Decrease in Bone Mechanical Properties after Exposure to Mixed Fields of Fast Neutrons and Extremely Low Frequency Electromagnetic Field

Aly F, Bishay A, Atef M and Khafagy M

Published on: 2020-12-31

Abstract

The aim of the present study is to investigate the effect of exposure of rat’s bone to extremely low frequency magnetic field (0.5 Gauss), fast neutrons FN (10µSV/h) and combined fields of both for a period of 4 weeks at a rate of 8 h/day, 5 days /week. For this purpose, 80 adult male albino rats were divided into four equal groups: group A, (control group), group B (exposed to magnetic field), group C (exposed to FN), group D (exposed to combined fields of magnetic field and fast neutron). After each experiment, bone samples were subjected to the following studies: bone density measurement, evaluation of Ca++ concentration following atomic absorption technique, measurement of mineral content, degree of calcification using FTIR technique & biomechanical testing of bone. Results reveal high significance decrease in bone quality causing brittle bone resulting in osteoporosis.

Keywords

Extremely low frequency; Electromagnetic field; Fast neutrons; Atomic absorption; Mechanical; Properties bone

Introduction

Usage of ionizing and non-ionizing radiation in many applications are in increase more and more every day. Such behavior attracts attention to study their effects on living cells. Previously, it was believed that low frequency fields are weak to an extent that cause significant heating could not possibly have any biological effect (1). Despite this opinion among researchers, evidences have accumulated to supports the existence of complex biological effects of weak extremely low electromagnetic fields, (including weak ELF magnetic field, and modulated RF) and microwave field (thermal energy) [2,3].The fast growing uses of nuclear accelerators in medicine, manufacture and research in addition to the use of nuclear reactors for generating electric power, all increased the health risk associated with mixed exposures to ionizing and non-ionizing radiation. Extremely low-frequency magnetic fields (ELF-MFs) are produced wherever electricity is generated, transmitted, or distributed, such as power lines, cables, subways, and electrical appliances. ELF-MFs include alternating current (AC) fields and other forms of non-ionizing radiation with frequencies ranging from 1 to 300 Hz. The major frequencies of ELF-MFs are 50/60 Hz, and humans in industrialized nations are continuously exposed to a few milli-tesla (mT) of ELF-MFs at these frequencies everyday [4,5]. The permissible doses of ionizing radiation, recommended by the ICRP, considered only its biological effects. The question now; does these recommended safe limits of exposures can be valid for mixed radiations of non-ionizing and ionizing fields? The skeletal system, important to the body both biomechanically and metabolically, is made up of Individual bones and the connective tissue that joins them. Bone is the main constituent of the system and differs from the connective tissues in rigidity and hardness. The rigidity and hardness of bone enable the skeleton to maintain the shape of the body; to protect the soft tissues of the cranial, thoracic, and pelvic cavities; to supply the framework for the bone marrow; and to transmit the force of muscular contraction from one part of the body to another during movement. The mineral contributes both to the mechanical strength of bone and to the ability of the skeleton to regulate mineral ion homeostasis. The body’s pools of ionic calcium (Ca++) and magnesium (Mg++) are maintained at critical levels by the coupled actions of hormones such as parathyroid hormone, Because the mineral crystals of bone are extremely small, they are relatively soluble [6]  and can be dissolved or reprecipitated in response to signals that reflect serum or even local cation concentrations .The techniques used for characterizing bone mineral provide information above and beyond the chemical and crystalline composition of bone. They are frequently used to indicate variations in bone composition with age and disease, to characterize mineral in cell and organ culture, and to study bone in growth into synthetic/implant materials. Mineral analyses have occasionally been used for forensic and archaeologic evaluations [7]. since a variety of measures of mechanical strength have been correlated with mineral content [8] A large number of studies have related the strength and stiffness of bone to its mineral content [9,10] but clearly, other chemical properties pertaining to both the organic and inorganic phases also contribute to bone’s mechanical properties. Crystal size, orientation, maturation, and perfection have been suggested to influence mechanical behavior [11,12]. Changes in carbonate content and carbonate substitution for phosphate ions alter crystal shape and the arrangement of the crystal lattice, leading to altered mechanical properties [13]. Collagen provides tensile strength, toughness, resilience to fracture, and ductility; and the strength of collagen-mineral bonding and the quality, maturity, and orientation of the collagen fibers have been implicated in bone’s mechanical behavior [14,15]. Stress-Strain curve figure 1 for bone is typically divided into two regions, the elastic and plastic, which are divided by yield point. In the first region, behavior is linearly/elastic. The second region an increase in strain is due to little or no change in stress. The point/region where the deformation changes from being elastic to at least partially plastic is the yield point.

Figure 1: shows a typical stress–strain curve.

Although bone reaching this point may still have far to go before it actually breaks, it is permanently damaged to some extent once it enters the plastic region. The amount of post yield strain that occurs in a material before fracture is a measure of the ductility of the material. A material showing a large post-yield strain is referred to as ductile, whereas a material showing little post-yield strain is described as brittle. Mechanical failure can be defined as the degradation of a material property beyond its elastic limits or loss of material continuity. The maximum stress the bone can sustain is called the ultimate stress, and the breaking stress is the stress at which the bone actually breaks catastrophically. Fatigue is the damage due to repetitive stresses below the ultimate stress. Fatigue is a slow progressive process, as opposed to an acute catastrophic process, which results when the ultimate strength of a material is surpassed. Typically, repetitive cyclical loading (smaller than ultimate stress) causes a crack through a material with subsequent separation of the object into pieces. The area under thestress-strain curve is a measure of the amount of energy needed to cause a fracture. This property is called energy absorption or toughness of bone and is an important property from a biomechanics point of view.Loading a bone specimen cyclically with progressively higher forces produces a highly nonlinear stress–strain curve. For example, in Figure 2, the bone specimen is loaded up to point A. When the force is removed, the specimen exhibits reversible behavior and returns to its original unloaded length. The load that causes the stress–strain curve up to point B also permits the material to return to its original length, although the time required to return to normal is longer.

Figure 2: shows a typical hysteresis loop.

Stress–strain up to point C produces a permanent change in the original length of the material and is not reversible. The loading and unloading curves do not overlap, but rather create a closed loop, known as a hysteresis loop, which is indicative of the inefficiency of storing and releasing of strain energy. The area under the unloading curve represents strain energy release during unloading. The area enclosed by the hysteresis loop represents energy dissipated within the material through mechanical damage and internal friction. Test-type influences the mechanical properties that bone will exhibit. Cyclic loading produces micro-damage that accumulates with each cycle and the damage increases as the intensity of testing increases. Intensity can be varied through change in the load magnitude and through the number of cycles to which the specimen is exposed. Lessons learned from cyclic loading include that, once a crack occurs, the number and cyclical load required for propagation of the fracture decreases rapidly and that there is a strong negative correlation between load intensity and number of cycles needed for failure [16].

Experimental Arrangement

252Cf source was put in a special designed drawer that was fixed on the inner top wall at the center of the solenoid chamber to allow average homogeneous fields to neutrons during magnetic field exposure, for group D as shown in figure 2.

Biochemical Study

Left tibia bone was defatted in chloroform /methanol mixture, dried at 110 oC for 18 hr for water elimination. The bone was then ashed at 560 0C for 72 hr and the ash residue of the tibia was digested with 1 ml of 60 % HNO3 and diluted up to 10 ml with distilled water. With such preparations, the concentration of Ca was measured following atomic absorption spectrophotometry technique (AAS). The analytical technique of metal measurement was checked by repeating measurements and using standard reference bone ash No.1400 (National Institute of Standard and Technology).    With the flame atomic absorption (AAS) technique the sample was aspirated into a flame using a nebulizer. The flame is lined up in a beam of light of appropriate wavelength depending upon measured element. The flame (thermal energy) causes the atoms to undergo a transition from the ground state to excited state. When the atoms perform their transition, they absorb some of the light from the beam. The more concentrated the solution, the lighter energy is absorbed.   In the meantime, the light beam generated by the lamp is specific for a target metal. The lamp must be perfectly aligned such that the beam crosses the hottest part of the flame. The light passing through the flame is received by a monochromator, which is prepared to accept and transmit radiation at the specified wavelength, and in turn travels to the detector. The detector measures the received intensity of the light beam. When some of the light is absorbed by the metal, the beam intensity is reduced. The detector records that reduction as absorption. That absorption is shown on an output device by the data system.  The concentration of   the metal in a sample can be found by running a series of calibration standards through the spectrophotometer. The spectrophotometer will record the absorption generated by a given concentration. By plotting the absorption versus the concentration of the standards, a calibration curve can be plotted. Looking at the absorption for the sample solution and using the calibration curve, one can determine the concentration of the sample.

Molecular Study

Bone mineral composition, of all exposed groups in addition to the normal bone were yielded to study by following base line technique spectral vibration infra-red Error! Reference source not found, [17]. Spectroscopic parameter that will be evaluated in the infrared spectra of bone is the CO3/ PO4 ratio (Relative area of sub-band at 873 cm-1/ 1030 cm-1), which represent Degree of HA crystal maturity or amount of carbonate substitution for phosphate in the mineral crystal.

Bone Density Measurement

A mediolateral view radiographs were taken for each bone sample from both control and the three different exposed groups according to the predetermined protocol just after animal scarification. For x-ray standardization, a fixed distance was measured that starting from the farthest end of rat’s leg and a point was marked. This point was placed at the center of the film each time. In addition, an aluminum step wedge was used. Aluminum step wedge was formed of fifteen steps of known thickness. Step wedge was fixed to the front of the film during X-ray projection. A mediolateral view of different groups was taken on occlusal radiograph with a standard 6x12 cm film at a 70 cm FFD, 48-52 KV and 15 mA Machine, After the predetermined stages of the experiment were radiographed, it was necessary to convert them into digital form to conduct further quantitative/qualitative image analysis. The conventional radiographs were scanned using an intensity-calibrated film digitizer. The radiographic films were scanned by illuminating the films from one side while scanned were received intensity from the other side of the film. The scanner performs self-calibration where a film is scanned to ensure that the light source and the light intensity detection front end providing the same result every time for the accuracy and reproducibility of the film scans.The scanner is connected to an IBM compatible computer for further processing and analysis of scanned film data. The scanner was adjusted to scan at a resolution of 200 dots per inch (dpi) for the full size of its active film scanning area at gray level resolution of 8 bits (256 gray shades). Once the image data became available on the scanner software, it was saved onto the hard drive on the computer in TIFF format. For determination of the bone density, Digora software allowed measuring of the mean density value along a line or an area of controlled dimensions. The term density refers to the degree of whiteness of the image or part thereof. These measurements were made for tibia bone of: -

  • The control group and the group exposed to 2-week 3-week magnetic field only.
  • The control group and the group exposed to 1 week, 2-week, 3-week, 4-week fast neutrons only.
  • The control group and the group exposed to 1 week, 2week, 3-week, and 4 weeks of both magnetic field and fast neutrons.

Bone density determined by software by marking a line at a fixed distance e.g. between the epiphysis and a diaphysis (i.e. approximately at the metaphysis point) and then the mean value was calculated. This procedure was repeated for each bone from each group. Finally, bone density of each line was compared with the corresponding value of aluminum step wedge (two successive steps); then, the bone density was calculated according to the following equation:

Thickness of step 1-x/thickness of step 2=density of step 1-density of bone/density of step 1-density of step 2.

Results

The concentration of Ca ion after two weeks, three weeks, and four weeks exposure to combined fields of both magnetic and neutron fields are shown in both table (1) as follows:

It can be observed that the average Ca ++ concentration for the exposed sub-groups namely B1,….D4 suffered highly significant decrease relative to control( A).

Molecular Analysis

The change in bone mineral content (PO4) and the degree of hydroxyapatite crystal maturity (PO4/CO3) for each studied group is shown in Table 2.

It can be observed that the average value of bone mineral content (PO4) and the degree of hydroxyapatite crystal maturity (PO4/CO3) for each exposed group shows highly significant increase relative to control (A). The increase is indicated by the degree of significance (P ≤0.005).

Table1: The concentration of Ca ion after two weeks, three weeks, and four weeks exposure to combined fields of both magnetic and neutron fields are shown in both as follows:

group

Ca++concentration (mg/g) ± S.D.

A

418.6 ± 0.53

B1

301.50 ± 0.39**

B2

128.10 ± 0.85**

C1

387.40 ± 4.12**

C2

372.70 ± 0.75**

C3

361.40 ± 5.32**

C4

204.10 ± 1.24**

D1

325.2 0 ± 1.41**

D2

236.60 ± 0.70**

D3

196.50 ± 1.14**

D4

178.40 ± 3.35**

Table2: The change in bone mineral content (PO4) and the degree of hydroxyapatite crystal maturity (PO4/CO3) for each studied group is shown.

Specimen Average

 

Average Area Enclosed load-unload curve(N/mm)

During first cycle ±SD

Average Area Enclosed load-unload curve((N/mm)

During second cycle ±SD

A

0.008 ± 0..08

0.0098±0.03

1B

0.117±0.69**

0.01±0.46**

2B

0.118±1.1*

0.015±0.22**

1C

0.0099±0.1*

0.017±.02*

2C

 

 

 

 

 

 

 

 

0.01225±0.34**

 

 

 

 

 

 

0.0132±0.9**

3C

0.0138±0.53**

0.0213±0.44**

4C

0.0198±0.68**

0.023±1.4**

Biomechanical Testing

The data in table (3) indicate that enclosed area in the first and second hysteresis cycle increases for all animals exposed to radiation, and the values of these increases showed to be time of exposure. This can be indicated by the degree of the significance, P ≤0.005 * means significant variations, ** means highly significant variations and n* means non -significant variations.

Table3: The data indicate that enclosed area in the first and second hysteresis cycle increases for all animals exposed to radiation, and the values of these increases showed to be time of exposure.

Specimen Average

 

Average Area Enclosed load-unload curve(N/mm)

During first cycle ±SD

Average Area Enclosed load-unload curve((N/mm)

During second cycle ±SD

1D

0.0103±1.5**

0.016±2.9**

2D

0.0112±3.3**

0.0168±0.05**

3D

0.0168±0.8**

0.0182±0.74**

4D

0.0192±0.03**

0.021±0.89**

Bone Density Measurement

Table (4) shows the average value tibia bone density (g/cm3) for each group demonstrated.It can be observed that the average bone density of the tibia for the different exposed groups namely B, C, D and E suffered highly significant decreases relative to control (A), which can be indicated by the degree of significance (P ≤0.005 ** means highly significant variations and * means -significant variations).

Table4: shows the average value tibia bone density (g/cm3) for each group demonstrated.

Group

Bone density(g/cm3)

SD±

A

3.2 ±0.19

1B

2.46±0.32**

2B

2.22±0.049**

1C

3.11±0.9*

2C

2.87±0.51**

3C

2.38±0.21**

4C

1.76±.07**

1D

3.08±.046**

2D

2.63±0.078**

3D

2.19±.012**

4D

1.98±0.44**

 

Discussion

In this work the effect of exposure 50 Hz, 0.5 Gauss magnetic field,, fast neutrons from Cf252 in the permissible limits of exposures according to the ICRP -60 and mixed radiation field of fast neutrons and magnetic field on the biomechanical properties of the rat bone is studied .As the area under the hysteresis loop represent the energy dissipated in bone during mechanical forcing .one may find from the result in table 3 that exposures to fast neutrons and/or magnetic field to the doses demonstrated causes increase of the area of the hystresses loop in the second cycle as compared with the control group, this result indicated that exposure to fast neutrons and/or magnetic field can lead to bone fatigue and damage even exposure to ionizing radiation are in the permissible dose limits(ICRP-60)[18].It is well understood that mechanical forcing on bone generate piezoelectricity on the bone. Which results in the formation of streaming potentials. This piezoelectric potential accelerates the ionic pumping through bone tubules which will generate ionic currents, the flow of this ionic currents will generate ionic potential to the bone marrow to generate blood and collect calcium in the stressed bone. Therefore, the damage of the tubules in the bone will result in deteriorate action potential and hence will affect the mechanism of generation of blood by bone marrow and collection of calcium in bone.The effects of exposures to magnetic field, neutrons and/or mixed field of them can be noticed from table 1 that shows the decrease in bone calcium level after exposure to different doses to one or both fields and supported by the observed decrease in the bone density measurements shown in table (4). Since crystal size, orientation, maturation and perfection have been suggested to influence mechanical behavior, changes in carbonate substitution for phosphate ions alter crystal shape and the arrangement of the crystal lattice, leading to altered mechanical properties.

Such decrease in the mechanical properties of bone, the Co3/Po4 ratio, bone density and the high shortage of calcium after exposures to fast neutrons and/or magnetic field indicate a serious damage in the exposed rats. However, more studies need to be followed to give a closer image about the changes occurred in bone after such exposures.

References

  1. BinhiVladimir N, Repiev A, Edelev M. Translators from russian. magnetobiology: underlying physical problems. San Diego: Academic Press. 2002; 1-16.
  2. Harland JD, Liburdy RP. Environmental magnetic fields inhibit the antiproliferative action of tamoxifen and melatonin in a human breast cancer cell line. Bioelectromagnetics.1997; 555-562.
  3. Aalto S, Haarala C, Bruck A, Sipila H, Hamalainen H, Rinne JO. Mobile phone affects cerebral blood flow in humans. J of Cerebral Blood Flow and Metabolism. 2006; 885-890.
  4. Moriyama K, Yoshitomi K. Apartment electrical wiring: A cause of extremely low frequency magnetic field exposure in residential areas. Bioelectromagnetics.2005; 26:238-241.
  5. Mezei G, Kheifets LI, Nelson LM. Milles KM, Iyire R, Et Al. Household appliance use and residential exposure to 60 Hz magnetic fields. J. Expo. Anal. Environ. 2001; 41-49.
  6. Neuman MW, Imai K, Kawase T, Saito SJ. The calcium-buffering phase of bone mineral: some clues to its form and formation. Bone miner. 1987; 171.
  7. Franc S, Marzin E, Boutillon MM, Lafont R, Lechene Porte LDL, Herbage D. Immuno histochemical and biochemical analyses of 20,000-25,000-year-old fossil cartilage. J.Biochem. 1995; 125.
  8. Alho A, Husby T, Hoiseth A.Bone mineral content and mechanical strength. An ex vivo study on human femora at autopsy. Clinical orthopaedics and related research. 1988; 292-297.
  9. Ding M, Dalstra M, Danielsen CC, Kabel J, Hvid I, Linde F. Age variations in the properties of human tibial trabecular bone. J Bone Joint Surg Br. 1997; 995-1002.
  10. Mc Calden RW, Mc Geough JA, Barker MB, Court-Brown CM. Age-related changes in the tensile properties of cortical bone. The relative importance of changes in porosity, mineralization, and microstructure. J Bone Joint Surg Am .1993; 1193-1205.
  11. Akkus O, Adar F, Schaffler MB. Age-related changes in physicochemical properties of mineral crystals are related to impaired mechanical function of cortical bone. Bone 2004; 443-453.
  12. Silva MJ, Ulrich SR. In vitro sodium fluoride exposure decreases torsional and bending strength and increases ductility of mouse femora. J Biomech. 2000; 231-234.
  13. Penel G, Leroy G, Rey C, Bres E. Micro raman spectral study of the po4 and co3 vibrational modes in synthetic and biological apatites. Calcif Tissue Int.1998; 475-481.
  14. Bailey AJ, Sims TJ, Ebbesen EN, Mansell JP, Thomsen JS, Mosekilde L. Age-related changes in the biochemical properties of human cancellous bone collagen: relationship to bone strength. Calcified tissue int. 1999; 203-210.
  15. Traub W, Arad T, Weiner S. Origin of mineral crystal growth in collagen fibrils. 1992; 12- 251.
  16. Burstein AH, Wright TM. Fundamentals of orthopedic biomechanics. Williams and Wilkins, Baltimore, MD. 1994.
  17. Bohic S, Heymann D, Pouezat JA, Gauthirer O, Daculsi G. Transimission ftir microscopy of mineral phases in calcified tissues. C.R.A cad Sci III: 1998; 865-876.
  18. ICRP publication 60 recommendation of the international commission on radiological protection annuals of the ICRP. 1991; 1-3.