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

Abstract

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 6.5 mg/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, 40 nm). 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.

Introduction

Copper (Cu) is one of three most abundant essential trace elements in the body of animals and humans, besides iron and zinc. It is required for normal functioning of all basic biochemical and physiological processes in the organism. The content of Cu in tissues changes throughout the lifetime in both animals and humans. Tissues in growing animals contain three times higher content of Cu than that in adults. However, bones contain the lowest amount of Cu [1], [2], [3]. It should be mentioned that Cu plays an important role in bone metabolism and turnover. [4]. It is essential for normal growth and development of the skeleton. Bone tissue is composed of organic (predominantly collagen type I) and inorganic (nanocrystal-line HA) components. Cu is needed for the action of lysyl oxidase, a Cu-dependent enzyme, which mediates the final step in the biosynthesis of collagen, elastin, and keratin (hair keratinisation) and normalizes the deposition of calcium and phosphorus in bones [5]. Studies on Cu supplementation in humans and animals indicate that a poor-Cu diet leads to bone loss and reduced bone mass, resulting in a decrease in its mechanical strength and subsequent fractures [6]. A possible underlying mechanism of these alterations is the functional defect of osteoblasts (bone tissue forming cells), while the activity of osteoclasts (bone tissue removing cells) remains unchanged [4]. Thus, the process of bone formation requires an adequate and continuous supply of Cu [7]. Since bones undergo nonstop remodeling, an inadequate supply of Cu cannot support the formation phase of bone remodeling and this may lead to the clinical risk of osteoporosis in later life. Inadequate Cu dietary intake may also be an important factor in the etiology of osteoarthritis [8].

The access to copper from the environment is limited in humans and animals. Moreover, the content of Cu in the diet varies because foodstuffs differ significantly in their natural content. Food, drinking water, and Cu-containing supplements are the main sources of Cu. A traditional dietary form of Cu is inorganic copper sulfate or carbonate and organic sources such as those present in a chelated form (complexes with amino acids) with higher Cu bioavailability [8], [9]. However, comparative results of organic vs. inorganic Cu sources are not consistent. On the other hand, the use of nanoparticles (NP) of Cu has recently been considered as an alternative to inorganic or organic Cu due to their higher physical activity, which can reduce Cu doses and decrease excretion of the element to the environment. Probably, Cu-NP has the same effect on animal health and performance as the other Cu sources. The results regarding application of Cu-NP as an alternative growth promoter are almost non-existent, except for a study demonstrating better performance of piglets supplied with Cu in nanoparticles and a study showing an altered metabolic rate in embryos and depressed development of organs [10], [11].

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 the biomechanical and morphometric properties of femur. The next aim was to evaluate the effects of the diet containing different levels of Cu in these forms, i.e. the standard dose and half the standard dose, on bone metabolism in a rat model. For this purpose, the present study involved a combined use of multiple methods, i.e. mechanical endurance and densitometry in combination with the X-ray diffraction method to characterize the role of Cu in nutrition.