Effect of whole body vibration on bone nanocomposites organisation and prevention of loss of bone mineral density under conditions of modeling obesity and sedentary lifestyle: experimental study

This study aimed to investigate the influence of high-frequency whole body vibration (WBV) on metabolic and structural responses of rats' bone tissue under the sedentary lifestyle and obesity. Obesity combined with a sedentary lifestyle can present the potential negative health effects. However, whole body vibration can be used as a means of non-pharmacological correction of bone mineral density. For characterization of bone nanocomposites organisation and prevention of mineral density loss, X-ray diffraction method was used. Markers of bone remodeling in the rats' blood: leptin, osteocalcin, tartarate resistant acid phosphatase 5b, alkaline phosphatase. Using a high-calorie diet and low-mobility model, we proved that bone mineral mass had been decreasing since 8th week. It should be noted that the decrease in the relative amount of crystalline phase (hydroxyapatite) continued throughout the experiment, up to 24 weeks (p<0.05). These structural changes were accompanied by changes in quantitative indicators of the bone remodeling markers. Rats had lower bone mineral density compared to the animals that were on the normal diet and were additionaly affected by WBV. We observed the increase of the crystalline phase volume fraction from 84% to 93% (p<0.05) in group with additional whole body vibration and the decrease of the mineral component in rats with limited mobility and high-calorie diet. Therefore, WBV could improve structural conditions of bone and prevent fat accumulation and obesity-associated biochemical markers in obese rats. This can be an effective method to improve the structural and functional state of the bones while preventing the loss of bone mineral density.

observed the increase of the crystalline phase volume fraction from 84% to 93% (p<0.05) in group with additional whole body vibration and the decrease of the mineral component in rats with limited mobility and high-calorie diet. Therefore, WBV could improve structural conditions of bone and prevent fat accumulation and obesity-associated biochemical markers in obese rats. This can be an effective method to improve the structural and functional state of the bones while preventing the loss of bone mineral density.
Obesity and a sedentary lifestyle are risk factors for chronic health disorders such as heart disease, diabetes type 2, osteoporosis, etc. [13]. However, there is evidence of these factors' positive impact on bone tissue formation through the increased mechanical stress [11,12], that can stimulate the bone tissue remodeling by reducing apoptosis of osteoblasts and osteocytes, increasing their proliferation and differentiation [10,17]. Therefore, the assumption that obesity leads to an increase of mechanical stress on the bone and facilitates the prevention of osteopenia is fallacious. Reduced functional stress leads to the decrease both of the bioelectric potentials and circulation intensity, which inhibits bone formation and stimulates resorption of the bone tissue [2, 14,15,16]. Lack of exercise causes reduced microvasculature capacity and capillary network, when the first signs of bone atrophy and osteoporosis are manifested, and long-term reduction of functional stress may cause irreversible changes in the bone tissue. As a result, the regressive transformation of bone leads to increased porosity and osteoporotic fractures of bone structures, even under minimal stress [7,9]. Despite conflicting data, the pathophysiological connection between obesity, sedentary lifestyle and bone mineral density (BMD) loss is rather complicated and requires further study. The majority of researches is devoted to studying the influence of high frequency vibration acceleration ≤ 0.5 g. In particular, Rubin and McLeod proved high bone sensitivity to mechanical stimuli. By modelling general vibration fluctuations with the frequency of 30 Hz and vibratory acceleration of 0.3 g for 5 min daily within 30 days, the scholars have determined bone mass acquisition in the trabecular layer of turkeys' tibia. Much later, Rubin et al., 1994, proved anabolic effect of the aforementioned fluctuations and deceleration of remodelling bone tissue by means of retardation of osteoclastogenesis processes (downregulation of RANKL and cytokines related to osteoclastogenesis).
The aim was to study the influence of highfrequency whole body vibration on the process of remodeling of the rats' bone tissue under the sedentary lifestyle and obesity conditions. For characterization of bone nanocomposites organisation and prevention of bone mineral density loss Xray diffraction method was used [3,4]. We determined markers of bone remodeling in the rats' blood, which allow the definition of actual bone metabolism.

MATERIALS AND METHODS OF RESEARCH
The experimental study was performed on 54 male rats of the Wistar line weighing 180-200 g, kept under the same vivarium conditions. All animal experiments were conducted in compliance with The experimental rats were divided into 3 groups, 18 rats in each: control group -standard vivarium conditions, I experimental group -limited mobility condition + high-calorie diet (LMC+HCD), II experimental group -LMC+HCD+WBV. Obesity condition was modeled through a high-calorie diet (C 11024, Research Diets, New Brunswick, NJ); limited mobility condition was modeled using partition cages to restrict the rats' mobility. All experimental rats were weighed every two weeks. Vertical vibration oscillations were modeled using a 250 W vibrating table with the maximum pressure of 7 bar and 50 Hz frequency, g -0.3. After the 8 th , the 16 th and the 24 th week, six animals from each group were removed from the experiment by decapitation under general intraperitoneal anesthesia at 0.3 g/kg.
Concentrations of cytokines and bone remodeling markers in blood plasma were determined using commercial immune-enzyme analysis kits (ELISA). We determined leptin (anti-Leptin (rat), pAb, Adi-poGen Life Sciences, osteocalcin (DRG® Mouse Osteocalcin ELISA); Mouse Tartarate Resistant Acid Phosphatase 5B (TRAP5B) ELISA Kit, MyBioSource. The activity of alkaline phosphatase in blood serum was performed through photocolorimetry. All analyses were performed according to the manufacturer's instructions [5,8].
To study the ultrastructure of the bone mineral component we used the method of X-ray diffraction analysis. The femur was dried at 110°C in a drying cabinet. The X-ray diffraction spectra of the samples were obtained on an automated X-ray diffractometer DRON-3 in Cu Kα radiation (λ=1.5418 Å), monochromatized by reflection from a plane (002) of a single pyrographite crystal, mounted on a diffracted beam. We used the Bragg-Brentano focusing scheme (θ-2θ). [3,4,6,8].The diffraction patterns were recorded in the continuous movement mode of the detector with an angular velocity of 2 0 /min, a constant value of the integration time τ=1 s., x-ray tube voltage at U=26 kV, and anode current at I=15 mA. To estimate the quantitative content of the amorphous and crystalline phases we used the following ratios:    (I am -the maximum intensity from the amorphous phase, measured at 2θ≈21,5 0 I cr -the maximum intensity of the crystalline phase, measured at 2θ≈32,1 0 , while taking into account the background scattering).
Statistical analysis of the data was performed in StatSoft STATISTICA 8.0.360. In the STATISTICA package, the comparison of two average samples of normally distributed features (Student's t-criterion) was implemented in the Basic Statistics/Tables module. The t-test, independent, by variable submodule, was used for two different general summations. One-way ANOVA is implemented in the Breakdown & one-way ANOVA submenu of the Basic Statistics and Tables module [1].

RESULTS AND DISCUSSION
The rats' weight in I group (HCD+LMC) increased from 194.63±6.1 g to 340.82±8.62 g in the 24 th week, which indicates a statistically significant increase in weight compared to the control group (p<0.014). In the HCD+LMC+WBV group, the rats' weight increased from 198.3±6.61 g to 304.93±5.07 g in the 24 th week, respectively (p>0,05), the experimental group was not statistically different, Fig. 1.
Leptin is the primary hormone involved in the regulation of body weight. While increasing weight by 10 %, the level of serum leptin may become more than 3 times higher. Therefore, to assess energy metabolism, it is advisable to determine its concentration. In the 8 th week of the experiment, the leptin level in control group was 5.25±0.42 ng/ml, in I experimental group it amounted to 15.01±1.19 ng/ml (p=0.000007), and in II -11.13±1.71 ng/ml (p=0.004). In the 16th week of the experiment, the dynamics of leptin levels was the following: in the control group it remained nearly unchanged at 5.91±0.35 ng/ml, in I experimental group the average level was at 21.01±1.95 ng/ml (р=0.000008), in II -16.07±1.84 ng/ml (p=0.00014). In the 24 th week of the experiment: the control group showed 4.93±0.25 ng/ml, I experimental group -24.51±2.29 ng/ml (р=0.000003), and II -18.07±1.67 ng/ml (p<0.032) ( Fig. 2A). Regression analysis found that the mass depends on the level of leptin and is described by the equation: Weight =179.51-6.4 * Leptin, with a correlation coefficient of 0.98.
To determine bone remodeling we investigated bone formation markers -osteocalcin, alkaline phosphatase and bone resorption marker -TRAP-5b. Blood analysis showed significant differences in osteocalcin levels between the control and experimental groups of rats (Fig. 2B)  The diffraction patterns of the femur series samples are shown in Fig. 4 compared to the theoretical diffraction pattern of the Ca 10 P 6 O 26 H 2 chemical compound (hexagonal syngony, space group P 63/m, unit cell parameters a=9.42 Å, c=6.88 Å). Significant erosion of the diffraction maxima of the Ca 10 P 6 O 26 H 2 crystalline phase indicates a low degree of crystallinity of the compound due to the small size of the coherent scattering regions (the crystallite size does not exceed 10 nm). Also, a wide diffuse halo is observed in the diffraction patterns around the diffraction angle 2θ≈21 0 , indicating the presence of an amorphous (disordered) phase represented by collagen fibers in the samples. In the series of samples, the highest content of the amorphous phase is observed in the control group. The decrease in the intensity of the diffuse maximum of I experimental group sample indicates a decrease in the content of the amorphous phase ( fig. 3). At the same time, in the sample of II experimental group we observe both a decrease of the diffuse maximum and an increase in the intensity of the maxima of the crystalline phase, which is especially pronounced in the region of the most intense lines (211), (121), (112) and (300) of the Ca 10 P 6 O 26 H 2 phase.
The calculations results are shown in Table 1. In the 8 th wk. → 16 th wk. → 24 th wk. sequence, we can observe the increase of the crystalline phase volume fraction from 84% to 93%, (p<0.05) in II experimental group and the decrease of the mineral component in I experimental group, (p<0.05), (Fig. 4, Table). Using a high-calorie diet and low-mobility model, we proved that bone mineral mass had been decreasing since week 8. These structural changes were accompanied by changes in quantitative indicators of the bone remodeling markers. In our experimental model, rats had lower bone mineral density compared to the animals that were on the normal diet and were further affected by WBV. It is clear that high-frequency mechanical oscillations activate osteoblasts, accelerate metabolic processes, and slow BMD decline. The obvious competitive impact of obesity and mechanical stress on bone 21/ Том XXVІ / 1 metabolism requires further study [11,12]. Our experiment suggests that excess fat does not prevent bone mineral decline and is often associated with an increased risk of osteoporosis and osteoporotic fractures, due to excessive pressure on the musculoskeletal system. It is also known that obesity can affect bone metabolism through multiple mechanisms. Since adipocytes and osteoblasts originate from a common multipotent mesenchymal stem cell, obesity may increase adipocyte differentiation and fat accumulation, thus reducing differentiation of osteoblasts and bone formation [14]. A slight increase in proinflammatory cytokines which may also occur under obesity, may increase osteoclast activity and bone resorption through changes in the RANK/RANKL/OPG system. In the HCD+LMC group there was an increase in serum leptin and TRAP-5b, while bone formation markers, osteocalcin and alkaline phosphatase were reduced. Leptin effect on bone is ambiguous, in particular, there are reports on both positive and negative effects. According to the literature, increased leptin level (as observed in our animal models with obesity) may have a negative impact on bone metabolism. We also found out that increased leptin level in blood serum is a negative regulator of bone mass that may occur through the cytokine inhibition of osteoblasts. The activated osteoclasts form the socalled "resorption holes" with low pH level, which are the areas of the inorganic matrix destruction. It should be noted that this process occurs due to the lysosomal enzymes, namely tartrate-resistant acid phosphatase (TRAP) and cathepsin K. It allows the efficient digestion of Type I collagen and its degradation products. Osteoblasts are drawn to the ruined surface and begin to form new osteoid. Type I collagen is secreted in the form of moleculeprecursor of procollagen into the extracellular space where it breaks down to amino-and carboxylterminal propeptides with their subsequent release into the bloodstream, accompanied by an increase in the alkaline phosphatase level. First crystals of hydroxyapatite are deposited in osteoid, then undergo a process of mineralization, which lasts a few months (for adults) and then this is followed by a period of physiological respite of the bone. We must note that about 70-90% of the osteoblast-synthesized osteocalcin is fused in the bone matrix, and the rest gets into the bloodstream. Therefore, it is considered the most particular protein of bone tissue. It is also known that bone consists of organic matrix and mineral phase structural units which are composed of hydroxyapatite crystals. Osteocalcin is located predominantly in mineralized tissue acting as a mediator for matrix mineralization and has a high affinity for calcium. After separation from osteoblasts, osteocalcin is deposited in the bone matrix and released into the blood, and therefore this marker may indicate the rate of bone remodeling. A rapid increase of the osteocalcin level in the rats' venous blood in II experimental group indicates the effect of high-frequency oscillation on bone metabolism and increased osteoblasts activity.

CONCLUSIONS
Thus, whole body vibration, with acceleration of 0.3 g, have a positive effect on body weight, biochemical laboratory indicators of obesity and leads to normalization of body weight. Vibration can be used as a potential non-pharmacological correction of bone mineral density and has an antiresorptive effect for preventing bone loss in obesity. In particular, for individuals with obesity and sedentary lifestyle it is important not only to correct diet, but also to apply mechanical loads on to the На умовах ліцензії CC BY 4.0 musculoskeletal system, which was modelled by whole-body vibration platform. Therefore, vibration of the whole body with a vibration acceleration of 0,3 g can be considered as the method of weight correction and has a positive effect on the remodelingand structural state of bone nanocomposites.
Conflict of interest.