N B m R A a b c a A R R A K O S B B 1 b p a n a F I m t b c R h 0 Journal of Trace Elements in Medicine and Biology 39 (2017) 169–175 Contents lists available at ScienceDirect Journal of Trace Elements in Medicine and Biology jo ur nal ho me page: www.elsev ier .com/ locate / j temb utrition oron supplementation improves bone health of non-obese diabetic ice enata Dessordia,∗, Adriano Levi Spirlandelib, Ariane Zamarioli c, José Batista Volponc, nderson Marliere Navarrob Department of Food and Nutrition, Faculty of Pharmaceutical Sciences, State University of São Paulo-UNESP, Brazil Department of Clinical Medicine, Ribeirão Preto Medical School, University of São Paulo-FMRP/USP, Brazil Biomechanics, Medicine and Rehabilitation, School of Medicine of Ribeirão Preto, University of São Paulo, Brazil r t i c l e i n f o rticle history: eceived 18 November 2015 eceived in revised form 8 September 2016 ccepted 30 September 2016 eywords: steoporosis upplementation one oron a b s t r a c t Diabetes Mellitus is a condition that predisposes a higher risk for the development of osteoporosis. The objective of this study was to investigate the influence of boron supplementation on bone microstructure and strength in control and non-obese diabetic mice for 30 days. The animals were supplemented with 40 �g/0,5 ml of boron solution and controls received 0,5 ml of distilled water daily. We evaluated the bio- chemical parameters: total calcium, phosphorus, magnesium and boron; bone analysis: bone computed microtomography, and biomechanical assay with a three point test on the femur. This study consisted of 28 animals divided into four groups: Group water control - Ctrl (n = 10), Group boron control - Ctrl±B (n = 8), Group diabetic water - Diab (n = 5) and Group diabetic boron - Diab±B (n = 5). The results showed that cortical bone volume and the trabecular bone volume fraction were higher for Diab±B and Ctrl±B compared to the Diab and Ctrl groups (p ≤ 0,05). The trabecular specific bone surface was greater for the Diab±B group, and the trabecular thickness and structure model index had the worst values for the Diab group. The boron serum concentrations were higher for the Diab±B group compared to non- supplemented groups. The magnesium concentration was lower for Diab and Diab±B compared with controls. The biomechanical test on the femur revealed maintenance of parameters of the bone strength in animals Diab±B compared to the Diab group and controls. The results suggest that boron supplemen- tation improves parameters related to bone strength and microstructure of cortical and trabecular bone in diabetic animals and the controls that were supplemented. © 2016 Elsevier GmbH. All rights reserved. . Introduction The role of nutrition in the development of the bone tissue has een the focus of many studies, which investigate dietary com- onents required for the maintenance of healthy bone functions, s well as there proper development. Nutrients such as calcium, Abbreviations: DM, Diabetes Mellitus; NOD, non-obese diabetic mice; Ctrl, on-diabetic animals; Ctrl±B, non-diabetic animals supplemented; Diab, diabetic nimals non-supplemented; Diab±B, diabetic animals supplemented; Fcfrp/USP, aculty of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo; CP-MS, inductively coupled plasma mass spectrometry; �CT, bone computed icrotomography; BV, bone volume; BV/TV, bone volume fraction; Ct.Th, cortical hickness; BS/BV, specific bone surface; SMI, structure model index; Tb.Th, tra- ecular thickness; Tb.N, trabecular number; Tb.Sp, trabecular separation; Conn.D, onnectivity density. ∗ Corresponding author at: Bandeirantes Avenue, 3900, Monte Alegre, 14049-900, ibeirão Preto, São Paulo, Brazil. E-mail address: re dessordi@hotmail.com (R. Dessordi). ttp://dx.doi.org/10.1016/j.jtemb.2016.09.011 946-672X/© 2016 Elsevier GmbH. All rights reserved. phosphorus, magnesium, vitamin D, fluoride, zinc, copper and boron are known to promote normal development of bone functions, ensuring the gain of mass and strength. Inadequate con- sumption of these nutrients or changes in metabolism, can cause an increase of excretion and absorption losses due to the presence of the disease, can promote losses in bone structure, and consequently the development of related bone diseases like osteoporosis [1]. Osteoporosis is a systemic skeletal disease characterized by an increase in susceptibility to fracture. Most cases are associated with post menopause or aging, but it can also develop as a result of any pathological situation [2]. Diabetes Mellitus (DM) is a condition that predisposes a higher risk for the development of osteoporosis [3]. A chronic condition of DM can negatively affect several parts of the body, such as the bones, muscles, retina, kidneys, and the cardiovascular system. The effects of this disease in the bone cells are very complex, and many studies have been conducted in order to explore the exact mechanisms in which DM induces osteoporosis and, consequently the increase in the rate of bone fractures [4]. dx.doi.org/10.1016/j.jtemb.2016.09.011 http://www.sciencedirect.com/science/journal/0946672X http://www.elsevier.com/locate/jtemb http://crossmark.crossref.org/dialog/?doi=10.1016/j.jtemb.2016.09.011&domain=pdf mailto:re_dessordi@hotmail.com dx.doi.org/10.1016/j.jtemb.2016.09.011 1 ts in M t r c f b d f i i a S i i t b c b m m f d b m r s m a w m m 2 2 w t R m a m s o m e g a o m b fi s f t a t s t 70 R. Dessordi et al. / Journal of Trace Elemen Hyperglycemia deregulates the proper functioning of osteoblas- ic cells, and negatively regulates the function of osteoclastic cells, elated to bone resorption. This condition can facilitate the pro- ess of the development of osteoporosis and cause dysfunction and ailure in different organs [5–7]. The main complication of insulin deficiency is related to the one formation. Experimental models of type 1 DM found a ecrease in the number of osteoblasts and bone turnover occurs rom a reduction in mineral content [5,8]. In diabetic patients decreases in levels of minerals, which exert mportant functions in the bone metabolism are common. Boron s an essential element for plants, but its role in the human body s well as in other mammals still needs to be further addressed. tudies are suggesting that this mineral is essential for maintain- ng bone health and has a vital role in embryogenesis, bone growth, mmunity, and psychomotor functions [9]. Gorustovich et al. [10], hrough an experimental study with mice, reported that boron is eneficial for bone growth and maintenance. Hakki et al. [11], con- luded that the deprivation of this mineral can affect bone growth y inhibiting its formation. Also, this mineral is linked to the for- ation of hormone steroids, and influences the metabolism of icronutrients such as calcium, magnesium and vitamin D. There- ore, it can be involved in preventing losses in calcium and bone emineralization [12,13]. Thus supplementation with minerals, as boron, is essential to one metabolism and can be an important factor in preserving bone ass. The available studies are still limited in their evaluation of the eal participation of boron in bone metabolism and the effects of upplementation. Consequently, it has become necessary to obtain ore results with regards to bone metabolism markers in the ssessment of bone microarchitecture. Hence, the aim of this study as to investigate the influence of boron supplementation on bone icrostructure and strength in control and non-obese diabetic ice. . Materials and methods .1. Animals For this study, we used female non-obese diabetic (NOD) mice eighing between 18 and 20 g and 16 weeks old, from the Cen- er for the Breeding of Special Mice, at the Faculty of Medicine of ibeirão Preto (FMRP) −University of São Paulo (USP). The diabetes odel used a model system. The development of diabetes in this nimal model are similar to that observed in humans, since these ice exhibit spontaneous autoimmunity which leads to progres- ive destruction of insulin-producing pancreatic cells. This process f cell destruction begins at 3 to 4 weeks and extends to 4 to 6 onths of age. The onset of diabetes is characterized by mod- rate glycosuria and blood glucose greater than 250 mg/dL. The lycosuria and hyperglycemia became progressively more severe round the 34th week of weight loss; polydipsia and polyuria both ccurred. Without treatment with exogenous insulin, the diabetic ice became severely hyperglycemic and ketosis, but they did not ecome ketoacidosis, and had a survival rate of 3–4 weeks after the rst detection of glycosuria. The animals were kept in the USP Animal Testing Laboratory, taying there for about 16 weeks for observation, with follow-ups rom the first day of accommodation with water and feed ad libi- um. There was no therapeutic intervention to control glycemic nimals because the objective was to maintain the high glucose o induce the bone loss process. Diabetic animals were a group elected from the moment their blood glucose levels were equal o or greater than 250 mg/dL. The animals that showed no changes edicine and Biology 39 (2017) 169–175 in their blood sugar levels after 16 weeks, were selected for the control groups. After the development of diabetes these animals were divided into four groups: Water Control − Ctrl (n = 10): ani- mals that received 0.5 ml/day of distilled water, Boron Control − Ctrl±B (n = 8): animals that received 40 �g/0.5 ml of boron, Dia- betic Water − Diab (n = 5): diabetic animals that received 0.5 ml of distilled water, and Diabetic Boron − Diab±B (n = 5): diabetic animals that received 40 �g/0.5 ml of boron. Based on literature reports about NOD mice, we selected a sample of 40 animals. After 16 weeks half of the sample developed monitoring glucose levels above 250 mg/dL. Diabetic animals (n = 10) were allocated to each group, but due to high blood sugar levels, only five animals sur- vived until the end of the experiment. The sample may not have the paired n, but it is in line with the minimum established n sam- ple size calculation. The administration of distilled water and boron solution was held once a day by gavage for 30 days. 2.2. Experiment The animals were kept according to the recommendations of the Commission of ethics in the use of Animals, according to the precepts of the law 11.794/2008 Resolution n◦ 879/2008 of the FMRP/USP. The project was approved by the Ethics Committee on Animal Research, FMRP registration number 074/2013. For this study a boron solution was prepared with 40 �g of boron in 0.5 ml of solution using boron chelate H 5% (chelating glycine). The boron solution was prepared at the Laboratory of the Phar- macy Course at the Ribeirão Preto Medical School/USP. Chelated minerals, like boron, have the advantage of being better bioavail- able (90% absorption), without interfering with the absorption of other nutrients and without having side effects. The diet given to the animals (Nuvital Nutrients S/A) contained in its composition ground whole corn, soybean meal, wheat bran, calcium carbonate, dicalcium phosphate, sodium chloride, mineral and vitamin premix amino acids (lysine and methionine). The min- erals present in the diet were: iron (50 mg), boron (0.5 mg), zinc (60 mg), copper (10 mg), iodine (2 mg), manganese (60 mg), sele- nium (0.05 mg), cobalt (1,50 mg) and for the supplemented animals 40 �g of boron was also added, which were administrated by gav- age. Throughout the experiment a bi-weekly monitoring of the glu- cose levels and the weight of control and diabetic animals were conducted. The blood glucose was measured through the use of a blood-glucose Monitor Accu-Chek Advantage II (Roche Diagnostics, São Paulo, Brazil) with the utilization of the test strip and weight on the scale Filizola ® Star precision (São Paulo, Penha, Brazil) of 0.5 g. After the trial period of 30 days the animals, were euthanized by decapitation for the 0.5 ml of blood collection. The blood sam- ple was collected in a tube Eppendorf® of 2 ml and centrifuged at 2.500 rpm, for twenty minutes. The serum used to determine the concentrations of total calcium, phosphorus, magnesium, and boron were obtained from the Laboratory of Toxicology and Essen- tiality of Metals − Faculty of Pharmaceutical Sciences of Ribeirão Preto (FCFRP). Blood samples were manipulated immediately after collection in a sterile environment, with sterile tips so that there was no contamination of the sample. The realization of mineral concentrations was frozen, as the serum was stored at −80 ◦C. The laboratory used for the concentrations has a sterile environ- ment and is suitable for contamination of the sample not to occur. The determination of the concentration of minerals was performed according to Batista et al. [14], by an Inductively Coupled Plasma Mass Spectrometry (ICP-MS), fitted with a dynamic reaction cell (DRC) (Perkin Elmer Sciex Norwalk, CT USA) and operated with high purity argon (99.999% Praxaair, Brazil). Samples were diluted to the ratio 1:50 with a solution containing Triton X-100 0.01% ts in Medicine and Biology 39 (2017) 169–175 171 ( s r 2 e e p r b r o U m r s e s s 2 l P w c m w d s a u C t t o a t a A 2 b t t a l e w M t d a o d ( l R. Dessordi et al. / Journal of Trace Elemen v/v), HNO3 0.05% (v/v), and 10 mg/L-1 rhodium (Rh) as an internal tandard. The concentration of the analytical calibration standards anged from 0 to 50 �g/L. .3. Bone computed microtomography This method evaluated the left femur, as suggested by De Paula t al. [15]. The left femur of the animals was preserved in 70% thanol, for 21 days after the end of the experiment. During this eriod the bones remained in this period in alcohol to facilitate emoval of soft tissue in order to carry out the analyses. Later the ones were cleaned, from the withdrawal of all the muscle that sur- ounded them and stored again in 70% alcohol until the completion f the analyses, which were held at the Faculty of Dentistry of the niversity of São Paulo in Ribeirão Preto. The bone samples were scanned by a high resolution 1172 odel Microtomography, brand SkyScan (Belgium, Bruker Corpo- ation), consisting of a tube of a Micro Focus x-ray high voltage ource (100 kV), each sample had precision handling by a CCD cam- ra detector with 11Mp connected to a computer data acquisition ystem and controlled by a cluster of networked computers with oftware for the reconstruction, visualization and quantification of D and 3D images (software CT Analyser v.1.13). The scan was performed at a low resolution, a 55k Vp energy evel, and intensity of 145 �A. The unit was calibrated weekly with hantom supplied by the manufacturer. Two regions of interest ere delimited, one at the distal femoral metaphysis, which mainly ontains trabecular bone, and the other at the mid-diaphysis, which ainly includes cortical bone. The reconstruction of the metaphysis as selected manually starting at approximately 0.6 mm from the istal growth plate for an extension of 2.5 mm (Fig. 1). The recon- truction of the diaphysis was defined by a 1 mm region starting t the third throcanter. Cortical and trabecular bone were isolated sing manually drawn contouring. CTAn software (Bruker-micro T), version 2.2.1, was used for the determination of the optimal hreshold from the image histograms and was set to exclude soft issue, but did include poorly mineralized bone. The same thresh- ld was used in all of the samples but differed between trabecular nd cortical bone. The parameters analyzed for trabecular, and cor- ical bone are in Table 1. All bone-morphometric measurements nd nomenclature are in agreement with recommendations of the SBMR [16]. .4. Biomechanical assay at three point test in femur The right femur of the animals was used for the three point iomechanical assay test done through an analysis performed by he material resistance equipment (Universal Testing Machine) of he Bioengineering Laboratory at the FMRP (USP). The load on the bone was applied in the central region in the nterior-posterior direction, through an accessory attached to the oad cell. The bone was placed next to the device so that the xtremety of the femur was put against the supports, and the sides ould aim at the base of the machine (Fig. 2). A dial indicator gauge itutoyo®, with an accuracy of 0.01 mm was used for the collec- ion of the deflection intervals during all mechanical testing. The eflection values were measured every 0.05 mm at the same time s the measurement of the value of the applied load on the bridge f the extensometer. The vertical load was applied until the bone fractured. Load and eformation curves were recorded in real time. The Tesc® software EMIC, São José dos Pinhais, PR, Brazil) was used to obtain ultimate oad, stiffness, and resilience. Fig. 1. Reconstruction of the metaphysis (femur), starting at approximately 0.6 mm from the distal growth plate for an extension of 2.5 mm. 2.5. Statistic analysis Exploratory analysis of data for an overview of the features of the variables were obtained, describing them through tables with descriptive measures. Median, minimum, and maximum statistics were calculated with a reliability rate of 95%. Statistical tests were performed to test the equality between groups. We used the SPSS Statistics software version 22.0 for statistical analysis. For a comparison between all groups, we used the two- way nonparametric ANOVA test with Tukey as a post hoc test. The statistical significance was defined as p ≤ 0.05. 3. Results The measurements of weight and blood glucose of groups Ctrl, Ctrl±B, Diab and Diab±B, are presented in Table 2. The analysis revealed that there was no difference in body weight of diabetic animals and controls in the first (one day before the start of the experiment) and second measurements (fifteen days afther the first measurement), but at the end of the experiment there was significant weight loss in the diabetic animals (Diab and Diab±B) compared to the Ctrl group (p < 0.05). The glycemic values revealed that Diab and Diab±B groups had glucose values significantly higher compared to Ctrl and Ctrl±B at the first, second, and third measurements showing evidence of diabetes. 172 R. Dessordi et al. / Journal of Trace Elements in Medicine and Biology 39 (2017) 169–175 Table 1 Definition and description of outcomes for cortical and trabecular bone morphology. BONE PARAMETERS VARIABLE DESCRIPTION STANDARD UNIT Trabecular Bone BV (bone volume) Volume of the region segmented as bone mm3 BV/TV (bone volume fraction) Ratio of the segmented bone volume to the total volume of the region of interest % BS/BV (specific bone surface) Ratio of the segmented bone volume to the total volume of the region of interest mm3 SMI (structure model index) An indicator of the stucture of trabeculae; SMI will be 0 for parallel plates and 3 for cylindrical rods – Tb.Th (trabecular thickness) Mean thickness of trabeculae, assessed using direct 3D methods mm Tb.N (trabecular number) Measure of the average number of trabeculae per unit length 1/mm Tb.Sp (trabecular separation) Mean distance between trabeculae, assessed using direct 3D methods mm Conn.D (connectivity density) A measure of the degree of connectivity of trabecular normalized by total volume 1/mm3 Cortical Bone Ct.Th Ct.Ar/Tt.Ar Ct.V Average cortical thickness Cortical area fraction Cortical volume mm % mm2 Fig. 2. Universal Machine strength and biomechanical simulation test three-point bendin tibia are placed up against a machine support with a dedicated front face to the base of th Table 2 The effect of boron on body weight and blood glucose in control and diabetic mice supplemented and non-supplemented with boron during the experimental period of 30 days. Crtl (n = 10) Crtl±B (n = 8) Diab (n = 5) Diab±B (n = 5) Weight (g) Day 01 26 [24–26] 25 [22–28] 26 [26–28] 24 [24–26] Day 15 24 [24–26] 25 [24–28] 24 [22–24] 22 [20–26] Day 30 26a [24–26] 24ab [22–28] 22b [20–24] 20b [16–24] Blood Glucose (mg/dl) Day 01 113.5a [96–128] 108a [98–117] 360b [308–500] 390b [320–550] Day 15 114.5a [94–154] 118.5a [111–130] 585b [440–593] 551b [545–593] Day 30 109.5a [98–161] 120a [101–140] 556b [403–560] 580b [550–593] Note: Two way nonparametric ANOVA. The values are expressed in median, min- imum and maximum. Different letters (a, b, ab) p ≤ 0,05. Crtl = control animal non-supplemented; Ctrl±B = control animal supplemented; Diab = diabetic animal non-supplemented; Diab±B = diabetic animal supplemented. g. The picture illustrates the correct positioning of the bone (colon of the femur and e machine) to the test. The results of a determination of minerals in the serum of dia- betic animals and controls are in Table 3. The determination of boron showed that the Diab±B group presented greater values than the Ctrl group. There was no difference for levels of calcium and phosphorus. Magnesium was higher for the Ctrl and Ctrl±B groups compared with Diab and Diab±B (p < 0.05). The results of the three point biomechanical assay test on the femur of diabetics and controls animals are in Table 4. The Diab group showed less strength than the Ctrl, Ctrl±B and Diab±B group (p < 0.05). The stiffness and energy absorption in the Diab group were lower than Ctrl, and the Diab±B group maintained the values similar to the control groups. The results of the microtomography of the cortical bone are in Table 5. Cortical bone from Ctrl±B and Diab±B groups displayed more cortical volume (Ct.V) than Ctrl and Diab groups. Animals of the Ctrl±B group showed higher values of cortical area fraction R. Dessordi et al. / Journal of Trace Elements in Medicine and Biology 39 (2017) 169–175 173 Table 3 The effect of boron on concentrations of calcium, phosphorus, boron and magnesium in the serum of control and diabetic mice supplemented and non-supplemented with boron during the experimental period of 30 days. Crtl (n = 10) Crtl±B (n = 8) Diab (n = 5) Diab±B (n = 5) Boron (�g/l) 81.5a [53.3–97.7] 98.7ab [74.1–126.4] 83.4ab [37.2–107.1] 114.9b [93.3–160.8] Calcium (mg/dl) 25.0 [22.9–28.9] 25.6 [18.7–32.5] 24.3 [17.4–25.6] 22.2 [18.1–28.3] Phosphorus (mg/dl) 17.2 [15.4–20.0] 18.4 [13.8–21.9] 16.1 [13.4–21.4] 18.4 [13.5–23.1] Magnesium (mg/dl) 2.4a [2.2–2.8] 2.6a [2.2–2.9] 1.8b [1.8–1.9] 2.1b [1.9–2.3] Note: Two way nonparametric ANOVA. The values are expressed in median, minimum and maximum. Different letters (a, b, ab) p ≤ 0,05. Crtl = control animal non- supplemented; Ctrl±B = control animal supplemented; Diab = diabetic animal non-supplemented; Diab±B = diabetic animal supplemented. Table 4 The effect of boron on bone strength parameters, by performing the biomechanical test of three-point bending on femurs of control and diabetic mice supplemented and non-supplemented with boron during the experimental period of 30 days. Crtl (n = 10) Crtl±B (n = 8) Diab (n = 5) Diab±B (n = 5) Maximum Strengh (N) 23.8a [2.03–24.7] 21.8a [20.3–22.6] 17.76b [14.8–19.1] 21.9a [19.3–25.1] Displacement (mm) 0.4 [0.3–0.4] 0.3 [0.2–0.5] 0.3 [0.2–0.3] 0.3 [0.3–0.3] Stiffness (N/mm) 109.0a [106.1–112.5] 104.1ab [93.9–109.9] 89.9b [64.4–99.6] 100.9ab [93.2–112.7] Energy Absorption (N.mm) 7.8a [5.9–9.3] 6.3ab [3.4–8.7] 3.4b [1.8–4.5] 5.3ab [4.1–9.8] Note. Two way nonparametric ANOVA. The values are expressed in median, minimum and maximum. Different letters (a, b, ab) p ≤ 0,05. Crtl = control animal non- supplemented; Ctrl±B = control animal supplemented; Diab = diabetic animal non-supplemented; Diab±B = diabetic animal supplemented. Table 5 The effect of boron on bone parameters computed microtomography of cortical bone on femurs of control and diabetic mice supplemented and non-supplemented with boron during the experimental period of 30 days. Crtl (n = 10) Crtl±B (n = 8) Diab (n = 5) Diab±B (n = 5) Ct.V (mm2) 1.03a [1.02–1.06] 9.02b [9.31–9.64] 1.04a [1.03–1.10] 9.61b [9.23–9.99] Ct.Ar/Tt.Ar (%) 40,6a [38.2–43.4] 48.6b [44.1–57.9] 37.4ab [35.9–43.1] 42.5b [40.6–44.4] Ct.Th (mm) 1.96 [1.5–2.2] 1.5 [1.4–1.9] 1.7 [1.6–1.9] 1.5 [1.6–1.7] N inimu s upple v ( D r o t o a s v D D C g g t l i t ( i a a b 4 m o m b ote: Two way nonparametric ANOVA. The values are expressed in median, m upplemented; Ctrl±B = control animal supplemented; Diab = diabetic animal non-s olume fraction; Ct.Th = average cortical thickness. Ct. Ar/Tt.Ar) higher compared with the Ctrl and Diab (p < 0.05) and iab±B showed values similar with Ctrl±B. Table 6 presents the results of the analysis of the microtomog- aphy of the trabecular bone. Fig. 3 shows a representative image f the trabecular bone of each group of the study. The choice of he figures was based on the analysis of the qualitative variables f each animal and which profile was in common among diabetics nd control groups. Therefore, a picture of an animal had been cho- en that best represented the characteristics of the group. The bone olume fraction was higher for the Ctrl±B group than for the Ctrl, iab and Diab±B. The specific bone surface (BS/BV) was greater in B group compared to Ctrl. The trabecular thickness for the Ctrl and trl±B groups presented values significantly higher than the Diab roup (p < 0.05). There were no significant differences between the roups for the parameters of bone volume, SMI, trabecular number, rabecular separation, or connectivity density. It is noted that the analysis parameters related to trabecu- ar and cortical bone have a significant overall effect of boron n the diabetic state of animals and with boron supplementa- ion for parameters Ct.V, Ct.Ar/Tt.Ar, BV/TV, BS/BV, SMI and Tb.Th p < 0.001). The same can be observed for the three points bend- ng test for parameters of maximum strengh, stiffness, and energy bsorption (p < 0.001). Regarding the concentration of minerals nd diabetic state, there was no significant overall effect of boron etween groups (p = 0.582). . Discussion The present study investigated the possible changes in bone etabolism between control and diabetic animals supplemented r not with boron through tests that enabled the evaluation of bone icrostructure, strength and concentration of minerals related to one health. m and maximum. Different letters (a, b, ab) p ≤ 0,05. Crtl = control animal non- mented; Diab±B = diabetic animal supplemented; BV = bone volume; BV/TV = bone According to Olofsson [17], the bone complications arising from chronic hyperglycemia caused by diabetes show that in diabetic patients there is also the risk of deficiencies in vitamins and min- erals. The literature shows that these patients have increases in urinary excretion of some minerals such as calcium, phosphorus and magnesium, which directly relate to bone health. The choice of this nutrient was based on studies that suggest the essentiality of this mineral in bone structure and mineralization, as well as the influence it exerts on the metabolism of some bone minerals matrix components [13,1]. The concentration of boron that was given in serum form to the control and diabetic animals showed that the Diab±B group presented boron concentrations sig- nificantly higher compared to the Ctrl group, suggesting that this increase could be due to supplementation. The analysis of the boron concentration in plasma or serum may be indicative of the nutri- tional status of the boron. A study in humans showed 1.5 times higher plasma levels in response to the increase of this mineral in the diet [18]. Boron supplementation for 60 days in perimenopause women showed an increase in their plasma levels [19,20]. In this study, we did not observe a significant difference in calcium levels in the serum of animals in both groups. Studies conducted by Derivan; Volpe [19] showed that boron supplemen- tation reduced urinary excretion of calcium and increased plasma concentrations of this mineral in humans. However, some studies have shown that boron does not significantly affect the levels of calcium and phosphorus, even in cases of deprivation and in this sense where the bone changes relate to a lower bone turnover lead- ing to insufficient strength and loss of bone structure in rodents [11,18,21]. The levels of magnesium in animals Diab and Diab±B showed a significant reduction compared to the Ctrl and Ctrl±B groups, possibly because of the diabetes. In diabetic animals, boron sup- plementation was ineffective for increasing magnesium levels, 174 R. Dessordi et al. / Journal of Trace Elements in Medicine and Biology 39 (2017) 169–175 Table 6 The effect of boron on bone parameters computed microtomography of trabecular bone on femurs of control and diabetic mice supplemented and non-supplemented with boron during the experimental period of 30 days. Crtl (n = 10) Crtl±B (n = 8) Diab (n = 5) Diab±B (n = 5) BV (mm3) 0.03 [0.02–0.06] 0.05 [0.03–0.10] 0.03 [0.01–0.05] 0.04 [0.03–0.06] BV/TV(%) 2.13a [1.93–2.28] 4.58b [3.20–6.74] 1.91a [0.98–2.69] 2.3a [2.3–2.7] BS/BV (mm3) 84.6a [62.6–92.9] 86.7ab [80.2–93.2] 94.7ab [77.6–99.3] 95.8b [89.4–111.6] SMI 2.70 [2.60–2.80]a 2.52 [2.40–2.80]ab 2.34 [2.10–2.50]b 2.5 [2.1–2.8]ab Tb.Th (mm) 0.04a [0.04–0.05] 0.05a [0.04–0.06] 0.03b [0.03–0.04] 0.04ab [0.03–0.05] Tb.N (1/mm) 0.49 [0.37–0.68] 0.59 [0.43–0.99] 0.51 [0.21–0.70] 0.54 [0.35–0.70] Tb.Sp (mm) 0.43 [0.40–0.52] 0.41 [0.37–0.46] 0.46 [0.38–0.66] 0.54 [0.46–0.58] Conn. D. (1/mm3) 43.2 [31.3–59.7] 46.2 [34.4–64.6] 30.4 [19.2–67.5] 46.7 [30.9–93.1] Note: Two way nonparametric ANOVA. The values are expressed in median, minimum and maximum. Different letters (a, b, ab) p ≤ 0,05. Crtl = control animal non- s upple v b.Th = C p n r r c u m s s n s t s a i t c S i p t h r m F ( upplemented; Ctrl±B = control animal supplemented; Diab = diabetic animal non-s olume fraction; BS/BV = specific bone surface; SMI = structure model index; T onn.D.= connectivity density. ossibly in response to diabetes that causes an increase in the uri- ary excretion of some minerals. Derivan; Volpe [19] studied the elation between magnesium and boron supplementation and the esults showed the opposite because boron increases magnesium oncentrations in plasma in animals supplemented and also atten- ated urinary excretion. It must be pointed out that the animals entioned in the study of Derivan; Volpe were not diabetic. The results of the biomechanical test performed on the femur howed that diabetic supplemented animals presented higher bone trength compared to the Diab group and observed the mainte- ance parameters of stiffness and energy absorption for diabetic upplemented animals compared to controls. It was observed hat diabetic animals have susceptibility to present loss of bone trength, which can be ameliorated by supplementation with boron nd even animals that are not ill may benefit from supplementation. Armstrong et al. [22] evaluated the boron supplementation abil- ty to increase bone strength in femurs of pigs. The results showed hat animals supplemented with boron had higher bone strength ompared to the animals that received a non-supplemented diet. heng et al. [23] evaluated the effects of boron supplementation n femurs and tibias of rats and observed no increase in strength arameters in the supplemented animals however it was found hat boron deprivation impairs bone strength parameters. Thus, a igher intake of boron for animals deprived of this mineral can esult in improvements in bone strength. Hakki et al. [11] found a similar result in femurs of rabbits sub- itted to a biomechanical test after boron supplementation, and ig. 3. �CT of trabecular bone, in the secondary spongy to 0.6 mm proximal growth plate u Ctrl±B) and diabetic animals non-supplemented (Diab) and supplemented (Diab±B). mented; Diab±B = diabetic animal supplemented; BV = bone volume; BV/TV = bone trabecular thickness; Tb.N = trabecular number; Tb.Sp = trabecular separation; Ghanizadeh et al. [1] verified increases in maximum strength and resistance of rat femurs supplemented with boron and fluorine, sustaining the results of this study. The analysis of bone computed microtomography was done to evaluate the bone microstructure, an essential study to assess the action of boron on the trabecular and cortical bone in the femur. Studies suggest that boron action is directed toward the improve- ment of the trabecular bone [13,24,25]. In this study, in addition to the improvements in the parameters for the trabecular bone, it can also be noted that there was a significant improvement in the parameters of the cortical bone of supplemented diabetic and control animals compared to those not supplemented, groups (Diab and Ctrl). The supplemented animals showed a significant gain in bone volume compared to the Diab and Ctrl groups and the Diab±B animals presented values of BV/TV similar to controls. The same was not observed in the Diab group. While analyzing the effects of diabetes and supplementation with boron, it was observed that diabetics animals are susceptible to presenting a change in the microstructure of the bone, leading to bone fragility and consequently increasing the probablility of a fracture to occur. Supplements with boron given to diabetic animals were able to improve the change in bone microarchitecture in comparison to the diabetic animals that received no supplementation. In addition, control animals that received the supplementation also showed improved bone health compared to the unsupplemented group. The gain in bone volume and maintenance of BV/TV is similar to the study conducted by Nielsen and Stoecker [24] and with the p to 2.5 mm proximal of control animals non-suppplemented (Ctrl), supplemented ts in M r b r p p t c w i a t t S h m m d b e t s d C F A t R [ [ [ [ [ [ [ [ [ [ [ [ [ [ [24] F.H. Nielsen, B.J. Stoecker, Boron and fish oil have different beneficial effects on strength and trabecular microarchitecture of bone, J. Trace Elem. Med. Biol. R. Dessordi et al. / Journal of Trace Elemen esults of the biomechanical, which showed that the femur of dia- etic supplemented animals presented higher bone strength. With espect to the trabecular bone, we observed higher maintenance of arameter values of BV/TV and Tb. Th in control and diabetic sup- lemented animals compared to the controls, respectivaly. Also, he animals of the Diab±B group showed higher values of BS/BV ompared to the Ctrl group. Similar results to the trabecular bone ere observed in the study of Nielsen and Stoecker [24], by evaluat- ng the influence of deprivation of this mineral. Microtomography nalysis performed by these researchers at the fourth vertebra of he lumbar spine indicated that boron deprivation led to a reduc- ion in the fraction of bone volume, reduced trabecular thickness, MI and, increased the trabecular separation. This study shows that igher boron intakes improve bone microarchitecture. From the results obtained, we concluded that boron supple- entation improved parameters related to bone strength and icrostructure of cortical and trabecular bone in supplemented iabetic and controlled animals. 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dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x dx.doi.org/10.1111/j.1753-4887.2008.00023.x http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 http://refhub.elsevier.com/S0946-672X(16)30308-X/sbref0125 Boron supplementation improves bone health of non-obese diabetic mice 1 Introduction 2 Materials and methods 2.1 Animals 2.2 Experiment 2.3 Bone computed microtomography 2.4 Biomechanical assay at three point test in femur 2.5 Statistic analysis 3 Results 4 Discussion Conflict of interest Funding Acknowledgement References