. Our findings reveal critical periods of HFD-induced glucose dysregulation with striking sex differences in Swiss Webster mice, highlighting the importance of careful consideration of HFD timing relative to critical developmental periods.     

Assessment of Femoral Neck Strength by 3-Dimensional X-ray Absorptiometry

Hip fractures due to osteoporosis are accompanied with increased mortality and morbidity. Bone mineral density (BMD [g/cm²]) measured by dual-energy X-ray absorptiometry (DXA) is the most important risk factor. However, an overlap exists between results of fractured and nonfractured populations. Macro-architectural parameters of the femur are independent risk factors of fracture. They have been evaluated in two dimensions using X-ray films or DXA scans; therefore, they are highly dependent on patient positioning and interindividual anatomical variations. To overcome this problem, we have previously shown the possibility to reconstruct human femurs using two perpendicular DXA scans and to calculate 3-dimensional (3D) geometric parameters from these reconstructions by a method called 3-dimensional X-ray absorptiometry (3D-XA). The aim of this article is to assess whether the combination of areal BMD and 3D geometric parameters calculated from 3D-XA improves failure load prediction of human proximal femurs in stance phase configuration. Twelve femurs (11 women, 1 man; aged 88 ± 9 yr; range: 72–103 yr) were included in this study. The BMD was measured using a Hologic Delphi-W device (Hologic, Waltham, MA) and 3D reconstruction of the femurs was done using two perpendicular DXA scans as previously published. The calculated 3D geometric parameters included femoral neck axis length (FNAL), mid-femoral neck cross-sectional area (mid-FN CSA), neck shaft angle (NSA), and femoral head diameter (FHD). Mechanical testing was performed using stance phase configuration, which resulted in subcapital fractures. The FHD was correlated to mid-FN CSA and FNAL (r = 0.68 and 0.76, respectively; p < 0.001). Failure load was correlated to age, FHD, NSA, and BMD measurements. Multiple regression analysis showed that femoral neck BMD, FHD, and mid-FN CSA gave the best statistical model for failure load prediction (r² = 0.84; p < 0.002). This is the first study suggesting that combining areal BMD to 3D geometric parameters obtained by 3D-XA improve failure load prediction in human femurs.     

Evaluation of Compressive Strength Index of the Femoral Neck in Caucasians and Chinese

Compressive strength index (CSI) of the femoral neck is a parameter that integrates the information of bone mineral density (BMD), femoral neck width (FNW), and body weight. CSI is considered to have the potential to improve the performance of assessment for hip fracture risk. However, studies on CSI have been rare. In particular, few studies have evaluated the performance of CSI, in comparison with BMD, FNW, and bending geometry, for assessment of hip fracture risk. We studied two large populations, including 1683 unrelated U.S. Caucasians and 2758 unrelated Chinese adults. For all the study subjects, CSI, femoral neck BMD (FN_BMD), FNW, and bending geometry (section modulus [Z]) of the samples were obtained from dual-energy X-ray absorptiometry scans. We investigated the age-related trends of these bone phenotypes and potential sex and ethnic differences. We further evaluated the performance of these four phenotypes for assessment of hip fracture risk by logistic regression models. Chinese had significantly lower FN_BMD, FNW, and Z, but higher CSI than sex-matched Caucasians. Logistic regression analysis showed that higher CSI was significantly associated with lower risk of hip fracture, and the significance remained after adjusting for covariates of age, sex, and height. Each standard deviation (SD) increment in CSI was associated with odds ratios of 0.765 (95% confidence interval, 0.634, 0.992) and 0.724 (95% confidence interval, 0.569, 0.921) for hip fracture risk in Caucasians and Chinese, respectively. The higher CSI in Chinese may partially help explain the lower incidence of hip fractures in this population compared to Caucasians. Further studies in larger cohorts and/or longitudinal observations are necessary to confirm our findings.     

Femoral and Vertebral Strength Improvements in Postmenopausal Women With Osteoporosis Treated With Denosumab

In the randomized, placebo‐controlled FREEDOM study of women aged 60 to 90 years with postmenopausal osteoporosis, treatment with denosumab once every 6 months for 36 months significantly reduced hip and new vertebral fracture risk by 40% and 68%, respectively. To gain further insight into this efficacy, we performed a nonlinear finite element analysis (FEA) of hip and spine quantitative computed tomography (QCT) scans to estimate hip and spine strength in a subset of FREEDOM subjects (n = 48 placebo; n = 51 denosumab) at baseline, 12, 24, and 36 months. We found that, compared with baseline, the finite element estimates of hip strength increased from 12 months (5.3%; p < 0.0001) and through 36 months (8.6%; p < 0.0001) in the denosumab group. For the placebo group, hip strength did not change at 12 months and decreased at 36 months (–5.6%; p < 0.0001). Similar changes were observed at the spine: strength increased by 18.2% at 36 months for the denosumab group (p < 0.0001) and decreased by –4.2% for the placebo group (p = 0.002). At 36 months, hip and spine strength increased for the denosumab group compared with the placebo group by 14.3% (p < 0.0001) and 22.4% (p < 0.0001), respectively. Further analysis of the finite element models indicated that strength associated with the trabecular bone was lost at the hip and spine in the placebo group, whereas strength associated with both the trabecular and cortical bone improved in the denosumab group. In conclusion, treatment with denosumab increased hip and spine strength as estimated by FEA of QCT scans compared with both baseline and placebo owing to positive treatment effects in both the trabecular and cortical bone compartments. These findings provide insight into the mechanism by which denosumab reduces fracture risk for postmenopausal women with osteoporosis. © 2014 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals, Inc. on behalf of the American Society for Bone and Mineral Research. This is an open access article under the terms of the Creative Commons Attribution–NonCommercial–NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.     

Bone Quality and Bone Strength in BXH Recombinant Inbred Mice

The relationship between bone quality and strength was studied in 11 BXH recombinant inbred (RI) strains of mice. The bone quality parameters studied were bone mineralization, microhardness, architecture, and connectivity. Previous studies have demonstrated considerable variability in bone density, biomechanical properties, and microstructure among inbred strains of mice. In particular, C3H/HeJ (C3H) mice exhibit thicker femoral and vertebral cortices and fewer trabeculae in the vertebral body compared with C57BL/6J (B6) mice, despite having similar vertebral bone strength. A set of RI mouse strains has been generated from B6 and C3H (denoted BXH) in an attempt to isolate genetic regulation of numerous traits, including bone. The objective of this study was to investigate relationships among bone quality and bone strength in femurs and vertebrae among BXH RI mice. The study involved 11 BXH RI strains of female mice (n = 5−7) as well as the B6 and C3H progenitor strains. Parameters contributing to bone quality were evaluated, including BMD, bone mineralization, microhardness, architecture, and connectivity. There was a strong correlation between femoral and vertebral BMD in all strains (P < 0.001) except in BXH-9 and -10 (P < 0.001). Within the vertebrae, cortical bone was more mineralized than trabecular bone, and a strong correlation existed between the two (P < 0.001). However, cortical microhardness did not differ from trabecular microhardness. Cortical bone was more mineralized in the femur than in the vertebrae and significantly harder, by 30%. There was a wide range in trabecular connectivity, architecture, and femur geometry among BXH RI strains. BMD explained 43% of vertebral bone strength but only 11% of femoral bone strength. Trabecular connectivity explained an additional 8% of vertebral strength, while mineralization and femur geometry explained 7% and 50% of femoral strength, respectively. Different bone quality parameters had varying influences on bone mechanical properties, depending on bone site. BMD may play a larger role in explaining bone strength in the vertebrae than in the femur. Moreover, cortical bone in the femur is harder than in vertebrae. The control of cortical bone material properties may be site-dependent.     

Hip Fractures and the Contribution of Cortical Versus Trabecular Bone to Femoral Neck Strength

Osteoporotic fractures are caused by both cortical thinning and trabecular bone loss. Both are seen to be important for bone fragility. The relative contributions of cortical versus trabecular bone have not been established. The aim of this study was to test the contribution of cortical versus trabecular bone to femoral neck stability in bone strength. In one femur from each pair of 18 human cadaver femurs (5 female; 4 male), trabecular bone was completely removed from the femoral neck, providing one bone with intact and the other without any trabecular structure in the femoral neck. Geometrical, X‐ray, and DXA measurements were carried out before biomechanical testing (forces to fracture). Femoral necks were osteotomized, slices were analyzed for cross‐sectional area (CSA) and cross‐sectional moment of inertia (CSMI), and results were compared with biomechanical testing data. Differences between forces needed to fracture excavated and intact femurs (ΔF/F mean) was 7.0% on the average (range, 4.6–17.3%). CSA of removed spongiosa did not correlate with difference of fracture load (ΔF/F mean), nor did BMD. The relative contribution of trabecular versus cortical bone in respect to bone strength in the femoral neck seems to be marginal and seems to explain the subordinate role of trabecular bone and its changes in fracture risk and the effects of treatment options in preventing fractures.     

Vitamin D Deficiency and Ovariectomy Reduced the Strength of the Femoral Neck in Rats

Considerable sample to sample variability in deoxypyridinoline crosslink/creatinine (Dpd/CREAT) ratio values was confirmed when twice-weekly sampling for 77 days was performed in C57 mice. Analysis of samples from individual mice indicated that, in the majority of mice in a given group (54–67%), phasic changes occurred with periodic peaks as much as 4–5 times basal values. Alignment of peaks in the individual time courses of mice revealed a clear cyclic crosslink production (periodicity 12–14 days) for the population, although not all mice gave a peak in every case. Ovariectomy (OVX) (compared to sham-operated mice) increased mean values of crosslink production by either C57 or C57 × 129 F1 mice from about 10 days after operation with highest values between 21 and 35 days, and then a decrease in the difference between sham and OVX, particularly in C57 mice. Analysis of both time courses for individual mice and distribution curves of the data from groups of mice indicated that OVX consistently increased basal crosslink values (6–9 ratio units) with phasic peak values superimposed. The peak alignment analysis reinforced this observation. The influence of the background variability was avoided by pooling data over one cycle time and the use of nonparametic statistics, and the effect of OVX was analyzed in several strains of mice. Crosslink levels and phasic production declined with age but were also influenced by manipulation of the mice, suggesting that experimental conditions should be rigorously controlled when this urinary crosslink measure is used as a parameter in mouse models of OVX-induced osteoporosis.     

Genetic Regulation of Vitamin D Levels

Vitamin D plays several roles in the body, influencing bone health as well as serum calcium and phosphate levels. Further, vitamin D may modify immune function, cell proliferation, differentiation, and apoptosis. Vitamin D deficiency has been associated with numerous health outcomes, including bone disease, cancer, autoimmune disease, infectious disease, type 1 and type 2 diabetes, hypertension, and heart disease, although it is unclear whether or not these associations are causal. Various twin and family studies have demonstrated moderate to high heritability for circulating vitamin D levels. Accordingly, many studies have investigated the genetic determinants of this hormone. Recent advances in the methodology of large-scale genetic association studies, including coordinated international collaboration, have identified associations of CG, DHCR1, CYP2R1, VDR, and CYP24A1 with serum levels of vitamin D. Here, we review the genetic determinants of vitamin D levels by focusing on new findings arising from candidate gene and genomewide association studies.     

Age-, Site-, and Sex-Specific Normative Centile Curves for HR-pQCT-Derived Microarchitectural and Bone Strength Parameters in a Chinese Mainland Population

High-resolution peripheral quantitative computed tomography (HR-pQCT) is an advanced 3D imaging technology that has the potential to contribute to fracture risk assessment and early diagnosis of osteoporosis. However, to date no studies have sought to establish normative reference ranges for HR-pQCT measures among individuals from the Chinese mainland, significantly restricting its use. In this study, we collected HR-pQCT scans from 863 healthy Chinese men and women aged 20 to 80 years using the latest-generation scanner (Scanco XtremeCT II, Scanco Medical AG, Brüttisellen, Switzerland). Parameters including volumetric bone mineral density, bone geometry, bone microarchitecture, and bone strength were evaluated. Age-, site-, and sex-specific centile curves were established using generalized additive models for location, scale, and shape with age as the only explanatory variable. Based on established models, age-related variations for different parameters were also quantified. For clinical purposes, the expected values of HR-pQCT parameters for a defined age and a defined percentile or Z-score were provided. We found that the majority of trabecular and bone strength parameters reached their peak at 20 years of age, regardless of sex and site, then declined steadily thereafter. However, most of the cortical bone loss was observed after the age of 50 years. Among the measures, cortical porosity changed most dramatically, and overall, changes were more notable at the radius than the tibia and among women compared with men. Establishing such normative HR-pQCT reference data will provide an important basis for clinical and research applications in mainland China aimed at elucidating microstructural bone damage driven by different disease states or nutritional status. © 2020 American Society for Bone and Mineral Research.