[ research report ] Effects of Low-Voltage Microamperage Stimulation on Tendon Healing in Rats

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1 Helen K.F. Chan, PT, MSc 1 Dicky T.C. Fung, PT, PhD 2 Gabriel Y.F. Ng, PT, PhD 3 Effects of Low-Voltage Microamperage Stimulation on Tendon Healing in Rats Achilles tendon ruptures are common in athletes and the middleaged, and there have been increasing reports of Achilles tendon ruptures in recent decades. 25,28 Despite the intrinsic capacity of spontaneous recovery in injured tendons, 26,27 the process often takes a long time and the qualities of the repaired tendons are inferior to those of intact tendons. 16,2,19,31,32 To restore the optimal function of injured tendons it is important to develop therapeutic interventions t Study Design: Randomized controlled prospective experimental study. t Objectives: To examine the effects of transcutaneous low-voltage microamperage stimulation () on the mechanical strength of Achilles tendon repair in rats at 4 weeks after injury. t Background: Understanding the effect of on the healing of injured tendons is hampered by the lack of related experimental studies, especially from the aspect of biomechanical outcome measures. that facilitate the process of healing. Low-voltage microamperage stimulation (), characterized as an electrical stimulation with very low current, is advocated to promote tissue healing. It is theorized that healthy tissue is the result of the direct flow of electrical current throughout the body. 6,4,39 It is the opinion of Becker and Sheldon 7 that when the body is injured, the electrical balance is disrupted in that particular region, causing the electrical current to change course. The use of over the injured site is thought to realign this t Methods and Measures: Fourteen 3-month-old male Sprague-Dawley rats received surgical transection to the medial portion of their right Achilles tendon. The rats were divided into a group (n = 7) and control group (n = 7). From day 6 postsurgery onwards, the group received daily treatment of transcutaneous (2.5 V, 1 μa/cm 2, 1 pulses per second, positive current) for a total of 22 sessions, while the control group received placebo by the same investigator during that period. On day 31, the Achilles tendons were harvested for biomechanical testing for load relaxation, stiffness, and ultimate tensile strength along the longitudinal direction. t Results: The normalized Achilles tendon ultimate tensile strength of the group (mean 6 SD, 11.5% 6 25.%) was higher than that of the control group (75.3% 6 2.8%) (P =.14), but no significant difference was found in normalized stiffness and load relaxation between the 2 groups (P =.239 and.35, respectively). t Conclusion: The results of this study suggest that the administration of transcutaneous could improve healing and consequently the tensile strength of partially transected Achilles tendons of rats at 4 weeks after injury. J Orthop Sports Phys Ther 27;37(7): doi:1.2519/jospt t Key Words: asymmetrical biphasic, biomechanical testing, electrical stimulation, tendon injuries flow, thus aid in tissue repair. 8 It has been shown that could increase adenosine triphosphate (ATP) concentration, promote amino acid uptake, and enhance protein synthesis in human fibroblasts. 9,14 In animal studies, was shown to facilitate osteogenesis in bone fractures of rabbit fibulas, 1,17 regeneration of transected sciatic nerves in rats, 36 and epithelization of surgically induced skin wounds in pigs and rats. 2,4 Clinical trials have been conducted on human subjects to evaluate the effects of on wound healing. The results indicated that the application of electric currents with intensities between 5 and 1 μa facilitated healing in various skin ulcers. 12,21 Despite the fact that the physiological effects of have not been thoroughly investigated, reports on the therapeutic actions of have suggested that this treatment modality is beneficial for soft tissue healing such as tendons and ligaments. 1,13,18,29,34,38 A few investigators have evaluated the efficacy of in tendons and ligaments healing. 1,13,18,29,34,38 Some studies have demonstrated that with implanted electrodes improves both biomechanical and histological properties of healing tendons in animals. 1,18,29,34,38 However, in a clinical environment, it is rare to perform on injured tendons and ligaments with implanted electrodes. A recent study by Chapman-Jones 13 demonstrated that a clinical type of transcutaneous could improve the clinical 1 Physical Therapist, Tuen Mun Hospital, Hong Kong SAR; Graduate student, Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hong Kong SAR, China. 2 Tutor, Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hong Kong SAR, China. 3 Professor, Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hong Kong SAR, China. The Animal Ethics Review Committee of the Hong Kong Polytechnic University reviewed and approved the procedures of this study. Address correspondence to Gabriel Ng, Department of Rehabilitation Sciences, The Hong Kong Polytechnic University, Hung Hom, Hong Kong, China. Gabriel.Ng@inet.polyu.edu.hk journal of orthopaedic & sports physical therapy volume 37 number 7 july

2 outcome after Achilles tendon injury in humans; but it is not known whether the strength of the healing tendon was restored. Therefore, the present study investigated the effect of transcutaneous on the biomechanical properties of Achilles tendon healing in rats at 4 weeks postinjury. METHODS Animals Fourteen 3-month-old male Sprague-Dawley rats were used in this study. The average mass at the beginning of the study was g (range, g). The Animal Ethics Review Committee of the Hong Kong Polytechnic University reviewed and approved the procedures of this study. The animals were randomly divided into 2 groups: LV- MAS (n = 7) and control (n = 7). Surgical Procedures All rats in both groups received the same surgical hemitransection injury to the right Achilles tendon of the back leg. The surgical procedures were carried out under general anesthesia with an intraperitoneal injection of a mixture of 1 mg/kg of ketamine and 2 mg/kg of xylazine. The skin on the medial aspect of the right calf was shaved, incised, and retracted to expose the Achilles tendon, and the medial and lateral bands were identified and separated with a probe. The medial band was then transected at the middle portion and the lateral band was left intact so as to simulate a partial tendon rupture and prevent retraction of the transected ends. 3,37 The skin wound was then sutured immediately. After the surgery, the animals were randomly assigned into either group by a technician not involved in the surgery. Two animals of the same group were kept in a cage of cm, with unlimited activities. All cages were kept inside a room with a 12-hour light/dark cycle, temperature regulated at about 2 C, and relative humidity at 5%. Tap water and food were given ad libitum throughout the study. Treatment The treatment started on day 6 postsurgery so as to let the skin wounds heal. Each animal received either or placebo stimulation according to its group allocation. All animals in both groups received 3 minutes of treatments, 6 times a week except on Sundays, until a total of 22 treatment sessions were received. This treatment regime was based on previous protocols that demonstrated that daily treatment of on injured tendons and ligaments significantly improved the mechanical properties of the tissues after 2 to 8 weeks. 1,13,34,38 A MICRO Plus electric stimulator (Bio- Medical Life Systems Inc, Vista, CA) was used for the treatment. During each session, the rat was kept in a light restrainer to enhance the placement of stimulation electrodes. Skin preparation was done by shaving the hair over the sites of the electrode placement. The anode electrode (1. 1. cm) was placed over the tendon injury site, while the cathode electrode was placed proximally on the calf of the same side, 1 cm apart. Both electrodes were secured by adhesive plaster. Conductive gel was applied as a coupling medium between the electrodes and skin. Animals in the group received electrical stimulation using the following parameters: waveform, asymmetrical square biphasic; pulse rate, 1 pulses per second; voltage, 2.5 V; current density, 1 μa/cm 2. The current output of the stimulator was calibrated with an amperage meter prior to the study. The batteries of the stimulator were fully charged before each treatment session to ensure adequate current output. To measure the waveform of the machine, an oscilloscope (MS614A Mixed Signal Oscilloscope; Agilent Technologies, Santa Clara, CA) was used to capture output pulse through a 1-k load at 2.5-V output. With the anode stimulation mode selected, the pulse was observed to be an asymmetrical biphasic square wave with an amplitude of 1 μa and pulse width of 25 μs (Figure 1). Because the machine delivered 1 pulse bursts per second with equal on/off time, the electric charges were present in half of the stimulation time. Each burst of stimulation contains short pulses of 25- μs duration, thus the accumulated charge was calculated to be 25 microcoulumbs (μc) per second. Because the negative phase of the stimulation was not square wave, the peak current was 1 µa and decayed exponentially to zero within 25 µs; therefore, the total charge of the negative current was estimated to be about 25% of the positive current. The control group received a comparable treatment but with the stimulator power turned off. This placebo treatment was applied to ensure that the electrical stimulation was the only treatment variable that differed between the and control groups. All other factors, such as restraining the animals and tactile input from the electrodes, were similar. To standardize the handling and testing procedures, only 1 person was responsible to provide treatment and the final testing for all the animals. Even though the examiner was not blinded to the grouping, examiner bias did not exist because the outcome measures were done objectively with a machine. Biomechanical Testing All rats were sacrificed on postsurgical day 31 with an overdose injection of ketamine and xylazine. Both lower limbs were harvested by disarticulation at the hip joints. The lower limb specimens were then sealed in plastic bags 25 µs 25 µs 1 µa.5 s 1 s FIGURE 1. Waveform of the low-voltage microamperage stimulation machine. The current is pulsed at.5- second intervals, with each pulse containing a train of asymmetrical biphasic electrical currents of 25 µs in duration and 1 µa in amplitude. 4 july 27 volume 37 number 7 journal of orthopaedic & sports physical therapy

3 and stored in a freezer at 4 C for later biomechanical testing. Before testing, the lower limb specimens were retrieved from the freezer and allowed to thaw for at least 6 hours inside the plastic bags at room temperature. Each leg specimen was carefully dissected. The lateral band of the Achilles tendon was removed at the musculotendinous junction, leaving only the medial band and calcaneus intact. The intramuscular tendinous fibers were mounted and secured between 2 strips of plastics with a quick-setting superglue (Aron Alpha; Toagosei Co Ltd, Tokyo, Japan). The specimen was then mounted on a MTS Synergie 2 machine (MTS Systems Corporation, Cary, NC), with an extensometer (MTS model F-24; MTS Systems Corporation) attached on the side. Room temperature was controlled at 25 C and the specimen was kept moist with normal saline throughout testing. Before the actual testing, the specimen was preconditioned with 1 oscillation cycles of 2.5% strain at a rate of 1 mm/min to minimize the effect of deep freezing on the tissue. 2,3,31,37,41 After preconditioning, the specimen was elongated to 2.5% strain and maintained at that elongation for 5 minutes. 2,3,31,33,37 The initial and final loads were recorded, and the change in load was expressed as a percentage of the original load, which denoted load relaxation. After the load relaxation test, the specimen was unloaded and left undisturbed for 5 minutes. It was then subjected to the failure test at a loading rate of 5 mm/ min. 2,3,31,37 Load and displacement data of the specimen during the test were recorded at a sampling rate of 5 Hz. A load displacement curve was then plotted. The maximum load on the curve represented the ultimate tensile strength (UTS), and the slope of the linear portion immediately after the toe region of the curve represented the stiffness of the specimen. Data Analysis The values of load relaxation, stiffness, and UTS of the transected medial portion of the right Achilles tendons were normalized against those of the intact medial portion of the left Achilles tendons of the same animal. These normalized values were then compared between groups using independent t tests with a set at.5. RESULTS On gross examination, the Achilles tendons in both groups showed signs of healing, with thick fibrotic scar formation around the injury sites. The healing Achilles tendons in both groups appeared to be larger than the intact tendons. Based on the t tests, the normalized UTS of the group (mean 6 SD, 11.5% 6 25.%) was significantly higher (P =.14) than for the control group (mean 6 SD, 75.3% 6 2.8%) (Figure 2). The effect size on UTS was calculated to be.61, based on the sample-specific mean and standard deviations (95% confidence interval of the difference between the 2 groups for the UTS data, 8.4%-61.9%). Statistical comparisons of the normalized load relaxation and stiffness between the 2 groups showed no significant differences (P =.35 and.239, respectively) (Figures 3 and 4). Calculation for the value of Cohen s d using the means and standard deviations of both groups revealed that the effect size was.32 for normalized stiffness and.27 for load relaxation. DISCUSSION The healing effects of with implanted electrodes have been demonstrated in tendons and ligaments of animals. 1,34,38 However, it is uncommon in clinical practice to deliver treatment with implanted electrodes. To our knowledge, this study is the first report on the effects of transcutaneous on the biomechanical properties of Achilles tendon healing in rats. The findings may, therefore, have clinical implications on the application of for treating tendons and ligaments. We found that the UTS of injured Achilles tendons improved significantly with treatment at 4 weeks postinjury when compared with the controls. This finding is in agreement with previous studies using with electrode implants. 1,34,38 The previously reported physiological effects of include promoting ATP production, increasing amino acid uptake, 14 enhancing active secretion of tenocytes, 17 and facilitating collagen synthesis. 14,29 These may explain the underlying mechanisms of in tissue healing, leading to the better Normalized Ultimate Tensile Strength (%) Mean = 11.5* SD = 25. Mean = 75.3 SD = 2.8 FIGURE 2. Data for normalized ultimate tensile strength of the low-voltage microamperage stimulation () and control groups. * The mean for the group was significantly higher than that of the control group (P =.14). Normalized Load-relaxation (%) Mean = 11.2 SD = 25.5 Mean = 13. SD = 42.6 FIGURE 3. Data for normalized load relaxation of the low-voltage microamperage stimulation () and control groups. No significant difference between groups (P =.35). Normalized Stiffness (%) Mean = 94.3 SD = 52. Mean = 67.7 SD = 22.8 FIGURE 4. Data for normalized stiffness of the low-voltage microamperage stimulation () and control groups. No significant difference was found between groups (P =.239). journal of orthopaedic & sports physical therapy volume 37 number 7 july 27 41

4 biomechanical recovery of the tendons reported in the current study. In this study, the 95% CI of the difference in normalized UTS between groups was 8.4% to 61.9%. This raises a question of whether the magnitude of difference at the low end (8.4%) has reached a minimal clinically important difference. 22 However, because the value of minimal important difference in tensile strength of rat tendons is not available in the literature, the above question cannot be answered in this study. For the parameter of strain, it is reported that the normal functional strain of tendons is between 3% to 4%. 23 The other structural property studied was stiffness, which is a measurement of the resilience of the specimen under submaximal loading, whereas UTS is the measurement of its maximal failure load. 2,31,33 The mean stiffness of tendons of the group was higher than the control group (Figure 3), but the difference was not statistically significant (P =.239). Besides structural properties, one should also consider the viscoelastic properties, such as load relaxation of the healing tissues when subject to continuous loading. 35 The group exhibited less load relaxation than the control group (Figure 2), but the difference was also not significant (P =.35). For the stiffness and load relaxation findings in the present study, the post hoc power analysis for both outcome measures indicated very low power (.32 and.27, respectively); thus the chance of committing a type II error cannot be ruled out. In this study, due to the lack of information in the literature on a minimal clinically important difference for stiffness and load relaxation, the post hoc power analyses were derived from the sample specific variance and effect size using the statistical software package PASS (NCSS, Kaysville, Utah). Substituting the observed effect sizes for the unknown minimal clinically important difference may have underestimated or overestimated the relevant statistical power to detect a nontrivial difference. But the data would suggest that the positive patterns of change in both stiffness and load relaxation in the group warrant further study with larger sample size to make the findings more conclusive. The efficacy of the on tissue healing may be closely related to the selected treatment parameters. A few researchers reported that cathodal ( with the cathode on the wound) could enhance tendon healing. 1,29,38 However, when comparing the effects of different polarities, Owoeye et al 34 demonstrated that tendons treated with anodal ( with the anode on the wound) had a significantly greater breaking strength than those treated with cathodal stimulation. Based on this finding, 34 the anodal mode was chosen in the present study, but there is no literature on the effects of polarity on collagen formation or fibroblast activity. In previous studies, the current intensities ranged from a few microamperes 1,38 to several hundred microamperes 2 for treating the collagenous tissues. Studies with implant electrodes used current amplitudes at 7 µa, 29 1 µa, 1 and 2 µa, 38 which were substantially lower than those of the present study of 1 µa. However, comparison with these studies may not be very meaningful because of the factor of skin impedance cannot be accounted for. In a study with rats, Cheng et al 14 examined the effects of at various current intensities on ATP generation, protein synthesis, and membrane transport. They reported that the majority of the biostimulatory effects occurred within the lower range from 1 to 5 μa. Based on this report, we chose the current intensity of about 1 μa for this study. The healing effects of this low current intensity was also supported by 2 other studies on rat skin and tenotomized rat Achilles tendon. 3,34 In both studies, the biomechanical properties of the healing skin and Achilles tendon were found to be significantly improved after treatment. However, this current intensity is substantially lower than that used by Feedar et al, 15 who reported that a current of 29.2 ma had significant beneficial effect on ulcer wound healing in humans. The discrepancies could be due to the difference in pathologies, as over 7% of the subjects in Feedar et al s 15 study suffered from changes in tissue vascularity, such as pressure sores. To fully describe the dosage of LV- MAS, the current density should be considered along with the current intensity. Due to the different sizes of the electrodes, the actual dosages may differ among studies with the same current intensity. For example, both Byl et al 11 and Leffmann et al 24 reportedly applied 1 μa of on surgically induced wounds. However, they used different electrode sizes, thus the actual current densities were.96 and 297 μa/cm 2, respectively. Therefore, to clarify the actual dosage applied in the treatment, both the current intensity as well as the sizes of the electrodes were described in the present study. Recently, protocols for have been changed from the continuous mode to the pulsed mode, 5 but there is a lack of studies that investigate the pulsing effect of on tissue healing. Some studies reported negative findings on LV- MAS at very low pulse rates of between.1 to.3 pulses per second in induced wounds. 11,24 For a higher pulse rate of 1 pulses per second, Owoeye et al 34 reported a positive result, which has provided the foundation on the choice of pulse rate used in the present study. There are some limitations in this study that should be considered when extrapolating the results to humans. The very low range of current intensities in would not be perceived by humans. Whether it is the same in animals is not known. However, based on our observations that both groups had no difference in their behaviors during the treatment, it was believed that the placement of electrodes was the only perceivable sensory input to both groups. 42 july 27 volume 37 number 7 journal of orthopaedic & sports physical therapy

5 To standardize the injury mode in both groups, a surgical transection of the Achilles tendon was performed. It should be noted that the healing of a surgically induced wound may differ from a tendon rupture as a result of loading, because the collagen fibrils would be disrupted during the injury loading and the broken ends would appear irregular instead of uniform across the surgical incision. This issue needs to be studied in future research. CONCLUSION We conclude that the application of daily transcutaneous (2.5 V, 1 μa/cm 2, 1 pulses per second) over 22 treatment sessions improved the tensile strength of partially transected Achilles tendons of rats at 1 month after injury. This study has provided a clinical basis for future study of noninvasive application of LV- MAS on soft tissue repair in humans. t references 1. Akai M, Oda H, Shirasaki Y, Tateishi T. Electrical stimulation of ligament healing. An experimental study of the patellar ligament of rabbits. 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