AIP Conference Proceedings 2092, 020028 (2019); https://doi.org/10.1063/1.5096696 2092, 020028
© 2019 Author(s).
Design and development of pulse
electromagnetic fields (PEMF) as adjuvant therapy for fracture healing: A preliminary study on rats
Cite as: AIP Conference Proceedings 2092, 020028 (2019); https://doi.org/10.1063/1.5096696 Published Online: 09 April 2019
Umiatin, Ismail Hadisoebroto Dilogo, Sastra Kusuma Wijaya, Puji Sari, and Andika Dwiputra Djaja
ARTICLES YOU MAY BE INTERESTED IN
ELM (extreme learning machine) method for detecting acute ischemic stroke using conventional and specific asymmetry BSI (brain symmetry index) features based on EEG signals
AIP Conference Proceedings 2092, 020023 (2019); https://doi.org/10.1063/1.5096691 Comparison study of human mononuclear cells presence in rat blood using flow cytometry and polymerase chain reaction
AIP Conference Proceedings 2092, 030010 (2019); https://doi.org/10.1063/1.5096714
Reduction of blood IL-6 level on aged Sprague-Dawley rats treated with Acalypha indica Linn ethanolic extract
AIP Conference Proceedings 2092, 030014 (2019); https://doi.org/10.1063/1.5096718
Design and Development of Pulse Electromagnetic Fields (PEMF) as Adjuvant Therapy for Fracture Healing: a
preliminary study on rats
Umiatin
1,2,a), Ismail Hadisoebroto Dilogo
3,b), Sastra Kusuma Wijaya
4), Puji Sari
5), Andika Dwiputra Djaja
3)1Doctoral Program of Biomedical Science, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
2Department of Physics, Faculty of Mathematics and Natural Science, Universitas Negeri Jakarta, Pulo Gadung, East Jakarta, Jakarta 13220, Indonesia
3Department of Orthopaedic and Traumatology, Cipto Mangunkusumo General Hospital, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
4Department of Physics, Faculty of Mathematics and Natural Science, Universitas Indonesia, Kampus UI Depok, West Java, 16424 Indonesia
5Department of Biology, Faculty of Medicine, Universitas Indonesia, Jl. Salemba Raya No. 6, Central Jakarta 10430 Indonesia
Corresponding author: a)[email protected], b)[email protected]
Abstract. This study aims to design and develop the pulsed electromagnetic fields (PEMF) device which can be utilized as an adjuvant therapy in delayed union fracture. In the first stage, Arduino-based PEMF device has been built with four main modules: pulse generator, user interface, Helmholtz coil and data acquisition system. Pulse generator allowing user to operate the physical parameters: frequency, duty cycle, burst duration and exposure duration. The Helmholtz coil could produce 1.6 mT of homogenous magnetic field. In the next stage, preliminary study was done to evaluate the effect of static magnetic field (SMF) and PEMF exposure on the healing of delayed union fracture model on Spraque Dawley (SD) rats.
Six SD rats were fracturized at the femoral shaft by non-union model, then they were grouped into 2 groups, 1 (n=3, SMFexposure, Bmax = 1.6 mT) and 2 (n=3, PEMF exposure, Bmax = 1.6 mT frequency = 50 Hz, duty cycle = 90%, burst cycle = 50%). Both groups had received the exposure for 4 hours per day and 7 hours per week. The evaluations of fracture healing were conducted by the histomorphometry analysis at 14, 21, and 28 days fracturization. PEMF group signal a better healing process through higher osseous tissue area.
Keywords: delayed union, fracture healing, histomorphometry
,
PEMFINTRODUCTION
The non-unions fracture, known as delayed union was indicated by the absence of the healing in period of six – nine months. Non-union treatment has become the main problem in orthopedic field since it impacted socio economic problem, decreased productivity, and prolonged hospitalization duration. Moreover, it takes higher risk during the treatment due to the complex strategy management, longer treatment period and prompt to cause permanent disability.
Besides that, most of non-union complication requires repeated surgical intervention that cost up to US$ 50,000 per patient in USA covering direct and indirect expenses [1].
Several methods are being developed to solve non-union in long bone such as the combination of autologous bone marrow derived mesenchymal stem cells (BMMSCs) and hydroxyapatite (HA), bone morphogenetic protein 2 and 7
(BMP 2 and BMP 7) which can induce the bone formation [2-4]. Moreover, the usage of mechanical and electrical biophysics stimulation were reported to be able to accelerate the fracture healing. Besides, mechanical stimulation in low intensity pulsed ultrasound (LIPUS) could improve the healing rate of fracture further [5].
Electrical stimulation is a popular adjunctive therapy aimed to promote bone healing since the discovery of strain- generated potentials in bone and their potential influence on bone healing formation and remodeling [6]. Faraday’s discovery in changes of magnetic field that induces a current in a conductor has contributed to various applications, such as the electromagnetic stimulation of body tissue and organs.
The effects of direct and indirect electrical stimulation (PEMF stimulation) on bone metabolism have been studied in numerous in vitro, in vivo and clinical studies. Ongaro et al. showed that the PEMF stimulation could affect the fracture healing by the induction of osteogenic differentiation of mesenchymal stem cells [7]. Li et al. concluded that PEMF with drug could prevent the disuse osteoporosis by changing the bone microstructure and promoting the level of osteocalcin [8].
However, the clinical evidence supporting the use of PEMF stimulation for bone healing has been inconclusive.
The main limitation of PEMF stimulation is the working mechanism which had not been yet explained regarding the cell response toward the induction that potential to produce positive effect in fracture healing. Several researchers reported EMF could affect the Resting Membrane Potential (RMP) resulting in the difference of ion concentration from intracellular to extracellular fluid, in which the order was around 70mV. Besides the unexplained mechanism, the absence of gold standard that related to the physical parameter in PEMF reduces the exposure area. The major electrical stimulation parameters are frequency, intensity of the field, waveform and duration of exposure.
Electromagnetic field which has the frequency range between 3-300 Hz is categorized as extremely low frequency (ELF). The recommended occupational exposure of the static magnetic fields (SMF) for continuous exposure is 40 mT referring to International Commission on Non-Ionizing Radiation Protection (ICNIRP) [9].
MATERIAL AND METHOD
This study consisted of two main stages; the first stage was design and development of the PEMF device. The second stage was the preliminary study of SMF and PEMF exposure to the animal model of delayed union fracture.
Hardware and Software Architecture
The aim of the first stage in this study was to create the PEMF device. The device was generally separated into four modules: A. Pulse generator, B. Helmholtz Coils, C. User Interface, and D. Data Acquisition System which were described into the diagram below:
FIGURE 1. Principle block diagram of microcontroller-based pulsed electromagnetic fields (PEMF) device
Pulse generator
The Arduino Uno R3 AT Mega 328 Microcontroller was programmed using LabVIEW to produce and set the pulse waveform, frequency, the duty cycle and the exposure duration. The interface was made to ease the user to operate the PEMF device. The acceptable frequency range was around 1-100 Hz while the acceptable duty cycle range was 0-100%.
FIGURE 2. Signal (pulse) generator of PEMF device.
The pulse which was produced by the Arduino had maximum voltage of 5V and maximum current of 0.5A whereas in order to produce 1.6 mT B which used in the experiment, it required the voltage up to 60V and 5A. For those requirements, the outside of Arduino was connected with the high current module with DC Mosfet 22A IRF5305 switch pwm driver to be traversed by the 60 V DC Power Supply and converted into pulse by the pulse generator. The pulses were driven through the coil, resulting in time varying magnetic field.
User Interface
The user interface made by LabVIEW software consists of two main functions. The first function is to set the pulse based on the desirable output with frequency parameter, duty cycle, burst time, and adjustable delay time. The other function was to measure the real time of magnetic field intensity and temperature in which the measurement result could be visualized and automatically saved into CSV format files. Moreover, timer was added to facilitate the user in adjusting the exposure duration during the experiment as the additional feature.
FIGURE 3. PEMF user interface was designed by LabVIEW consisting of pulse generator, Gauss meter, temperature meter, and timer.
Helmholtz Coil
The Helmholtz Coil (HC) is a pair of coils consisting of several copper wires coils which were flowed by an electrical current. Based on Biot Savart law, the electromagnetic field intensity (B) produced by HC in the core was calculated by the following equation:
3
4
25
o
NI
B R
(1)This study used a ∅60 cm HC with N=500 copper wire coil (18 AWG), the current at I = 1.3 A and the distance between coils is 30cm. The magnetic field intensity which was produced by the HC was measured by IDR 324 Gauss
meter (Integrity Design Research USA) resulting in Bmax of 1.6 mT. Figure 4 illustrated the PEMF configuration used in this study.
(a) (b)
FIGURE 4. Schematic representation of the PEMF exposure system: (a) PEMF consisting of Helmholtz coil, pulse generator and user interface. (b). Experimental set up of PEMF exposure on rats.
Data Acquisition System
The PEMF device was equipped with magnetic field sensor (UN 3505 of linear ratio metric magnetic sensor) and real time temperature sensors (KY-013 component temperature sensor) that connected to the Arduino at the same time. The measurement data was analyzed by the program to visualize the graphic and recorded into the text file in CSV format.
Animal Model of Non Union Fracture Healing
Preliminary study to the effect of SMF and PEMF exposure on fracture healing of delayed union model was done in the second stage of this study. Six adult male of Spraque Dawley rats (aged 12-16 weeks, weight 250-350 gr) were purchased from Animal laboratory of Litbangkes and acclimatized for a week prior for the experiment. Those rats were housed at 23-25oC with 12h light/dark cycle and given free access to water and standard laboratory pellets.
Finally, the rats were maintained and killed in a manner approved by the Ethics Committee of Faculty of Medicine, University of Indonesia No. 17-05-0489.
Surgical Procedures
The rats were anesthetized with an intraperitoneal injection of ketamine (70 mg/kg body weight) and xylazine (35 mg/kg body weight) as pre-operative treatment. Aseptic and antiseptic procedure was done to the left thigh and an incision of approximately 2 cm in length was performed at the posterolateral side of the left femur, by the 1/3 middle of the shaft. The fracture site was exposed and 5 mm of the periosteum was stripped on each side to create the delayed union model. Besides, a ∅1.2 mm K-wire was inserted into the femur in a retrograde fashion through the knee joint to stabilize the fracture. Lastly, soft tissues (fascia, subcutaneous, and skin) were sutured with absorbable sutures at the end of the procedure. Postoperatively, the rats received an intramuscular dose of penicillin and streptomycin (0.1 ml/kg of body weight) and acetaminophen (15 mg/kg of body weight) [3,10,11]. Clinical follow up of the surgical site was done every day to oversee any infection or other adverse events [3,10,11].
Exposure to PEMF
The rats were randomly divided into two groups of three rats as follow: group 1 treated by PEMF exposure (B max 1.6 mT, frequency 50 Hz, duty cycle 90%, burst cycle 50%) and group 2 treated by SMF exposure (B=1.6 mT). The treatment last for four hours per day, seven days per week until the end of 14, 21 and 28 days.
Histopathologic Examination by Histomorphometry
Upon the end of each week, one specimen of each group was dispatched for histologic analysis. After euthanasia, the left femur was resected immediately. The harvested femur was fixed in 10% neutral buffered formalin for 48 hours by maintaining the K wire. The subject was decalcified with Hydrochloric acid (ShandonTM TBD-1TM Rapid Decalcifier, Thermo Scientific). Subsequently, the femur of each rat was dehydrated in ascending alcohol concentrations. The specimens were embedded in paraffin and cut longitudinally with a microtome 5 μm section.
Histomorphometric analysis was done by imbuing Hematoxylin Eosin to a section of each specimen. Standardized histological images were obtained with Nikon Eclipse Microscope and analyzed by Image J software. Measurements were performed as follow: total area callus, osseous, cartilage and fibrous tissue.
RESULTS AND DISCUSSION Output Characterization of PEMF Device
(a) (b)
FIGURE 5. The output characteristic of PEMF device: (a) time varying (pulse) magnetic fields pattern (b) Magnetic field strenght to the duty cycle.
Histomorphometry Evaluation
Endochondral bone healing which predominates in recovery of long bone fracture requires the correct temporal and spatial coordination series of molecular and cellular events. Typically, bone fracture healing involves four overlapping phase: (1) inflammation, (2) soft callus (cartilaginous) formation, (3) hard (woven bone) callus formation and (4) woven bone remodeling [12]. Quantitative analysis of the histologic sample (histomorphometry) has proven to be a prominent tool for bone diseases assessment [13]. In the present study, the healing progression was evaluated at day 14, 21 and 28 by histomorphometry analysis. The result of the preliminary study was present in Fig. 6 and 7.
In the 14th day, calluses were recently dominated by the fibrous and cartilage tissues. However, at 21st day, the area of woven bone were dominant.
0 500 1000 1500 2000
1 40 79 118 157 196 235 274 313 352 391 430 469 508 547
Magnetic Fields (μT)
Time(ms)
y = 8.3185x + 366.17
0 200 400 600 800 1000 1200 1400
0 50 100 150
Magnetic Field (μT)
Duty Cycle (%)
(a) (b)
FIGURE 6. Histological image of the left femur longitudinal sections with delayed union fracture model treat by (a) SMF Exposure, (b) PEMF Exposure at 40x magnification. Tb: trabecular bone, Ca: Cartilage, Fc: fibrous connective tissue. Bar =
1000μ. Black arrow indicated the fracture site.
During the beginning of endochondral ossification, MSCs converted into chondroblasts thus started proliferate from the seventh to twenty-first days formed soft callus formation. It was shown that cartilaginous area in the PEMF group was smaller than the static group. Previous studies claimed that callus was replaced by new bone on the fourteenth days in the fracture site hence small cartilage regions were left. Diniz et al., suggested that continuation of a large cartilaginous along deficiency of trabecular bone ensues a delayed healing and premature callus formation in rodents.
Bone histomorphometry is a newly developed quantitative research method of bone tissues that brings possibility to convert 2D images of bone tissue slices into quantity data which facilitated the investigation of SMF and PEMF exposure towards bone callus during periods of fracture healing. The inspection of the callus tissue composition shows the difference area of fibrous tissue, cartilage and bone at each time. Fibrous tissues observed to dominate over 50%
of the callus area at 14th days post fracture. However, significant decrease occurred at 21st day. Besides, the specimen exposed to PEMF exhibit notable reduction of fibrosis area compared to the specimen exposed to SMF. Later,
D 14
D 21
D 28
substantial improvement in the fracture healing process derived from the depreciation of cartilaginous area past the 21st day. Overall, the percentage of bone tissue area in PEMF group was higher at 28 days post fracture compared to SMF group.
(a) (b) (c)
FIGURE 7. Calluses tissue composition of each specimen at (a) fibrous area, (b) cartilage area, and (c) bone area.
CONCLUSIONS
The PEMF prototype had successfully been designed and implemented in the preliminary study of animal model of delayed union fracture healing. The histomorphometry evaluation shows that PEMF exposure had greater osseous tissue area compared to SMF exposure, thus improve the healing process in delayed union fracture model. However, further examination for the PEMF mechanism need to be done and the effect of the exposure in molecular level has not been justified yet. Nonetheless, it has potential in bio electromagnetics application after refinement.
ACKNOWLEDGEMENTS
This study was supported by Doctoral Research Grant 2018 from Universitas Indonesia No.
1309/UN2.R3.1/HKP.05.00/2018.
REFERENCES
1. E. Antonova, T.K. Le, R. Burge, and J. Mershon, BMC Musculoskelet Disord 14, 42 (2013).
2. H.D. Ismail , P. Phedy, E. Kholinne, Y.P. Djaja, Y. Kusnadi, M. Merlina, N.D. Yulisa, Bone Joint Res 5(7), 287–293 (2016).
3. M. Orth, N.J. Kruse, B.J. Braun, C. Scheuer, J.H. Holstein, A. Khalil, X. Yu, W.L. Murphy, T. Pohlemann, M.W. Laschke, M.D. Menger, Eur Cell Mater 33, 1–12 (2017).
4. R. Singh, S. Bleibleh, N.K. Kanakaris, P.V. Giannoudis, Injury 47 Suppl 6, S33–S39 (2016).
5. R. Zura, G.J. Della Rocca, S. Mehta, A. Harrison, C. Brodie, J. Jones, R.G. Steen, Injury 46(10), 2036–2041 (2015).
6. R.M. Kowalchuk, S.R. Pollack, J Orthop Res 11(6), 874–883 (1993).
7. A. Ongaro, A. Pellati, L. Bagheri, C. Fortini, S. Setti, M. De Mattei, Bioelectromagnetics 35(6), 426–436 (2014).
8. B. Li, J. Bi, W. Li, S. Huang, S. Zhang, J. Zhao, Q. Meng, J. Fei, Technol Health Care 25(S1), 13–20 (2017).
9. K. Schaap, Y. Christopher-De Vries, S. Crozier, F. De Vocht, H. Kromhout, Ann Occup Hyg 58(9), 1094–1110 (2014).
10. J.C. Esteves, E. Jr. Marcantonio, A.P. de Souza Faloni, F.R. Rocha, R.A. Marcantonio, K. Wilk, G. Intini, J Transl Med 11, 221 (2013).
11. A. Fauzi, A.F. Kamal, A. Kurniawan, E. Kodrat, Br J Med Med Res 8(5), 419–428 (2015).
12. A. Schindeler, M.M. McDonald, P. Bokko, D.G. Little, Semin Cell Dev Biol 19(5), 459–466 (2008).
13. L.C. Gerstenfeld, T.J. Wronski, J.O. Hollinger, T.A. Einhorn, J Bone Miner Res 20(10), 1715–1722 (2005).
14. S.F. Diniz, F.P. Amorim, F.F. Cavalcante-Neto, A.L. Bocca, A.C. Batista, G.E. Simm, T.A. Silva, Braz J Med Biol Res 41(5), 373–379 (2008).
Day 14 Day 21 Day 28 SMF 0.23 0.89 0.75 PEMF 0.33 0.88 0.92 0.00
0.20 0.40 0.60 0.80 1.00
% Bone Area
Day 14 Day 21 Day 28
SMF 0.52 0.07 0.04
PEMF 0.56 0.05 0.03 0.00
0.10 0.20 0.30 0.40 0.50 0.60
% Fibrouss Area
Day 14 Day 21 Day 28
SMF 0.25 0.04 0.21
PEMF 0.11 0.07 0.05 0.00
0.10 0.20 0.30
% Cartilage Area
