Aims In this study we examined the effect of electroacupuncture (EA) stimulation on the mechanical strength of the rat Achilles tendon after long-term recovery.
Methods Using 20 rats, an Achilles tendon rupture model was created in an invasive manner. The rats were assigned to one of three groups, that received EA treatment (EA group), minimal acupuncture (MA group) or remained untreated (Control group). In the EA group, EA stimulation (5 ms, 50 Hz, 20 µA, 20 min) was applied to the rupture region over a period of 90 days (five times/week). In the MA group, needles were inserted into the same positions as in the EA group but no electrical current was applied. After 90 days the tendon was measured to calculate the cross-sectional area of the rupture region. Then, the mechanical strength of the tendon was measured by tensile testing.
Results No significant differences were observed between the three groups in cross-sectional area of the injured tendon. For maximum breaking strength, the EA group showed a significantly higher threshold compared with the Control group (P<0.05) but not the MA group (P=0.24). No significant difference was seen between the MA group and the Control group (P=0.96).
Conclusion Given the EA group showed a significant increase in maximum breaking strength, it is likely that EA stimulation increases the mechanical strength of a repaired tendon after long-term recovery, and EA stimulation could be useful for preventing re-rupture.
- tendon repair
- mechanical strength
- long term recovery
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Tendon rupture is a common injury. In addition to occurring while engaging in athletic activities, it can occur even while performing normal daily activities due to degeneration of the tendinous tissue. Of all types of tendon rupture, Achilles tendon rupture has the highest frequency of occurrence and is cited as a typical example of tendon rupture. In the past, Achilles tendon rupture was mainly treated surgically, but presently, many experts recommend conservative treatment.1 However, if immobilisation due to conservative treatment is prolonged, the ruptured tendon may conglutinate to the surrounding tissue and the joint may contract. To avoid these complications, the tendon repair post-rupture must be prompt and complete to allow movement and weight-bearing soon after injury. For that reason, several physical therapies have been attempted to accelerate the restoration of the tendon.2 3
We have reported in our previous studies of the effects of acupuncture and moxibustion on tissue regeneration and restoration4–7 that direct current electroacupuncture (EA) stimulation accelerates the regeneration of peripheral nerves as well as ossification. From such a background, we began our study with the aim of developing the application of EA as a method for accelerating tendon repair. In our previous study in a rat Achilles tendon rupture model, we verified that EA stimulation to the injured region soon after the tendon was ruptured accelerated tendon repair.8 However, regardless of the treatment method applied, it is known that a tendon that has ruptured once is highly likely to re-rupture. Therefore, interventions capable of protecting against re-rupture of the repaired tendon would be desirable. To prevent the re-rupture of the tendon, the repaired tendon needs to regain high mechanical strength. To determine whether or not physical treatments, including EA, are effective not only in the early repair of the ruptured tendon but also in increasing the mechanical strength of the repaired tendon, a long-term follow-up observational study in a tendon rupture model is required. Accordingly, using a rat model similar to our preceding study, we aimed to perform EA stimulation under the same conditions and, after rupturing the rats’ Achilles tendons, measure the mechanical strength of the repaired tendons once the repair was expected to be complete.
This study was conducted with the approval of the Ethics Committee of Meiji University of Integrative Medicine (approval no. 20111014). This research was carried out in accordance with guidelines equivalent to the NIH ‘Guide for the Care and Use of Laboratory Animals’. Twenty Wistar rats (male, weighing 270–290 g) were used as experimental animals. Over the course of the experiments, except when treatments were administered, the rats were allowed to eat, drink and move freely in a plastic cage under a 12/12 hour light/dark cycle in a room maintained at a constant temperature. The number of experimental animals was determined based on the results of our prior studies.8
A rat model of an Achilles tendon rupture in the right hind leg was created. A mixture of ketamine hydrochloride (90 mg/kg) and xylazine hydrochloride (10 mg/kg) was administered to the abdominal cavity for general anaesthesia. Before the creation of the Achilles tendon rupture, to avoid motion at the ankle joint, a 23 gauge needle (diameter 0.60 mm, length 60 mm, Terumo Corp, Japan) was percutaneously inserted from the tibial tuberosity to the medullary cavity to completely fix the ankle joint in a position of plantar flexion. The skin of the facies posterior cruris was cut open about 10 mm along the vertical axis to expose the Achilles tendon. The Achilles tendon was separated from the plantaris muscle tendon and sharply cut free at proximally 5 mm from its attachment. A disposable surgical scalpel was used to separate the Achilles tendon, which was completely ruptured in the traverse direction at proximally 5 mm from its insertion into the calcaneus. The two ends of the cut Achilles tendon were brought together and the skin was sutured under a stereoscopic microscope. After the skin was closed, the wound region was sterilised with a povidone iodine solution. The method used in our preceding study8 was followed when creating this model. The Achilles tendon rupture model rats were randomly assigned to three experimental groups by investigators who were not involved in creating the models.
Electroacupuncture group (EA group, n=7)
EA stimulation was applied to the EA group while their extremities were restrained, from the day after the model was created until the day of evaluation at a frequency of 5 days/week. Two needles (diameter 0.25 mm, length 30 mm, stainless steel, Seirin Corp, Japan) were percutaneously inserted such that the tip of one needle would touch the inside and the tip of another needle would touch the outside of the Achilles tendon rupture region. For electrical stimulation, an electronic stimulator (SEN-3301; Nihon Kohden, Japan) and an isolator (SS-104J; Nihon Kohden) were used. Intermittent direct current EA was applied using the inner side and the outer side as a cathode and an anode, respectively. The conditions set for electrical stimulation were a pulse width of 5 ms, single polarity square wave, stimulation frequency of 50 Hz, stimulation strength of 20 µm, and stimulation time of 20 min/day.
Minimal acupuncture group (MA group, n=6)
As with the EA group, needles were inserted at the same locations for the same time period to the same depth as the EA group from the day after the model was created until the day of evaluation at a frequency of 5 days/week, with the extremities restrained. No electrical stimulation was provided.
Control group (n=7)
For the Control group, the extremities were restrained (without needle insertion or electrical stimulation) from the day after the model was created until the day of evaluation at a frequency of 5 days/week.
In this study, it was decided to measure the weight of each rat approximately once a week. Assuming the weight before creation of the model was 100%, it was determined a priori that, if any rat lost more than 15% of their weight or displayed abnormal behaviour (such as an inability to eat or drink or a self-inflicted injury), the rat would be removed from evaluation; however, no rats showed weight loss exceeding the pre-specified threshold or abnormal behaviour during the course of the study.
At the end of the study, all experimental animals were euthanised with an overdose of intraperitoneal pentobarbital solution (≥100 mg/kg) and the following parameters were evaluated.
Longitudinal and transverse diameters of Achilles tendon rupture region
Using a digital calliper (IP67 Absolute: Mitutoyo Corp, Japan), the longitudinal and transverse diameters of the Achilles tendon rupture region were measured and its cross-sectional area was calculated (assuming an elliptical shape).
Maximum breaking strength of repaired tendon
Ninety days after the model was created, the maximum breaking strength of the repaired tendon was measured by tensile testing, using a small desk-top test instrument (EZ Graph: Shimadzu Corp, Japan). After euthanasia, the repaired tendon was collected along with the triceps surae muscle and calcaneus. In order to attach the repaired tendon to a clamp for tensile testing, muscular tissue proximal to the repaired tendon was removed from the tendon fibre by blunt dissection. The tendon fibres from which the muscular tissue was removed were gathered together and attached to the clamp. A maximum load capacity of 100 N and testing rate of 0.05 mm/s were set as the conditions for tensile testing and the tendon was stretched until it ruptured, at which point the maximum breaking strength measurement was recorded. Investigators were blinded to experimental group allocation.
Results are presented as mean±SD. For comparison of the longitudinal and transverse diameters, the cross-sectional area and the maximum breaking strength of the Achilles tendon rupture region between groups, a one-way analysis of variance (ANOVA) was used and followed up with Bonferroni/Dunn multiple comparison tests as post hoc analysis. Statview version 4.5 (SAS Institute, Japan) was used for statistical processing and the significance level was set at 5%.
Longitudinal diameter, transverse diameter and cross-sectional area of the Achilles tendon rupture region
Figure 1 shows the measurements of the most swollen area of the Achilles tendon rupture site. The longitudinal diameter was 2.70±0.32 mm in the EA group, 2.63±0.32 mm in the MA group, and 2.61±0.31 mm in the Control group. No significant differences were observed between the three groups (EA vs MA: P=0.668, EA vs Control: P=0.655, MA vs Control: P=0.668). The transverse diameter was 2.77±0.59 mm in the EA group, 2.77±0.31 mm in the MA group, and 2.71±0.51 mm in the Control group and no significant differences were observed between the three groups (EA vs MA: P=0.617, EA vs Control: P=0.898, MA vs Control: P=0.721). The cross-sectional area of the tendon rupture site calculated from the longitudinal and transverse diameters was 5.92±1.68 mm2 in the EA group, 5.77±1.41 mm2 in the MA group, and 5.47±0.72 mm2 in the Control group. Although the EA group showed the highest value, no significant differences were observed between the three groups (EA vs MA: P=0.775, EA vs Control: P=0.749, MA vs Control: P=0.886) (figure 2).
Maximum breaking strength of the repaired tendon
The maximum breaking strength of the repaired tendon was 48.09±7.43 N in the EA group, 32.67±8.22 N in the MA group, and 32.98±15.56 N in the Control group. Compared with the MA and Control groups, the EA group showed a significantly higher value (EA vs MA: P<0.05, EA vs Control: P<0.05). No significant difference was observed between the MA group and the Control group (MA vs Control: P=0.962) (figure 2).
In our preceding study using a similar rat model, the increased expression of various growth factors (transforming growth factor-β1 (TGF-β1), basic fibroblast growth factor (b-FGF)) at the repaired site resulting from EA stimulation applied shortly after the rupture was verified by applying direct current EA stimulation to the tendon rupture region.8 In addition, an increase in mechanical strength of the repaired tendon was confirmed.8 The results of our preceding study suggest that EA stimulation has the potential to accelerate the speed of tendon repair. If the tendon adhesion is completed promptly, the prevalence of various complications caused by a prolonged healing process may be suppressed. While this is valuable in its clinical application, the issue of re-rupturing long after the initial rupture still remains. Hence, we aimed to investigate whether or not EA stimulation could be useful in resolving this issue. In this study, we examined the effect of continuous EA stimulation over a long period of time under the same stimulation conditions as the preceding study. During the tendon repair process, the tendon passes through the stages of inflammation, growth and remodelling, and it is said that this process takes about 2–3 months after the rupture in the case of a rat’s Achilles tendon. Based on this, in the current study we set an evaluation date of 90 days after the rupture, which is considered to be a sufficiently long period of time for tendon adhesion to have completed. The weaker mechanical strength of a repaired tendon is considered to be a possible cause of re-rupturing of said tendon, compared with an intact tendon, so as an index to verify the mechanical strength of the repaired tendon, we measured the maximum rupture strength—that is, the amount of energy required to rupture the tendon by applying tensile force to the repaired tendon.
In this study, since the EA group showed a significant increase in the maximum rupture strength of the repaired tendon, EA stimulation was considered likely to have had a positive effect on the mechanical strength of the repaired tendon after long-term recovery following the rupture. In clinical practice, re-rupture of the tendon after the completion of repair and joint contracture are major issues. The results of this study suggest that EA stimulation can promote tendon recovery, not only by accelerating the repair process but also by acting beneficially on the ultimate mechanical strength of the repaired tendon. The mechanical strength of a tendon is thought to depend on the gross weight of the constituent elements of tendinous tissues including fibroblasts in the repair region or the extracellular matrix, such as collagen synthesised by these cells.9 10 Almeida et al reported that the diameter of collagen fibres of damaged tendons increases with EA at acupuncture points ST36 and BL57 in the lower extremity of a damaged Achilles tendon in a rat model.11 However, in the current study, there was no significant difference compared with the Control group in terms of the cross-sectional area of the repaired tendon at the same time.8 Also, this time no significant difference was observed among the groups in the cross-sectional area of the repaired tendon, which indicates that it is unlikely that EA, as a means of increasing the mechanical strength of the repaired tendon after a long period of time has elapsed, acts on the quantity of tendinous tissue, but instead influences the density or arrangement of collagen fibres. To verify this, a histological study is required, and additionally it is necessary to investigate the current application period that causes this effect.
As mentioned above, in our preceding study, a histological change in the early stages after the rupture was confirmed. Some growth factors, such as cytokines involved in the process of tendon repair, are persistently expressed and affect the repair. In particular, TGF-β1 is recognised to reach its peak expression during the inflammatory stage.9 It is also known that b-FGF is produced and acts during the growth stage together with platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF).10 The effect of these growth factors on the expression in the early stages of the repair process may have improved the mechanical strength of the repaired tendon after a long-term recovery; on the other hand, as we did in this study, applying acupuncture stimulation continuously over a long period of time until the repair process is completed may have directly acted on the structure of collagen fibres, leading to positive results. It is important to clarify what current application period would be required for the purposes of clinical application, and this could be accomplished in the future by comparing EA stimulation only during the early stages after the tendon ruptured against the results of this study. In addition, in this study, EA was applied under the stimulation conditions that produced positive results in our preceding study, but an in-depth investigation is required to determine optimal conditions.
In studies related to tissue regeneration/repair, in the case of peripheral nerve regeneration, peripheral cathode acupuncture stimulation (in which needles are inserted at a nerve tract sandwiching the injured region and the needle farther away from the injured region is set as the cathode) has shown good results.4 5 In the case of bone fusion, Yasuda discovered that new bone is created at the cathode side by applying direct current electrical stimulation, and Friedenberg et al compared the effect of the position of the electrodes on the rate of healing in bone fractures12 13; moreover, in the study conducted by these authors, direct current EA stimulation using the needle inserted at the fracture site as the cathode has shown good results.6 7 While the effects of electrical stimulation such as those in this case have been shown, the possible appearance of neurotransmitter and neurotrophic factors as well as a potential reduction in mechanical hypersensitivity by neuromodulation have been suggested as possible effects of acupuncture without the application of an electrical current.14 15 However, in our earlier study and the studies by Almeida et al, a significant difference was observed between manual needle acupuncture and EA. Based on this, the effects of EA are considered greater than the effects of needle insertion alone in the case of tendon repair. When these findings are considered alongside the results obtained so far regarding the regeneration of peripheral nerves and promotion of bone fusion, it is inferred that the effects due to the electrical polarity of electrical stimulation are greater. However, regarding the tendon repair, both in the preceding study and this study, the locations in which two needles were inserted and used as electrodes were very close to each other and a clear difference due to the difference in electrodes was not confirmed, so we cannot assess the effect of polarity.
Even in past reports in which the effect of electrical stimulation was investigated using embedded type electrodes for tendon repair, none of them conducted comparison tests; therefore, we think further study is required to establish the ideal stimulation conditions, including direct versus alternating current and the insertion locations of needle electrodes. Furthermore, in this study, it was only possible to grasp the potential effect of EA stimulation on the mechanical strength of a repaired tendon after a long-term recovery as a phenomenon, and at this moment it is difficult to clarify the mechanism. Further investigation in this area is also required.
We conducted an investigation using a rat Achilles tendon rupture model for the purpose of studying the effect of direct current EA on the mechanical strength of a repaired tendon after long-term recovery following the tendon rupture. We have verified that continuous application of EA stimulation can increase the maximum breaking strength of the repaired tendon. From the results of this study, we feel that direct current EA stimulation has the potential to improve the mechanical strength of a tendon that has been repaired long after the initial rupture.
We are very grateful to Professor Naoto Ishizaki (Department of Clinical Acupuncture and Moxibustion, Meiji University of Integrative Medicine) for his valuable suggestions. We would also like to thank Mr Keisuke Yamamoto and Mr Yohei Suido (graduate students, Doshisha University) for their help in performing the force testing.
Contributors MI designed the study, conducted the research, analysed the data and wrote the manuscript. TH and KT analysed and interpreted the data. MI and HK analysed the data and supervised the study. MI critically revised the article for important intellectual content and retained overall control. All authors approved the final version of the manuscript accepted for publication.
Funding This research was carried out with funding from the scientific research fund for 2014–2015 (Young researchers research B, Research Subject Number: 26870719) and the Meiji University of Integrative Medicine research fund for 2015 (Research Classification: Focused research).
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.
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