Indian Journal of Experimental Biology Vol. 41 , June 2003 , pp. 581-586

Effects of low level pulsed radio frequency fields on induced osteoporosis in rat bone

layanand, litendra Behari* & Rajeev Lochan

31 1, School of Environmental Sc iences, l awaharl al Nehru University, New Del hi 110067, Indi a

Received 17 July 2002; revised 13 March 2003

Effect of modul ated pulsed electromagnetic fi e lds (PEMFs; carrier frequency, 14 MHz. modulated at 16 Hz of ampli ­tude 10 V peak to peak) on sciati c neurectomy induced osteoporosis in rat femur and tibi a resulted in stati stically significant increase in bone mineral density, and deceleration in bone resorption process and consequently further osteoporosis in rat bone. Thesc results suggest that such an effect ive window of pu lsed radio frequency fie lds may be used therapeutica ll y for the treatment of osteoporosis.

Keywords: Bone mineral density , Pulsed e lectromagnetic fie ld, Osteoporosis.

Bone formation and resorption are continuous proc­esses occurs throughout life. As a living and con­stantly changing ti ssue normally there is a balance between the amount of old bone being removed and the amount of new bone replacing itl . When the re­sorption is more than the formation , the bone loss oc­curs resulting in osteoporosis. Osteoporosis, or porous bone, is a disabling disease characterized by low bone mass and structural deterioration of bone tissue. It reduces the density and strength of bones, leading to bone fragility and an increased susceptibility to frac­tures2

. Osteoporosis is probably the most common metabolic disorder controlled by several systemic and local factors, which regulate the formation and activ­ity of bone cells (osteoclasts and osteoblasts). People over 45 years of age experience bone fractures due to osteoporosis (senile or osteoblast mediated) in which fracture of proximal femur and inter trochantric frac­tures are common. Around 250 million women worldwide have osteoporosis now. By the year 2020, the number of women affected will double3

. Clinical surveys have demonstrated that adult bone mass di ­minishes at a mean rate of 0 .5% per year4

, and it can reach a loss of 2% per year after menopause. As the probability of fracture is related to a person's effective bone mass , a modality that could prevent or retard loss of bone may provide a substanti al reduction in the incidence of skeletal morbidity .

*Correspondcnt au thor Phone: 9 1-0 11-26704323. Fax: 91- 11 -26 165886. E-mail: jbehari@hotmail.co m

Several prophylactic measures to prevent loss of bone are available; these include estrogen therapy, calcitonin, parathyroid hormone, bisphosphonates, vitamin D, prostaglandin E2, supplemental dietary calcium and exercise. There are also treatment regi­mens that stimulate formation of bone, such as so­dium fluoride and parathyroid hormone. Although these regimens have been effective in the treatment of osteoporosis, limitations, cautions, and dangers are inherent in their extended use. The clinical potential for increasing bone mass or simply preventing bone loss, by alternative non-invasive means is therefore substantial.

According to Brighton et al. 5 a capacitatively cou­pled electrical signal, delivered through gel-coated electrodes, could largely reverse an established disuse osteoporosis due to neurectomy in the rat tibia. Bio­electric effects therefore appear to provide a link be­tween mechanical stimuli and cellular behavior. The application of electricity, later on as a treatment for delayed unions and non-unions of bone has received attention6

. Rubin et al.7 concluded that there is an ef­fective window of pulsed electromagnetic fi elds which could control the bone mass. Mcleod and Rubin8 also demonstrated that a simple, low power sinusoidal field at 15 Hz is the most osteogenic of any induced field they studied . This is achieved by com­paratively short daily exposure generated within a physiological intensity and frequency. These electri­cal fields can slow, inhibit or even reverse the osteo­porotic processes that normally accompany disuse in animal model. Behari et al.9 and Behari 10 showed that

582 INDI AN J EXP SIOL, JUNE 2003

pul sed radi o frequency e lectrica l fi e ld can acce lerate bone fracture healing in rats. Rubin and McLeod 11

reported th at th e mechani cal stimuli at 15-30 Hz fre­quencies results in a bone formati on rate than do sti muli at 1-10 Hz frequencies . Wang e l al.12 sug­gested that direct current sti m ul ation pro moted the osteogeni c processes in bone metabo li sm and ex­panded osteobl ast-li ke ce ll s abl e to be entering in to mass ive ske leta l defec ts to pro mote ce ll medi ated re­generation of ske letal ti ssues.

As there is no establi shed trea tment of osteoporo­sis, the effo rts are o n fo r preventi on o f bone loss and frag ility. The obj ecti ve o f the present study is to ex­am ine the extent bone aff li cted with os teoporosis re­sponds to the stimulatio n of a no n-in vas ive method of pul sed electro magneti c f ie ld (PE M F).

Materials and Methods Three months o ld 24 male Wi star rats we ighin g

approximate ly 200 g each were di vided into 3 groups (l 0, 10, and 4). Each rat of first group (10 rats) un­derwent only one-s ided sc iati c neurecto my on the first day of 70 days ex pe ri ment. Afte r the animal had been anaes theti zed with phenobarbito ne sod ium (30 mg/kg of body wt, ip), one hind li mb was shaved over the thigh and di sinfec ted . A n inc isio n was made on the upper thi gh just poste rio r to the fe mora l trochanteri c reg ion. The sc iati c nerve was mobili zed within the incision and about 0 .5cm sec ti o n was exc ised . The inc ised skin was closed with sterili zed ethicon thread by using surgical needle. Sc iati c neurecto my was d · f h 2nd one I n rats 0 t e grou p (1 0 rats) in both legs by the same procedure (one leg "sham-exposed" and other as "exposed") o n the same day as the f irst group. Antibioti c (anthrocin 250; equi va lent of 250 mg of erythro mycin esto late IP) was g iven (as needed) through drinking wate r for early healing of wou nd and inhibition of any infec tio n. Third group (4 rats) was left as "contro l" without any surgical

procedures. Each rat was ho used in 17 x 18 x 24 cm sized pl astic cage and a llowed free access to tap wate r and pe lle ted commercia l die t.

Afte r 30 days of sc iati c neurectomy, rats of first group were scanned in vivo by DEXA (Dual Energy X-ray Absorptio metry) . Then all rats of the first g roup were sacri ficed and fe mur and tibi a were resected fo r further analys is. After 30 days of denervation, a ll rats of the 2nd g roup received the ex posure of pulsed radio frequency signal. For the exposure, we used a bone stimulator with fo ll owing spec i ficati on 10.

Carri er frequency Modulating frequ ency A mplitude O ut put wave shape Elec trode di ameter A verage e lec tri c fi e ld between e lec trodes

14 .0 MHz 16.0 Hz 10 V (peak to peak) Square i cm 7.8 Volt 1m (10)

Calibrati on was do ne with the he lp o f Iwatsu Oscil lo­scope SS -57 11 C, Japan.

Output of bone stimul ator was given to each rat separate ly by a pair of e lectrodes (Fig. 1) in only o ne leg da ily for 2 hr. C urrent density at the point of ap­pI icatio n was 80 p., A/c lTt Other leg was ti ed with sa me type of e lec trodes without any connecti on to stimul ato r (Sham-exposed). Rats were lightly anaes­the ti zed, before g iving the exposure, so that they could not di sturb the ex perimental process. In vivo DEXA scan of rats of second group was do ne after a 30 days of exposure. After the in vivo DEXA scan of rats of third group, all rats of 2nd and 3rd group were sac ri f iced and femur and tibi a were resected for fur­the r ana lys is.

DEXA scanning - A ll DEXA scans were per­fo rmed on Holog ic QDR-4500A scanner (Holog ic, Waltham, M A) by us in g software for small animal at Nuc lear Medic ine Department, Indraprastha Apo llo Hos pita l, New Delhi , India. Before scanning rats were li ghtly anaestheti zed with phenobarbitone sodium (ip) to make them stable and immovable . Then they were fixed o n hard paper ( 150 gs m) in supine positi on with the he lp of surgical tape. Afte r the scanning, we care­full y selected the regio ns of interest (RO! , i.e. tibi a and fe mur) on the mo nitor of the instrument and ob­ta ined the bo ne minera l content and bone minera l density of the areas under in vesti gati on.

Scanning electron microscopy - Resected femur and tibia of fi ve rats of 1 s l and 2nd g roup were made free of a ll soft ti ssues. Transverse sections of fe mur

Fig. I - Schemati c diagram of exposu re set up.

JA YANAND el or.· LOW LEVEL PULSED RADIO FREQUENCY & OSTEOPOROSIS 583

and tibia of each leg ('normal' and 'denerved' of 1st

group, 'exposed' and 'sham-exposed' of 2nd group) of 0.5 cm thickness were made by fine saw edged knife very carefully as osteoporotic bone was fragile and generally could brake during cutting. They were separately put into fixative (glutaraldehyde; 2ml-25% glutaraldehyde solution, 3 ml-37 % formaldehyde solution, 1.58g-dehydrated calcium acetate, aqua ad 100 ml) for minimum fixation time (8-24 hr for anatomical details of bone) at room temperature. Then these samples were washed carefully with phosphate buffer solution and dehydrated till the critical point drying to keep the surface intact without swelling and shrinking. With silver paint these samples were mounted on a circular stub. The samples were coated with a thin layer (50-300 A) of gold prior to being placed in the microscope to make the sample conductive for taking image. Same procedure was repeated with femur and tibia of two rats of 3rd group. SEM photography was performed on 'low vacuum SEM' Leo 435 VP (Cambridge, England).

Bone ash - Resected femur and tibia of rest of the rats of all groups were made free from soft tissues and their wet weights were determined by electronic bal­ance. Femur and tibia were next placed in individual porcelain crucibles and dried for twenty-four hours in oven at 100°e. After dry weights were determined, femur and tibia were placed in a muffle furnace for 24 hr at 800°C and ash weights were determined.

Results The results of DEXA measurements in the region

of interest of femur and tibia are summarized in Table l. A significant decrease in the BMD was observed in the femur and tibia of de nerved leg after one month of denervation, which was further lowered in ROI of sham-exposed legs, whereas, BMD increased in the femur and tibia of the legs exposed to PEMF. Although, it was not up to the normal level but it was certainly more than the osteoporotic and sham-

exposed legs. P-value in one month sham-exposed versus one-month exposed rat bone was less than 0.05 but versus denerved it was more than 0.05 (Table I). Results of percentage of bone ash content also confirms the results of DEXA.

Images of various parts of transverse section of fe­mur and tibia show the clear differences in normal , osteoporotic and exposed bone. Compactness of can­cellus part of bone in transverse section indicates the mineral deposition in normal and exposed bone with respect to osteoporotic bone (Fig. 2a-c). Moreover in normal tibia, bone marrow is attached to the cortex (Fig. 3a) and cortical thickness was more than that of the osteoporotic tibia in which marrow was also de­tached from the cortex (Fig. 3b). But after the PEMF exposure, new growth in the endosteal part of the cor­tex was clearly observed (Fig. 3c). Denervation in­duced the osteoclastic activities in bone and made it porous (Fig. 4b) with respect to normal (Fig. 4a) . After exposure of low-level PEMF for 30 days, the amount of porosity started to reduce and pores were being filled with minerals (Fig. 4c) .

Discussion The study of the bioelectric effects in bone had its

modern origin in 1957, when Fukada and Yasuda l3

presented their experimental and theoretical work demonstrating that bone possessed piezoelectric properties. Their work indicated that when external forces were applied to bone it generated electrical potential. The bone contains mechanosensing cells (osteocytes) distributed throughout the bone matrix, monitor mechanical strain and activate corrective biological processes 14. These osteocytes produce a electrical signal proportional to · mechanical loading either by sensing strain on bone surfaces through stretch-activated ion channels 15 or electrical potentials 16

or flow of interstitial fluid I? or some other phenomenon. Cell to cell communication of electrical signals and small molecules through gap junctions has been

Table I - Bone mineral density (BMD) and bone ash (%; in relation to dry wt) of femur and tibia at one month of exposure to PEMF

Normal Denerved Sham ex posed Exposed

[Values are mean ± SE]

Femur Tibia 0.1568±0.01l8 0.1I14±0.0108 0.1425 ± 0.0083 0.0968 ± 0.0086 0.1402 ± 0.0 101 0.0957 ± 0.0021 0.1526 ± 0.0122 0.1065 ± 0.0085

% of bone ash in relation to dry wt Femur Tibia

54.21±5.81 51.l1±2.88 39.53 ± 5.16 36.99 ± 3.25 37.43 ± 6.65 35.61 ± 2.58 46.02 ± 1.55 43.91 ± 3.07

P < 0.05 sham exposed vs. exposed; » 0.05 sham ex posed vs. one month denerved .

584 INDIAN J EXP SIOL, JUNE 2003

d d . bl 18 19 S" I .. emonstrate In osteo asts . . Iml ar gap Junctions in osteocytes20 participate in such communication with osteoblasts, and bone lining cells as well. Mechanical loading is converted to an electrical signal that can be transmitted intracellularly to the bone

, " ".

{ ':." .,~.

" \ -' , . ""'

Fig. 2 - T. S. of cancellus part of femur bone showing compact­ness, (a) normal , (b) osteoporotic and (c) exposed .

lining cells, creating intracellular or transmembrane potential changes in the osteoblasts21

. According to above-mentioned works it is established that mechanical stress generates electrical signals, which induces bone remodeling. In osteoporotic bone we

~ .. f:~ ., ... &;J

'.>,#'

i.. , t.;

" : \ ' ~ .

" ;,'f

Fig. 3 - T. S. of tibi a showing (a) normal, (b) osteoporotic , and (c) exposed bone. In exposed bone (c) new growth observed in endosteal part of the cortex.

JA YANAND et al.: LOW LEVEL PULSED RAD IO FREQUENCY & OSTEOPOROSIS 585

couldn't induce mechanically stress-generated potential as bone was fragile. Hence we induced osteogenic potential in bone through non-invas ive capacitor plates. It is yet to be settled that which form of

Fig. 4 - T. S. or remur bone showing porosity, (a) normal , (b) osteoporotic, and (c) ex posed. Porosity in osteoporotic bone (b) is more than exposed (c) and normal (a) bonc.

electrical energy (AC, DC, Pulsed) is most efficient stimulator of osteogenesis. In an attempt to quantitate these a series of experiments have been carried out to study the high frequency responses22

. Stimulation caused by the PEMFs is like ly to communicate this signal to osteocytes and may well as perturb the fluid fl ow in bone. Experimental and clinical research studies have shown positive effects of PEMFs on endochondral bone formation and on osteogenesis. It is shown that extremely low frequency EMF ex posure

'+ C '+ can alter Ca- transport, probably through a-channel without hav ing any such effect on non­activated cells23

. The temporary application of a 60 Hz sinusoidal E-fields causes some dynamic changes in ce ll membrane components and/or within the vicinity of cellular me mbrane, reflecting in reduced or induced Ca2

+ influx respective ly through ATP (cyclic AMP) o r hi stamine induced ion channel s. Even though the observed effects have been temporary, it is poss ible that chronic exposure of low intensity EMFs could have long las ting effects on cell physiology, through changes of Ca2

+ distribution within the ce ll s9

.24

. Increased BMD and increased percentage of bone ash of exposed bone in comparison to sham­exposed bone (Tab le 1) suggest that PEMF exposure reduces the process of induced osteoporosi s and retains the minerali zation inside bone by the same mechani sm.

Bone strength depends on the mechanical quality and the spatial distribution of the mineralized matri x. Thus, the effects of treatments for bone-weakening diseases should not be evaluated according to the changes elicited in the bone mass, but in the stiffness of bone material and/or in the architectural design of the bone as an 0 1'­

gan25. Although, it is clear that mineralization occurs in

the osteoporotic bone and bone mass and bone mineral density (BMD) increase after the PEMFs exposure, it is not as much compact as the original structure. But it cer­tainly gives the support to the osteoporotic bone by fill­ing its pores and reduces the ri sk of fractures . lncrease in duration of PEMFs exposure in hours as well as in days may provide compactness and strength to bone. As the begin ning of ti ssue regeneration in endosteal part of tibia makes the cortical bone thick and must have positi ve effect on the bone strength.

Acknowledgement The authors are thankfu l to Dr. Uma Ravishankar

(Senio r Consultant , Nuclear Med icine Department, Indraprastha Apo ll o Hospital, New Delhi , Indi a) for he lp in DEXA Scans.

586 INDIAN J EX P BIOL, JUN E 2003

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