Skeletal Integrity Research Articles

Vitamin D and bone health

Throughout the life-cycle, the skeleton requires optimum development and maintenance of its integrity to prevent fracture. Bones break because the loads placed upon them exceed the ability of the bone to absorb the energy involved. It is now estimated that 1:3 women and 1:12 men over the age of 55 years will suffer from osteoporosis in their lifetime and in the UK, at a cost in excess of £1.7 billion per annum to the exchequer. The pathogenesis of osteoporosis is multi-factorial. Both the development of peak bone mass and the rate of bone loss are determined by key endogenous and exogenous factors. Calcium supplements appear to be effective in reducing bone loss in late menopausal women (>5 years post-menopause), particularly in those with low habitual calcium intake (< 400mg/d). In younger postmenopausal women, who are not vitamin D deficient, vitamin D supplementation has little effect on BMD. However, vitamin D and calcium supplementation studies have been shown to reduce fracture rates in the institutionalized elderly but there remains controversy as to whether supplementation is effective in reducing fracture in free-living populations. Re-defining vitamin D requirements in the UK is urgently needed since there is evidence of extensive hypovitaminosis D in the UK. Low vitamin D status is associated with an increased risk of falling and a variety of other health outcomes and is an area that requires urgent attention. The role of other micronutrients on bone remains to be fully defined, although there are promising data in the literature for a clear link between vitamin K nutrition, dietary protein and dietary alkali on skeletal integrity including fracture reduction.

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye.

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

Insight into the transgenerational effect of benzo[a]pyrene on bone formation in a teleost fish (Oryzias latipes)

Recent cross-generational studies in teleost fish have raised the awareness that high levels of benzo[a]pyrene (BaP) could affect skeletal integrity in the directly exposed F0 and their F1–F2. However, no further details were provided about the causes for abnormalities on the molecular and cellular level and the persistence of such sub-organismal impairments at the transgenerational scale (beyond F2). Adult Oryzias latipes were exposed to 1μg/L BaP for 21days. The F1–F3 were examined for skeletal deformities, histopathological alterations of vertebral bodies and differential expression of key genes of bone metabolism. Significant increase of dorsal–ventral vertebral compression was evident in ancestrally exposed larvae. Histopathological analysis revealed abnormal loss of notochord sheath, a lack of notochord epithelial integrity, reduced bone tissue and decreased osteoblast abundance. A significant downregulation of ATF4 and/or osterix and a high biological variability of COL10, coupled with a significant deregulation of SOX9a/b in the F1–F3 suggest that ancestral BaP exposure most likely perturbed chordoblasts, chondroblast and osteoblast differentiation, resulting in defective notochord sheath repair and rendering the vertebral column more vulnerable to compression. The present findings provide novel molecular and cellular insights into BaP-induced transgenerational bone impairment in the unexposed F3. From the ecological risk assessment perspective, BaP needs to be regarded as a transgenerational skeletal toxicant, which exerts a far-reaching impact on fish survival and fitness. Given that basic mechanisms of cartilage/bone formation are conserved between medaka and mammals, the results may also shed light on the potential transgenerational effect of BaP on the genesis of skeletal diseases in humans.

Homeostasis-based aging model for trabecular changes and its correlation with age-matched bone mineral densities and radiographs

PurposeThis paper aims (1) to propose a novel bone adaptation model for age-related trabecular changes by adopting two implicit parameters in optimization, (2) to compare the simulated bone volume fraction (BV/TV) with the reported bone mineral density (BMD), and (3) to review the simulated trabecular architectures with the age-matched radiographs. Materials and methodsThe proposed model simulated the trabecular changes for an age span of 32–80 years. Quantitative comparison was conducted in terms of BMD and other morphometric indices. Then, two radiologists scored the simulated trabecular architectures using the age-matched radiographs. This protocol was approved by the hospital institutional review board. ResultsThe simulated BV/TV was well correlated with BMD reported in the literature (R2=0.855; p<0.05). In comparison with age-matched radiographs, the consensus scores of agreement of the trabeculae were higher in age groups over the 50s, and the means of the Ward's triangle areas were strongly correlated with those in the age-matched radiographs (R2=0.982; p<0.05). ConclusionThe proposed model could reflect the targeted trabecular changes in proximal femur with age. With further follow-up measurements, this research would contribute to the development of patient-specific models that assist radiologists in predicting skeletal integrity with aging.

Growth hormone replacement therapy regulates microRNA-29a and targets involved in insulin resistance.

Replacement of growth hormone (GH) in patients suffering from GH deficiency (GHD) offers clinical benefits on body composition, exercise capacity, and skeletal integrity. However, GH replacement therapy (GHRT) is also associated with insulin resistance, but the mechanisms are incompletely understood. We demonstrate that in GH-deficient mice (growth hormone-releasing hormone receptor (Ghrhr)lit/lit), insulin resistance after GHRT involves the upregulation of the extracellular matrix (ECM) and the downregulation of microRNA miR-29a in skeletal muscle. Based on RNA deep sequencing of skeletal muscle from GH-treated Ghrhrlit/lit mice, we identified several upregulated genes as predicted miR-29a targets that are negative regulators of insulin signaling or profibrotic/proinflammatory components of the ECM. Using gain- and loss-of-function studies, five of these genes were confirmed as endogenous targets of miR-29a in human myotubes (PTEN, COL3A1, FSTL1, SERPINH1, SPARC). In addition, in human myotubes, IGF1, but not GH, downregulated miR-29a expression and upregulated COL3A1. These results were confirmed in a group of GH-deficient patients after 4 months of GHRT. Serum IGF1 increased, skeletal muscle miR-29a decreased, and miR-29a targets were upregulated in patients with a reduced insulin response (homeostatic model assessment of insulin resistance (HOMA-IR)) after GHRT. We conclude that miR-29a could contribute to the metabolic response of muscle tissue to GHRT by regulating ECM components and PTEN. miR-29a and its targets might be valuable biomarkers for muscle metabolism following GH replacement.Key messagesGHRT most significantly affects the ECM cluster in skeletal muscle from mice.GHRT downregulates miR-29a and upregulates miR-29a targets in skeletal muscle from mice.PTEN, COL3A1, FSTL1, SERPINH1, and SPARC are endogenous miR-29a targets in human myotubes.IGF1 decreases miR-29a levels in human myotubes.miR-29a and its targets are regulated during GHRT in skeletal muscle from humans.Electronic supplementary materialThe online version of this article (doi:10.1007/s00109-015-1322-y) contains supplementary material, which is available to authorized users.

Age-related T2 changes in hindlimb muscles of mdx mice.

Magnetic resonance imaging (MRI) was used to monitor changes in the transverse relaxation time constant (T2) in lower hindlimb muscles of mdx mice at different ages. Young (5 weeks), adult (44 weeks), and old mdx (96 weeks), and age-matched control mice were studied. Young mdx mice were imaged longitudinally, whereas adult and old mdx mice were imaged at a single time-point. Mean muscle T2 and percent of pixels with elevated T2 were significantly different between mdx and control mice at all ages. In young mdx mice, mean muscle T2 peaked at 7-8 weeks and declined at 9-11 weeks. In old mdx mice, mean muscle T2 was decreased compared with young and adult mice, which could be attributed to fibrosis. MRI captured longitudinal changes in skeletal muscle integrity of mdx mice. This information will be valuable for pre-clinical testing of potential therapeutic interventions for muscular dystrophy.

MicroRNA-29a mitigates glucocorticoid induction of bone loss and fatty marrow by rescuing Runx2 acetylation

Glucocorticoid treatment reportedly increases the morbidity of osteoporotic or osteonecrotic disorders. Exacerbated bone acquisition and escalated marrow adipogenesis are prominent pathological features of glucocorticoid-mediated skeletal disorders. MicroRNAs reportedly modulate tissue metabolism and remodeling. This study was undertaken to investigate the biological roles of microRNA-29a (miR-29a) in skeletal and fat metabolism in the pathogenesis of glucocorticoid-induced osteoporosis. Transgenic mice overexpressing miR-29a precursor or wild-type mice were given methylprednisolone. Bone mass, microarchitecture and histology were assessed by dual energy X-ray absorptiometry, μCT and histomorphometry. Differential gene expression and signaling components were delineated by quantitative RT-PCR and immunoblotting. Glucocorticoid treatment accelerated bone loss and marrow fat accumulation in association with decreased miR-29a expression. The miR-29a transgenic mice had high bone mineral density, trabecular microarchitecture and cortical thickness. miR-29a overexpression mitigated the glucocorticoid-induced impediment of bone mass, skeletal microstructure integrity and mineralization reaction and attenuated fatty marrow histopathology. Ex vivo, miR-29a increased osteogenic differentiation capacity and alleviated the glucocorticoid-induced promotion of adipocyte formation in primary bone-marrow mesenchymal progenitor cell cultures. Through inhibition of histone deacetylase 4 (HDAC4) expression, miR-29a restored acetylated Runx2 and β-catenin abundances and reduced RANKL, leptin and glucocorticoid receptor expression in glucocorticoid-mediated osteoporosis bone tissues. Taken together, glucocorticoid suppression of miR-29a signaling disturbed the balances between osteogenic and adipogenic activities, and thereby interrupted bone formation and skeletal homeostasis. miR-29a inhibition of HDAC4 stabilized the acetylation state of Runx2 and β-catenin that ameliorated the detrimental effects of glucocorticoid on mineralization and lipogenesis reactions in bone tissue microenvironments. This study highlighted emerging skeletal-anabolic actions of miR-29a signaling in the progression of glucocorticoid-induced bone tissue destruction. Sustaining miR-29a actions is beneficial in protecting against glucocorticoid-mediated osteoporosis.

Denosumab and teriparatide transitions in postmenopausal osteoporosis (the DATA-Switch study): extension of a randomised controlled trial

Unlike most chronic diseases, osteoporosis treatments are generally limited to a single drug at a fixed dose and frequency. Nonetheless, no approved therapy is able to restore skeletal integrity in most osteoporotic patients and the long-term use of osteoporosis drugs is controversial. Thus, many patients are treated with the sequential use of two or more therapies. The DATA study showed that combined teriparatide and denosumab increased bone mineral density more than either drug alone. Discontinuing teriparatide and denosumab, however, results in rapidly declining bone mineral density. In this DATA-Switch study, we aimed to assess the changes in bone mineral density in postmenopausal osteoporotic women who transitioned between treatments. This randomised controlled trial (DATA-Switch) is a preplanned extension of the denosumab and teriparatide administration study (DATA), in which 94 postmenopausal osteoporotic women were randomly assigned to receive 24 months of teriparatide (20 mg daily), denosumab (60 mg every 6 months), or both drugs. In DATA-Switch, women originally assigned to teriparatide received denosumab (teriparatide to denosumab group), those originally assigned to denosumab received teriparatide (denosumab to teriparatide group), and those originally assigned to both received an additional 24 months of denosumab alone (combination to denosumab group). Bone mineral density at the spine, hip, and wrist were measured 6 months, 12 months, 18 months, and 24 months after the drug transitions as were biochemical markers of bone turnover. The primary endpoint was the percent change in posterior-anterior spine bone mineral density over 4 years. Between-group changes were assessed by one-way analysis of variance in our modified intention-to-treat population. This study is registered with ClinicalTrials.gov, number NCT00926380. Between Sept 27, 2011, and Jan 28, 2013, eligible women from the DATA study were enrolled into DATA-Switch. Of 83 potential enrollees from the DATA study, 77 completed at least one post-baseline visit. After 48 months, the primary outcome of mean spine bone mineral density increased by 18·3% (95% CI 14·9-21·8) in 27 women in the teriparatide to denosumab group, 14·0% (10·9-17·2) in 27 women the denosumab to teriparatide group, and 16·0% (14·0-18·0) in 23 women in the combination to denosumab group, although this increase did not differ significantly between groups (for between-group comparisons, p=0·13 for the teriparatide to denosumab group vs the denosumab to teriparatide group, p=0·30 for the teriparatide to denosumab group vs the combination to denosumab group, and p=0·41 for the denosumab to teriparatide group vs the combination to denosumab group). For the bone mineral density secondary outcomes, total hip bone mineral density increased more in the teriparatide to denosumab group (6·6% [95% CI 5·3-7·9]) than in the denosumab to teriparatide group (2·8% [1·3-4·2], p=0·0002), but had the greatest increase in the combination to denosumab group (8·6% [7·1-10·0]; p=0·0446 vs the teriparatide to denosumab group, p<0·0001 vs the denosumab to teriparatide group). Similarly, femoral neck bone mineral density increased more in the teriparatide to denosumab group (8·3% [95% CI 6·1-10·5]) and the combination to denosumab group (9·1% [6·1-12·0]) than in the denosumab to teriparatide group (4·9% [2·2-7·5]; p=0·0447 for teriparatide to denosumab vs denosumab to teriparatide, p=0·0336 for combination to denosumab vs denosumab to teriparatide). Differences between the combination to denosumab group and the teriparatide to denosumab group did not differ significantly (p=0·67). After 48 months, radius bone mineral density was unchanged in the teriparatide to denosumab group (0·0% [95% CI -1·3 to 1·4]), whereas it decreased by -1·8% (-5·0 to 1·3) in the denosumab to teriparatide group, and increased by 2·8% (1·2-4·4) in the combination to denosumab group (p=0·0075 for the teriparatide to denosumab group vs the combination to denosumab group; p=0·0099 for the denosumab to teriparatide group vs the combination to denosumab group). One participant in the denosumab to teriparatide group had nephrolithiasis, classified as being possibly related to treatment. In postmenopausal osteoporotic women switching from teriparatide to denosumab, bone mineral density continued to increase, whereas switching from denosumab to teriparatide results in progressive or transient bone loss. These results should be considered when choosing the initial and subsequent management of postmenopausal osteoporotic patients. Amgen, Eli Lilly, and National Institutes of Health.

Hypothesis: Coupling between Resorption and Formation in Cancellous bone Remodeling is a Mechanically Controlled Event.

Coupling is the process that links bone resorption to formation in a temporally and spatially coordinated manner within the remodeling cycle. In order to maintain skeletal integrity, it is of crucial importance that the amount of bone resorbed matches the amount of newly formed bone in each remodeling site. Although a number of different explanatory models have been developed, the mechanisms that couple bone resorption and formation in bone remodeling are still a matter of controversy. Here, I propose a model in which coupling is achieved by biomechanical strain sensed by osteocytes within the newly built bone package. In this model, the resorption cavity created by osteoclasts results in mechanical weakening of the structural element, and, thus, in increased strain under constant loading conditions. Subsequent bone formation is initiated by strain-sensitive osteocytes in the underlying bone matrix. After osteoblastic bone formation has started, the newly built osteocyte–osteoblast network detects strain. Once the mechanical strain within the newly built bone structural unit falls below a certain threshold, bone formation stops. In this biomechanical strain-driven model, osteoblasts do not need to “know” how much bone was previously resorbed in a given site. In addition, this model does not require the transfer of any information from bone-resorbing osteoclasts to bone-forming osteoblasts, because biomechanical strain “guides” osteoblasts through their job of re-filling the resorption cavity.

Immediate effects of retinoic acid on gene expression in primary murine osteoblasts.

Consistent with clinical observations demonstrating that hypervitaminosis A is associated with increased skeletal fracture risk, we have previously found that dietary retinol deprivation partially corrects the bone mineralization defects in a mouse model of X-linked hypophosphatemic rickets. That retinol-dependent signaling pathways impact the skeleton is further supported by various findings demonstrating a negative influence of retinoic acid (RA) on bone-forming osteoblasts. We hypothesized that RA would directly regulate the expression of specific target genes in osteoblasts, and we aimed to identify these by genome-wide expression analyses. Here we show that high dietary retinol intake in mice causes low bone mass associated with increased osteoclastogenesis and decreased osteoblastogenesis, but intact bone matrix mineralization. We additionally found that short-term treatment of primary osteoblasts with RA causes a rapid induction of specific genes involved in either retinol-dependent signaling (i.e. Rara, Crabp2) or skeletal remodeling (i.e. Twist2, Tnfsf11). In contrast, neither expression of established osteoblast differentiation markers nor the proliferation rate was immediately affected by RA administration. Collectively, our data suggest that the negative effects of vitamin A on skeletal integrity are explainable by an immediate influence of RA signaling on specific genes in osteoblasts that in turn influence bone remodeling.

The mammalian lectin galectin-8 induces RANKL expression, osteoclastogenesis, and bone mass reduction in mice.

Skeletal integrity is maintained by the co-ordinated activity of osteoblasts, the bone-forming cells, and osteoclasts, the bone-resorbing cells. In this study, we show that mice overexpressing galectin-8, a secreted mammalian lectin of the galectins family, exhibit accelerated osteoclasts activity and bone turnover, which culminates in reduced bone mass, similar to cases of postmenopausal osteoporosis and cancerous osteolysis. This phenotype can be attributed to a direct action of galectin-8 on primary cultures of osteoblasts that secrete the osteoclastogenic factor RANKL upon binding of galectin-8. This results in enhanced differentiation into osteoclasts of the bone marrow cells co-cultured with galectin-8-treated osteoblasts. Secretion of RANKL by galectin-8-treated osteoblasts can be attributed to binding of galectin-8 to receptor complexes that positively (uPAR and MRC2) and negatively (LRP1) regulate galectin-8 function. Our findings identify galectins as new players in osteoclastogenesis and bone remodeling, and highlight a potential regulation of bone mass by animal lectins.

Sexual Dimorphism in MAPK-Activated Protein Kinase-2 (MK2) Regulation of RANKL-Induced Osteoclastogenesis in Osteoclast Progenitor Subpopulations

Osteoclasts (OCs) are bone-resorptive cells critical for maintaining skeletal integrity through coupled bone turnover. OC differentiation and activation requires receptor activator of NF-kB ligand (RANKL) signaling through the p38 MAPK pathway. However the role of the p38 MAPK substrate, MAPK-activated protein kinase 2 (MK2), is not clearly delineated. Within the bone marrow exists a specific subpopulation of defined osteoclast progenitor cells (dOCPs) with surface expression of B220-Gr1-CD11blo/-CD115+ (dOCPlo/-). In this study, we isolated dOCPs from male and female mice to determine sex-specific effects of MK2 signaling in osteoclastogenesis (OCgen). Male Mk2-/- mice display an increase in the dOCPlo cell population when compared to Mk2+/+ mice, while female Mk2-/- and Mk2+/+ mice exhibit no difference. Defined OCPs from male and female Mk2+/+ and Mk2-/- bone marrow were treated with macrophage colony stimulation factor (M-CSF) and RANKL cytokines to promote OCgen. RANKL treatment of dOCPlo cells stimulated p38 and MK2 phosphorylation. Tartrate-resistant acid phosphatase (TRAP) assays were used to quantify OC number, size, and TRAP enzyme activity post-RANKL stimulation. MK2 signaling was critical for male dOCPlo OCgen, yet MK2 signaling regulated OCgen from female dOCP- and CD11bhi subpopulations as well. The functional gene, Ctsk, was attenuated in both male and female Mk2-/- dOCPlo-derived OCs. Conversely, MK2 signaling was only critical for gene expression of pre-OC fusion genes, Oc-stamp andTm7sf4, in male OCgen. Therefore, these data suggest there is a sexual dimorphism in MK2 signaling of OCP subpopulations.

Duck gait: Relationship to hip angle, bone ash, bone density, and morphology

The rapid growth meat birds, including ducks, undergo requires skeletal integrity; however, fast growth may not be conducive to adequate bone structure. A relationship likely exists between skeletal changes and duck mobility. Reduced mobility in meat ducks may have impacts on welfare and production. This study examined the relationships among gait score, bone parameters, and hip angle. Commercial Pekin ducks, ages 14 d (n = 100), 21 d (n = 100), and 32 d (n = 100) were weighed and gait scored with a 3-point gait score system by an observer as they walked over a Tekscan gait analysis system. Gait was scored as GS0, GS1, or GS2 with a score of GS0 defined as good walking ability and a score of GS2 as poorest walking ability. Ducks were humanely euthanized, full body scanned using quantitative computed tomography (QCT), and the right femur and tibia were extracted. Leg bones were cleaned, measured, fat extracted, and ashed. QCT scans were rendered to create computerized 3D models where pelvic hip angles and bone density were measured. Statistical analysis was conducted using PROC MIXED with age and gait score in the model. Body weight increased with age, but within an age, body weight decreased as walking ability became worse (P < 0.01). As expected, linear increases in tibia and femur bone width and length were observed as the ducks aged (P < 0.01). Right and left hip angle increased with duck age (P < 0.01). Additionally, ducks with a GS2 had wider hip angles opposed to ducks with a GS0 (P < 0.01). Bone density increased linearly with both age and gait score (P < 0.05). Femur ash content was lowest in 32-day-old ducks and ducks with GS1 and GS2 (P < 0.0001). Tibia ash content increased with age, but decreased as gait score increased (P < 0.001). The observation that right hip angle changed with gait scores merits further investigation into the relationship between duck mobility and skeletal changes during growth.

Interactive effect of vitamin D and strontium on performance and bone composition in broiler chickens

Vitamin D and strontium have a close physiological interrelationship in respect to mineral homeostasis and skeletal integrity which is not well defined. To elucidate this relationship a total of 960-day-old male broiler chickens were fed for 40 days one of eight diets comprising of two levels of vitamin D3 (5000 or 10,000IU/kg) and four levels of strontium (0, 400, 800 or 1200mg/kg). Bodyweight gain, feed intake, bone composition and ileal nutrient digestibilities were analysed.All treatments improved BWG in excess of breed Ross 308 expectations and there was no statistical difference in overall performance between treatments. In the starter period, higher vitamin D3 interacted with the highest level of strontium supplementation to reduce BWG (P<0.05) and increase FCR (P<0.05). There was no change in percentage tibia ash content between treatments. Increasing concentrations of dietary strontium resulted in a reduced Ca content (P<0.01) and an increase in strontium (P<0.0001), Mg (P<0.01) and K content (P<0.05) of bone. The ileal digestibility of sodium was reduced in birds fed higher vitamin D3 with the highest level of strontium supplementation (P<0.05).There was no improvement in BWG or FCR by day 40 with higher levels of vitamin D3 and various levels of supplemental strontium however performance was negatively affected in the starter phase. Strontium supplementation in the presence of additional vitamin D3 altered tibia bone composition and reduced Na ileal digestibility. The ratio of 30:1 for calcium to sodium in bone was maintained across all treatments as did the total percentage content of calcium, sodium, strontium, potassium and magnesium at 43%. The higher level of vitamin D3 (10,000IU/kg) with no added strontium (0mg/kg) produced the heaviest BWG per bird by day 40 (3157g) however it was not statistically significant. The higher level of vitamin D3 significantly increased mortality. The feeding of lower doses of strontium and vitamin D3, particularly in the starter phase, requires further investigation.

The effects of 1α, 25-dihydroxyvitamin D3 and transforming growth factor-β3 on bone development in an ex vivo organotypic culture system of embryonic chick femora.

Transforming growth factor-beta3 (TGF-β3) and 1α,25-dihydroxyvitamin D3 (1α,25 (OH) 2D3) are essential factors in chondrogenesis and osteogenesis respectively. These factors also play a fundamental role in the developmental processes and the maintenance of skeletal integrity, but their respective direct effects on these processes are not fully understood. Using an organotypic bone rudiment culture system the current study has examined the direct roles the osteotropic factors 1α,25 (OH)2D3 and TGF-β3 exert on the development and modulation of the three dimensional structure of the embryonic femur. Isolated embryonic chick femurs (E11) were organotypically cultured for 10 days in basal media, or basal media supplemented with either 1α,25 (OH) 2D3 (25 nM) or TGF-β3 (5 ng/mL & 15 ng/mL). Analyses of the femurs were undertaken using micro-computed tomography (μCT), histology and immunohistochemistry. 1α,25 (OH)2D3 supplemented cultures enhanced osteogenesis directly in the developing femurs with elevated levels of osteogenic markers such as type 1 collagen. In marked contrast organotypic femur cultures supplemented with TGF-β3 (5 ng/mL & 15 ng/mL) demonstrated enhanced chondrogenesis with a reduction in osteogenesis. These studies demonstrate the efficacy of the ex vivo organotypic embryonic femur culture employed to elucidate the direct roles of these molecules, 1α,25 (OH) 2D3 and TGF-β3 on the structural development of embryonic bone within a three dimensional framework. We conclude that 1α,25(OH)2D and TGF-β3 modify directly the various cell populations in bone rudiment organotypic cultures effecting tissue metabolism resulting in significant changes in embryonic bone growth and modulation. Understanding the roles of osteotropic agents in the process of skeletal development is integral to developing new strategies for the recapitulation of bone tissue in later life.

Two-Year Changes in Bone Density After Roux-en-Y Gastric Bypass Surgery

Bariatric surgery is increasingly popular but may lead to metabolic bone disease. The objective was to determine the rate of bone loss in the 24 months after Roux-en-Y gastric bypass. This was a prospective cohort study conducted at an academic medical center. The participants were adults with severe obesity, including 30 adults undergoing gastric bypass and 20 nonsurgical controls. We measured bone mineral density (BMD) at the lumbar spine and proximal femur by quantitative computed tomography (QCT) and dual-energy x-ray absorptiometry at 0, 12, and 24 months. BMD and bone microarchitecture were also assessed by high-resolution peripheral QCT, and estimated bone strength was calculated using microfinite element analysis. Weight loss plateaued 6 months after gastric bypass but remained greater than controls at 24 months (-37 ± 3 vs -5 ± 3 kg [ mean ± SEM]; P < .001). At 24 months, BMD was 5-7% lower at the spine and 6-10% lower at the hip in subjects who underwent gastric bypass compared with nonsurgical controls, as assessed by QCT and dual-energy x-ray absorptiometry (P < .001 for all). Despite significant bone loss, average T-scores remained in the normal range 24 months after gastric bypass. Cortical and trabecular BMD and microarchitecture at the distal radius and tibia deteriorated in the gastric bypass group throughout the 24 months, such that estimated bone strength was 9% lower than controls. The decline in BMD persisted beyond the first year, with rates of bone loss exceeding controls throughout the second year at all skeletal sites. Mean serum calcium, 25(OH)-vitamin D, and PTH were maintained within the normal range in both groups. Substantial bone loss occurs throughout the 24 months after gastric bypass despite weight stability in the second year. Although the benefits of gastric bypass surgery are well established, the potential for adverse effects on skeletal integrity remains an important concern.

The effect of locomotion on the mobilization of minerals from the maternal skeleton.

Bone is a dynamic tissue from which minerals are deposited or withdrawn according to the body’s demand. During late pregnancy and lactation, female mammals mobilize mineral from bone to support the ossification of offspring skeleton(s). Conversely, in response to mechanical loading, minerals are deposited in bone enabling it to develop a stronger architecture. Despite their central importance to reproductive performance and skeletal integrity, the interactions between these potentially opposing forces remains poorly understood. It is possible that inter-individual differences in the loading imposed by different forms of locomotion may alter the amount of mineral mobilized during reproduction. Here, the impact of vertical versus horizontal locomotion on bone mobilization was examined during reproduction in the laboratory mouse. The vertical, or climbing, group had access to a 60-cm tower, increasing strain on their appendicular skeleton. The horizontal, or tunnel, group had access to a 100-cm tunnel, which encouraged movements within the horizontal plane. Form of locomotion did not impact the amount of bone females mobilized during reproduction or the amount of mineral females deposited in the litter, but maternal bone architecture differed between groups. The climbing group displayed more trabeculae than the tunnel group, whereas the tunnel group displayed greater cortical bone mineral density mid-shaft. Interestingly, pups born to mothers in the climbing group had a higher concentration of total body calcium at 16 days than pups of mothers in the tunnel group. As maternal total body calcium composition and the amount of calcium invested in the full litter were not different between groups, the difference in the relative calcium content of pups between groups is not suspected to reflect difference in mineral allocation. Future research should consider the impact of maternal activity on the efficiency of offspring skeletal ossification via hormones and other bioactive factors transferred in utero and in milk.

Monitoring in vivo (re)modeling: A computational approach using 4D microCT data to quantify bone surface movements

Bone undergoes continual damage repair and structural adaptation to changing external loads with the aim of maintaining skeletal integrity throughout life. The ability to monitor bone (re)modeling would allow for a better understanding in how various pathologies and interventions affect bone turnover and subsequent bone strength. To date, however, current methods to monitor bone (re)modeling over time and in space are limited. We propose a novel method to visualize and quantify bone turnover, based on in vivo microCT imaging and a 4D computational approach. By in vivo tracking of spatially correlated formation and resorption sites over time it classifies bone restructuring into (re)modeling sequences, the spatially and temporally linked sequences of formation, resorption and quiescent periods on the bone surface. The microCT based method was validated using experimental data from an in vivo mouse tibial loading model and ex vivo data of the mouse tibia. In this application, the method allows the visualization of time-resolved cortical (re)modeling and the quantification of short-term and long-term modeling on the endocortical and periosteal surface at the mid-diaphysis of loaded and control mice tibiae. Both short-term and long-term modeling processes, independent formation and resorption events, could be monitored and modeling (spatially not correlated formation and resorption) and remodeling (resorption followed by new formation at the same site) could be distinguished on the bone surface. This novel method that combines in vivo microCT with a computational approach is a powerful tool to monitor bone turnover in animal models now and is waiting to be applied to human patients in the near future.

Characterization of damage mechanisms associated with reference point indentation in human bone

Measurement of bone mineral density (BMD) is the clinical gold standard in cases of compromised skeletal integrity, such as with osteoporosis. While BMD is a useful measurement to index skeletal health, it is also limited since it cannot directly assess any mechanical properties. The ability to directly assess mechanical properties of bone tissue would be clinically important. Reference point indentation (RPI) is a technology that has been designed to try and achieve this goal. While RPI has been shown to detect altered bone tissue properties, the underlying physical mechanism of these measurements has not been characterized. Thus, we designed a study whereby the contribution of (1) test cycle number and (2) test load level to RPI test-induced sub-surface damage was characterized and quantified. Standardized specimens were prepared from cadaveric human tibiae (n=6), such that 12 replicates of each testing condition could be carried out. A custom rig was fabricated to accurately position and map indentation sites. One set of tests was carried out with 1, 5, 10, 15 and 20cycles (Max Load: 8N, Freq: 2Hz), and a second set of tests was carried out with Load levels of 2, 4, 6, 8 or 10N (Cycle number: 20, Freq: 2Hz). The RPI parameter Loading Slope (LS) was cycle dependent at 5, 10, 15 and 20cycles (p<0.05). First Cycle Indentation Distance (ID 1st), Total Indentation Distance (TID), Mean Energy Dissipation (ED), First Cycle Unloading Slope (US 1st), Mean Unloading Slope (US) and LS were significantly different at 6, 8 and 10N compared to 2N (p<0.05). From the histomorphometric measurements, damage zone span was significantly different after 5, 10, 15 and 20cycles compared with 1cycle while indent profile width and indent profile depth were significantly different at 10, 15 and 20cycles (p<0.05). With the load varying protocol, each of these parameters differed significantly at each increased load level (4, 6, 8, 10N) compared with the basal level of 2N (p<0.05). The damage area parameter in both protocols was significantly different from baseline at the three upper levels tested (i.e. 10, 15, 20cycles and 6, 8, 10N, in cycle and load variant protocols, respectively). Specimens were scanned by micro-computed tomography, which showed no material or microstructural differences between samples, and processed for histological analyses and damage quantification. Consistent microdamage patterns were present with evidence of damage via compaction at the indented regions. While damage in the direction of loading was established early, the damage area then increased radially with cycle number. These data help to further understand the physical manifestations of RPI parameters and will help to further facilitate its use as a clinical diagnostic tool.