Bone Mineral Composition Research Articles

7216 Absence Of Weight Gain In C57Bl/67 Female Mice On A High-Fat Diet Protects The Skeleton From Bone Loss

Abstract Disclosure: C. McAndrews: None. C. Chlebek: None. S. Costa: None. C.J. Rosen: None. Background: Obesity is associated with both systemic metabolic changes that increase the risk of comorbidities and reduced bone quality that increases the risk of fracture. The effects of high-fat diet consumption have been well studied in preclinical models where high-fat diet-feeding induces obesity, but there have been no studies to investigate the effects of high-fat diet-feeding in animals that do not become obese. Therefore, we characterized components of systemic metabolism and bone mineral content in non-obese C57Bl/6J mice that were fed a high-fat diet. Methods: Female C57Bl/6J mice aged 8 weeks received either a high-fat (60% kcal fat, n = 31) or control (10% kcal fat, n = 17) diet for 12 weeks. Body composition, areal bone mineral density (BMD) and bone mineral composition (BMC) were assessed after the feeding period using dual energy x-ray absorptiometry (Faxitron UltraFocus-DXA). Glucose (GTT) and insulin (ITT) tolerance testing were performed at the conclusion of the study. Changes in body weight were calculated as the difference between baseline and experiment end. High-fat diet-fed animals were classified as obese (HFD-O, n = 24) if they weighed more than 25 g or had greater than 25% body fat as evaluated via DXA, and were otherwise considered non-obese (HFD-NO, n = 7). In a small subset of animals, subcutaneous inguinal fat pads were harvested and weighed (n=5-7/group). Differences between groups were assessed with one-way ANOVAs and Tukey post-hoc analyses. Results: HFD-O mice weighed more than HFD-NO and control animals, whereas the body weights of HFD-NO mice were not significantly different from control (p < 0.001). Fat percent was increased in HFD-O mice compared to HFD-NO and control animals (p < 0.001). Subcutaneous inguinal fat mass was increased in both HFD-O and HFD-NO mice compared to control (p < 0.001). Fasting blood glucose did not significantly differ between groups. Glucose tolerance was worsened in both HFD-O and HFD-NO mice compared to control (p < 0.001), but there were no significant differences in insulin tolerance between groups. BMD was decreased in HFD-O mice compared to HFD-NO and control animals (p < 0.001). DISCUSSION: The consumption of a high-fat diet in mice induces deleterious metabolic changes irrespective of weight gain. However, high-fat diet-induced bone loss does not occur in the absence of weight gain. These findings suggest that certain adipokines derived from larger adipose depots may drive high-fat diet-induced bone loss and warrant further investigation. Acknowledgements: This work was supported by the 2023 Endocrine Society Research Experiences for Graduate and Medical Students (REGMS) Program. Presentation: 6/1/2024

Different dietary branched-chain amino acid ratios, crude protein levels, and protein sources can affect the growth performance and meat yield in broilers

Balanced ratios of branched-chain amino acids (BCAAs) can enhance chicken growth, immunity, and muscle synthesis. However, these ratios can be affected by changes in crude protein (CP) levels or the substitution of protein sources, leading to BCAA antagonism. This, in turn, can have a negative impact on chicken growth. In Experiment 1, a total of 960 0-d-old male Cobb 500 broilers were divided into 6 treatments with 8 replicates. Three different BCAA ratios were used in High or Low CP diets as follows: 1) Low Leu group (Low level of leucine with increased valine and isoleucine levels), 2) Med Leu group, and 3) High Leu group (High level of leucine with reduced valine and isoleucine levels) for a total of 6 diets. In Experiment 2, a total of 640 0-d-old male Cobb 500 broilers were divided into 4 treatments with 8 replicates. The four diets had either High or Low CP and one of two protein sources with the same medium levels of BCAAs: 1) the soybean meal (SBM) group, which had SBM as the main protein source (protein bound AA), and 2) the wheat middlings with non-bound AAs (WM+AA) group (non-bound AA), which had additional non-bound AAs to replace SBM. The High Leu diet had a negative effect on overall growth performance, carcass weight, breast muscle weight, and body mineral composition compared to the Low Leu and Med Leu groups, particularly in the High CP diet (P < 0.05). The SBM group showed increased growth performance, breast muscle weight, expression levels of genes promoting muscle growth, and improved bone mineral composition compared to the WM+AA group, and the High CP group intensified the negative effect of the WM+AA diet (P < 0.05). In summary, balanced BCAA ratios and SBM-based diets have positive effects on chicken growth and muscle accretion, whereas excessive leucine and non-bound AA levels in the diets may negatively affect growth performance and meat yield in chickens.

Effects of dietary free fatty acid content and degree of fat saturation on tibia bone properties of laying hens

Acid oils and fatty acid distillates are fat by-products of the refining process of edible oils and are characterized by their high proportion of free fatty acids (FFA). While lipids are essential in poultry diets, their chemical structure may interfere with calcium absorption. Therefore, this study investigated the effects of dietary FFA content and the degree of fat saturation on bone metabolism in commercial layers. For 15-wk, a total of 144 laying hens (19-wk-old) were randomly assigned to 8 treatments (6 replicates with 3 birds each), which were obtained by gradually replacing crude soybean oil (rich in unsaturated fatty acids [UFA]) with soybean acid oil (rich in UFA and FFA), or crude palm oil (rich in saturated fatty acids [SFA]) with palm fatty acid distillate (rich in SFA and FFA). Following a 2 × 4 factorial design, 4 UFA-rich and 4 SFA-rich diets were created with varying FFA content: 10, 20, 30, and 45%. Tibiae (6 birds/treatment) were collected at the end of the trial for the assessment of mineral composition, morphological properties, and mechanical characteristics. The data were analyzed using a 2-way ANOVA with the GLM procedure. Orthogonal polynomial contrasts were employed to determine the linear effect of increasing %FFA, with statistical significance set at P < 0.05. The degree of saturation was found to negatively impact on calcium and phosphorus bone content, with higher levels found in soybean-based diets (P < 0.001). A significant interaction was observed for medullary bone mineral content, showing a linear decrease as the dietary %FFA increased (P < 0.05) in palm diets. In contrast, morphological and mechanical bone traits, total ash content, and cortical bone mineral composition remained unaffected (P > 0.05). These results suggest that the degree of fat saturation exerts a greater impact than FFA content on bone mineral metabolism, supporting the commercial use of fat by-products rich in FFA in laying hen diets, at least during the early stages of the laying cycle.

PSV-19 Impact of precision feeding and bump feeding strategy during gestation on the performance of post-weaning pigs

Abstract The objective of this study was to evaluate the post-weaning growth performance of piglets from sows of parities 1 to 3 fed with 4 different feeding programs during gestation; Flat feeding (FF, constant concentration and feed intake), Bump feeding (BF, constant concentration and variable feed intake), precision feeding by parity (PFP, variable concentration and variable feed intake using the average weight per parity), individual precision feeding (PFI, variable concentration and variable feed intake using the weight of each sow at breeding). At weaning of each parity, five litters from each of the dietary treatments were selected and five average piglets from each of these litters were transferred to a nursery building with 20 pens. All pens received the same feeding program in 3 phases of 14 d each. Piglets were weighed at the beginning and end of each feeding phase. The quantity of feed was noted for each phase. At the end of phases 1 and 3, one piglet per pen was scanned on the DXA to assess its body lean, fat, and bone mineral composition. During phase 1, average daily gain (ADG) was greater for piglets from parity 1 sows than those from parity 2 and 3 while average daily feed intake (ADFI) and feed conversion (FC) were greater for piglets from parity 3 sows (P &amp;lt; 0.05; Table). Dietary treatments during gestation did not affect performances in phase 1. In phase 2, ADG and ADFI were greater while FC was decreased for piglets from sows of parity 2 and 3 (P&amp;lt; 0.001). Piglets from FF treatment had a greater ADG and a lesser FC (P &amp;lt; 0.05) than piglets from BF, piglets from PFP and PFI being intermediate. In phase 3, piglets from parity 2 sows and those from FF treatment had greater ADG and ADFI than piglets from PFI treatment, piglets from PFP and BF treatments being intermediate (P &amp;lt; 0.005). For all post-weaning period, piglets from parity 2 sows had greater ADG, ADFI and body weight at 42 d (P &amp;lt; 0.001) while piglets from the FF treatment had better ADG and weight at 42 d (P &amp;lt; 0.05) than those from BF and PFI (P &amp;lt; 0.05), PFP being intermediate. The lean ADG was greater for piglets from FF treatment (P &amp;lt; 0.021) than piglets from PFI, piglets from BF and PFP being intermediate. Piglets from sows of parity 2 and 3 had also greater lean ADG (P &amp;lt; 0.001). Fat ADG was also higher for piglets from parity 2 sows (P &amp;lt; 0.010) than piglets from parity 1 sows, piglets from parity 3 sows being intermediate. Piglets from parity 2 sows clearly had better post-weaning performances. The FF treatment during gestation led to better post-weaning performance, suggesting that energy or nutrient intake in early gestation affects offspring growth and development in post-weaning period.

Dosimetry in cranial photobiomodulation therapy: effect of cranial thickness and bone density

This research aims to examine the influence of human skull bone thickness and density on light penetration in PBM therapy across different wavelengths, focusing on how these bone characteristics affect the absorption of therapeutic light. Analyses explored the effect of skull bone density and thickness on light penetration in PBM, specifically using Low-Level Laser Therapy (LLLT) for efficacy prediction. Measurements of bone thickness and density were taken using precise tools. This approach emphasizes LLLT's significance in enhancing PBM outcomes by assessing how bone characteristics influence light penetration. The study revealed no significant correlation between skull bone density and thickness and light penetration capability in photobiomodulation (PBM) therapy, challenging initial expectations. Wavelengths of 405 nm and 665 nm showed stronger correlations with bone density, suggesting a significant yet weak impact. Conversely, wavelengths of 532 nm, 785 nm, 810 nm, 830 nm, 980 nm, and 1064 nm showed low correlations, indicating minimal impact from bone density variations. However, data variability (R2 < 0.4) suggests that neither density nor thickness robustly predicts light power traversing the bone, indicating penetration capability might be more influenced by bone thickness at certain wavelengths. The study finds that the effectiveness of photobiomodulation (PBM) therapy with bone isn't just based on bone density and thickness but involves a complex interplay of factors. These include the bone's chemical and mineral composition, light's wavelength and energy dose, treatment duration and frequency, and the precise location where light is applied on the skull.

Multi-scale cortical bone traits vary in females and males from two mouse models of genetic diversity.

Understanding the genetic basis of cortical bone traits can allow for the discovery of novel genes or biological pathways regulating bone health. Mice are the most widely used mammalian model for skeletal biology and allow for the quantification of traits that cannot easily be evaluated in humans, such as osteocyte lacunar morphology. The goal of our study was to investigate the effect of genetic diversity on multi-scale cortical bone traits of 3 long bones in skeletally-mature mice. We measured bone morphology, mechanical properties, material properties, lacunar morphology, and mineral composition of mouse bones from 2 populations of genetic diversity. Additionally, we compared how intrabone relationships varied in the 2 populations. Our first population of genetic diversity included 72 females and 72 males from the 8 inbred founder strains used to create the Diversity Outbred (DO) population. These 8 strains together span almost 90% of the genetic diversity found in mice (Mus musculus). Our second population of genetic diversity included 25 genetically unique, outbred females and 25 males from the DO population. We show that multi-scale cortical bone traits vary significantly with genetic background; heritability values range from 21% to 99% indicating genetic control of bone traits across length scales. We show for the first time that lacunar shape and number are highly heritable. Comparing the 2 populations of genetic diversity, we show that each DO mouse does not resemble a single inbred founder, but instead the outbred mice display hybrid phenotypes with the elimination of extreme values. Additionally, intrabone relationships (eg, ultimate force vs. cortical area) were mainly conserved in our 2 populations. Overall, this work supports future use of these genetically diverse populations to discover novel genes contributing to cortical bone traits, especially at the lacunar length scale.

SYNTHESIS AND CHARACTERIZATION OF Mg, Cu-HAp USING CALCIUM FROM LOKAN SHELL (Geloina expansa) AND ITS ANTIBACTERIAL ACTIVITY

Hydroxyapatite (HAp) is the primary inorganic constituent found in bones and teeth. It possesses biocompatibility and bioactivity within the human body, while its mechanical qualities are somewhat inferior. Magnesium is a viable alternative to calcium in the composition of bone minerals. Copper is both an antibacterial agent and a vital metal within our bodies. This study aims to produce Mg, and Cu-HAp by employing the sol-gel process in situ utilising Lokan shell (Geloina. Expansa) as the source material. Additionally, the antibacterial properties of Mg and Cu-HAp will be evaluated. The lokan shell is utilized as a calcium source, containing 94.626% calcium oxide (CaO), for synthesizing hydroxyapatite (HAp) with a calcium-to-phosphorus ratio of 1.67. The X-ray diffraction (XRD) analysis revealed a distinct pattern at a specific angle of 2θ, following the ICDD (No.96-101-1243) standard. The FT-IR spectra suggest that the bending vibration of the PO4 3- functional group occurs at a wave number of 610 cm-1, whereas the asymmetrical stretching occurs at wave numbers between 1010-1036 cm-1. These observations suggest the presence of HAp content. The antibacterial test results demonstrated that Mg, Cu-HAp3, and Mg, Cu-HAp4 exhibited good antibacterial activity, as evidenced by inhibition zone diameters of 0.9 mm for Escherichia coli and 0.8 mm for Staphylococcus aureus. These findings indicate that Mg and Cu-HAp effectively possess antibacterial properties.

Rat bone responses to hindlimb unloading-reloading: Composition, BMD and mechanical properties

IntroductionBone loss in lower limbs under space microgravity condition can persist even after the astronauts return to Earth's gravitational environment. It is crucial to determine the extent and timing of bone recovery after spaceflight, especially for long-term flights. However, there is a lack of comprehensive descriptions of the recovery of various aspects of bone quality, particularly in terms of compositional characteristics that reflect changes in bone quality. Therefore, the objective of this study is to investigate the alterations in bone mineral density, composition characteristics, and mechanical properties of rat lower limbs during unloading (tail suspension) and the subsequent recovery loading period. MethodsRats were subjected to tail suspension for four weeks (TS4) followed by reloading for an additional four weeks. Bone mineral density (BMD) was measured by μ-CT and analyzed. The mechanical properties of the tibia were assessed by a three-point bending test, and the composition characteristics of bone tissue were evaluated using Raman spectroscopy. Regression analysis was performed to identify main component related to mechanical property. ResultsIn the metaphysis, BMD of the tibia significant decreased, particularly in trabecular bone. Upon reloading, only the cortical bone showed recovery. In the diaphysis, cortical bone remained unchanged during unloading but demonstrated delayed bone loss during the reloading period. Simultaneously, the mechanical properties of tibia experienced a substantial reduction after four weeks of unloading, The stiffness and maximum strength did not fully recover, though the elastic modulus and the ultimate strength were restored following the reloading process. Raman spectroscopy characterization found an increase in the carbonate substitution and crystal crystallinity of the diaphysis after 4 weeks of unloading. There was an intriguing alteration in the mineralized collagen ratio, that an increase was observed in the diaphysis while a decrease was observed in the metaphysis. During 4 weeks of reloading, the compositional characteristics of the diaphysis recovered earlier than those of the metaphysis, with the exception of crystallinity which remained unrecovered. Regression analysis indicated that carbonate replacement has a strong correlation to bone mechanical properties during the 4 weeks of unloading and subsequent 4 weeks of reloading. ConclusionsThe data obtained in this study highlight the incomplete recovery of bone mineral density, mechanical properties, and compositional characteristics during reloading in tail-suspended rats. These findings deepen our understanding of microgravity-induced bone loss and provide valuable information to develop interventions not only for astronauts but also for persons on earth during and after prolonged periods of unloading.

Decellularization of bone tissue in a supercritical carbon dioxide environment

The use of pig bone tissue as a graft makes it possible to obtain high-quality, relatively cheap and biocompatible ECM. This is since the mineral composition of pork bone is most similar to the mineral composition of human bone. Decellularization in SC-CO2 is a promising direction, since carbon dioxide easily diffuses into the depth of the cell and is a good solvent for lipids, it is non-flammable, non-toxic and chemically non-aggressive. In this paper, the ECM in SC-CO2 was obtained in three ways: 1) in dynamic mode with large SC-CO2 flows; 2) in static and dynamic modes with large SC-CO2 flows; 3) in static and dynamic modes with large SC-CO2 flows, with preliminary exposure in ethyl alcohol as a co-solvent. According to the histological examination, the removal of ICC by the first method is 55%. The use of a co-solvent before starting the decellularization process increases the percentage of ICC removal to 98%, which allows the use of ECM as a transplant.

Application of Optical Photothermal Infrared (O-PTIR) Spectroscopy for Assessment of Bone Composition at the Submicron Scale.

The molecular basis of bone structure and strength is mineralized collagen fibrils at the submicron scale (∼500 nm). Recent advances in optical photothermal infrared (O-PTIR) spectroscopy allow the investigation of bone composition with unprecedented submicron spatial resolution, which may provide new insights into factors contributing to underlying bone function. Here, we investigated (i) whether O-PTIR-derived spectral parameters correlated to standard attenuated total reflection (ATR) Fourier transform infrared spectroscopy spectral data and (ii) whether O-PTIR-derived spectral parameters, including heterogeneity of tissue, contribute to the prediction of proximal femoral bone stiffness. Analysis of serially demineralized bone powders showed a significant correlation (r = 0.96) between mineral content quantified using ATR and O-PTIR spectroscopy, indicating the validity of this technique in assessing bone mineralization. Using femoral neck sections, the principal component analysis showed that differences between O-PTIR and ATR spectra were primarily attributable to the phosphate ion (PO4) absorbance band, which was typically shifter toward higher wavenumbers in O-PTIR spectra. Additionally, significant correlations were found between hydrogen phosphate (HPO4) content (r = 0.75) and carbonate (CO3) content (r = 0.66) quantified using ATR and O-PTIR spectroscopy, strengthening the validity of this method to assess bone mineral composition. O-PTIR imaging of individual trabeculae at 500 nm pixel resolution illustrated differences in submicron composition in the femoral neck from bones with different stiffness. O-PTIR analysis showed a significant negative correlation (r = -0.71) between bone stiffness and mineral maturity, reflective of newly formed bone being an important contributor to bone function. Finally, partial least squares regression analysis showed that combining multiple O-PTIR parameters (HPO4 content and heterogeneity, collagen integrity, and CO3 content) could significantly predict proximal femoral stiffness (R2 = 0.74, error = 9.7%) more accurately than using ATR parameters. Additionally, we describe new findings in the effects of bone tissue orientation in the O-PTIR spectra. Overall, this study highlights a new application of O-PTIR spectroscopy that may provide new insights into molecular-level factors underlying bone mechanical competence.

Raman spectroscopy and U-Net deep neural network in antiresorptive drug-related osteonecrosis of the jaw.

Application of an optical method for the identification of antiresorptive drug-related osteonecrosis of the jaw (ARONJ). We introduce shifted-excitation Raman difference spectroscopy followed by U-Net deep neural network refinement to determine bone tissue viability. The obtained results are validated through established histological methods. Discrimination of osteonecrosis from physiological tissues was evaluated at 119 distinct measurement loci in 40 surgical specimens from 28 patients. Mean Raman spectra were refined from 11,900 raw spectra, and characteristic peaks were assigned to their respective molecular origin. Then, following principal component and linear discriminant analyses, osteonecrotic lesions were distinguished from physiological tissue entities, such as viable bone, with a sensitivity, specificity, and overall accuracy of 100%. Moreover, bone mineral content, quality, maturity, and crystallinity were quantified, revealing an increased mineral-to-matrix ratio and decreased carbonate-to-phosphate ratio in ARONJ lesions compared to physiological bone. The results demonstrate feasibility with high classification accuracy in this collective. The differentiation was determined by the spectral features of the organic and mineral composition of bone. This merely optical, noninvasive technique is a promising candidate to ameliorate both the diagnosis and treatment of ARONJ in the future.

Microwave-Assisted Hydrothermal Treatment of Multifunctional Substituted Hydroxyapatite with Prospective Applications in Bone Regeneration.

Orthopedic bone graft infections are major complications in today's medicine, and the demand for antibacterial treatments is expanding because of the spread of antibiotic resistance. Various compositions of hydroxyapatite (HAp) in which Calcium (Ca2+) ions are substituted with Cerium (Ce3+) and Magnesium (Mg2+) are herein proposed as biomaterials for hard tissue implants. This approach gained popularity in recent years and, in the pursuit of mimicking the natural bone mineral's composition, over 70 elements of the Periodic Table were already reported as substituents into HAp structure. The current study aimed to create materials based on HAp, Hap-Ce, and Hap-Mg using hydrothermal maturation in the microwave field. This route has been considered a novel, promising, and effective way to obtain monodisperse, fine nanoparticles while easily controlling the synthesis parameters. The synthesized HAp powders were characterized morphologically and structurally by XRD diffraction, Dynamic light scattering, zeta potential, FTIR spectrometry, and SEM analysis. Proliferation and morphological analysis on osteoblast cell cultures were used to demonstrate the cytocompatibility of the produced biomaterials. The antimicrobial effect was highlighted in the synthesized samples, especially for hydroxyapatite substituted with cerium. Therefore, the samples of HAp substituted with cerium or magnesium are proposed as biomaterials with enhanced osseointegration, also having the capacity to reduce device-associated infections.

Genetic markers associated with bone composition in Rhode Island Red laying hens

BackgroundBone damage has welfare and economic impacts on modern commercial poultry and is known as one of the major challenges in the poultry industry. Bone damage is particularly common in laying hens and is probably due to the physiological link between bone and the egg laying process. Previous studies identified and validated quantitative trait loci (QTL) for bone strength in White Leghorn laying hens based on several measurements, including bone composition measurements on the cortex and medulla of the tibia bone. In a previous pedigree-based analysis, bone composition measurements showed heritabilities ranging from 0.18 to 0.41 and moderate to strong genetic correlations with tibia strength and density. Bone composition was measured using infrared spectroscopy and thermogravimetry. The aim of this study was to combine these bone composition measurements with genotyping data via a genome-wide association study (GWAS) to investigate genetic markers that contribute to genetic variance in bone composition in Rhode Island Red laying hens. In addition, we investigated the genetic correlations between bone composition and bone strength.ResultsWe found novel genetic markers that are significantly associated with cortical lipid, cortical mineral scattering, medullary organic matter, and medullary mineralization. Composition of the bone organic matter showed more significant associations than bone mineral composition. We also found interesting overlaps between the GWAS results for tibia composition traits, particularly for cortical lipid and tibia strength. Bone composition measurements by infrared spectroscopy showed more significant associations than thermogravimetry measurements. Based on the results of infrared spectroscopy, cortical lipid showed the highest genetic correlations with tibia density, which was negative (− 0.20 ± 0.04), followed by cortical CO3/PO4 (0.18 ± 0.04). Based on the results of thermogravimetry, medullary organic matter% and mineral% showed the highest genetic correlations with tibia density (− 0.25 ± 0.04 and 0.25 ± 0.04, respectively).ConclusionsThis study detected novel genetic associations for bone composition traits, particularly those involving organic matter, that could be used as a basis for further molecular genetic investigations. Tibia cortical lipids displayed the strongest genetic associations of all the composition measurements, including a significantly high genetic correlation with tibia density and strength. Our results also highlighted that cortical lipid may be a key measurement for further avian bone studies.

Effect of stress on the dissolution/crystallization of apatite in aqueous solution: a thermochemical equilibrium study.

Bone mineralization is critical to maintaining tissue mechanical function. The application of mechanical stress via exercise promotes bone mineralization via cellular mechanotransduction and increased fluid transport through the collagen matrix. However, due to its complex composition and ability to exchange ions with the surrounding body fluids, bone mineral composition and crystallization is also expected to respond to stress. Here, a combination of data from materials simulations, namely density functional theory and molecular dynamics, and experimental studies were input into an equilibrium thermodynamic model of bone apatite under stress in an aqueous solution based on the theory of thermochemical equilibrium of stressed solids. The model indicated that increasing uniaxial stress induced mineral crystallization. This was accompanied by a decrease in calcium and carbonate integration into the apatite solid. These results suggest that weight-bearing exercises can increase tissue mineralization via interactions between bone mineral and body fluid independent of cell and matrix behaviours, thus providing another mechanism by which exercise can improve bone health. This article is part of a discussion meeting issue 'Supercomputing simulations of advanced materials'.

Long-term effects of canagliflozin treatment on the skeleton of aged UM-HET3 mice.

Sodium glucose cotransporter-2 inhibitors (SGLT2is) promote urinary glucose excretion and decrease plasma glucose levels independent of insulin. Canagliflozin (CANA) is an SGLT2i, which is widely prescribed, to reduce cardiovascular complications, and as a second-line therapy after metformin in the treatment of type 2 diabetes mellitus. Despite the robust metabolic benefits, reductions in bone mineral density (BMD) and cortical fractures were reported for CANA-treated subjects. In collaboration with the National Institute on Aging (NIA)-sponsored Interventions Testing Program (ITP), we tested skeletal integrity of UM-HET3 mice fed control (137 mice) or CANA-containing diet (180ppm, 156 mice) from 7 to 22months of age. Micro-computed tomography (micro-CT) revealed that CANA treatment caused significant thinning of the femur mid-diaphyseal cortex in both male and female mice, did not affect trabecular bone architecture in the distal femur or the lumbar vertebra-5 in male mice, but was associated with thinning of the trabeculae at the distal femur in CANA-treated female mice. In male mice, CANA treatment is associated with significant reductions in cortical bone volumetric BMD by micro-CT, and by quantitative backscattered scanning electron microscopy. Raman microspectroscopy, taken at the femur mid-diaphyseal posterior cortex, showed significant reductions in the mineral/matrix ratio and an increased carbonate/phosphate ratio in CANA-treated male mice. These data were supported by thermogravimetric assay (TGA) showing significantly decreased mineral and increased carbonate content in CANA-treated male mice. Finally, the sintered remains of TGA were subjected to X-ray diffraction and showed significantly higher fraction of whitlockite, a calcium orthophosphate mineral, which has higher resorbability than hydroxyapatite. Overall, long-term CANA treatment compromised bone morphology and mineral composition of bones, which likely contribute to increased fracture risk seen with this drug.

MASS PRODUCTION OF OSTEOGENICALLY ENHANCED HA/CC SCAFFOLDS FOR BONE REPAIR

Accidents, osteoporosis or cancer can cause severe bone damage requiring grafts to heal. All current grafting methods have disadvantages including scarcity and infection/rejection risks. An alternative is therefore needed. Hydroxyapatite/calcium carbonate (HA/CC) scaffolds mimic the mineral bone composition but lack growth factors present in auto- and allografts, limiting their osteoinductive capacity. We hypothesize that this will increase the osteogenicity and osteoinductivity of scaffolds through the presence of growth factors. The objectives of this study are to develop and mass-produce grafts with enhanced osteoinductive capacity.HA/CC scaffolds were cultured together with umbilical cord mesenchymal stem cells in bioreactors so that they adhere to the surface and deposit growth factors. Cells growing on the scaffolds are confirmed by Alamar blue assays, SEM, and confocal microscopy. ELISA and IHC are used to assess the growth factor content of the finished product.It has been confirmed that cells attach to the scaffolds and proliferate over time when grown in bioreactors. Dynamic seeding of cells is clearly advantageous for cell deposits, equalizing the amount of cells on each scaffold granule.Hydroxyapatite/calcium carbonate scaffolds support cell-growth. This should be confirmed by further research, including Quantification of BMPs and other indicators of osteogenic differentiation such as Runx2, osteocalcin and ALP is pending, and amounts are expected to be increased in enhanced scaffolds and in-vivo implantation.

Betaine and Soluble Fiber Improve Body Composition and Plasma Metabolites in Cats with Chronic Kidney Disease.

Chronic kidney disease (CKD) is a common condition in cats and cachexia (loss of lean body mass) is a concern. A nutrition-based intervention was investigated in cats with CKD for its effects on body composition, the plasma metabolome, and possible implications on health. After a 4-week prefeed period with the control food, cats with CKD (N = 24) were randomized to one of six groups to consume a control food; a food supplemented with 0.5% betaine, 0.39% oat beta-glucan, and 0.27% short-chain fructooligosaccharides (scFOS, test food 1); and a food supplemented with 0.5% betaine, 0.59% oat beta-glucan, and 0.41% scFOS (test food 2) in a William's Latin Square design, each for 10 weeks. Body composition was assessed via dual-energy X-ray absorptiometry measurements, and the plasma metabolome was characterized. Despite no significant differences in daily intake among the three foods, significant increases in total body mass, lean body mass, and lean plus bone mineral composition were observed when cats with CKD consumed test food 1 compared with the control food; numerical increases were seen with test food 2 versus the control food. Plasma metabolomics indicated increased one-carbon metabolism following consumption of test food 1 and/or 2, with significant increases in sarcosine and numerical increases in methionine. Lower levels of plasma trans-4-hydroxyproline and N-methylproline following consumption of test foods 1 and 2 indicates reduced collagen breakdown and perhaps reduced fibrosis. Several acylcarnitines and branched-chain fatty acids associated with CKD were also reduced when cats ate test food 1 or 2 versus the control food. Higher plasma levels of sphingomyelins with consumption of test food 1 or 2 may reflect less severe CKD. Consumption of foods with supplemental betaine and fibers by cats with CKD led to improvements in body composition and changes in the plasma metabolome that correspond to better kidney health.

Triple-gene deletion for osteocalcin significantly impairs the alignment of hydroxyapatite crystals and collagen in mice.

Osteocalcin (Ocn), also known as bone Gla protein, is synthesized by osteoblasts and thought to regulate energy metabolism, testosterone synthesis and brain development. However, its function in bone is not fully understood. Mice have three Ocn genes: Bglap, Bglap2 and Bglap3. Due to the long span of these genes in the mouse genome and the low expression of Bglap3 in bone, researchers commonly use Bglap and Bglap2 knockout mice to investigate the function of Ocn. However, it is unclear whether Bglap3 has any compensatory mechanisms when Bglap and Bglap2 are knocked out. Considering the controversy surrounding the role of Ocn in bone, we constructed an Ocn-deficient mouse model by knocking out all three genes (Ocn-/-) and analyzed bone quality by Raman spectroscopy (RS), Scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and MicroCT (μCT). The RS test showed that the alignment of hydroxyapatite crystals and collagen fibers was significantly poorer in Ocn-/- mice than in wild-type (WT) mice. Ocn deficiency resulted in a looser surface structure of bone particles and a larger gap area proportion. FTIR analysis showed few differences in bone mineral index between WT and Ocn-/- mice, while μCT analysis showed no significant difference in cortical and trabecular regions. However, under tail-suspension simulating bone loss condition, the disorder of hydroxyapatite and collagen fiber alignment in Ocn-/- mice led to more obvious changes in bone mineral composition. Collectively, our results revealed that Ocn is necessary for regulating the alignment of minerals parallel to collagen fibrils.

Effect of Nano- and Microzinc Supplementation on the Mineral Composition of Bones of Rats with Induced Mammary Gland Cancer.

The aim of this study was to determine changes in the mineral composition of the bones of rats with chemically induced mammary gland cancer and to attempt to establish whether a specific diet modification involving the inclusion of zinc ions in two forms-nano and micro-will affect the mineral composition of the bones. Female Sprague-Dawley rats were used for the research. The animals were randomly assigned to three experimental groups. All animals were fed a standard diet (Labofeed H), and selected groups additionally received zinc nanoparticles or microparticles in the amount of 4.6 mg/mL. To induce mammary cancer, the animals were given 7,12-dimethyl-1,2-benz[a]anthracene. The content of Ag, As, B, Ba, Cd, Cr, Cu, Mn, Ni, Pb, Rb, Se, Sr, Tl, U, and V was determined using ICP-MS, while that of Ca, Fe, K, Mg, Na, and Zn was determined using FAAS. The use of a diet enriched with zinc nano- or microparticles significantly influenced the content of the elements tested. In the bones of rats fed a diet with zinc nanoparticles, changes were found in the content of Ca, Mg, Zn, Cd, U, V, and Tl, while in the case of the diet supplemented with zinc microparticles, there were differences in six elements-Ca, Mg, B, Cd, Ag, and Pb-compared to animals receiving an unsupplemented diet. The content of elements in the bone tissue of rats in the experimental model indicates disturbances of mineral metabolism in the tissue at an early stage of mammary cancer.

Effects of Dietary Supplementation of L-Carnitine and Mannan-Oligosaccharides on Growth Performance, Selected Carcass Traits, Content of Basic and Mineral Components in Liver and Muscle Tissues, and Bone Quality in Turkeys.

The study aimed to determine the effect of L-carnitine and Bio-Mos administration on selected production performance, slaughter parameters, elemental and mineral content of liver, breast and thigh muscles, and physical, morphometric, strength and bone mineral composition parameters of turkeys. The experiment was conducted on 360 six-week-old Big-6 turkey females, randomly divided into three groups of 120 birds each (six replicates of 20 birds). The turkeys of the control group were fed standard feed without additives; group II was fed with drinking water, a preparation containing L-carnitine at a dose of 0.83 mL/L, while group III was provided mixed feed with 0.5% Bio-Mos. The addition of L-carnitine and Bio-Mos increased body weight at 16 weeks (p = 0.047) and reduced the proportion of fat in the breast muscle (p = 0.029) and liver (p = 0.027). It also modified the content of some minerals in breast muscle, thigh muscle, liver, and bone. Furthermore, the addition of L-carnitine and Bio-Mos increased bone mass and length and modified the value of selected morphometric and strength parameters. The results indicate a positive effect of the applied feed additives on selected rearing indices and carcass quality while improving the elasticity and fracture toughness of the femur. There is a need for further research to determine optimal doses of L-carnitine and Bio-Mos in poultry nutrition.