Effects of Low-Dose Microwave on Healing of Fractures with Titanium Alloy Internal Fixation: An Experimental Study in a Rabbit Model

Background Microwave is a method for improving fracture repair. However, one of the contraindications for microwave treatment listed in the literature is surgically implanted metal plates in the treatment field. The reason is that the reflection of electromagnetic waves and the eddy current stimulated by microwave would increase the temperature of magnetic implants and cause heat damage in tissues. Comparing with traditional medical stainless steel, titanium alloy is a kind of medical implants with low magnetic permeability and electric conductivity. But the effects of microwave treatment on fracture with titanium alloy internal fixation in vivo were not reported. The aim of this article was to evaluate the security and effects of microwave on healing of a fracture with titanium alloy internal fixation. Methods Titanium alloy internal fixation systems were implanted in New Zealand rabbits with a 3.0 mm bone defect in the middle of femur. We applied a 30-day microwave treatment (2,450MHz, 25W, 10 min per day) to the fracture 3 days after operation. Temperature changes of muscle tissues around implants were measured during the irradiation. Normalized radiographic density of the fracture gap was measured on the 10th day and 30th day of the microwave treatment. All of the animals were killed after 10 and 30 days microwave treatment with histologic and histomorphometric examinations performed on the harvested tissues. Findings The temperatures did not increase significantly in animals with titanium alloy implants. The security of microwave treatment was also supported by histology of muscles, nerve and bone around the implants. Radiographic assessment, histologic and histomorphometric examinations revealed significant improvement in the healing bone. Conclusion Our results suggest that, in the healing of fracture with titanium alloy internal fixation, a low dose of microwave treatment may be a promising method.


Introduction
Internal fixation of broken bones was considered to be the most prevalent methods of fracture treatment [1]. However, a part of patients suffered from painful symptoms such as implant-related pain, delayed union or nonunion so forth after the surgery [2]. Typically, clinical hyperthermia induced by focused microwave has been applied to patients with muscleskeletal injuries [3][4][5][6][7]. The physiological effects of microwave are those of heat in deep the body generally. These include increased temperature over 40°C [8], increased blood flow [9,10], decreased pain [11], and alterations in the physical properties of fibrous tissues [12,13]. The clinic application of microwave can accelerate the resolution of haematoma and fracture healing, and increase range of movements of a joint by correcting contractures and decreasing viscosity of body fluids [3]. According to its effects, microwave treatment is clinically applied by doctors and therapist to the cure and rehabilitation in injuries bone [13][14][15][16][17][18]. However, a contraindication for microwave extensively documented in the literature is surgically implanted metal plates, screws, and pins in the treatment field [14]. As a kind of electromagnetic wave, microwave irradiation can be reflected, refracted or transmitted at boundaries [14,19]. Additionally, the eddy current stimulated by electromagnetic field could cause Joule heating of implants as well. All these factors would result in rapid temperature rise, even heat damages in local tissues [20,21]. The previous studies in vitro found that the temperature increase of implanted metallic plates might cause tissues ambustion in a frequency field of 900 MHz [22] and 27 MHz [23]. However, some doctors believed that this recommendation of contraindication appeared to be based on "common sense" and consensus rather than evidence-based practice in vivo. The findings of researches in vitro on radiofrequency (RF) electromagnetic field indicated that the presence of implantations should seldom be a risk, though 1800 [24] and 2450MHz [25,26] microwave irradiation could enhance the values of specific absorption rate (SAR). Besides, Seiger [27] and Draper [28] improved ankle range of motion (ROM) for patients with titanium alloy metal implants by pulsed shortwave diathermy. No discomfort, pain, or burning sensation was complained meanwhile. With regard to patient's safety, invasive temperature monitoring was not applied during the microwave treatment in the two clinic studies mentioned above. However, doctors and physical therapists were more concerned with the efficiency and safety of microwave exposure in patients who were implanted titanium and its alloys since such kind of implants are widely used in clinic.
The current study was based on the hypothesis that, due to its physical characteristics [29], a titanium alloy dose not disrupt the uniformity of the field in an electromagnetic field and cause no heat accumulation under a proper dose of microwave irradiation. Accordingly, we applied a low-dose microwave treatment on the fracture models of rabbit femurs with internal fixation titanium alloy. The security of the treatment was assessed by temperature measurement and histodiagnosis of the tissues adjacent to the implants. Moreover, to assess whether such a dose of microwave therapy might accelerate the fracture healing, radiographic assessment, histomorphometery and histologic grade of callus were investigated. The preclinical research we did was to provide evidence for microwave treatment on fractures with titanium alloy internal fixation devices.

Ethics statement
All the experimental procedures involving animals were conducted under a protocol reviewed and approved by the Animal Welfare and Ethics Committee of Shanghai Sixth People's Hospital (Permit Number: SYXK(沪) 2011-0128). All animal work was carried out in accordance with national and international guidelines to minimize suffering to animals.

Animal, treatment and grouping
Thirty-eight (18 females and 20 males) New Zealand healthy adult white rabbits weighing from 2.0 to 3.2 kilograms (an average of 2.5 kilograms) were used in this experiment. The animals were fed in Laboratory Animal Center at Shanghai sixth people's hospital, and were housed in a temperature, humidity, and light controlled environment.
After one week feeding adaptation, the animals were accurately weighed and randomly divided into three groups. Both the microwave treatment group (n=19) and implanted control group (n=16) were operatively implanted with a titanium alloy implants on bone fractures, while only the microwave treatment group received microwave treatment after the surgery. The non-implanted control group (n=3) was of bone lesions without titanium alloy implants, which was only used in the temperature measurement study (Table 1).

Animal model construction
The rabbits were anesthetized with an intravenous injection of pentobarbital sodium (30 mg/kg). The femur was exposed via longitudinal skin incision on the outside of right hind limb. Transverse osteotomy with a 3.0 mm bone defect was created in the middle of femur. The internal fixation system (LCP, Tplate 2.4, Synthes Company, USA) was composed of a titanium alloy plate (4.60 ± 0.24 X 0.42 ± 0.08cm) and screws, which were planted on the right femoral upper end of the implanted control group and microwave treatment group ( Figure 1). To act as an operated control, the non-implanted control animals underwent the same surgical procedure without titanium alloy implants. All rabbits were injected intramuscularly with penicillin (800,000 units) once per day during the 3 days following surgery.

Microwave treatment
Three days after operation, microwave therapy was applied on the treatment group. The treatment regimen consisted of the right upper thigh. The applicator (RM-170A, ITO Company, Japan) was connected to a 2450 MHz microwave generator (PM-800, ITO Company, Japan) with power output ranging from 0-200W. A 25W continuous-wave microwave exposure lasted for 10 min per day [15,30]. The non-contact applicator was perpendicular to the lesion and 10 centimeters away from the skin. Similar procedure was repeated for the implanted control group with the microwave apparatus in the off position. In the non-implanted control groups, the legs were observed without further microwave treatment. In order to exclude the influence of body temperature changes and other factors in one day, we provided all the microwave treatment at the same time of the day.

Temperature measurements
In all, 3 animals from the microwave treatment group and 3 animals from the non-implanted control group were used in this experiment. Anti-interference couple thermometer (FHC, ME-04008, BOWDOINHAM, USA), which is not influenced by the magnetic field and the radio frequency irradiation, was used for temperature measurements. The thermal probes of 8 centimeters length were placed into two sites for temperature measurements, including deep muscles (5mm above the middle hole of the titanium alloy plate) and superficial muscles (15mm above the hole). For placement in muscle, the vastus lateralis and the biceps femoris muscles were separated by blunt dissection, and the probes were placed with the recording side facing biceps femoris. The temperatures of non-implanted control group were tested in the same positions of the treatment regimen as well. The temperature was recorded every minute for 15 minutes while the microwave treatment was taking. The temperature of the laboratory was maintained at 24°C.

Radiographic assessment
The healing of fracture was assessed by anteroposterior plain radiographs. The same X-ray machine and settings were used for all radiographs just after the operation and on the 10th and 30th day of the microwave treatment. Also, the same light source was used for the optical densitometry. An optical densitometer was used to measure radiographic density of the radiographic films. However, there may be variations within radiographs with respect to film processing. A standard aluminum step wedge was used to calibrate the optical densitometer and check for its reproducibility. Radiographs of the wedge were taken 10 times, and the film's densities at each layer were measured by the optical densitometer (Table 2).

Histology and Histomorphometery
After 10 days of microwave treatment, four rabbits from each group were sacrificed and eight intact samples were obtained for histologic examination. The remaining animals were killed at the end of 30-day treatments. Four intact samples from each group in addition to 5 samples from the implanted control group and 6 samples from the microwave treatment group were included for the same examination. The specimens were fixed in 10% formalin and decalcified with 10% nitric acid. After being embedded in paraffin, 5µm-thick longitudinal sections were prepared and stained with hematoxylin and eosin or Masson's trichrome. Two slides from each specimen, one stained with hematoxylin-eosin and the other stained with trichrome, were examined. A panel of pathologists (LZW,JQ, DJX) who were blinded to the treatment allocation scored all the stained sections according to mineralization amounts of the fracture gap, using a grading system described by Perry et al [31], in which grade 1 indicated fibrous union, grade 2 indicated predominantly fibrous tissue with some cartilage, grade 3 indicated equal amounts of fibrous tissue and cartilage, grade 4 indicated all cartilage, grade 5 indicated predominantly cartilage with some woven bone, grade 6 indicated equal amounts of cartilage and woven bone, grade 7 indicated predominantly woven bone with some cartilage, grade 8 indicated woven bone, grade 9 indicated woven bone and some lamellar bone, and grade 10 indicated lamellar bone. The mean score was calculated for each group.
For histomorphometric evaluations of bone, undecalcified sections of 4 intact samples from each group were prepared after 10 and 30 days of microwave treatment. The specimens were fixed in 10% buffered formalin, dehydrated in increasing concentrations of ethanol, from 70% to 99% during 12 days, and embedded in methylmethacrylate. The 50-µm-thick sagittal sections were prepared using an electric diamond saw and grinding system, and stained with toluidine blue. Digital images of the sections were obtained by a digital camera attached to an microscope at a magnification rate of ×25. The images were transferred to a personal computer, and measurements at the fracture gap were made by histomorphometry software (simple PCI system; Hamamatsu, Sewickley, PA, USA). The fracture gap was selected for examination. The nomenclature and calculations for bone histomorphometry were applied in accordance with the report of the American Society for Bone and Mineral Research [32].
Muscular tissue and nervous tissue around implants were removed and fixed in 10% formalin at the end of the scheduled. The 5 µm sections were prepared and stained with haematoxylin and eosin following standard techniques for microscopic examination. For transmission electron microscopy, skeletal muscle tissue and nervous tissue were fixed with 2.5% glutaraldehyde for 16 h at 4°C, postfixed with 2% OsO 4 for 2 h at 4°C, dehydrated by an ascending ethanol series, passed through propylene oxide, and then embedded in resin. Ultrathin sections (90 nm thick) on mesh grids were

Statistic
Statistical analyses were performed with SPSS 19.0 for Windows (SPSS, Chicago, USA). All the results are presented as the mean value ± standard deviation. Differences between groups were tested using t-test or analysis of variance (ANOVA). For all analyses, P values were two-tailed, and P<0.05 was considered statistically significant.

Microwave Treatment Caused No Significant Heated Damage In Tissues Around The Implants
During the microwave irradiation, temperatures were measured in deep and superficial muscle tissues meanwhile. Temperature changes were show in table 3. No difference was found between non-implanted control group and microwave treatment group in peak temperatures of the deep muscles (39.73 ± 0.23°C vs. 39.83 ± 0.25, P=0.639) and superficial muscles (39.90 ± 0.10°C vs. 39.67 ± 0.21°C, P=0.155). Although the temperature changes (T peak -T vally ) of microwave treatment group in deep muscles were higher than those of non-implanted control group (3.57 ± 0.44 and 3.50 ± 0.36 respectively), no statistic difference was observed between the two groups (P =0.82). So was the temperature increasing in the superficial muscles (control group vs. treatment group: 3.77 ± 0.36 and 3.60 ± 0.41, P =0.28). We next investigated whether a long-term course of the on titanium implanted subjects could cause heat damage to the tissues around the implants. After 30-day microwave treatment, skeleton muscle tissues adjacent to the titanium alloy were investigated histologically. The muscles from the treatment and control group were stained with hematoxylin and eosin ( Figure  2 a, b). For the treatment group, most myocytes displayed normal morphology though swelling myocytes were observed occasionally in the treatment field. We further performed transmission electron microscopic analysis of the skeletal muscle. The muscle of the treatment group showed mitochondrial swelling and mitochondrial cristae loss, which were not observed in control group (Figure 2 c, d). The nerve tissues adjacent to the implants were observed with both light microscope and transmission electronic microscope. No abnormal morphological changes were investigated in either treatment or control group (Figure 2 e-h).

Microwave Treatment Accelerated Fracture Healing
Radiographic examinations showed that all injured femora were in the process of normal bone healing. Callus formation was seen in the control group as well as treatment group. But it was more evident in the microwave treatment group compared with the implanted control group, indicating a positive effect of microwave treatment ( Figure 3A). Within-group statistical analysis showed that, within the implanted control group and within the treatment group, this measure increased as time point increased (control group: P=0.001; treatment group: P<0.001; Figure 3B). Differences between implanted control and treatment groups were significant on the 10th day of the microwave treatment (P=0.03; Figure 3B). However, normalized radiographic density was not significantly different on the 30th day in the treatment group compared with that in the control group (P=0.12; Figure 3B).
Histological changes of bone were studied microscopically. There was no evidence of ambustion in osseous tissues after 30-day microwave treatment ( Figure S1). The histologic grade of callus in fracture gap was studied microscopically at two time points. At day 10, the mean histological grade was 2.75 ± 0.50 for the microwave treated group and 1.75 ± 0.50 for the implanted control group. There was a significant difference between the two groups (P=0.03). At day 30, the mean histologic grade was 7.08 ± 0.52 for the microwave treated group compared with 6.45 ± 0.69 for the implanted control group (Figure 4 and Table 4). The difference between the two groups was significant (P=0.025).
The histomorphometric data regarding the mean values of bone volume, trabecular thickness, trabecular separation, and node-terminus ratio were given in Table 4. Bone volume and node-terminus ratio were significantly different between implanted control and treatment groups after 10-day and 30day microwave treatment (all P<0.05). Additionally, significant difference in trabecular thickness were also observed between the two groups after 10-day microwave treatment (P=0.048)

Discussion
Metallic implants in treated regions are described as a contraindication for RF treatment because of the intense and highly heating occurrence. Two mechanisms have been proposed for such an undesirable heating. The reflection of electromagnetic wave from metal surfaces was thought to be  one reason [33]. Double dose of RF irradiation would heat the tissues adjacent to the metal implants. Comparing with osseous tissue, muscle tissues are of high water content which characteristically absorbs microwaves strongly because that microwave heats water preferentially [34]. Therefore, the present study was concerned with the temperature changes of muscles adjacent to the metal implants. Additionally, the eddy current stimulated by electromagnetic field is the other reason. The difference of eddy current intensity derived from the physical properties of the materials. Comparing with traditional medical stainless steel, titanium alloy is a kind of medical implants with low magnetic permeability and electric conductivity. For these reasons, lower RF heating was observed at the border of titanium alloy implants and phantom in vitro [35,36]. Implants made by titanium alloy are nonferromagnetic and produce much less artifacts than ferromagnetic implants made of stainless steel at magnetic resonance (MR) imaging [37][38][39]. RF heating of cobaltchromium alloy implants and titanium alloy implants was evaluated in vitro by Muranaka. The maximum temperature rise for titanium implant was obviously lower than that for cobaltchromium [35,40]. Hence, the current study was based on the assumption that there is no dramatic temperature raise in tissues around titanium alloy implants resulting from a low dose microwave exposure. The findings of our research showed that such a dose of microwave irradiation did not dramatically increase the temperatures in the skeletal muscle tissues around titanium alloy.
In the current research, the results show that the temperature increase of muscles adjacent to titanium alloy implants was lower than 40°C. A previous study found that enzyme and proteins in the cell began to degeneration when the body temperature goes over 43°C [41,42]. Cumulative equivalent minutes at 43°C (CEM 43 ) are the accepted metric for thermal dose assessment that correlates well with thermal damage in a variety of tissues. The calculation of CEM 43 involves knowledge of thermal history: CEM 43 = Δt * R 43-T Where Δt signifies summation over the length of exposure, T is the average temperature during time interval t, and R is a constant equal to 0.25 for T < 43°C and 0.5 for T >43°C [43,44]. The histology of skeleton muscle in pig revealed that a minor damage appeared at 30 CEM 43 [45]. The thermal dose scale was 240 CEM 43 for leg muscle of rabbits and pigs, which corresponded to an irreversible lethal dose for cells [46][47][48]. In the current study, the thermal doses (CEM 43 ) of microwave treatment group were 0.08 CEM 43 in deep muscles and 0.03 CEM 43 in superficial muscles (the data in table 3 analysed). However, we observed swelling myocyte occasionally though the peak temperatures were lower than 40°C in muscles. The reason might be the mitochondrial swelling and cristae loss, which prevented mitochondrial ATP/ADP exchange [49]. It was noteworthy that the sign of mitochondrial swelling was typical features of mitochondrial stress. The previous studies found that the mitochondria could change their size in a phasic manner, swelling when cells were exposed to the temperature of 40°C, and all these shape changes were reversible when the temperature returns to normal [50,51]. Thus we can conclude that such a dose of microwave treatment on the subject with titanium alloy can't cause irreversible damages to the muscle around the implants. However, burn injuries could happen with the increase of microwave dose. In our preliminary experiment, heat effects of different doses of microwave irradiation on the muscles adjacent to the titanium alloy implants were studied. We found that if the dose increased to 2,450MHz, 60W, the temperature of muscles around the implants would reach 43°C within 10min (data not shown). Moreover, microwave is relatively insensitive to high impedance and has a deeper penetration profile [52]. Since bone has low conductivity (high impedance), less Joule heat is caused by reflection of electromagnetic wave and electrical resistance compared with muscles. On the other hand, bone, especially cortical bone, has low heat-sink strength from tissue volume by blood perfusion. It means that a long duration of hyperthermia might induce heat damages in bones. Therefore, we adopted a shorter duration of microwave exposure mentioned by Chang [15]. The microscopical results of the current research provided confirmation that such a short-duration microwave treatment caused no heat damage in osseous tissue and bone marrow around the titanium alloy ( Figure S1).
The results of our current studies examine the effects of microwave continuous irradiation on promoting femoral fracture healing in rabbit. Many technical obstacles, such as temperature control, burns, and the fact that microwaves from an external source penetrate only 3-4 cm, are some of the reasons that microwave is not yet a clinically accepted modality of treatment. However, the use of microwave for extracorporeal heating of bone seems feasible. Using a dedicated microwave hyperthermia system, Leon et al. systematically examined the effect of microwave hyperthermia on bone both in vitro and in vivo [16,17]. They demonstrated that hyperthermia promoted bone deposition. The effect of microwave is mainly induced by the alternating electromagnetic fields, which activates the dipoles in the molecule of the material [16]. The collisions of the molecules cause generation of heat inside the object [53]. Due to the dose of microwave we  used, temperate changes were not dramatic in the treatment field. Although this heating effect is small, some enzymes are exquisitely sensitive to small variations in temperature [54]. It was reported that 0.24 W/cm 2 pulsed shortwave diathermy was safe for human with titanium implants [28,55], and 0.5 W/cm 2 continuous microwave could produce thermal effect on fracture rehabilitation in rabbit [15,16]. In the present study, the effective treatment area of the applicator we used was 138.9 cm 2 and the dose of continuous microwave was 0.18 W/cm 2 , which derived from a randomized clinical trial of microwave stimulation of bone healing we did previously. It was recognized as the minimum dose which has an effect on the femoral fracture patients without metal implants. In current study, we confirmed that the healing of fracture with titanium alloy implants could be accelerated by such low-dose of microwave treatment in animals. Although the dose we used was lower than that previous studies did, its security of femoral fracture fixed with titanium alloy plate was confirmed in rabbits. A favorable effect of microwave irradiation on bone union was also found, especially in the first 10 days treatment. The similar results were reported by Olchowik [56]. The results indicated that the low-dose of microwave therapy bring positive impacts in the early stage of fracture repair. But the mechanisms remained unclear. There seem to be three mechanisms involved. First of all, the most important physiological response induced by microwave is the regional increase in the blood flow [10]. Thus an increase in nutrients and oxygen in the treatment region would likely benefit to the bone healing. Additionally, microwave treatment has been advocated by several researchers to accelerate the resolution of haematoma which is diffusely observed near the fracture site in the early stage of bone trauma [9,57]. Moreover, at the molecular level, it is possible that microwave treatment accelerates or increases the release of local growth factors such as transforming growth factor β (TGF-β), fibroblast growth factor and platelet-derived growth factor -all of which have been reported to accelerate the earlier appearance and promote the proliferation of chondrocytes during the early phase of fracture healing [58,59]. A number of limitations of this study warrant mention. First, although there was no heat damage shown in histology, the temperature changes of bone should be observed in advanced researches because of the report that it could damage at 80 CEM43 [60]. Second, the small sample size of rabbits in each group, the limited number of specimens in temperature measurement and histomorphometery might not enable us to reach any firm conclusions. However, as all the results in this study were consistent with each other, we feel confident in our findings. Meanwhile, our further studies in the future with ample numbers of subjects and long-term follow-up may strengthen these findings and confirm the superiority of this treatment.

Conclusion
Our study in vivo proved that the continuous-wave microwave treatment, at 2,450MHz, 25W, 10 minutes per day, did not dramatically increase the temperature in muscle tissues around titanium alloy implants. It did not cause irreversible heat damage in the tissues for a 30-day treatment as well. Additionally, such dose of microwave improved the femoral fracture healing in rabbit. Our results suggest that, in the healing of fracture with titanium alloy internal fixation, a low dose of microwave treatment may be a promising method.