Effect of Cu-nanoparticles versus copper carbonate salt supplementation in low-copper diet: analysis of humerus biomechanical response in a rat model study

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Department of Physics, Faculty of Production Engineering, University of Life Sciences in Lublin

Effect of Cu-nanoparticles versus copper carbonate salt supplementation in low-copper diet: analysis of humerus biomechanical response in a rat model study

Siemowit Muszynski, Katarzyna Ognik,

Maigorzata Kwiecien, Piotr Dobrowolski,

Ewelina Cholewinska, Agnieszka Tomczyk,

Katarzyna Kwiatkowska, Natalia Kowal, Marta Ejtel,

Agnieszka Leus, Estera Kaminska

Copper is required for normal bone growth and metabolism. The purpose of this study was to assess the effect of adding of Cu (in form of copper carbonate or nanoparticles) at the reduced level of 50% of daily demand to growing rats evaluated on the basis of mechanical and geometric parameters of humerus. Twenty four 5-weeks old male Wistar rats were divided into control group fed diet without Cu supplementation and two experimental groups fed either a diet supplemented with Cu as copper carbonate at 3.25 mg kg1 of the premix or a diet with a Cu nanoparticles (40 nm) at the same dosage. The supplementation lasted 4 weeks. At the end of experiment, there were no changes in rats body weight and generally in bone geometry, but significant alteration was noted in the structural parameters, which was partially dependent on Cu source. Our study showed an increase in work to fracture and toughness significantly in both Cu-supplemented groups, while the increase of elastic energy was observed only when Cu was supplemented in form of nanoparticles. On contrary, the yield load and stiffness decreased in Cu-Salt-treated rats. The values of most material parameters were also altered after Cu supplementation, irrespective of its source, as increase of yield strain, ultimate strain and yield stress as well as an decrease of Young modulus in both Cu-treated groups was observed. 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.

Keywords: COPPER, NANOPARTICLES, HUMERUS, MECHANICAL TESTING, RAT.

Вплив наночастинок Cu в порівнянні з карбонатом міді в раціоні з низьким вмістом міді: аналіз біомеханічної реакції плечової кістки в модельному досліді на щурах

Анотація

Мідь потрібна для нормального росту і метаболізму кісток. Метою дослідження було оцінити ефект додавання Cu (у формі карбонату міді або наночастинок) при зниженому рівні 50 % добової потреби для щурів, що ростуть, на основі механічних та геометричних параметрів плечової кістки. Двадцять чотири 5-тижневі самці щурів Wistar ділили на контрольні групи, що годували раціоном без додавання Cu, і дві експериментальні групи з раціоном з Cu у формі преміксу карбонату міді на рівні 3,25 мг / кг-1 або раціону з наночастинками Cu (40 нм) однакової дози. Вигодовування тривало 4 тижні. Наприкінці експерименту не було змін у масі тіла щурів та загалом в геометрії кістки, але значні зміни зазначалися в структурних параметрах, що частково залежать від джерела Cu. У щурів із CuSalt-раціоном знизився приріст маси.. Значення більшості матеріальних параметрів також змінювалися після додавання Cu, незалежно від його джерела, в обох групах, що отримували Cu. Це дослідження показало, що Cu-NP, що вводиться в низьких дозах, збільшує механічну витривалість кістки, без змін деформації та напруженості в порівнянні з низькою дозою Cu, що дається в традиційній формі.

Ключові слова: МІДЬ, НАНОЧАСТИНКИ, ПЛЕЧОВА КІСТКА, МЕХАНІЧНЕ ВИПРОБУВАННЯ, ЩУРИ.

Влияние наночастиц Cu по сравнению с карбонатом меди в рационе с низким содержимым меди: анализ биомеханической реакции плечевой кости в модельном опыте на крысах

Аннотация

Медь нужна для нормального роста и метаболизма костей. Целью исследования было оценить эффект добавления Cu (в форме карбоната меди или наночастиц) при сниженном уровне 50 % суточной потребности для крыс, которые растут, на основе механических и геометрических параметров плечевой кости. Двадцать четыре 5-недельных самца крыс Wistar разделили на контрольные группы, которых кормили рационом без добавления Cu, и две экспериментальных группы с рационом с Cu в форме премикса карбоната меди на уровне 3,25 мг / кг- 1 или рациону с наночастицами Cu (40 нм) одинаковой дозы. Выкармливание длилось 4 недели. В конце эксперимента не было изменений в массе тела крыс и в целом в геометрии кости, но значительные изменения отмечались в структурных параметрах, которые частично зависят от источника Cu. У крыс из CuSalt- рационом снизился прирост массы. Значения большинства материальных параметров также изменялись после добавления Cu, независимо от его источника, в обеих группах, которые получали Cu. Это исследование показало, что Cu - NP, что вводится в низких дозах, увеличивает механическую выносливость кости, без изменений деформации и напряженности по сравнению с низкой дозой Cu, которая дается в традиционной форме.

Ключевые слова: МЕДЬ, НАНОЧАСТИЦЫ, ПЛЕЧЕВАЯ КОСТЬ, МЕХАНИЧЕСКОЕ ИСПЫТАНИЕ, КРЫСЫ.

Copper (Cu) is an essential metal micronutrient closely linked to bone metabolism [1, 2]. Cu-dependent enzyme, lysyl oxidase, mediates in the biosynthesis of collagen, elastin, and keratin and normalizes the deposition of calcium and phosphorus in bones [3]. Studies on animals indicate that a poor-Cu diet leads to bone loss and reduced bone mass, resulting in a reduction of its mechanical strength and increases the risk of fractures [4]. Since bones undergo nonstop remodeling, bone formation requires an adequate and continuous supply of Cu.

Food, drinking water, and mineral supplements are the main sources of Cu. A traditional dietary form of Cu presents in supplemental mineral mixture is inorganic copper sulfate or carbonate. Recently, the use of elements in form of nanoparticles of has been considered as an alternative to inorganic forms due to their higher physical activity. To date, the results regarding application of Cu nanoparticles as an alternative growth promoter in livestock animals are limited. However, in pigs feeding, some researchers demonstrated better performance of piglets supplied with Cu in nanoparticles, showed an altered metabolic rate in embryos and depressed development of organs [5, 6]. Further, the study performed on broiler chickens showed that femoral bones from the group supplemented in ovo with Cu nanoparticles were characterised by a higher weight and volume and by significantly greater resistance to fractures compared to the non-supplemented group [7]. On the other hand, copper nanoparticles did not affect immunoglobulin concentrations or humoral responses [6].

Essentially copper nanoparticles at smaller concentrations can demonstrate positive responses in the microbiota while promoting growth and performance [8]. However, there are no scientific analyses involving the use of Cu nanoparticles in animal feeding and their effect on the bone structure when included in diet.

The objective of the present study was to investigate the geometrical, biomechanical and densitometric traits of the humerus in growing male rats given Cu in two different chemical forms (carbonate and nanoparticles) at the reduced level of 3.25 mgkg¹ of the premix. With regard to the important role of Cu in the development of bones, it is hypothesized that the use of Cu in a more assailable form of nanoparticles might improve the development of the skeletal system, even if it is administered at the reduced level of 50% of daily recommended dose.

Materials and methods. Animal protocol and dietary treatments

All animal care and experimental protocols were approved by the respective Local Institutional Animal Care and Use Committee in Lublin.

In the experiment, twenty four healthy male, albino Wistar rats of 5 weeks were randomly allocated to three dietary treatments: control group fed without additional Cu supplementation in premix (the CONT group), first experimental group fed with lowered level of inorganic copper as cooper carbonate (the CuSalt group) in premix and the second experimental group fed with lowered level of copper as nanoparticles (Sky Spring Nanomaterials, Inc. USA) of the size of 40 nm (the CuNano group). Rats were housed individually in stainless steel cages under a stable temperature of 21-22 °C and 12-h light-dark cycle. For 4 weeks, the rats had ad libitum access to tap water and experimental semi-purified diets. The diets were modifications of a casein diet for laboratory rodents recommended by the American Institute of Nutrition. After 4 weeks, at the end of the experiment, the rats were fasted for 24 hours and euthanized one by one with carbon dioxide inhalation and by dislocation of the spine.

Bone collection, geometrical analysis and densitometric evaluation. The bone length and weight were measured after removal of soft tissues from left humerus. Geometric properties such as the cross-section area, mean relative wall thickness, and cortical index were estimated on the basis of horizontal and vertical diameter measurements of the mid-diaphyseal cross-section of bone [9]. The second moment of inertia and radius of gyration about medial-lateral axis were also calculated [9]. Bone mineral density was assessed using the dual-energy X-ray absorptiometry (DXA) method on a Discovery W Hologic densitometer (Bedford, MA, USA) [10]. The measurement of bone tissue density was performed with an AccuPyc 1330 helium gas pycnometer (Micromeritics, Inc., Norcross, GA, USA) [11].

Mechanical testing. The mechanical properties of the humerus were determined using the three-point bending test of right humerus on a Zwick Z010 universal testing machine (Zwick GmbH & Company KG, Ulm, Germany). The distance between the supports was set at 40 % of the total bone length. Ultimate load was determined as the force causing bone fracture and the yield load as maximal force under elastic deformation of bone [9]. On the basis of measured geometric and mechanical traits the following material properties of the mid-diaphyseal fragment of the bone were calculated: bending moment, indicating bone elastic load capability [9]; yield stress, reflecting the elastic strength of midshaft cortical bone; the ultimate stress, equal to the maximum stress a bone can withstand in bending before fracture; Young modulus of elasticity, describing bending resistance of the bone [9]; yield strain, indicating the maximum strain the bone can withstand for reversible deformation and ultimate strain, describing the relative deformation occurring when the fracture load is applied. The determined bone structural traits were: stiffness, describing the bone resistance to deformation; toughness, as a bone resistance to fracture; elastic energy, as the energy absorbed by bone in elastic region and work to fracture, as a total work done to break the bone [9].

Statistical analysis. The results of the experiment are expressed as means values. As our aim was to evaluate the effect of an addition of carbonate salt or nanoparticles copper mineral supplements on the physical traits (geometry and mechanical properties) of humerus, statistical analysis was carried out with one-way ANOVA. Contrast analysis was applied in order to compare the effect of Cu supplementation source and the control group. Analyzed contrasts were as follows: (1) the control diet (CONT) vs. the diet containing inorganic form of Cu (CuSalt) and (2) CONT vs. the diets containing Cu in forms of nanoparticles (CuNano). Significance was declared at P<0.05. All statistical analyses were carried out by means of Statistica 12.0 software (StatSoft Inc., Tulsa, OK, USA).

Results and discussion. There was no effect of the form of Cu used on feed intake or weight gain during 4 weeks of the study (data not shown). Table presents the effects of copper supplementation in low-copper diet on measured bone physical properties.

copper biomechanical humerus

The effect of a diet containing copper in inorganic or nanoparticles form at level of 50% of recommended dosage on bone general properties, midshaft geometrical properties and biomechanical properties of humerus obtained from 9-weeks-old rats