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Journal of Human Nutrition & Food Science

Cite this article: Perez-Torrero E, Zamarron-Alvarez SS, Rodriguez-Garcia ME (2018) Trabecular Structural Pattern of Femur from Perinatal Food Restricted Adult Rats. J Hum Nutr Food Sci 6(2): 1125.

*Corresponding authorEsther Perez-Torrero, Division de Investigacion y Posgrado, Facultad de Ingenieria, Universidad Autonoma de Queretaro. Cerro de las Campanas s/n, Queretaro, Qro, Mexico, CP 76010, Tel: 52- 442- 192 12 00. Ext. 65422; Fax: 52-442- 192 12 00. Ext. 6005; Email:

Submitted: 09 June 2018

Accepted: 03 July 2018

Published: 05 July 2018

ISSN: 2333-6706

Copyright© 2018 Perez-Torrero et al.

OPEN ACCESS

Research Article

Trabecular Structural Pattern of Femur from Perinatal Food Restricted Adult RatsEsther Perez-Torrero1*, Sinuhe Salvador Zamarron-Alvarez1 and Mario Enrique Rodriguez-Garcia2

1Division de Investigacion y Posgrado, Facultad de Ingenieria, Universidad Autonoma de Queretaro. Cerro de las Campanas s/n, Queretaro, Qro, Mexico, CP 76010, Tel: 52-442- 192 12 00. Ext. 65422; Fax: 52-442- 192 12 00. Ext. 60052Departamento de Nanotecnologia, Centro de Fisica Aplicada y Tecnologia Avanzada, Universidad nacional Autonoma de Mexico. Campus Juriquilla Boulevard Juriquilla No. 3001 Queretaro, Qro, Mexico, CP 76230

Abstract

Male rats control (C) and food restricted (R) feeding with different decrement percentage of daily intake, during gestation and, during lactating period rats were maintained with access to milk only 12 hrs. At 9 week of age rats were sacrificed under profound anesthesia and were submit to 3-point bending mechanical test like indicators of bone strength, after that the samples were observed to scanning electron microscope. The restriction diets during perinatal period have long lasting effects in the general physiology of the rat. Neither, the three-point bending mechanical properties, nor the tensile mechanical properties differed significantly between the groups, while there was a trend towards decreasing bending mechanical properties in restricted diet group. It was demonstrated that compared to R group, restricted diet group produces a significant decrease in body weight, femur weight and length. The C group was stronger as measured by three-point bending test and more resistant compared to R group. Additionally, the microscopy result shows that the general microstructure was altered in the R group and the bundles of collagen fibers are sparser relative to control group. The result provides evidence to adaptive changes of bone particularly the bundles collagen fibers in young expose to sever gestation food restriction and can contribute to detrimental of the bone function and induce physiopathology and metabolic bone disease in adulthood.

Keywords•Rat bone•Breaking resistance•Development•Bundlecollagenfibers•Food restriction

ABBREVIATIONSC: Control Group; R: Food Restricted Group; Ca: Calcium; SEM:

Scanning Electronic Microscope; BMD: Bone Mineral Density; P: Phosphorous

INTRODUCTIONNutritional conditions are determinant for preventing and

maintain good health throughout life. It is well known its role as important factors of chronic non-transmissible diseases, and that makes them fundamental components of prevention activities [1,2]. Nutritional deficiencies are great health problem around the world human populations, having a dramatic effect on the economic productivity [3]. Under nutrition during pregnancy has been identified as an important risk factor in many adult diseases [4,5]. The bone growth and their development are a phenomenon that occurs from the intrauterine development and remains along life [6]. Epidemiological evidence indicates that skeletal growth is programmed during intrauterine and early postnatal life [7-11].

During the first years of life (through adolescence), bone production exceeds losses remodeling. In young people exists a balance between adsorption and resorption. This process may be affected by nutritional events (deficiencies and excesses), which may result in bone illness such as osteoporosis, rickets or osteomalacia affecting a high percentage of people in the world. Bone development depends mainly on genetic and epigenetic factors. It is well known that combined genetic and epigenetic aspects define the ultimate profile and force of the bone structure [12,13]. Which means that proper organized food early in life provides considerable support for the skeletal healthy [6,13-15]. It has been established that Ca deposition in the bone at an early stage contributes to the prevention of bone diseases [16,17]. The changes that occur in any of these factors have valuable implications for the growth, development and adult bone establishment.

Studies of animals provide crucial evidence that exposure to relatively short periods of malnutrition or endocrine disturbance in fetal life can programmed risk of osteoporosis [18,19]. The range

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of nutritional exposure capable of altered the bone health in the developing offspring is broad, including micronutrient deficiency and protein restriction [20-24]. Protein deficiency in adult male rats show cortical and trabecular thinning, additionally decrease bone strength [22], protein restriction during late pregnancy and early post-natal periods show that affect thyroid status, bone formation, and mineral metabolism of their offspring profoundly, isocaloric protein undernutrition decreased bone mineral mass and strength in spongy and cortical bone [21]. Until now is little unknown the environmental modulation in the perinatal period over the skeletal growth trajectory and propensity to later osteoporotic fracture.

Bone tissue is constituted by organic (collagen) and inorganic phases (mineral), the organic phase is composed of blood, vessels, nerves, collagen, and living cells including osteoblasts, and osteoclasts [25]. The collagen fibers have high tensile strength, while the calcium (Ca) confers great compression strength of bone. These combined properties, plus the degree of bondage between the collagen fibers and the crystals, provide a bone structure that has both extreme tensile compression strength. Until now little is known about the regular spacing of the bundle of collagen fiber in an undernourished model. Later in life depends upon the peak bone mass obtained during skeletal growth, and the subsequent rate of bone loss [7]. In the present paper, we report findings from a model of caloric restriction global nutrient during the perinatal period, with the aim to consider the impact of perinatal caloric restriction in young animal upon most previous studies tended to focus in adult. To assessed bone strength and pores pattern distribution of femur adult males were performed following the “Principles of Laboratory Animal Care” [26]. The protocol was approved by the local Ethics Committees of the Universidad Autonoma de Queretaro and Universidad Nacional Autonoma de Mexico and Mexican Guide for Management of Laboratory Animals [27].

Groups

All rats were housed in appropriate environments 12:12 h light-dark cycle, humidity about 45-55% and temperature 23 ± 1°C. A total of 16 virgin adult female Wistar rats from Indianapolis Minnesota strain, weighing approximately 200 g each, were housed 3 female and 1 male during 1 to 5 days, the presence of semen in vaginal smears was considered day zero of gestation (G0). At this time, pregnant dams were singly housed and allocated to either a C group with standard diet or an R group with caloric restriction diet. At birth, all pups were mixed and culled to a maximum of eight pups per litter to prevent variation in neonatal growth.

In this study 5 male at 9 weeks of age in both groups were used. The group C with control diet the pregnant dams were fed throughout gestation and lactation period with pellets from Lab Diet (5001 rodent diet) and water ad libitum. The group R with caloric restricted diet the pregnant dam was limited with different percentages of control diet during pregnancy: G0-G6=100%, G7-G12=70%, G13-22=70% and in G23 100% of control diet in order to avoid cannibalism. At birth, two mother rats were paired, and galactophorous conducts were ligated in one of them and interchanged with each other every 12h. During suckling period, all mother rats had diet and water ad libitum. After weaning the

offspring were housed in the group of four male’s whit free access to lab diet and water ad libitum throughout the period of study.

Preparation of bone specimens

Five male rats were sacrificed with carbon dioxide aspiration by group. The body weight was recorded; both left and right femurs were removed, cleaned of surrounding soft tissue and cartilage. Femur weighed in grams using digital balance (Precision balance Sartorius TE2101) and femur length (from the longitudinal epicondyle to the distal end) and width and thickness in mid-diaphysis were recorder using Vernier calipers (Mitutoyo, Japan).

Biomechanical test

The biomechanical properties of bone were measured using a three-point-bending test. Ten femurs from each group were used. After immersing in paraformaldehyde solution (4%) a buffer solution was introduced to eliminate paraformaldehyde excess and finally distilled water was used to eliminate the residual buffer before and completely thawed before mechanical testing. Three-point bending test was used for measuring the mechanical properties of the bone as previously described in both groups [28]. Specimens were tested at temperature 25.2ºC, humidity 46% using a universal texture analyzer model TA-XT 2 (Texture Technologies Corp). Bones were placed with their anterior side down on two horizontal supports spaced 22 mm; the central loading point contacted the posterior surface of the diaphysis at the midpoint of the bone length. The loading point was displaced downward (transverse to the long axis of the bone) at a constant speed of 1mm/min until failure. Load-displacement data were recorded at TXT Universal Texturometer and test curves were analyzed to determine measures of whole-bone strength: ultimate load at fracture (N), is the applied load that leads the specimen to rupture, failure displacement is the maximum flexion of the specimen to the maximum load and tensile modulus (Young`s modulus), the Young`s modulus is a measure of the intrinsic stiffness of the material.

Scanning electron microscopy analysis

After the mechanical test, it was performed bone morphological analysis on sections obtained with a precision saw. Bones were sectioned in the middle diaphysis, and in the femoral head neck in order to obtain compact and trabecular bone respectively. Bones were cleaned and defatted in order to eliminate all organic material and to have clear images. In order to make the bone microanalysis, was used a scanning electron microscope model EVO-50, Brand Carl Zeiss, the bone samples of 9 weeks of age of both groups were placed in a sample holder and sputter coated, group n = 5 per group were used. For trabecular bone analyses, we use one specimen and ten trabecular bones randomly selected, after that, were measure with public domain image processing software (Image J, NIH).

Bone trabecular pores distribution

Analyses have been conducted to find patterns that govern trabecular structures by a method that involve image processing techniques, by most computer using software Image J. Trabecular structures showed different patterns according to the nutritional

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Figure 1 Sections of the femur head visualized under electron microscopy. Sections are from C (A) and R (B) rats at 9 weeks of age. Micrographs showed that the trabecular array and the porosity were damaged in R group. 200 X magnification.

Figure 2 Processing image from SEM micrograph using Image J.

state, whether it is that there are deficiencies which alter the skeletal system.

To evaluate the pore distribution, used micrographs of trabecular femur bone in rats from C and R groups (see Figures 3A and 3B). These pores distribution were built in order to search for patterns comparing the C and R samples, performance the trabeculae.

Statistics

The biological experiment data was analyzed by using the Statistical Package for Social Sciences (SPSS) version 17.0. The differences of the evaluated parameters were compared with ANOVA by nutrimental condition (C and R groups). Post hoc

comparisons for specific parameter of the study were evaluated with the Feisher test. The probability of ≤ 0.05 was considered statistically significant.

RESULTS AND DISCUSSION

Body weight

The body weight in R group was always less than in those in C group. The statistical analyses show that comparison of the body weight between C and R group significant effects by the diet, F (1,16) = 845.76, (p ≤0.0001). Comparison at each group indicated consistently low body weight in R rats throughout the study as compared to C rats, (p ˂0.05) and the proportion of increasing R group is reduced. In summary, the data showed that food

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Figure 3 Processing image from SEM micrograph using ImageJ. Model from trabecular pattern of femur head micrographs, visualized under optic microscopy. Sections of R group rats (A), section of C rats (B) at 9 weeks of age.

restriction during gestation and early ages paradigm interfered with the physical growth of R group. Additionally, the values for the femur length show significant differences by diet, F (1,16) = 26.51, (p ≤0.0001.

Femur weight

Additionally, the values for the femur weight show significant differences by diet, F (1,16) = 14.52, (p ≤0.001). The statistical analysis for the femur width show significant differences by diet, F (1,36) = 50.69, (p≤0.001). In the case of femur thickness show significant differences by diet, F (1,36) = 67.22, (p ≤0.001) (Table 1).

Bone mechanical properties

The mechanical test shows that all fractures occurred in mid half femur, additionally in R group the fracture is extensive to head femur. The statistical analysis of Young’s modulus shows effects by the nutritional condition F (1,36) 10.40, p ˂0.002. The ultimate load at fracture show effect by diet F (1,36) 19.12, p˂0.0001. The effect of the diet is shown by less values of failure displacement in R in contrast to C in 9 weeks of age (P ˂0.05).

MORPHOLOGICAL ANALYSISThe coronal sections of the proximal femur from C showed

a femoral head with a spherical shape and which was formed by well-preserved trabeculae with the outer surface. In the R, the main changes occurred in the femoral head, which had lost its

spherical shape and presented a flat appearance additionally the amount of trabecular bone was clearly reduce in when compared with the controls, with a collapsed and disorganized trabecular pattern, loss of the intra-trabecular space, the trabecular were thin and discontinuous, while the controls were thick and continuous. In the C group, all morphological aspects were preserved shape, height, and trabecular arrangement in the femoral head in contrast in R there are a loss of horizontal trabecular (Figures 1-5). For the diaphyseal cross-sections of the femur, there was a significant reduced area in R with reference to control group. The femoral head shows a significant decrease of R versus C, F (1,8) 30.93, p ˂0.0001, respect to circularity the femoral head of R show significant less values versus C, F (1,8) 22.04, p ˂0.0001, the trabecular thickness of the femoral head show significant differences by the diet, F (1,17) 40.86, p ˂0.0001. In more detail of the morphological of the trabecula, the intra-lamellar space is more densely package in C the group F (1,6) 15.28, p ˂0.007. The size of pores is significantly major in R versus C group, F (1,6) 18.67, p ˂0.004. Finally, the cortex thickness in the medial diaphysis of the femur in R was thinner than the C group, F (1,8) 16.80, p ˂0.003 (Table 2).

Pores distribution pattern

Table 1: Body and femur weight, length, width and thickness of femur diaphysis from C and R groups.

C R p valuesBody weight 295.05±3.01 160.05±5.68* 0.0001

FemurWeight (g) 0.74±0.04 0.63±0.02* 0.0010

Lenght (cm) 3.21±0.48 2.82±0.03* 0.0411Width (mm) 4.22±0.04 3.90±0.05* 0.0001

Thickness (mm) 3.12±0.09 2.88±0.03* 0.0020Values are in means ± SEM. * Significant differences (p≤0.05 by nutritional conditions R vs C

Table 2: Comparison of mechanical properties, ultimate load fracture, failure displacement, Young`s modulus and percentage of maximum deformation.

C RYoung`s modulus (N/mm2) 3006.93±661.25 3740.32±2.92*Ultimate load fracture (N) 73.19±6.33 40.93±2.92*

Failure displacement (mm) 0.41±0.05 0.37±0.05Maximum deformation (%) 1.19±0.05 1.19±0.10

Values are in means ± SEM. *Significant differences (p≤0.05 by nutritional conditions R vs C

Table 3: Effects of nutritional condition of pores measures from SEM micrographs of femur rats.

C R p values

Area 3966.26±582.74 (mµ2) 4972.07±1094.92 (mµ2)* 0.017

Perimeter 242.38±24.43 (mµ) 249.67±17.93 (mµ)* 0.005

Width 54.09±4.73 (mµ) 89.676±9.84 (mµ) 0.005

High 89.67±9.84 (mµ) 86.24±6.90 (mµ)* 0.001Values are in means ± SEM. * Significant differences (p≤0.05 by nutritional conditions R vs C

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Bone distribution was different according to nutritional condition, the size of pores from C rats, showed an increment in the measure for pores (Table 3). The used model showed the pore distribution patterns in the trabecular bone from micrographs of SEM, the dimensions of the pores were reproduced for the construction of a model reaching a design of a pore from these geometries using Blender Program (Figure 3,4). This was done using to obtain the geometry of pores of different sizes for the construction of a distribution model. In addition, for analysis of fractals, ImageJ program used (see Figure 4 and 5), this program achieved a design of a pore from these geometries using Blender Program (Figure 5).

The results show that body weight was reduced in R group and

this result is in line with previous reports the reason is that the gestational and lactation period are critical for cell differentiation and development [29,30]. Protein-energy undernutrition early in life may cause a delay in growth and a decrease in the cortical bone formation and thereby interfere in the acquisition of a suitable bone mineral peak [31].

Treatment with malnutrition had a significant impact on the microarchitecture of the rat femur. These observed changes occurred in 9 weeks old suggest a delayed effect of R on mechanical properties of bone. Thus, changes in cortical bone beyond cancellous bone might further increase fracture risk in malnourishment patients [32].

Figure 4 Trabecular pores pattern from micrographs of the femur head, visualized under SEM in C group.

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Figure 5 Trabecular pores distribution of the femur head, from micrographs under SEM. Section of R group rats.

In this study showed sparser bundle collagen fibers per area in R group, could be explained by increasing the regular spacing of collagen bundle fibers was not preserved and may be responsible for the decrease in structural organization. Changes on the spacing orientation may give rise to different mechanism responses of fibrils array [33,34]. The trabecular bone has a different arrangement of the lamellae in comparison with cortical bone, which are organized longitudinally along the trabeculae instead of concentrically [35,36]. However, the several layers of lamellae may have different mineral contents, the arrangement of collagen fibers [36,37].

The mechanical properties of bone depend on the

arrangement of collagen fibers which determine the deposition of mineral crystals and influences bone strength [37]. Woven bone composed by unorganized collagen fibers has lower mechanical properties than lamellar bone with a well-defined arrangement of mineralized collagen fibers, which reveals the importance of the collagen disposition. Related to other nutrients, such as vitamins, proteins, and amino acids, have been shown that promote bone remodeling and improves calcium absorption, thereby inhibiting bone loss [38]. Findings for breaking resistance are consistent with the other factors such as the size of the bone. Besides, the increment of bone mineralization degree and microstructures together define the bone properties. The observed decrements of diaphysis thickness can explain the decreases of the bone

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breaking resistance. Another factor is the collagen and probably, in this research, the arrangement of the cross-linking of collagen fibers was altered, presenting irregular arrangement, changing the tensile strength and increasing the bone fragility [39]. This was observed, in the thickness diaphysis, of R group, the physical development of rats is related to the changes in the skeletal and muscle, which depends mainly on four factors: genetic, hormonal, living style, physical activity, and feeding conditions. On the other hand, it has been demonstrated that there exists a direct relationship between weight and bone mineral density [40-42] osteocalcin is an important protein for bone formation and to be diminished by malnutrition affected bone mineral density (BMD) by a decrease in bone mineralization.

Furthermore, it was found that bone loss occurs frequently in children and in rats that have suffered protein-energy malnutrition. The results presented here can be explained because hormonal levels may be involved due to the intestinal calcium absorption dependent of estradiol and 25-hydroxyvitamin D, when these are low, consequently it is observed a marked decrease in BMD.

This also was proved with the bone microanalysis and the mineral content, and even the group with experimental diet presented higher quantity of Ca, the Ca phosphorous (P) ratio (Ca/P) was not the adequate, suggesting that bones mineralization does not reach the optimum levels [43]. Several previous studies showed similar results, which reports a high P content in diet has a negative effect in Ca deposition in bones [44-46].

CONCLUSIONThe optimal nutritional condition associated with weight

loss is critical for bone diseases [40]. Considering this bulk of knowledge, if early life the individual obtains adequate diet, it could ensure that even during adulthood is passed through periods of poor diet, the risk of demineralization would be less and consequently preventing degenerative diseases such as osteopenia or osteoporosis. The number of trabecular pores and size determine the bone fragility, when increases the pore dimensions, the bone break resistance decrease. Additionally, the increasing size of pores could be associated with decrease of support the body mass.

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