Tomasz Blicharski

Comparison of the effect of dietary copper nanoparticles with copper (II) salt on bone geometric and structural parameters as well as material characteristics in a rat model

Copper is required for normal functioning of all basic biochemical and physiological processes in the body. The objective of this study was to compare the effect of two different chemical forms (carbonate and nanoparticles) of Cu administered in feed mixtures to growing rats on bone geometric and structural parameters as well as material characteristics in a rat model. For this purpose, five experimental treatments were used to evaluate the effects of different levels of Cu applied in the diet as a mineral mixture (the standard dose of 6.5mg/kg diet, half the standard dose, and no Cu in mineral mixture as a negative control) and two Cu sources (CuCO3 - commonly used in rodent laboratory diets and a Cu-NP nanoparticle preparation, 40nm). There were no changes in body weight and bone morphology, but significant alteration was noted in the geometry and mechanical parameters, which was Cu-NP-dose dependent. Our study showed an increase in the ultimate load and toughness in Cu-NP-treated rats at the standard concentration. The higher values of the studied parameters prove that there was a change in bone mineralization. Although bone mineral density and content were not changed, bone tissue density and ash increased. The XRD analysis revealed that some peaks did not originate from hydroxyapatite, and they indicated existence of other mineral phases. No studies conducted so far have provided a detailed mechanical X-ray diffraction analysis of bone tissue of growing rats administered with diet containing Cu nanoparticles. This study showed that Cu-NP given in low dose increased mechanical endurance of bone, without the changes in strain and stress compared to low dose of Cu given in traditional form.

Biofortification of soy (Glycine max (L.) Merr.) with strontium ions.

Soy (Glycine max (L.) Merr.) is an annual plant cultivated worldwide mostly for food. Moreover, due to its pharmacological properties it is widely used in pharmacy for alleviating the symptoms of osteoporosis. The aim of the present study was to investigate the biofortification of soy treated with various concentrations of strontium. Soy was found to have a strong capacity to absorb Sr(2+) (bioconcentration factor higher than 1). A positive linear correlation (R(2) > 0.98) between the amount of strontium in the growth medium and its content in the plant was also observed. Moreover, at a concentration of 1.5 mM, strontium appeared to be nontoxic and even stimulated plant growth by approximately 19.4% and 22.6% of fresh weight for shoots and roots, respectively. Our research may be useful to obtain vegetable products or herbal preparations containing both phytoestrogens and strontium to prevent postmenopausal osteoporosis.

The Stimulatory Effect of Strontium Ions on Phytoestrogens Content in Glycine max (L.) Merr

The amount of secondary metabolites in plants can be enhanced or reduced by various external factors. In this study, the effect of strontium ions on the production of phytoestrogens in soybeans was investigated. The plants were treated with Hoagland’s solution, modified with Sr2+ with concentrations ranging from 0.5 to 3.0 mM, and were grown for 14 days in hydroponic cultivation. After harvest, soybean plants were separated into roots and shoots, dried, and pulverized. The plant material was extracted with methanol and hydrolyzed. Phytoestrogens were quantified by HPLC. The significant increase in the concentration of the compounds of interest was observed for all tested concentrations of strontium ions when compared to control. Sr2+ at a concentration of 2 mM was the strongest elicitor, and the amount of phytoestrogens in plant increased ca. 2.70, 1.92, 3.77 and 2.88-fold, for daidzein, coumestrol, genistein and formononetin, respectively. Moreover, no cytotoxic effects were observed in HepG2 liver cell models after treatment with extracts from 2 mM Sr2+-stressed soybean plants when compared to extracts from non-stressed plants. Our results indicate that the addition of strontium ions to the culture media may be used to functionalize soybean plants with enhanced phytoestrogen content.

The effect of supplementation of a glutamine precursor on the growth plate, articular cartilage and cancellous bone in fundectomy-induced osteopenic bone

The aim of the study was to investigate the effect of 2-oxoglutaric acid (2-Ox) supplementation (a precursor of glutamine and hydroxyproline, the most abundant amino acid of collagen) on cartilage and bone in pigs after fundectomy. Pigs at the age of forty days were subjected to fundectomy and divided into two groups depending on 2-Ox supplementation (at the daily dosage of 0.4 g/kg of body weight). Other pigs were sham operated. Pigs were euthanized at the age of eight months. An analysis of the morphometry of trabeculae, growth plate and articular cartilage in fundectomy-induced osteopenic bone was performed. Moreover, the levels of expression of osteocalcin, osteopontin and osteoprotegerin in trabecular bone and osteocalcin in articular cartilage were evaluated. Articular cartilage was thinnest in fundectomized pigs and thickest in 2-Ox-supplemented animals after fundectomy. Moreover, 2-Ox supplementation after fundectomy enhanced the total thickness of the growth plate and trabeculae in fundectomized pigs. The most evident signal for osteocalcin and osteoprotegerin in trabecular bone was in sham-operated and 2-Ox-supplemented pigs; a low reaction was observed in the fundectomized group. Additionally, as a long-term postoperative consequence, a change was observed in the expression of osteocalcin in articular cartilage. It seems that 2-Ox is suitable for use in preventing the negative effects of fundectomy on cancellous bone and cartilage.

A metabolite of leucine (β-hydroxy-β-methylbutyrate) given to sows during pregnancy alters bone development of their newborn offspring by hormonal modulation

The effects of dietary β-hydroxy-β-methylbutyrate (HMB) supplementation during gestation on bone, growth plate, and articular cartilage in newborns were determined. Thermal analysis of articular cartilage was performed to examine the structural changes in collagen. At day 70 of gestation, a total of 12 sows (Large White Polish breed, at the second parity) were randomly assigned to two groups, with each group receiving either a basal diet or the same diet supplemented with 0.2 g/day HMB until the 90th day. Maternal HMB supplementation enhanced body weight, bone length, and diameter in males. It also improved geometric and mechanical properties contributing to increased bone morphology and endurance. In turn, alteration of the length was only observed in females. The positive effects were mediated by increased serum concentrations of insulin-like growth factor-1 and leptin. HMB-treatment enhanced the concentration of FSH, LH, estradiol, and testosterone. Serum TAP was enhanced by the HMB-treatment by 34% in females and 138% in males. Beneficial effects of the HMB-treatment on trabecular bone and content of proteoglycans in articular cartilage were shown. The HMB-treatment significantly changed the collagen structure in cartilages, especially in the females, which was demonstrated by the PSR analysis. Differences between the HMB-supplemented and the control females in the calorimetric peak temperatures were presumably related to different collagen fibril density in the articular cartilage structure. In summary, maternal HMB supplementation in the mid-gestation period significantly improved general growth and mechanical endurance of long bones by the influence on the somatotropic and pituitary-gonadal axes in the offspring.

Effect of sample pre-treatment on isoflavones quantification in soybean

High performance liquid chromatography is the most common technique for analysing isoflavones in soy; however, the appropriate sample pre-treatment is required, including extraction and, often, hydrolysis. In the present work, the kinetics of extraction was investigated with regard to the number of extraction steps and their duration for the exhaustive extraction of bonded forms of isoflavones and aglycones. In addition, a systematic experimental study of hydrolysis depending on the temperature and acidity of the medium was conducted. The results showed that the assisted methods were more effective for the isolation of isoflavones. Moreover, ^the differences between the results obtained using ultrasound-assisted extraction (UAE), microwave-assisted extraction and pressurised liquid extraction were not statistically significant; however, the appropriate number of extraction steps and their duration for UAE were required. The investigation also revealed that, due to the varying susceptibility of glycosides on conversion to free forms and the stability of the aglycones obtained, two parallel hydrolysis procedures should be conducted to obtain the highest hydrolysis efficiency. The optimised conditions, taking into account the shortest time combined with the highest efficiency, were as follows: 4 M HCl and 60 min for genistein; 8 M HCl and 30 min for glycitein and daidzein.

A Phase III Randomized Placebo-Controlled Trial to Evaluate Efficacy and Safety of Romosozumab in Men With Osteoporosis

Context Globally, one in five men aged >50 years is predicted to experience an osteoporotic fracture. Because of the treatment gap in osteoporosis and the paucity of bone-forming agents for men, new osteoporosis treatments are needed. Objective To evaluate the safety and efficacy of romosozumab in men with osteoporosis. Design Phase III randomized BRIDGE study (placebo-controlled double-blind study evaluating the efficacy and safety of romosozumab in treating men with osteoporosis; ClinicalTrials.gov identifier, NCT02186171) for 12 months. Setting Thirty-one centers in Europe, Latin America, Japan, and North America. Patients Men aged 55 to 90 years with a baseline bone mineral density (BMD) T-score at the lumbar spine (LS), total hip (TH), or femoral neck of ≤-2.5 or ≤-1.5 with a history of a fragility nonvertebral or vertebral fracture. Interventions The subjects were randomized 2:1 to receive romosozumab 210 mg subcutaneously monthly or placebo for 12 months. Main Outcome Measures The primary efficacy endpoint was percentage change from baseline in LS BMD at month 12. Results In 245 subjects (163 romosozumab, 82 placebo), at month 12, the mean percentage change from baseline in the LS and TH BMD was significantly greater for the romosozumab group than for the placebo group (LS, 12.1% vs 1.2%; TH, 2.5% vs -0.5%; P < 0.001). Adverse events and serious adverse events were balanced between the two groups, with a numerical imbalance in the positively adjudicated cardiovascular serious adverse events [romosozumab, 8 (4.9%) vs placebo, 2 (2.5%)]. Conclusions Treatment with romosozumab for 12 months increased the spine and hip BMD compared with placebo and was well tolerated in men with osteoporosis.

Dietary 2-oxoglutarate prevents bone loss caused by neonatal treatment with maximal dexamethasone dose

Synthetic glucocorticoids (GCs) are widely used in the variety of dosages for treatment of premature infants with chronic lung disease, respiratory distress syndrome, allergies, asthma, and other inflammatory and autoimmune conditions. Yet, adverse effects such as glucocorticoid-induced osteoporosis and growth retardation are recognized. Conversely, 2-oxoglutarate (2-Ox), a precursor of glutamine, glutamate, and collagen amino acids, exerts protective effects on bone development. Our aim was to elucidate the effect of dietary administered 2-Ox on bone loss caused by neonatal treatment with clinically relevant maximal therapeutic dexamethasone (Dex) dose. Long bones of neonatal female piglets receiving Dex, Dex+2-Ox, or untreated were examined through measurements of mechanical properties, density, mineralization, geometry, histomorphometry, and histology. Selected hormones, bone turnover, and growth markers were also analyzed. Neonatal administration of clinically relevant maximal dose of Dex alone led to over 30% decrease in bone mass and the ultimate strength (P < 0.001 for all). The length (13 and 7% for femur and humerus, respectively) and other geometrical parameters (13–45%) decreased compared to the control (P < 0.001 for all). Dex impaired bone growth and caused hormonal imbalance. Dietary 2-Ox prevented Dex influence and vast majority of assessed bone parameters were restored almost to the control level. Piglets receiving 2-Ox had heavier, denser, and stronger bones; higher levels of growth hormone and osteocalcin concentration; and preserved microarchitecture of trabecular bone compared to the Dex group. 2-Ox administered postnatally had a potential to maintain bone structure of animals simultaneously treated with maximal therapeutic doses of Dex, which, in our opinion, may open up a new opportunity in developing combined treatment for children treated with GCs. Impact statement The present study has showed, for the first time, that dietary 2-oxoglutarate (2-Ox) administered postnatally has a potential to improve/maintain bone structure of animals simultaneously treated with maximal therapeutic doses of dexamethasone (Dex). It may open the new direction in searching and developing combined treatment for children treated with glucocorticoids (GCs) since growing group of children is exposed to synthetic GCs and adverse effects such as glucocorticoid-induced osteoporosis and growth retardation are recognized. Currently proposed combined therapies have numerous side effects. Thus, this study proposed a new direction in combined therapies utilizing dietary supplementation with glutamine derivative. Impairment caused by Dex in presented long bones animal model was prevented by dietary supplementation with 2-Ox and vast majority of assessed bone parameters were restored almost to the control level. These results support previous thesis on the regulatory mechanism of nutrient utilization regulated by glutamine derivatives and enrich the nutritional science.

Gut-bone axis response to dietary replacement of soybean meal with raw low-tannin faba bean seeds in broiler chickens

It seems that faba bean (FB) seeds could be a good protein-energy component in animal feed, but the presence of anti-nutritional substances limits their use as a substitute of soybean meal. The aim of the study was to examine the influence of different concentrations of raw, low-tannin, FB seeds on the gut-bone axis in Ross 308 broilers. One-day old chickens were randomly subjected to one of the 3 dietary treatments: the control group was fed standard diet based on soybean meal and without FB seeds, and two groups were fed 8%/15% and 16%/22% of raw low-tannin FB seeds in the starter and grower, respectively. On the 35th day, hematological and serum biochemical analyses as well histomorphometry of the small intestine and liver tissue and bone mechanical tests were performed. The diet type had no effect on the body weight gain and feed conversion ratio. However, the basal intestinal structures were significantly reduced in birds fed the lower concentration of FB. The enlargement of nerve plexuses was dependent on the concentration used in the diet and, additionally, on the kind of plexus and location in the intestinal tract. The liver was characterized by an increase in non-hepatocytes. There was no influence of the low-tannin FB seeds on most of the analyzed serum parameters in the 35-day-old broiler chickens, except the decreased concentration of total cholesterol and Ca in both experimental groups, triglycerides in group I, and P and uric acid in group II. Furthermore, the increasing concentration of the dietary low-tannin FB did not influence the activities of AspAT (except the group fed the higher amount of FB), ALAT, and LDH. The broiler chickens had no visible leg lesions and no problem in the locomotor function, but the tibiae were lighter mainly in birds fed the higher concentration of FB seeds. Geometric analysis revealed reduction of the cross section area and wall thickness, indicating a decline in the bone midshaft, which influenced the densitometric parameters and the results of mechanical tests. In conclusion, the dietary inclusion of raw low-tannin FB seeds had no negative effects on broiler growth, but disturbed the intestine structure and tibia characteristics. Therefore, all these negative effects necessitate additional examinations before inclusion of raw low-tannin FB seed into poultry diet.

DON-induced changes in bone homeostasis in mink dams

Abstract Introduction: The aim of the study was to investigate the mechanical and geometric properties as well as bone tissue and mineral density of long bones in mink dams exposed to deoxynivalenol (DON) since one day after mating, throughout gestation (ca. 46 d) and lactation to pelt harvesting. Material and Methods: Thirty clinically healthy multiparous minks (Neovison vison) of the standard dark brown type were used. After the mating, the minks were randomly assigned into two equal groups: nontreated control group and DON group fed wheat contaminated naturally with DON at a concentration of 1.1 mg·kg-1 of feed. Results: The final body weight and weight and length of the femur did not differ between the groups. However, DON contamination decreased mechanical endurance of the femur. Furthermore, DON reduced the mean relative wall thickness and vertical wall thickness of the femur, while vertical cortical index, midshaft volume, and cross-sectional moment of inertia increased. Finally, DON contamination did not alter bone tissue density, bone mineral density, or bone mineral content, but decreased the values of all investigated structural and material properties. Conclusion: DON at applied concentration probably intensified the process of endosteal resorption, which was the main reason for bone wall thinning and the weakening of the whole bone.